Williams Textbook of Endocrinology

Larsen: Williams Textbook of Endocrinology, 10th ed., Copyright © 2003 Elsevier Section 1 - Hormones and Hormone Actio...

Author: P. Reed Larsen MD FACP FRCP | Henry M. Kronenberg MD | Shlomo Melmed MD | Kenneth S. Polonsky MD | Jean D. Wilson | Daniel W. Foster | Henry M. Kronenberg

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Larsen: Williams Textbook of Endocrinology, 10th ed., Copyright © 2003 Elsevier

Section 1 - Hormones and Hormone Action 1 - Principles of Endocrinology 2 - The Endocrine Patient 3 - Genetic Control of Peptide Hormone Formation 4 - Mechanism of Action of Hormones That Act on Nuclear Receptors 5 - Mechanism of Action of Hormones That Act at the Cell Surface 6 - Laboratory Techniques for Recognition of Endocrine Disorders Section 2 - Hypothalamus and Pituitary 7 - Neuroendocrinology 8 - Anterior Pituitary 9 - Posterior Pituitary Gland Section 3 - Thyroid 10 - Thyroid Physiology and Diagnostic Evaluation of Patients with Thyroid Disorders 11 - Thyrotoxicosis 12 - Hypothyroidism and Thyroiditis 13 - Nontoxic Goiter and Thyroid Neoplasia Section 4 - Adrenal Cortex and Endocrine Hypertension 14 - The Adrenal Cortex 15 - Endocrine Hypertension Section 5 - Reproduction 16 - The Physiology and Pathology of the Female Reproductive Axis 17 - Fertility Control: Current Approaches and Global Aspects 18 - Disorders of the Testes and the Male Reproductive Tract 19 - Sexual Dysfunction in Men and Women Section 6 - Endocrinology and the Life Span 20 - Endocrine Changes in Pregnancy 21 - Endocrinology of Fetal Development 22 - Disorders of Sex Differentiation 23 - Normal and Aberrant Growth 24 - Puberty: Ontogeny, Neuroendocrinology, Physiology, and Disorders 25 - Endocrinology and Aging Section 7 - Mineral Metabolism 26 - Hormones and Disorders of Mineral Metabolism 27 - Metabolic Bone Disease 28 - Kidney Stones Section 8 - Disorders of Carbohydrate and Lipid Metabolism 29 - Type 2 Diabetes Mellitus 30 - Type 1 Diabetes Mellitus 31 - Complications of Diabetes Mellitus 32 - Glucose Homeostasis and Hypoglycemia 33 - Obesity 34 - Disorders of Lipid Metabolism Section 9 - Polyendocrine Disorders 35 - Pathogenesis of Endocrine Tumors 36 - Multiple Endocrine Neoplasia 37 - The Immunoendocrinopathy Syndromes Section 10 - Paraendocrine and Neoplastic Syndromes 38 - Gastrointestinal Hormones and Gut Endocrine Tumors 39 - Endocrine-Responsive Cancer 40 - Humoral Manifestations of Malignancy 41 - Carcinoid Tumors, Carcinoid Syndrome, and Related Disorders Appendix: Reference Values

Larsen: Williams Textbook of Endocrinology, 10th ed. , Copyright © 2003 Elsevier

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Williams Textbook of Endocrinology

TENTH EDITION P. Reed Larsen MD, FACP, FRCP Professor of Medicine Harvard Medical School Chief, Division of Endocrinology, Diabetes and Hypertension Brigham and Women's Hospital Boston, Massachusetts

Henry M. Kronenberg MD Professor of Medicine Harvard Medical School Chief, Endocrine Unit Massachusetts General Hospital Boston, Massachusetts

Shlomo Melmed MD Senior Vice President, Academic Affairs Cedars Sinai Research Institute Professor of Medicine and Associate Dean University of California, Los Angeles, School of Medicine Los Angeles, California

Kenneth S. Polonsky MD Adolphus Busch Professor and Chairman Department of Medicine Professor, Department of Cell Biology and Physiology Washington University School of Medicine St. Louis, Missouri

SAUNDERS An Imprint of Elsevier Science

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SAUNDERS An Imprint of Elsevier Science The Curtis Center Independence Square West Philadelphia, Pennsylvania 19106 WILLIAMS TEXTBOOK OF ENDOCRINOLOGY ISBN 0-7216-9184-6 Copyright © 2003, 1998, 1992, 1985, 1981, 1974, 1968, 1962, 1955 by Elsevier Science (USA). Copyright 1950 by W.B. Saunders Company Copyright renewed 1990 by A.B. Williams, R.I. Williams Copyright renewed 1983 by William H. Daughaday Copyright renewed 1978 by Robert H. Williams All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Notice Medicine is an ever-changing field. Standard safety precautions must be followed, but as new research and clinical experience broaden our knowledge, changes in treatment and drug therapy may become necessary or appropriate. Readers are advised to check the most current product information provided by the manufacturer of each drug to be administered to verify the recommended dose, the method and duration of administration, and contraindications. It is the responsibility of the treating physician, relying on experience and knowledge of the patient, to determine dosages and the best treatment for each individual patient. Neither the publisher nor the editor assumes any liability for any injury and/or damage to persons or property arising from this publication. THE PUBLISHER

Library of Congress Cataloging-in-Publication Data Williams textbook of endocrinology/P. Reed Larsen ... [et al.].10th ed. p.; cm. ISBN 0-7216-9184-6 1. Endocrinology. 2. Endocrine glandsDiseases. I. Williams, Robert Hardin. II. Larsen, P. Reed. RC648 .T48 2002 616.4dc21 DNLM/DLC 2002019193 Acquisitions Editor: Catherine Carroll Senior Developmental Editor: Faith Voit Project Manager: Norman Stellander Designer: Steven Stave PIT/MVB Printed in the United States of America. Last digit is the print number: 9 8 7 6 5 4 3 2 1



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Contributors

Eli Y. Adashi

Presidential Professor of Obstetrics and Gynecology and John A. Dixon Professor and Chair, Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, Salt Lake City, Utah The Physiology and Pathology of the Female Reproductive Axis; Fertility Control: Current Approaches and Global Aspects Lloyd P. Aiello

Associate Professor of Ophthalmology, Harvard Medical School; Assistant Director, Beetham Eye Institute; Investigator and Head, Section of Eye Research, Joslin Diabetes Center, Boston, Massachusetts Complications of Diabetes Mellitus Andrew Arnold

Murray-Heilig Chair in Molecular Medicine and Professor of Medicine and Genetics, University of Connecticut School of Medicine; Director, Center for Molecular Medicine, and Chief, Division of Endocrinology and Metabolism, University of Connecticut, Farmington, Connecticut Pathogenesis of Endocrine Tumors Jennifer Berman

Assistant Professor, Department of Urology, David Geffen School of Medicine at University of California, Los Angeles; Attending Surgeon, UCLA Medical Center; Co-Director, Female Sexual Medicine Center at UCLA, Los Angeles, California Sexual Dysfunction in Men and Women

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Laura Berman

Assistant Professor, Department of Urology, David Geffen School of Medicine at University of California, Los Angeles; Co-Director, Female Sexual Medicine Center at UCLA, Los Angeles, California Sexual Dysfunction in Men and Women Shalender Bhasin

Professor of Medicine, University of California, Los Angeles, School of Medicine; Chief, Division of Endocrinology, Metabolism, and Molecular Medicine, Charles Drew University and King-Drew Medical Center, Los Angeles, California Sexual Dysfunction in Men and Women Andrew J. M. Boulton

Professor of Medicine, University of Manchester; Consultant Physician, Manchester Royal Infirmary, Manchester, United Kingdom Complications of Diabetes Mellitus Robin P. Boushey

General Surgery Resident, Department of Surgery, University of Toronto, Toronto, Ontario, Canada Gastrointestinal Hormones and Gut Endocrine Tumors Glenn D. Braunstein

Professor of Medicine, University of California, Los Angeles, School of Medicine; Chairman, Department of Medicine, The James R. Klinenberg, MD, Chair in Medicine, Cedars-Sinai Medical Center, Los Angeles, California Endocrine Changes in Pregnancy F. Richard Bringhurst

Associate Professor of Medicine, Harvard Medical School; Physician, Massachusetts General Hospital, Boston, Massachusetts Hormones and Disorders of Mineral Metabolism

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Michael Brownlee

Anita and Jack Saltz Professor of Diabetes Research and Professor of Medicine and Pathology, Albert Einstein College of Medicine, Bronx, New York Complications of Diabetes Mellitus Serdar E. Bulun

Associate Professor of Obstetrics-Gynecology and Molecular Genetics; Director, Division of Reproductive Endocrinology and Infertility, University of Illinois, Chicago, Illinois The Physiology and Pathology of the Female Reproductive Axis Charles F. Burant

Associate Professor, Department of Internal Medicine, University of Michigan; Attending Physician, University of Michigan Hospitals, Ann Arbor, Michigan Type 2 Diabetes Mellitus John B. Buse

Associate Professor of Medicine; Chief, Division of General Medicine and Clinical Epidemiology; Director, Diabetes Care Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina Type 2 Diabetes Mellitus; Type 1 Diabetes Mellitus David A. Bushinsky

Professor of Medicine and of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry; Chief, Nephrology Unit, Strong Memorial Hospital, Rochester, New York Kidney Stones Judy L. Cameron

Associate Professor, Department of Psychiatry, University of Pittsburgh, Pittsburgh, Pennsylvania; Associate Scientist, Oregon National Primate Research Center, Oregon Health and Science University, Portland, Oregon Neuroendocrinology

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Christin Carter-Su

Professor of Physiology; Associate Director, Michigan Diabetes Research and Training Center, University of Michigan Medical School, Ann Arbor, Michigan Mechanism of Action of Hormones That Act at the Cell Surface Roger D. Cone

Senior Scientist, Vollum Institute; Associate Professor, Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, Oregon Neuroendocrinology Felix A. Conte

Professor of Pediatrics, Department of Pediatrics, University of California, San Francisco, School of Medicine, San Francisco, California Disorders of Sex Differentiation Philip E. Cryer

Irene E. and Michael M. Karl Professor of Endocrinology and Metabolism in Medicine, Washington University School of Medicine; Chief of Endocrinology, Diabetes, and Metabolism, Barnes-Jewish Hospital, St. Louis, Missouri Glucose Homeostasis and Hypoglycemia Terry F. Davies

Professor of Medicine and Director, Division of Endocrinology, Diabetes and Bone Diseases, Mount Sinai School of Medicine; Attending Physician, Mount Sinai Hospital, New York, New York Thyroid Physiology and Diagnostic Evaluation of Patients with Thyroid Disorders; Thyrotoxicosis; Hypothyroidism and Thyroiditis Marie B. Demay

Associate Professor of Medicine, Harvard Medical School; Associate Physician, Massachusetts General Hospital, Boston, Massachusetts Hormones and Disorders of Mineral Metabolism

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Robert G. Dluhy

Professor of Medicine, Harvard Medical School; Clinical Chief, Division of Endocrinology, Diabetes and Hypertension, Brigham and Women's Hospital, Boston, Massachusetts Endocrine Hypertension Daniel J. Drucker

Professor of Medicine, University of Toronto Faculty of Medicine; Director, Banting and Best Diabetes Centre, Toronto General Hospital, Toronto, Ontario, Canada Gastrointestinal Hormones and Gut Endocrine Tumors George S. Eisenbarth

Professor of Pediatrics, Medicine, and Immunology, University of Colorado School of Medicine; Executive Director, Barbara Davis Center for Childhood Diabetes, Denver, Colorado Type 1 Diabetes Mellitus; The Immunoendocinropathy Syndromes Joel K. Elmquist

Associate Professor of Medicine and Neurology, Harvard Medical School, Division of Endocrinology, Beth Israel Deaconess Medical Center, Boston, Massachusetts Neuroendocrinology Robert V. Farese Jr.

Professor of Medicine, University of California, San Francisco; Senior Investigator, Gladstone Institute of Cardiovascular Disease, San Francisco, California Disorders of Lipid Metabolism Daniel D. Federman

Carl W. Walter Distinguished Professor of Medicine and Medical Education, Harvard Medical School; Senior Physician, Brigham and Women's Hospital and Massachusetts General Hospital, Boston, Massachusetts The Endocrine Patient

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Sebastiano Filetti

Professor of Medicine, Department of Internal Medicine, University of Sapienza, and Chairman of Internal Medicine, and Policlinico Umberto I, Rome, Italy Nontoxic Goiter and Thyroid Neoplasia Delbert A. Fisher

Professor of Pediatrics and Medicine Emeritus, University of California, Los Angeles, School of Medicine; Vice President, Science and Innovation, Quest Diagnostics Inc., Nichols Institute, San Juan Capistrano, California Endocrinology of Fetal Development Eli Friedman

Distinguished Teaching Professor of Medicine, State University of New York; Chief, Division of Renal Disease, Department of Medicine, Downstate Medical Center; Attending Physician, University Hospital of Brooklyn and Kings County Hospital, Brooklyn, New York Complications of Diabetes Mellitus Robert F. Gagel

Professor of Medicine, Division of Internal Medicine, University of Texas/M.D. Anderson Cancer Center; Adjunct Professor of Medicine, Internal Medicine, and Cell Biology, Baylor College of Medicine, Houston, Texas Multiple Endocrine Neoplasia Peter A. Gottlieb

Assistant Professor of Pediatrics and Medicine, Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Denver, Colorado The Immunoendocrinopathy Syndromes James E. Griffin

Professor of Internal Medicine and Diana and Richard C. Strauss Professor in Biomedical Research, The University of Texas Southwestern Medical Center, Dallas, Texas Disorders of the Testes and Male Reproductive Tract

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Melvin M. Grumbach

Edward B. Shaw Emeritus Professor of Pediatrics and Emeritus Chairman, Department of Pediatrics, University of California, San Francisco, San Francisco, California Disorders of Sex Differentiation; Puberty: Ontogeny, Neuroendocrinology, Physiology, and Disorders Joel F. Habener

Professor of Medicine, Harvard Medical School; Associate Physician and Chief, Laboratory of Molecular Endocrinology, Massachusetts General Hospital; Investigator, Howard Hughes Medical Institute, Boston, Massachusetts Genetic Control of Peptide Hormone Formation Ian D. Hay

Professor of Medicine, Mayo Medical School; Consultant in Endocrinology and Internal Medicine, Mayo Clinic, Rochester, Minnesota Thyroid Physiology and Diagnostic Evaluation of Patients with Thyroid Disorders; Nontoxic Goiter and Thyroid Neoplasia Wayne J. G. Hellstrom

Professor of Urology and Chief, Section of Andrology and Male Dysfunction, Tulane University Health Sciences Center, New Orleans, Louisiana Sexual Dysfunction in Men and Women Ieuan A. Hughes

Professor of Paediatrics and Head, Department of Paediatrics, University of Cambridge; Honorary Consultant, Paediatric Endocrinology, Addenbrooke Hospital, Cambridge, United Kingdom Disorders of Sex Differentiation Michael Kafrissen

Adjunct Professor, Maternal and Child Health, University of North Carolina School of Medicine, Chapel Hill, North Carolina; Vice President, Ortho-McNeil Pharmaceutical Co., Raritan, New Jersey Fertility Control: Current Approaches and Global Aspects

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George G. Klee

Professor of Laboratory Medicine, Department of Laboratory Medicine, Mayo Medical School; Consultant in Clinical Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinical Hospitals, Rochester, Minnesota Laboratory Techniques for Recognition of Endocrine Disorders Samuel Klein

Danforth Professor of Medicine, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri Obesity David L. Kleinberg

Professor of Medicine and Director, Neuroendocrine Research Medicine, New York University School of Medicine; Attending, Medicine, New York University Medical Center; Consultant in Medicine, New York Harbor Healthcare Veterans Administration Hospital, New York, New York Anterior Pituitary Barbara E. Kream

Professor, Departments of Medicine and Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut Metabolic Bone Disease Henry M. Kronenberg

Professor of Medicine, Harvard Medical School; Chief, Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts Principles of Endocrinology; Hormones and Disorders of Mineral Metabolism Steven W. J. Lamberts

Professor of Medicine, Erasmus University Rotterdam, Rotterdam, The Netherlands Endocrinology and Aging

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P. Reed Larsen

Professor of Medicine, Harvard Medical School; Chief, Division of Endocrinology, Diabetes and Hypertension, and Senior Physician, Brigham and Women's Hospital, Boston, Massachusetts Principles of Endocrinology; Thyroid Physiology and Diagnostic Evaluation of Patients with Thyroid Disorders; Thyrotoxicosis; Hypothyroidism and Thyroiditis Jennifer E. Lawrence

Director, South Georgia Medical Center Diabetes Center; Chief, Division of Endocrinology, South Georgia Medical Center, Valdosta, Georgia Endocrine Hypertension Mitchell A. Lazar

Sylvan H. Eisman Professor of Medicine and Genetics and Director, Penn Diabetes Center, University of Pennsylvania School of Medicine; Chief, Division of Endocrinology, Diabetes, and Metabolism, Hospital of The University of Pennsylvania, Philadelphia, Pennsylvania Mechanism of Action of Hormones That Act on Nuclear Receptors Joseph A. Lorenzo

Professor of Medicine, University of Connecticut School of Medicine; Attending Physician, John Dempsey Hospital/University of Connecticut Health Center, Farmington, Connecticut Metabolic Bone Disease Malcolm J. Low

Professor, Department of Behavioral Neuroscience, Oregon Health and Science University; Scientist, Vollum Institute, Oregon Health and Science University, Portland, Oregon Neuroendocrinology Robert W. Mahley

Professor of Pathology and Medicine, University of California, San Francisco; Director, Gladstone Institute of Cardiovascular Disease, San Francisco, California Disorders of Lipid Metabolism

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Stephen J. Marx

Chief, Metabolic Diseases Branch, and Chief, Genetics and Endocrinology Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland Multiple Endocrine Neoplasia Shlomo Melmed

Senior Vice President, Academic Affairs, Cedars Sinai Medical Center; Professor of Medicine and Associate Dean, University of California, Los Angeles, School of Medicine, Los Angeles, California Principles of Endocrinology; Anterior Pituitary Rebeca D. Monk

Assistant Professor of Medicine, University of Rochester School of Medicine and Dentistry; Head, Urolithiasis Clinic, Strong Memorial Hospital, Rochester, New York Kidney Stones Richard W. Nesto

Associate Professor of Medicine, Harvard Medical School, Boston; Chairman, Department of Cardiovascular Medicine, Lahey Clinic Medical Center, Burlington, Massachusetts Complications of Diabetes Mellitus Kjell E. Öberg

Chairman, Department of Medical Sciences, Medical Faculty, Uppsala University; Professor of Endocrine Oncology, University Hospital, Uppsala, Sweden Carcinoid Tumors, Carcinoid Syndrome, and Related Disorders Kenneth S. Polonsky

Adolphus Busch Professor and Chairman, Department of Medicine, and Professor, Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri Principles of Endocrinology; Type 2 Diabetes Mellitus; Type 1 Diabetes Mellitus

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Lawrence G. Raisz

Professor of Medicine, University of Connecticut School of Medicine and Health Center, Farmington; Physician, Hartford Hospital and St. Francis Medical Center, Hartford, Connecticut Metabolic Bone Disease Edward O. Reiter

Professor of Pediatrics, Tufts University School of Medicine, Boston; Chairman, Department of Pediatrics, Baystate Medical Center, Children's Hospital, Springfield, Massachusetts Normal and Aberrant Growth Alan G. Robinson

Vice Provost, Medical Sciences, and Executive Associate Dean and Professor of Medicine, University of California, Los Angeles, School of Medicine, Los Angeles, California Posterior Pituitary Gland Johannes A. Romijn

Professor of Medicine and Endocrinology, Department of Endocrinology, Leiden University School of Medicine and Medical Center, Leiden, The Netherlands Obesity Ron G. Rosenfeld

Credit Union Endowment Professor and Chairman, Department of Pediatrics, and Professor, Cell and Developmental Biology, Oregon Health and Science University; Physician-in-Chief, Doermbecher Children's Hospital, Portland, Oregon Normal and Aberrant Growth Richard Santen

Professor of Medicine, University of Virginia School of Medicine; Member, Division of Endocrinology, Department of Internal Medicine, University of Virginia Health System, Charlottesville, Virginia Endocrine-Responsive Cancer

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Martin-Jean Schlumberger

Professor, University of Paris, Sud; Chief, Department of Nuclear Medicine and Endocrine Tumors, Institute Gustave Roussy, Villejuif, France Thyroid Physiology and Diagnostic Evaluation of Patients with Thyroid Disorders; Nontoxic Goiter and Thyroid Neoplasia Allen Spiegel

Director, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland Mechanism of Action of Hormones That Act at the Cell Surface Paul M. Stewart

Professor of Medicine, School of Medicine, University of Birmingham; Consultant Physician, Queen Elizabeth Hospital, Birmingham, United Kingdom The Adrenal Cortex Gordon J. Strewler

Professor of Medicine and Master, Walter Bradford Cannon Society, Harvard Medical School; Physician, Beth Israel Deaconess Medical Center, Boston, Massachusetts Humoral Manifestations of Malignancy Dennis M. Styne

Professor and Chief, Pediatric Endocrinology, University of California, Davis, School of Medicine, Sacramento, California Puberty: Ontogeny, Neuroendocrinology, Physiology, and Disorders Simeon I. Taylor

Vice President, Discovery Biology, Bristol-Myers Squibb, Hopewell, New Jersey Mechanism of Action of Hormones That Act at the Cell Surface

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Joseph G. Verbalis

Professor of Medicine, Department of Medicine, Georgetown University School of Medicine; Chief, Division of Endocrinology and Metabolism, Georgetown University Medical Center, Washington, D.C. Posterior Pituitary Gland Aaron I. Vinik

Professor, Internal Medicine, Pathology, and Neurobiology, Eastern Virginia Medical School; Director, Strelitz Diabetes Research Institute, Norfolk, Virginia Complications of Diabetes Mellitus Karl H. Weisgraber

Professor of Pathology, University of California, San Francisco, School of Medicine; Deputy Director and Senior Investigator, Gladstone Institute of Cardiovascular Disease, San Francisco, California Disorders of Lipid Metabolism Gordon H. Williams

Professor of Medicine and Director of Scholars in Clinical Science Program, Harvard Medical School; Director, General Clinical Research Center and Center for Clinical Investigation, Brigham and Women's Hospital, Boston, Massachusetts Endocrine Hypertension Jean D. Wilson

Charles Cameron Sprague Distinguished Chair in Biomedical Research, The University of Texas Southwestern Medical Center, Dallas, Texas Disorders of the Testes and the Male Reproductive Tract

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Foreword

Robert H. Williams The publication of the tenth edition comes 52 years after the first edition of Williams' Textbook of Endocrinology. There had been other large textbooks of endocrinology, such as Biedl's Innere Sekretion in 1916 1 and Rolleston's The Endocrine Organs in Health and Disease in 1936,2 but only a handful of physicians could be identified as endocrinologists by the middle of the twentieth century. Consequently, Robert H. Williams exercised "powerful persuasion" to overcome the reluctance of the sales staff of the W. B. Saunders Company to publish a book that had no visible audience. 3 In fact, however, Williams was correct in predicting a large readership, because its publication coincided with an explosive increase in basic endocrine science and in the application of this basic information to patients, and with the evolution of endocrinology into a recognized subspecialty of several branches of medicine. Indeed, the book has had a profound impact on endocrine science and on the development of the clinical discipline, and it is appropriate at this time to remember Robert Williams and his contributions to the field and to Textbook of Endocrinology. Williams described in a memoir the training and the background that led to his development of the textbook. 4 After his graduation from Washington and Lee University, he obtained the M.D. degree at Johns Hopkins University Medical School in 1934. His house staff training was spread between the Mallory Institute of Pathology at the Boston City Hospital, the Department of Medicine at Vanderbilt (where he did research with Tinsley R. Harrison), and the Department of Medicine at Johns Hopkins (where he worked with Warren Longcope). He finished his training at the Massachusetts General Hospital as an endocrine fellow at a time when there were "many quacks in this area throughout the world." 4 He and his mentor, Fuller Albright, became good friends and maintained close contact over the years. In 1940, Williams was appointed to the staff of the Endocrine Unit of the Thorndike Laboratory in the Harvard Medical Unit at the Boston City Hospital. In 1942, he became head of the Unit, where his research focused principally on the biochemistry and physiology of thyroid disease, including pioneering work on the treatment of thyrotoxicosis with thionamide drugs and with radioactive iodine. In addition, he described the syndrome of biotin deficiency and published papers on adrenal physiology, obesity, and nephrogenic diabetes insipidus. To attract students and fellows into the field, he developed the concept that "endocrinology is the backbone of metabolism and metabolism is the interstitium of medicine." His students and trainees included at least three future contributors to his textbook, Sidney Ingbar, Peter Forsham, and William H. Daughaday. Daughaday describes Williams as a man of extraordinary exuberance and enthusiasm who took great pleasure in lecturing and in bedside teaching and whose motto was "B(bright) and E(early) and on the B(ball)." 5 Williams considered himself first and foremost an educator, and in 1948 he moved to the University of Washington as Chairman of the Department of Medicine, where his extraverted and outgoing personality made him a superb teacher, recruiter, administrator, and institution builder. The Endocrine Division in Seattle was very broad and encompassed diabetes mellitus, clinical nutrition, and metabolism, as well as endocrinology. Williams served as President of the Endocrine Society, the American Society for Clinical Investigation, and the Association of American Physicians, and he was the founder of the Association of the Professors of Medicine. In brief, he was an academic giant of twentieth century medicine. The founding of the Textbook of Endocrinology evolved from his interest in education: "In view of the rapid progress in endocrinology and metabolism, and the fact that our unit at Boston City Hospital had registered very high in undergraduate, graduate, and postgraduate teaching, I decided that there was a great need for a new textbook in endocrinology." 4 The arrangements for the textbook were completed before Williams left Boston, and the aims were clearly described in the preface to the first edition: The rapidity and extent of advances in endocrinology have made it increasingly difficult for the student and physician to take full advantage of information available for understanding, diagnosis and treatment of clinical disorders. It is the realization of these difficulties that prompted the writing of this book. The main

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objective is to provide a condensed and authoritative discussion of the management of clinical endocrinopathies, based upon the application of fundamental information obtained from chemical and physiologic investigations. The product was a book that over the years has served as an effective bridge between clinical medicine and the science of endocrinology. There may be no other arena of medicine in which the basic and clinical sciences are so tightly interwoven into one discipline. On the one hand, the clinical discipline profits immensely from scientific advances; on the other hand, clinical observations often raise important questions for investigation and on occasion provide answers that have an impact on basic science. By reflecting advances in both areas, the Williams Textbook was designed to convey the intellectual excitement of a rapidly changing scientific base and, at the same time, to promote the integration of a spectrum of disciplines ranging from molecular genetics to patient care into a unified discipline. The achievement of this aim was possible because, from the initial edition, Williams chose contributors who were at the forefront of the field, thereby ensuring the freshness of each edition. Now, of course, there are several textbooks of endocrinology, but Williams' pioneering book continues to enjoy a growing readership of both the English and the foreign language editions. Williams edited the first five editions, and Edwin L. Bierman completed the editing of the sixth edition after Williams' death in 1979. Jean D. Wilson and Daniel W. Foster edited the seventh and eighth editions and were joined by Henry M. Kronenberg and P. Reed Larsen as editors for the ninth edition. For the tenth edition, Larsen and Kronenberg are joined by editors Kenneth S. Polonsky and Shlomo Melmed, and continuing the tradition set by Williams, the editors of the tenth edition have enlisted an outstanding group of new and former contributors. Saunders continues as publisher.

Endocrinology has changed in many ways during the past 50 years, and the editorial challenges likewise change with each edition. On one level, these challenges reflect scientific advances, such as the explosion of knowledge about hormone action, the development of new and improved diagnostic techniques and imaging modalities, and the application of molecular genetics to biology. On another level, the concept that the discipline of endocrinology was defined by the concept of humoral control mechanisms has become blurred by recognition that the endocrine, immune, and neurologic signaling systems constitute a single integrated system rather than separate control mechanisms. The most significant challenge now, however, is the same as that faced by all textbooks at a time when the volume of published information is rapidly increasing, namely the dilemma of how to take full advantage of developments in electronic publishing and the evolving revolution in information retrieval systems to devise effective learning systems for the near and remote futures. The fundamental educational issues are the same that Williams faced in 1948, namely the need to integrate rapidly evolving basic and clinical science in a cohesive format appropriate for undergraduate, graduate, and continuing medical education. Now, however, the prose text can be enhanced by multimedia additions, and updating can be done in a continuum. How these tools will be utilized to create new types of teaching materials by academicians faced with multiple demands on their time is not entirely clear, but the response to this challenge will determine whether Williams Textbook of Endocrinology will continue to have the same impact over the next 50 years. Jean D. Wilson

References 1. 2. 3. 4.

Biedl A. Innere Sekretion. Ihre Physiologischen Grundlagen und Ihre Bedeutung für die Pathologie. Berlin: Urban & Schwarzenberg, 1916. Rolleston HD. The Endocrine Organs in Health and Disease with an Historical Review. Oxford, Oxford University Press, 1936. Dusseau JL. An Informal History of W. B. Saunders Company. Philadelphia: W. B. Saunders Company, 1988, pp 9899. Williams RH. My experiences in endocrinology: 19401948. In Finland M (ed). The Harvard Medical Unit at Boston City Hospital, Vol I. Boston, Harvard Medical School, 1982, pp 455481. 5. Daughaday WH. Personal communication.



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Preface

This tenth edition of Williams Textbook of Endocrinology is a milestone in many respects. A tenth edition per se is testimony to the enduring accomplishments of our predecessors, who have consistently created a product that remains the most popular textbook in this field. It is also now a half century since the publication of the first edition of Williams, a landmark suitably celebrated in a foreword by our coauthor and former editor, Dr. Jean Wilson. Two internationally renowned endocrinologists, Drs. Shlomo Melmed and Kenneth Polonsky, have joined Drs. Reed Larsen and Henry Kronenberg to formulate, co-edit, and assemble this volume. We will strive to meet the high standards maintained by Drs. Wilson and Daniel Foster during their editorial leadership. Our goal for this first edition of the new millennium was to emulate the achievements of our predecessors by producing a definitive and fresh approach to the presentation of the essentials of clinical endocrinology. Accordingly, we invited a number of new authors, including several European colleagues, to prepare 23 of the 41 chapters. Our challenge to them and to those updating their material was to distill the burgeoning molecular and physiological knowledge into a complete, but relevant, scholarly presentation. Where appropriate, this would include a practical experienced guide as to how the author uses this information in the diagnosis and management of his or her own patients. Achieving such relevance, thoroughness, and practicality requires a unique combination of scientific knowledge and total clinical familiarity best encapsulated in the term "physician-scientist." We believe that our physician-scientist authors have again met Robert Williams' stipulation that this text should provide "a condensed and authoritative discussion of the management of clinical endocrinopathies based upon the application of fundamental information obtained from chemical and physiologic investigation." We hope our readers will agree. Both new and revised chapters are replete with tables and figures. Highlights of this edition include a new and expanded diabetes section, new chapters on many old and new topics including endocrinology and aging, female reproduction and fertility control, sexual function and dysfunction, kidney stones, the adrenal cortex, endocrine hypertension, endocrine-responsive tumors, and non-insulin-secreting tumors of the gastroenteropancreatic system. A largely new, concise introductory section includes several new chapters discussing mechanisms of hormone action and the clinical approach to the endocrine patient, as well as a thorough guide to the intricacies of the rapidly changing laboratory techniques. The entire section containing chapters on the hypothalamus and both anterior and posterior pituitary disorders is original, and the thyroid section has been thoroughly revised and divided into expanded disorder-based presentations. Stylistic innovations include page numbers in the introductory outlines for each chapter, which we hope will permit the reader ready access to specific topics. We have also introduced algorithms and clinical guidelines for diagnostic and treatment strategies to crystallize recommendations for each disease. Readers will also note that this edition is published only five years following its predecessor, reflecting the all-too-familiar rapidity with which new knowledge is accumulating in the biomedical disciplines. Its timely appearance despite so much new material reflects the diligent efforts of our editorial staff and especially the new authors. We would like to express our deep gratitude to the coworkers in our offices, Anita Nichols, Debra Hession, Lynn Moulton, Grace Labrado, Linda Walker, Louise Ishibashi, and Sherri Turner, without whose dedication this project could not have been completed. We also wish to thank our colleagues at Elsevier, Richard Zorab and Cathy Carroll, and our tireless and effective developmental editors, Faith Voit and Joanne Husovski. Their painstaking attention to every detail is a major contribution to this new edition. P. Reed Larsen Henry M. Kronenberg Shlomo Melmed Kenneth S. Polonsky XX



Section 1 - Hormones and Hormone Action

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Chapter 1 - Principles of Endocrinology Henry Kronenberg Shlomo Melmed P. Reed Larsen Kenneth Polonsky

INTRODUCTION Roughly a hundred years ago, Starling coined the term hormone to describe secretin, a substance secreted by the small intestine into the blood stream to stimulate pancreatic secretion. In his Croonian Lectures, Starling considered the endocrine and nervous systems as two distinct mechanisms for coordination and control of organ function. Thus, endocrinology found its first home in the discipline of mammalian physiology. Work over the next several decades by biochemists, physiologists, and clinical investigators led to the characterization of many hormones secreted into the blood stream from discrete glands or other organs. These investigators showed for the first time that diseases such as hypothyroidism and diabetes could be treated successfully by replacing specific hormones. These initial triumphs formed the foundation of the clinical specialty of endocrinology. Advances in cell biology, molecular biology, and genetics over the ensuing years began to help explain the mechanisms of endocrine diseases and of hormone secretion and action. Although these advances have embedded endocrinology into the framework of molecular cell biology, they have not changed the essential subject of endocrinologythe signaling that coordinates and controls the functions of multiple organs and processes. Here we would like to survey the general themes and principles that underpin the diverse approaches used by clinicians, physiologists, biochemists, cell biologists, and geneticists to understand the endocrine system.



THE EVOLUTIONARY PERSPECTIVE Hormones can be defined as chemical signals secreted into the blood stream that act on distant tissues, usually in a regulatory fashion. Hormonal signaling represents a special case of the more general process of signaling between cells. Even unicellular organisms such as baker's yeast, Saccharomyces cerevisiae, secrete short peptide mating factors that act on receptors of other yeast cells to trigger mating between the two cells. These receptors resemble the ubiquitous family of mammalian 7-transmembrane spanning receptors that respond to ligands as diverse as photons and glycoprotein hormones. Because these yeast receptors trigger activation of heterotrimeric G proteins just as mammalian receptors do, this conserved signaling pathway must have been present in the common ancestor of yeast and humans. Signals from one cell to adjacent cells, so-called paracrine signals, often trigger cellular responses that use the same molecular pathways used by hormonal signals. For example, the sevenless receptor controls the differentiation of retinal cells in the Drosophila eye by responding to a membrane-anchored signal from an adjacent cell. Sevenless is a membrane-spanning receptor with an intracellular tyrosine kinase domain that signals in a way that closely resembles the signaling by hormone receptors such as the insulin receptor tyrosine kinase. Since paracrine factors and hormones can share signaling mechanisms it is not surprising that hormones can, in some settings, act as paracrine factors. Testosterone, for example, is secreted into the blood stream but also acts locally in the testes to control spermatogenesis. Insulin-like growth factor I (IGF-I) is a hormone secreted into the blood stream from the liver and other tissues, but it is also a paracrine factor made locally in most tissues to control cell proliferation. Further, one receptor can mediate the actions of a hormone, such as parathyroid hormone, and of a paracrine factor, such as parathyroid hormonerelated protein. Target cells respond similarly to signals that reach them from the blood stream (hormones) or from the cell next door (paracrine factors); the cellular response machinery does not distinguish the sites of origin of signals. The shared final common pathways used by hormonal and paracrine signals should not, however, obscure important differences between hormonal and paracrine signaling system (Fig. 1-1) . Paracrine signals do not travel very far; consequently, the specific site of origin of a paracrine factor determines where it will act and provides specificity to that action. When the paracrine factor BMP4 is secreted by cells in the developing kidney, it regulates the differentiation of renal cells; when BMP4 is secreted by cells in bone, it regulates bone formation. Thus, the site of origin

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Figure 1-1 Comparison of determinants of endocrine (A) and paracrine (B) signaling.

of BMP4 determines its physiologic role. In contrast, since hormones are secreted into the blood stream, their sites of origin are often divorced from their functions. We know nothing about thyroid hormone function, for example, that requires that the thyroid gland to be in the neck. Because the specificity of action of paracrine factors is so dependent on their precise site of origin, elaborate mechanisms have evolved to regulate and constrain the diffusion of paracrine factors. Paracrine factors of the hedgehog family, for example, are covalently bound to cholesterol to constrain the diffusion of these molecules in the extracellular milieu. Most paracrine factors interact with binding proteins that block their action and control their diffusion. Chordin, noggin, and many other distinct proteins all bind to various members of the BMP family to regulate their action, for example. Proteases such as tolloid then destroy the binding proteins at specific sites to liberate BMPs so that the BMPs can act on appropriate target cells. Hormones have rather different constraints. Because they diffuse throughout the body, they must be synthesized in enormous amounts relative to the amounts of paracrine factors needed at specific locations. This synthesis usually occurs in specialized cells designed for that specific purpose. Hormones must then be able to travel in the blood stream and diffuse in effective concentrations into tissues. Therefore, for example, lipophilic hormones bind to soluble proteins that allow them to travel in the aqueous media of blood at relatively high concentrations. The ability of hormones to diffuse through the extracellular space means that the local concentration of hormone at target sites will rapidly decrease when glandular secretion of the hormone stops. Because hormones diffuse throughout extracellular fluid quickly, hormonal metabolism can occur in specialized organs such as the liver and kidney in a way that determines the effective concentration of the hormones in other tissues. Paracrine factors and hormones thus use several distinct strategies to control their biosynthesis, sites of action, transport, and metabolism. These differing strategies may partly explain why a hormone such as IGF-I, unlike its close relative insulin, has multiple binding proteins that control its action in tissues. As noted earlier, IGF-I has a double life as both a hormone and a paracrine factor. Presumably, the local actions of IGF-I mandate an elaborate binding protein apparatus. All the major hormonal signaling programsG proteincoupled

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receptors, tyrosine kinase receptors, serine/threonine kinase receptors, ion channels, cytokine receptors, nuclear receptorsare also used by paracrine factors. In contrast, several paracrine signaling programs are used only by paracrine factors and are probably not used by hormones. For example, Notch receptors respond to membrane-based ligands to control cell fate, but no bloodborne ligands use Notch-type signaling (at least none is currently known). Perhaps the intracellular strategy used by Notch, which involves cleavage of the receptor and subsequent nuclear actions of the receptor's cytoplasmic portion, is too inflexible to serve the purposes of hormones. The analyses of the complete genomes of multiple bacterial species, the yeast Saccharomyces cerevisiae, the fruit fly Drosophila melanogaster, the worm Caenorhabitis elegans, the plant Aradopsis thaliana, and humans have allowed a comprehensive view of the signaling machinery used by various forms of life. As noted already, S. cerevisiae uses G proteinlinked receptors; this organism, however, lacks tyrosine kinase receptors and nuclear receptors that resemble the estrogen/thyroid receptor family. In contrast, the worm and fly share with humans the use of each of these signaling pathways, although with substantial variation in numbers of genes committed to each pathway. For example, the Drosophila genome encodes 20 nuclear receptors, the C. elegans genome encodes 270, and the human genome encodes (tentatively) more than 50. These patterns suggest that ancient multicellular animals must have already established the signaling systems that are the foundation of the endocrine system as we know it in mammals. Even before the sequencing of the human genome, sequence analyses had made clear that many receptor genes are found in mammalian genomes for which no clear ligand or function was known. The analyses of these "orphan" receptors has succeeded in broadening the current understanding of hormonal signaling. For example, the liver X receptor (LXR) was one such orphan receptor found when searching for unknown nuclear receptors. Subsequent experiments showed that oxygenated derivatives of cholesterol are the ligands for LXR, which regulates genes involved in cholesterol and fatty acid metabolism. [1] The example of LXR and many others raise the question of what constitutes a hormone. The classical view of hormones is that they are synthesized in discrete glands and have no function other than activating receptors on cell membranes or in the nucleus. In contrast, cholesterol, which is converted in cells to oxygenated derivatives that activate the LXR, uses a hormonal strategy to regulate its own metabolism. Other orphan nuclear receptors respond similarly to ligands, such as bile acids and fatty acids. These "hormones" have important metabolic roles quite separate from their signaling properties, although the hormone-like signaling serves to allow regulation of the

metabolic function. The calcium-sensing receptor is an example from the G proteinlinked receptor family of receptors that responds to a nonclassical ligand, ionic calcium. Calcium is released into the blood stream from bone, kidney, and intestine and acts on the calcium-sensing receptor in parathyroid cells, renal tubular cells, and other cells to coordinate cellular responses to calcium. Thus, many important metabolic factors have taken on hormonal properties as part of a regulatory strategy.



ENDOCRINE GLANDS Hormone formation may occur either in localized collections of specific cells, in the endocrine glands, or in cells that have additional roles. Many protein hormones, such as growth hormone, parathyroid hormone, prolactin, insulin, and glucagon, are produced in dedicated cells by standard protein synthetic mechanisms common to all cells. These secretory cells usually contain specialized secretory granules designed to store large amounts of hormone and to release the hormone in response to specific signals. Formation of small hormone molecules initiates with commonly found precursors, usually in specific glands such as the adrenals, gonads, or thyroid. In the case of the steroid hormones, the precursor is cholesterol, which is modified by various hydroxylations, methylations, and demethylations to form the glucocorticoids, androgens, and estrogens, and their biologically active derivatives. In contrast, the precursor of vitamin D, 7-dehydrocholesterol, is produced in skin keratinocytes, again from cholesterol, by a photochemical reaction. Leptin, which regulates appetite and energy expenditure, is formed in adipocytes, thus providing a specific signal reflecting the organism's nutritional state to the central nervous system. Thyroid hormone synthesis occurs via a unique pathway. The thyroid cell synthesizes a 660,000-kd homodimer, thyroglobulin, which is then iodinated at specific iodotyrosines. Certain of these "couple" to form the iodothyronine molecule within thyroglobulin, which is then stored in the lumen of the thyroid follicle. In order for this to occur, the thyroid cell must concentrate the trace quantities of iodide from the blood and oxidize it via a specific peroxidase. Release of thyroxine (T 4 ) from the thyroglobulin requires its phagocytosis and cathepsin-catalyzed digestion by the same cells. Hormones are synthesized in response to biochemical signals generated by various modulating systems. Many of these systems are specific to the effects of the hormone product; for example, parathyroid hormone synthesis is regulated by the concentration of ionized calcium, whereas gonadal, adrenal, and thyroid hormone synthesis is achieved by the hormonostatic function of the hypothalamicpituitary axis. Cells in the hypothalamus and pituitary monitor the circulating hormone concentration and secrete trophic hormones that activate specific pathways for hormone synthesis and release. Typical examples are luteinizing (LH) follicle-stimulating (FSH), thyroid-stimulating (TSH), and adrenocorticotrophic (ACTH) hormones. These trophic hormones increase rates of hormone synthesis and secretion and also may induce target cell division, thus causing enlargement of the various target glands. For example, in hypothyroid individuals living in iodine-deficient areas of the world, TSH secretion causes a marked hyperplasia of thyroid cells. In such regions, the thyroid gland may be 20- to 50-fold its normal size. Adrenal hyperplasia occurs in patients with genetic deficiencies in cortisol formation. Hypertrophy and hyperplasia of parathyroid cells, in this case initiated by an intrinsic response to the stress of hypocalcemia, occur in patients with renal insufficiency or calcium malabsorption. Hormones may be fully active when released into the blood stream (e.g., growth hormone or insulin) or may require activation in specific cells to produce their biological effects. These activation steps are often highly regulated. For example, the T 4 released from the thyroid cell is a prohormone that must undergo a specific deiodination to form the active 3,5,3' triiodothyronine (T 3 ). This deiodination reaction can occur in target tissues, such as in the central nervous system; in the thyrotrophs, where T3 provides feedback regulation of TSH production; or in hepatic and renal cells, from which T 3 is released into the circulation for uptake by all tissues. A similar post secretory activation step catalyzed by a 5-reductase causes tissue-specific activation of testosterone to dihydrotestosterone in target tissues, including the male urogenital tract and genital skin, as well as in the liver. Vitamin D undergoes hydroxylation at the 25 position in the liver and in the 1 position in the kidney. Both hydroxylations must occur to produce the active hormone 1,25(OH) 2 vitamin D. The activity of the 1-hydroxylase, but not that of the 25-hydroxylase,

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is stimulated by parathyroid hormone and reduced plasma phosphate but is inhibited by calcium and 1,25(OH) 2 vitamin D. Hormones are synthesized as required on a daily, hourly, or minute-to-minute basis with minimal storage, but there are significant exceptions. One such exception is the thyroid gland, which contains enough stored hormone to last for about two months. This permits a constant supply of this hormone despite significant variations in the availability of iodine. If iodine deficiency is prolonged, however, the normal reservoirs of thyroxine can be depleted. The various feedback signaling systems exemplified above provide the hormonal homeostasis characteristic of virtually all endocrine systems. Regulation may include the central nervous system or local signal recognition mechanisms in the glandular cells, such as the calcium-sensing receptor of the parathyroid cell. Superimposed, centrally programmed increases and decreases in hormone secretion or activation through neuroendocrine pathways also occur. Examples include the circadian variation in the secretion of ACTH directing the synthesis and release of cortisol. The monthly menstrual cycle exemplifies a system with much longer periodicity that requires a complex synergism between central and peripheral axes of the endocrine glands. Disruption of hormonal homeostasis due to glandular or central regulatory system dysfunction has both clinical and laboratory consequences. Recognition and correction of these are the essence of clinical endocrinology.



TRANSPORT OF HORMONES IN BLOOD Protein hormones and some small molecules such as the catecholamines are water-soluble and readily transported by the circulatory system. Others are nearly insoluble in water (e.g., the steroid and thyroid hormones), and their distribution presents special problems. Such molecules are bound to 50 to 60-kd carrier plasma glycoproteins such as thyroxine-binding globulin (TBG), sex hormonebinding globulin (SHBG), and corticosteroid-binding globulin (CBG), as well as to albumin. These ligandprotein complexes serve as reservoirs of these hormones, ensure ubiquitous distribution of their water-insoluble ligands, and protect the small molecules from rapid inactivation or excretion in the urine or bile. Without these proteins, it is unlikely that hydrophobic molecules would be transported much beyond the veins draining the glands in which they are formed. The protein-bound hormones exist in rapid equilibrium with the often minute quantities of hormone in the aqueous plasma. It is this "free" fraction of the circulating hormone that is taken up by the cell. It has been shown, for example, that if tracer thyroid hormone is injected into the portal vein in a protein-free solution, it is bound to hepatocytes at the periphery of the hepatic sinusoid. When the same experiment is repeated with a protein-containing solution, there is a uniform distribution of the tracer hormone throughout the hepatic lobule. [2] Despite the very high affinity of some of the binding proteins for their ligands, one specific protein may not be essential for hormone distribution. For example, in humans with a congenital deficiency of TBG, other proteins, transthyretin and albumin, subsume its role. Because the affinity of these secondary thyroid hormone transport proteins is several orders of magnitude lower than that of TBG, it is possible for the hypothalamicpituitary feedback system to maintain free thyroid hormone in the normal range at a much lower total hormone concentration. The fact that the "free" hormone concentration is normal in subjects with TBG deficiency indicates that it is this free moeity that is defended by the hypothalamicpituitary axis and is the active hormone. [3] The availability of gene-targeting techniques has allowed specific tests of the physiologic role of several hormone-binding proteins. For example, mice with targeted inactivation of the vitamin Dbinding protein (DBP) have been generated. [4] Although the absence of DBP markedly reduces the circulating concentration of vitamin D, the mice are otherwise normal. However, they show enhanced susceptibility to a vitamin Ddeficient diet because of the reduced reservoir of this sterol. In addition, the absence of DBP markedly reduces the half-life of 25(OH)D 2 by accelerating its hepatic uptake, making the mice less susceptible to vitamin D intoxication. In rodents, transthyretin (TTR) carries retinol-binding protein and is also the principal thyroid hormonebinding protein. This protein is synthesized in the liver and in the choroid plexus. It is the major thyroid hormonebinding protein in the cerebrospinal fluid of both rodents and humans and was thought to perhaps serve an important role in thyroid hormone transport into the central nervous system. This hypothesis has been disproven by the fact that mice without TTR have normal concentrations of T 4 in the brain as well as of free T 4 in the plasma.[5] [6] To be sure, the serum concentrations of vitamin A and total T 4 are decreased, but the knockout mice have no signs of vitamin A deficiency or hypothyroidism. Such studies suggest that these proteins primarily serve distributive/reservoir functions. Protein hormones and some small ligands (e.g., catecholamines) produce their effects by interacting with cell surface receptors. Others, such as the steroid and thyroid hormones, must enter the cell to bind to cytosolic or nuclear receptors. In the past, it has been thought that much of the transmembrane transport of hormones was passive. Evidence is now in hand that there are specific organic anion transporters involved in cellular uptake of thyroid hormone (see reference [7] ). This may be found to be the case for other small ligands as well, revealing yet another mechanism for ensuring the distribution of a hormone to its site of action.



TARGET CELLS AS ACTIVE PARTICIPANTS Hormones determine cellular target actions by binding with high specificity to receptor proteins. Whether a peripheral cell is hormonally responsive depends to a large extent on the presence and function of specific and selective hormone receptors. Receptor expression thus determines which cells will respond, as well as the nature of the intracellular effector pathways activated by the hormone signal. Receptor proteins may be localized to the cell membrane, cytoplasm, and nucleus. Broadly, polypeptide hormone receptors are cell-membrane associated, whereas soluble intracellular proteins selectively bind to steroid hormones (Fig. 1-2) (Figure Not Available) . This idea of selective localization has recently been challenged, however, because related sequences can be found in multiple cellular compartments. Membrane-associated receptor proteins usually consist of extracellular sequences that recognize and bind ligand, transmembrane anchoring hydrophobic sequences, and intracellular sequences, which initiate intracellular signaling. Intracellular signaling is mediated by soluble second messengers (e.g., cyclic AMP) or by activation of intracellular signaling molecules (e.g., signal transduces and activates of transcription [STAT] proteins). Receptor-dependent activation of heterotrimeric G-proteins, comprising , , and subunits, may either induce or suppress effector enzymes or ion channels.

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Figure 1-2 (Figure Not Available) Hormonal signaling by cell-surface and intracellular receptors. The receptors for the watersoluble polypeptide hormones, LH, and IGF-I; are integral membrane proteins located at the cell surface. They bind the hormone-utilizing extracellular sequences and transduce a signal by the generation of second messengers, cAMP for the LH receptor, and tyrosine-phosphorylated substrates for the IGF-I receptor. Although effects on gene expression are indicated, direct effects on cellular proteins, for example, ion channels, are also observed. In contrast, the receptor for the lipophilic steroid hormone progesterone resides in the cell nucleus. It binds the hormone and becomes activated and capable of directly modulating target gene transcription. (Tf = transcription factor; R = receptor molecule.) (Reproduced from Mayo K. In Conn PM, Melmed S (eds). Endocrinology: Basic and Clinical Principles. Totowa, NJ, Humana Press, 1997, p. 11.)

Several growth factors and hormone receptors (e.g., for insulin) behave as intrinsic tyrosine kinases or activate intracellular protein tyrosine kinases. Ligand activation may cause receptor dimerization (e.g., growth hormone [GH]) or heterodimerization (e.g., interleukin-6 [IL-6]), followed by activation of intracellular phosphorylation cascades. These activated proteins ultimately determine specific nuclear gene expression. Both the number of receptors expressed per cell and their responses are also regulated, thus providing a further level of control for hormone action. Several mechanisms account for altered receptor function. Receptor endocytosis causes internalization of cell surface receptors; the hormonereceptor complex is subsequently dissociated, resulting in abrogation of the hormone signal. Receptor trafficking may then result in recycling back to the cell surface (e.g., as for insulin), or the internalized receptor may undergo lysosomal degradation. Both these mechanisms triggered by activation of receptors effectively lead to impaired hormone signaling by down-regulation of these receptors. The hormone signaling pathway may also be down-regulated by receptor desensitization (e.g., as for epinephrine); ligand-mediated receptor phosphorylation leads to a reversible deactivation of the receptor. Desensitization mechanisms can be activated by a receptor's ligand (homologous desensitization) or by another signal (heterologous desensitization), thereby attenuating receptor signaling in the continued presence of ligand. Receptor function may also be limited by the action of specific phosphatases (e.g., Src homology 2 domain-containing protein tyrosine phosphatase [SHP]) or by intracellular negative regulation of the signaling cascade (e.g., suppressor of cytokine signaling [SOCS] proteins inhibiting Janus kinase [JAK]-STAT signaling). Mutational changes in receptor structure can also determine hormone action. Constitutive receptor activation may be induced by activating mutations (e.g., TSH receptor), leading to endocrine organ hyperfunction, even in the absence of hormone. Conversely, inactivating receptor mutations may lead to endocrine hypofunction (e.g., testosterone or vasopressin receptors). These syndromes are now well characterized and are well described in this volume (Fig. 1-3) . The functional diversity of receptor signaling also results in overlapping or redundant intracellular pathways. For example, both GH as well as cytokines activate JAK-STAT signaling, whereas the distal effects of these stimuli clearly differ. Thus, despite common signaling pathways, hormones elicit highly specific cellular effects. Tissue or cell-type genetic programs or receptorreceptor interactions at the cell surface (e.g., dopamine D2 with somatostatin receptor hetero-oligonization) may also confer specific cellular response to a hormone and provide an additive cellular effect. [8]



CONTROL OF HORMONE SECRETION Anatomically distinct endocrine glands are composed of highly differentiated cells that synthesize, store, and secrete hormones. Circulating hormone concentrations are a function of glandular secretory patterns and hormone clearance rates. Hormone secretion is tightly regulated to attain circulating levels that are most conducive to elicit the appropriate target tissue response. For example, longitudinal bone growth is initiated

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Figure 1-3 Diseases caused by mutations in G-protein-coupled receptors. All are human conditions with the exception of the final two entries, which refer to the mouse. (AD = autosomal dominant; AR = autosomal recessive inheritance.) Loss of function refers to inactivating mutations of the receptor, and gain of function to activating mutations. Abbreviations for G-protein-coupled receptors: ACTH = adrenocorticotropic hormone; LH = luteinizing hormone; TSH = thyroid-stimulating hormone; PTH-PTHrP = parathyroid hormone and parathyroid hormone-related peptide; MSH = melanocyte-stimulating hormone; GHRH = growth hormone-releasing hormone; FSH = follicle-stimulating hormone. (Reproduced from Mayo K. In Conn PM, Melmed S (eds), Endocrinology: Basic and Clinical Principles. Totowa, NJ, Humana Press, 1997, page 27.)

and maintained by exquisitely regulated levels of circulating GH, whereas mild GH hypersecretion results in gigantism and GH deficiency causes growth retardation. Ambient circulating hormone concentrations are not uniform, and secretion patterns determine appropriate physiologic function. Thus, insulin secretion occurs in short pulses elicited by nutrient and other signals and gonadotrophin secretion is episodic, determined by a hypothalamic pulse generator, whereas prolactin secretion appears to be relatively continuous, with secretory peaks elicited during suckling. Hormone secretion also adheres to rhythmic patterns. Circadian rhythms serve as adaptive responses to environmental signals and are controlled by a circadian timing mechanism.[9] Light is the major environmental cue adjusting the endogenous clock. The retinohypothalamic tract entrains circadian pulse generators situated within hypothalamic suprachiasmatic nuclei. These signals subserve timing mechanisms for the sleepwake cycle and determine patterns of hormone secretion and action. Disturbed circadian timing results in hormonal dysfunction and may also be reflective of entrainment or pulse generator lesions. For example, adult GH deficiency due to a damaged hypothalamus or pituitary is associated with elevations in integrated 24-hour leptin concentrations, decreased leptin pulsatility, and yet preserved circadian rhythm of leptin. GH replacement restores leptin pulsatility, followed by loss of body fat mass. [10] Sleep is also an important cue regulating hormone pulsatility. About 70% of overall GH secretion occurs during slow-wave sleep, and increasing age is associated with declining slow-wave sleep and concomitant decline in GH and elevation of cortisol secretion. [11] Most pituitary hormones are secreted in a circadian (daynight) rhythm, best exemplified by ACTH peaks before 9 AM, whereas ovarian steroids follow a 28-day menstrual rhythm. Disrupted episodic rhythms are often a hallmark of endocrine dysfunction. Thus, loss of circadian ACTH secretion with high midnight cortisol levels is a feature of Cushing's disease. Hormone secretion is induced by multiple specific biochemical and neural signals. Integration of these stimuli results in the net temporal and quantitative secretion of the hormone (Fig. 1-4) . Thus, signals elicited by hypothalamic hormones (GHRH, somatostatin), peripheral hormones (IGF-I, sex steroids, thyroid hormone), nutrients, adrenergic pathways, stress, and other neuropeptides, all converge on the somatotroph cell, resulting in the ultimate pattern and quantity of GH secretion. Networks of reciprocal interactions allow for dynamic adaptation and shifts in environmental signals. These regulatory systems embrace the hypothalamic, pituitary, and target endocrine glands, as well as the adipocyte and lymphocyte. Peripheral inflammation and stress elicit cytokine signals, which interface with the neuroendocrine system, resulting in hypothalamicpituitary axis activation. The parathyroid and pancreatic secreting cells are less tightly controlled by the hypothalamus, but their functions are tightly regulated by the effects they elicit. Thus, parathyroid hormone (PTH) secretion is induced when serum calcium levels fall, and the signal for sustained PTH secretion is abrogated by rising calcium levels. Several tiers of control subserve the ultimate net glandular secretion. First, central nervous system signals including stress, afferent stimuli, and neuropeptides signal the synthesis and secretion of hypothalamic hormones and neuropeptide (Fig. 1-5) . Four hypothalamic releasing hormones (GHRH, corticotropin-releasing hormone [CRH], TRH, and gonadotrophin releasing hormone [GnRH]) traverse the hypothalamic portal vessels and impinge on their respective transmembrane trophic hormone-secreting cell receptors. These distinct cells express GH, ACTH, TSH, and gonadotrophins. In contrast, hypothalamic somatostatin and dopamine suppress GH, prolactin (PRL), or TSH secretion. Trophic hormones also maintain the structuralfunctional integrity of endocrine organs, including the thyroid and adrenal glands, and the gonads. Target hormones, in turn, serve as powerful negative feedback regulators of their respective trophic hormone and often also suppress secretion of hypothalamic releasing hormones. In certain circumstances, for example during puberty, peripheral sex steroids may positively induce the hypothalamicpituitarytarget gland axis. Thus, luteinizing hormone (LH) induces ovarian estrogen

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Figure 1-4 Peripheral feedback mechanism and a million-fold amplifying cascade of hormonal signals. Environmental signals are transmitted to the central nervous system, which innervates the hypothalamus, which responds by secreting nanogram amounts of a specific hormone. Releasing hormones are transported down a closed portal system, pass the blood-brain barrier at either end through fenestrations, and bind to specific anterior pituitary cell membrane receptors to elicit secretion of micrograms of specific anterior pituitary hormones. These enter the venous circulation through fenestrated local capillaries, bind to specific target gland receptors, trigger release of micrograms to milligrams of daily hormone amounts, and elicit responses by binding to receptors in distal target tissues. Peripheral hormone receptors enable widespread cell signaling by a single initiating environmental signal, thus facilitating intimate homeostatic association with the external environment. Arrows with a black dot at their origin indicate a secretory process. (Reproduced from Normal AW, Litwack G. Hormones, 2nd edn. New York, Academic Press, 1997, p 14.)

Figure 1-5 Model for regulation of anterior pituitary hormone secretion by three tiers of control. Hypothalamic hormones impinge directly on their respective target cells. Intrapituitary cytokines and growth factors regulate tropic cell function by paracrine (and autocrine) control. Peripheral hormones exert negative feedback inhibition of respective pituitary trophic hormone synthesis and secretion. (Reproduced with permission from Ray D, Melmed S. Pituitary cytokine and growth factor expression and action. Endocrin Rev 1997; 18:206228.)

secretion, which feeds back positively to induce further LH release. Pituitary hormones themselves, in a short feedback loop, may also regulate their own respective hypothalamic controlling hormone. Hypothalamic releasing hormones are secreted in nanogram amounts and have short half-lives of a few minutes. Anterior pituitary hormones are produced in microgram amounts and have longer half-lives, whereas peripheral hormones can be produced in up to milligram amounts daily, with much longer half-lives.

A further level of secretion control occurs within the gland itself. Thus, intraglandular paracrine or autocrine growth peptides serve to autoregulate pituitary hormone secretion, as exemplified by epidermal growth factor (EGF) control of prolactin or IGF-I control of GH secretion. Molecules within the endocrine cell may also subserve an intracellular feedback loop. Thus, corticotrope SOCS-3 induction by gp 130-linked cytokines serves to abrogate the ligand-induced JAK-STAT cascade and to block pro-opiomelanocortin gene transcription and ACTH secretion. This rapid on-off regulation of ACTH secretion provides a plastic endocrine response to changes in environmental signaling and serves to maintain homeostatic integrity. [12] In addition to the central-neuroendocrine interface mediated by hypothalamic chemical signal transduction, the CNS directly controls several hormonal secretory processes. Posterior pituitary hormone secretion occurs as direct efferent neural

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extensions. Postganglionic sympathetic nerves also regulate rapid changes in renin, insulin, and glucagon secretion, and preganglionic sympathetic nerves signal to adrenal medullary cells, eliciting adrenaline release.



HORMONE MEASUREMENT Endocrine function can be assessed by measuring levels of basal circulating hormone, evoked or suppressed hormone, or hormone-binding proteins. Alternatively, peripheral hormone receptor function can be assessed. Meaningful strategies for timing hormonal measurements vary from system to system. In some cases, circulating hormone concentrations can be measured in randomly collected serum samples. This measurement, when standardized for fasting, environmental stress, age, and gender, is reflective of true hormone concentrations only when levels do not fluctuate appreciably. For example, thyroid hormone, prolactin, and IGF-I levels can be accurately assessed in fasting morning serum samples. On the other hand, when hormone secretion is clearly episodic, timed samples may be required over a defined time course to reflect hormone bioavailability. Thus, early morning and late evening cortisol measurements are most appropriate. Although 24-hour sampling for GH measurements, with samples collected every 2, 10, or 20 minutes, are expensive and cumbersome, they may yield valuable diagnostic information. Random sampling may also reflect secretion peaks or nadirs, thus confounding adequate interpretation of results. In general, confirmation of failed glandular function is made by attempting to evoke hormone secretion by recognized stimuli. Thus, testing of pituitary hormone reserve may be accomplished by injecting appropriate hypothalamic releasing hormones. Injection of trophic hormones, including TSH and ACTH, evokes specific target gland hormone secretion. Pharmacologic stimuli, for example metoclopromide for induction of prolactin secretion, may also be useful tests of hormone reserve. In contrast, hormone hypersecretion can be diagnosed by suppressing glandular function. Thus, failure to appropriately suppress GH levels after a standardized glucose load implies inappropriate GH hypersecretion. Radioimmunoassays utilize highly specific antibodies unique to the hormone, or a hormone fragment, to quantify hormone levels. Enzyme-linked immunoabsorbent assays (ELISA) employ enzymes instead of radioactive hormone markers, and enzyme activity is reflective of hormone concentration. This sensitive technique has allowed ultrasensitive measurements of physiologic hormone concentrations. Hormone-specific receptors may be employed in place of the antibody in a radioreceptor assay.



ENDOCRINE DISEASES Endocrine diseases fall into four broad categories: (1) hormone overproduction; (2) hormone underproduction; (3) altered tissue responses to hormones; and (4) tumors of endocrine glands. Hormone Overproduction

Occasionally, hormones are secreted in increased amounts because of genetic abnormalities that cause abnormal regulation of hormone synthesis or release. In glucocorticoid-remediable hyperaldosteronism, for example, an abnormal chromosomal crossing-over event puts the aldosterone synthetase gene under the control of the ACTH-regulated 11 -hydroxylase gene. More often, diseases of hormone overproduction are associated with an increase in the total number of hormone-producing cells. For example, the hyperthyroidism of Graves' disease, in which antibodies mimic TSH and activate the TSH receptors on thyroid cells, is associated with dramatic increase in thyroid cell proliferation, as well as with increased synthesis and release of thyroid hormone from each thyroid cell. In this example, the increase in thyroid cell number represents a polyclonal expansion of thyroid cells, in which large numbers of thyroid cells proliferate in response to an abnormal stimulus. Most endocrine tumors are not polyclonal expansions, however, but instead represent monoclonal expansions of one mutated cell. Pituitary and parathyroid tumors, for example, are usually monoclonal expansions in which somatic mutations occur in multiple tumor suppressor genes and proto-oncogenes. These mutations lead to an increase in proliferation and/or survival of the mutant cells. Sometimes this proliferation is associated with abnormal secretion of hormone from each tumor cell as well. For example, mutant G proteins in somatotrophs can lead to both increased cellular proliferation and increased secretion of growth hormone from each tumor cell.



Hormone Underproduction

Underproduction of hormone can result from a wide variety of processes, ranging from surgical removal of parathyroid glands during neck surgery, to tuberculous destruction of adrenal glands, or to iron deposition in -cells in hemochromatosis. A frequent cause of destruction of hormone-producing cells is autoimmunity. Autoimmune destruction of beta cells in type 1 diabetes mellitus and autoimmune destruction of thyroid cells in Hashimoto's thyroiditis are two of the most common disorders treated by endocrinologists. More uncommonly, a host of genetic abnormalities can also lead to decreased hormone production. These disorders can result from abnormal development of hormone-producing cells (e.g., hypogonadotrophic hypogonadism caused by KAL gene mutations), from abnormal synthesis of hormones (e.g., deletion of the growth hormone gene), or from abnormal regulation of hormone secretion (e.g., the hypoparathyroidism associated with activating mutations of the parathyroid cell's calcium-sensing receptor).



Altered Tissue Responses

Resistance to hormones can be caused by a variety of genetic disorders. Examples include mutations in the growth hormone receptor in Laron dwarfism and mutations in the G gene in the hypoparathyroidism of pseudohypoparathyroidism, type 1a. The insulin resistance in muscle and liver central to the etiology of type 2 diabetes mellitus appears to be polygenic in origin. Type 2 diabetes is also an example of a disease in which end organ insensitivity is worsened by signals from other organs, in this case by signals originating in fat cells. In other cases, the target organ of hormone action is more directly abnormal, as in the parathyroid hormone (PTH) resistance of renal failure. Increased end organ function can be caused by mutations in signal reception and propagation. For example, activating mutations in TSH, LH, and PTH receptors can cause increased activity of thyroid cells, Leydig cells, and osteoblasts, even in the absence of ligand. Similarly, activating mutations in the G s protein can cause precocious puberty, hyperthyroidism, and acromegaly in McCune-Albright syndrome.



Tumors of Endocrine Glands

Tumors of endocrine glands, as noted above, often result in hormone overproduction. Some tumors of endocrine glands

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produce little if any hormone but cause disease by their local compressive symptoms or by metastatic spread. Examples include so-called nonfunctioning pituitary tumors, which are usually benign but can cause a variety of symptoms due to compression on adjacent structures, and thyroid cancer, which can spread throughout the body without causing hyperthyroidism.



THERAPEUTIC STRATEGIES In general, hormones are employed pharmacologically for both their replacement and their suppressive effects. Hormones may also be used for diagnostic stimulatory effects (e.g., hypothalamic hormones) to evoke target organ responses or to diagnose endocrine hyperfunction by suppressing hormone hypersecretion (e.g., T 3 ). Ablation of endocrine gland function due to genetic or acquired causes can be restored by hormone replacement therapy. In general, steroid and thyroid hormones are replaced orally, where as peptide hormones (e.g., insulin, DH) require injection. Gastrointestinal absorption and firstpass kinetics determine oral hormone dosage and availability. Physiologic replacement can achieve both appropriate hormone levels (e.g., thyroid), as well as approximate hormone secretory patterns (e.g., GnRH delivered intermittently via a pump). Hormones can also be used to treat diseases associated with glandular hyperfunction. Long-acting depot preparations of somatostatin analogs suppress GH hypersecretion in acromegaly or 5-HIAA hypersecretion in carcinoid syndrome. Estrogen receptor antagonists (e.g., tarmoxilen) are useful for some patients with breast cancer, and GnRH analogs may downregulate the gonadotrophin axis and benefit patients with prostate cancer. Novel formulations of receptor-specific hormone ligands are now being clinically developed (e.g., estrogen agonists/antagonists, somatostatin receptor subtype ligands), resulting in more selective therapeutic targeting. Modes of hormone injection (e.g., for PTH) may also determine therapeutic specificity and efficacy. Improved hormone delivery systems, including computerized minipumps, intranasal sprays (e.g., for 1-desamino-8- D-arginine vasopression [DDAVP]), pulmonary inhalations, and depot intramuscular injections, will also allow added patient compliance and ease of administration. Despite this tremendous progress, some therapies, such as insulin delivery to rigorously control blood sugar, still require tremendous patient involvement and await innovative approaches.



References 1. Chawla

A, Repa, JJ, Evans RM et al. Nuclear receptors and lipid physiology: Opening the X-files. Science 2001; 294: 18661870.

2. Mendel

CM, Weisiger RA, Jones AL, et al. Thyroid hormone-binding proteins in plasma facilitate uniform distribution of thyroxine within tissues: A perfused rat liver study. Endocrinology 1987; 120:17421749. 3. Mendel 4. Safadi

CM. The free hormone hypothesis: A physiologically based mathematical model. Endocr Rev 1989; 10(3):232274.

FF, Thornton P, Magiera H, et al. Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein. J Clin Invest 1999; 103:239251.

5. Palha

JA, Fernandes R, de Escobar GM, et al. Transthyretin regulates thyroid hormone levels in the choroid plexus, but not in the brain parenchyma: Study in a transthyretin-null mouse model. Endocrinology 2000; 141:32673272. 6. Palha

JA, Episkopou V, Maeda S, et al. Thyroid hormone metabolism in a transthyretin-null mouse strain. J Biol Chem 1994; 269:3313533139.

7. Hennemann 8. Rochevill 9. Moore

G, Docter R, Friesema ECH, et al. Plasma membrane transport of thyroid hormones and its role in thyroid hormone metabolism and bioavailability. Endocr Rev 2001; 22:451476.

M, Lange DC, Kumar U, et al. Receptors for dopamine and somatostatin: formation of hetero-oligomers with enhanced functional activity. Science 2000; 288:154157.

RY. Circadian rhythms: Basic neurobiology and clinical applications. Ann Rev Med 1997; 48:253266.

10.

Aftab MA, Guzder R, Wallace AM, et al. Circadian and ultradian rhythm and leptin pulsitility in adult GH deficiency: Effects of GH replacement. J Clin Endocr Metab 2001; 86:34993506.

11.

Cauter EV, Leproult R, Plat L. Age-related changes in slow wave sleep and REM sleep and relationship with growth hormone and cortisol levels in healthy men. JAMA 2000; 284:861868.

12.

Melmed S: The immuno-neuroendocrine interface. J Clin Invest 2001; 108:15631566.

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11

Chapter 2 - The Endocrine Patient Daniel D. Federman

A textbook of medicine is inevitably about disease, but the practice of medicine deals with illness, that is, a person experiencing a disease. It is for that reason that the present chapter has been entitled "The Endocrine Patient." It is my intention to lay out the general issues and approaches applicable to caring for patients with endocrine disorders. The topics to be discussed include initial evaluation and the nature of referral, the fact finding required in clinical evaluation, the use of the laboratory and imaging, the formulation of a differential diagnosis, decision making, and management. In each case, the steps are portrayed from the patient's point of view. It is worth noting that, except for acute adrenal insufficiency, endocrine disorders are seldom life-threatening. They have enormous effect on the quality of life, however, and successful intervention can be extremely important to both patient and family.



GENERAL CONSIDERATIONS Many features of being an endocrine patient are common to all experiences of illness. Most often, a perceived change in bodily function, a symptom, gets one to the doctor. Although generations of medical students have described new patients as being "in no acute distress," most patients are, in fact, worried and anxious when they see a physician, the more so when the physician is not known to them. A few minutes spent in getting to know the patient can pay enormous dividends in the accuracy of the history obtained and in setting the stage for further cooperation with testing and treatment. Inasmuch as most endocrine consultation is elective rather than emergent, I favor asking a few simple questions, such as "Where are you from?" "What do you do?" "How did you come to us?" "Were you referred?" and so on. Almost always, some common experience or acquaintance is discovered that provides the basis for a rapport that does not emerge from formal medical questioning. This step also immediately conveys that you are interested in the patient as a person and not just as a disease.



SPECIAL FEATURES OF ENDOCRINE ILLNESS Discovery through Screening

Numerous special features of endocrine disease make patient presentation quite different from that seen in general medicine. One is the discovery of abnormality through screening of asymptomatic individuals, for example, a high serum calcium level discovered through multiphasic screening or a high blood glucose level discovered in a shopping mall kiosk. The very absence of symptoms lends an unreality to the moment and should become an explicit topic of the patient-doctor interaction. In this circumstance, it is worth emphasizing the value of early discovery and prevention of greater morbidity.



Quantitative Rather Than Qualitative Abnormalities

A second special feature of endocrine disorders is that they are all quantitative, rather than qualitative, departures from normal. No endocrine disorder is due to a novel hormone. Everyone has cortisol circulating as a determining feature of his or her life. Hypercorticism and adrenal insufficiency represent just more or less of the hormone. Similarly, all hormones found in excess or in deficiency in disease are physiologic determinants of stature, weight, complexion, hairiness, temperament, and behavior. In contrast, no one has a little pneumonia or a little inflammatory bowel disease as a constitutive status. In addition, most endocrine glands have both a basal and a stimulable or reserve function. It is common to have partial diminution of capacity in which the basal function is

12

adequate but a reserve called upon during part of each dayor, more dramatically, in emergenciesis not available.



Overlap with Other Diseases

The symptoms of endocrine disorders overlap a great range of normal characteristics, including body contour, facial configurations, weight distributions, skin and hair coloring, and muscular capacity. They also overlap with other conditions that are far more common, including depression and normal aging. The added adipose tissue of hyperadrenocorticism is more difficult to recognize in a person who is already obese. The nervousness associated with hyperthyroidism is less apparent in a thin, hyperkinetic man than in a person of moderate body weight. The effects of an androgen-producing adrenal tumor are less likely to be noticed in a family of swarthy, hirsute individuals. Finally, most endocrine disorders evolve gradually over months to years instead of appearing suddenly, such as a heart attack or an acute infection. This combination of varied host background and slow evolution of disease leads to considerable delay in diagnosis: both the patient and primary care physician adapt to the changes as part of the person, and definitive evaluation, now relatively easy for most disorders, is not undertaken. Hypothyroidism and acromegaly are good examples of this phenomenon. All series show a remarkable delay in diagnosis despite sometimes disabling symptoms. Hormones have more distant effects than local effects. This, of course, reflects their messenger status. Unlike an abscess, a myocardial infarction, or an esophageal cancer, endocrine disorders seldom produce symptoms near the gland of origin. (Subacute thyroiditis and large pituitary tumors, of course, are exceptions.) But because in most endocrinopathies the excess or missing hormone works on several or many systems, the resulting syndrome can be enigmatic. Several endocrine disorders are important not because of their incidence but because of their curability: Cushing's disease, acromegaly, and pheochromocytoma are cases in point. Although these disorders enter the differential diagnosis of common problems such as diabetes, their occurrence is so rare that the primary care physician does not easily think of them.



Unique Features of Reproductive Disorders

Reproductive disorders have symptoms and signs that have no parallel in other areas. This is the one system in which sexual dimorphism is inherent rather than epiphenomenal; it is also the one with the greatest span of developmental change. Once the heart starts beating in the embryo, it goes on doing so until the last moment of life; but puberty, adult sexual functioning, and menopause establish time lines against which all symptoms are to be assessed. Thus, vaginal bleeding has entirely different meanings whether it occurs on the first day of life, as a natural appearance at age 12, between menstrual periods at age 25 years, as a harbinger of menopause at age 46 years, or as a highly probable symptom of cancer at age 66 years. Physical appearance and function are important features of self-image. Thus, hirsutism, thinness, obesity, sexual arousal, and erectile capacity bear considerable psychological import to the endocrine patient. The clinician should be constantly aware of both spoken and unspoken thoughts that may be troubling the patient. The Couple as a Clinical Unit

The ultimate goal of reproductive capacity is, of course, a fertile union. This means that the couple, rather than the individual, is the unit of clinical concern. It is thus the principal area in medicine whereby two people and their interaction, rather than a single person and her capacities, are studied and treated. In addition, there are dimensions of successful sexual function that are important at other times than when fertility is sought. Sex drive, erotic responsiveness, affection, and tenderness are all important aspects of life whether or not fertility is an issue. These areas are notoriously difficult to evaluate in a society that treats sexual function as such a special topic, particularly enshrouded in personal issues of such importance.



EVALUATION OF PATIENTS WITH ENDOCRINE DISORDERS I have emphasized previously the belief that establishing an interested and warm relationship is the beginning of excellence in any elective medical interaction. In addition to its affective power, the relationship elicits a more informative history, establishes better cooperation in both testing and treatment, and provides a platform for informed decision making by the patient. History

As in most areas of medicine, precision of diagnosis and economy of investigation begin with a carefully wrought history. An open-ended question, combined with an attentive silence, allows the patient to provide the background for the clinical moment. After the patient has spoken spontaneously, the physician provides a guided expansion of the information. Details of timing, sequence, changes of diet or activity, relationship to the menstrual cycle, changes in weight or size, and alterations in mood or sleep patternall of these may provide clues to underlying endocrine abnormality. A good example of the power of the history is the interpretation of irregular periods in a woman of reproductive age. The simple statement, "I've never been regular," points to a presumptive diagnosis of polycystic ovary syndrome in a way that a very convoluted sequence of questions might actually fail to do. That statement is to be contrasted with this one: "I used to be regular, but in the last year or so, I never know when my period is going to come." If the presenting symptom is irregular periods, the simple invitation, "Tell me about your periods," is likely to be the key to the diagnosis. Careful questioning about use of complementary and alternative medicines is an important and, occasionally, a very revealing step. A thorough family history has become increasingly important as the genetic basis for more and more endocrine diseases becomes established. For practical purposes, I favor diagramming a pedigree of the first-order relativesparents, siblings, childrenof all patients, not just those for whom a genetic disorder is already suspected. Known disorders are readily revealed this way, and unknown conjunctions of clinical and genetic factors may also be disclosed (Fig. 2-1) .



Physical Examination General Examination

It is said that the history is 80% or more of clinical diagnosis, and that is no less true in endocrine disorders than in general medicine. Yet the physical examination is a critical element in the process of arriving at a diagnosis, and here I want to call particular attention to the first impression.

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Figure 2-1 Simple pedigree of the propositi (arrow) and first-order relatives should be the standard family history in a new patient workup. If the patient has children, their health status should be included as well.

The possibility of Cushing's syndrome, Addison's disease, hyperthyroidism and hypothyroidism, acromegaly, polycystic ovary syndrome, hypogonadism, and Turner's syndromethese and other endocrine disorders should be considered from the first moment one encounters a new patient. Otherwise, one risks accepting that the appearance of the patient is just that and no more. In other words, as soon as one accepts that the initial impression is what the person looks like naturally, the quantitative departure from normal that is the essence of endocrine disease fails to impress one. This, incidentally, is why both families and primary care physicians often miss a diagnosis that seems obvious to the consultant endocrinologist. A quantification of this last point may be helpful. If the signs of hypothyroidism or acromegaly, for example, take 3 years to become striking, the person living with the patient is exposed to 1/1095th of fractional change per daywell below the threshold of just noticeable difference. Similarly, a primary care physician seeing the patient perhaps four times a year for a general checkup and management of hypertension is exposed to 91/1095 fractional change. This can sometimes lead to a diagnosis but often does not. When one sees the patient for the first time, however, the imprint of the disease catches attention and the constitutional appearance is in the background. Although a consultant participates because of a special area of interest and expertise, he or she is a general physician first and should be alert to all dimensions of the physical examination: What is the height/weight ratio? What is the basic degree of muscularity? Is there evidence of heart disease to explain the chest pain and dyspnea one has heard about in the history? What is the degree of hirsutism? Are there signs of liver disease, malnutrition, or poor or excellent physical training? What is the blood pressure with the patient standing as well as lying or sitting? These and many other points of a general examination begin to modify the thinking one has undertaken on the basis of history. Targeted Examination

The targeted physical examination of any consultant is an interesting interplay of general and specific goals. Theoretically, any experienced clinician should undertake a general examination and come to all the findings pertinent to an underlying endocrine disorder. In fact, however, the physical examination is greatly influenced by the hypotheses generated in the history. Let us look at a few examples. If a patient reports weight loss despite a good appetite, there is only a very restricted differential diagnosis, principally malabsorption or hypermetabolism. In doing a physical examination, therefore, I would pay particular attention to signs of malabsorption (muscular wasting, vitamin deficiencies, purpura) and to signs of thyroid disease with its generalized hypermetabolism and localized autoimmune phenomena, including ophthalmopathy. Similarly, if a patient complains of hirsutism or other signs of androgen excess, one is immediately thrust into a consideration of ethnic hair distribution and quality. Is there temporal recession of the hairline? Does the hair on the abdomen come up over the umbilicus? Is hair present on the back (rare without marked hyperandrogenism)? How much acne is there? At the extreme, is there evidence of clitoral enlargement? Finally, and most important, does the patient look like or unlike the other women in her family? Direct Assessment of Endocrine Glands

Three endocrine glands are palpablethe thyroid, the testis, and the ovary. Specific attention should be given to each of these. The thyroid gland should be approached first by inspectionwhile the patient swallowsfor size, symmetry, or localized enlargement. Many thyroid nodules are visible, and inspection often calls attention to lesions that would be missed on palpation. The thyroid should then be felt while the patient swallows, from the front with your thumbs or from behind the patient with the index and third fingers. It is crucial to keep your own fingers from moving while the patient is swallowing. The principal observation is whether there is diffuse enlargement of the thyroid gland (most often Graves' diffuse hyperplasia or Hashimoto's thyroiditis) or one or more nodules. Although the consistency of the gland is to be noted, in fact it is often not concordant with the pathology. Functioning tumors of the testis may be too small to be felt with the fingers, and most internists and general physicians are not skilled in palpation of the ovaries. For this reason, ultrasound and other forms of imaging have become key features of gonadal evaluation and are discussed later. The size of one other endocrine gland, the pituitary, can be inferred from physical examination for what Cushing called "neighborhood signs." As a pituitary tumor or diffuse enlargement proceeds, it pushes up on the optic chiasm from below, producing a bitemporal hemianopsia first manifested in the upper quadrants, often to a blinking or flashing red light. This finding is too subtle for the generalist's confidence, however, and pituitary assessment depends on formal visual fields and imaging. Indirect Assessment of Endocrine Status

Many consequences of hormone action can be detected on physical examination; the results combine with the history to produce a highly reliable differential diagnosis and thus an informed basis for laboratory evaluation and imaging. Among the things to be looked for are the eye signs and dermopathy of Graves' disease, acanthosis nigricans as a clue to insulin resistance, muscular wasting and tremor, changes in the voice due to hypothyroidism or acromegaly, and a general impression of nutrition and its adequacy or excess. Each of these findings is described in more detail with the specific disorder in subsequent chapters.



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LABORATORY TESTING OF ENDOCRINE FUNCTION Modern endocrine laboratory evaluation began with the introduction of radioimmunoassay by Berson and Yalow. The precise measurement of hormone concentrations, determined by competitive displacement of specific antibodies, was soon succeeded by competitive binding assays and, more recently, by immunofluorescent and radioluminescent determinations of even greater sensitivity and specificity. It should therefore be possible to enter the name of a hormone on a laboratory slip and expect to get back a definitive reflection of the status of the patient for that gland. For practical purposes, that has become true of thyroid-stimulating hormone (TSH). Reliable determinations of elevated, normal, and suppressed levels of this hormone by radioor chemiluminescent determination have made it the standard of care for thyroid disease and a model for all endocrine laboratory tests. However, it is an exception rather than the rule, and it is worthwhile reviewing why other testing is not as easy and why considerable judgment is required. The following examples illustrate this point. Pulsatile Hormone Secretion

Many hormones are secreted in pulses rather than steadily. The peaks or valleys of hormones secreted in pulsatile fashion, such as luteinizing hormone or growth hormone, may fall above or below the ostensibly normal range. If such a value is obtained by chance, it can erroneously suggest hypofunction or hyperfunction. Repeating the test with three samples drawn at 30-minute intervals and pooled can clarify this type of problem.



Diurnal Variation

The hypothalamic-pituitary-adrenal axis of cortisol secretion is typically maximal during the day and lower in the evening and night. A plasma cortisol level of 12 µg/dL is normal at 8 AM, but the same value at 8 PM reflects a loss of diurnal rhythm resulting from either stress or hypercorticism. A plasma cortisol sample drawn at midnight is an excellent test for evaluation of overactive adrenal function.



Cyclic Variation

The menstrual cycle provides the most extreme "normal variation" of any hormone level. From the first day of a menstrual period, when estrogen levels may be indistinguishable from those of a normal man, the level rises extraordinarily rapidly and at the 14th day can be as high as in early pregnancy. As a consequence, an estrogen level must be evaluated in the light of the stage of the cycle at which it is drawn.



Age

All clinicians are aware that gonadal hormones show marked differences reflective of the individual's stage of life. It is not as widely known that the adrenal hormone dehydroepiandrosterone (DHEA) is barely secreted during childhood, is actively put out by the adrenal glands from age 8 or so to age 55, and then disappears as mysteriously as it came. At present, there is no clear understanding of the physiologic role of its presence or absence.



Sleep Entrainment

Both prolactin and growth hormone have a sleep-entrained secretory pulse shortly after sleep begins. In people who work at night and sleep during the day, this secretion is clearly related to sleep and not to clock time.



Hormone Antagonism

Certain hormones antagonize the effects of other hormones; it is thus necessary to know the value of each hormone to interpret the clinical phenomenon. The opposite effects of estrogen and androgen on the male breast are a good example. A normal testosterone level combined with an elevated estrogen level, or a normal estrogen level but a decreased androgen level, easily accounts for gynecomastia.



Dynamic Testing

Many endocrine glands have a basal secretory level and a reserve secretion elicited by either a tropic hormone or a change in metabolic or physiologic state. Cortisol secretion can increase fivefold to 10-fold in response to stress or adrenocorticotropic hormone (ACTH). Insulin release is stimulated by both glucose and amino acids and by distinct pathways. Baseline hormone levels can be misleading. The test results in Table 2-1 were obtained on a 30-year-old woman who complained of fatigue and amenorrhea 6 months after a pregnancy during which she had been markedly anemic (hemoglobin, 9 g/dL); she had never been in shock and had received no transfusions.



Hormone and Metabolite Interaction

Insulin is a good example of a hormone whose absolute level is less meaningful than its relationship to the blood glucose level. A plasma insulin of 70 is a normal response to a meal, when the blood glucose level is rising. In contrast, an insulin value of 10 or 12 is abnormal (is not appropriately suppressed) if the glucose level is 40 mg/dL. Indeed, the lower insulin level in a hypoglycemic patient is distinct evidence of spontaneous hyperinsulinism, such as in an islet cell tumor. Growth hormone represents another instance in which a single random sample cannot be given much meaning. During a day, plasma growth hormone levels vary from values that, if sustained, would be diagnostic of acromegaly to values that, again if sustained, would point to hypopituitarism. In normal people, growth hormone secretion is suppressed by glucose intake. A plasma growth hormone of 8 within an hour of a standard meal containing glucose would be pathologically elevated; it should be less than 2. Similarly, however, a growth hormone value of 2 in a fasting person who had run up a flight of stairs suggests deficient pituitary function. Most hormones are part of a feedback loop in which an artificial increase, especially by ingestion of the hormone in a medication, decreases endogenous secretion. If a normal person takes 0.1 to 0.3 mg of thyroxine (T 4 ), hypothalamic secretion of thyrotropin-releasing hormone (TRH) and pituitary secretion of thyrotropin (TSH, or thyroid-stimulating hormone) are suppressed. Plasma levels of T 4 and triiodothyronine (T 3 ) may not change, but TSH levels would be decreased and reflections of TSH effect, such as radioactive iodine uptake, would similarly be suppressed. Although ultrasensitive TSH testing has replaced tests of suppressibility for the diagnosis of thyroid disease, tests of suppressibility are the standard approach to evaluating growth hormone and ACTH/cortisol regulation, respectively.



Protein Binding

Hormones such as T4 and cortisol are compartmentalized into a fraction attached to a transport protein (and thus physiologically

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Time

Glucose

0 80 mg/dL

Cortisol 7.7 µg/dL

TSH

TABLE 2-1 -- Baseline Hormonal Values * Prolactin FSH

3.2 mU/L

Insulin IV

6.6 ng/mL

8.9 mIU/mL

TRH IV

LH 6.7 mIU/mL GnRH IV

15 47

8.2

5.9

9.3

30 23

8.6

5.6

7.6

45 30

7.7

60 38

13.0

90 50

9.7

120 63

9.6

14.3

13.0

15.4

*In this study, all basal values are within normal limits. Note, however, that intravenous insulin lowers the blood glucose levels but does not elicit an adequate release of cortisol. Thyrotropin-releasing hormone (TRH) does not induce a normal rise in thyrotropin (TSH) or prolactin. Gonadotropin-releasing hormone (GnRH) evokes a submaximal increase in follicle-stimulating hormone (FSH) and luteinizing hormone (LH).

unavailable) and a free portion able to diffuse into cells and initiate a hormone effect. It is the free or unbound portion that is physiologically regulated; the level of the binding protein may be increased or decreased without physiologic consequence if the free portion is unchanged. The measurement of free T 4 or a free T4 index (FT4 I) (see Chapter 10) has become the standard second step if a screening TSH value is abnormally high or low. Testosterone is even more complicated because it is trebly partitioned among sex hormonebinding globulin, albumin, and a free portion. Measurement of the free hormone level is often necessary, particularly when the binding protein level has been artifactually raised or lowered (see Chapter 6) .



Laboratory Error

Laboratory error may seem too obvious a source of confusion to mention, but it provides a reminder for an important caution about laboratory testing. It is easy to be seduced by numbers and to consider the laboratory report the final arbiter. In fact, it is the history and physical examination, plus the clinician's judgment, that establish the prior probability of a given diagnosis. Both in choosing and in interpreting laboratory tests, the endocrinologist should establish his or her own expectations before testing. If the physician feels strongly that a particular condition is present, discordant initial laboratory results should not be dissuasive. More detailed testing, as discussed in subsequent chapters, is then appropriate. The clinician's judgment is still a key component of the process.



IMAGING The extraordinary power of modern imaging, particularly ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI), has enriched endocrinology as it has all of medicine. However, the role of imaging in endocrinology is, to my mind, different from its contribution elsewhere. For one thing, several endocrine glands (the thyroid, the pituitary, and the adrenals in particular) frequently contain clinically insignificant, nonfunctioning adenomas and cysts. Second, functioning and nonfunctioning lesions other than in the thyroid gland can be very difficult to distinguish from each other. Thus, except in an emergency (e.g., suspected pituitary apoplexy), the clinician should define the functional state of the gland before requesting imaging. In other words, one should be clear from hormone measurements, including dynamic testing, whether the gland is overactive, underactive, or normal. In addition, one should have a clear idea of how the radiologist can be expected to help. Such clarity reduces costs by targeting the selection of imaging and making the radiologic findings a truly complementary element of evaluation. The best imaging modalities for the various glands are discussed in their respective chapters. The approach suggested here, however, is broadly applicable.



CONVEYING RESULTS Both sophisticated imaging and thorough laboratory testing produce results after the actual office visit. Patients are understandably anxious about the findings and deserve a prompt response. The best approach depends on the circumstance. A new patient with Cushing's syndrome or acromegaly should be given an early in-person visit. A patient with hypothyroidism who understands the disease well and just needs a slight change in T 4 dose can easily be informed with a telephone call. Someone with negative results can be left a message of reassurance and can be encouraged to call back, both to confirm receipt of the information and to get questions answered.



SOME NEW FEATURES OF CLINICAL ENDOCRINOLOGY Genetics

The decoding of the human genome promises to change the face of medical practice. Ironically, the first human disease in which cancer was prevented by application of genetic testing in susceptible families was the screening for medullary thyroid carcinoma in pedigrees of multiple endocrine neoplasia type 2 (MEN-2). The screening at that time was done by pentagastrin or calcium provocation of calcitonin release. Now the screening for endocrine manifestations of MEN-2 is secondary to screening families for the RET proto-oncogene defect that is the basis of the disease. Endocrine testing, such as measurement of calcitonin or plasma catecholamines, is restricted to patients who have the genetic abnormality. In the dangerous variant of the MEN-2 syndrome in which medullary thyroid carcinomas appear in the first year of life, aggressive genetic screening is done during

16

that year, and endocrine testing in patients at risk can justify surgical thyroidectomy before the first birthday. Hereditary predispositions will certainly emerge for other endocrine disorders and will make it crucial for the clinician to take a revealing family history and follow up even minor clues.



The Internet

Never in history has so much medical information been available to patients. I therefore now routinely ask patients what they already know about their condition or their symptoms. A bit sheepishly in some cases, many patients admit to looking up topics on the World Wide Web and are about to compare what I tell them with what they have already read. Much of that information is accurate, but some is nonsense, and it requires patience and clear explanation before such patients go away satisfied.



Electronic Mail

Although opinions differ widely, I find e-mail an extremely useful advance in communicating with patients who have computers. They have access to you between appointments and on a time frame of mutual convenience. The computer thus reduces anxiety on the patient's part, particularly regarding questions or findings for which they might otherwise hesitate to make an appointment. Reporting laboratory test results is expedited, and accompanying the report with a few sentences of interpretation can be as useful as a telephone call. It is wise to keep copies of e-mails so that a clear record of the exchange is available. There are, however, several important caveats. Never let an e-mail exchange substitute for a true evaluation, including history and physical examination. I believe that one should not prescribe for a patient whom one has not seen, and one should not provide much interpretation of history or laboratory tests without very fundamental disclaimers. On the whole, however, e-mail can be used to initiate a new relationship and can certainly support an ongoing one.



Managed Care

The effort to control health care costs by limiting reimbursement for physician services, laboratory testing, and imaging has had a profound impact throughout medicine. Without taking on the whole issue, I want to comment on several practical consequences. "Curbsiding"the request by a physician for patient guidance without being asked to see the patienthas increased strikingly. Consultants can provide some general help to primary care physicians without seeing the patient; however, much hinges on the history and physical examination done by the primary care physician. The failure to realize that hyperthyroidism is due to a hot nodule, for example, totally distorts the picture and will lead to an erroneous recommendation for treatment. The failure to distinguish a recent onset of amenorrhea and virilization from a polycystic ovary-like syndrome may hide the presence of a readily curable virilizing tumor. The failure to recognize hypoglycemic unresponsiveness may perpetuate a dangerous degree of overinsulinization and elicit inappropriate advice from the consultant. Thus, the consultant must set bounds and at some point indicate that it is important for a formal consultation to take place.



Costs

Some endocrine work-ups can be expensive and invite challenge from third-party insurers. The best approach to this concern is a careful history and physical examination, clear establishment of the prior probabilities of certain diagnoses, and then effective use of screening tests before embarking on an unnecessarily extensive evaluation. For example, in a patient with suspected Cushing's syndrome, it is mandatory to establish the presence of hypercorticism before embarking on a search for its cause. Once this lethal but curable disorder has been properly diagnosed, however, no cost should deter one from finding the cause and correcting it. One argument I make is that expensive tests and imaging should be amortized rather than considered an extravagant or unnecessary expense. If a young woman age 30, with a life expectancy of 80 years or more, has a husband and two children to whom her life matters, the $3000 evaluation breaks down to $20 per loved one per year of life expectancy. Any plan manager has to see that this is an appropriate cost.



MANAGEMENT There are few more gratifying experiences in medicine than recognizing and correcting an endocrine disorder. Patients feel that they have been rescued from a mysterious overtaking of their identity. Body contour, facial appearance, temperament, and well-being are restored to the patient's constitutive status. Deterioration attributed to aging or depression or chronic disease is reversed. In brief, something almost miraculous takes place. Even when these goals cannot be achieved, as in diabetes, a major impact on mortality and morbidity can be. Of course, these optimal outcomes require accuracy of diagnosisbut that is only the beginning. A true sharing by patient and physician, based on a sound knowledge of normal physiology, provides the best foundation for choice of therapy and maintenance of a continuing program. The result can be, simply put, wonderful.



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Chapter 3 - Genetic Control of Peptide Hormone Formation Joel F. Habener

Advances in the fields of molecular and cellular biology have provided new insights into the mechanistic workings of cells. Recombinant deoxyribonucleic acid (DNA) technology and the sequencing (decoding) of the entire human and mouse genomes now make it possible to analyze the precise structure and function of DNA, the genetic substance that is the basis for life. The discovery of the unique biochemical and structural properties of DNA provided the conceptual framework with which to begin a systematic investigation of the origins, development, and organization of life forms. [1] The near completion of the entire sequences of the human and mouse genomes was accomplished in the years 2000 and 2001, 5 years ahead of the originally anticipated schedule. The availability of a complete blueprint of the structure and organization of all expressed genes now provides profound insights into the basis of genetically determined diseases. Within the next decades, genotyping of individuals shortly after birth will be possible. Therapeutic approaches for the correction of genetic defects by techniques of gene replacement are likely to become a reality. The polypeptide hormones constitute a critically important and diverse set of regulatory molecules encoded by the genome whose functions are to convey specific information among cells and organs. This type of molecular communication arose early in the development of life and evolved into a complex system for the control of growth, development, and reproduction and for the maintenance of metabolic homeostasis. These hormones consist of approximately 400 or more small proteins ranging from as few as three amino acids (thyrotropin-releasing hormone, TRH) to 192 amino acids (growth hormone). In a broader sense, these polypeptides function both as hormones, whose actions on distant organs are mediated by way of their transport through the blood stream, and as local cell-to-cell communicators (Fig. 3-1) . The latter function of the polypeptide hormones is exemplified by their elaboration and secretion within neurons of the central, autonomic, and peripheral nervous systems, where they act as neurotransmitters. These multiple modes of expression of the polypeptide hormone genes have aroused great interest in the specific functions of these peptides and the mechanisms of their synthesis and release. This chapter reviews the diverse structures of genes encoding peptide hormones and the multiple mechanisms that govern their expression. The synthesis of nonpeptide hormones (e.g., catecholamines, thyroid hormones, steroid hormones) involves the action of multiple enzymesand hence the expression of multiple genesand is discussed in the individual chapters devoted to such hormones.



EVOLUTION OF PEPTIDE HORMONES AND THEIR FUNCTIONS Peptide hormones arose early in the evolution of life. Indeed, polypeptides that are structurally similar to mammalian peptides are present in lower vertebrates, insects, yeasts, and bacteria. [2] An example of the early evolution of regulatory peptides is the factor (mating pheromone) of yeast, which is similar in structure to mammalian luteinizing hormone-releasing hormone (also called gonadotropin-releasing hormone [GnRH]). [3] The oldest member of the cholecystokinin-gastrin family of peptides appeared at least 500 million years ago in the protochordate Ciona intestinalis. [4] Thus, the genes encoding polypeptide hormones, and particularly regulatory peptides, evolved early in the development of life and initially fulfilled the function of cell-to-cell communication to cope with problems concerning nourishment, growth, development, and reproduction. As specialized organs connected by a circulatory system developed during evolution, similar, if not identical, gene products became hormones for purposes of organ-to-organ communication.

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Figure 3-1 Different modes of utilization of polypeptide hormones in expression of their biologic actions. The peptide hormones are expressed in at least four ways in fulfilling their functions as cellular messenger molecules: (1) endocrine mode, for purposes of communication among organs (e.g., pituitary-thyroid axis); (2) paracrine mode, for communication among adjacent cells, often located within endocrine organs; (3) neuroendocrine mode, for synthesis and release of peptides from specialized peptidergic neurons for action on distant organs through the blood stream (e.g., neuroendocrine peptides of hypothalamus); and (4) neurotransmitter mode, for action of peptides in concert with classical amino acid-derived aminergic transmitters in the neuronal communication network. Identical polypeptides are often utilized in the nervous system both as neuroendocrine hormones and as neurotransmitters. In some instances, the same gene product is used in all four modes of expression.



STEPS IN EXPRESSION OF A PROTEIN-ENCODING GENE The steps involved in transfer of information encoded in the polynucleotide language of DNA to the poly-amino acid language of biologically active proteins involve gene transcription, post-transcriptional processing of ribonucleic acids (RNAs), translation, and post-translational processing of the proteins. The expression of genes and protein synthesis can be considered in terms of several major processes, any one or more of which may serve as specific control points in the regulation of gene expression (Fig. 3-2) : 1. Rearrangements and transpositions of DNA segments. These processes occur over many years (eons) in evolution, with the exception of uncommon mechanisms of somatic gene rearrangements such as the rearrangements in the immunoglobulin genes during the lifetime of an individual. 2. Transcription. Synthesis of RNA results in the formation of RNA copies of the two gene alleles and is catalyzed by the basal RNA polymerase II-associated transcription factors. 3. Post-transcriptional processing. Specific modifications of the RNA include the formation of messenger RNA (mRNA) from the precursor RNA by way of excision and rejoining of RNA segments (introns and exons) and modifications of the 3' end of the RNA by polyadenylation and of the 5' end by addition of 7-methylguanine "caps." 4. Translation. Amino acids are assembled by base pairing of the nucleotide triplets (anticodons) of the specific "carrier" aminoacylated transfer RNAs to the corresponding codons of the mRNA bound to polyribosomes and are polymerized into the polypeptide chains. 5. Post-translational processing and modification. Final steps in protein synthesis may involve one or more cleavages of peptide bonds, which result in the conversion of biosynthetic precursors (prohormones), to intermediate or final forms of the protein; derivatization of amino acids (e.g., glycosylation, phosphorylation, acetylation, myristoylation); and the folding of the processed polypeptide chain into its native conformation. Each of the specific steps of gene expression requires the integration of precise enzymatic and other biochemical reactions. These processes have developed to provide high fidelity in the reproduction of the encoded information and to provide control points for the expression of the specific phenotype of cells. The post-translational processing of proteins creates diversity in gene expression through modifications of the protein. Although the functional information contained in a protein is ultimately encoded in the primary amino acid sequence, the specific biologic activities are a consequence of the higher order secondary, tertiary, and quaternary structures of the polypeptide. Given the wide range of possible specific modifications of the amino acids, such as glycosylation, phosphorylation, acetylation, and sulfation, [5] any one of which may affect the conformation or function of the protein, a single gene may ultimately encode a wide variety of specific proteins as a result of post-translational processes. Polypeptide hormones are synthesized in the form of larger precursors that appear to fulfill several functions in biologic systems (Fig. 3-3) , including (1) intracellular trafficking, by which the cell distinguishes among specific classes of proteins and directs them to their sites of action, and (2) the generation of multiple biologic activities from a common genetically encoded protein by regulated or cell-specific variations in the post-translational modifications (Fig. 3-4) . All the peptide hormones and regulatory peptides studied thus far contain signal or leader sequences at the amino termini; these hydrophobic sequences recognize specific sites on the membranes of the rough endoplasmic reticulum, which results in the transport of nascent polypeptides into the secretory pathway of the cell ( see Fig. 3-2 and Fig. 3-3 ).[6] The consequence of the specialized signal sequences of the precursor proteins is that proteins destined for secretion are selected from a great many other cellular proteins for sequestration and subsequent packaging into secretory granules and export from the cell. In addition, most, if not all, of the smaller hormones and regulatory peptides are produced as a consequence of post-translational cleavages of the precursors within the Golgi complex of secretory cells.



SUBCELLULAR STRUCTURE OF CELLS THAT SECRETE PROTEIN HORMONES Cells whose principal functions are the synthesis and export of proteins contain highly developed, specialized subcellular organelles for the translocation of secreted proteins and their

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Figure 3-2 Steps in the cellular synthesis of polypeptide hormones. Steps that take place within the nucleus include transcription of genetic information into a messenger ribonucleic acid (mRNA) precursor (pre-mRNA) followed by post-transcriptional processing, which includes RNA cleavage, excision of introns, and rejoining of exons, resulting in formation of mRNA. Ends of mRNA are modified by addition of methylguanosine caps at the 5' end and addition of poly(A) tracts at the 3' ends. The cytoplasmic mRNA is assembled with ribosomes. Amino acids, carried by aminoacylated transfer RNAs (tRNAs), are then polymerized into a polypeptide chain. The final step in protein synthesis is that of post-translational processing. These processes take place both during growth of the nascent polypeptide chain (cotranslational) and after release of the completed chain (post-translational), and they include proteolytic cleavages of polypeptide chain (conversion of pre-prohormones or prohormones to hormones), derivatizations of amino acids (e.g., glycosylation, phosphorylation), and cross-linking and assembly of the polypeptide chain into its conformed structure. The diagram depicts post-translational synthesis and processing of a typical secreted polypeptide, which requires vectorial, or unidirectional, transport of the polypeptide chain across the membrane bilayer of the endoplasmic reticulum, thus resulting in sequestration of the polypeptide in the cisterna of the endoplasmic reticulum, a first step in the export of proteins destined for secretion from the cell (see Fig. 3-6) . Most translational processing occurs within the cell as depicted (presecretory) and in some instances outside the cell, when further proteolytic cleavages or modifications of the protein may take place (postsecretory). CHO, carbohydrate.

packaging into secretory granules. The subcellular pathways utilized in protein secretion have been elucidated largely through the early efforts of Palade [7] and colleagues (reviewed by Jamieson [8] ). Secretory cells contain an abundance of endoplasmic reticulum, Golgi complexes, and secretory granules (Fig. 3-5) . The proteins that are to be secreted from the cells are transferred during their synthesis into these subcellular organelles, which transport the proteins to the plasma membrane. Protein secretion begins with translation of the mRNA encoding the precursor of the protein on the rough endoplasmic reticulum, which consists of polyribosomes attached to elaborate membranous saccules that contain cavities (cisternae). The newly synthesized, nascent proteins are discharged into the cisternae by transport across the lipid bilayer of the membrane. Within the cisternae of the endoplasmic reticulum, proteins are carried to the Golgi complex by mechanisms that are incompletely understood. The proteins gain access to the Golgi complex either by direct transfer from the cisternae, which are in continuity with the membranous channels of the Golgi complex, or by way of shuttling vesicles known as transition elements (see Fig. 3-5) . Within the Golgi complex, the proteins are packaged into secretory vesicles or secretory granules by their budding from the Golgi stacks in the form of immature granules. Immature granules undergo maturation through condensation of the proteinaceous material and application of a specific coat around the initial Golgi membrane. On receiving the appropriate extracellular stimuli (regulated pathway of secretion), the granules migrate to the cell surface and fuse to become continuous with the plasma membrane, which results in the release of proteins into the extracellular space, a process known as exocytosis. The second pathway of intracellular transport and secretion involves the transport of proteins contained within secretory vesicles and immature secretory granules (see Fig. 3-5) . Although the use of this alternative vesicle-mediated transport pathway remains to be demonstrated conclusively (it is generally considered to be a constitutive, or unregulated, pathway), different extracellular stimuli may modulate hormone secretion differently, depending on the pathway of secretion. For example, in the parathyroid gland and in the pituitary cell line derived from corticotropic cells (AtT-20), newly synthesized hormone is released more rapidly than hormone synthesized

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Figure 3-3 Diagrammatic depiction of two configurations of precursors of polypeptide hormones. Diagrams represent polypeptide backbones of protein sequences encoded in mRNA. One form of precursor consists of the NH 2 -terminal signal, or presequence, followed by the apoprotein portion of the polypeptide that needs no further proteolytic processing for activity. A second form of precursor is a pre-prohormone that consists of the NH 2 -terminal signal sequence followed by a polyprotein, or prohormone, sequence consisting of two or more peptide domains linked together that are subsequently liberated by cleavages during post-translational processing of the prohormone. The reason for synthesis of polypeptide hormones in the form of precursors is only partly understood. Clearly, NH2 -terminal signal sequences function in the early stages of transport of polypeptide into the secretory pathway. Prohormones, or polyproteins, often serve to provide a source of multiple bioactive peptides (see Fig. 3-4) . However, many prohormones contain peptide sequences that are removed by cleavage and have no known biologic activity, and they are referred to as cryptic peptides. Other peptides may serve as spacer sequences between two bioactive peptides (e.g., the C peptide of proinsulin). In instances in which a bioactive peptide is located at the COOH terminus of the prohormone, the NH 2 -terminal prohormone sequence may simply facilitate cotranslational translocation of polypeptide in endoplasmic reticulum (see Fig. 3-6) .

earlier. These findings suggest that the newly synthesized hormone may be transported by way of a vesicle-mediated pathway without incorporation into mature storage granules.



INTRACELLULAR SEGREGATION AND TRANSPORT OF POLYPEPTIDE HORMONES Specific amino acid sequences encoded in the proteins serve as directional signals in the sorting of proteins within subcellular organelles. [6] [9] [10] A typical eucaryotic cell synthesizes an estimated 5000 different proteins during its life span. These different proteins are synthesized by a common pool of polyribosomes. However, each of the different proteins is directed to a specific location within the cell, where its biologic function is expressed. For example, specific groups of proteins are transported into mitochondria, into membranes, into the nucleus, or into other subcellular organelles, where they serve as regulatory proteins, enzymes, or structural proteins. A subset of proteins is specifically designed for export from the cell (e.g., immunoglobulins, serum albumin, blood coagulation factors, and protein and polypeptide hormones). This process of directional transport of proteins involves sophisticated informational signals. Because the information for these translocation processes must reside either wholly or in part within the primary structure or in the conformational properties of the protein, sequential post-translational modifications may be crucial for determining the specificity of protein function.

Figure 3-4 Diagrammatic illustration of primary structures of several prohormones. The darkly shaded areas of prohormones denote regions of sequence that constitute known biologically active peptides after their post-translational cleavage from prohormones. Sequences indicated by hatching denote regions of precursor that alter the biologic specificity of that region of precursor. For example, the precursor contains the sequence of -melanocyte-stimulating hormone (-MSH), but when the latter is covalently attached to the clip peptide, it constitutes adrenocorticotropic hormone (corticotropin, ACTH). Somatostatin-28 (SS-28) is an NH 2 -terminally extended form of somatostatin-14 (SS-14) that has higher potency than somatostatin-14 on certain receptors. The neurophysin sequence linked to the COOH terminus of vasopressin (ADH) functions as a carrier protein for hormone during its transport down the axon of neurons in which it is synthesized. Precursor proenkephalin represents a polyprotein that contains multiple similar peptides within its sequence, either met-enkephalin (M) or leu-enkephalin (L). Procalcitonin and procalcitonin gene-related product (CGRP) share identical NH 2 -terminal sequences but differ in their COOH-terminal regions as a result of alternative splicing during the post-transcriptional processing of the RNA precursor. -LPH, -lipotropin; GLP, glucagon-like peptide; IP, intervening peptide. Signal Sequences in Peptide Prohormone Processing and Secretion

The early processes of protein secretion that result in the specific transport of exported proteins into the secretory pathway are now becoming better understood. [6] [10] [11] [12] Initial clues to this process came from determinations of the amino acid sequences of the proteins programmed by the cell-free translation of mRNAs encoding secreted polypeptides. [13] Secreted proteins are synthesized as precursors that are extended at their NH 2 termini by sequences of 15 to 30 amino acids, called signal or leader sequences. Signal sequence extensions, or their functional equivalents, are required for targeting the ribosomal or nascent protein to specific membranes and for the vectorial transport of the protein across the membrane of the endoplasmic reticulum. On emergence of the signal sequence

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Figure 3-5 Schematic representation of subcellular organelles involved in transport and secretion of polypeptide hormones or other secreted proteins within a protein-secreting cell. (1) Synthesis of proteins on polyribosomes attached to endoplasmic reticulum (RER) and vectorial discharge of proteins through the membrane into the cisterna. (2) Formation of shuttling vesicles (transition elements) from endoplasmic reticulum followed by their transport to and incorporation by the Golgi complex. (3) Formation of secretory granules in the Golgi complex. (4) Transport of secretory granules to the plasma membrane, fusion with the plasma membrane, and exocytosis resulting in the release of granule contents into the extracellular space. Note that secretion may occur by transport of secretory vesicles and immature granules as well as mature granules. Some granules are taken up and hydrolyzed by lysosomes (crinophagy). Golgi, Golgi complex; RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum. (From Habener JF. Hormone biosynthesis and secretion. In Felig P, Baxter JD, Broadus AE, et al. [eds]. Endocrinology and Metabolism. New York, McGraw-Hill, 1981, pp 2959. Copyright © 1981 by McGraw-Hill, Inc. Used by permission of McGraw-Hill Book Company.)

from the large ribosomal subunit, the ribosomal complex specifically makes contact with the membrane, which results in translocation of the nascent polypeptide across the endoplasmic reticulum membrane into the cisterna as the first step in the transport of the polypeptide within the secretory pathway. These observations initially left unanswered the question of how specific polyribosomes that translate mRNAs encoding secretory proteins recognize and attach to the endoplasmic reticulum (Fig. 3-6) . Because microsomal membranes in vitro reproduce the processing activity of intact cells, it was possible to identify macromolecules responsible for processing of the precursor and for translocation activities. [14] The endoplasmic reticulum and the cytoplasm contain an aggregate of molecules, called a signal recognition particle complex, that consists of at least 16 different proteins, including three guanosine triphosphatases to generate energy [15] and a 7S RNA.[6] [10] [16] This complex, or particle, binds to the polyribosomes involved in the translation of mRNAs encoding secretory polypeptides when the NH 2 -terminal signal sequence first emerges from the large subunit of the ribosome. The specific interaction of the signal recognition particle with the nascent signal sequence and the polyribosome arrests further translation of mRNA. The nascent protein remains in a state of arrested translation until it finds a high-affinity binding protein on the endoplasmic reticulum, the signal recognition particle receptor, or docking protein. [6] On interaction with the specific docking protein, the translational block is released and protein synthesis resumes. The protein is then transferred across the membrane of the endoplasmic reticulum through a proteinaceous tunnel. At some point, near the termination of synthesis of the polypeptide chain, the NH 2 -terminal signal sequence is cleaved from the polypeptide by a specific signal peptidase located on the cisternal surface of the endoplasmic reticulum membrane. The removal of the hydrophobic signal sequence frees the protein (prohormone or hormone) so that it may assume its characteristic secondary structure during transport through the endoplasmic reticulum and the Golgi apparatus. Interestingly, after its cleavage from the protein by signal peptidase, the signal peptide may sometimes be further cleaved in the endoplasmic reticulum membrane to produce a biologically active peptide. The signal sequence of preprolactin of 30 amino acids, for example, is cleaved by a signal peptide peptidase to give a charged peptide of 20 amino acids that is released into the cytosol, where it binds to calmodulin and inhibits Ca 2+ -calmodulin-dependent phosphodiesterase. [17] This sequence in the directional transport of specific polypeptides ensures optimal cotranslational processing of secretory proteins, even when synthesis commences on free ribosomes. The presence of a cytoplasmic form of the signal recognition particle complex that blocks translation guarantees that the synthesis of the presecretory proteins is not completed in the cytoplasm; the efficient transfer of proteins occurs only after contact has been made with the specific receptor or docking protein on the membrane. Although the identification of the signal recognition particle and the docking protein explains the specificity of the binding of ribosomes containing mRNAs encoding the secretory proteins, it does not explain the mode of translocation of the nascent polypeptide chain across the membrane bilayer. Further dissection and analysis of the membrane have identified other macromolecules that are responsible for the transport process. [6]

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Figure 3-6 Diagram depicting cellular events in initial stages of synthesis of a polypeptide hormone according to the signal hypothesis. In this schema, a signal recognition particle, consisting of a complex of six proteins and an RNA (7S RNA), interacts with the NH 2 -terminal signal peptide of the nascent polypeptide chain after approximately 70 amino acids are polymerized, which results in the arrest of further growth of the polypeptide chain. The complex of the signal recognition particle and the polyribosome nascent chain remains in a state of translational arrest until it recognizes and binds to a docking protein, which is a receptor protein located on the cytoplasmic face of the endoplasmic reticular membrane. This interaction of the signal recognition particle complex with docking protein releases the translational block, and protein synthesis resumes. The nascent polypeptide chain is discharged across the membrane bilayer into the cisterna of the endoplasmic reticulum and is released from the signal peptide by cleavage with a signal peptidase located in the cisternal face of the membrane. In this model, the signal peptide is cleaved from the polypeptide chain by signal peptidase before the chain is completed (cotranslational cleavage). The configuration of the polypeptide during transport across the membrane and the forces and mechanisms responsible for its translocation are unknown. The loop, or hairpin, configuration of the chain that is shown is an arbitrary model; other models are equally possible.



Cellular Processing of Prohormones

The signal sequences of prehormones and pre-prohormones are involved in the transport of these molecules, but the function of the intermediate hormone precursors (prohormones) is not fully understood. The conversion of prohormones to their final products begins in the Golgi apparatus. For example, the time that elapses between the synthesis of pre-proparathyroid hormone and the first appearance of parathyroid hormone correlates closely with the time required for radioautographic grains to reach the Golgi apparatus. [18] Similarly, the conversion of proinsulin to insulin takes place about an hour after the synthesis of proinsulin is complete, and processing of proinsulin to insulin and C peptide takes place during the transport within the secretory granule. [19] The conversion of prohormones to hormones can also be blocked by inhibitors of cellular energy production such as antimycin A and dinitrophenol [20] and by drugs that interfere with the functions of microtubules (vinblastine, colchicine). [21] Thus, the translocation of the prohormone from the rough endoplasmic reticulum to the Golgi complex depends on metabolic energy and probably involves microtubules. There is no evidence that sequences that are specific to the prohormone contribute to or are chemically involved in transport of the newly synthesized protein from the rough endoplasmic reticulum to the Golgi apparatus or that they are involved in the packaging of the hormone in the vesicles or granules. Analyses of the structures of the primary products of translation of mRNAs encoding secretory proteins indicate that many of these are not synthesized in the form of prohormone intermediates (see Fig. 3-3) . It remains puzzling that some secretory proteins (e.g., parathyroid hormone, insulin, serum albumin) are formed by way of intermediate precursors, whereas others (e.g., growth hormone, prolactin, albumin) are not. Size constraints may be placed on the length of a secretory polypeptide. When the bioactivity of peptides resides at the COOH termini of the precursors (e.g., somatostatin, calcitonin, gastrin), NH 2 -terminal extensions may be required to provide a sufficient "spacer" sequence to allow the signal sequence on the growing nascent polypeptide chain to emerge from the large ribosome subunit for interaction with the signal recognition particle and to provide adequate polypeptide length to span the large ribosomal subunit and the membrane of the endoplasmic reticulum during vectorial transport of the nascent polypeptide across the membrane (see Fig. 3-6) . When the final hormonal product is 100 amino acids long or longer (e.g., growth hormone, prolactin, or the and subunits of the glycoprotein hormones), there may be no requirement for a prohormone intermediate. Although the exact functions of prohormones remain unknown, certain details of their cleavages have been established. Unlike

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Figure 3-7 Regulatory feedback loops of the hypothalamic-pituitary-target organ axis. Being a combination of both stimulatory and inhibitory factors, hormones often act in concert to maintain homeostatic balance in the presence of physiologic or pathophysiologic perturbations. The concerted actions of hormones typically establish closed feedback loops by stimulatory and inhibitory effects coupled to maintain homeostasis.

the situation with prehormones, in which the amino acids at the cleavage site between the signal sequence and the remainder of the molecule (hormone or prohormone) vary from one hormone to the next, the cleavage sites of the prohormone intermediates consist of the basic amino acid lysine or arginine, or both, usually two to three in tandem. This sequence is preferentially cleaved by endopeptidases with trypsin-like activities. Specific prohormone-converting enzymes (PCs) consist of a family of at least eight such enzymes. [22] [23] [24] The most studied of the isozymes are PC2 and PC1/3, which are responsible for the cleavages of proinsulin between the A chain/C peptide and B chain/C peptide, respectively. A rare patient missing PC1 presented with childhood obesity, hypogonadotropic hypogonadism, and hypercortisolism and was found to have elevated proinsulin levels and presumably widespread abnormalities in neuropeptide modification. [25] Targeted disruption of the PC2 gene in mice resulted in incomplete processing of proinsulin, leaving the A chain and C peptide intact. [26] Notably, proglucagon in the pancreas remains completely unprocessed, indicating that PC2 is required for the formation of glucagon. As a consequence of defective PC2 activity and low levels of glucagon, the mice have severe chronic hypoglycemia. After endopeptidase cleavage, the remaining basic residues are selectively removed by exopeptidases with activity resembling that of carboxypeptidase B. In the instances in which the COOH-terminal residue of the peptide hormone is amidated, a process that appears to enhance the stability of a peptide by conferring resistance to carboxypeptidase, specific amidation enzymes in the Golgi complex work in concert with the cleavage enzymes for modification of the COOH terminal of the bioactive peptides. [27] [28] All proproteins and prohormones are cleaved by PC enzymatic processes within the Golgi complex of cells of diverse origins. The significance of specific cleavages of specific prohormones remains incompletely understood, as does the reason for the existence of prohormone intermediates in some but not all secretory proteins. As indicated earlier, precursor peptides removed from the prohormones may have intrinsic biologic activities that are as yet unrecognized.



PROCESSES OF HORMONE SECRETION Specific extracellular stimuli control the secretion of polypeptide hormones. The stimuli consist of changes in homeostatic balance; the hormonal products released in response to the stimuli act on the respective target organs to reestablish homeostasis (Fig. 3-7) . Endocrine systems typically consist of closed-loop feedback mechanisms such that, if hormones from organ A stimulate organ B, organ B in turn secretes hormones that inhibit the secretion of hormones from organ A. The concerted actions of both positive and negative hormonal influences thereby maintain homeostasis. For example, an increase in the concentration of plasma electrolytes as a consequence of dehydration stimulates the release of arginine vasopressin (also called antidiuretic hormone [ADH]) in the neural lobe of the pituitary gland, and vasopressin in turn acts on the kidney to increase the reabsorption of water from the renal tubule, thereby readjusting serum electrolyte concentrations toward normal levels. These regulatory processes commonly include inhibitory feedback loops in which the products elaborated by the target organs in response to the actions of a hormone inhibit further endocrine secretion. An example of such negative feedback regulation is the control of the secretion of adrenocorticotropic hormone (ACTH) by the anterior pituitary gland. Increased ACTH stimulates the adrenal cortex to produce and secrete cortisol, which in turn feeds back to suppress further pituitary secretion of ACTH. In many instances, endocrine regulation is complex and involves the responses of several endocrine glands and their respective target organs. After a meal, the release of a dozen or more hormones is triggered as a result of gastric distention, variations in the pH of the contents of the stomach and duodenum, and increased concentrations of glucose, fatty acids, and amino acids in the blood. The rise in plasma glucose and amino acid levels stimulates the release of insulin and the incretin hormones glucagon-like peptide 1 and glucose-dependent insulinotropic peptide and suppresses the release of glucagon from the pancreas. Both effects promote the net uptake of glucose by the liver; insulin increases cellular transport and uptake of glucose, and the lower blood levels of glucagon decrease the outflow of glucose because of diminished rates of glycogenolysis and gluconeogenesis.



STRUCTURE OF A GENE ENCODING A POLYPEPTIDE HORMONE Structural analyses of gene sequences have resulted in at least three major discoveries that are important for understanding

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Figure 3-8 Diagrammatic structure of a "consensus" gene encoding a prototypical polypeptide hormone. Such a gene typically consists of a promoter region and a transcription unit. The transcription unit is the region of deoxyribonucleic acid (DNA) composed of exons and introns that is transcribed into a messenger ribonucleic acid (mRNA) precursor. Transcription begins at the cap site sequence in DNA and extends several hundred bases beyond the poly(A) addition site in the 3' region. During post-transcriptional processing of the RNA precursor, the 5' end of mRNA is capped by addition of methylguanosine residues. The transcript is then cleaved at the poly(A) addition site approximately 20 bases 3' to the AATAAA signal sequence, and the poly(A) tract is added to the 3' end of the RNA. Introns are cleaved from the RNA precursor, and exons are joined together. Dinucleotides GT and AG are invariably found at the 5' and 3' ends of introns. Translation of mRNA invariably starts with the codon ATG for methionine. Translation is terminated when the polyribosome reaches the stop codon TGA, TAA, or TAG. The promoter region of the gene located 5' to the cap site contains numerous short regulatory DNA sequences that are targets for interactions with specific DNA-binding proteins. These sequences consist of the basal constitutive promoter (TATA box), metabolic response elements that modulate transcription (e.g., in response to cAMP, steroid hormone receptors, and thyroid hormone receptors), and tissue-specific enhancers and silencers that permit or prevent transcription of the gene, respectively. The enhancer and silencer elements direct expression of specific subsets of genes to cells of a given phenotype. Whether a gene is or is not expressed in a particular cellular phenotype depends on complex interactions of the various DNA-binding proteins among themselves and, most important, with the TATA box proteins of the basal constitutive promoter.

the expression of peptide-encoding genes. First, sequences of almost all the known biologically active hormonal peptides are contained within larger precursors that often encode other peptides, many of which are of unknown biologic activity. Second, the transcribed regions of genes (exons) are interrupted by sequences (introns) that are transcribed but subsequently cleaved from the initial RNA transcripts during their nuclear processing and assembly into specific mRNAs. Third, specific regulatory sequences reside in the regions of DNA flanking the 5' ends of structural genes, and these DNA sequences constitute specific targets for the interactions of DNA-binding proteins that determine the level of expression of the gene. The DNA of higher organisms is wound into a tightly and regularly packed chromosomal structure in association with a number of different proteins organized into elements called nucleosomes.[29] [30] Nucleosomes are composed of four or five different histone subunits that form a core structure about which approximately 140 base pairs of genomic DNA are wound. The nucleosomes are arranged similarly to beads on a string, and coils of nucleosomes form the fundamental organizational units of the eucaryotic chromosome. The nucleosomal structure serves several purposes. For example, nucleosomes enable the large amount of DNA (2 × 10 9 pairs) of the genome to be compacted into a small volume. Nucleosomes are involved in the replication of DNA and gene transcription. In addition to histones, other proteins are associated with DNA, and the complex nucleoprotein structure provides specific recognition sites for regulatory proteins and enzymes involved in DNA replication, rearrangements of DNA segments, and gene expression. The acetylation and deacetylation of histone-rich chromatin is involved in the regulation of gene transcription. The topography of a typical protein-encoding gene consists of two functional units (Fig. 3-8) : A transcriptional region A promoter or regulatory region Transcriptional Regions

The transcriptional unit is the segment of the gene that is transcribed into an mRNA precursor. The sequences corresponding to the mature mRNA consist of the exon sequences that are spliced from the primary transcript during the posttranscriptional processing of the precursor RNA; these exons contain the code for the mRNA sequence that is translated into protein and for untranslated sequences at the 5'- and 3'-flanking regions. The 5' sequence typically begins with a methylated guanine residue known as the cap site. The 3'-untranslated region contains within it a short sequence, AATAAA, that signals the site of cleavage of the 3' end of the RNA and the addition of a poly(A) tract of 100 to 200 nucleotides located approximately 20 bases from the AATAAA sequence. Although the functions of these modifications of the ends of mRNAs are not completely understood, they appear to provide signals for leaving the nucleus; enhance stability, perhaps through providing resistance to degradation by exonucleases; and stimulate initiation of mRNA translation. The protein-coding sequence of the mRNA begins with the codon AUG for methionine and ends with the codon immediately preceding one of the three nonsense, or stop, codons (UGA, UAA, and UAG). The nature of the enzymatic splicing mechanisms that result in the excision of intron-coded sequences and the rejoining of exon-coded sequences is incompletely understood. Short "consensus"

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sequences of nucleotides reside at the splice junctionsfor example, the bases GT and AG at the 5' and 3' ends of the introns, respectively, are invariantand a polypyrimidine stretch is found near the AG. [31] Splicing involves a series of cleavage and ligation steps that remove the introns as a lariat structure with its 5' end ligated near the 3' end of the introns and ligate the two adjacent exons together. An elaborate machinery (the spliceosome) consisting of five small nuclear RNAs (snRNAs) and roughly 50 proteins direct these steps, guided by base pairing between three of the snRNAs and the mRNA precursor.



Regulatory Regions

The regulation of the expression of genes that encode polypeptides is beginning to be understood in some detail. As a result of experiments involving the deletion of 5' sequences upstream from structural genes, followed by analyses of the expression of the genes after introduction into cell lines, several insights have been obtained. These regulatory sequences, termed promoters and enhancers, consist of short polynucleotide sequences (see Fig. 3-8) . They can be divided into at least four groups with respect to their functions and distances from the transcriptional initiation site. First, the sequence TATAA (TATA, or Goldberg-Hogness, box) is usually present in the more proximal promoter within 25 to 30 nucleotides upstream from the point of transcriptional initiation. The TATA sequence is required to ensure the accuracy of initiation of transcription at a particular site. The TATA box directs the binding of a complex of several proteins, including RNA polymerase II. The proteins, referred to as TATA box transcription factors (TFs), number six or more basal factors (IIA, IIB, IID, IIE, IIF, IIH) and, along with RNA polymerase II, form the general or basal transcriptional machinery required for the initiation of RNA synthesis. [32] The other three groups of regulatory sequences consist of tissue-specific silencers (TSSs), which function by binding repressor proteins; tissue-specific enhancers (TSEs), which are activated by the binding of transcriptional activator proteins; and metabolic response elements (MREs), which are regulated by the binding

Figure 3-9 Diagram of the pancreatic glucagon gene and its encoded messenger RNA (mRNA) (complementary DNA). The glucagon gene is an example of a gene in which exons precisely encode separate functional domains. The gene consists of six exons (E1 to E6) and five introns (1A to 1E). The mRNA encoding pre-proglucagon, the protein precursor of glucagon, consists of 10 specific regions: from left to right, a 5'-untranslated sequence (UN-TX, unshaded), a signal sequence (S, stippled), an NH2 -terminal extension sequence (N, hatched), glucagon (Gluc, shaded), a first intervening peptide (IP-I, hatched), a first glucagon-like peptide (GLP-I, shaded), a second intervening peptide (IP-II, hatched), a second glucagon-like peptide (GLP-II, shaded), a dilysyl dipeptide (hatched) after the glucagon-like peptide II sequence, and an untranslated region (UN-TX, unshaded). Exons from left to right encode the 5'-untranslated region, signal sequence, glucagon, glucagon-like peptide I, glucagon-like peptide II, and 3'-untranslated sequence. Letters shown above the mRNA denote amino acids located at positions in pre-proglucagon that are cleaved during cellular processing of precursor. The amino acid methionine (M) marks the initiation of translation of mRNA into pre-proglucagon. H, histidine; K, lysine; Q, glutamine; R, arginine.

of specialized proteins whose transcriptional activities (repressor or activator) are regulated by metabolic signaling, often involving changes in their phosphorylation.



Introns and Exons

Genes encoding proteins and ribosomal RNAs in eukaryotes are interrupted by intervening DNA sequences (introns) that separate them into coding blocks (exons). [33] In bacterial genes the nucleotide sequences of the chromosomal genes match precisely the corresponding sequences in the mRNAs. Interruption of the continuity of genetic information appears to be unique to nucleated cells. The reasons for such interruption are not completely understood, but introns appear to separate exons into functional domains with respect to the proteins that they encode. An example is the gene for proglucagon, a precursor of glucagon in which five introns separate six exons, three of which encode glucagon and the two glucagon-related peptides contained within the precursor (Fig. 3-9) .[34] A second example is the growth hormone gene, which is divided into five exons by four introns that separate the promoter region of the gene from the protein-coding region and the latter into three partly homologous repeated segments, two coding for the growth-promoting activity of the hormone and the third for its carbohydrate metabolic functions. [35] As a rule, the genes for the precursors of hormones and regulatory peptides contain introns at or about the region where the signal peptides join the apoproteins or prohormones, thus separating the signal sequences from the components of the precursor that are exported from the cell as hormones or peptides. There are exceptions to the one exon, one function theory in mammalian cells. The genes of several precursors of peptide hormones are not interrupted by introns in a manner that corresponds to the separation of the functional components of the precursor. Notable in this regard is the precursor proopiomelanocortin, from which the peptides ACTH, -melanocyte-stimulating hormone, and -endorphin are cleaved during the post-translational processing of the precursor. The proteincoding region of the pro-opiomelanocortin gene is devoid of introns. Likewise, no introns interrupt the protein-coding region

26

of the gene for the proenkephalin precursor, which contains seven copies of the enkephalin sequences. It is possible that, in the past, introns separated each of these coding domains and were lost during the course of evolution. A precedent for the selective loss of introns appears to be exemplified by the rat insulin genes. The rat genome harbors two nonallelic insulin genes: one containing two introns and the other containing a single intron. The most likely explanation is that an ancestral gene containing two introns was transcribed into RNA and spliced; then that RNA was copied back into DNA by a cellular reverse transcriptase and inserted back into the genome at a new site.



REGULATION OF GENE EXPRESSION The regulation of expression of genes encoding polypeptide hormones can take place at one or more levels in the pathway of hormone biosynthesis (Fig. 3-10)

[36] [37]

DNA synthesis (cell growth and division) Transcription Post-transcriptional processing of mRNA Translation Post-translational processing In different endocrine cells, one or more levels may serve as specific control points for regulation of production of a hormone (see also Generation of Biologic Diversification later).

Figure 3-10 Diagram of an endocrine cell showing potential control points for regulation of gene expression in hormone production. Specific effector substances bind either to plasma membrane receptors (peptide effectors) or to cytosolic or nuclear receptors (steroids), which leads to initiation of a series of events that couple the effector signal with gene expression. In the illustration shown, peptide effector-receptor complex interactions act initially through activation of adenylate cyclase (AC) coupled with a guanosine triphosphate-binding protein (G). Coupling factors and substances such as glucose, cyclic adenosine monophosphate, and cations activate protein kinases, resulting in a series of phosphorylations of macromolecules. As discussed in the text, specific effectors for various endocrine cells appear to act at one or more of the indicated five levels of gene expression, with the possible exception of post-translational processing of prohormones, for which no definite examples of metabolic regulation have yet been found. Levels of Gene Control

Newly synthesized prolactin transcripts are formed within minutes after exposure of a prolactin-secreting cell line to TRH. [38] Cortisol stimulates growth hormone synthesis in both somatotropic cell lines and pituitary slices through increases in rates of gene transcription and enhancement of the stability of mRNA. [39] [40] The time required for cortisol to enhance transcription of the growth hormone gene is 1 to 2 hours, which is considerably longer than the time required for the action of TRH on prolactin gene transcription. Regulation of proinsulin biosynthesis appears to take place primarily at the level of translation. [41] [42] Within minutes after raising the plasma glucose level, the rate of proinsulin biosynthesis increases fivefold to 10-fold. Glucose acts either directly or indirectly to enhance the efficiency of initiation of translation of proinsulin mRNA. Rapid metabolic regulation at the level of post-transcriptional processing of mRNA precursors is not yet clearly established. However, alternative exon splicing plays a major role in the regulation of the formation of mRNAs during development (see next section in chapter on Generation of Biologic Diversification). For example, the primary RNA transcripts derived from the calcitonin gene are alternatively spliced to provide two or more tissue-specific mRNAs that encode chimeric protein precursors with both common and different amino acid sequences, suggesting that regulation might take place at the level of processing of the calcitonin gene transcripts. In many instances, the level of gene expression under regulatory control is optimal for meeting the secretory and biosynthetic demands of the endocrine organ. For example, after a meal there is an immediate requirement for the release of large amounts of insulin. This release depletes insulin stores of

27

the pancreatic beta cells within a few minutes, and increasing the translational efficiency of preformed proinsulin mRNA provides additional hormone rapidly.


:


Tissue-Specific Gene Expression

Differentiated cells have a remarkable capacity for selective expression of specific genes. In one cell type, a single gene may account for a large fraction of the total gene expression, and in another cell type the same gene may be expressed at undetectable levels. When a gene can be expressed in a particular cell type, the associated chromatin is loosely arranged; when the same gene is never expressed in a particular cell type, the chromatin organization is more compact. Thus, the DNA within the chromatin of expressed genes is more susceptible to cleavage by deoxyribonuclease than is the DNA in tissues in which the genes are quiescent. [43] [44] [45] This looseness may facilitate access of RNA polymerase to the gene for purposes of transcription. In addition, inactive genes appear to have a higher content of methylated cytosine residues than the same genes in tissues in which they are expressed. [46] [47] Determinants for the tissue-specific transcriptional expression of genes exist in control sequences usually residing within 1000 base pairs of the 5'-flanking region of the transcriptional sequence. Enhancer sequences in animal cell genes were first described for immunoglobulin genes, a finding that extended the earlier observations of enhancer control elements in viral genomes. [48] However, the first clear demonstrations of these elements directing transcription to cells of distinct phenotypes came from studies of the comparative expression of two model genes, insulin and chymotrypsin, in the endocrine and exocrine pancreas, respectively. [49] The restricted expression of genes in a cell-specific manner is determined by the assembly of specific combinations of DNA-binding proteins on a predetermined array of control elements of the promoter regions of genes to create a transcriptionally active complex of proteins that includes the components of the general or basal transcriptional apparatus.



Transcription Factors in Developmental Organogenesis of Endocrine Systems

Certain families of transcription factors are critical for organogenesis and the development of the body plan. Among these factors are the homeodomain proteins [50] and the nuclear receptor proteins. [51] [52] [53] The family of homeotic selector, or homeodomain, proteins are highly conserved throughout the animal kingdom from flies to humans. The orchestrated spatial and temporal expression of these proteins and the target genes that they activate determine the orderly development of the body plan of specific tissues, limbs, and organs. Similarly, the actions of families of nuclear receptors (steroid and thyroid hormones, retinoic acid, and others) are critical for normal development to occur. Inactivating mutations in the genes encoding these essential transcription factors predictably result in loss or impairment of the development of the specific organ whose development they direct. Three examples are described of impaired organogenesis attributable to mutations in essential transcription factors: Partial anterior pituitary agenesis (Pit-1) Adrenal and gonadal agenesis (SF-1, DAX-1) Pancreatic agenesis (IDX-1) Partial Pituitary Agenesis

The transcription factor Pit-1 is a member of a family of pou-homeodomain proteins, which is a specialized subfamily of the larger family of homeodomain proteins. Pit-1 is a key transcriptional activator of the promoters of the growth hormone, prolactin, and thyroid-stimulating hormone genes, produced in the anterior pituitary somatotrophs, lactotrophs, and thyrotrophs, respectively. Pit-1 is also the major enhancer activating factor for the promoter of the growth hormone-releasing factor receptor gene. [55] Mutations in Pit-1 that impair its DNA-binding and transcriptional activation functions are responsible for the phenotype of the Jackson and Snell dwarf mice.[54]

[54]

Mutations in the gene encoding Pit-1 have been found in patients with combined pituitary hormone deficiency in which there is no production of growth hormone, prolactin, or thyroid-stimulating hormone, resulting in growth impairment and mental deficiency. [56] Notably, the production of the other two of the five hormones secreted by the anterior pituitary gland, adrenocorticotropin and the gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH), is unaffected. [56] In these human Pit-1 mutations, Pit-1 can bind to its cognate DNA control elements but is defective in trans-activating gene transcription. Furthermore, the mutated Pit-1 acts as a dominant negative inhibitor of Pit-1 actions on the unaffected allele. Pancreatic Agenesis

The homeodomain protein islet duodenum homeobox 1 or IDX-1 (somatostatin transcription factor 1 [STF-1], insulin promoter factor 1 [IPF-1]) appears to be responsible for the development and growth of the pancreas. Targeted disruption of the IDX-1 gene in mice resulted in a phenotype of pancreatic agenesis. [57] A child born without a pancreas was shown to be homozygous for inactivating mutations in the IDX-1 gene. [58] Notably, the parents and their ancestors who are heterozygous for the affected allele have a high incidence of maturity-onset (type 2) diabetes mellitus, suggesting that a decrease in gene dosage of IDX-1 may predispose to the development of diabetes. The possibility that a mutated IDX-1 allele may be one of several "diabetes genes" is supported by the observation that IDX-1 and the helix-loop-helix transcription factors E47 and beta-2 appear to be key up-regulators of the transcription of the insulin gene. [59] Agenesis of the Adrenal Gland and Gonads

Two nuclear receptor transcription factors have been identified as critical for the development of the adrenal gland, gonads, pituitary gonadotrophs, and the ventral medial hypothalamus. These nuclear receptors are SF-1 (steroidogenic factor 1) [60] and DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, X chromosome).[61] SF-1 binds to half-sites of estrogen response elements that bind estrogen receptors in the promoters of genes. DAX-1 binds to retinoic acid receptor (RAR) binding sites in promoters and inhibits RAR actions. Targeted disruption of SF-1 in mice results in a phenotype of adrenal and gonadal agenesis. In addition, pituitary gonadotrophs are absent and the ventral medial hypothalamus is severely underdeveloped. X-linked adrenal hypoplasia congenita is an X-linked, developmental disorder of the human adrenal gland that is lethal if untreated. The gene responsible for adrenal hypoplasia congenita has been identified by positional cloning and encodes DAX-1, a member of the nuclear receptor proteins related to RAR. [61] Several inactivating mutations identified in the DAX-1 gene result in the syndrome of adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Thus, genetically defined and transmitted defects in the genes encoding the transcription factors SF-1 and DAX-1 result in profound arrest in the development of the target organs regulated by the hypothalamic-pituitary-adrenal

28

Figure 3-11 Diagram showing three cell-surface receptor-coupled signal transduction pathways involved in the activation of a superfamily of nuclear transcription factors. Peptide hormone molecules (H1, H2, and H3) interact with sensor receptors (R1, R2, and R3) coupled to the diacylglycerol (DAG)-protein kinase C (PKC), the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA), and the calcium-calmodulin pathways in which small diffusible second messenger molecules are generated (DAG, cAMP, Ca 2+ ). The third messengers or effector protein kinases are generated and phosphorylate transcription factors such as members of the CREB/ATF and jun/AP-1 families of DNA-binding proteins to modulate DNA-binding affinities or transcriptional activation, or both. The various proteins bind as dimers determined by a poorly understood code that is not promiscuous in as much as only certain homodimer or heterodimer combinations are permissible. AP-1, activator protein 1; ATF, activating transcription factor; CaMK, calcium/calmodulin-dependent protein kinase; CREB, cAMP response element-binding protein.

axis involved in steroidogenesisthe adrenal gland (glucocorticoids, mineral corticoids) and the gonads (estrogens and androgens).



Coupling of Effector Action to Cellular Response

Another mode of gene control consists of the induction and suppression of genes that are normally expressed in a specific tissue. These processes are at work in the minute-to-minute and day-to-day regulation of rates of production of the specific proteins produced by the cells (e.g., production of polypeptide hormones in response to extracellular stimuli). At least two classes of signaling pathwaysprotein phosphorylation and activation of steroid hormone receptors by hormone bindingappear to be involved in the physiologic regulation of hormone gene expression. These two pathways mediate the actions of peptide and steroid hormones, respectively. Peptide ligands bind to receptor complexes on the plasma membrane, which results in enzyme activation, mobilization of calcium, formation of phosphorylated nucleotide intermediates, activation of protein kinases, and phosphorylation of specific regulatory proteins such as transcription factors (see Chapter 5) . [62] [63] Steroidal compounds, because of their hydrophobic composition, readily diffuse through the plasma membrane, bind to specific receptor proteins, and interact with other macromolecules in the nucleus, including specific domains on the chromatin located in and around the gene that is activated (see Chapter 4) .[51] [52] [53] Phosphorylated nucleotides such as cyclic adenosine monophosphate (cAMP), adenosine triphosphate, and guanosine triphosphate, as well as calcium, appear to have important functions in secretory processes. In particular, fluxes of calcium from the extracellular fluid into the cell and from intracellular organelles (e.g., endoplasmic reticulum) into the cytosol are closely coupled to secretion. [64] [65] The cellular signaling pathways that involve protein phosphorylations are multiple and complex. They typically consist of sequential phosphorylations and dephosphorylations of molecules referred to as protein kinase or phosphatase cascades.[66] These cascades are initiated by hormones, sensor molecules known as ligands, that bind to and activate receptors located on the surface of cells, resulting in the generation of small second messenger molecules such as cAMP, diacylglycerol, or calcium ions. These second messengers then activate protein kinases that phosphorylate and thereby activate key target proteins (Fig. 3-11) . The final step in the signaling pathways is the phosphorylation and activation of important transcription factors, resulting in gene expression (or repression). Insight has been gained into the identities of some of the phosphoproteins. As discussed earlier, a specific group of transcription factors, DNA-binding proteins, interacts with cAMP-responsive and phorbol ester-responsive DNA elements to stimulate gene transcription mediated by the cAMP-protein kinase A, diacylglycerol-protein kinase C, and calcium-calmodulin signal transduction pathways (see Fig. 3-11) . These proteins are encoded by a complex family of genes and bind to the DNA elements in the form of heterodimers or homodimers through a coiled coil helical structure known as a leucine zipper motif. [67] There is evidence that phosphorylation of these proteins modulates dimerization, DNA recognition and binding, and transcriptional trans-activation activities. Phosphorylation of the protein substrates might change their conformations and activate the proteins, which, in turn, interact with coactivator proteins such as the cAMP response element-binding protein (CREB) and the protein components of the basal transcriptional machinery, thereby allowing RNA polymerase to initiate gene transcription. [68] Generally, the second messengers activate serine/threonine kinases, which phosphorylate serine or threonine residues, or both, on proteins, whereas the receptor kinases are tyrosine-specific kinases that phosphorylate tyrosine residues. [66] [69] Examples of receptor tyrosine kinases are growth factor receptors such as those for insulin, insulin-like growth factor (IGF),

29

Figure 3-12 Schema indicating levels in expression of genetic information at which diversification of information encoded in a gene may take place. The three major levels of genetic diversification are (1) gene duplication, a process that occurs in terms of evolutionary time; (2) variation in the processing of ribonucleic acid (RNA) precursors, which results in formation of two or more messenger RNAs (mRNAs) by way of alternative pathways of splicing of transcript ( see Fig. 3-13 and Fig. 3-14 ); and (3) use of alternative patterns in processing of protein biosynthetic precursors (polyproteins, or prohormones). These three levels in gene expression provide a means for diversification of gene expression at levels of deoxyribonucleic acid (DNA), RNA, or protein. One or a combination of these processes leads to formation of the final biologically active peptide or hormone. In the diagram, loops depicted in transcripts denote introns; in diagrammatic structures of proteins, the stippled, shaded, and unshaded areas denote exons. See text for details.

epidermal growth factor, and platelet-derived growth factor. Receptors in the cytokine receptor family, which include leptin, growth hormone, and prolactin, activate associated tyrosine kinases in a variation on the theme. The different types of signal transduction pathways are described as more or less distinct pathways for semantic purposes. In reality, there is considerable cross-talk among the different pathways that occur developmentally and in cell typespecific settings. An active area of research in endocrine systems is attempting to understand these complex interactions among different signal transduction pathways. Although the growth factor and cytokine receptors are similar in some respects, they differ in other respects. For example, growth factor receptor tyrosine kinases activate transcription factors through cascades that involve both tyrosine phosphorylation and serine/threonine kinases such as mitogen-activated protein kinases, whereas the Janus kinases (JAKs) activated by cytokine receptors directly tyrosine phosphorylate the signal transducer and activator of transcription (STAT) factors. [69] [70]



GENERATION OF BIOLOGIC DIVERSIFICATION In addition to providing control points for the regulation of gene expression, the various steps involved in transfer of information encoded in the DNA of the gene to the final bioactive protein are a means for diversification of information stored in the gene (Fig. 3-12) . Five steps in gene expression can be arbitrarily described: (1) gene duplication and copy number, (2) transcription, (3) post-transcriptional RNA processing, (4) translation, and (5) post-translational processing. Gene Duplications

At the level of DNA, diversification of genetic information comes about by way of gene duplication and amplification. Many of the polypeptide hormones are derived from families of multiple, structurally related genes. Examples include the growth hormone family, consisting of growth hormone, prolactin, and placental lactogen; the glucagon family, consisting of glucagon, vasoactive intestinal peptide, secretin, gastric inhibitory peptide, and growth hormonereleasing hormone; and the glycoprotein hormone family, thyrotropin, luteinizing hormone, follicle-stimulating hormone, and chorionic gonadotropin. A remarkable example of diversification at the level of gene amplifications is the extraordinarily large number of genes encoding the pheromone and odorant receptors. [71] It is estimated that as many as 1000 such receptor genes may exist in mouse and rat genomes, each receptive to a particular odorant ligand. Over the course of evolution, an ancestral gene encoding a prototypic polypeptide representative of each of these families was duplicated one or more times and, through mutation and selection, the progeny proteins of the ancestral gene assumed different biologic functions. The exonic-intronic structural organization of the genomes of higher animals lends itself to gene recombination and RNA copying of genetic sequences with subsequent reintegration of DNA reverse-transcribed sequences

30

back into the genome, resulting in rearrangement of transcriptional units and regulatory sequences.


[ 72] [73]


Transcription

In addition to duplication of genes and their promoters, another way to create diversity in expression is at the level of gene transcription by providing genes with alternative promoters[74] and by utilizing a large array of cis-regulatory elements in the promoters regulated by complex combinations of transcription factors. Alternative Promoters

Many of the genes encoding hormones and their receptors utilize more than one promoter during development or when expressed in different tissue types. The employment of alternative promoters results in the formation of multiple transcripts that differ at their 5' ends (Fig. 3-13) . It is presumed that some genes have multiple promoters because they provide flexibility in the control of expression of the genes. For example, in some cases, expression of genes in more than one tissue or developmental stage may require distinct combinations of tissue-specific transcription factors. This flexibility enables genes in different cell types to respond to the same signal transduction pathways or genes in the same cell type to respond to different signal transduction pathways. A single promoter may not be adequate to respond to a complex array of transcription factors and a changing environment of cellular signals. The organization of alternative promoters in genes is manifested in several patterns within exons or introns in the 5' noncoding sequence or the coding sequence (see Fig. 3-13) . The most common occurrence of alternative promoters is within the 5' noncoding or leader exons. The utilization of different promoters in the 5' untranslated region of a gene, often accompanied by alternative exon splicing, results in the formation of mRNAs with different 5' sequences. The alternative usage of promoters in 5' leader exons can affect gene expression and generate diversity in several different ways. These include the developmental stagespecific and temporal expression of genes, the tissue-type specificity of expression, the levels of expression, the responsivity of gene expression to specific metabolic signals conveyed through signal transduction pathways, the stability of the mRNAs, the efficiencies of translation, and the structures of the amino termini of proteins encoded by the genes.[74] Examples of genes that use alternative 5' leader promoters during development are those encoding IGF-I, IGF-II, the retinoic acid receptors, and glucokinase, all of which are regulated by multiple promoters that are active in a variety of embryonic and adult tissues and are subject to developmental and tissue-specific regulation. [74] During fetal development, promoters P2, P3, and P4 of the IGF-II gene are active in the liver. These promoters are shut off after birth, at which time the P1 promoter is activated. The P1 and P2 promoters of the IGF-I gene are differentially responsive to growth hormone: P2 expressed in liver is responsive to growth hormone, whereas P1 expressed in muscle is not. The retinoic acid receptor exists in three isoforms (RAR, RAR, and RAR) encoded by separate genes that give rise to at least 17 different mRNAs generated by a combination of multiple promoters and alternative splicing. [75] The RAR isoforms appear to differ in their specificity for retinoic acidresponsive promoters, in their affinities for ligand isoforms, and in trans-activating capabilities. The different RAR isoforms are expressed at different times in different tissues during development. It has been proposed that the different RAR isoforms provide a means of achieving a diverse set of cellular responses to a single, simple ligand, retinoic acid. [75]

Figure 3-13 Utilization of alternative promoters in the expression of genes as a means to generate biologic diversification of gene expression. The use of alternative promoters allows a gene to be expressed in a variety of unique contexts that alter the properties of the messenger ribonucleic acid (mRNA) that is expressed. Such alternative promoter usage may render the mRNA more or less stable, affect translational efficiencies, or switch the translation of one protein isoform to another. The use of alternative promoters in genes characteristically occurs during development, or after development is completed, to designate tissue-specific patterns of expression of the gene. Exons are shown as boxes whose protein-coding regions are shaded. Introns are designated by horizontal lines. Dashed lines indicate introns that are spliced out. (Adapted from Ayoubi TAY, Van De Ven WJM. Regulation of gene expression by alternative promoters. FASEB J 1996; 10:453460.)

Glucokinase is an example of the alternative use of 5' leader promoters that have different metabolic responsiveness. [76] Expression of glucokinase in pancreatic beta cells and some other neuroendocrine cells utilizes an upstream promoter (1), whereas in liver a promoter (IL) 26 kb downstream of the 1 promoter is used exclusively. In beta cells, expression of the glucokinase gene is apparently not responsive to hormones. In contrast, in liver expression mediated by the IL promoter is intensely up-regulated by insulin and down-regulated by glucagon. The -amylase gene provides an example in which two alternative promoters in the 5' noncoding exons expressed in

31

two different tissues have dramatically different strengths of expression. [74] A strong upstream promoter directs expression within the parotid gland, contrasting with weak expression directed by an alternative downstream promoter in liver. Examples of the alternative usage of promoters in the coding regions of genes are the progesterone receptor (PR) and the transcription factor cAMP response element modulator (CREM). In both of these examples, different protein isoforms are produced that have markedly different functional activities. The genes encoding the chicken and human progesterone receptors express two isoforms of the receptor (isoforms A and B). [77] Isoform A initiates translation at a methionine residue located 164 amino acids downstream from the methionine that initiates the translation of the longer form B. Analyses of the mechanisms responsible for the synthesis of two different isoforms revealed that two promoters exist in the human PR gene: one upstream of the 5' leader exon and the other in the first protein coding exon. The two isoforms of the human PR differ markedly in their capabilities to trans-activate transcription from different progesterone responsive elements (PRE). Both human PR isoforms equivalently activate a canonical PRE. Isoform B is much more efficient than A at activating the PRE in the mouse mammary tumor virus promoter, whereas isoform A, but not B, activates transcription from the ovalbumin promoter. [77] The utilization of an alternative intronic promoter within the protein coding sequence of a gene is exemplified by the CREM gene. [78] The CREM gene employs a constitutively active, unregulated promoter (P1) that encodes predominantly activator forms of CREM and an internal promoter (P2) located in the fourth intron that is regulated by cAMP signaling and encodes a repressor isoform, ICER (inducible cAMP early response). The remarkable complexity of the alternative mechanisms of expression of the CREM and CREB genes is discussed subsequently. Diversity of Transcription Factors

Another mechanism to create diversity at the level of gene transcription is that of the interplay of multiple transcription factors on multiple cis-regulatory sequences. The promoters of typical genes may contain 20 or 30 or more cis-acting control elements, either enhancers or silencers. These control elements may respond to ubiquitous transcription factors found in all cell types and to cell typespecific factors. Unique patterns of control of gene expression can be affected by several different mechanisms acting in concert. The spacing, relative locations, and juxtapositioning of control elements with respect to each other and to the basal transcriptional machinery can influence levels of expression. Transcription factors often act in the form of dimers or higher oligomers among factors of the same or different classes. A given transcription factor may act as either an activator or a repressor as a consequence of the existing circumstances. The ambient concentrations of transcription factors within the nucleus in conjunction with their relative DNA-binding affinities and trans-activation potencies may determine the levels of expression of genes.



Post-transcriptional Processing (Alternative Exon Splicing)

Identification of the mosaic structure of transcriptional units encoding polypeptide hormones and other proteins that consist of exons and introns raised the possibility that the use of alternative pathways in RNA splicing could provide informationally distinct molecules. Different proteins could arise either by inclusion or exclusion of specific exonic segments or by

Figure 3-14 Alternative exon splicing provides a means to generate biologic diversification of gene expression. Mechanisms of exon skipping or switching and intron slippage are frequently utilized in the alternative processing of premessenger ribonucleic acids (mRNAs) to provide unique mRNAs and encoded proteins during development and in a tissue-specific pattern of expression in the fully differentiated tissues or organs. Exons are shown as boxes with protein-coding regions shaded to designate origin of protein isoforms. Introns are depicted as horizontal lines. Dashed lines denote spliced-out introns.

utilization of parts of introns in one mRNA as exons in another mRNA. In addition, differences in the splice sites would result in expression of new translational reading frames. Alternative splicing utilizes two distinct mechanisms (Fig. 3-14) . One is that of exon skipping or switching in or out of exons. The other mechanism, known as intron slippage, is to include part of an intron in an exon, to splice out part of an exon along with the intron, or to include a "coding" intron. There are many examples of both mechanisms used to generate diversity in endocrine systems. Included among the genes encoding prohormones in which the pre-mRNAs are alternatively spliced by exon skipping or switching are those for procalcitonin/calcitonin gene-related peptide, prosubstance P/K, and the prokininogens. Alternative processing of the RNA transcribed from the calcitonin gene results in production of an mRNA in neural tissues that is distinct from that formed in the C cells of the thyroid gland. [79] The thyroid mRNA encodes a precursor to calcitonin, whereas the mRNA in the neural tissues generates a neuropeptide known as calcitonin generelated peptide. Immunocytochemical analyses of the distribution of the peptide in brain and other tissues suggest functions for the peptide in perception of pain, ingestive behavior, and modulation of the autonomic and endocrine systems. The splicing of the RNA precursor that encodes substance P can take place in at least two ways. [80] One splicing pattern results in the mRNA that encodes substance P and another peptide, called substance K, in a common protein precursor. Other mRNAs are apparently spliced so as to exclude the coding sequence for substance K. An alternative RNA splicing pattern also occurs in the processing of transcripts arising from the gene encoding bradykinin. [81] The high-molecular-weight and low-molecular-weight kininogens are translated from mRNAs that differ by the alternative use of 3'-end exons encoding the COOH termini of the prohormones, a situation similar to that found in the transcription of the calcitonin gene.

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Other examples of genetic diversification arise from the programmed flexibility in the choice of splice acceptor sites within coding regions (intron slippage), which allows an array of coding sequences (exons) to be put together in a number of useful combinations. For example, the coding sequences of the growth hormone, lutropin-choriogonadotropin, [82] and leptin receptors [83] can be brought together in two different ways, one to include, the other to exclude, an exonic coding sequence specifying the transmembrane spanning domains of the polypeptide chains that anchor the receptors to the surface of cells. If mRNA splicing excludes the anchor's peptide sequence, a secreted rather than a surface protein is produced.



Translation

The process of translation provides a fourth level for the creation of diversity of gene expression. As discussed earlier in the section of the chapter on Regulation of Gene Expression, the rate of translational initiation can be regulated as typified by the proinsulin and prohormone convertase mRNAs, in which translation is augmented by glucose and cAMP. Molecular diversity of translation, however, is generated by the developmentally regulated utilization of alternative translation initiation (start) codons (methionine codons, AUGs). The mechanism of translation initiation involves the assembly of the 40S ribosome subunit on the 5' methyl guanosine cap of the mRNA. [84] The ribosome subunit then scans 5' to 3' along the mRNA until it encounters an AUG sequence in a context of surrounding nucleotides favorable for the initiation of protein synthesis. Upon encountering such a favorable AUG, the subunit pauses and recruits the 60S subunit plus a number of other essential translation initiation factors, allowing the polymerization of amino acids. The use of an alternative downstream start codon for translation can occur by mechanisms of loose scanning and reinitiation (Fig. 3-15) .[85] Loose scanning is believed to occur when the most 5' AUG codon is not in a strongly favorable context and allows the 40S ribosomal subunit to continue scanning until it encounters a more favorable AUG downstream. Thus, in the loose scanning mechanism, both translational start codons are used. In contrast, the mechanism of translational reinitiation involves the termination of translation followed by the reinitiation of translation at a downstream start codon. Thus, two proteins are encoded from the same mRNA by a start and stop mechanism. This process of translational reinitiation can occur either by continued scanning of the 40S ribosomal subunit after termination of translation followed by reinitiation, as in loose scanning, or by complete dissociation of the ribosomal subunits at

Figure 3-15 Alternative translational initiation sites are used to change the coding sequences of messenger ribonucleic acids to encode different protein isoforms. The two mechanisms illustrated involve loose scanning and reinitiation of translation. See text.

the time of termination followed by complete reassembly at a downstream start codon, referred to as an internal ribosomal entry site (IRES). Such utilization of alternative translation start codons occurs in mRNAs encoding certain classes of transcription factors illustrated by the basic leucine zipper (bZIP) proteins CREB, CREM, and certain of the CCAAT/enhancer binding proteins (C/EBPs), the C/EBP and C/EBP isoforms. In all four of these DNA-binding proteins, the alternative use of internal start codons results in a switch from activators to repressors. The CREB gene uses translational reinitiation by the somewhat novel mechanism of alternative exon switching that occurs during spermatogenesis. [86] At developmental stages IV and V of the seminiferous tubule of the rat, an exon (exon W) is spliced into the CREB mRNA. Exon W introduces an inframe stop codon, thereby terminating translation approximately 40 amino acids upstream of the DNA-binding domain. [87] [88] The termination of translation then permits reinitiation of translation at each of two downstream start codons, resulting in the synthesis of two repressor or inhibitor isoforms of CREB known as I-CREBs that are powerful dominant negative inhibitors of activator forms of CREB and CREM because they consist of the DNA-binding domain devoid of any trans-activation domains.[86] [87] [88] The function, if any, of the amino-terminal truncated protein consisting of the activation domains devoid of the DNA-binding domain is unknown. It has been postulated that the role of the alternative splicing of exon W in the CREB pre-mRNA is to interrupt a forward positive feedback loop during spermatogenesis. CREM, C/EBP, and C/EBP mRNAs utilize alternative downstream start codons to synthesize repressors during development. Like the I-CREBs, these repressors consist of the DNA-binding domains and lack trans-activation domains. The CREM repressor (S-CREM) is expressed during brain development. [78] The C/EBP-30 and C/EBP-20 isoforms are expressed during the differentiation of adipoblasts to adipocytes, and the C/EBP repressor liver inhibitory protein (LIP) is expressed during the development of the liver. [78]



Post-translational Processing

A fifth level of gene expression at which diversification of biologic information can take place is that of post-translational processing. Many precursors of polypeptide hormones, particularly those encoding small peptides, contain multiple peptides that are cleaved during post-translational processing of the prohormones. [89] Certain polyprotein precursors, however, contain several copies of the peptide. Examples of prohormones that contain multiple identical peptides are the precursors encoding TRH[90] and the mating factor of yeast,[91] each of which contains four copies of the respective peptide. Polyproteins that contain several distinct peptides include proenkephalins, [92] pro-opiomelanocortin, [93] and proglucagon. [94] In many instances, biologic diversification at the level of post-translational processing occurs in a tissue-specific manner. The processing of pro-opiomelanocortin differs markedly in the anterior compared with the intermediate lobe of the pituitary gland. In the anterior pituitary the primary peptide products are ACTH and -endorphin, whereas in the intermediate lobe of the pituitary one of the primary products is -melanocyte-stimulating hormone. The smaller peptides produced are extensively modified by acetylation and phosphorylation of amino acid residues. The processing of proglucagon in the pancreatic A cells and that in the intestinal L cells are also different (see Fig. 3-15) . [34] In the pancreatic A cells, the predominant bioactive product of the processing of proglucagon is glucagon itself; the two glucagon-like peptides are not processed efficiently from proglucagon in the A cells and are biologically inactive by virtue of

33

having NH2 -terminal and COOH-terminal extensions. On the other hand, in the intestinal L cell the glucagon immunoreactive product is a molecule, called glicentin, that consists of the NH 2 -terminal extension of the proglucagon plus glucagon and the small COOH-terminal peptide known as intervening peptide I. Glicentin has no glucagon-like biologic activity, and therefore the bioactive peptide (or peptides) in the intestinal L cells must be one or both of the glucagon-like peptides. In fact, glucagon-like peptide I in its shortened form of 31 amino acids, GLP-I (737), is a potent insulinotropic hormone in its actions of stimulating insulin release from pancreatic beta cells. [95] This peptide is released from the intestines into the blood stream in response to oral nutrients and appears to be a potent intestinal incretin factor implicated in the augmented release of insulin in response to oral compared with systemic (intravenous) nutrients. This potential for diversification of biologic information provided by the alternative pathways of gene expression is impressive when one considers that these pathways can occur in multiple combinations.



Unexpectedly Low Numbers of Expressed Genes in Genomes of Mammals (Humans and Mice)

A somewhat surprising initial conclusion, heralded in the lay press when the results of the sequencing of the human and mouse genomes were revealed, was that the number of genes in the human and mouse was approximately 30,000. This number was viewed as remarkably low because the number of genes in yeast (Saccharomyces cerevisiae), worm (Caenorhabditis elegans), and fly (Drosophila melanogaster) is about 20,000. However, it seems quite clear from the complexities of the mRNAs expressed in humans and mice, as exemplified by the growing database of expressed sequence tags, that tissue-specific alternative exon splicing and alternative promoter usage occur much more frequently in humans and mice than in yeast, worms, and flies. Considering the as yet incomplete database of expressed genes at the mRNA level, it seems reasonable to extrapolate that the human genome may actually express as many as 100,000 to 200,000 mRNAs that encode proteins with distinct, specific functions. This extrapolation is based on the observation that alternative exon splicing and promoter usage appear to be on the order of 5 to 10 times more frequent in higher vertebrate mammals than in yeasts and flies.



Acknowledgments

I am indebted to the members of the laboratory whose forbearance and helpful discussions of this chapter were invaluable. I thank Townley Budde for help in the preparation of the manuscript. J. F. H. is an Investigator with the Howard Hughes Medical Institute.



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35

Chapter 4 - Mechanism of Action of Hormones That Act on Nuclear Receptors Mitchell A. Lazar

Hormones can be divided into two groups on the basis of where they function in a target cell. The first group includes hormones that do not enter cells; instead, they signal via second messengers generated by interacting with receptors at the cell surface. All polypeptide hormones, as well as monoamines and prostaglandins, utilize cell surface receptors ( see Chapter 5 , "Mechanism of Action of Hormones That Act at the Cell Surface"). The second group, the focus of this chapter, includes hormones that can enter cells. These hormones bind to intracellular receptors that function in the nucleus of the target cell to regulate gene expression. Classical hormones that utilize intracellular receptors include thyroid and steroid hormones. Hormones serve as a major form of communication between different organs and tissues that allows specialized cells in complex organisms to respond in a coordinated manner to changes in the internal and external environments. Classical endocrine hormones, such as thyroid and steroid hormones, are secreted by ductless glands and are distributed throughout the body via the blood stream. These hormones were discovered by purifying the biologically active substances from clearly definable glands. It is now recognized that numerous other signaling molecules share with thyroid and steroid hormones the ability to function in the nucleus to convey intercellular and environmental signals. Not all of these molecules are produced in glandular tissues. Further, whereas some of these signaling molecules arrive at target tissues via the blood stream like classical endocrine hormones, others have paracrine functions (i.e., they act on adjacent cells) or autocrine functions (i.e., they act on the cell of origin). Lipophilic signaling molecules that utilize nuclear receptors include the following: Derivatives of vitamins A and D Endogenous metabolites such as oxysterols and bile acids Non-natural chemicals encountered in the environment ( xenobiotics) These molecules are referred to generically as ligands for nuclear receptors. The nuclear receptors for all of these signaling molecules are structurally related and collectively referred to as the nuclear receptor superfamily.



LIGANDS THAT ACT VIA NUCLEAR RECEPTORS General Features of Nuclear Receptor Ligands

Unlike polypeptide hormones that function via cell surface receptors, no ligands for nuclear receptors are directly encoded in the genome. To the contrary, all nuclear receptor ligands are small (molecular weight < 1000 daltons [d]) and lipophilic, enabling them to enter cells. Cellular uptake of nuclear receptor ligands may be a passive process, but in some cases a membrane transport protein is involved. For example, the oatp3 organic anion transporter mediates thyroid hormone entry into cells. [1] The lipophilicity of nuclear receptor ligands also allows them to be absorbed from the gastrointestinal tract, thus facilitating their use in replacement or pharmacologic therapies of disease states. Another common feature of nuclear receptor ligands is that all are derived from dietary, environmental, and metabolic precursors. In this sense, the function of these ligands and their receptors is to translate cues from the external and internal environments into changes in gene expression. Their critical role in maintaining homeostasis in multicellular organisms is highlighted by the fact that nuclear receptors are found in all vertebrates as well as insects but not in single-cell organisms such as yeast.[2]



Subclasses of Nuclear Receptor Ligands

One classification of nuclear receptor ligands is outlined in Table 4-1 and is described next. Classical Hormones

The classical hormones that utilize nuclear receptors for signaling are thyroid hormone and steroid hormones. Steroid hormones include receptors for cortisol, aldosterone, estrogen, progesterone, and testosterone. In some cases (e.g., thyroid hormone receptor [TR] and genes, estrogen receptor [ER] and ), there are multiple receptor genes, encoding multiple receptors. Multiple receptors for the same hormone can also derive from a single gene either by alternative promoter usage or alternative splicing (e.g., TR 1 and 2). Finally, some receptors can mediate the signal of multiple hormones. For example, the mineralocorticoid (aldosterone) receptor (MR) has equal affinity for cortisol and probably functions as a glucocorticoid receptor in some tissues, such as the brain. [3] The androgen receptor (AR) binds and responds to both testosterone and dihydrotestosterone (DHT). Vitamins

Vitamins were discovered as essential constituents of a healthful diet. Two fat-soluble vitamins, A and D, are precursors of important signaling molecules that function as ligands for nuclear receptors.

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TABLE 4-1 -- Nuclear Receptor Ligands and Their Receptors Classical Hormones Thyroid hormone: thyroid hormone receptor (TR), subtypes , Estrogen: estrogen receptor (ER), subtypes , Testosterone: androgen receptor (AR) Progesterone: progesterone receptor (PR) Aldosterone: mineralocorticoid receptor (MR) Cortisol: glucocorticoid receptor (GR) Vitamins 1,25-(OH)2 -vitamin D3 : vitamin D receptor (VDR) All-trans-retinoic acid: retinoic acid receptor, subtypes , , 9-cis-retinoic acid: retinoid X receptor (RXR), subtypes , , Metabolic Intermediates and Products Oxysterols: liver X receptor (LXR), subtypes , Bile acids: bile acid receptor (BAR) Fatty acids: peroxisome proliferatoractivated receptor (PPAR), subtypes , , Xenobiotics Pregnane X receptor (PXR), constitutive androstane receptor (CAR)

Precursors of vitamin D are synthesized and stored in skin and activated by ultraviolet light; vitamin D can also be derived from dietary sources. Vitamin D is then converted in the liver to 25(OH) vitamin D and in the kidney to 1,25-(OH) 2 -vitamin D3 , the most potent natural ligand of the vitamin D receptor (VDR). 1,25-(OH)2 -vitamin D3 acts as a circulating endocrine hormone. Vitamin A is stored in the liver and is activated by metabolism to all- trans-retinoic acid, which is a high affinity ligand for retinoic acid receptors (RARs). Retinoic acid is likely to function as a signaling molecule in paracrine as well as endocrine pathways. Retinoic acid is also converted to its 9- cis-isomer, which is a ligand for another nuclear receptor called the retinoid X receptor (RXR).[4] These retinoids and their receptors are essential for normal life and development of multiple organs and tissues.[5] They also have pharmaceutical utility for conditions ranging from skin diseases to leukemia. [6] Metabolic Intermediates and Products

Certain nuclear receptors have been discovered to respond to naturally occurring, endogenous metabolic products. One, called liver X receptor (LXR), is activated by oxysterol intermediates in cholesterol biosynthesis. Mice lacking LXR- have dramatically impaired ability to metabolize cholesterol. [7] Another "orphan receptor," bile acid receptor (BAR) also known as FXR, or "Farnesyl X receptor", is thus likely to play a role in regulation of bile synthesis and circulation in normal as well as disease states. [8] The peroxisome proliferator-activated receptors (PPARs) constitute another subfamily of nuclear receptors. [9] There are three subtypes, and all are activated by polyunsaturated fatty acids. No single fatty acid has particularly high affinity for any PPAR, and it is possible that these receptors may function as integrators of the concentration of a number of fatty acids. PPAR is expressed primarily in liver; to date, the natural ligand with highest affinity for PPAR is an eicosanoid, 8(S)-hydroxyeicosatetraenoic acid. The most potent PPAR ligands are the fibrate class of lipid-lowering pharmaceuticals. The name PPAR derives from the fact that compounds such as fibrates induce proliferation of hepatic peroxisomes, organelles involved in -oxidation of fatty acids. The other PPARs ( and ) are structurally related but are not activated by peroxisome proliferators. PPAR- is ubiquitous, and its ligandsother than fatty acidsare not well characterized. PPAR is expressed primarily in fat cells (adipocytes) and is necessary for differentiation along the adipocyte lineage. [10] [11] PPAR is also expressed in other cell types, including colonocytes, macrophages, and vascular endothelial cells, where it may play physiologic as well as pathologic roles. The natural ligand for PPAR is not known, although prostaglandin J derivatives have the highest affinity (in the micromolar range). It is exciting news that PPAR appears to be the target of thiazolidinedione antidiabetic drugs that improve insulin sensitivity. [12] [13] These pharmaceutical agents bind to PPAR with nanomolar affinities, and

non-thiazolidinedione PPAR ligands are also insulin sensitizers, further implicating PPAR in this physiologic role. Xenobiotics

Other nuclear receptors appear to function as integrators of exogenous environmental signals, including natural endobiotics (e.g., medicinals and toxins found in plants) and xenobiotics (compounds that are not naturally occurring). [14] In these cases, the role of the activated nuclear receptor is to induce cytochrome P450 enzymes that facilitate detoxification of potentially dangerous compounds in the liver. Receptors in this class include: SXR, or sterol and xenobiotic receptor [15] CAR, or constitutive androstane receptor [16] PPAR, which is also activated by certain environmental chemicals Unlike other nuclear receptors that have high affinity for very specific ligands, xenobiotic receptors have low affinity for a large number of ligands, reflecting their function in defense from a varied and challenging environment. Although these xenobiotic compounds are clearly not "hormones" in the classical sense, the function of these nuclear receptors is consistent with the general theme of helping the organism to cope with environmental challenges. Orphan Receptors

The nuclear receptor superfamily is one of the largest families of transcription factors. The hormones and vitamins just described account for the functions of only a fraction of the total number of nuclear receptors. The remainder have been designated as orphan receptors because their putative ligands are not known. [17] [18] From analyses of mice and humans with mutations in various orphan receptors, it is clear that many of these receptors are required for life or development of specific organs ranging from brain nuclei to endocrine glands. Some orphan receptors appear to be active in the absence of any ligand ("constitutively active") and may not respond to a natural ligand. Nevertheless, all of the receptors now known to respond to metabolites and environmental compounds were originally discovered as orphans. Thus, it is likely that future research will find that additional orphan receptors function as receptors for physiologic, pharmacologic, or environmental ligands. Variant Receptors

As to be discussed later, the carboxyl (C-) terminus of the nuclear receptors is responsible for hormone binding. In the case of a few nuclear receptors, including TR and the glucocorticoid receptor, alternative splicing leads to the production of variant receptors with unique C-termini that do not bind ligand. [19] [20] These variant receptors are normally expressed, but their biologic relevance is uncertain. It has been speculated that they modulate the action of the classical receptor to which they are related by inhibiting its function. Another type of normally occurring variant nuclear receptors

37

lacks a classical deoxyribonucleic acid (DNA) binding domain (see later). These include DAX-1, which is mutated in human disease, any, are not known, and it is likely that DAX-1 and SHP-1 bind to and repress the actions of other receptors.

[21]

and SHP-1.[22] Their ligands, if

Rare, naturally occurring mutations of hormone receptors can cause hormone resistance in affected patients, for instance: 1. Inheritance of the hormone resistance phenotype can be dominant if the mutant receptor inhibits the action of the normal receptor, as with generalized resistance to thyroid hormone. [23] 2. Inheritance is recessive if the mutation results in a complete loss of receptor function, as with the syndrome of hereditary 1,25-dihydroxyvitamin D-resistant rickets.[24] 3. Inheritance can be X-linked, as with the mutated androgen receptor in androgen insensitivity syndromes, including testicular feminization. [25]



Regulation of Ligand Levels

Ligand levels can be regulated in a number of ways (Table 4-2) . A dietary precursor may not be available in required amounts, as occurs in hypothyroidism due to iodine deficiency. Pituitary hormones (e.g., thyroid-stimulating hormone) regulate the synthesis and secretion of classical thyroid and steroid hormones. When the glands that synthesize these hormones fail, hormone deficiency can occur. Many of the nuclear receptor ligands are enzymatically converted from inactive prohormones to the biologically active hormone (e.g., 5' deiodination of thyroxine [T 4 ] to triiodothyronine [T 3 ]). In other cases, one hormone is precursor for another (e.g., aromatization of testosterone to estradiol). Biotransformation may occur in a specific tissue that is not the main target of the hormone (e.g., renal 1-hydroxylation of vitamin D) or may occur primarily in target tissues (e.g., 5-reduction of testosterone to DHT). Deficiency or pharmacologic inhibition of such an enzyme can also reduce hormone levels. Hormones can be inactivated by standard hepatic or renal clearance mechanisms or by more specific enzymatic processes. In the latter case, reduction in enzyme activity due to gene mutations or pharmacologic agents can result in hormone excess syndromes, for example, the renal deactivation of cortisol by 11--hydroxysteroid dehydrogenase (11-OHSD). Since, as noted earlier, cortisol can activate the mineralocorticoid receptor, insufficient 11-OHSD activity due to licorice ingestion, gene mutation, or extremely high cortisol levels causes syndromes of apparent mineralocorticoid excess. [26]



NUCLEAR RECEPTOR SIGNALING MECHANISMS Nuclear receptors are multifunctional proteins that transduce the signals of their cognate ligands. General features of nuclear receptor signaling are illustrated in Figure 4-1 . First and foremost, the ligand and the nuclear receptor must get to the nucleus. The nuclear receptor must also bind its ligand with high affinity. Because a major function of the receptor is to selectively regulate target gene transcription, it must recognize and bind to promoter elements in appropriate target genes. One discriminatory mechanism is dimerization of a receptor with a second copy of itself or with another nuclear receptor. The DNA-bound receptor must also work in the context of chromatin to signal the basal transcription machinery to increase or decrease transcription of the target gene.

TABLE 4-2 -- Regulation of Nuclear Receptor Ligand Levels Precursor availability Synthesis Secretion Activation (prohormone active hormone) Deactivation (active hormone inactive hormone) Elimination (hepatic, renal clearance)

Throughout the following discussion on the mechanisms and regulation of signaling by nuclear receptors, it should be kept in mind that some basic mechanisms are generally used by many or all members of the nuclear receptor superfamily, whereas other mechanisms impart the specificity that is crucial to the vastly different biologic effects of the many hormones and ligands that utilize these related receptors. Domain Structure of Nuclear Receptors

The nuclear receptors are proteins whose molecular weights are generally between 50,000 and 100,000 d. They all share a common series of domains, referred to as A to F (Fig. 4-2) . This linear depiction of the receptors is useful for describing and comparing the receptors, but it does not capture the role of protein folding and tertiary structure in mediating the various receptor functions. As of this writing, no full-length nuclear hormone receptor has been crystallized, but structures of individual domains have been extremely revealing, as will be clear from the discussions of specific receptor functions that follow.



Nuclear Localization

The nuclear receptors, like all cellular proteins, are synthesized on ribosomes that reside outside the nucleus. Import of

Figure 4-1 Signal transduction by hormones and other ligands that act via nuclear receptors. HRE, hormone response element; mRNA, messenger ribonucleic acid.

38

Figure 4-2 Domain structure of nuclear receptors.

the nuclear receptors into the nucleus requires the nuclear localization signal (NLS), located near the border of the C and D domains (see Fig. 4-2) . As a result of their nuclear localization signals, most of the nuclear receptors reside in the nucleus in the absence, as well as in presence, of ligand. A major exception is the glucocorticoid receptor (GR), which, in the absence of hormone, is tethered in the cytoplasm to a complex of chaperone molecules, including heat shock proteins (hsps). Hormone binding to GR induces a conformational change that results in dissociation of the chaperone complex, thereby allowing the hormone-activated GR to translocate to the nucleus via its nuclear localization signal.



Hormone Binding

High-affinity binding of a lipophilic ligand is a shared characteristic of many nuclear receptors. This defining function of the receptor is mediated by the C-terminal ligand-binding domain (LBD), domains D and E in Figure 4-2 . This region of the receptor also has many other functions, including dimerization and transcriptional regulation (see "Receptor Dimerization" and "Receptor Regulation of Gene Transcription" below). The structure of the LBD has been solved for a number of receptors. All share a similar overall structure consisting of 12 -helical segments in a highly folded tertiary structure (Fig. 4-3) . The ligand binds within a hydrophobic pocket composed of amino acids in helix 3 (H3), H4, and H5. The major structural change induced by ligand binding is an internal folding of the most C-terminal helix (H12), which forms a cap on the ligand-binding pocket. Although the overall mechanism of ligand binding is similar for all receptors, the details are crucial in determining ligand specificity. [27] [28] Although the molecular details of ligand binding are beyond the scope of this chapter, this is the most critical determinant of receptor specificity.



Target Gene Recognition by Receptors

Another crucial specificity factor for nuclear receptors is their ability to recognize and bind to the subset of genes that are to be regulated by their cognate ligand. Target genes contain specific DNA sequences that are called hormone response elements (HREs). Binding to the HRE is mediated by the central C domain of the nuclear receptors (see Fig. 4-2) . This region is typically composed of 66 to 68 amino acids, including two subdomains called zinc fingers because the structure of each subdomain is maintained by four cysteine residues that coordinate with a zinc atom. The first of these zinc-ordered modules contains basic amino acids that contact DNA; as with the LBD, the overall structure of the DNA-binding domain (DBD) is very similar for all members of the nuclear receptor superfamily. The specificity of DNA binding is determined by multiple factors (Table 4-3) . All steroid hormone receptors, except for the estrogen receptor (ER), bind to the double-stranded DNA sequence AG AACA (Fig. 4-4) . By convention, the double-stranded sequence is described by the sequence of one of the complementary strands, with the bases ordered from the 5' to the 3' end. Other nuclear receptors recognize the sequence AG GTCA. The primary determinant of this specificity is a group of amino acids residues in the so-called P-box of the DBD (see Fig. 4-4) . These hexamer DNA sequences are referred to as half-sites. The only two differences between these hexameric half-sites are the central two base pairs (underlined). For some nuclear receptors, the C-terminal extension of the DBD contributes specificity for extended half-sites containing additional, highly specific DNA sequences 5' to the hexamer (see Fig. 4-2) . [29] Another source of specificity for target genes is the spacing and orientation of these half-sites, which in most cases are bound by receptor dimers.



Receptor Dimerization

As noted earlier, the nuclear receptor DBD has affinity for the hexameric half-site, or extended half-sites; many HREs, however, are composed of repeats of the half-site sequence, and most nuclear receptors bind such HREs as dimers. Steroid receptors, including ER, function primarily as homodimers, which preferentially bind to two half-sites oriented toward each other (inverted repeats) with three base pairs in between (IR3) (Fig. 4-4A) . The major dimerization domain in steroid receptors is within the C-domain, although the LBD contributes. Ligand-binding facilitates dimerization and DNA binding of steroid hormone receptors. Most other receptors, including

39

Figure 4-3 Structural basis of nuclear receptor ligand binding and cofactor recruitment.

TR, RAR, VDR, PPAR, LXR, and VDR, bind to DNA as heterodimers with RXR (Fig. 4-4B) . Heterodimerization is mediated by two distinct interactions. The receptor LBD mediates the strongest interaction, which occurs even in the absence of DNA. These receptor heterodimers bind to two half-sites arranged as direct repeats (DRs) with variable numbers of base pairs in between. The spacing of the half-sites is a major determinant of target gene specificity. This is due to the second receptor-receptor interaction, which involves the DBDs and is highly sensitive to the spacing of the half-sites. For example, VDR/RXR heterodimers bind preferentially to direct repeats separated by three bases (DR3 sites), TR/RXR binds DR4, and RAR/RXR binds DR5 with highest affinity. [30] The structural basis of this restriction on DNA binding is related to the fact that the RXR binds to the upstream half-site (farthest from the start of transcription). As a result of the periodicity of the DNA helix, each base pair separating the half-sites leads to a rotation of about 36° of one half-site relative to the other. Subtle differences in the structure of the receptor LBDs make the DBD interactions more or less favorable at the different degrees of rotation. [31]



Receptor Regulation of Gene Transcription

Nuclear receptors mediate a variety of effects on gene transcription. The most common modes of regulation (Table 4-4) are: Ligand-dependent gene activation Ligand-independent repression of transcription Ligand-dependent negative regulation of transcription The remainder of this chapter describes these mechanisms. Ligand-Dependent Activation

Ligand-dependent activation is the most well-understood function of nuclear receptors and their ligands. In this case, the ligand-bound receptor increases transcription of a target gene to which it is bound. The DBD serves to bring the receptor domains that mediate transcriptional activation to a specific gene. Transcriptional activation itself is mediated primarily by the LBD, which can function in the same way even when it is transferred to a DNA-binding protein that is not

40

Specificity

TABLE 4-3 -- Determinants of Target Gene Specificity Of Nuclear Receptors Region of Receptor

1. Binding to DNA

1. DNA-binding domain (DBD, C domain)

2. Binding to specific hexamer (AGGTCA vs. AGAACA)

2. P-box in C-domain

3. Binding to sequences 5' to hexamer

3. C-terminal extension of DBD

4. Binding to hexamer repeats

4. Dimerization domain (C domain for steroid receptors, D-E-F for others)

5. Recognition of hexamer spacing

5. Heterodimerization with retinoid X receptor (RXR) (non-steroid receptors, C domain)

related to nuclear receptors. The activation function (AF) of the LBD is referred to as AF-2 (see Fig. 4-2) . Gene transcription is mediated by a large complex of factors that ultimately regulate the activity of ribonucleic acid (RNA) polymerase, the enzyme that uses the chromosomal DNA template to direct the synthesis of messenger RNA. Most mammalian genes are transcribed by RNA polymerase II, utilizing a large set of cofactor proteins, including basal transcription factors, and associated factors collectively referred to here as general

Figure 4-4 Structural basis of nuclear receptor (NR) DNA binding specificity. Ribbon diagrams of receptor DNA-binding domains (DBDs) are shown. A, Steroid hormone receptor binding as homodimer to inverted repeat ( arrows) of AGAACA half-site. B, RXR-NR heterodimer binding to direct repeat of AGGTCA. The position of the P-box, the region of the DBD that makes direct contact with DNA, is shown. N, number of base pairs between the two half-sites; RXR, retinoid X receptor.

TABLE 4-4 -- Regulation of Gene Transcription By Nuclear Receptors 1. Ligand-dependent gene activation: DNA binding and recruitment of coactivators 2. Ligand-independent gene repression: DNA binding and recruitment of corepressors 3. Ligand-dependent negative regulation of gene expression: DNA binding and recruitment of corepressors or recruitment of coactivators off DNA transcription factors (GTFs). Details about GTFs are of fundamental importance and are available elsewhere. [32] [33] The ligand-bound nuclear receptor communicates stimulatory signals to GTFs on the gene to which it is bound. This process involves recruiting positively acting cofactors, called coactivators.[34] These coactivators bind to the nuclear receptor on DNA only when hormone or ligand is bound. Thus, these coactivators specifically recognize the ligand-bound conformation of the LBD. The most important determinant of coactivator binding is the position of H12, which changes dramatically when ligands bind receptors (see Fig. 4-3) . Along with H3, H4, and H5, H12 forms a hydrophobic cleft that is bound by short polypeptide regions of the coactivator molecules. [35] [36] [37] These polypeptides, called NR boxes, have characteristic sequences of LxxLL, where L is leucine and xx can be any two amino acids. [38] A number of coactivator proteins containing LxxLL

41

TABLE 4-5 -- Nuclear Receptor Coactivators and Corepressors Coactivators 1. Chromatin remodeling Swi/Snf complex 2. Histone acetyl transferase p160 family (SRC-1, GRIP-1, pCIP) p300/CBP pCAF (p300/CBPassociated factor) 3. Activation TRAP/DRIP (thyroid receptorassociated proteins/D receptor interacting proteins)

Corepressors N-CoR (nuclear receptor corepressor) SMRT (silencing mediator for retinoid and thyroid hormone receptors) CBP, calcium-binding protein; SRC-1, steroid receptor coactivator 1; GRIP-1, glucocorticoid receptor interacting protein-1; pCIP, CBP interacting protein. motifs that bind to liganded nuclear receptors have been described (Table 4-5) . [34] [39] Coactivators increase the rate of gene transcription. This is accomplished by enzymatic functions, including DNA unwinding activity as well as histone acetyltransferase (HAT) activity. [40] HAT activity is critically important for activation because chromosomal DNA is tightly wrapped around nucleosomal units composed of core histone proteins. Acetylation of lysine tails on histones "opens up" this chromatin structure. [41] The best understood class of coactivator proteins is the socalled p160 family, whose name is based on their size (160 kd). [42] There are at least three such molecules, each with numerous names (see Table 4-5) .[34] [43] These factors possess HAT activity and also recruit other coactivators (CBP and p300), which are also HATs. [44] A third HAT, called p300/CBP-associated factor (pCAF), is also recruited by liganded receptors. [45] Together these HATs along with a complex of molecules called Swi/Snf, which directs adenosine triphosphate (ATP)-dependent DNA unwinding, create a chromatin structure that favors transcription (Fig. 4-5) . It is possible that the recruitment of multiple HATs reflects different specificities for core histones and potentially other, nonhistone proteins. Some HATs also interact directly with GTFs and further enhance their activities. An important complex that also links nuclear receptors to GTFs is the TRAP (TR-associated proteins) or DRIP (D receptor-associated proteins) complex. [46] [47]

Figure 4-5 Coactivators and corepressors in transcriptional regulation by nuclear receptors. CBP, calcium-binding protein; DRIP, D receptor-interacting protein; HRE, hormone response element; HAT, histone acetyltransferase; HDAC, histone deacylase; N-CoR, nuclear receptor corepressor; NR, nuclear receptor; PCAF, p300/CBP-associated factor; SMRT, silencing mediator of retinoid and thyroid receptors; TRAP, thyroid hormone receptor-associated protein. Repression of Gene Expression by Unliganded Receptor

Although DNA binding is ligand-dependent for steroid hormone receptors, other nuclear receptors are bound to DNA even in the absence of their cognate ligand. The unliganded DNA-bound receptor is not passively waiting for hormone; instead, it actively represses transcription of the target gene. This repression both "turns off" the target gene and amplifies the magnitude of the subsequent activation by hormone or ligand. For instance, if the level of gene transcription in the repressed state is 10% of the basal level in the absence of receptor, a hormone-activation to 10-fold above that basal level represents a 100-fold difference of transcription rate between hormone-deficient (repressed) genes and hormone-activated genes (Fig. 4-6) . [48] In many ways, the molecular mechanism of repression is the mirror image of ligand-dependent activation. The unliganded nuclear receptor recruits negatively acting factors (corepressors) to the target gene. The two major corepressors are large (270 kd) proteins [49] [50] : Nuclear receptor corepressor (N-CoR) Silencing mediator for retinoid and thyroid receptors (SMRT) N-CoR and SMRT specifically recognize the unliganded conformation of nuclear receptors and use an amphipathic helical sequence similar to the NR box of coactivators to bind to a hydrophobic pocket in the receptor. For corepressors, the peptide responsible for receptor binding is called the CoRNR box and contains the sequence (I or L) xx (I or V)I (where I is isoleucine, L is leucine, V is valine, and xx represents any two amino acids). [51] The receptor utilizes helices 3 to 5 to form the hydrophobic pocket, as in coactivator binding, but H12 does not promote and even hinders corepressor binding. This negative role of H12 highlights the role of the ligand-dependent change in the position of H12 as the switch that determines repression and activation by nuclear receptors (see Fig. 4-5) .[52] The transcriptional functions of N-CoR and SMRT are the opposite of those of the coactivators. The corepressors themselves do not possess enzyme activity but do recruit multiple histone deacetylases (HDAC) to the target gene, thereby reversing the effects of histone acetylation described earlier and leading to a compact, repressed state of chromatin. The corepressors also interact directly with GTFs to inhibit their transcriptional activities.

42

Figure 4-6 Repression and activation functions augmenting the dynamic range of transcriptional regulation by nuclear receptors. HRE, hormone response element. Ligand-Dependent Negative Regulation of Gene Expression (Transrepression)

The ligand-dependent switch between the repressed and activated receptor conformations explains how hormones activate gene expression. However, many of the most important gene targets of hormones are turned off in the presence of the ligand. This is referred to as ligand-dependent negative regulation of transcription, or transrepression, to distinguish it from the repression of basal transcription by unliganded receptors. The mechanism of negative regulation is less well understood than ligand-dependent activation, and, indeed, there may be more than one mechanism. One mechanism involves nuclear receptor binding to DNA binding sites that reverse the paradigm of ligand-dependent activation ( negative response elements). Ligand-bound receptors recruit corepressors and HDAC activity to such binding sites. [53] For example, when the unliganded TR binds to the negative response element of the gene for the subunit of thyroid-stimulating hormone (TSH), transcription is activated. Ligand binding recruits corepressors and HDAC to the TR and leads to suppression of transcription. [54] In other cases, it has been postulated that negative regulation may result from ligand binding to nuclear receptors that bind to other transcription factors without binding DNA. This TABLE 4-6 -- Factors Modulating Receptor Activity in Different Tissues Receptor concentration Ligand concentration Ligand function (agonist, partial agonist, antagonist) Concentrations and types of coactivators and corepressors Phosphorylation state of nuclear receptor interaction leads to removal of coactivators such as p300 and CBP from the other transcription factors that positively regulate the gene. the activity of the positively acting factors results in the observed negative regulation.

[55]

In this model, inhibition of

Role of Other Nuclear Receptor Domains

The N-terminal A/B domain of the nuclear receptors is the most variable region among all members of the superfamily in terms of length and amino acid sequence. Even subtypes of the same receptor often have completely different A/B domains. The function of this domain is least well defined. It is not required for unliganded repression or ligand-dependent activation. In many receptors, the A/B domain contains a positive transcriptional activity, often referred to as AF-1 (see Fig. 4-2) , that is ligand-independent but probably interacts with coactivators [56] and may influence the magnitude of activation by agonists or partial agonists (see later). This activation function is tissue-specific and tends to be more important for steroid hormone receptors, whose A/B domains are notably longer than those of other members of the superfamily. The F domain of the nuclear receptors is hypervariable in length and sequence, and its function is not known. Cross-talk with Other Signaling Pathways

Hormones and cytokines that signal via cell surface receptors also regulate gene transcription, often by activating protein kinases that phosphorylate transcription factors such as cAMP-response element-binding protein (CREB). Such signals can also lead to phosphorylation of nuclear receptors. Multiple signal-dependent kinases can phosphorylate nuclear receptors,

43

leading to conformational changes that regulate function. [57] Phosphorylation can lead to changes in DNA binding, ligand binding, or coactivator binding; these variable consequences depend on the specific kinase, receptor, and domain of the receptor that is phosphorylated. The properties of coactivators and corepressor molecules are also regulated by phosphorylation. Receptor Antagonists

Certain ligands function as receptor antagonists by competing with agonists for the ligand-binding site. In the case of steroid hormone receptors, the position of H12 in antagonist-bound conformation is not identical to that in the unliganded receptor or the agonist-bound receptor. H12, which itself has a sequence that resembles the NR box, binds to the coactivator-binding pocket and thereby prevents coactivator binding. [37] [58] This antagonist-bound conformation also favors corepressor binding to steroid hormone receptors. Tissue-Selective Ligands

Some ligands function as antagonists in some tissues but as full or partial agonists in others. These selective receptor modulators include compounds such as tamoxifen, a selective estrogen receptor modulator (SERM). SERMs are estrogen receptor antagonists with respect to the functions of AF-2, including coactivator binding, and require the AF-1 function for their agonist activity. [59] Such agonism, like AF-1 activity, tends to be tissue-specific and therefore has great therapeutic utility. In addition to drugs, certain endogenous ligands (e.g., testosterone, DHT) also mediate tissue-specific effects. The molecular basis of tissue-specific activity is not well understood but is probably due to the expression or activity of transcriptional cofactors that differentiate between receptors bound to different ligands. Table 4-6 summarizes factors contributing to tissue-specificity of receptor activity.



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Chapter 5 - Mechanism of Action of Hormones That Act at the Cell Surface Allen Spiegel Christin Carter-Su Simeon Taylor

Hormones are secreted into the blood and act upon target cells at a distance from the secretory gland. In order to respond to a hormone, a target cell must contain the essential components of a signaling pathway. First, there must be a receptor to bind the hormone. Second, there must be an effectorfor example, an enzymatic activitythat is regulated when the hormone binds to its receptor. Finally, there must be appropriate downstream signaling pathways to mediate the physiologic responses to the hormone. In fact, this type of mechanism involving receptors, effectors, and downstream signaling pathways is quite general and also functions in nonendocrine systems such as neurotransmitters, cytokines, and paracrine and autocrine factors. This chapter reviews several examples of endocrine signaling pathways, with particular attention to the molecular mechanisms that function in normal physiology and to the molecular pathology causing disease.



RECEPTORS Definition and Classification

There are two essential functions that define hormone receptors: (1) the ability to bind the hormone and (2) the ability to couple hormone binding to hormone action. Both components of the definition are essential; for example, many hormones bind to binding proteins, which are distinct from receptors because the binding proteins do not trigger the signaling pathways that mediate hormone action. Many classes of receptors are of interest in endocrinology. Some receptors are located within the cell and function as transcription factors (e.g., receptors for steroid and thyroid hormones). Other receptors are located on the cell surface and function primarily to transport their ligands into the cell by a process referred to as receptor-mediated endocytosis (e.g., low-density lipoprotein receptors). In this chapter, we focus upon cell-surface receptors that trigger intracellular signaling pathways. These cell-surface receptors can be classified according to the molecular mechanisms by which they accomplish their signaling function: 1. 2. 3. 4. 5. 6.

Ligand-gated ion channels (e.g., nicotinic acetylcholine receptor). Receptor tyrosine kinases (e.g., receptors for insulin and insulin-like growth factor I). Receptor serine/threonine kinases (e.g., receptors for activins and inhibins). Receptor guanylate cyclase (e.g., atrial natriuretic factor receptor). G protein-coupled receptors (e.g., receptors for adrenergic agents, muscarinic cholinergic agents, glycoprotein hormones, glucagon, and parathyroid hormone). Cytokine receptors (e.g., receptors for growth hormone, prolactin, and leptin).

The receptors belonging to classes 1 to 4 are bifunctional molecules that can bind hormone and also serve as effectors by functioning either as ion channels or as enzymes. In contrast, the receptors belonging to classes 5 and 6 have the ability to bind the hormone but must recruit a separate molecule to catalyze the effector function. For example, as the name implies, G protein-coupled receptors utilize G proteins to regulate downstream effector molecules. Similarly, cytokine receptors recruit cytosolic tyrosine kinases (e.g., Janus family

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tyrosine kinases, JAKs) as effectors to trigger downstream signaling pathways.



Hormone Binding

As predicted by the fact that hormones circulate in relatively low concentrations in the plasma, the binding interaction between a hormone and its receptor is characterized by high binding affinity. Furthermore, hormone binding has a high degree of specificity. Generally, the receptor binds its cognate hormone more tightly than it binds other hormones. However, some receptors may bind structurally related hormones with lower affinity. For example, the insulin receptor binds insulin-like growth factors (IGFs) with approximately 100-fold lower affinity than it binds insulin. Similarly, the thyrotropin receptor binds human chorionic gonadotropin with lower affinity than it binds thyrotropin. This phenomenon has been referred to as specificity spillover and provides an explanation of several pathologic conditions, such as hypoglycemia caused by tumors secreting IGF-II and hyperthyroidism associated with choriocarcinoma. [1] Binding of a hormone (H) to its receptor (R) can be described mathematically as an equilibrium reaction: H+R HR

At equilibrium, K a = (RH)/(H)(R), where Ka is the association constant for the formation of the hormone receptor complex (HR). As originally shown by Scatchard, it is possible to rearrange this equation in terms of the total concentration of receptor binding sites, R 0 = (R) + (RH), as follows: Ka = (RH)/{[Ro (RH)](H)} (RH) = Ka [Ro (RH)](H) (RH)/(H) = Ka Ro Ka (RH)

A straight line is obtained when (RH)/(H) (i.e., the ratio of bound to free hormone) is plotted as a function of (RH) (the concentration of bound hormone). The slope of the line is -K a , and the line intercepts the horizontal axis at the point where (HR) = R 0 = the total number of binding sites. This type of plot is referred to as a Scatchard plot and has been used as a graphic method to estimate the affinity with which a receptor binds its hormone. Although the binding properties of some receptors are described more or less accurately by these simple equations, other receptors exhibit more complex properties. This simple algebraic derivation of the Scatchard equation implicitly assumes that there is only one class of receptors and that the binding sites on the receptors do not interact with one another. If these assumptions do not apply to the interaction of a particular hormone with its receptor, the Scatchard plot may not be linear. Several molecular mechanisms may contribute to nonlinearity of the Scatchard plot. For example, there may be more than one type of receptor that binds the hormone (e.g., a high-affinity, low-capacity site and a low-affinity, high-capacity site). Alternatively, some receptors have more than one binding site, and there may be cooperative interactions among the binding sites (e.g., the insulin receptor). In addition, the interaction between a G protein and a G protein-coupled receptor may affect the affinity with which the receptor binds its ligand; moreover, the effect on binding affinity depends on whether guanosine diphosphate (GDP) or guanosine triphosphate (GTP) is bound to the G protein. However, a detailed discussion of these complexities is beyond the scope of this chapter.



REGULATION OF HORMONE SENSITIVITY Early in the history of endocrinology, attention was focused on the regulation of hormone secretion as the most important mechanism for regulating physiology. However, it has become apparent that the target cell is not passive. Rather, there are many influences that can alter the sensitivity of the target cell's response to a given concentration of hormone. For example, the number of receptors can be regulated. All things being equal, hormone sensitivity is directly related to the number of hormone receptors expressed on the cell surface. In addition, post-translational modifications of the receptor can modify either the affinity of hormone binding or the efficiency of coupling to downstream signaling pathways. Moreover, all of the downstream components in the hormone action pathway are subject to similar types of regulatory influences, which can have a significant impact on the ability of the target cell to respond to hormone. Just as hormone sensitivity is subject to normal physiologic regulation, pathologic influences can cause disease by targeting components of the hormone action pathway. Multiple etiologic factors can impair the hormone action pathway, such as genetic, autoimmune, and exogenous toxins. For example, disease mechanisms can alter the functions of cell-surface receptors, effectors such as G proteins, and other components of the downstream signaling pathways. This chapter describes several examples illustrating these principles.



RECEPTOR TYROSINE KINASES Receptor tyrosine kinases have several structural features in common: an extracellular domain containing the ligand-binding site, a single transmembrane domain, and an intracellular portion that includes the tyrosine kinase catalytic domain (Fig. 5-1) . Analysis of the sequence of the human genome suggests that there are approximately 100 receptor tyrosine kinases. The tyrosine kinase domain is the most highly conserved sequence among all the receptors in this family. In contrast, there is considerable variation among the sequences of the extracellular domains. Indeed, the family of receptor tyrosine kinases can be classified into 16 subfamilies, primarily on the basis of the differences in the structure of the extracellular domain. [2] Furthermore, receptor tyrosine kinases mediate the biologic actions of a wide variety of ligands, including insulin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and vascular endothelial cell-derived growth factor. The variation in the sequences of the extracellular domains enables the receptors to bind this structurally diverse collection of ligands. The EGF receptor was the first cell-surface receptor demonstrated to possess tyrosine kinase activity [3] and also the first receptor tyrosine kinase to be cloned. [4] Like most receptor tyrosine kinases, the EGF receptor exists primarily as a monomer in the absence of ligand. However, binding of ligand induces receptor dimerization. As discussed later in this chapter, ligand-induced dimerization is central to the mechanism whereby the receptor mediates the biologic activity of EGF. In addition to the ability to form homodimers, the EGF receptor can form heterodimers with other members of the same subfamily of receptor tyrosine kinases. Because a small number of receptors can combine in a large number of pairings, heterodimer

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Figure 5-1 Receptor tyrosine kinases. This diagram illustrates 3 of the 16 families of receptor tyrosine kinases. [2] [ 99] All receptor tyrosine kinases possess an extracellular domain containing the ligand-binding site, a single transmembrane domain, and an intracellular domain containing the tyrosine kinase domain. Several structural motifs (i.e., cysteine-rich domain, immunoglobulin-like domain, tyrosine kinase domain) in these receptor tyrosine kinases are indicated on the right side of the figure. Cys, cysteine; EGF, epidermal growth factor; Ig, immunoglobulin; PDGF, platelet-derived growth factor.

formation has the potential to fine-tune the specificity of receptors with respect to both ligand binding and downstream signaling. The insulin receptor is of special interest to endocrinologists because diabetes is among the most common diseases of the endocrine system. Furthermore, the insulin receptor closely resembles the type 1 receptor for IGFs. [5] This is the receptor that mediates the biologic actions of IGF-I and therefore also plays an important role in the physiology of growth hormone (GH) in vivo. Although the kinase domains of receptors for insulin and IGF-I closely resemble other receptor tyrosine kinases, at least two distinctive features set them apart. First, the receptors are synthesized as proreceptors that undergo proteolytic cleavage into two subunits ( and ). The subunit contains the ligand-binding site; the subunit includes the transmembrane and tyrosine kinase domains. Second, both receptors exist as 2 2 heterotetramers that are stabilized by intersubunit disulfide bonds. In contrast to other receptor tyrosine kinases, which are thought to dimerize in response to ligand binding, the insulin receptor exists as a dimer of

Figure 5-2 Ligand-induced dimerization of receptors. Two molecular mechanisms of ligand-induced receptor dimerization are illustrated. In the case of the platelet-derived growth factor, the ligand is dimeric and therefore contains two receptor binding sites. [7] [ 8] In the case of growth hormone, a single ligand molecule contains two binding sites so that it can bind simultaneously to two receptor molecules. [10] [11] [ 12]

monomers even in the absence of ligand. The remainder of this section reviews the molecular mechanisms whereby receptor tyrosine kinases mediate biologic action, with special emphasis on the insulin receptor as an illustrative example. Receptor Activation: Role of Receptor Dimerization

Dimerization plays a central role in the mechanism whereby most receptor tyrosine kinases are activated by their cognate ligands. [2] [6] Although receptor dimerization is a common theme, the detailed molecular mechanisms differ from receptor to receptor. The following are three examples of the mechanisms of receptor dimerization (Fig. 5-2) : Dimeric Ligand

PDGF and vascular endothelial cell-derived growth factor are examples of dimeric ligands (see Fig. 5-2) . [7] [8] [9] Because each subunit of ligand can bind one receptor molecule, simultaneous

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Figure 5-3 Phosphorylation of tyrosine residues in the activation loop leads to activation of the insulin receptor tyrosine kinase. A hypothetical mechanism for ligand-stimulated activation of the insulin receptor tyrosine kinase is illustrated. The model is based on the three-dimensional structure of the isolated insulin receptor tyrosine kinase as determined by x-ray crystallography.[18] [ 19] [ 22] In the inactive insulin receptor kinase (left), Tyr1162 blocks the active site so that substrates cannot bind. In contrast, when the tyrosine residues in the activation loop (including Tyr1162) become phosphorylated (right), Tyr1162 moves out of the way, and there is a conformational change that allows binding of adenosine triphosphate (ATP) and protein substrate so that the kinase reaction can proceed.

binding of two receptor molecules drives receptor dimerization. Direct support for this type of mechanism is provided by the crystal structure of vascular endothelial cell-derived growth factor bound to its receptor (Flt-1). [9] Two Receptor Binding Sites on a Monomeric Ligand

Although this mechanism is important for many receptor tyrosine kinases, it was first shown rigorously for the GH receptor, which is not a member of the receptor tyrosine kinase family (see Fig. 5-2) .[10] [11] [12] As illustrated by the crystal structure of GH bound to its receptor, one molecule of ligand can bind two molecules of receptor. In fact, there are two distinct receptor-binding sites on each GH molecule, and this enables the ligand to promote receptor dimerization. This observation has an important implication for pharmacology. By abolishing one of the two receptor-binding sites, it is possible to design mutant ligands that lack the ability to promote receptor dimerization and therefore lack the ability to trigger hormone action. Nevertheless, by binding to receptors, the mutant ligand acquires the ability to inhibit the

action of the endogenous hormone. Such mutant GH molecules are being evaluated as potential therapeutic agents, for example, in conditions such as acromegaly. Preexisting Receptor Dimers

The insulin receptor represents a paradox. The insulin receptor exists as a dimer even in the absence of ligand. (Actually, it is an 2 2 heterotetramer, which is a dimer of monomers.) If the receptor is already dimerized, why is it not active? Although the molecular details remain to be elucidated, it seems likely that the two halves of the insulin receptor are not oriented in an optimal way to permit receptor activation in the absence of ligand. Perhaps, insulin binding triggers a conformational change that somehow mimics the effects of dimerization in other receptor tyrosine kinases. In any case, several studies have demonstrated that receptor dimerization is necessary for the ability of insulin to activate its receptor. For example, monomers retain the ability to bind insulin but are not activated in response to insulin binding. [13] [14] Furthermore, indirect evidence suggests that a single insulin molecule binds simultaneously to both subunits of the insulin receptor [15] [16] ; the ability to bind simultaneously to both halves of the dimeric receptor appears to be essential to the ability of insulin to activate its receptor.



Receptor Activation: Conformational Changes in the Kinase Domain

When ligand binds to the extracellular domain, it stimulates the tyrosine kinase activity of the intracellular domain. Although the detailed mechanisms of transmembrane signaling are not completely understood, considerable progress has been made in elucidating the molecular mechanisms of receptor activation. Investigations of the three-dimensional structure of the insulin receptor tyrosine kinase domain help to explain why the receptor is maintained in a low-activity state in the absence of insulin (Fig. 5-3) .[17] [18] [19] In the inactive form of the insulin receptor kinase, Tyr1162 is located in a position so that it blocks protein substrates from binding to the active site. Furthermore, in the inactive state of the tyrosine kinase domain, the active site assumes a conformation that does not accommodate magnesium adenosine triphosphate (ATP). Thus, the tyrosine kinase is inactive because the active site cannot bind either of its substrates. How does insulin activate the receptor? Insulin binding triggers autophosphorylation of three tyrosine residues (Tyr1158, Tyr1162, and Tyr1163) in the "activation loop." When the three tyrosine residues in the activation loop become phosphorylated, an important conformational change occurs. As a result of the movement of the

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activation loop, the active site acquires the ability to bind both ATP and protein substrates. Thus, the conformational change induced by autophosphorylation activates the receptor to phosphorylate other substrates. [20] [21] It remains unclear how this process is initiated. Because the inactive state of the tyrosine kinase cannot bind ATP, it seems unlikely that phosphorylation of Tyr1162 proceeds by a true autophosphorylation mechanism. Rather, it is likely that Tyr1162 in one subunit is transphosphorylated by the second subunit in the 2 2 heterotetramer molecule. [2] [22] However, this proposed mechanism poses a "chicken and egg" problem. It requires that at least one of the subunits is active before the Tyr residues in the activation loop become phosphorylated. Perhaps the activation loop is somewhat mobile so that some molecules of unphosphorylated tyrosine kinase can assume an active conformation and initiate a chain reaction of transphosphorylation and receptor activation.



Receptor Tyrosine Kinases Phosphorylate Other Intracellular Proteins

Once activated, tyrosine kinases are capable of phosphorylating other protein substrates. Several factors determine which proteins are phosphorylated under physiologic conditions within the cell. Amino Acid Sequence Context of Tyr Residue

Tyrosine kinases do not exhibit strict specificity with respect to the amino acid sequence of the phosphorylation site. Nevertheless, most tyrosine phosphorylation sites are located in the vicinity of acidic amino acid residues (i.e., Glu or Asp). [2] Binding to the Tyrosine Kinase

Some protein substrates bind directly to the intracellular domain of the receptor. The binding interaction brings the substrate into close proximity to the kinase, thereby promoting phosphorylation of the substrate. For example, the insulin receptor substrate (IRS) proteins are characterized by a highly conserved phosphotyrosine-binding (PTB) domain that binds to a conserved motif (Asn-Pro-Xaa-pTyr) in the juxtamembrane domain of the insulin receptor. [23] [24] [25] Binding of the PTB domain to the insulin receptor requires phosphorylation of the Tyr residue in the Asn-Pro-Xaa-pTyr motif. This provides another mechanism (in addition to activation of the intrinsic receptor tyrosine kinase) whereby autophosphorylation of the receptor enhances phosphorylation of IRS proteins. Similarly, substrates for some tyrosine kinases contain Src homology 2 (SH2) domains, highly conserved domains that bind phosphotyrosine residues (see later). For example, the activated PDGF receptor contains a phosphotyrosine residue near its C-terminus that binds the SH2 domain of phospholipase C. This enables the PDGF receptor to phosphorylate and activate phospholipase C. [2] [26] Subcellular Localization

Because receptor tyrosine kinases are located in the plasma membrane, they are in close proximity to other plasma membrane proteins. This colocalization has the potential to promote phosphorylation. For example, the insulin receptor has been reported to phosphorylate pp120/hepatocyte antigen-4 (HA4). [27] [28] Like the insulin receptor, pp120/HA4 is an integral membrane glycoprotein associated with the plasma membrane of hepatocytes. Similarly, FGF receptor substrate-2 (FRS2), a substrate of the fibroblast-derived growth factor receptor, is targeted to the plasma membrane by an N-terminal myristoylation site. [29]



Functional Significance of Tyrosine Phosphorylation

There are at least two distinct mechanisms whereby tyrosine phosphorylation regulates protein function. First, tyrosine phosphorylation can induce a conformational change in a protein, thereby altering its function. For example, as discussed earlier, phosphorylation of the three Tyr residues in the activation loop of the insulin receptor changes the conformation of the active site, thereby facilitating binding of substrates and activating the receptor tyrosine kinase. [17] [18] [19] However, most of the effects of tyrosine phosphorylation on protein function are mediated indirectly by regulating protein-protein interactions. In order to understand how tyrosine phosphorylation regulates protein-protein interactions, it is useful to review the biochemistry of c-src, the prototype of a nonreceptor tyrosine kinase. When the amino acid sequence of c-src is analyzed, it is apparent that there are three highly conserved domains in the molecule: the kinase catalytic domain and two noncatalytic domains that are referred to as src homology domains 2 and 3 (SH2 and SH3, respectively). SH2 Domains

SH2 domains consist of conserved sequences (approximately 100 amino acid residues) that are present in many proteins that function in signaling pathways. From a functional point of view, SH2 domains share the ability to bind pTyr residues. However, individual SH2 domains vary with respect to their binding specificity. The binding affinity of an SH2 is determined by the three amino acid residues downstream from the pTyr residue. For example, the SH2 domains of phosphatidyl-inositol (PI) 3-kinase exhibit a preference for pTyr-(Met/Xaa)-Xaa-Met, whereas the SH2 domain of growth factor receptor binding protein 2 (Grb-2) prefers to bind pTyr-Xaa-Asn-Xaa. Thus, a given SH2 domain binds to a tyrosine-phosphorylated protein if and only if the pTyr residue is located in a context that corresponds to the binding specificity of the SH2 domain. SH3 Domains

SH3 domains consist of conserved sequences (approximately 50 amino acid residues) that bind to proline-rich sequences. Like SH2 domains, SH3 domains are found in many proteins that function in signaling pathways.



Downstream Signaling Pathways

Receptor tyrosine kinases mediate the action of a wide variety of ligands in a wide variety of cell types. The bewildering complexity of the downstream signaling pathways corresponds to the huge number of physiologic processes that are regulated by receptor tyrosine kinases. Although it is beyond the scope of this chapter to attempt an encyclopedic review of all the downstream signaling pathways, we have selected examples to illustrate general principles. As discussed earlier, the activated insulin receptor phosphorylates multiple substrates including IRS-1, IRS-2, IRS-3, and IRS-4. [30] Each of these substrates contains multiple tyrosine phosphorylation sites, many of which correspond to consensus sequences for SH2 domains in important signaling molecules. Thus, IRS proteins serve as docking proteins that bind SH2 domain-containing proteins. Among these, two of the most important are PI 3-kinase and Grb-2. As discussed subsequently, binding of SH2 domains triggers multiple downstream signaling pathways.

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Phosphatidylinositol 3-Kinase

The catalytic subunit of PI 3-kinase (p110; molecular mass approximately 110,000) is bound to a regulatory subunit. The classical isoforms of the regulatory subunit (p85; molecular mass approximately 85,000) contain two SH2 domains, both of which bind to pTyr in the context of pTyr-(Met/Xaa)-Xaa-Met motifs. Binding of pTyr residues to both SH2 domains of p85 leads to maximal activation of PI 3-kinase catalytic activity. (Submaximal activation can be achieved with occupancy of a single SH2 domain in p85.) Because all four IRS molecules (IRS-1, IRS-2, IRS-3, and IRS-4) contain multiple tyrosine phosphorylation sites that conform to the Tyr-(Met/Xaa)-Xaa-Met consensus sequence, insulin-stimulated phosphorylation promotes binding of IRS proteins to the SH2 domains in the regulatory subunit PI 3-kinase, thereby increasing the enzymatic activity of the catalytic subunit. [31] [32] [33] [34] Activation of PI 3-kinase triggers activation of a cascade of downstream kinases, beginning with phosphoinositide-dependent kinases 1 and 2. These phosphoinositide-dependent kinases phosphorylate and activate multiple downstream protein kinases including protein kinase B and atypical isoforms of protein kinase C. [35] [36] [37] [38] [39] [40] [41] A large body of evidence demonstrates that the pathways downstream from PI 3-kinase mediate the metabolic activities of insulin (e.g., activation of glucose transport into skeletal muscle, activation of glycogen synthesis, and inhibition of transcription of the phosphoenolpyruvate carboxykinase gene). Among other lines of evidence, PI 3-kinase inhibitors (e.g., LY294002 and wortmannin) block the metabolic actions of insulin. [42] Similarly, overexpression of dominant negative mutants of the p85 regulatory subunit of PI 3-kinase also inhibits the metabolic actions of insulin. [35] Although it is generally agreed that activation of PI 3-kinase is necessary, it is controversial whether it is sufficient to trigger the metabolic actions of insulin. For example, a second parallel pathway may also be required. The latter pathway involves tyrosine phosphorylation of Cbl, another protein that can be phosphorylated by the insulin receptor in some cell types. [43] [44] [45] Grb-2 and the Activation of Ras

Grb-2 is a short adaptor molecule that contains an SH2 domain [46] capable of binding to pTyr residues in several signaling molecules, for example, IRS-1 and Shc, another PTB domain-containing protein that is phosphorylated by several receptor tyrosine kinases including the insulin receptor. [47] [48] The SH2 domain of Grb-2 is flanked by two SH3 domains[46] which bind to proline-containing sequences in mSos (the mammalian homologue of Drosophila son-of-sevenless). [49] mSos is capable of activating Ras, a small G protein that plays an important role in intracellular signaling pathways. mSos activates Ras by catalyzing the exchange of GTP for GDP in the guanine nucleotide-binding site of Ras. This, in turn, triggers the activation of a cascade of serine/threonine-specific protein kinases including Raf, mitogen-activated protein/extracellular signal-regulated kinase (MEK), and mitogen-activated protein (MAP) kinase. These pathways downstream from Ras contribute to the ability of tyrosine kinases to promote cell growth and regulate the expression of various genes. We have focused on the signaling pathways downstream from the insulin receptor because of the importance of insulin and IGF-I in endocrinology (Fig. 5-4) . In many ways, the molecular mechanisms closely resemble those downstream from other receptor tyrosine kinases. However, the insulin signaling pathway is atypical in at least one respect. The insulin receptor phosphorylates docking proteins (e.g., IRS-1), which bind SH2 domain-containing proteins (e.g., PI 3-kinase and Grb-2). In contrast, the intracellular domains of most receptor tyrosine kinases contain binding sites for SH2 domains. For example, the SH2 domain of Grb-2 binds to pTyr716 in the activated PDGF receptor. [2] Similarly, the PDGF receptor contains two Tyr-(Met/Xaa)-Xaa-Met motifs in the kinase insert domain that bind to the two SH2 domains in the p85 subunit of PI 3-kinase. [2] [50] It is not clear why some tyrosine kinases (e.g., the PDGF receptor) activate PI 3-kinase through a direct binding interaction, whereas others (e.g., the insulin receptor) utilize an indirect mechanism involving docking proteins. However, in contrast to PDGF receptors, which are associated with the plasma membrane, IRS proteins appear to be associated with the cytoskeleton. [51] Perhaps this differential subcellular localization contributes to signaling specificity. In other words, if insulin and PDGF receptors trigger translocation of PI 3-kinase to different locations within the cell, this compartmentation may permit two different receptors to elicit different biologic responses even though both responses are mediated by the same signaling molecule (i.e., PI 3-kinase).



Off Signals: Termination of Hormone Action

Just as there are complex biochemical pathways that mediate hormone action, there are also mechanisms to terminate the biologic response. The necessity for these mechanisms is illustrated by the following example. After we eat a meal, the concentration of plasma glucose increases. This elicits an increase in insulin secretion, which in turn leads to a decrease in plasma glucose levels. If these processes went on unchecked, the level of glucose in the plasma would eventually fall so low that it would lead to symptomatic hypoglycemia. How is insulin action terminated? The answers to this question are not yet entirely clear, but several mechanisms contribute to turning off the insulin signaling pathway. Receptor-Mediated Endocytosis

Insulin binding to its receptor triggers endocytosis of the receptor. Although most of the internalized receptors are recycled to the plasma membrane, some receptors are transported to lysosomes, where they are degraded. [52] [53] As a result, insulin binding accelerates the rate of receptor degradation, thereby down-regulating the number of receptors on the cell surface. Furthermore, endosomes contain proton pumps, which acidify the lumen; the acidic pH within the endosome promotes dissociation of insulin from its receptor. Ultimately, insulin is transported to the lysosome for degradation. In fact, receptor-mediated endocytosis is the principal mechanism whereby insulin is cleared from the plasma. [54] Binding of ligands to other receptor tyrosine kinases also triggers receptor-mediated endocytosis by similar mechanisms. Protein Tyrosine Phosphatases

Protein phosphorylation is a dynamic process. Tyrosine kinases catalyze the phosphorylation of tyrosine residues, but there are also protein tyrosine phosphatases (PTPases) to remove the phosphates. [2] Thus, PTPases antagonize the action of tyrosine kinases. Studies with knockout mice have demonstrated that the absence of PTPase-1B is associated with increased insulin sensitivity and also protects against weight gain. [55] [56] Nevertheless, the human genome encodes a large number of PTPases, and it is an important goal of research to elucidate their physiologic functions. If one could develop selective inhibitors of the PTPases that oppose the effects of the insulin receptor tyrosine kinase, it is possible that these inhibitors would provide novel therapies for diabetes.

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Figure 5-4 Simplified model of signaling pathways downstream from the insulin receptor. Insulin binds to the insulin receptor, thereby activating the receptor tyrosine kinase to phosphorylate tyrosine residues on insulin receptor substrates (IRSs) including IRS-1 and IRS-2. [30] Consequently, phosphotyrosine residues in IRS molecules bind to Src homology 2 (SH2) domains in molecules such as growth factor receptor-binding protein 2 (Grb-2) and the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase. These SH2 domain-containing proteins initiate two distinct branches of the signaling pathway. Activation of PI 3-kinase leads to activation of phosphoinositide-dependent kinases (PDKs) 1 and 2, which activates multiple protein kinases including Akt/protein kinase B, atypical protein kinase C (PKC) isoforms, and serum/glucocorticoid-activated protein kinases (Sgk). [100] Grb-2 interacts with m-SOS, a guanine nucleotide exchange factor that activates Ras.[ 101] Activation of Ras triggers a cascade of protein kinases leading to the activation of mitogen-activated protein (MAP) kinase. Serine/Threonine Kinases

Most receptor tyrosine kinases, including the insulin receptor, are substrates for phosphorylation by Ser/Thr-specific protein kinases. Interestingly, the Ser/Thr phosphorylation appears to inhibit the action of the tyrosine kinase. Similarly, other phosphotyrosine-containing proteins are subject to inhibitory influences of Ser/Thr phosphorylation resistance. For example, it has been reported that Ser/Thr phosphorylation of IRS-1 may inhibit insulin action, thereby causing insulin resistance. [57] [58] [59] [60] [61]



Mechanisms of Disease

The simplest forms of endocrine disease are caused by either a deficiency or an excess of a hormone. However, hormone resistance syndromes resulting from defects in the signaling pathways can masquerade as hormone deficiency states. Similarly, diseases associated with constitutively activated receptors can mimic a state of hormone excess. In some cases, the abnormality in hormone action is genetic in origin, resulting from a mutation in a gene encoding one of the proteins in the signaling pathway. Similar syndromes can also be caused by other mechanisms; for example, there are autoimmune syndromes caused by autoantibodies directed against cell-surface receptors. These clinical syndromes illustrate the principle that understanding the biochemical pathways of hormone action can provide important insights into the pathophysiology of human disease. Genetic Defects in Receptor Function

At least two distinct major types of genetic defects can cause hormone resistance. [62] First, mutations can lead to a decrease in the number of receptors. For example, in the case of the insulin receptor, mutations have been identified that decrease receptor number by at least three mechanisms: (1) impairing receptor biosynthesis, (2) inhibiting the transport of receptors to their normal location in the plasma membrane, and (3) accelerating the rate of receptor degradation. Second, mutations can impair the intrinsic activities of the receptor. In the case of the insulin receptor, mutations have been reported that decrease the affinity of insulin binding or inhibit receptor tyrosine kinase activity. Receptor dimerization is known to play a central role in the mechanisms whereby ligands activate many cell-surface receptors. This role has been shown most convincingly in the case of the GH receptor (a member of the family of cytokine receptors) but has also been postulated for receptor tyrosine kinases. The syndromes of multiple endocrine neoplasia types 2A and 2B and familial medullary carcinoma of the thyroid are caused by mutations in the gene encoding the ret tyrosine kinase (a subunit of the receptor for glial cell-derived growth factor). [63] Ordinarily, there are cysteine residues in the extracellular

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Figure 5-5 Mutations leading to constitutive activation of Ret. "Wild-type" Ret has intramolecular disulfide bonds formed by two cysteine residues in the same receptor molecule (left). When one of the two cysteine residues is mutated, the unpaired cysteine residue is available to form an intermolecular disulfide bond with a cysteine residue on another receptor molecule. This leads to receptor dimerization (right), which in turn leads to constitutive activation of the receptor tyrosine kinase. [63] [102] [103] This type of mutation has been identified in patients with multiple endocrine neoplasia type 2.

domain of Ret that participate in the formation of intramolecular disulfide bonds. Mutation of one of the cysteine residues leaves an unpaired cysteine residue that promotes dimerization of Ret molecules, thereby activating the Ret receptor tyrosine kinase (Fig. 5-5) . Activation of the Ret tyrosine kinase through this germ line mutation converts Ret into an oncogene. Autoantibodies Directed against Cell-Surface Receptors

Inhibitory antireceptor autoantibodies were first identified in myasthenia gravis. [64] In this neurologic disease, antibodies to the nicotinic acetylcholine receptor impair neuromuscular transmission, apparently by accelerating receptor degradation. Subsequently, autoantibodies to the insulin receptor were demonstrated to block insulin action in the syndrome of type B extreme insulin resistance. [65] Insulin resistance is caused by at least two mechanisms: (1) the antireceptor antibodies inhibit insulin binding to the receptor, [66] and (2) the antibodies accelerate receptor degradation. [67] Graves' disease provided the first example of stimulatory antireceptor autoantibodies. [68] In Graves' disease, there are autoantibodies directed against the thyroid-stimulating hormone (TSH) receptor. These antireceptor antibodies activate the TSH receptor, thereby stimulating growth of the thyroid gland as well as hypersecretion of thyroid hormone. This "experiment of nature" demonstrates that the receptor can be activated by ligands other than the physiologic ligand and that the normal spectrum of biologic actions can be triggered by this unphysiologic ligand (i.e., the antireceptor antibody). Similarly, antibodies to the insulin receptor have been demonstrated to activate the insulin receptor by mimicking insulin action. Although it is more common for a patient with anti-insulin receptor autoantibodies to present with insulin resistance, patients with anti-insulin receptor autoantibodies have also been reported to experience fasting hypoglycemia. [69] [70] [71]



RECEPTORS THAT SIGNAL THROUGH ASSOCIATED TYROSINE KINASES Overview

Members of the cytokine family of receptors resemble receptor tyrosine kinases in their mechanism of action, with one important difference. Instead of the tyrosine kinase being intrinsic to the receptor, enzymatic activity resides in a protein that associates with the cytokine receptor. As with receptor tyrosine kinases, ligand binding to the cytokine receptor activates the associated kinase. The more than 25 known ligands that bind to members of the cytokine receptor family have diverse functions. Three of the ligands are hormones: GH, which is vital for normal body height; prolactin (PRL), which is required for reproduction and lactation; and leptin, which is a potent appetite suppressant and a regulator of rates of metabolism. Other ligands of cytokine receptors, for example, erythropoietin, most interleukins, and interferons , and , regulate hematopoiesis or the immune response. A number of genetic diseases can be traced to defects in cytokine receptors. For example, Laron dwarfism is caused by autosomal recessive mutations of the GH receptor [72] and autosomal recessive mutations of the leptin receptor can cause morbid obesity. [73]



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Cytokine Receptors Are Composed of Multiple Subunits

Members of the cytokine family of receptors share homology in both the extracellular and cytoplasmic domains. Some cytokine receptors, including the receptors for GH, PRL, and leptin, are thought to be composed of dimers of a single receptor subunit (Fig. 5-6) . One ligand is thought to bind to both receptor subunits, as discussed earlier for the GH receptor. However, most cytokine receptors are composed of two or more different subunits, with as many as six subunits constituting a single receptor. [74] [75] Some of these receptors are thought to bind ligand dimers. One or more of these receptor subunits is shared by receptors for other cytokines. This phenomenon of "mixing and matching" receptor subunits is an efficient way for the cell to fine-tune its cellular responses and increase the number of ligands a group of receptor subunits can bind. For example, a receptor composed of gp130 and leukemia inhibitory factor receptor subunit binds leukemia inhibitory factor, a pleiotropic cytokine with multiple functions that appears to serve as a molecular interface between the neuroimmune and endocrine systems. [76] The same receptor subunits, when combined with a ciliary neurotrophic factor receptor subunit, show a preference for ciliary neurotrophic factor, a trophic factor for motor neurons in the ciliary ganglion and spinal cord and a potent appetite suppressor. [77] Combine two gp130 subunits with an interleukin-6 (IL-6) receptor subunit, and the new receptor shows a preference for IL-6, an inducer of the acute phase response with additional anti-inflammatory properties. [78]



Cytokine Receptors Activate Members of the Janus Family of Tyrosine Kinases

Members of the cytokine family of receptors do not themselves exhibit enzymatic activity. Rather, they bind members of the Janus family of tyrosine kinases (JAKs) through a proline-rich region (see Fig. 5-6) . There are four known JAKs, designated JAK1, JAK2, JAK3, and TYK2. As do the cytokine receptors, the JAKs mix and match in that some receptors show a strong preference for a single JAK, some require two different JAKs, and others appear to activate multiple JAK family members. For example, GH, PRL, and leptin preferentially activate JAK2. Interferon- activates JAK1 and JAK2, and IL-2 activates JAK1 and JAK3. [74] [79] Binding of ligand to a cytokine receptor activates the appropriate JAK family member or members. In some cases (e.g., PRL), the JAKs appear to be constitutively associated with the cytokine receptor and ligand binding increases their activity. [80] In other cases (e.g., the GH receptor), ligand binding increases both the affinity of JAKs for the cytokine receptor and the activity of the associated JAKs. [81] Activation of JAKs requires receptor oligomerization, presumably to bring two or more JAKs into sufficiently close proximity to transphosphorylate each other on the activating tyrosine in the kinase domain, as described previously in the chapter for the receptor tyrosine kinases. Although receptor dimerization appears to be required for receptor activation, a conformational change in receptor may also be required. [82] [83] Transphosphorylation is believed to cause a conformational change that exposes the ATP- or substrate-binding site, or both. Once the JAKs are activated, they phosphorylate themselves and their associated receptor subunits on multiple tyrosines. JAKs appear to be vital for normal human function. Mutations in the JAK3 gene have been linked to an autosomal recessive form of severe combined immunodeficiency disease. [84] Targeted disruption of the JAK2 gene in mice is embryonic lethal. [85]



Signaling Pathways Initiated by Cytokine Receptor-JAK Complexes

Phosphorylated tyrosines within the cytokine receptor subunits and their associated JAKs form binding sites for various signaling proteins containing phosphotyrosine binding domains, such as SH2 and PTB domains. Each cytokine receptor-JAK complex would be expected to have some tyrosine-containing motifs shared with many other cytokine receptor-JAK complexes (e.g., tyrosines within JAKs) and some specific tyrosine-containing motifs (e.g., tyrosines within a specific combination of receptor subunits). Thus, ligand binding to cytokine receptors would be expected to initiate some signaling pathways that are shared by many cytokines and some that are more specialized to a particular cytokine receptor. The signaling proteins known to be recruited to subsets of cytokine receptor-JAK complexes are generally the same as those recruited to receptor tyrosine kinases. Examples include the IRS proteins, the adapter proteins Shc and Grb-2 that lead to activation of the Ras-MAP kinase pathway, phospholipase C, and PI 3-kinase. However, there is one family of signaling proteins that appears to be particularly important for the function of cytokinessignal transducers and activators of transcription (STATs) (Fig. 5-7) . STAT proteins are latent cytoplasmic transcription factors. STATs bind, through their SH2 domains, to one or more phosphorylated tyrosines in activated receptor-JAK complexes. Once bound, they themselves are tyrosyl phosphorylated, presumably by the receptor-associated JAKs. STATs then dissociate from the receptor-JAK complexes, homodimerize or heterodimerize with other STAT proteins, move to the nucleus, and bind to gamma-activated sequence-like elements in the promoters of cytokine-responsive genes. [86] The transcriptional response depends on how many STAT binding sites exist in the receptor-JAK complex, with which of the seven known STATs a particular STAT heterodimerizes, to what other proteins a particular STAT binds, the degree of serine or threonine phosphorylation of the STAT, and what other transcription factors are also activated. For example, leukemia inhibitory factor, whose receptor contains seven STAT3 binding motifs (YXXQ, where Y = tyrosine, X = any amino acid, and Q = glutamine) is a particularly potent activator of STAT3. [87] The transcriptional activity of STAT5 is enhanced by its forming a complex with the glucocorticoid receptor. [88]



Precise Regulation of the Cytokine Receptors Is Required for Normal Function

Ligand binding to cytokine receptors normally activates JAKs rapidly and transiently. Conversely, constitutively activated JAKs and STATs are associated with cellular transformation. For example, in cells transformed by the Abl oncoprotein v-Abl, JAK1 is constitutively activated and inhibition of JAK1 blocks the ability of v-Abl to transform bone marrow cells.[89] Constitutively active JAKs and STATs are a common characteristic of leukemias, [90] and both JAK2 and STAT5b have been identified as fusion partners in translocations in leukemias. The Tel-JAK2 fusion protein is constitutively active, leading to constitutively active STAT proteins. Thus, an understanding of what turns off cytokine receptor signaling is of utmost importance in understanding normal signaling through cytokine receptors. As with the receptor tyrosine kinases, several steps have been hypothesized to serve as points of signal termination for cytokine signaling. These include receptor degradation (e.g., through a ubiquitination-proteosome pathway) and dephosphorylation

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Figure 5-6 Cytokine receptors are composed of multiple subunits and bind to one or more members of the Janus kinase (JAK) family of tyrosine kinases. A, Growth hormone (GH), like prolactin and leptin, binds to receptor homodimers and activates JAK2. B, Interferon (IFN) homodimers bind to their ligand-binding R1 subunits. The R2 subunits are then recruited, leading to activation of JAK1, which binds to R1 subunit, and JAK2, which binds to R2 subunit. Both subunits and both JAKs are necessary for responses to IFN. C, Interleukin-2 (IL-2) binds to receptors composed of three subunits: a c subunit shared with receptors for ILs 4, 7, 9, and 15; an IL-2R subunit shared with the IL-15 receptor; and a noncytokine receptor subunit, IL-2R subunit. IL-2 activates both JAK3, bound to the c subunit, and JAK1, bound to IL2-R. Extracellular regions of homology are indicated by the black lines and patterns. Intracellular regions of homology are indicated by the white boxes.

of tyrosines within JAK or receptor (e.g., by an SH2 domain containing tyrosine phosphatase recruited to receptor-JAK complexes). The suppressors of cytokine-signaling (SOCSs) are thought to be particularly important players in the termination or suppression of cytokine-signaling pathways. SOCS proteins are an excellent example of an effective negative feedback loop. They are generally synthesized in response to cytokines. The newly synthesized SOCS proteins in turn bind, through their SH2 domain, to phosphorylated tyrosines within the cytokine receptor-JAK complex and inhibit further cytokine signaling. In some cases (i.e., SOCS1), SOCS proteins are thought to bind to phosphotyrosines in the kinase domain of JAK and inhibit kinase activity. [91] [92] In other cases (i.e., SOCS 3), SOCS proteins bind to phosphorylated tyrosines in the receptor and inhibit JAK activity. [93] Finally, in some cases (i.e., cytokine-inducible SH2 protein [CIS]), SOCS proteins bind to phosphorylated tyrosines in the receptor and block STAT binding and activation. [94] SOCS proteins can also be synthesized in response to noncytokine receptors, suggesting a mechanism whereby prior exposure to one ligand suppresses subsequent responses to another. For example, SOCS proteins have been implicated in the well-known ability of endotoxin to cause resistance to GH. [95]



Summary

Hormones, growth factors, and cytokines that bind to members of the cytokine family of receptors activate JAK family tyrosine kinases. The activated kinases in turn phosphorylate tyrosines in themselves and associated receptors. The phosphorylated tyrosines form binding sites for other signaling proteins, including STAT proteins and a variety of other phosphotyrosine-binding proteins. STAT proteins promote the regulation of cytokine-sensitive genes, including SOCS proteins that serve a negative feedback function of terminating ligand activation of JAKs or STATs, or both. Although this gives the general picture, it should be recognized that the picture is becoming much more complex every day. For example, there are reports that members of the Src family of tyrosine kinases can also be activated by some cytokine receptors (e.g., PRL receptor), [96] that some JAK-binding proteins (e.g., SH2-B) are potent activators of JAK2, [97] and that other proteins contribute to the down-regulation of cytokine-signaling pathways, including protein inhibitors of activated STAT (PIAS), that bind and inhibit specific STATs. [98]



G PROTEIN-COUPLED RECEPTORS Overview

G protein-coupled receptors (GPCRs) are an evolutionarily conserved gene superfamily with members in all eucaryotes from yeast to mammals. They transduce a wide variety of extracellular signals including photons of light; chemical odorants; divalent cations; monoamine, amino acid, and nucleoside neurotransmitters; lipids; and peptide and protein hormones. [104] All members of the GPCR superfamily share a common structural feature, seven membrane-spanning helices, but various subfamilies diverge in primary amino acid sequence and in the domains that serve in ligand binding, G protein coupling, and interaction with other effector proteins (Fig. 5-8) . All GPCRs act as guanine nucleotide exchange factors. In their activated (agonist-bound) conformation, they catalyze exchange of GDP tightly bound to the subunit of heterotrimeric G proteins for GTP (Fig. 5-9) . This in turn leads to activation of the subunit and its dissociation from the G protein dimer. Both G protein subunits are capable of regulating effector activity. [105] Identified G protein-regulated

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Figure 5-7 Cytokines activate signal transducers and activators of transcription (STATs). STAT proteins are latent cytoplasmic transcription factors. STATs bind, through their Src homology 2 (SH2) domains, to one or more phosphorylated tyrosines in activated receptor-JAK complexes. Once bound, they themselves are tyrosyl phosphorylated, presumably by the receptor-associated JAKs. STATs then dissociate from the receptor-JAK complexes, homodimerize or heterodimerize with other STAT proteins, move to the nucleus, and bind to gamma-activated sequence-like elements (GLEs) in the promoters of cytokine-responsive genes. (Adapted from figure by J. Herrington, with permission.)

effectors include enzymes of second messenger metabolism such as adenylyl cyclase and phospholipase C- and a variety of ion channels. Agonist binding to GPCRs thus alters intracellular second messenger and ion concentrations with resultant rapid effects on hormone secretion, muscle contraction, and a variety of other physiologic functions. Long-term changes in gene expression are also seen as a result of second messenger-stimulated phosphorylation of transcription factors. The G protein subunits are encoded by three distinct genes. The subunit binds guanine nucleotides with high affinity and specificity and has intrinsic guanosine triphosphatase (GTPase) activity. The and polypeptides are tightly but noncovalently associated in a functional dimer subunit. The three-dimensional structures of the individual and associated subunits have been determined. [105] [106] [107] There is considerable diversity in G protein subunits, with multiple genes encoding all three subunits and alternative gene splicing resulting in additional polypeptide products. There are at least 16 distinct subunit genes in mammals. These vary widely in range of expression. Some such as Gs-, which couples many GPCRs to stimulation of adenylyl cyclase, are ubiquitous; others such as Gtl-, which couples the GPCR rhodopsin to cyclic guanosine monophosphate phosphodiesterase in retinal rod photoreceptor cells, are highly localized. Because multiple distinct GPCRs, G proteins, and effectors are expressed within any given cell, the degree and basis for specificity in G protein coupling to GPCRs and to effectors are major subjects of investigation with implications for drug action and disease mechanisms. [108] Since the pioneering work of Rodbell [109] in discovering G proteins and showing that G protein-mediated signal transduction involves three separable components (receptor, G protein, and effector), additional complexity has emerged. A large new gene family termed RGS (for regulators of G protein signaling) has been identified. RGS proteins bind to a transition state of the GTP-activated G protein subunit and accelerate its GTPase activity, thus helping deactivate the subunit. RGS domains have also been found in modular proteins with additional functions, in certain cases linking heterotrimeric G protein signaling with the function of low-molecular-weight GTP-binding proteins in the ras superfamily. [110] Lefkowitz[111] has shown that a family of GPCR kinases and of arrestin proteins is involved in GPCR desensitization after agonist binding. In addition, it is now clear that GPCRs interact directly with a number of other proteins in addition to G proteins. Not only are GPCRs important targets for treatment of many diseases, but also mutations in genes encoding GPCRs have been identified as the cause of a number of endocrine as well as nonendocrine disorders.



G Protein-Coupled Receptor Structure and Function Structure

Hydropathy analysis of the primary sequence of all GPCRs predicts seven membrane-spanning helices connected by three intracellular loops and three extracellular loops with an

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Figure 5-8 The G protein-coupled receptor (GPCR) superfamily: diversity in ligand binding and structure. Each panel depicts various members of the GPCR superfamily in cartoon form. The seven membrane-spanning helices are shown as cylinders with the extracellular amino terminus and three extracellular loops above and the intracellular carboxyl terminus and three intracellular loops below. The superfamily can be divided into three subfamilies on the basis of amino acid sequence conservation within the transmembrane helices. Family 1 includes (A) the opsins, in which light (jagged arrow) causes isomerization of retinal covalently bound within the pocket created by the transmembrane helices (bar); (B) monoamine receptors, in which agonists (arrow) bind noncovalently within the pocket created by the transmembrane helices (bar); (C) receptors for peptides such as vasopressin, in which agonist binding (arrow) may involve parts of the extracellular amino terminus and loops as well as the transmembrane helices (bar); and (D) glycoprotein hormone receptors, in which agonists (oval) bind to the large extracellular amino terminus, thereby activating the receptor through as yet undefined interactions with the extracellular loops or transmembrane helices (arrow). Family 2 includes receptors for peptide hormones such as parathyroid hormone (PTH) and secretin. Agonists (arrow) may bind to residues in the extracellular amino terminus and loops as well as transmembrane helices (bar). Family 3 includes the extracellular Ca 2+ sensing receptor and metabotropic glutamate receptors. Agonists (sphere) bind in a cleft of the Venus flytrap-like domain in the large extracellular amino terminus, thereby activating the receptor through as yet undefined interactions with the extracellular loops or transmembrane helices (arrow).

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Figure 5-9 The G protein guanosine triphosphatase (GTPase) and G proteincoupled receptor (GPCR) desensitization-resensitization cycle. In each panel, the stippled region denotes the plasma membrane with extracellular above and intracellular below. In the basal state, the G protein is a heterotrimer with guanosine diphosphate (GDP) tightly bound to the subunit. The agonist-activated GPCR catalyzes release of GDP, which permits guanosine triphosphate (GTP) to bind. The GTP-bound subunit dissociates from the dimer. Arrows from subunit to effector and from dimer to effector indicate regulation of effector activity by the respective subunits. Arrow from effector to subunit indicates regulation of its GTPase activity by effector interaction. Under physiologic conditions, effector regulation by G protein subunits is transient and is terminated by the GTPase activity of the subunit. The latter converts bound GTP to GDP, thus returning the subunit to its inactivated state with high affinity for the dimer, which reassociates to form the heterotrimer in the basal state. In the basal state, the receptor kinase and arrestin are shown as cytosolic proteins. Dissociation of the GTP-bound subunit from the dimer permits the latter to facilitate binding of receptor kinase to the plasma membrane ( arrow from dimer to receptor kinase). Plasma membrane binding permits the receptor kinase to phosphorylate the agonist-bound GPCR (depicted here as occurring on the carboxyl-terminal tail of the GPCR, but sites on intracellular loops are also possible). GPCR phosphorylation in turn facilitates arrestin binding to GPCR, resulting in desensitization. Endocytic trafficking of arrestin-bound GPCR and recycling to the plasma membrane during resensitization are not depicted here.

extracellular amino terminus and an intracellular carboxyl terminus (see Fig. 5-8) . This basic structure has now been verified by x-ray crystallography for rhodopsin. [112] Although there was already evidence that visual transduction in the retina and hormone activation of adenylyl cyclase shared common features, the discovery that the -adrenergic receptor has the same topographic structure as rhodopsin came as a surprise. [111] Cloning of the complementary deoxyribonucleic acids (cDNAs) for a vast number of GPCRs followed elucidation of the primary sequence of the -adrenergic receptor, and in every case the same core structure was predicted by hydropathy analysis. In addition to the predicted core structure, certain other common features (with exceptions in some subsets of the GPCR superfamily) were noted [104] : (1) a disulfide bridge connecting the first and second extracellular loops; (2) one or more N-linked glycosylation sites, usually in the amino terminus but occasionally in extracellular loops; (3) palmitoylation of one or more cysteines in the carboxyl terminus, effectively creating a fourth intracellular loop; (4) potential phosphorylation sites in the carboxyl terminus and occasionally the third intracellular loop. Glycosylation appears to be important for proper folding and trafficking to the plasma membrane rather

58

than for ligand binding. The disulfide bridge may help in proper arrangement of the transmembrane helices. Superimposed on the basic structure of GPCRs are a number of variations relevant to differences in ligand binding, G protein coupling, and interaction with other proteins.[104] First, there are major differences in amino acid sequence among members of the GPCR superfamily. Sequence alignment, especially of the transmembrane helices, allows one to divide the superfamily into subfamilies (see Fig. 5-8) . Of these, family 1 is the largest and itself can be subdivided. The largest subset includes opsins, odorant receptors, and monoamine, purinergic, and opiate receptors. These are characterized by a short amino terminus. The next subset includes chemokine, protease-activated, and certain peptide hormone receptors characterized by a slightly longer amino terminus. The last subset comprises receptors for the large glycoprotein hormones, TSH, luteinizing hormone, and follicle-stimulating hormone. These have an approximately 400-residue extracellular amino terminus. Family 2 shows essentially no sequence homology to family 1 even within the transmembrane helices and is characterized by an approximately 100-residue amino terminus. Members include receptors for a number of peptide hormones such as parathyroid hormone (PTH), calcitonin, vasoactive intestinal peptide, and corticotropin-releasing hormone. Family 3, in addition to a unique primary sequence, has other unique features such as an approximately 200-residue carboxyl terminus and an approximately 600-residue amino terminus. The latter consists of a putative "Venus flytraplike" domain and a cysteine-rich domain. Members include the metabotropic glutamate receptors, an extracellular Ca 2+ -sensing receptor, and putative taste and pheromone receptors. [113] The determination of the three-dimensional crystal structure of part of the extracellular amino terminus of one of the metabotropic glutamate receptors verifies the Venus flytrap structure. [113] Ligand Binding

Given the diversity of ligands (>1000) that bind to GPCRs, it is not surprising that considerable diversity is evident in both the sequence and structure of presumptive GPCR ligand-binding domains. The opsins are unique among GPCRs in that the ligand, retinal, is covalently bound to a lysine in the seventh transmembrane helix. [112] Ligand binding for other members of family 1 with a short extracellular amino terminus, for example, adrenergic and other monoamine receptors, probably involves a

pocket within the transmembrane helices as demonstrated for rhodopsin (see Fig. 5-8) . For other family 1 GPCRs, the extracellular amino terminus, perhaps together with extracellular loops and portions of the transmembrane helices, is involved in ligand binding. In the case of the glycoprotein hormone receptors, the large extracellular amino terminus plays the principal role in hormone binding. Likewise, in family 2 receptors, the extracellular amino terminus is largely responsible for ligand binding. For family 3 GPCRs, the three-dimensional structure of the type 1 metabotropic glutamate receptor shows that agonist binding occurs within a cleft between the lobes of the Venus flytrap. [113] G Protein Coupling

Because the number of potential G proteins to which GPCRs couple is much more limited than the number of ligands that bind GPCRs, more conservation of the domains involved in G protein coupling would be expected. Although GPCRs can be broadly divided into those that couple to Gs, those that couple to the Gq subfamily, and those that couple to the Gi-Go subfamily, the situation is probably more complicated. Specificity of coupling to the most recently identified G proteins, G12 and G13, is still uncertain. Also, some GPCRs evidently can couple to both Gs and Gq. A vast number of studies have been performed to define the sites of ligand binding and G protein coupling of GPCRs. [114] Considerable evidence points to the third intracellular loop (particularly its membrane-proximal portions) and to the membrane-proximal portion of the carboxyl terminus as key determinants of G protein coupling specificity. For example, exchanging only the third intracellular loop between different GPCRs confers the G protein coupling specificity of the exchanged loop upon the recipient GPCR. [115] In contrast, the second intracellular loop, although important for G protein coupling, appears to play a role in the activation mechanism rather than in determining specificity of coupling. [115] A tripeptide motif (D/E, R, Y/W) at the start of the second intracellular loop that is highly conserved in family 1 GPCRs is critical for G protein activation. [114] Mechanism of Activation

The precise mechanism of activation after agonist binding remains to be defined for most GPCRs, but studies of rhodopsin provide the clearest picture available. In the ground state, retinal covalently bound to the seventh transmembrane helix in rhodopsin holds the transmembrane helices in an inactive conformation. Isomerization of retinal upon absorption of light of the appropriate wavelength converts an antagonist ligand into an agonist. The rhodopsin crystal structure identifies the residues in the transmembrane helices that interact with retinal and suggests a mechanism for movement of the helices upon photoactivation of retinal. [112] Movement of the transmembrane helices in turn leads to changes in conformation of cytoplasmic loops that promote G protein activation. For family 1 receptors related to rhodopsin, the determination of its three-dimensional structure validates the idea that a change in conformation of transmembrane helices is the direct result of agonist versus antagonist binding to residues within the helices. Further refinements in understanding the mechanism of activation for opsin-related GPCRs should come as additional three-dimensional structures are determined. Until then, molecular modeling by computer on the basis of the rhodopsin structure and then experimental testing offer a useful approach. [116] For other GPCRs whose presumptive site of agonist binding does not involve direct contact with transmembrane helices (families 2 and 3 and the glycoprotein hormone receptors in family 1), much remains to be learned about the mechanism of activation. Specifically, determining how agonist binding to the extracellular domain of such GPCRs leads to presumptive changes in conformation of transmembrane helices requires further studies of structure and function. A general hypothesis of GPCR activation postulates that GPCRs are in equilibrium between an activated state and an inactive state. These states presumably differ in the disposition of the transmembrane helices and, in turn, the cytoplasmic domains that determine G protein coupling. Agonists, according to this model, are viewed as stabilizing the activated state. Antagonists may be neutral, that is, they simply compete with agonist for receptor binding but their binding does not influence this equilibrium. Alternatively, they may be "inverse" agonists; that is, their binding stabilizes the inactive state of the receptor. Naturally occurring, activating mutations of GPCRs lend support to this hypothesis. Dimerization

Members of the tyrosine kinase receptor family have long been known to require dimerization as part of their activation mechanism. It is now apparent that many GPCRs likewise form homodimers and heterodimers.[104] Residues within transmembrane

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helix 6 may foster dimerization of small family 1 GPCRs, [117] and intermolecular disulfide bonds in the extracellular amino-terminal domain are involved in homodimerization of most family 3 GPCRs.[113] [118] A coiled-coil interaction in the carboxyl terminus of -aminobutyric acid B receptor subtypes is responsible for heterodimerization, and this is critical for proper receptor function. [119] Modifications of ligand binding, signaling, and receptor sequestration have been demonstrated upon heterodimerization of angiotensin with bradykinin receptors, of with opioid receptors, and of opioid with -adrenergic receptors. [120] [121] Further studies are needed to elucidate the role of homodimerization and heterodimerization in GPCR function.



G ProteinCoupled Receptor Desensitization

Pharmacologists long ago appreciated that continued exposure to agonist leads to a diminished response, so-called desensitization. This phenomenon has been extensively studied in GPCRs. Two forms are defined: heterologous, in which binding of agonist to one GPCR leads to a diminished response of a different GPCR to its agonist, and homologous, in which desensitization occurs only for the GPCR to which agonist is bound. Both forms of desensitization involve GPCR phosphorylation but by different kinases and at different sites. Stimulation of cyclic adenosine monophosphate formation by agonist binding to a Gs-coupled GPCR leads to activation of protein kinase A, which in turn can phosphorylate and desensitize the GPCR. Such phosphorylation may also alter G protein coupling specificity. [104] Similarly, protein kinase C activation resulting from GPCR coupling to Gq family members may cause protein kinase Ccatalyzed phosphorylation of GPCRs with desensitization. In retinal photoreceptors, a specific rhodopsin kinase and a protein termed arrestin were implicated in attenuation of the light response. Just as parallels were identified between rhodopsin and GPCR structure, so were parallels identified in this desensitization mechanism. Rhodopsin kinase is but one member of a family of GPCR kinases and arrestin only one of a family of related proteins that function in desensitization of many members of the GPCR superfamily. [111] GPCR kinases preferentially phosphorylate the agonist-bound form of a GPCR, thus ensuring homologous desensitization. Upon GPCR phosphorylation by GPCR kinase, arrestins bind to the third intracellular loop and carboxyl-terminal tail of the GPCR, thereby blocking G protein binding (see Fig. 5-9) . There is evidence that GPCR kinases and arrestins not only act to desensitize GPCRs but also mediate other functions including receptor internalization and interaction with other effectors (see next section).



G ProteinCoupled Receptor Interactions with Other Proteins

The initial paradigm of GPCR function postulated that G protein activation is the sole outcome of agonist binding to GPCRs. With the identification of GPCR interactions with GPCR kinases and arrestins, this concept was modified to include these proteins involved in GPCR desensitization. Later evidence, however, suggests that GPCR interaction with arrestins may also permit recruitment of other proteins to the GPCR. For example, the src tyrosine kinase may interact with the -adrenergic receptor with -arrestin serving as an adaptor. [122] Arrestins may also recruit proteins involved in endocytosis. GPCR kinases may also serve to recruit additional signaling proteins to the GPCR. [122] Other classes of proteins may interact with specific GPCRs without recruitment by GPCR kinases and arrestins. These include SH2 domaincontaining proteins, small GTP-binding proteins, and PDZ (for postsynaptic density protein-95/discs large/zona occluden-1) domaincontaining proteins. Examples of the latter include binding of the Na+ /H+ exchanger regulatory factor to the carboxyl terminus of the -adrenergic receptor. [122] The long carboxyl terminus of family 3 GPCRs such as metabotropic glutamate receptors contains polyproline motifs involved in binding members of the Homer family. The latter can facilitate functional interactions with yet other proteins such as the inositol trisphosphate receptor. [123] Receptor activitymodifying proteins (RAMPs), a new family of single-transmembrane-domain proteins, appear to heterodimerize with certain GPCRs, assisting them in proper folding and membrane trafficking. [124] Interestingly, when the calcitonin receptorlike GPCR associates with RAMP1, it forms a calcitonin generelated peptide receptor, whereas when it associates with RAMP2, it becomes an adrenomedullin receptor. Clearly, this rapidly evolving aspect of GPCR function holds many further interesting developments in store.



G ProteinCoupled Receptors in Disease Pathogenesis and Treatment

Because of their diverse and critical roles in normal physiology, their accessibility on the cell surface, and the ability to synthesize selective agonists and antagonists, GPCRs have long been a major target for drug development. One estimate is that about 65% of prescription drugs are targeted against GPCRs. With the cloning of GPCR cDNAs, much greater diversity of receptor subclasses became evident than had been anticipated on the basis of pharmacologic studies. For example, five muscarinic receptor subtypes and an even greater number of serotoninergic GPCRs were identified. [114] This has allowed the development of highly specific, subtype-selective drugs that have fewer side effects than those produced by previously available agents. Another result of the cloning of GPCR cDNAs by homology screening and polymerase chain reactionbased approaches is the identification of "orphan" GPCRs, that is, receptors with the canonical, predicted seven-transmembrane-domain structure of GPCRs but without knowledge of their physiologic agonist. There have been substantial efforts to identify the relevant ligands for such orphan receptors. An example of the success of such efforts is the identification of an orphan GPCR as the neuromedin U receptor involved in regulation of feeding. [125] This will permit testing of candidate drugs targeting this receptor for obesity prevention and treatment. Beyond drug development, defects in GPCRs are an important cause of a wide variety of human diseases. [126] GPCR mutations can cause loss of function by impairing any of several steps in the normal GPCR-GTPase cycle (see Fig. 5-9) . These include failure to synthesize GPCR protein altogether, failure of synthesized GPCR to reach the plasma membrane, failure of GPCR to bind or be activated by agonist, and failure of GPCR to couple to or activate G protein. Because in most cases clinically significant impairment of signal transduction requires loss of both alleles of the GPCR gene, most such diseases are inherited in autosomal recessive fashion (Table 5-1) . Most of these diseases are manifested as resistance to the action of the normal agonist and thus mimic deficiency of the agonist. For example, TSH receptor loss-of-function mutations cause a form of hypothyroidism mimicking TSH deficiency, but serum TSH is actually elevated in such cases, reflecting resistance to the hormone's action caused by defective receptor function. Nephrogenic diabetes insipidus (renal vasopressin resistance)

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TABLE 5-1 -- Diseases Caused by G ProteinCoupled Receptor Loss-of-Function Mutations Disease

Receptor

Inheritance

V2 vasopressin Nephrogenic diabetes insipidus

X-linked

ACTH

Familial ACTH resistance

Autosomal recessive

GHRH

Familial GH deficiency

Autosomal recessive

GnRH

Hypogonadotropic hypogonadism

Autosomal recessive

FSH

Hypergonadotropic ovarian dysgenesis

Autosomal recessive

LH

Male pseudohermaphroditism

Autosomal recessive

TSH

Familial hypothyroidism

Autosomal recessive

Ca2+ sensing

Familial hypocalciuric hypercalcemia, neonatal severe primary hyperparathyroidism

Autosomal dominant, autosomal recessive

Melanocortin 4

Obesity

Autosomal recessive

PTH/PTHrP

Blomstrand chondrodysplasia

Autosomal recessive

ACTH, adrenocorticotropic hormone; FSH, follicle-stimulating hormone; GH, growth hormone; GHRH, growth hormonereleasing hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; PTH, parathyroid hormone; PTHrP, parathyroid hormonerelated protein; TSH, thyroid-stimulating hormone. is caused by loss-of-function mutations in the V2 vasopressin receptor gene located on the X chromosome. Thus, males with a single copy of the gene experience the disease when they inherit a mutant gene, whereas most females do not show overt disease because random X inactivation leaves them with on average 50% of normal gene function. Most V2 vasopressin receptor mutations associated with nephrogenic diabetes insipidus cause loss of function by impairing normal synthesis or folding of the receptor, or both. A novel mechanism for receptor loss of function elucidated for a V2 vasopressin receptor missense mutation associated with nephrogenic diabetes insipidus involves constitutive arrestin-mediated desensitization. [127] The extracellular Ca 2+ -sensing receptor appears to be an interesting exception to the association between GPCR loss-of-function mutations and hormone resistance. Loss-of-function mutations of the Ca 2+ -sensing receptor mimic a hormone hypersecretion state, primary hyperparathyroidism. In fact, Ca 2+ -sensing receptor loss-of-function mutations do cause hormone resistance, but in this case extracellular Ca 2+ is the hormonal agonist that acts through this receptor to inhibit PTH secretion. A loss-of-function mutation of one copy of the receptor gene typically causes mild resistance to extracellular Ca 2+ manifested as familial hypocalciuric hypercalcemia. If two defective copies are inherited, extreme Ca 2+ resistance causing neonatal severe primary hyperparathyroidism results (see Table 5-1) . In some cases, a heterozygous receptor loss-of-function mutation may be associated with neonatal severe primary hyperparathyroidism, perhaps reflecting a dominant negative effect caused by dimerization of wild-type and mutant receptors. [128] GPCR gain-of-function mutations (Table 5-2) are also an important cause of disease. [126] Given the dominant nature of activating mutations, most such diseases are inherited in an autosomal dominant manner. Activating TSH receptor mutations may be inherited in autosomal dominant fashion and cause diffuse thyroid enlargement in familial nonautoimmune hyperthyroidism, or they may occur as somatic mutations causing focal, sporadic hyperfunctional thyroid nodules. [129] Unlike

Receptor

TABLE 5-2 -- Diseases Caused by G ProteinCoupled Receptor Gain-of-Function Mutations Disease Inheritance

LH

Familial male precocious puberty

Autosomal dominant

TSH

Sporadic hyperfunctional thyroid nodules

Noninherited (somatic)

TSH

Familial nonautoimmune hyperthyroidism

Autosomal dominant

Ca2+ sensing

Familial hypocalcemic hypercalciuria

Autosomal dominant

PTH/PTHrP

Jansen's metaphyseal chondrodysplasia

Autosomal dominant

LH, luteinizing hormone; PTH, parathyroid hormone; PTHrP, parathyroid hormonerelated protein; TSH, thyroid-stimulating hormone. loss-of-function mutations, which may be missense as well as nonsense or frameshift mutations that truncate the normal receptor protein, GPCR gain-of-function mutations are almost always missense mutations. The location and nature of naturally occurring, disease-causing mutations offer important insights into GPCR structure and function. The basis for defective receptor function is clear with mutations that truncate receptor synthesis prematurely. More subtle missense mutations may impair function if they involve highly conserved residues in transmembrane helices critical for normal protein folding. Activating missense mutations often involve residues within or bordering transmembrane helices and are thought to disrupt normal inhibitory constraints that maintain the receptor in its inactive conformation. [130] Mutations disrupting these constraints mimic the effects of agonist binding and shift the equilibrium toward the activated state of the receptor.

Clinically, diseases caused by activating GPCR mutations therefore mimic states of agonist excess, but direct measurement shows that agonist concentrations are actually low, reflecting normal negative feedback mechanisms. Again, the Ca 2+ -sensing receptor is an apparent exception, with activating mutations causing functional hypoparathyroidism. For most GPCRs, disease-associated gain-of-function mutations cause constitutive, agonist-independent, activation but with rare exceptions,[131] the Ca 2+ -sensing receptor gain-of-function mutations cause increased sensitivity to extracellular Ca 2+ rather than to Ca 2+ -independent activation. Naturally occurring animal models of human disease have revealed additional examples of etiologic GPCR mutations. For example, a loss-of-function mutation in the hypocretin (orexin) type 2 receptor gene was identified in canine narcolepsy. [132] Dozens of mouse GPCR gene knockout models have been created, many revealing interesting and in some cases unexpected phenotypes. Characterization of the phenotype resulting from disruption of a mouse GPCR gene may accurately predict the clinical picture resulting from the corresponding mutation in humans, such as with disruption of the melanocortin-4 receptor gene resulting in obesity in mouse [133] and human[134] and disruption of the PTH/PTH-related protein receptor gene impairing normal bone growth and development in mouse [135] and in the human disease Blomstrand chondrodysplasia. [136] Further knockout models and further detailed studies of these models can be expected to increase substantially our understanding of GPCR function and to address questions such as the unique roles of multiple subtypes of various GPCR subclasses, for example, the 3-adrenergic receptor subtype.[137] Availability of mouse knockout models of human diseases such as nephrogenic diabetes insipidus [138] should also facilitate testing of novel therapies including gene transfer.

61

Screening of GPCR genes for mutations as the potential cause of additional human disorders may continue to turn up new examples, but it is also becoming clear that variations in GPCR gene sequence can have profound consequences beyond mutations causing diseases. One of the most striking examples is the discovery that homozygous loss-of-function mutations of the type 5 chemokine receptor (CCR5) confer resistance to human immunodeficiency virus (HIV) infection in individuals with this genotype. [139] The reason is that CCR5 serves as a coreceptor for HIV entry into cells. In the roundworm, two isoforms of a neuropeptide receptor are associated with profound differences in feeding behavior. [140] As more polymorphisms are discovered in the human genome, many examples of variations in GPCR gene sequence will be found and the challenge will be to elucidate their possible functional significance. In vitro studies may reveal functional differences, such as differences in G protein coupling seen with a four-amino-acid polymorphism in the third intracellular loop of the 2C -adrenergic receptor, [141] but further studies are required to determine whether such differences are important in individual variation in response to various drugs (pharmacogenomics) or in other subtle physiologic differences that could confer susceptibility to disease (complex disease genes). Given the high proportion of the human genome devoted to GPCR genes, it is clear that studies of this gene superfamily will play a prominent role in the postgenomic era.



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Chapter 6 - Laboratory Techniques for Recognition of Endocrine Disorders George Klee

Endocrinology is a practice of medicine that is highly dependent on accurate laboratory measurements because small changes in hormone levels often may be more specific and more sensitive for early disease than the classic physical signs and symptoms. Because most endocrinologists currently do not have facilities to develop and validate laboratory assays, they rely on commercial analytic assays or send a patient's specimen to specialized laboratories. Even most hospital and commercial laboratories have minimal expertise for developing analytic assays. This critical dependence on quality laboratory measurements, combined with minimal information about the performance of these tests, places endocrinologists in a potentially vulnerable position. This chapter attempts to provide an overview of the strengths and weaknesses of the analytic techniques typically used for endocrine measurements in blood and urine. Concentrations of most hormones are much lower than those of general chemistry analytes, and specialized techniques are necessary to measure these low concentrations. Three major types of assays for measuring hormones are described: Immunoassays (both competitive and sandwich) Chromatography Mass spectrometry Nucleic acid measurements for evaluation of genetic alterations also are reviewed. The minimal analytic performance validation required by the federal government for laboratories testing specimens of Medicare patients, along with explanations of these performance parameters, is outlined. This information should help endocrinologists better assess the performance of the analytic systems that they are using. Techniques to investigate discordant laboratory test values also are presented to help clinicians work with their laboratories to reconcile test values that do not match clinical presentations. Hormone concentrations are reported in molar units, mass units, or standardized units, such as World Health Organization (WHO) International Units (IU). When these measurements are expressed in molar units, most hormones in blood and urine are present in concentrations of 10 -6 to 10-12 M/L (Fig. 6-1) . The terms used to describe these concentrations are micromolar (10 -6 M/L), nanomolar (10 -9 M/L), and picomolar (10-12 M/L). The rangefrom the lowest to highest concentrationsis more than a million-fold difference. Therefore, laboratory techniques must be targeted to the levels of each given hormone. The major techniques for measuring picomolar concentrations are immunoassay and mass spectroscopy, whereas nanomolar and micromolar concentrations can be measured by these methods as well as chromatography and chemical detection systems. Some hormones, such as thyrotropin (TSH), have very low concentrations in the femtomolar (10-15 M/L) range in patients with diseases such as thyrotoxicosis. Exquisitely sensitive immunometric assays are usually needed to measure these very low concentrations.[1] [2]



TYPES OF ASSAYS The four major techniques used for endocrine measurements are as follows: Antibody-based immunologic assays, of which there are two subcategories: competitive immunoassays and immunometric (sandwich) assays Chromatographic assays Mass spectroscopy Nucleic acid-based assays Competitive Immunoassays

The term competitive radioimmunoassay refers to a measurement method in which an antigen (e.g., a hormone) in a specimen competes with radiolabeled reagent antigen for a limited number of binding sites on a reagent antibody. The three basic components of a competitive immunoassay are [3] [4] : 1. Antiserum specific for a unique epitope on a hormone or antigen. 2. Labeled antigen that binds to this antiserum. 66

3. Unlabeled antigen in the specimen or standard that is to be measured.

Figure 6-1 Seven-logarithm range of normal concentrations for the plasma concentrations of endocrine tests. DHEA, dehydroepian-drosterone; FSH, follicle-stimulating hormone; FT 4 , free thyroxine; FT 3 , free triiodothyronine; LH, luteinizing hormone; T 3 , triiodothyronine; T 4 , thyroxine; TSH, thyrotropin.

The antiserum is diluted to a concentration in which the number of binding sites available on the antibodies is fewer than the number of antigen molecules (labeled and unlabeled) in the reaction mixture. The labeled and unlabeled antigens compete for these limited number of binding sites on the antiserum. The competition is not always equal because the labeled antigen (tracer) may react differently with the antibody compared with the native antigen. This disparity in reactivity may be caused by alteration of the antigen due to the chemical attachment of the label or by differences in the endogenous antigen versus the form of the antigen used in the reagents. As long as the reactions are reproducible, these differences in reactivity are not important because the reaction can be calibrated with standard reference materials having known concentrations. Figure 6-2 illustrates the concepts of a competitive immunoassay. In the schematic diagram, 8 units of antibody react with 16 units of labeled antigen and 4 units of native antigen. At equilibrium (assuming equal reactivity), 6 units of label and 2 units of native antigen are bound to the limited supply of antibody. The antigen bound to the antibody is separated from the liquid antigen by any of several methods, and the amount of labeled antigen in the bound portion is quantitated. The assay is calibrated by measuring standards with known concentrations and cross-plotting the signal (i.e., counts of the gamma rays emitted from the radioactive label) versus the concentration of the standards to generate a dose-response curve. As the concentration increases, the signal decreases exponentially. Typically, the antiserum used in a competitive assay is diluted to a titer that binds between 40% to 50% of the labeled antigen when no unlabeled antigen is present. Further dilution of the antiserum increases the analytic sensitivity but decreases the signal and range of the assay. The precision of competitive immunoassays is related to the rate of change of the signal compared with the rate of change of concentration (i.e., the slope of the dose-response curve). [5] In Figure 6-2 , the slope is much lower at higher concentrations, causing the assay precision to be less at higher concentrations. Most competitive immunoassays also have a relatively flat dose-response curve at very low concentrations, causing poor precision at the low end of the assay. Consequently, the precision profile for most immunoassays is U-shaped, having the best coefficients of variation in the center of the dose-response curve. As shown in Figure 6-2 , the higher the concentration of the unlabeled antigen, the lower the amount of radiolabeled antigen that binds to the limited amount of antiserum. The signal decreases exponentially from the approximately half-maximum at zero concentration to a minimum value at high concentrations. This minimal binding, or nonspecific binding (NSB), is a valuable control parameter. Elevations in NSB usually signify impurities in the label that bind to the sides of the tubes and are not competitively displaced. Most assays add surfactants and proteins to minimize the NSB. Monitoring of changes in the NSB provides an early warning of potential assay problems. Statistical data-processing techniques are needed to translate the assay signals into concentrations. As illustrated, because these reactions are not linear, numerous curve-fitting algorithms have been developed. Before the introduction of micro-processors,

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Figure 6-2 A, Principles of competitive binding assays. B, Typical dose-response curve.

tedious error-prone, manual calculations were required to mathematically transform the data into linear models. A commonly used model was to cross-plot the logit of the normalized signal versus the logarithm of the concentration and to use linear regression lines to establish the dose-response curve. [5] Fortunately, today this procedure of curve fitting usually is accomplished electronically by using programs that automatically test the robustness of fit of multiparameter curves after statistically eliminating discordant data points. [6] However, users of these systems must understand the limitations and should pay attention to any warnings presented by the programs during processing of the data. In radioimmunoassays, radioactive iodine ( 125 I) is usually used to label the antigen. The immune complexes are separated from the unbound molecules by precipitation with centrifugation after reaction with secondary antisera and precipitating reagents (e.g., polyethylene glycol). [7] These radioimmunoassays may require special handling and licensure to ensure safety of the radioisotopes and are labor-intensive. The statistical counting errors associated with the relatively low radioactive counts and the poor reproducibility associated with the multiple manual steps generally necessitate that most laboratories perform the measurements in

duplicate. [8] Even when the averages of duplicate measurements are used, many manual radioimmunoassays have coefficients of variation between 10% and 15%. It is important that key quality control parameters for radio-immunoassays be carefully monitored. In addition to NSB, another key quality control parameter is the percentage binding of the radiolabel when zero antigen (Bo) is present. As the label deteriorates, because of aging, the binding often decreases, resulting in a less reliable assay. Another important quality control parameter is the slope of the dose-response curve. This parameter can be tracked by monitoring the concentration corresponding to half-maximum binding (50% of B/Bo). If this concentration increases significantly, the slope of the response curve decreases and the assay may not be capable of reliably measuring patient specimens at clinically important concentrations. Many commercial kits and automated immunoassays today use nonisotopic signal systems to measure hormone concentrations. These assays often use colorimetric, fluorometric, or chemiluminescent signals rather than radioactivity to quantitate the response. The advantages of these alternate signals are biosafety, longer reagent self-life, and ease of automation. On the other hand, these signals are more subject to matrix interferences than radioactive iodine. Radioactivity is not affected by changes in protein concentration, hemolysis, color, or drugs (except for other radioactive compounds), whereas many of the current signal systems may yield spurious results when such interferences are present. In addition, many of today's automated immunoassays are read kinetically before the reactions reach equilibrium. This step accentuates the effects of matrix differences between the reference standards and patient specimens. Later in this chapter potential trouble-shooting steps are outlined to help clinicians evaluate the integrity of test measurements when spurious results are suspected. Solid-phase reactions often are used in current immunoassays to facilitate the separation of the bound antibody-antigen complexes from the free reactants. [9] Three frequently used solid-phase materials are (1) microtiter plates, (2) polystyrene beads, and (3) paramagnetic particles. [10] Typically, the antibody is attached to the solid phase, and the separation of the immune complexes from the unbound moieties is accomplished by plate washers, bead washers, or magnetic wash stations, eliminating the need for centrifugation. Other novel ways of accomplishing this separation is to attach high-affinity linkers to antiserum, which then can be coupled to a complementary linker on the solid phase. An excellent pair of linkers are biotin and streptavidin. These compounds bind with affinity constants of approximately 10 15 L/M.[11] Biotin is a relatively small molecule that can easily be covalently attached to antiserum and used with streptavidin (a 70-kD tetrameric nonglycosylated protein) conjugated to microtiter plates, beads, or paramagnetic particles to facilitate separation. This technique allows the antibody-antigen reaction to proceed faster with less stearic hindrance than when the antibody is directly coupled to the solid phase. The antiserum used in these assays is a crucial component. Most earlier immunoassays used polyclonal antiserum produced in animals. The process of generating these antisera is a combination of art, science, and luck. Generally, a relatively pure form of the antigen is conjugated to a carrier protein (especially if the antigen is less than 10,000 d), mixed with adjuvant (e.g., Freud's complete adjuvant), and injected intradermally into the host animal. After several boosts with conjugated protein plus Freud's incomplete adjuvant, the host animal recognizes the material as foreign and develops immune responses. The antiserum then is harvested from the animal's blood. Under optimal conditions, moderate quantities of high-affinity antisera, which react only with the specific target antigen, are developed. The analytic sensitivity of a competitive immunoassay is approximately inversely related to the affinity of the antiserum, such that an antiserum with an affinity constant of

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109 L/M can be used to measure analytes in the nanomolar concentration range. The polyclonal antiserum developed by immunizing animals represents a composite of many immunologic clones, with each clone having a different affinity and different immunologic specificity. Most clones have affinities in the 10 7 to 108 L/M range, with only rare clones having affinities above 10 12 L/M. Various techniques are used to develop a specific antiserum, including (1) altering the form of the antigen by blocking cross-reacting epitopes and (2) purifying the antiserum using affinity chromatography to select antibodies directed toward the epitope of interest. Affinity-column purification can also be used for immunoextraction of higher-affinity antisera by selectively eluting antiserum from the column by means of a series of buffers with increasing acidity. [12] The major disadvantage of a polyclonal antiserum is the limited quantity. The large quantities needed by commercial suppliers of immunoassay reagents often require them to use multiple sources of antisera. These changes in antisera can cause significant changes in assay performance. In many instances, laboratories and clinicians are not informed about these changes, which may cause problems in medical decisions. Monoclonal antisera are used in many current immunoassays. These antisera are made by immunizing animals (usually mice) using techniques similar to those used for polyclonal antisera; instead of harvesting the antisera from the blood, however, the animal is killed and the spleen is removed. [13] The lymphocytes in the spleen are fused with myeloma cells to make cells that will grow in culture and produce antisera. These fused cells are separated into clones by means of serial plating techniques similar to those used in subculturing bacteria. The supernatant of these monoclonal cell lines (or ascites fluid if the cells are transplanted into carrier mice) contains monoclonal antisera. The selection processes used to separate the initial clones can be targeted to identify specific clones producing antisera with high affinities and low cross-reactivity to related compounds. The high specificity of monoclonal antisera can cause problems for some endocrine assays. Many hormones circulate in the blood as heterogeneous mixtures of multiple forms. Some of these forms are caused by genetic differences in patients, whereas other forms are related to metabolic precursors and degradation products of the hormone. Genetic differences cause some patients to produce variant forms of a hormone such as luteinizing hormone (LH). These genetic differences can cause marked variations in measurements made using assays with specific monoclonal antisera compared with more uniform measurements made using assays with polyclonal antisera that cross-react with the multiple forms. [14] Well-characterized monoclonal antisera can be mixed together to make an "engineered polyclonal antiserum" with improved sensitivity and specificity. [15] Cross-reactivity with precursor forms of the analytes and with metabolic degradation products can cause major differences in assays. For example, cross-reactivity with precursor forms causes differences in insulin assays, and cross-reactivity with metabolic fragments causes major differences in carboxyl-terminal parathyroid hormone (PTH) assays. [16] [17] Extraction of hormones from serum and urine specimens prior to measurement is a technique that can enhance both sensitivity and specificity of immunoassays. Numerous extraction systems have been developed, including (1) organic-aqueous partitioning to remove water-soluble interferences seen with steroids, (2) solid-phase extraction with absorption and selective elution from resins such as silica gels, and (3) immunoaffinity chromatography. [18] [19] Unfortunately, extraction and purification before immunoassay are seldom used in clinical assays. These techniques are difficult to automate and require skills and equipment not available in many clinical laboratories. Although

Figure 6-3 Comparison of an immunologic technique for measuring hormone concentration versus a receptor technique for measuring hormone activity. ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate.

commercial assays generally use reagents having adequate sensitivity and specificity to measure most patient specimens, some patient specimens may give spurious results and some disease states may require more analytic sensitivity to ensure sound clinical decisions. In these cases, extraction of specimens prior to measurement may provide more reliable information. Immunoassays measure concentrations rather than biologic activity. For most hormones, there is a strong correlation between the concentration of the protein or steroid being measured and the biologic activity, but this is not universally true. The reactive site for most antibodies is relatively small, about 5 to 10 amino acids for linear peptides. Some antiserum reactions are specific for the tertiary structure that corresponds to unique molecular configurations, but immunoassays seldom react

with the exact antigenic structure that confers biologic activity. Figure 6-3 presents a schematic illustration of the difference between immunologic binding site and biologic receptor binding site on a hormone. Indirect immunoassays have been developed using cultured cells that synthesize second messengers such as cyclic adenosine monophosphate (cAMP) at rates proportional to the concentration of hormone in the specimen. An example of this technique is the immunoassay measurement of cAMP produced by osteosarcoma cells to quantitate PTH bioactivity in serum. [20] Unfortunately, these assays are tedious and generally are not reproducible. More recent techniques using recombinant receptors as immunoassay binders may provide improved specificity with good reliability. [21] [22] [23]



Immunometric (Sandwich) Assays

A second immunologic technique used to measure hormones is the immunometric (sandwich) assay. The three basic components of a sandwich assay are: 1. An antigen large enough to allow two antibodies to bind concurrently on different binding sites. 2. A capture antiserum directed to one of the antigenic sites on the antigen. This antiserum is attached to a solid phase to permit immunologic extraction of the immune complexes. 3. A signal antiserum directed to a second antigenic site on the antigen. This antiserum is attached to an assay signal system. In contrast to competitive immunoassays, these assays use a large excess of antiserum binding sites compared with the concentration of antigen. The capture antibody immunoextracts the antigen from the sample and the signal antibody binds to

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Figure 6-4 A, Principles of immunometric assays. Ag, antigen; ATB1, capture antiserum; ATB2, signal antiserum. B, Typical dose-response curve. NSB, nonspecific binding.

the capture antibody-antigen complex to form a tertiary complex. As the antigen concentration increases, the signal increases progressively. Figure 6-4 schematically illustrates these concepts. The capture antiserum (ATB1) is attached to biotin (see solid circles). The signal antiserum (ATB2) is labeled with a detection system (see asterisks). The ATB1-antigen-ATB2 complexes are immunologically extracted using a streptavidin solid phase (see horizontal cups). After the complex is bound to the solid phase, most of the unbound signal antibody is washed away. As shown in Figure 6-4 , the signal increases progressively with the concentration. For lower concentrations, the signal generally increases proportional to the assay concentrations (after the offset caused by the NSB). At higher concentrations, the signal generally is less than proportional, so that nonlinear curve-fitting techniques are used to generate the dose-response curves. Again, the relative imprecision, expressed as a coefficient of variation, depends on the slope of the dose-response curve; consequently, the relative precision is less at higher concentrations. In immunometric assays, the background level of signal is associated with very low concentrations. This background signal is caused by the NSB. The analytic sensitivity of immunometric assays is related to the ratio of the true signal to the NSB signal. Therefore, assays can be made more sensitive either by increasing the response signal or by decreasing NSB. Inadvertent increases in NSB caused by specimen interference or reagent deterioration can significantly alter the assay performance. In immunometric assays, it is also important that a large excess of capture antibody be used. When the antigen concentration approaches the effective binding capacity of the capture antibody system, the signal no longer increases. If the antigen concentration exceeds the binding capacity of the capture antibody, the signal may actually decrease. Figure 6-5 illustrates this high-dose book effect for immunometric assays caused by insufficient amounts of capture or signal antiserum. [24] The signal increases progressively until the hormone concentration exceeds the binding capacity; the signal then decreases, apparently as a result of the removal of some of the weaker binding antigen-antibody complexes during the wash cycle on the assay. [25] [26] [27] This is a potentially dangerous phenomenon because very high concentrations can give the same "answer" as lower concentrations. If this artifact is suspected, the specimen can be diluted and reanalyzed. If the answer for the diluted specimen is higher than the original answer, a high-dose hook effect probably is present. Most manufacturers are aware of this potential problem and configure assays with relatively large amounts of capture antibody; however, some patients produce high concentrations of hormones or antigens that may exceed assay limits. Laboratories are able to detect this phenomenon by analyzing specimens at two dilutions, but this practice generally is not cost-effective. Therefore, feedback to the laboratory about results that are inconsistent with clinical findings is essential. Another potential problem for immunometric assays consists of endogenous heterophile antibodies that cross-react with reagent antiserum. [28] Normally, the signal antibody does not form a "sandwich" with the capture antibody unless the specific antigen is present; however, divalent heterophile antibodies may mimic the antigen by simultaneously binding to the signal and capture reagent antibodies. [29] [30] [31] Figure 6-6 schematically illustrates this situation. The problem is most common with monoclonal antibodies but may also occur with polyclonal antibodies. Immunoglobulins contain both a constant (Fc) region and a variable (Fab) region. As implied in the name, the Fc region is constant, or similar, for all immunoglobulins from that species. Therefore, if a patient receives immunotherapy or imaging reagents containing mouse immunoglobulin, they are likely to develop human antimouse antibodies (HAMAs) directed to the Fc fragment. [32] Some patients may develop heterophile antibodies after exposure to foreign proteins from domestic pets or food contaminants. When these endogenous antibodies are present in a patient's specimen, they may bridge across the reagent antibodies used

Figure 6-5 Immunometric "high-dose hook effect." The response signal reaches a maximum and then decreases when the antigen concentration exceeds the limit of the assay.

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Figure 6-6 Assay interferences caused by heterophile antibodies, which result in either false high or false low results. Ag, antigen; ATB1, capture antiserum; ATB2, signal antiserum.

in immunometric assays and may cause falsely high values. These antibodies also may bind to sites on the reagent antibodies, which sterically block the binding of the specific antigen and give falsely low test values. Most manufacturers include nonimmune immunoglobulin in the assays to help block these interferences; as with the high-dose hook effect, however, the amounts added are not always adequate and some patients with high titer antibodies thus may still show in vitro assay interference. The combined specificity of the two antibodies used in an immunometric assay can produce exquisitely sensitive and specific immunoassays. In the past, a common problem with early competitive immunoassays was cross-reactivity among the structurally similar gonadotropins: LH, follicle-stimulating hormone (FSH), TSH, and human chorionic gonadotropin (hCG). The subunits of each of these hormones are almost identical, and the subunits have considerable structural homology. Many individual antisera (especially polyclonal antisera) used for measuring one of these hormones may have cross-reactivity for the other gonadotropins. The cross-reactivity of a pair of antibodies is less than the cross-reactivity of each of the individual antibodies because any cross-reacting substance must contain both of the binding epitopes in order to simultaneously bind to both antibodies. For example, consider two antibodies for LH, each having 1% cross-reactivity with hCG. The cross-reactivity of the pair is less than the product of the two cross-reactivities or, in this case, less than 0.01%. Most current immunoassays for LH have cross-reactivity less than 0.01% because even this relatively low percentage of cross-reactivity would still cause significant assay interference in pregnant patients and patients with choriocarcinoma who have high hCG concentrations. Multiple forms of most hormones circulate in the blood. Some hormones (e.g., prolactin, growth hormone) circulate with macro forms, which can cause difficulty in their analysis if specimens are not pretreated. [7] For hormones composed of subunits (e.g., the gonadotropins), both the intact and the free subunits circulate in blood. Immunometric assays can be made specific for intact molecules by pairing an antibody specific for the - bridge site of the subunits with a second antibody specific for the subunit. Assays using these antibody pairs retain the two-antibody, low cross-reactivity needed for measuring gonadotropins and do not react with the free subunit forms of the hormones. The heterogenous specificity characteristics of immunoassays make calibration and harmonization difficult. Two immunoassays calibrated with the same reference preparation can give widely varying measurements on patient specimens. Consider the example of hCG in Table 6-1 . The three assays are calibrated TABLE 6-1 -- Effect of Immunoassay Specificity on Calibration of Human Chorionic Gonadotropin (hCG) Assay Assay 1 Assay 2 Specificity for intact hCG standard

Assay 3

100%

100%

100%

Cross-reactivity with free -hCG

0%

100%

200%

Value of specimen with no free -hCG, IU/L

10.0

10.0

10.0

Value of specimen with 10% free -hCG, IU/L

9.0

10.0

11.0

Value specimen with 50% free -hCG, IU/L

5.0

10.0

15.0

with a pure preparation of intact hCG, such as the WHO Third International Reference Preparation. [33] The three assays differ in their cross-reactivity with free -hCG (0, 100%, and 200%, respectively). These assays give identical measurements for a specimen containing only intact hCG but progressively disparate values as the percentage of free -hCG in the specimen increases. In reality, the standardization issue is much more complex because multiple forms of hormones (i.e., intact, free subunits, nicked forms, glycosylated forms, degradation products) circulate in patients and each assay has different cross-reactivities for these forms. [33]



Free (Unbound) Hormone Assays

Many hormones are tightly bound to specific plasma-binding proteins and loosely bound to albumin. The unbound (free) forms as well as some of the loosely bound forms are biologically active. Multiple methods are available to measure these free or biologically active forms of a hormone. Theoretically, the best procedure is direct measurement of the free hormone concentration after physical separation of free-form bound hormone by equilibrium dialysis, ultrafiltration, or gel filtration. Unfortunately, this method is difficult to perform and is thus not readily available and is subject to technical errors. The two major clinical applications for free hormone measurements are for thyroid hormones (thyroxine [FT 4 ] and triiodothyronine [FT 3 ] and steroids (testosterone and estradiol). Four techniques are commonly used to estimate free thyroid hormone concentrations: 1. Indirect index methods. The indirect indices involve two measurements: one for total hormone concentration and another for the thyroxine-binding globulin (TBG), followed by calculation of the ratio or a normalized index (FT4I or FT3I). These methods correct for routine changes in TBG associated with estrogen levels, but they may produce inappropriately abnormal values in patients with extreme variations in TBG levels found in patients with congenital disorders of the TBG gene, familial dysalbuminemic hyperthyroxinemia, thyroid hormone autoantibodies, and nonthyroidal illnesses. 2. Two-step labeled hormone methods. These methods immunologically bind the free and loosely bound thyroid hormone to a solid phase. The other serum components are washed away, and the residual binding sites are back-titrated with labeled hormone. When calibrated with appropriate serum standards, these methods are thought to pose fewer problems with binding protein abnormalities. 3. One-step labeled hormone analogue methods. These methods use synthetic analogues of T 4 and T3 that bind to the measurement 71

antibody but do not bind to normal TBG. These methods are seldom used because performance has been poor in patients with abnormal albumin concentrations, abnormal free fatty-acid concentrations, and all conditions that interfere with the indirect indices. [34] 4. Labeled antibody methods. These methods use kinetic reactions of antibodies with selected affinities that bind preferentially with the free form of the hormone. These methods work best for automated testing instruments and have become popular. Each of these methods works well for correcting for minor changes in TBG levels, but each has problems with some patient sera, especially those containing interfering substances such as inhibitors and heterophilic antibodies. Unfortunately, most manufacturers have not fully validated their methods in patients with these abnormalities. [35] Multiple methods are also available for measuring both the free and the biologically active forms of steroid hormones. The preferred method for measurement of free hormones consists of direct physical separation and high-sensitivity assays similar to those recommended for the thyroid hormones. One-step labeled hormone-analogue methods also have been developed, but these are associated with interference problems similar to the problems with free thyroid hormone assays. Another complexity in regard to steroid hormones is that in addition to the free hormones, testosterone and estrogen bound to albumin also are biologically active. The concentration of the biologically active forms can be estimated using indirect indices calculated from measurements of the total hormones and sex hormone-binding globulin (SHBG) or by measurement of the residual free and albumin-bound steroids after separation of the SHBG-bound forms after differential precipitation with ammonium sulfate. [36]



Chromatographic Assays

Another major method of measuring hormone concentrations involves chromatographically separating the various biochemical forms and quantitating specific characteristics of the molecules. High-performance liquid chromatography (HPLC) systems utilize multiple forms of detection, including light absorption, fluorescence, electrochemical properties, and mass spectrometry. [37] [38] There are two major advantages of these techniques: (1) they can be used to simultaneously measure multiple forms of an analyte, and (2) they are not dependent on unique immunologic reagents. Therefore, harmonization of measurements made with different assays is more feasible. The major disadvantages of these methods are their complexity and their limited availability. Many chemical separation techniques are based on chromatography, but the two most commonly used for liquid chromatography are (1) normal-phase HPLC and (2) reverse-phase HPLC. [25] In both systems, a bonded solid-phase column is made that interacts with the analytes as they flow past in a liquid solvent. In normal-phase HPLC, the functional groups of the stationary phase are polar (e.g., amino or nitrile ions) relative to the nonpolar stationary phase (e.g., hexane); in reverse-phase HPLC, a nonpolar stationary phase (e.g., C-18 octadecylsilane molecules bonded to silica) is used. More recently, polymeric packings made of mixed copolymers have been made with C4, C8, and C18 functional groups directly incorporated so that they are more stable over a wide pH range. The mobile and stationary phases are selected to optimize adherence of the analytes to the stationary phase. The adhered molecules can be eluted differentially from the solid phase after washing to separate specific forms of the analyte from interfering substances as follows: 1. When the composition of the mobile phase remains constant throughout the run, the process is called an isocratic elution. 2. If the mobile-phase composition is abruptly changed, a step elution occurs. 3. If the composition is gradually changed throughout the run, a gradient elution occurs. The efficiency of separation in a chromatography system is a function of the flow rates of the different substances. [39] The resolution of the system is a measure of the separation of the two solute bands in terms of their relative retention volumes (V r ) and their bandwidths (W). Resolution (R s ) of solutes A and B is shown as

Values of Rs less than 0.8 result in inadequate separation, and values greater than 1.25 correspond to baseline separation. The resolution of a chromatography column is a function of flow rates and thermodynamic factors. The simultaneous measurement of the three catecholamines (epinephrine, norepinephrine, and dopamine) can be performed with reverse-phase HPLC with a C-18 column and electrochemical detection system [40] or fluorometric detection. [41] Prior extraction by absorption on activated alumina and acid elution helps to improve specificity. Dihydroxybenzylamine, a molecule similar to endogenous catecholamines, can be used as an internal standard.



Mass Spectrometry

The technique of mass spectrometry involves fragmentation of target molecules, followed by separation and measurement of the mass to charge ratio of the components. [39] When coupled with liquid chromatography, a mass spectrometer can function as a unique detector to provide structural information about the composition of individual solutes. [42] Inclusion of internal standards in the specimens, which are molecularly similar to the measured compounds, allows precise quantitation of the concentration of the eluting analytes. The measurement of specific mass fragments makes possible the quantitation of multiple specific analytes in complex mixtures. A fundamental step in mass spectrometry is the fragmentation of the target compound into charged ions. Multiple techniques are used to generate these charged ions, including chemical ionization and electron-impact ionization. Chemical ionization uses reagent gas molecules, such as methane, ammonia, water, and isobutane, to transfer protons. This process produces less fragmentation than other techniques because the process is not highly excited. The electron impact bombards gas molecules from the sample, with electrons emitted from a heated filament. The process occurs in a vacuum to prevent the filament from burning out. Electron-spray ionization is a process in which a solution containing the analyte is introduced into a gas phase and is sprayed across an ionizing potential.[43] The charged droplets are desolvinated and analyzed in a mass spectrometer. A mass spectrum is a bar graph in which the heights of the bars correspond to the relative abundance of a particular ion plotted as a function of the mass/charge ratio. Modern mass spectrometers can measure molecular masses so accurately and precisely that the elemental composition of a compound can be predicted by comparison with stored spectral libraries. When these systems are used to measure only a few select compounds having known spectrums, the mass spectrometer can be programmed to focus only on these selected ions. Stable isotopes of the compounds of interest can be used as

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Figure 6-7 Mass spectrum illustrating the concurrent measurement of 10 cortisol-related compounds with one assay. cps, counts per second; LC, Supelcosil LC-18 column; MS/MS, tandem mass spectrometry. (Courtesy of Dr. R. Singh, Mayo Clinic.)

internal standards through a technique called isotope dilution mass spectrometry. Stable isotopes generally perform the same as the native compounds in terms of extraction, chromatography, and mass spectrometry and are thus ideal internal standards. However, they must have a sufficient number of isotopic atoms to ensure that their mass is different from naturally occurring substances that may be in the specimen. Tandem mass spectrometry (MS/MS) is a powerful new tool consisting of two mass analyzers separated by an ion-activation device. [44] [45] The first analyzer is used to isolate and dissociate the ion of interest by activation, and the second mass analyzer is used to analyze its dissociation products. This technique can be used to provide rapid, definitive measurements of multiple endocrine analytes. [42] For example, liquid chromatography and tandem mass-spectrometry can be used to simultaneously quantitate multiple glucocorticoid-related compounds. [46] [47] In Figure 6-7 , the chromatograph shows peaks for ten steroids that were first separated on reverse-phase liquid chromatography using a Supelcosil LC-18 column (Supelco, Bellefonte, Calif.) and a gradient elution of a 53% to 75% methanol/water mixture. The column eluate was fed directly into an electrospray ionization device in a triple-quadrapole mass spectrometer (API 3000, Perkin-Elmer Sciex, Foster City, Calif.). The stable isotopes were from Cambridge Isotope Laboratories (Andover, Mass.). A 10-minute analysis provides quantitation of the 10 compounds: cortisone, cortisol, 21-deoxycortisol, corticosterone, 11-deoxycortisol, androstenedione, deoxycorticosterone (DOC), 17-hydroxyprogesterone, progesterone, and pregnenolone. The sensitivity for cortisol using d 4 cortisol calibration is 0.1 µg/dL.



Nucleic Acid-Based Assays

The decoding of the human genome has set the stage for an enormous increase in nucleic acid-based gene assays. The basic principles of nucleic acid-based assays have been known for several decades, but the identification of specific genes and the mapping of gene defects to clinical disease states have now made these measurements clinically useful. [48] [49] Four concepts important for nucleic acid measurements are (1) hybridization, (2) amplification, (3) restriction fragment length polymorphisms (RFLPs), and (4) electrophoretic separation. [50] Hybridization

Nucleic acid molecules have a unique ability to fuse with complementary base-pair sequences. When a fragment of a known sequence (probe) is mixed under specific conditions with a specimen containing a complementary sequence, hybridization occurs. This feature is analogous to the antibody-antigen binding used in immunoassays. Many of the formats used for immunoassay have been adopted to nucleic acid assays, including some of the same signal systems (e.g., radioactivity, fluorescence, chemiluminescence) and the same solid-phase

73

capture systems (e.g., magnetic beads, biotin-streptavidin binding). In situ hybridization, which involves the binding of probes to intact tissue and cells, provides information about morphologic localization analogous to immunohistochemistry. Amplification

Nucleic acid assays have an advantage that low concentrations can be amplified in vitro prior to quantitation. The best known amplification procedure is the polymerase chain reaction (PCR), first reported by Mullis and Faloona. [51] The three steps in the process (denaturation, annealing, and elongation) occur rapidly at different temperatures. Each "cycle" of amplification can occur in less than 90 seconds by cycling the temperature. The target double-stranded DNA is denatured at high temperature to make two single-stranded DNA fragments. Oligonucleotide primers, which are specific for target region, are annealed to the DNA when the temperature is lowered. Addition of DNA polymerase allows the primer DNA to extend across the amplification region, thus doubling the number of DNA copies. At 85% to 90% efficiency, this process can amplify the DNA by about 250,000-fold in 20 cycles. This huge amplification is subject to major problems with contamination if special precautions are not taken. In one control technique, a psoralen derivative is used to prevent subsequent copying by polymerase during exposure to ultraviolet light. Restriction Fragment Length Polymorphisms

Some diseases (e.g., sickle cell anemia) are associated with a specific gene mutation; generally, however, a series of deletions and additions of DNA are involved with the disease. A number of restriction enzymes that cleave DNA at specific locations have been identified. Changes in the sequence of DNA result in different fragment lengths. This technique, or RFLP, is particularly helpful in family studies for disorders that have a unique genetic fingerprint. Electrophoretic Separation

E. M. Southern invented an electrophoretic separation technique known as Southern blotting.[52] Restriction enzymes are used to digest a sample of DNA into fragments, and the product is subjected to electrophoresis. The separated bands of DNA are then transferred to a solid support and hybridized. Northern blotting is a similar technique, in which RNA is used as the starting material. Western blotting refers to electrophoresis and transfer of proteins.



ANALYTIC VALIDATION Clinicians generally assume that laboratory methods have been validated and that they function correctly. Although this assumption is generally true, it is helpful to understand the level of assay validation performed and the appropriateness of the validation criteria for each clinical application of a test. [53] [54] In the United States, the federal government regulates all laboratories performing complex tests for patients receiving Medicare. [55] These regulations, published in the Federal Register, outline the validation requirements for both Food and Drug Administration (FDA)-approved instruments, kits, and test systems as well as methods developed in-house. Laboratories must document analytic accuracy, precision, reportable ranges, and reference ranges for all procedures. The regulations for in-house procedures and modifications of approved commercial procedures are more extensive and require laboratories to further document (1) analytic sensitivity; (2) analytic specificity, including interfering substances; and (3) other performance characteristics required for testing patient specimens. Although the details of method validation may be unique to a specific procedure, the following analytic validation studies have proved valuable for most procedures: (1) method comparison, (2) precision, (3) linearity, (4) recovery, (5) detection limit, (6) reportable range, (7) analytic interference, (8) carry-over, (9) reference interval, (10) specimen stability, and (11) specimen type. Laboratories should have documentation for each of these performance characteristics, either from the diagnostics manufacturer or from direct studies. Method Comparison

Ideally, the system should be compared with an established reference method; however, many endocrine tests do not have reference methods and many laboratories do not have the facilities to perform reference methods when they exist. As a minimum, the assay should be compared with an analytic system that has been clinically validated with specimens from healthy subjects and specimens from patients with the diseases being investigated. [56] The system should be traceable to established reference standards, such as those from the WHO and the National Institute of Standards and Technology (NIST). [57] [58] [59] Between 100 and 200 different specimens distributed over the assay range are recommended for method comparisons. [60] A cross-plot displaying the new method on the vertical axis versus the established method on the horizontal axis, along with the identity line, reference value lines, and regression statistics, is a useful way of displaying these comparisons. An alternative display method is the Bland-Altman difference plot, in which the difference between the test method and the reference method is plotted against the reference method values. Although acceptable performance criteria for method comparisons are not well established, some important characteristics to examine are as follows: 1. 2. 3. 4.

Any grossly discordant test values. The degree of scatter about the regression curve. The size of the regression off-set on the vertical axis. The number of points crossing between the low, normal, and high reference intervals for the two methods.

The European Union (EU) has enacted the In Vitro Diagnostics Directive, which requires manufacturers marketing in the EU after the year 2003 to establish that their products are "traceable to reference standards and reference procedures of a higher order" when these references exist. [61] This directive should serve to harmonize many test methods worldwide because most diagnostic companies market internationally. [62]



Precision

Precision is a measure of the replication of repeated measurements of the same specimen; it is a function of the time between repeats and the concentration of the analyte. Both short-term precision (within a run or within a day) and long-term precision (across calibrations and across batches of reagents) should be documented at clinically appropriate concentration levels. [63] In general, normal range, abnormally low range, and abnormally high range targets are chosen for precision studies; however, targets focused on critical medical decision limits may be more appropriate for some analytes. Twenty measurements are recommended at each level for both short-term and long-term precision validations. Precision generally is expressed as the

74

TABLE 6-2 -- Recommended Analytic Performance Limits Biologic CVi (%) Precision (%)

Analyte

Accuracy (%)

Calcium

1.8*

0.9

0.7*

Glucose

4.4*

2.2

1.9*

Thyroxine

7.6*

3.4

4.1*

Potassium

4.4*

2.4

1.6*

Triiodothyronine

8.7

4.0

5.5

Thyrotropin

20.2

8.1

8.9

Cortisol

15.2

(7.6)§

Estradiol

21.7

(10.9)

Follicle-stimulating hormone

30.8

(15.4)

Luteinizing hormone

14.5

(7.2)

Prolactin

40.5

(20.2)

Testosterone

8.3

(4.1)

Insulin

15.2

(7.6)

Dehydroepiandrosterone

5.6

(2.8)

11-deoxycortisol

21.3

(10.6) [ 65]

* Data from Stockl D, et al. Eur J Clin Chem Clin Biochem 1995; 33:157169. Data from Fraser CG. Arch Pathol Lab Med 1992; 116:916923. [66] Data from Fraser CG, et al. Eur J Clin Chem Clin Biochem 1992; 30:311317. [67] § Numbers in parentheses correspond to one-half of CVi (CVi = within individual coefficient of variation).

coefficient of variation, calculated as 100 times the standard deviation divided by the average of the replicate measurements.

[64]

There is no universal agreement on the performance criteria for analytic precision, although numerous recommendations have been put forth. Two major approaches to defining these criteria have been (1) comparison with biologic variation and (2) expert opinion of clinicians based on their perceived impact of laboratory variation on clinical decisions. The total variation clinically observed in test measurements is a combination of the analytic and biologic variations, for instance: 1. If the analytic standard deviation (SD) is less than one-fourth of the biologic SD, the analytic component increases the SD of the total error by less than 3%. 2. If the analytic precision is less than one-half of the biologic SD, the total error increases by only 12%. These observations have led to recommendations for maintaining precision less than one quarter or one-half of the biologic variation. The expert opinion precision recommendations are based on estimates of the magnitude of change of a test value that would cause clinicians to alter their clinical decisions. Table 6-2 lists some precision recommendations for selected endocrine tests. [65] [66] [67]



Linearity

Patient specimens commonly contain several different forms of the hormones to be measured compared with the pure form contained in the reference standards and calibrators used to establish the assay dose-response curve. [68] When a patient specimen is diluted, the measured value for these dilutions should parallel the dose-response curve and give results proportional to the dilution. Linearity can be evaluated by measuring serial dilutions of patient specimens with high concentrations diluted in the appropriate assay diluent. [69] [70] The product of the measured value multiplied by the dilution factor should be approximately constant. There are no performance standards for linearity, but a reasonable expectation for most hormones is that dilutions are comparable within 10% of the undiluted value.



Recovery

Two methods of assessing the recovery of assays are (1) measuring the increase in test values after the reference analyte is added and (2) measuring the proportional changes caused by mixing high-concentration and low-concentration specimens. Some analytes circulate in the blood in multiple forms, and some of these forms may be bound to carrier proteins. The recovery rate of pure substances added to a specimen may be low if the assay does not measure some of the bound forms. Mixtures of patient specimens may not be measured correctly if one of the specimens contains cross-reacting substances such as autoantibodies. A thorough understanding of the chemical forms of the analyte and their cross-reactivities in the assay is important during assessment of recovery data.



Detection Limit

The minimal analytic detection limit is the smallest concentration that can be statistically differentiated from zero. This concentration is mathematically determined as the upper 95% limit of replicate measurements of the zero standard, calculated from the average signal plus 2.0 SD. This minimal detection limit is valid only for the average of multiple replicate measurements. When individual determinations are performed on a specimen having a true concentration exactly at the minimal detection limit, the probability that the measurement is above the noise level of the assay is only about 50%. A second term for the lowest level of reliable measurement for an assay is the functional detection limit, or the limit of quantitation. For this parameter to be measured, multiple pools with low concentrations are made and analyzed in the replicate. A cross-plot of the coefficient of variation of the measurements versus the concentration allows one to generate a precision profile. The concentration corresponding to a coefficient of variation of 20% is the functional detection limit. [1] This term generally applies to across-assay variation, but it also can be calculated using within-assay variation if one uses the tests to evaluate results measured within one run (e.g., provocative and suppression tests).



Reportable Range

The reportable range of an assay generally spans from the functional detection limit to the concentration of the highest standard. Values above the highest standard may be reported if they are diluted and the measured value is multiplied by the dilution factor. The validity of the analytic range is documented by the linearity and recovery studies. Some laboratories erroneously report the exact values displayed by the test systems even if they are outside of the analytic range. Therefore, it is important for clinicians to understand the limitations of valid measurements and not inappropriately use meaningless numbers that may be reported. Another potential source of error is failure of the technologist to multiply the measured value of diluted specimens by the dilution factor to correct for the dilution. In addition, care should be taken to define the number of significant figures used for reporting test values and to establish an appropriate algorithm for rounding test values to the significant number of digits.



Analytic Interference

The cross-reactivity and potential interference of other analytes that may react in a test system should be documented.

[71]

The

75

Figure 6-8 Flow diagram illustrating appropriate preanalytic conditions for measuring plasma catecholamines. EDTA, edetate.

choice of potential interfering substances that must be evaluated requires an understanding of both the analytic system and the pathophysiology of the analyte being evaluated. In immunoassays, for example, compounds with similar structures as well as precursor forms and degradation products should be tested. [72] [73] [74] Drugs commonly prescribed for the diseases under evaluation should be assessed for interference both by addition of the drug to a specimen and by analysis of specimens from patients before and after receiving the drug. [75] [76] [77] Most assays also are evaluated for the effects of hemolysis, lipemia, and icterus.



Carry-over Studies

Many diagnostic systems use automated sample-handling devices. If a specimen to be tested is preceded by a specimen with a very high concentration, a trace amount of the first specimen may significantly increase the reported concentration of the second specimen. The choice of the concentration that should be tested for carry-over depends on the pathophysiology of the disease, but high values may need to be tested because some endocrine disorders may produce these high values. A prudent procedure would be to retest all specimens following a specimen with an extraordinarily high value. One also should document that carry-over from the sampling probe has not inadvertently contaminated subsequent specimen vials, thereby invalidating subsequently repeated measurements.



Reference Intervals

The development and validation of reference intervals for endocrine tests can be a very complex task. [78] [79] The normal reference interval for most laboratory tests is based on estimates of the central 95 percentile limits of measurements in healthy subjects. [80] A minimum of 120 subjects is needed to reliably define the 2.5 and 97.5 percentiles. The reference intervals for many endocrine tests depend on gender, age, developmental status, and other test values. Formal statistical consultation is recommended to determine the appropriate number of subjects to test and to develop statistical models for defining multivariate reference ranges. Full evaluation of the adrenal, gonadal, and thyroid axes requires simultaneous measurement of the trophic and target hormones. Bivariate displays of these hormone concentrations along with their multivariate reference intervals facilitate the interpretation. [81] Preanalytic conditions should be well defined and controlled during evaluation of both healthy reference subjects and patients. Figure 6-8 shows a recommended protocol to control preanalytic conditions for collection of plasma catecholamine specimens.



Specimen Stability

Analyte stability is a function of storage conditions and specimen type. [82] Although most hormones are relatively stable in serum or urine if they are rapidly frozen and stored in hermetically sealed vials at -70°C, multiple freeze/thaw cycles may damage analytes, and storage in frost-free freezers that repeatedly cycle through thawing temperatures can adversely affect stability. Blood specimens collected in edetate (EDTA) often are more stable than serum or heparinized specimens because edetate chelates calcium and magnesium ions, which function as coenzymes for some proteases. The addition of protease inhibitors (e.g., aprotinin) to blood specimens may also improve specimen stability. [83]



Types of Specimens

Most hormones are measured in blood or urine, but alternate testing sources, such as saliva and transdermal membrane monitors, are also used. Urine Specimens

The 24-hour urine specimen is used for many endocrine tests. Urine specimens represent a time average that integrates over the multiple pulsatile spikes of hormone secretion occurring throughout the day. The 24-hour urine specimen also has the advantage of better analytic sensitivity for some hormones. [84] [85] Urine often contains not only the original hormone but also key metabolites that may or may not have biologic activity. Drawbacks include the inconvenience of and delays in collecting the 24-hour specimen. Another limitation of urine specimens is the uncertainty of the completeness of the collection. Measurement of urinary creatinine concentrations helps in monitoring collection completeness, especially when it is compared with the patient's muscle mass. Many urinary hormones are conjugated to carrier proteins before excretion. Therefore, both hepatic function and, to a lesser degree, renal function may alter urinary hormone values. Blood Specimens

Blood specimens have both the advantage and the limitation of time dependency. The ability to direct rapid changes to a provocative stimulus is a strong advantage, whereas the unsuspected changes due to pulsatile secretions may be a major limitation. Most hormones undergo significant biologic variations, including ultradian, diurnal, menstrual, and seasonal changes. [86] [87] [88] Many hormones have short half-lives and are thus rapidly cleared from the blood. The half-life is particularly important when one is attempting to measure the response to a provocative drug, such as the effect of gonadotropin-releasing hormone (GnRH). [89] The development of rapid intraoperative methods for measuring PTH and growth hormone has highlighted the importance of plasma specimens, which do not require extra waiting time for the blood to clot to make serum. [90] [91]

76

Saliva Specimens

Saliva has been used to measure some hormones. Methods of stimulation, collection, and storage of saliva should be standardized in order to ensure that the measurements are reproducible and meaningful. [92] [93] Saliva measurements correlate with blood measurements in some hormones like cortisol, progesterone, estradiol and testosterone but do not correlate well for others, (e.g., thyroid and pituitary hormones). [94] [95] [96] Unconjugated steroid hormones enter saliva by diffusion, and their concentrations are relatively independent of the rate of saliva production. The saliva concentration of conjugated steroids, thyroxine, chorionic gonadotropin, and many protein hormones generally do no correlate well with plasma concentrations. [97] Blood Drops

Blood drops collected on filter paper from punctures of a finger or heel are a convenient system for collecting, transporting, and measuring hormones. [98] [99] If standardized collection conditions and extraction techniques are used, these measurements correlate well with serum measurements. Integration of immunochemistry with computer chip technology has also led to immunochips that can measure multiple analytes using a single drop of blood. [100] Noninvasive Measurements

Noninvasive transcutaneous measurements also have been developed for some endocrine tests. [101] Transcutaneous glucose measurements using near-infrared spectroscopy correlate well with blood measurements. [102] The GlucoWatch device is also being marketed for noninvasive monitoring of glucose. [103]



QUALITY ASSURANCE Quality Control Systems

Laboratory quality control programs are intended to ensure that the test procedures are being performed within defined limits. A critical component of control systems is the definition of acceptable performance criteria. [104] [105] Unfortunately, these criteria often are not well defined and many laboratories use floating criteria that change when assays change.[106] Control limits are often set at the mean ±2 or 3 SDs, where the mean and SD are arbitrarily assigned based on measurements made in that laboratory. When reagents or equipment change, new limits are assigned. These types of control systems provide some assurance that the laboratory is functioning at a level of performance similar to that of the recent past, but they provide little assurance that measurements are adequate for clinical decisions. Statistically, there are two major forms of analytic errors: random and systematic. Random error relates to reproducibility; systematic error relates to the offset or bias of the test values from the target or reference value. Performance criteria can be defined for each of these parameters, and quality control systems can be programmed to monitor compliance with these criteria. Control systems must have low false-positive rates as well as high statistical power to detect assay deviations. The multirule algorithms developed by Westgard and colleagues [107] use combinations of control rules, such as two consecutive controls outside of warning limits, one control outside of action limits, or moving average trend analyzers outside of limits to achieve good statistical error detection characteristics. [108]

Figure 6-9 Effect of analytic bias, or shift, on the number of patients with elevated levels of thyrotropin (TSH).

Traditionally, quality control programs have focused primarily on precision; however, analytic bias also can cause major clinical problems. When fixed decision levels are used to trigger clinical actions, such as therapy and additional investigations, changes in the analytic set-point of an assay can cause major changes in the number of follow-up cases. [108] This concept is illustrated in Figure 6-9 for TSH measurements. Under stable laboratory testing conditions, approximately 122 per 1000 patients tested have TSH values above 5.0 mIU/L. If the test shifts upward by 20%, the number of patients with TSH values above 5.0 mIU/L increases to 189, which equates to more than a 50% increase in the number of patients flagged as abnormal. These changes in test value distributions can often be sensed by clinicians who encounter multiple patients with unexpected elevated test values, causing them to call the laboratory and inquire whether the "test is running high today." Some modern quality control systems use moving averages of patient test values to help monitor changes in analytic bias. [109] Some medical facilities are linking together into networks to provide more integrated patient care. This crossover of both physicians and patients is increasing the importance of harmonized testing systems. For endocrine tests, harmonization is best achieved when all the laboratories in the network use the same test systems. Differences in analytic specificity may cause across-method differences in patient test distributions even when the methods use the same reference standard. Full harmonization of testing requires not only standardization of equipment but also standardization of reagents (including using the same lot numbers) and standardization of laboratory protocols. Real-time quality control monitors with peer group comparisons across the laboratories in the health care network are necessary to ensure uniformity of testing.



Investigation of Discordant Test Values

The practice of modern endocrinology depends extensively on reliable and accurate test values; even in the best laboratories, however, erroneous results sometimes are reported. Careful correlation of pathophysiology with test values can help to identify values that are "discordant." [81] Some of these discordant test values may be analytically correct, but others may be erroneous. Clinicians can help investigate these suspicious test values by requesting laboratories to perform a few simple validation procedures. Repeated testing of the same specimen is a valuable first step. If the specimen has been stored under stable conditions, the absolute value of the difference between the initial and the

77

Figure 6-10 Nonproportional dilutions. Discordant values are produced when samples do not dilute linearly. (Nt = undiluted [neat].)

repeated measurements should be less than 3 analytic SDs 95% of the time. Normally, the 95% confidence range is associated with the mean ±2 SDs; with repeated laboratory tests, however, errors are associated with the first as well as the second measurement. The confidence interval for the uncertainty of the difference between two measurements can be calculated using the statistical rules for propagation of errors. To better understand this propagation of error, consider that D = X 1 X2 where X1 is the first measurement, X2 is the repeated measurement, and D is the difference. The variance of D is the sum of the variance of X1 and the variance of X2 . The SD of D is the square root of the variance of D, or the square root of twice the variance of X1 . The SD of D equates to 2 multiplied by the SDs of S. Therefore, 95% of the absolute values for D should be within 22 SD(X), or approximately 3 SD(X). If a repeat measurement exceeds this 3 SD(X) limit, the initial (or reagent) measurement is probably in error. Linearity and recovery are valuable techniques for evaluating test validity. If the initial test value is elevated, serially diluting the specimen in the assay diluent and reassaying should be considered. If the specimen dilutes nonproportionally (Fig. 6-10) , no meaningful value can be reported with that assay. In the example, the undiluted specimen reads 22, the twofold dilution multiplies back to 34 (2 × 17), and the fourfold dilution multiplies back to 60 (4 × 15). Therefore, the result depends on the dilution factor, so that no reliable answer can be reported. If the initial value is low, one may consider adding known quantities of the analyte to part of the specimen. Analyzing these spiked or diluted specimens with the original specimen allows one to evaluate both reproducibility and recovery. It may be helpful to analyze the linearity or recovery of the assay standards at the same time to provide internal controls of the dilution or spiking procedures and the appropriateness of the diluent and spiking material. If the replication, dilution, or recovery experiment appears successful, further analytic troubleshooting will vary according to the method used. Immunoassays may be affected by interference caused by heterophile antibodies. Addition of nonimmune mouse serum or heterophile antibody-blocking solutions may neutralize these effects. [110] [111] Chromatographic assays are usually more robust than immunoassays. Specimens with suspected interference on one type of assay can be reanalyzed by means of an alternative methodology. Water-soluble interferences have been reported for some direct assays for steroid measurements. [18] [19] [112] Extraction of the hormones into organic solvents, followed by drying down and reconstitution in the assay zero standard, removes these interferences. Similarly, interferences with cross-reacting drugs and metabolic products can be minimized with selective extraction.



Summary The analytic methods of assessing endocrine problems in patients are continually expanding. The newer systems are often based on analytic techniques similar to those outlined in this chapter, but the configurations are generally more user-friendly. These advances make the systems more convenient, but they also become more of a "black box" that conceals most of the details of the system. The performance validation steps outlined in this chapter become important procedures for ensuring that these systems continue to provide the reliable measurements needed for quality medical care.



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Section 2 - Hypothalamus and Pituitary

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Chapter 7 - Neuroendocrinology Roger D. Cone Malcolm J. Low Joel K. Elmquist Judy L. Cameron

HISTORICAL PERSPECTIVE The field of neuroendocrinology can be most broadly defined as the interaction between the central nervous system (CNS) and endocrine systems in the control of homeostasis. Much of this field, of course, has focused on the control of pituitary hormone secretion by the hypothalamus. At the beginning of the 21st century, we now appreciate the fundamental role of the hypothalamus in controlling anterior pituitary function. It is noteworthy that this concept is relatively recent, although the intimate interaction of the hypothalamus and the pituitary gland has been appreciated for some time. [1] [2] [3] For example, at the end of the 19th century, clinicians including Alfred Fröhlich described an obesity and infertility condition (often referred to as the adiposogenital dystrophic syndrome) in patients with pituitary tumors. This condition subsequently became known as Fröhlich's syndrome and was most often associated with pituitary tumors and the accumulation of excessive subcutaneous fat and hypogonadism. [4] [5]

[ 4]

Whether this syndrome was due to injury to the pituitary gland itself or to the overlying hypothalamus was extremely controversial, however. Several leaders in the field of endocrinology, including Cushing and his colleagues, argued that the syndrome was due to disruption of the pituitary gland. [3] [6] [7] However, experimental evidence began to accumulate that the hypothalamus was somehow involved in the control of the pituitary gland. For example, Aschner [8] demonstrated in dogs that the precise removal of the pituitary gland without damage to the overlying hypothalamus did not result in obesity. Later, seminal studies by Hetherington and Ranson, using a stereotaxic apparatus, demonstrated that destruction of the medial basal hypothalamus with electrolytic lesions, without damage to the pituitary gland, resulted in morbid obesity and neuroendocrine derangements similar to those of the patients described by Fröhlich. [8] [9] This and subsequent studies clearly established that an intact hypothalamus is required for normal endocrine function. However, the mechanisms by which the hypothalamus was involved in endocrine regulation remained unsettled for years to come. We now know that the phenotypes of Fröhlich's syndrome and the ventromedial hypothalamic lesion syndrome are probably due to destruction of key hypothalamic neurons that respond to key metabolic signals including leptin [10] (see later). The field of neuroendocrinology took a major step forward when it was recognized by several groups, especially Ernst and Berta Scharrer, that neurons in the hypothalamus were the source of the axons that constitute the neural lobe (see "Neurosecretion"). The hypothalamic control of the anterior pituitary gland remained unclear, however. For example, Popa and Fielding [11] are credited with the identification of the pituitary portal vessels linking the median eminence of the hypothalamus and the anterior pituitary gland. Although they appreciated the fact that this vasculature provided a link between hypothalamus and pituitary gland, they hypothesized at the time that blood flowed from the pituitary up to the brain. Anatomic studies by Wislocki and King [12] supported the concept that blood flow was from the hypothalamus to the pituitary. Later

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studies, including the seminal work of Geoffrey Harris, [13] [14] [15] established the flow of blood from the hypothalamus at the median eminence to the anterior pituitary gland. This supported the concept that the hypothalamus controlled anterior pituitary gland function indirectly and led to the now accepted hypophyseal-portal chemotransmitter hypothesis. [1] [16] Later, several important studies, especially those from Schally and colleagues [17] [18] and the Guillemin group, [19] [20] [21] established that the anterior pituitary is tightly controlled by the hypothalamus. Both groups identified several putative peptide hormone releasing factors (see later sections). These fundamental studies resulted in the awarding of the Nobel Prize to Andrew Schally and Roger Guillemin. Of course, we now know that these releasing factors are the fundamental link between the CNS and the control of endocrine function. It is also established that these peptide hormones are highly conserved across species and are essential for reproduction, growth, and metabolism. The anatomy, physiology, and genetics of these factors constitute a major portion of this chapter. Over the past two decades, work in the field of neuroendocrinology has continued to advance across several fronts. Cloning and characterization of the specific G proteincoupled rereceptors used by the hypothalamic releasing factors [22] [23] [24] [25] have helped define signaling mechanisms utilized by the releasing factors. Furthermore, characterization of the distribution of these receptors has, in every case, demonstrated receptor expression in the brain and in peripheral tissues other than the pituitary, arguing for multifactorial roles for these factors. Finally, the last two decades have also seen tremendous advances in our understanding of both regulatory neuronal and humoral inputs to the hypophyseotropic neurons. The adipostatic hormone leptin, discovered in 1994, [26] is an example of a humoral factor that has profound effects on multiple neuroendocrine circuits as the factor that suppresses the thyroid and reproductive axes during the starvation response. The subsequent discovery of ghrelin, [27] [28] a stomach peptide that regulates appetite and also acts on multiple neuroendocrine axes, demonstrates that much remains to be learned regarding the regulation of the hypothalamic releasing hormones. Traditionally, it has been extremely difficult to study the regulation of releasing factor gene expression or the specific regulation of the releasing factor neurons as a consequence of their small numbers and, in some cases, diffuse distribution. Transgenic experiments have resulted in the production of mice in which expression of fluorescent marker proteins has been specifically targeted to gonadotropin-releasing hormone (GnRH) neurons [29] [30] and arcuate pro-opiomelanocortin (POMC) neurons.[31] This technology will allow detailed study of important neuroendocrine neurons in the more native context of slice preparations or organotypic cultures. For example, investigators have already used this method to characterize directly the electrophysiologic properties of individual GnRH neurons. As just described, much of the field of neuroendocrinology has focused on hypothalamic releasing factors and their control of reproduction, growth, development, fluid balance, and the stress response through their control of pituitary hormone production. More broadly, however, neuroendocrinology has become a rubric to define the study of interaction of the endocrine and nervous systems in the regulation of homeostasis. The rubric of neuroendocrinology has been further expanded, however, because many areas of basic research have often been fundamental to understanding the neuroendocrine system and thus championed by scientists in the field. These areas include studies of neuropeptide structure, function, and mechanism of action; neural secretion; hypothalamic neuroanatomy; G proteincoupled receptor structure, function, and signaling; transport of substances into the brain; and the action of hormones on the brain. Many homeostatic systems involve integrated endocrine, autonomic, and behavioral responses. Thus, many homeostatic systems exist in which the classical neuroendocrine axes are important but not autonomous pathways, such as energy homeostasis and immune function, and these subjects are also often studied in the context of neuroendocrinology. This chapter first presents the concepts of neural secretion, the neuroanatomy of the hypothalamic-pituitary unit, and the CNS structures most relevant to the control

of the neurohypophysis and hypophysis. The chapter then covers each classical hypothalamic-pituitary axis, followed by two homeostatic systems, energy homeostasis and immune function, which are heavily integrated with neuroendocrine function. Finally, the chapter reviews the pathophysiology of disorders of neural regulation of endocrine function.



NEURAL CONTROL OF GLANDULAR SECRETION A fundamental principle of neuroendocrinology is the concept of regulated secretion of hormones, neurotransmitters, or neuromodulators by secretory cells. [16] [32] Endocrine cells and neurons are prototypical secretory cells, and both are characterized by the ability to be stimulated to cause the release of their products. In addition, secretory cells exist that can be broadly classified by their mechanisms of secretion. For example, endocrine cells secrete their contents directly into the blood stream, allowing these substances to act globally as hormones. In contrast, secretory cells in exocrine glands secrete substances into ductal systems. Cells classified as paracrine secrete their contents and affect the function of cells in the immediate vicinity. Similarly, autocrine secretory cells affect their own function by the local actions of their own secretions. Neurosecretion

Neurons are specialized secretory cells that send their axons throughout the nervous system to release their neurotransmitters and neuromodulators into chemical synapses.[33] A specialized subset of neurons are the neurohumoral or neurosecretory cells. Two examples of neurosecretory cells are neurohypophyseal and hypophyseotropic cells. [34] The prototypical neurohypophyseal cells are the magnocellular neurons of the paraventricular and supraoptic nuclei in the hypothalamus. Hypophyseotropic cells are neurons that secrete their products into the pituitary portal vessels at the median eminence (Fig. 7-1) (see later). In the most basic sense, neurosecretory cells are neurons that secrete substances directly into the blood stream to act as hormones. This concept of release is often referred to as neurosecretion (Fig. 7-2) . The theory of neurosecretion evolved from the seminal work of Scharrer and Scharrer, [3] [32] [35] [36] who used morphologic techniques to identify stained secretory granules in the supraoptic and paraventricular hypothalamic neurons. They found that cutting the pituitary stalk led to an accumulation of these granules in the hypothalamus. [32] [35] These findings led them to hypothesize that the source of substances secreted by the neural lobe (posterior pituitary) was hypothalamic neurons. Of course, we now know that the axon terminals in the neural lobe arise from the supraoptic and paraventricular magnocellular neurons that contain oxytocin and arginine vasopressin (AVP). The modern definition of neurosecretion has evolved to include the release of any neuronal secretory product from a neuron. Indeed, a basic principle of neuroscience is that all neurons in the CNS, including neurons that secrete AVP and oxytocin in the neural lobe, receive multiple synaptic inputs largely onto their dendrites and cell bodies. In addition, neurons

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Figure 7-1 Three types of hypothalamic neurosecretory cells. Left, A magnocellular neuron that secretes arginine vasopressin or oxytocin (AVP, OXY). The cell body, which is located in the supraoptic or paraventricular hypothalamic nucleus (SON, PVH), projects its neuronal process into the neural lobe, and neurohormone is released from nerve endings. Center, Similar peptidergic neurons are located in the medial basal hypothalamus in nuclear groups including the PVH and arcuate nucleus of the hypothalamus (Arc). The neuropeptides in this case are released into the specialized blood supply to the pituitary to regulate its secretion. Similar in plan are neurosecretory neurons that terminate in relation to another neuron (right). These projection neurons are found in sites including the PVH, Arc, and lateral hypothalamic area (LHA) that project to autonomic preganglionic neurons in the brain stem and spinal cord. Such substances act as neurotransmitters or neuromodulators. ACTH, corticotropin; CART, cocaine and amphetamineregulated transcript; CRH, corticotropin-releasing hormone; FSH, follicle-stimulating hormone; GH, growth hormone; GHRH, growth hormonereleasing hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; MCH, melanin-concentrating hormone; ORX, orexin-hypocretin; POMC, pro-opiomelanocortin; TRH, thyrotropin-releasing hormone; TSH, thyrotropin.

have the basic ability to respond and integrate input from multiple neurons through specific receptors. [33] [34] They in turn fire action potentials that result in the release of neurotransmitters and neuromodulators into synapses formed with postsynaptic neurons. The vast majority of communication between neurons is accomplished by "classical" neurotransmitters (e.g., glutamate, -aminobutyric acid [GABA], acetylcholine) and neuromodulators (e.g., neuropeptides) acting at chemical synapses (see Fig. 7-2) .[33] [34] [37] [38] Thus, neurosecretion represents a fundamental concept in understanding the mechanisms used by the nervous system to control behavior and maintain homeostasis. In the era of the elucidation of the human genome, the importance of these early observations is often not fully appreciated. However, accounts of these early studies are illuminating. [3] Moreover, it is not an overstatement that the confirmation of the neurosecretion hypothesis represented one of the major advances in the field of neuroscience and neuroendocrinology. Indeed, this and other early experiments, including the pioneering work of Geoffrey Harris, [13] [15] [39] led to the fundamental concept that the hypothalamus releases hormones directly into the blood stream (neurohypophyseal cells). These observations provided the principles on which the modern discipline of neuroendocrinology is built.



The Autonomic Nervous System Contribution to Endocrine Control

One of these fundamental principles of neuroendocrinology is that the nervous system controls or modifies, or both, the

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Figure 7-2 Neurobiologic features of the peptidergic neuron. Neurosecretory neurons can be regarded as having secretory functions that are in many ways analogous to those of glandular cells. A secretory product, which is formed on the endoplasmic reticulum under the direction of messenger ribonucleic acid, is packaged in granules and transported along the axon by axoplasmic flow to reach nerve terminals, where the granules are released. Virtually all neurons carry out similar functions. Some secrete neurotransmitters, such as acetylcholine or norepinephrine; others, such as motor nerves, secrete acetylcholine and myotropic factors. In all neurons there is a constant orthograde (forward) flow of cytoplasm and formed elements such as mitochondria. Retrograde flow also takes place to bring substances that enter nerve endings back to the body of the cell. In typical neurotransmitter neurons, the neurotransmitters are synthesized by enzymes and are packaged into secretory granules. These granules are transported in a manner similar to that of neuropeptide-containing granules. In many neurons cosecretion of one or two peptides may occur in association with secretion of a classic neurotransmitter. (From Reichlin S. Summarizing comments. In Gotto AM Jr, Peck EJ Jr, Boyd AE III, et al [eds]. Brain Peptides: A New Endocrinology. New York, Elsevier/North-Holland, 1979, pp 379403.)

function of both endocrine and exocrine glands. The exquisite control of the anterior pituitary gland is accomplished by the release of releasing factor hormones (see later). Other endocrine and exocrine organs (e.g., pancreas, adrenal, pineal, salivary glands) are also regulated through direct innervation from the cholinergic and noradrenergic inputs from the autonomic nervous system. [40] [41] Although it is beyond the scope of this chapter, an appreciation of the functional anatomy and pharmacology of the parasympathetic and sympathetic nervous systems is fundamental in understanding the neural control of endocrine function. The efferent arms of the autonomic nervous system comprise the sympathetic and parasympathetic systems. Both limbs are a classical two-neuron chain. Both are characterized by a preganglionic neuron that innervates a postganglionic neuron that targets an end organ. Preganglionic and postganglionic parasympathetic neurons are cholinergic. In contrast, preganglionic sympathetic neurons are cholinergic and postganglionic neurons are noradrenergic (except for those innervating sweat glands, which are cholinergic). [40] [41] Another basic concept is that autonomic neurons coexpress several neuropeptides. This coexpression is a common feature in neurons in both the central and peripheral nervous systems. [37] [38] [42] For example, postganglionic noradrenergic neurons coexpress somatostatin and neuropeptide Y (NPY). Postganglionic cholinergic neurons coexpress neuropeptides including vasoactive intestinal polypeptide and calcitonin gene-related peptide. The majority of the sympathetic preganglionic neurons lie in the intermediolateral cell column in the thoracolumbar regions of the spinal cord. [40] [41] Most postganglionic neurons are located in sympathetic ganglia lying near the vertebral column (e.g., sympathetic chain and superior cervical ganglia). Postganglionic fibers, in turn, innervate target organs. Thus, as a rule, sympathetic preganglionic fibers are relatively short and the postganglionic fibers are long. In contrast, the parasympathetic preganglionic neurons lie in the midbrain (Edinger-Westphal nucleus of the third cranial nerve), the medulla oblongata (e.g., dorsal motor nucleus of the vagus and nucleus ambiguus), and the sacral spinal cord. Postganglionic neurons that innervate the eye and salivary glands arise from the ciliary, pterygopalatine, submandibular, and otic ganglia. Postganglionic neurons in thorax and abdomen typically lie in the target organs including the gut wall and pancreas.[40] [41] Thus, preganglionic neurons are relatively long and the postganglionic fibers are short. The importance of coordinated neural control of endocrine organs is illustrated by the innervation of the pancreas. The endocrine pancreas receives both parasympathetic (cholinergic) innervation and sympathetic (noradrenergic) innervation. [40] [41] [43] [44] [45] The cholinergic innervation is provided by the vagus nerve (dorsal motor nucleus of the vagus). The activity in this innervation is an excellent example of neural modulation as it is clear that the secretory activity of insulin-producing beta cells is affected by the cholinergic tone of the beta cell. [43] [44] For example, vagal input is thought to modulate insulin secretion before (cephalic phase), during, and after ingestion of food. [46] In addition, noradrenergic stimulation of the endocrine pancreas can alter the secretion of glucagon and inhibits insulin release.[43] [44] It should be noted, of course, that a major regulator of insulin secretion by beta cells is glucose concentrations. [47] In fact, glucose can induce insulin secretion in the absence of neural input. However, the exquisite control by the nervous system is illustrated by the fact that populations of neurons in the brain stem and hypothalamus, like the beta cell, have the ability to sense glucose levels in the blood stream. [45] [48] This information is integrated by the hypothalamus and ultimately results in alterations in the activity of the autonomic nervous system innervating the pancreas. Thus, neural control of the endocrine pancreas probably contributes to the physiologic control of insulin secretion and may contribute to the pathophysiology of disorders such as diabetes mellitus. Certainly, an increased understanding of this complex interplay between the CNS and endocrine function is needed to diagnose and clinically manage endocrine disorders.



HYPOTHALAMIC-PITUITARY UNIT The hypothalamus is one of the most evolutionarily conserved and essential regions of the mammalian brain. Indeed, the hypothalamus is the ultimate brain structure that allows mammals to maintain homeostasis, and destruction of the hypothalamus is not compatible with life. [2] [49] [50] Hypothalamic control of homeostasis stems from the ability of this collection of neurons to orchestrate coordinated endocrine, autonomic,

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and behavioral responses. A key principle is that the hypothalamus receives sensory inputs from the external environment (e.g., light, pain, temperature, odorants) and information regarding the internal environment (e.g., blood pressure, blood osmolality, blood glucose levels). In addition, of particular relevance to neuroendocrine control, hormones (e.g., glucocorticoids, estrogen, testosterone, thyroid hormone) exert negative feedback directly on the hypothalamus. [16] [49] [51] [52] These sensory and hormonal cues are examples of a fundamental concept of neuroendocrinology: the hypothalamus integrates sensory and hormonal inputs and provides coordinated responses through motor outputs to key regulatory sites. These include the anterior pituitary gland, the posterior pituitary gland, the cerebral cortex, premotor and motor neurons in the brain stem and spinal cord, and autonomic (parasympathetic and sympathetic) preganglionic neurons. The patterned hypothalamic outputs to these effector sites ultimately result in coordinated endocrine, behavioral, and autonomic responses that maintain homeostasis. The focus of this section, the hypothalamic control of the pituitary gland, is an exquisitely controlled system and underlies the ability of mammals to coordinate endocrine functions that are necessary for survival. Anatomy of the Hypothalamic-Pituitary Unit

The pituitary gland is regulated by three interacting elements: hypothalamic inputs (releasing factors or hypophyseotropic hormones), feedback effects of circulating hormones, and paracrine and autocrine secretions of the pituitary itself. [16] [51] In humans, the pituitary gland (hypophysis) can be divided into two major parts, the adenohypophysis and the neurohypophysis. The adenohypophysis in turn can be subdivided into three distinct lobes, the pars distalis (anterior lobe), pars intermedia (intermediate lobe), and pars tuberalis (Fig. 7-3) .[53] [54] [55] Whereas a well-developed intermediate lobe is found in most mammals, only rudimentary vestiges of the intermediate lobe are detectable in adult humans with the bulk of intermediate lobe cells being dispersed in the anterior and posterior lobes. [55] The neurohypophysis is composed of the pars nervosa (also known as the neural or posterior lobe), the infundibular stalk, and the median eminence. The infundibular stalk is surrounded by the pars tuberalis, and together they constitute the hypophyseal stalk. The pituitary gland lies in the sella turcica (the Turkish saddle) of the sphenoid bone and underlies the base of the hypothalamus. [1] [56] This anatomic location explains the hypothalamic damage described by Fröhlich. [4] In humans, the base of the hypothalamus forms a mound called the tuber cinereum, the central region of which gives rise to the median eminence ( Fig. 7-4 Fig. 7-4 ; see Fig. 7-3 ). The anterior and intermediate lobes of the pituitary are derived from an outgrowth of the pharyngeal cavity called Rathke's pouch and migrate during development to surround the neural lobe. The intermediate lobe is in contact with the neural lobe and is the least prominent of the three lobes. With age, the intermediate lobe in humans decreases in size and is represented in the adult as a relatively small collection of POMC cells. In some species, these cells are responsible for secreting the POMC-derived product -melanocyte-stimulating hormone (-MSH). [55] [57] In a strict sense, the neurohypophysis is made up of the neural lobe, the infundibular stalk, and the median eminence. The major component of the neural lobe is a collection of axon terminals arising from magnocellular secretory neurons from the paraventricular and supraoptic nuclei of the hypothalamus (Fig. 7-5 (Figure Not Available) ; see Fig. 7-8C ). These axon terminals are in close association with a capillary plexus, and they secrete substances including AVP and oxytocin into the hypophyseal veins and into the general circulation (Table 7-1) . [58] [59] The blood supply to the neurohypophysis arises from the inferior hypophyseal artery (a branch of the internal carotid artery). Scattered among the nerve terminals are glial-like cells called pituicytes. As the source of AVP to the general circulation, the paraventricular and supraoptic nuclei and their axon terminals in the neural lobe are the effector arms of the central regulation of blood osmolality, fluid balance, and blood pressure [60] [61] [62] (see "Circumventricular Organs"). The secretion of oxytocin by magnocellular neurons is also well characterized and is critical at the time of parturition, resulting in uterine myometrial contraction. In addition, the secretion of oxytocin is regulated by the classical milk let-down reflex. [63] [64] The exact neuroanatomic substrate underlying the milk let-down response is still unclear. However, it is apparent that mechanosensory information from the nipple reaches the magnocellular neurons, directly or indirectly, from the dorsal horn of the spinal cord, [50] [65] resulting in release of oxytocin into the general circulation. Oxytocin acts on receptors on myoepithelial cells in the mammary gland acini, leading to release of milk into the ductal system and ultimately the release of milk from the mammary gland.



The Median Eminence and Hypophyseotropic Neuronal System

The median eminence lies in the center of the tuber cinereum; it is composed of an extensive array of blood vessels and nerve endings and is the functional link between the hypothalamus and the anterior pituitary gland (Fig. 7-6 (Figure Not Available) Fig. 7-7 Fig. 7-8 Fig. 7-9 ; see Fig. 7-5 (Figure Not Available) ). [15] [36] [50] [65] [66] [67] [68] [69] [70] [ 71] [72] [ 73] [74] The median eminence can be considered the functional link between the hypothalamus and pituitary and the site of the hypothalamus from which the pituitary portal vessels arise. The median eminence is characterized by an extremely rich blood supply that arises from the superior hypophyseal artery (from the internal carotid artery). The artery sends off many small branches that form capillary loops. [52] The small capillary loops extend into the internal and external zones (see later), form anastomoses, and drain into sinusoids that become the pituitary portal veins that enter the vascular pool of the pituitary gland. [68] [75] [76] The flow of blood in these short loops is thought to be predominantly (if not exclusively) in a hypothalamic-to-pituitary direction. [12] [15] This well-developed plexus results in a tremendous increase in the vascular surface area. In addition, the vessels are fenestrated, allowing diffusion of the peptide-releasing factors to their site of action in the anterior pituitary gland. This vascular complex in the base of the hypothalamus and its "arteriolized" venous drainage to the pituitary compose a circulatory system analogous to the portal vein system of the liver, hence the term hypophysealportal circulation. Typically, three zones of the median eminence are discussed, the ependymal layer, the internal zone, and the external zone (see Fig. 7-8) . [1] The innermost zone is made up of ependymal cells that form the floor of the third ventricle. These ependymal cells are unique in that they have microvilli rather than cilia. The ependymal layer also contains specialized cells called tanycytes that send processes into the other layers of the median eminence. There are tight junctions between the ependymal cells lining the floor of the third ventricle forming a barrier between the cerebrospinal fluid (CSF) and the blood in the median eminence. In addition, tight junctions exist between tanycytes at the lateral edges of the median eminence that are thought to prevent the diffusion of releasing factors back into the medial basal hypothalamus. [51] The internal zone of the median eminence is composed of axons of passage of the supraoptic and paraventricular magnocellular neurons en route to the posterior pituitary (see Fig. 7-8C)

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Figure 7-3 A, Human hypothalamic-pituitary unit showing the relationship to the sella turcica, brain membranes, and optic chiasm. B, Midsagittal nuclear magnetic resonance scan of the brain of a normal woman, which corresponds to the diagram in A. Note the location of the pituitary stalk, the intense signal from the posterior pituitary, and the anatomic relationship to the optic commissure and the optic nerve. (See also Fig. 7-4A.) (Courtesy of Dr. Samuel Wolpert.)

and the axons of the hypophyseotropic neurons destined for the external layer of the median eminence (see Fig. 7-8A and B) . In addition, supportive cells are found in this layer. Finally, the external zone represents the exchange point of the hypothalamic releasing factors and the pituitary portal vessels. The external zone contains terminals from two general types of releasing factors, peptides (discussed in detail later) and monoamines (dopamine and norepinephrine). This zone represents the site of convergence where the peptides come in contact with portal vessels. [75] Two general types of tuberohypophyseal neurons project to the external zone of the median eminence: peptide-secreting (peptidergic) neurons (e.g., thyrotropin-releasing hormone [TRH], corticotropin-releasing hormone [CRH], and luteinizing hormone-releasing hormone [LHRH]; see Fig. 7-7 and Fig. 7-8 ) and neurons containing bioamines (e.g., dopamine and serotonin). [77] Although the secretion of these substances into the portal circulation is an important control mechanism, some peptides and neurotransmitters in nerve endings are not released into the hypophyseal-portal circulation [69] but instead

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Figure 7-4a A, Midsagittal view of the human brain showing the hypothalamus and neighboring structures.

function to regulate the secretion of other nerve terminals. The anatomic relationships of nerve endings, basement membranes, interstitial spaces, fenestrated (windowed) capillary endothelia, and glia in the median eminence are similar to those in the neural lobe. As in the case of the neurohypophysis, the release of neuropeptides is mediated by the depolarization of hypothalamic cells leading to secretion at the median eminence. [51] [52] Non-neuronal supporting cells in the hypothalamus also play a dynamic role in hypophyseotropic regulation. For example, nerve terminals in the neurohypophysis are enveloped by glia (in the neural lobe they are called pituicytes); when the gland is inactive they surround the nerve endings, whereas the nerve ending is exposed when vasopressin secretion is enhanced as in states of dehydration. Within the median eminence, LHRH nerve endings are enveloped by the specialized ependymal cells called tanycytes, which also cover or uncover neurons with changes in functional status. [78] Thus, supporting elements, with their own sets of receptors, can change the neuroregulatory milieu within the hypothalamus, median eminence, and pituitary. The site of production, the genetics, and the regulation of synthesis and release of peptide releasing factors are discussed in detail in the following. Briefly, the cell groups in the hypothalamus [79] that contain releasing factors that are secreted into the pituitary portal circulation are located in several cell groups of the medial hypothalamus (Table 7-2) . These cell groups include the arcuate (infundibular) nucleus (see Fig. 7-9D) , the paraventricular nucleus (see Fig. 7-9A and C) , the periventricular nucleus, and a group of cells in the medial preoptic area near the organum vasculosum of the lamina terminalis (OVLT) (Fig. 7-10) . [73] [80] As discussed, magnocellular neurons in the supraoptic and paraventricular nucleus send axon terminals that traverse the median eminence and make up the neural lobe of the pituitary. In addition, a projection from magnocellular neurons to the external zone of the median eminence has been described. However, its functional significance is not clear. The third structure often grouped as a component of the median eminence is the pars tuberalis. The pars tuberalis is a subdivision of the adenohypophysis and is a thin glandular sheet of tissue that lies around the infundibulum and pituitary stalk. In some animals, the epithelial component may make up as much as 10% of the total glandular tissue of the anterior pituitary. The pars tuberalis contains cells making pituitary tropic hormones including luteinizing hormone (LH) and thyrotropin. A definitive physiologic function of the pars tuberalis is not established, but melatonin receptors are expressed in the pars tuberalis.



CIRCUMVENTRICULAR ORGANS A fundamental principle of physiology and pharmacology is that the brain, including the hypothalamus, resides in an environment that is protected from humoral signals.[52] [60] [81] [82] [83] This exclusion of macromolecules is due to the structural vascular

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Figure 7-4b B, Base of the human brain, showing the hypothalamus and neighboring structures. On gross inspection, several landmarks outline the hypothalamus. It is bounded anteriorly by the optic chiasm, laterally by the sulci formed with the temporal lobes, and posteriorly by the mammillary bodies (in which the mammillary nuclei are located). Dorsally, the hypothalamus is delineated from the thalamus by the hypothalamic sulcus. The smooth, rounded base of the hypothalamus is the tuber cinereum; the pituitary stalk descends from its central region, which is termed the median eminence. The median eminence stands out from the rest of the tuber cinereum because of its dense vascularity, which is formed by the primary plexus of the hypophysealportal system. The long portal veins run along the ventral surface of the pituitary stalk. (From Nauta WJ, Haymaker W. Hypothalamic nuclei and fiber connections. In Haymaker W, Anderson E, Nauta WJ [eds]. The Hypothalamus. Springfield, Ill, Charles C Thomas, 1969, pp 136209.)

specializations that make up the blood-brain barrier. These include tight junctions of brain vascular endothelial cells that preclude the free passage of polarized macromolecules including peptides and hormones. In addition, astrocytic foot processes and perivascular microglial cells contribute to the integrity of the blood-brain barrier. [52] However, to exert homeostatic control, the brain, especially the hypothalamus, must assess key sensory information from the blood stream including hormone levels, metabolites, and potential toxins. [84] For example, to monitor key signals the brain has "windows on the circulation" or circumventricular organs (CVOs) that serve as a conduit of peripheral cues into key neuronal cell groups that maintain homeostasis. [52] [60] As the name implies, CVOs are specialized structures that lie on the midline of the brain along the third and fourth ventricles. These structures include the OVLT, subfornical organ (SFO), median eminence, neurohypophysis (posterior pituitary), subcommissural organ, and the area postrema (see Fig. 7-10) . Unlike the vasculature in the rest of the brain, the blood vessels in CVOs have fenestrated capillaries that allow relatively free passage of molecules such as proteins and peptide hormones. [52] [60] [81] [82] [83] Thus, neurons and glial cells that reside within the CVOs have access to these macromolecules. In addition to the distinct nature of the vessels themselves, the CVOs have an unusually rich blood supply, allowing them to act as integrators at the interface of the blood-brain barrier. As discussed in more detail later, several of the CVOs have major projections to hypothalamic nuclear groups that regulate homeostasis. [50] [79] Thus, the CVOs serve as a critical link between peripheral metabolic cues, hormones, and potential toxins with cell groups within the brain that regulate coordinated endocrine, autonomic, and behavioral responses. Detailed discussion of the physiologic roles of individual CVOs is beyond the scope of this chapter, but several in-depth reviews have assessed the function of each. [50] [60] [61] [79] [81] [82] [83] [85] [86] [87] [88] Median Eminence

The median eminence and neurohypophysis contain the neurosecretory axons that control pituitary function. The role of the median eminence as a link between the hypothalamus and the pituitary gland is covered in greater detail in other sections of this chapter ( see Fig. 7-8 and Fig. 7-9 and "Hypothalamic-Pituitary Unit"). However, it is important to understand that the anatomic location of the median eminence places it in a position to serve as an afferent sensory organ as well. Specifically, the median eminence is located adjacent to several neuroendocrine and autonomic regulatory nuclei at the tuberal level of the hypothalamus (see Fig. 7-9) . These nuclear groups include the arcuate, ventromedial, dorsomedial, and paraventricular nuclei. [10] [89] A role of cell groups surrounding the median eminence as afferent sensory centers is supported by several observations. For example, toxins such as monosodium glutamate and gold

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Figure 7-5 (Figure Not Available) Hypothalamic magnocellular neurons and the posterior pituitary gland. This drawing of a midsagittal view of the hypothalamus and pituitary gland illustrates the concept that magnocellular neurons in the paraventricular and supraoptic nuclei secrete oxytocin and arginine vasopressin directly into capillaries in the posterior lobe of the pituitary gland. (From Bear MF, Connors BW, Paradiso MA. Neuroscience: Exploring the Brain. Baltimore, Williams & Wilkins, 1996, p 408.)

thioglucose damage neurons in cell groups overlying the median eminence, resulting in obesity and hyperphagia. Experimental evidence suggests that the median eminence is a portal of entry for hormones such as leptin. Indeed, administration of radiolabeled peptides or hormones, such as -MSH or leptin, led to their accumulation around the median eminence. [90] [91] Moreover, leptin receptor messenger ribonucleic acid (mRNA) and leptin-induced gene expression are densely localized in the arcuate, ventromedial, dorsomedial, and ventral premammillary hypothalamic nuclei. [92] [93] [94] [95] [96] As discussed in detail in other sections of this chapter, leptin is an established mediator of body weight and neuroendocrine function that acts on several cells in the hypothalamus including POMC neurons that reside in the arcuate nucleus (see Fig. 8-67). [31] [97] [98] [99] [100] Notably, POMC TABLE 7-1 -- Sequences of the Principal Peptides of the Neurohypophysis 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Mammals (except pig)

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH 2 Oxytocin

Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH 2 Arginine vasopressin

Pig

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH 2 Oxytocin

Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH 2 Lysine vasopressin

Birds, reptiles, amphibians, lungfishes

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Ile-Gly-NH 2 Mesotocin

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Arg-Gly-NH 2 Vasotocin

Bony fishes (palcopteryglans and neopteryglans)

Cys-Tyr-Ile-Ser-Asn-Cys-Pro-Ile-Gly-NH 2 Isotocin

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Arg-Gly-NH 2 Vasotocin

neurons are also found embedded within the median eminence. Thus, it is likely that the median eminence is involved in conveying information from humoral factors such as leptin to key hypothalamic regulatory neurons in the medial basal hypothalamus.



Thyrotropin-Releasing Hormone Chemistry and Evolution

TRH, the smallest known peptide releasing hormone, is the tripeptide pyroGlu-His-Pro-NH 2 . The TRH peptide sequence is repeated six times within the human TRH pre-prohormone gene (Fig. 7-13) (Figure Not Available) . The rat pro-TRH precursor contains five TRH peptide repeats flanked by dibasic residues (Lys-Arg or Arg-Arg), along with seven or more non-TRH peptides. Two prohormone convertases, PC1 and PC2, cleave the TRH tripeptides at the dibasic residues within the regulated secretory pathway. Carboxypeptidase E then removes the dibasic residues, leaving the sequence Gln-His-Pro-Gly. This peptide is then amidated at the C-terminus by peptidylglycine alpha-amidating monooxygenase, with Gly acting as the amide donor. The amino-terminal pyro-Glu residue results from cyclization of the Gln. Although the TRH tripeptide is the only established hormone encoded within its large prohormone, the rat pro-TRH yields seven additional peptides that have unique tissue distributions. [175] Several biologic activities of these peptides have

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Figure 7-13 (Figure Not Available) Structure of human thyrotropin-releasing hormone (TRH) gene and peptide, showing six repeating codons for the TRH sequence. CPE, carboxypeptidase E; PAM, peptidylglycine alpha-amidating monooxygenase; PC1, prohormone convertase 1. (From Yamada M, Radovick S, Wondisford FE, et al. Cloning and structure of human genomic DNA and hypothalamic cDNA encoding human preprothyrotropin-releasing hormone. Mol Endocrinol 1990; 4:551556.)

been observed: pre-pro-TRH [160] [161] [162] [163] [164] [165] [166] [167] [168] [169] may be a hypophyseotropic factor because it is released from hypothalamic slices [176] and potentiates the thyrotropin-releasing effects of TRH. [177] Pro-TRH [177] [178] [179] [180] [181] [182] [183] [184] [185] [186] [187] [188] [189] [190] [191] [192] [193] [194] [195] [196] [197] [198] [199] is also released from the median eminence[176] and appears to inhibit ACTH release. [178] TRH is a phylogenetically ancient peptide, being found in primitive vertebrates, such as the lamprey, and even invertebrates such as the snail. TRH is widely expressed in both CNS and periphery in amphibians, reptiles, and fishes but does not stimulate thyrotropin release in these poikilothermic vertebrates. [179] Thus, TRH has multiple peripheral and central activities and was co-opted as a hypophyseotropic factor midway during the evolution of vertebrates, perhaps specifically as a factor needed for coordinate regulation of temperature homeostasis. Effects on the Pituitary Gland and Mechanism of Action

After intravenous injection of TRH in humans, serum thyrotropin levels rise within a few minutes (Fig. 7-14) ,[180] [181] [182] followed by a rise in serum triiodothyronine (T 3 ) levels; there is an increase in thyroxine (T 4 ) release as well, but a change in blood levels of T 4 is usually not demonstrable because the pool of circulating T 4 (most of which is bound to carrier proteins) is so large. The clinical applications of TRH testing are covered later in this chapter and in Chapter 10 . TRH action on the pituitary is blocked by previous treatment with thyroid hormone, which is a crucial element in feedback control of pituitary thyrotropin secretion. TRH is also a potent PRF (Fig. 7-15) . [180] [181] [182] The time course of response of blood PRL levels to TRH, the dose-response characteristics, and the suppression by thyroid hormone pretreatment (all of which parallel changes in thyrotropin secretion) suggest that TRH may be involved in the regulation of PRL secretion. Moreover, TRH is present in the hypophyseal-portal blood of lactating rats. [183] However, it is unlikely to be a physiologic regulator of PRL secretion [184] [185] because the PRL response to nursing in humans is unaccompanied by changes in plasma thyrotropin levels. [185] Nevertheless, TRH may occasionally cause hyperprolactinemia (with or without galactorrhea) in patients with hypothyroidism. In normal individuals TRH has no influence on the secretion of pituitary hormones other than thyrotropin and PRL, but it enhances the release of human growth hormone (hGH) in acromegaly and of corticotropin in some patients with Cushing's disease. Furthermore, prolonged stimulation of the normal pituitary with GHRH can sensitize it to the hGH-releasing effects of TRH. [186] [187] TRH also causes the release of hGH in some patients with uremia, hepatic disease, anorexia

Figure 7-14 Effect of intravenous injection of thyrotropin-releasing hormone on serum thyrotropin levels in humans. TRF, thyrotropin-releasing hormone; TSH, thyrotropin. (From Hershman JM, Pittman JA Jr. Control of thyrotropin secretion in man. N Engl J Med 1971; 285:9971006. Reprinted by permission of The New England Journal of Medicine.)

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Figure 7-15 Prolactin (PRL) and thyrotropin (TSH) secretory responses to intravenous injection of 800 µg of thyrotropin-releasing hormone (TRH) in humans. This figure shows that TRH induces discharge of both PRL and thyrotropin, that the effect in females is greater than that in males (presumably owing to estrogen sensitization of the pituitary), and that thyrotoxicosis inhibits the response of both PRL and thyrotropin to TRH. An inhibitory effect on the TRH response is noted at the upper limit of the normal range of thyroid hormone levels and is a sensitive test of minor degrees of thyroid hormone excess. Although TRH is a potent prolactin-releasing factor (PRF), there is evidence that there is another PRF physiologically connected to PRL regulation. (Replotted from data of Bowers C, Friesen HG, Hwang P, et al. Prolactin and thyrotropin release in man by synthetic pyroglutamylhistidyl-prolinamide. Biochem Biophys Res Commun 1971; 45:10331041.)

nervosa, and psychotic depression [182] and in children with hypothyroidism. TRH inhibits sleep-induced hGH release through its actions in the CNS (see later in the section on extrapituitary actions of TRH). Stimulatory effects of TRH are initiated by binding of the peptide to specific receptors on the plasma membrane of the thyrotroph. [24] [188] Neither thyroid hormone nor somatostatin, both of which antagonize the effects of TRH, interfere with its binding. TRH was originally thought to activate membrane adenylate cyclase to stimulate formation of cAMP,[182] [189] and cAMP in turn was thought to stimulate thyrotropin secretion. However, cAMP does not increase under all conditions of TRH-induced thyrotropin release, [190] and it is now clear that TRH action is mediated mainly through hydrolysis of phosphatidylinositol, with phosphorylation of key protein kinases and an increase in intracellular free Ca 2+ as the crucial step in postreceptor activation (see Chapter 5) . [190] [191] [192] TRH effects can be mimicked by exposure to a Ca2+ ionophore and are partially abolished by a Ca 2+ -free medium. TRH stimulates the formation of mRNAs coding for thyrotropin [193] and PRL,[194] confirming that this peptide is trophic as well as a releasing factor. TRH is degraded to acid TRH and to the dipeptide histidylprolineamide, which cyclizes nonenzymatically to histidylproline diketopiperazine (cyclic His-Pro).

[195]

Acid

TRH has some behavioral effects in rats that are similar to those of TRH but no other proven actions. Cyclic His-Pro is reported to act as a PRF and to have other neural effects, including reversal of ethanol-induced sleep (TRH is also effective in this system), elevation of brain cyclic guanosine monophosphate levels, an increase in stereotypical behavior, modification of body temperature, and inhibition of eating behavior. Some of the effects of TRH may be mediated through cyclic His-Pro, but the fact that cyclic His-Pro is abundant in some areas and is not proportional to the amount of TRH suggests that the peptide may not be derived solely from TRH.[196] [197] Extrapituitary Function

TRH is present in virtually all parts of the brain: cerebral cortex, circumventricular structures, neurohypophysis, pineal gland, and spinal cord. [182] [198] [199] [200] [201] [202] TRH is also found in pancreatic islet cells and in the gastrointestinal tract. [203] Although it exists in low concentration, the total amount in extrahypothalamic tissues exceeds the amount in the hypothalamus. The extensive extrahypothalamic distribution of TRH, its localization in nerve endings, and the presence of TRH receptors in brain tissue suggest that TRH serves as a neurotransmitter or neuromodulator outside the hypothalamus. TRH is a general stimulant [182] [204] [205] [206] and induces hyperthermia on intracerebroventricular injection, suggesting a role in central thermoregulation. Clinical Applications

The use of TRH for the diagnosis of hyperthyroidism is less common since the development of ultrasensitive assays for thyroid-stimulating hormone (TSH) [182] (see Chapter 10) ; its use to discriminate between hypothalamic and pituitary causes of thyrotropin deficiency has also declined because of the test's poor specificity, [182] [207] but the application of ultrasensitive assays in conjunction with the TRH test has not been fully evaluated. [208] TRH testing is also not of value in the differential diagnosis of causes of hyperprolactinemia [209] but is useful for the demonstration of residual abnormal somatotropin-secreting cells in acromegalic patients who release hGH in response to TRH before treatment. Studies of the effect of TRH on depression have shown inconsistent results, [210] possibly because of poor blood-brain barrier penetration. [211] Intrathecal administration of TRH may improve responses in depressed patients, [211] [212] but its clinical utility is unknown. Although a role for TRH in depression is not established, many depressed patients have a blunted thyrotropin response to TRH and changes in TRH responsiveness correlate with the clinical course. [210] The mechanism by which blunting occurs is unknown. [213] TRH has been proposed as a treatment for women with threatened premature labor to stimulate the production of lung surfactant in the preterm fetus. Despite encouraging results in early studies, several large-scale trials failed to show improvement in the survival of babies so treated. [214] [215] TRH has been evaluated for the treatment of spinal muscle atrophy and amyotrophic lateral sclerosis; transient improvement in strength was reported in both disorders,[216] [217] [218] [219] but the combined experience at many centers using a variety of treatment protocols including long-term intrathecal administration failed to confirm efficacy. [220] [221] [222] TRH administration also reduces the severity of experimentally induced spinal and ischemic shock [223] [224] [225] ; preliminary studies in humans suggest that TRH treatment may improve recovery after spinal cord injury [226] and head trauma.[227] TRH has been used to treat children with neurologic disorders including West's syndrome, Lennox-Gastaut syndrome, early infantile epileptic encephalopathy, and intractable epilepsy. [228] TRH has been proposed to be an analeptic agent. Sleeping or drug-sedated animals were awakened by the administration of TRH, [229] TRH reportedly reversed sedative effects of ethanol in humans,[230] and TRH is said to have awakened a patient with a profound sleep disorder caused by a hypothalamic and midbrain eosinophilic granuloma. [231] Regulation of Thyrotropin Release

The secretion of thyrotropin is regulated by two interacting elements: negative feedback by thyroid hormone and open-loop neural control by hypothalamic hypophyseotropic factors

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Figure 7-16 Regulation of the hypothalamic-pituitary-thyroid axis. AGRP, agouti-related protein; CART, cocaine and amphetamineregulated transcript; CRH, corticotropin-releasing hormone; NPY, neuropeptide Y; POMC, proopiomelanocortin; T 3 , triiodothyronine; T 4 , thyroxine; TRH, thyrotropin-releasing hormone; TSH, thyrotropin; OB-R, leptin receptor.

(Fig. 7-16) . Thyrotropin secretion is also modified by other hormones, including estrogens, glucocorticoids, and possibly GH, and is inhibited by cytokines in the pituitary and hypothalamus. Aspects of the pituitary-thyroid axis are also considered in Chapter 10 .[182] [198] [232] [233] [234] [235] [236] Feedback Control: Pituitary-Thyroid Axis

In the context of a feedback system, the level of thyroid hormone in blood or of its unbound fraction is the controlled variable and the set-point is the normal resting level of plasma thyroid hormone. Secretion of thyrotropin is inversely regulated by the level of thyroid hormone so that deviations from the set-point of control lead to appropriate changes in the rate of thyrotropin secretion (Fig. 7-17) . Factors that determine the rate of thyrotropin secretion required to maintain a given level of thyroid hormone include the rate at which thyrotropin and thyroid hormone disappear from the blood (turnover rate) and the rate at which T 4 is converted to its more active form, T3 .

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Figure 7-17 Relationship between plasma thyrotropin levels and thyroid hormone as determined by plasma protein-bound iodine (PBI) measurements in humans and rats. These curves illustrate, in the human ( A) and the rat (B), that plasma thyrotropin levels are a curvilinear function of plasma thyroid hormone level. Human studies were carried out by giving myxedematous patients successive increments of thyrotropin T 4 at approximately 10-day intervals. Each point represents simultaneous measurements of plasma PBI and plasma thyrotropin at various times in the six patients studied. The rat studies were performed by treating thyroidectomized animals with various doses of T 4 for 2 weeks before assay of plasma thyrotropin and plasma PBI. These curves illustrate that the secretion of thyrotropin is regulated over the entire range of thyroid hormone levels. At the normal set point for T 4 , the small changes above and below the control level are followed by appropriate increases or decreases in plasma thyrotropin. TSH, thyrotropin; T 4 , thyroxine. (A from Reichlin S, Utiger RD. Regulation of the pituitary thyroid axis in man: relationship of TSH concentration to concentration of free and total thyroxine in plasma. J Clin Endocrinol Metab 1967; 27:251255, copyright by The Endocrine Society. B from Reichlin S, Martin JB, Boshans RL, et al. Measurement of TSH in plasma and pituitary of the rat by a radioimmunoassay utilizing bovine TSH: effect of thyroidectomy or thyroxine administration on plasma TSH levels. Endocrinology 1970; 87:10221031, copyright by The Endocrine Society.)

Thyroid hormones act on both the pituitary and the hypothalamus. Feedback control of the pituitary by thyroid hormone is remarkably precise. Administration of small doses of T3 and T4 inhibited the thyrotropin response to TRH, [237] and barely detectable decreases in plasma thyroid hormone levels were sufficient to sensitize the pituitary to TRH. [238] TRH stimulates thyrotropin secretion within a few minutes through its action on a membrane receptor, whereas thyroid hormone actions,

mediated by intranuclear receptors, require several hours to take effect (see Chapter 10) . The secretion of hypothalamic TRH is also regulated by thyroid hormone feedback. Systemic injections of T 3 [239] or implantations of tiny T 3 pellets in the paraventricular nucleus of hypothyroid rats [240] (Fig. 7-18) (Figure Not Available) reduced the concentration of TRH mRNA and TRH prohormone in TRH-secreting cells. Thyroid hormone also suppressed TRH secretion into hypophyseal-portal blood in sheep. [241] T4 in the blood gains access to TRH-secreting neurons in the hypothalamus by way of the CSF. The hormone is taken up by epithelial cells of the choroid plexus of the lateral ventricle of the brain, bound within the cell to locally produced transthyretin (T 4 -binding prealbumin), and then secreted across the blood-brain barrier. [242] Within the brain, T 4 is converted to T 3 by type II deiodinase, and T 3 interacts with subtypes of the thyroid hormone receptor, TR 1 , TR1 , and TR2 , in the paraventricular nucleus and other brain cells (see Chapter 10) .[243] Thereby the set-point of the pituitary-thyroid axis is determined by thyroid hormone levels within the brain. [244] T 3 in the circulation is not transported into brain in this manner but presumably gains access to the paraventricular TRH neurons across the blood-brain barrier. The brain T4 transport and deiodinase system account for the fact that higher blood levels of T 3 are required to suppress pituitary-thyroid function after administration of T 3 than after administration of T 4 . [244] Transthyretin is present in the brain of early reptiles and in addition is synthesized by the liver in warm-blooded animals. [242] During embryogenesis in mammals, transthyretin is first detected when the blood-brain barrier appears, ensuring thyroid hormone access to the developing nervous system. Neural Control

The hypothalamus determines the set-point of feedback control around which the usual feedback regulatory responses are elicited. Lesions of the thyrotropic area lower basal thyroid hormone levels and make the pituitary more sensitive to inhibition by thyroid hormone, [232] and high doses of TRH raise thyrotropin and thyroid hormone levels. [245] Synthesis of TRH in the paraventricular nuclei is regulated by feedback actions of thyroid hormones. [246] The hypothalamus can override normal feedback control through an open-loop mechanism involving neuronal inputs to the hypophyseotropic TRH neurons (see Fig. 7-16) . For example, cold exposure caused a sharp increase in thyrotropin release in animals [247] [248] and in human newborns.[249] Circadian changes in thyrotropin secretion are another example of brain-directed changes in the set-point of feedback control, but if thyroid hormone levels are sufficiently elevated, as in hyperthyroidism, TRH cannot overcome the inhibition. Hypothalamic regulation of thyrotropin secretion is also influenced by two inhibitory factors, somatostatin and dopamine. [235] [250] [251] Antisomatostatin antibodies increase basal thyrotropin levels and potentiate the response to stimuli that normally induce thyrotropin release in the rat, such as cold exposure and TRH administration. [250] [251] Thyroid hormone in turn inhibits the release of somatostatin, [252] implying coordinated, reciprocal regulation of TRH and somatostatin by thyroid hormone. GH stimulates hypothalamic somatostatin synthesis [253] and can inhibit thyrotropin secretion. The role of

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Figure 7-18 (Figure Not Available) A and B, Direct effects of triiodothyronine (T 3 ) on thyrotropin-releasing hormone (TRH) synthesis in the rat hypothalamic paraventricular nucleus (parvicellular division) were shown in this experiment by immunohistochemical detection of pre-pro TRH(2550) after implantation of a pellet of either T 3 (B) or inactive diiodotyrosine (T 2 ) as a control (A). The T 2 pellet had no effect on the concentration of pre-proTRH (A). In contrast, the TRH prohormone (B) concentrations were markedly reduced. These studies indicate that thyroid hormone regulates the hypothalamic component of the pituitary-thyroid axis as well as the pituitary thyrotrope itself. C and D, Effects of 1 hour at 4°C on TRH messenger ribonucleic acid (mRNA). E to G, Effects on TRH mRNA levels of starvation (F) and leptin replacement during starvation (G). (Photographs courtesy of Dr. R. M. Lechan. From Dyess EM, Segaerson TP, Liposits Z, et al. Triiodothyronine exerts direct cell-specific regulation of thyrotropin-releasing hormone gene expression in the hypothalamic paraventricular nucleus. Endocrinology 1988; 123:22912297, copyright by The Endocrine Society.)

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somatostatin in the regulation of thyrotropin secretion in humans is uncertain. Dopamine has modest effects on thyrotropin secretion, and blockade of dopamine receptors (in the human) stimulates thyrotropin secretion slightly. [235] Changes in the metabolism of thyroid hormone also influence T 3 homeostasis within the brain. In states of thyroid hormone deficiency, brain T 3 levels are maintained by an increase in the deiodinase that converts T 4 to T3 . [254] The pineal gland has been reported to inhibit thyroid function in some [255] but not all [256] studies. The pineal gland contains TRH, and in the frog its content changes with the season and with light and dark cycles independently of hypothalamic thyrotropin. [257] Circadian Rhythm

Plasma thyrotropin in humans is characterized by a circadian periodicity, with a maximum between 9 PM and 5 AM and a minimum between 4 PM and 7 PM. [258] Smaller ultradian thyrotropin peaks occur every 90 to 180 minutes, [259] probably because of bursts of TRH release from the hypothalamus, and are physiologically important in controlling the synthesis and glycosylation of thyrotropin. [260] Glycosylation is a determinant of thyrotropin potency. Temperature

External cold exposure activates and high ambient temperature inhibits the pituitary-thyroid axis in animals, [248] and analogous changes occur in humans under certain conditions. Exposure of infants to cold at the time of delivery causes an increase in blood thyrotropin levels, possibly because of alterations in the turnover and degradation of the thyroid hormones. [249] Blood thyroid hormone levels are higher in the winter than in the summer in individuals in cold climates [261] but not in other climates. [249] However, it is difficult to show that changes in environmental or body temperature in adults influence thyrotropin secretion. For example, exposure to cold ambient temperature or central hypothalamic cooling does not modify thyrotropin levels in young men. [262] Behavioral changes, activation of the sympathetic nervous system, and shivering appear to be more important in temperature regulation in adults than the thyroid response. [248] The autonomic nervous system and the thyroid axis work together to maintain temperature homeostasis in mammals, and TRH plays a role in both pathways. [263] Hypothalamic TRH release is rapidly (30 to 45 minutes) increased in rats exposed to cold. [247] Rapid inhibition of somatostatin release in the median eminence has also been documented, and both changes appear to play important roles in the rise in plasma TSH induced by cold exposure. TRH mRNA is elevated within an hour of cold exposure (see Fig. 7-18 C and D) (Figure Not Available) . The regulation of hypophyseotropic TRH release and expression by cold is largely mediated by catecholamines. Noradrenergic and adrenergic fibers, originating in the brain stem, are found in close proximity to TRH nerve endings in the median eminence, and a rapid rise in TRH release was seen after norepinephrine treatment of hypothalamic fragments containing mainly median eminence. [264] Brain stem adrenergic and noradrenergic fibers also make synaptic contacts with TRH neurons in the PVH (see Fig. 7-16) , [265] [266] and thus catecholamines are likely to be involved in the regulation of TRH gene expression by cold. TRH neurons in the PVH are densely innervated by NPY terminals, [267] and a portion of the NPY terminals arising from the C1, C2, C3, and A1 cell groups of the brain stem and projecting to the PVH are known to be catecholaminergic. [268] Somatostatin, dopamine, and serotonin also play a variety of roles in the regulation of TRH. Stress

Stress is another determinant of thyrotropin secretion. [232] [234] In humans physical stress inhibits thyrotropin release, as indicated by the finding that in the euthyroid sick syndrome low T 3 and T4 do not cause compensatory increases in thyrotropin secretion as would occur in normal individuals. [269] [270] [271] A number of observations demonstrate interactions between the thyroid and adrenal axes. Physiologically, the bulk of evidence suggests that glucocorticoids in humans[272] and rodents[273] act to blunt the thyroid axis through actions in the CNS. Some actions may be direct because the TRH gene (see Fig. 7-13) (Figure Not Available) contains the glucocorticoid response element consensus sequence [196] and hypophyseotropic TRH neurons appear to contain glucocorticoid receptors. [274] The diurnal rhythm of cortisol is opposite that of TSH (see Fig. 7-12) (Figure Not Available) and acute administration of glucocorticoids can block the nocturnal rise in

TSH, but disruption of cortisol synthesis with metyrapone only modestly affects the TSH circadian rhythm. [275] Several lines of evidence, however, identify conditions in which elevated glucocorticoids are associated with stimulation of the thyroid axis. Human depression is often associated with hypercortisolism and hyperthyroxinemia, [276] and TRH mRNA levels are elevated by glucocorticoids in a number of cell lines as well as in cultured fetal hypothalamic TRH neurons from the rat. [277] Thus, although glucocorticoids probably stimulate TRH production in TRH neurons, their overall inhibitory effect on the thyroid axis results from indirect glucocorticoid negative feedback on structures such as the hippocampus. Disruption of hippocampal suppression of the hypothalamic-pituitary-adrenal (HPA) axis is proposed to be involved in the hypercortisolemia commonly seen in affective illness, [278] and disruption of hippocampal inputs to the hypothalamus have been shown to produce a rise in hypophyseotropic TRH in the rat. [279] Starvation

The thyroid axis is depressed during starvation, presumably to help conserve energy by depressing metabolism (see Fig. 7-18 E to G) (Figure Not Available) . In humans, reduced T 3 , T4 , and TSH are seen during starvation or fasting. [280] There are also changes in the thyroid axis in anorexia nervosa, such as low blood levels of T 3 and low normal levels of T 4 (see Chapter 33) . Inappropriately low levels of TSH are found, suggesting defective activation of TRH production by low thyroid hormone levels. During starvation in rodents, reduced TRH release into hypophyseal portal blood [281] and reduced pro-TRH mRNA levels [282] are seen, despite lowered thyroid hormone levels. Reduced basal TSH levels are also usually present. The hypothyroidism seen in fasting or in the leptin-deficient Lepob /Lepob mouse can be reversed by administration of leptin, [283] and the evidence suggests that the mechanism involves leptin's ability to up-regulate TRH gene expression in the PVH (see Fig. 7-18 E to G) (Figure Not Available) . [284] Leptin appears to act both directly through leptin receptors on hypophyseotropic TRH neurons and indirectly through its actions on other hypothalamic cell groups, such as arcuate nucleus POMC and NPY-agouti-related peptide (AgRP) neurons. [285] TRH neurons in the PVH receive dense NPY-AgRP[267] and POMC projections [286] from the arcuate and express NPY and melanocortin-4 receptors, [287] and -MSH administration partially prevents the fasting-induced drop in thyroid hormone levels. [286] Indeed, the TRH promoter contains a signal transducer and activator of transcription (STAT) response element and a cAMP response element that have been demonstrated to mediate induction of TRH gene expression by leptin and -MSH, respectively, in a heterologous cell system (see Fig. 7-13) (Figure Not Available) . [287] The regulation of TRH by metabolic state is likely to be under redundant control,

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however, because, unlike rodents, leptin-deficient children are euthyroid, euthyroid.[290]

[ 288]

and both melanocortin-4 receptor (MC4R)deficient rodents [289] and humans are

Central TRH outside the paraventricular nucleus also plays a role in thermoregulation through the autonomic nervous system. Infection and Inflammation

The molecular basis of infection- or inflammation-induced thyrotropin suppression is now established. Sterile abscesses or the injection of interleukin-1 (IL-1; endogenous pyrogen, a secretory peptide of activated lymphocytes) [291] or of tumor necrosis factor (TNF-) inhibits thyrotropin secretion, [292] and IL-1 stimulates the secretion of somatostatin.[293] TNF- inhibits thyrotropin secretion directly and induces functional changes in the rat characteristic of the "sick euthyroid" state. [294] It is likely that the thyrotropin inhibition in animal models of the sick euthyroid syndrome is due to cytokine-induced changes in hypothalamic and pituitary function. [295] IL-6, IL-1, and TNF- contribute to the suppression of TSH in the sick euthyroid syndrome. [296]



Area Postrema

The area postrema lies at the caudal end of the fourth ventricle adjacent to the nucleus of the solitary tract ( see Fig. 7-10 and Fig. 8-66D). In experimental animals such as the rat and mouse, it is a midline structure lying above the nucleus of the solitary tract. [50] [52] [60] [112] However, in humans the area postrema is a bilateral structure. As the area postrema overlies the nucleus of the solitary tract, it also receives direct visceral afferent input from the glossopharyngeal nerve (including the carotid sinus nerve) and the vagus nerve. In addition, the area postrema receives direct input from several hypothalamic nuclei. The efferent projections of the area postrema include projections to the nucleus of the solitary tract, ventral lateral medulla, and the parabrachial nucleus. Consistent with a role as a sensory organ, the area postrema is enriched with receptors for several peptide hormones including glucagon-like peptide-1 [113] and cholecystokinin. [114] It also contains chemosensory neurons that include osmoreceptors. [61] [82] Notably, the area postrema is thought to be critical in the detection of potential toxins and can induce vomiting in response to foreign substances. In fact, the area postrema is often referred to as the chemoreceptor trigger zone. [112] The best-described physiologic role of the area postrema is probably the coordinated control of blood pressure. [52] [61] [82] [115] [116] For example, the area postrema contains binding sites for angiotensin II, AVP, and atrial natriuretic peptide. Moreover, lesions of the area postrema in rats blunt the rise in blood pressure induced by angiotensin II. [85] [115] [116] Finally, administration of angiotensin II induces the expression of Fos in neurons of the area postrema. [81] [117] The area postrema has also been hypothesized to play a role in responding to inflammatory cytokines during the acute febrile response ( see Fig. 7-50 and Fig. 7-51 and "Neuroendocrine-Immune Interactions").



Subcommissural Organ

The subcommissural organ (SCO) is located below the posterior commissure near the junction of the third ventricle and cerebral aqueduct below the pineal gland (see Fig. 7-10) . [82] [83] The SCO is composed of specialized ependymal cells that secrete a highly glycosylated protein of unknown function. The secretion of this protein leads to aggregation and formation of the so-called Reissner fibers. [87] The glycoproteins are extruded through the aqueduct, the fourth ventricle, and the spinal cord lumen to terminate in the caudal spinal canal. In humans, intracellular secretory granules are identifiable in the SCO but Reissner's fibers are absent. The SCO secretion in

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Figure 7-9 The tuberoinfundibular system is revealed by retrograde transport of cholera toxin subunit B (CtB). The location of hypothalamic cell bodies of neurons projecting to the median eminence (ME) and the posterior pituitary can be identified by microinjecting a small volume of retrograde tracer (CtB) into the median eminence of the rat (see Wiegand, [73] Lechan [ 80] ). A, Retrogradely labeled cells can be seen in the paraventricular and supraoptic nuclei of the hypothalamus (PVH, SON). B, Magnocellular neurons are observed in the SON. C, Labeled neurons are found in the posterior magnocellular group (pm) as well as the medial parvicellular subdivision (mp). The labeled cells in the PVH include those that contain corticotrophin-releasing hormone and thyrotropin-releasing hormone. D, Retrogradely labeled cells are also found in the arcuate nucleus of the hypothalamus (Arc). These include neurons that release growth hormonereleasing hormone and dopamine. 3v, third ventricle; ot, optic tract.

humans is therefore presumed to be more soluble and to be absorbed directly from the CSF. Compared with other CVOs, relatively little is known about the physiologic role of the SCO. Hypothesized roles for the SCO include clearance of substances from the CSF. [86] [87]



PINEAL GLAND Historically, the functional significance of the pineal gland has been difficult to discern. For example, Descartes called the pineal gland the "seat of the soul." The pineal gland is both an endocrine and a circumventricular organ; it is derived from cells from the roof of the third ventricle and lies above the posterior commissure near the level of the habenular complex and the sylvian aqueduct. [118] [119] [120] The pineal gland is composed of two cell types, pinealocytes and interstitial (glial-like) cells. Histologic studies suggest that the pineal gland cells are secretory in nature, and indeed the pineal is the source of melatonin in mammals. [121] As discussed subsequently, the pineal gland integrates information encoded by light into coordinated secretions that underlie biologic rhythmicity. [122] [123] The pineal is an epithalamic structure and consists of primordial photoreceptive cells. The pineal retains its light sensitivity in lower vertebrates such as fish and amphibians but lacks photosensitivity in mammals and has evolved as a strictly secretory organ in higher vertebrates. [118] [120] [121] However, neuroanatomic studies have established that light-encoded information is relayed to the pineal in an indirect and multisynaptic fashion. [124] This series of synapses ultimately results in innervation of the gland by noradrenergic sympathetic nerve terminals that are critical regulators of melatonin production and release. Specifically, the retina provides direct innervation to the suprachiasmatic nucleus (SCN) of the hypothalamus through the retinohypothalamic tract. [125] The SCN in turn provides input to the paraventricular nucleus of the hypothalamus (PVH), a key cell group in neuroendocrine and autonomic control. This input is provided through direct and indirect pathways by intrahypothalamic projections. [126] [127] The PVH in turn provides direct innervation to sympathetic preganglionic neurons in the intermediolateral cell column of the thoracic regions of the spinal cord. [50] [128] Sympathetic preganglionic neurons innervate postganglionic neurons in the superior cervical ganglion, [129] which in turn provide the noradrenergic innervation to the pineal (see "Hypothalamic-Pituitary Unit"). This rather circuitous pathway is thought to represent the anatomic substrate for light to regulate the secretion of melatonin. It is important to note that in the absence of light input, the pineal gland rhythms persist but are not entrained to the external light-dark cycle. The Pineal Is the Source of Melatonin

The predominant hormone secreted by the pineal gland is melatonin (Fig. 7-11) . However, the pineal also contains biogenic amines, peptides, and GABA. Pineal-derived melatonin is synthesized from tryptophan, through serotonin, with the rate-limiting

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TABLE 7-2 -- Neuroactive Materials in the Paraventricular Nucleus and the Arcuate Nucleus Paraventricular Nucleus Magnicellular Division Angiotensin II Cholecystokinin Glucagon Oxytocin Peptide 7B2 Proenkephalin B (dynorphin, rimorphin, -neoendorphin) Vasopressin Nitric oxide (NO) Parvicellular Division -Aminobutyric acid (GABA) Angiotensin II Atrial natriuretic factor Cholecystokinin Corticotropin-releasing hormone Dopamine Follicle-stimulating hormonereleasing factor Galanin Glucagon Neuropeptide Y Neurotensin Peptide 7B2 Proenkephalin A (met-enkephalin, leu-enkephalin, BAM 22P, metorphamide, met-enkephalin-Arg 6 -Phe7 , met-enkephalin-Arg6 -Gly 7 -Leu8 ) Somatostatin Thyrotropin-releasing hormone (TRH) Vasopressin Interleukin-1 (IL-1) Vasoactive intestinal peptide (VIP)peptide-histidine-isoleucine (PHI) Nitric oxide Arcuate Nucleus Acetylcholine (?) -Aminobutyric acid Dopamine Galanin Growth hormonereleasing hormone (GHRH) Luteinizing hormonereleasing hormone (LHRH)

Neuropeptide Y Neurotensin Pancreatic polypeptide Proenkephalin A Prolactin Melanocortins (corticotropin, -melanocyte-stimulating hormone [-MSH], -melanocyte-stimulating hormone [-MSH]) Endogenous opioids (-endorphin, -lipotropin [-LPH]) Somatostatin Substance P Modified from Lechan RM. Neuroendocrinology of pituitary hormone regulation. Endocrinol Metab Clin North Am 1987; 16:475502. step being catalyzed by the enzyme N-acetyltransferase (NAT)[130] [131] [132] (Fig. 7-12) (Figure Not Available) . The final step of melatonin synthesis is catalyzed by hydroxyindole-O-methyltransferase (HIOMT). These enzymes are expressed in a pineal specific manner; however, hydroxyindole- O-methyltransferase is also expressed in the retina and red blood cells. It is now established that melatonin plays a key role in regulating a myriad of circadian rhythms, and a fundamental principle of circadian biology is that the synthesis of melatonin is exquisitely controlled. [122] [133] This control is exerted at several levels. NAT mRNA levels, NAT activity, and melatonin synthesis and release are regulated in a circadian fashion and are entrained by the light-dark cycle, with darkness thought to be the most important signal. [130] [131] [134] [135] For example, melatonin and NAT levels are highest during the dark and decrease sharply with the onset of light. Melatonin is not thought to be stored to any degree and thus

Figure 7-10 Median sagittal section through the human brain to show the circumventricular organs (black). AP, area postrema; ME, median eminence; NH, neurohypophysis; OVLT, organum vasculosum of the lamina terminalis; PI, pineal body; SFO, subfornical organ; SCO, subcommissural organ; CP, choroid plexus. (From Weindl A. Neuroendocrine aspects of circumventricular organs. In Ganong WF, Martini L [eds]. Frontiers in Neuroendocrinology, vol 3. New York, Oxford University Press, 1973, pp 332.)

is released into blood or CSF directly. [136] Thus, this regulatory mechanism results in melatonin levels in the blood that are highest in the dark when NAT mRNA and activity are highest. The CNS control of melatonin secretion during the dark is mediated by the neuroanatomic pathway already outlined. Lack of light ultimately results in the release of norepinephrine from postganglionic sympathetic nerve terminals that act on -adrenergic receptors in pinealocytes, resulting in an increase in adenylate cyclase activity.[130] [137] The resultant increased levels of cyclic adenosine monophosphate (cAMP) activate signal transduction cascades, including increased protein kinase activity and phosphorylation of cAMP response elementbinding protein. Notably, cAMP response elements have been identified in the promoter of NAT. [130] [138] [139] Thus, light (or lack of it) acting through the sympathetic nervous system induces an increase in cAMP, representing a fundamental regulator of NAT transcription and melatonin synthesis that ultimately results in a dramatic change of melatonin levels across the day.



Physiologic Roles of Melatonin

One of the best characterized roles of melatonin is the regulation of the reproductive axis, including gonadotropin secretion [140] [141] and the timing and onset of puberty (see "Gonadotropin-Releasing Hormone and Control of the Reproductive Axis"). The potent regulation of the reproductive axis by melatonin is established in rodents and domestic animals such as the sheep. It was observed experimentally with the demonstration that removal of the pineal leads to precocious puberty and ameliorates the effects of constant darkness to induce gonadal involution. In addition, male rats exposed to constant darkness or made blind experimentally display testicular atrophy and decreased levels of testosterone. These profound effects are normalized by removal of the pineal gland. [136] [142] The physiologic significance of melatonin is probably most important in species referred to as seasonal breeders. Indeed, the role of

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Figure 7-11 Biosynthesis of melatonin from tryptophan in the pineal gland. Step 1 is catalyzed by tryptophan hydroxylase, step 2 by aromatic- L-amino acid decarboxylase, step 3 by N-acetylating enzyme, and step 4 by hydroxyindole-O-methyltransferase. (From Wurtman RJ, Axelrod J, Kelly DE [eds]. Biochemistry of the pineal gland. In The Pineal. New York, Academic Press, 1968, pp 4775.) Figure 7-12 (Figure Not Available) Diurnal rhythms of corticotropin-releasing hormone (CRH), cortisol, leptin, melatonin, thyrotropin (TSH), and luteinizing hormone (LH). CSF, cerebrospinal fluid; IR, immunoreactive. (From Kling et al. J Clin Endocrinol Metab 1994; 79:233, Fig 3; Van Coevorden et al. Am J Physiol 1991; 260:E651, Fig 1A; Sinha et al. J Clin Invest 1996; 97:1344, Fig 2; Van Coevorden et al. Am J Physiol 1991; 260:E651, Fig 1C; Brabant et al. J Clin Endocrinol Metab 1990; 70:403, Fig 2B; Clarke and Cummins. Endocrinology 1982; 111:1737, Fig 2A.)

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melatonin in regulating reproductive capacity in species such as the sheep and the horse is now established. [143] This type of reproductive strategy probably evolved to synchronize the length of day with the gestational period of the species to ensure that the offspring are born at favorable times of the year and maximize the viability of the young. Despite the potent effects of day length on reproduction in these species, exact mechanisms of melatonin regulation of GnRH release are unsettled. However, melatonin inhibits LH release from the rat pars tuberalis. [141] The role of the pineal in human reproduction is even more unsettled. [144] Earlier onset of menarche in blind women has been reported. In addition, a decline in melatonin at puberty has been described. [145] However, it was not found in other studies. [144] [146] [147] [148] Thus, the role of melatonin in human reproduction is not clear. Nonetheless, the therapeutic potential of melatonin in regulating and shifting biologic rhythms in humans has received great attention. [130] [133]



Melatonin Receptors

It is now established that melatonin mediates its effects by acting on a family of G proteincoupled receptors, which have been characterized by pharmacologic, neuroanatomic, and molecular approaches. [122] [149] [150] The first member of the family, Mel1a , is a high-affinity receptor that was isolated originally from Xenopus melanophores. The second, Mel 1b , has approximately 60% homology with the Mel 1a receptor. A third receptor, the Mel 1c melatonin receptor, has been cloned from zebra fish, Xenopus, and chickens but not as yet from mammals. The mechanisms for the effects of melatonin on regulating and entraining circadian rhythms are becoming increasingly understood. For example, melatonin inhibits the activity of neurons in the SCN of the hypothalamus, the master circadian pacemaker in the mammalian brain. [122] [150] [151] [152] [153] Melatonin can entrain several mammalian circadian rhythms, probably by the inhibition of neurons in the SCN. Neuroanatomic evidence suggests that many of the effects of melatonin on circadian rhythms involve actions on Mel 1a receptors, as the distribution of Mel 1a mRNA overlaps with radiolabeled melatonin binding sites in the relevant brain regions. These sites include the SCN, the retina, and the pars tuberalis of the adenohypophysis. The Mel 1b melatonin receptor is also expressed in retina and brain; however, this is thought to be at much lower levels. [122] [150] [152] Genetic studies in mice have also helped to illuminate the relative roles of each melatonin receptor in mediating the effects of this hormone. Targeted deletion (knockout) of the Mel 1a receptor abolished the ability of melatonin to inhibit the activity of SCN neurons. [152] Several studies have suggested that the inhibition of SCN neurons by melatonin is of great physiologic significance. [122] [152] For example, Reppert and colleagues have suggested that elevations of melatonin at night could decrease the responsiveness of the SCN to activity-related stimuli that could result in phase shifts. As noted, light potently inhibits melatonin synthesis and release. Thus, melatonin may underlie the mechanism by which light induces phase shifts. However, it should be noted that lack of the Mel 1a gene does not block the ability of melatonin to induce phase shifts. These unexpected and somewhat confusing results have resulted in the hypothesis that Mel 1b is involved in melatonin-induced phase shifts, as this receptor may be expressed in the human brain. [152]



Melatonin Therapy in Humans

The role of melatonin as a "wonder drug" has received great attention from the lay press. [133] [154] The proposed beneficial and therapeutic uses of melatonin include treatment of jet lag, slowing or reversing the progression of aging, and enhancing immune function. As noted earlier, the most studied and established role of melatonin is that of phase shifting and resetting circadian rhythms. In this context, melatonin has been used to treat jet lag and may be effective in treating circadian-based sleep disorders. [154] In addition, melatonin administration has been shown to regulate sleep in humans. [130] [133] Specifically, melatonin has a hypnotic effect at relatively low doses. Melatonin therapy has also been suggested as a way to treat seasonal affective disorders. It is important to note that melatonin is now available over the counter and without a prescription throughout the United States. However, there is a striking paucity of controlled clinical studies of the relative efficacy and safety of melatonin administration. This should be viewed as problematic because melatonin is an endocrine hormone, and most hormones are not widely available without a prescription. [133] [152] [154] Clearly, controlled clinical studies are needed to assess fully the therapeutic potential and safety of melatonin in humans.



HYPOPHYSEOTROPIC HORMONES AND NEUROENDOCRINE AXES With the demonstration by the first half of the 1900s that pituitary secretion was controlled by hypothalamic hormones released into the portal circulation, the search was on for the hypothalamic releasing factors. The search for hypothalamic neurohormones with anterior pituitary regulating properties focused on extracts of stalk, median eminence, neural lobe, and hypothalamus from sheep and pigs. To give some idea of the herculean nature of this effort, approximately 250,000 hypothalamic fragments were required to purify and characterize the first such factor, TRH. [20] [155] The identification and characterization of TRH in 1970 and of other releasing hormones ultimately led to the Nobel Prize in Medicine in 1977 for Andrew Schally and Roger Guillemin. [38] [156] Such hypophyseotropic substances were initially called releasing factors but are now more commonly called releasing hormones. All of the hypothalamic-pituitary regulating hormones are peptides with the exception of dopamine, which is a biogenic amine that is the principal prolactin-inhibiting factor (PIF) (Table 7-3) . All are now available for human investigation and treatment, and therapeutic analogues have been synthesized for dopamine, GnRH, and somatostatin. In addition to regulating hormone release, some hypophyseotropic factors control pituitary cell differentiation and proliferation and hormone synthesis. Somatostatin and dopamine are inhibitory, and some act on more than one pituitary hormone. For example, TRH is a potent releaser of prolactin (PRL) and of thyrotropin and under some circumstances releases corticotropin and growth hormone (GH). GnRH releases both LH and follicle-stimulating hormone (FSH). Somatostatin inhibits the secretion of GH, thyrotropin, and a wide variety of nonpituitary hormones. The principal inhibitor of PRL secretion, dopamine, also inhibits secretion of thyrotropin, gonadotropin, and, under certain conditions, GH. Dual control is exerted by the interaction of inhibitory and stimulatory hypothalamic hormones. For example, somatostatin interacts with growth hormonereleasing hormone (GHRH) and TRH to control secretion of GH and thyrotropin, respectively, and dopamine interacts with prolactin-releasing factors (PRFs) to regulate PRL secretion. Some hypothalamic hormones act synergistically; for example, CRH and vasopressin act together to regulate the release of pituitary adrenocorticotropic hormone (ACTH). Secretion of the releasing hormones in turn is regulated by

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TABLE 7-3 -- Structural Formulas of Principal Human Hypothalamic Peptides Directly Related to Pituitary Secretion Vasopressin Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH 2 (MW = 1084.38) Oxytocin Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH 2 (MW = 1007.35) Thyrotropin-releasing hormone pGlu-His-Pro-NH 2 (MW = 362.42) Gonadotropin-releasing hormone pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 (MW = 1182.39) Corticotropin-releasing hormone Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH (MW = 4758.14) Growth hormonereleasing hormone (GHRH 140, 144-NH2 , Human) Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala (MW = 4544.73), [-Arg-Ala-Arg-Leu-NH 2 ] (MW = 5040.4) Somatostatin Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys (MW = 1638.12) Somatostatin-28 Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys (MW = 3149.0) Somatostatin-28 (112) Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu (MW = 1244.49) Vasoactive intestinal peptide (human, pig, rat) His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH 2 (MW = 3326.26) Prolactin-releasing peptide (PrRP31, PrRP20) Ser-Arg-Thr-His-Arg-His-Ser-Met-Glu-Ile-Arg-Thr-Pro-Asp-Ile-Asn-Pro-Ala-Trp-Tyr-Ala-Ser-Arg-Gly-Ile-Arg-Pro-Val-Gly-Arg-Phe-NH

2

(MW = 3665.16; 2273.58)

Ghrelin Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg (MW = 3314.9) [Ser 3 is n-octanoylated] MW, molecular weight. neurotransmitters and neuropeptides released by a complex array of neurons synapsing with hypophyseotropic neurons. Control of secretion is also exerted through feedback control by hormones such as glucocorticoids, gonadal steroids, thyroid hormone, anterior pituitary hormones (short-loop feedback control), and hypophyseotropic factors themselves (ultrashortloop feedback control). The distribution of the hypophyseotropic hormones is not limited to the hypothalamus. Most are also found in nonhypophyseotropic hypothalamic neurons, in extrahypothalamic regions of the brain, and in other organs where they may have functions (e.g., effects on behavior or homeostasis) unrelated to pituitary regulation. Most, although not all, of the peptides, hormones, and neurotransmitters involved in the regulation of hypothalamic-pituitary control belong to the G proteincoupled receptor family (Table 7-4) . Feedback Concepts in Neuroendocrinology

In order to understand the regulation of each hypothalamicpituitary-target organ axis, it is important to understand some basic concepts of homeostatic systems. A simplified account of feedback control in relation to neuroendocrine regulation is presented in this section. [157] [158] [159] [160] Hormonal systems form part of a feedback loop in which the controlled variable (generally the blood hormone level or some biochemical surrogate of the hormone) determines the rate of secretion of the hormone. In negative feedback systems the controlled variable inhibits hormone output, and in positive feedback control systems the controlled variable increases

hormone secretion. Both negative and positive endocrine feedback control systems can be part of a closed loop, in which regulation is entirely restricted to the interacting regulatory glands, or an open loop, in which the nervous system influences the feedback loop. All pituitary feedback systems have nervous system inputs that either alter the set-point of the feedback control system or introduce open-loop elements that can influence or override the closed-loop control elements. In engineering formulations of feedback, three controlled variables can be identified: a sensing element that detects the concentration of the controlled variable, a reference input that defines the proper control levels, and an error signal that determines the output of the system. The reference input is the set-point of the system. Hormonal feedback control systems resemble engineering systems in that the concentration of the hormone in the blood (or some function of the hormone) regulates the output of the controlling gland. Hormonal feedback differs from engineering systems in that the sensor element and the reference input element are not readily distinguishable. The set-point of the controlled variable is determined by a complex cascade beginning with the kinetics of binding to a receptor and the activities of successive intermediate messengers. Sophisticated models incorporating control elements, compartmental analysis, and hormone production and clearance rates have been developed for many systems.



Endocrine Rhythms

Virtually all functions of living animals (regardless of their position on the evolutionary scale) are subject to periodic or cyclic changes, many of which are influenced mainly by the nervous system ( see Table 7-5 for definitions). [161] [162] [163] [164] Most periodic changes are free-running; that is, they are intrinsic to the organism independent of the environment and are driven by a biologic "clock." Most free-running rhythms can be coordinated ( entrained) by external signals (cues), such as light-dark changes, meal patterns, cycles of the lunar periods, or the ratio of the length of day to the length of night. External signals of this type ( zeitgeber or time givers) do not bring about the rhythm but provide

97

TABLE 7-4 -- Receptors for Neurotransmitters and Neuropeptides Involved in Hypothalamic-Pituitary Control * Group Name General Structure Transmembrane Sequences

Mode of Action

Classical Neurotransmitters Biogenic amines 1>A

2A

-Adrenoreceptors

-Adrenoreceptors

-Adrenoreceptors

Serotonin (5-OH-tryptamine)

Dopamine

Histamine

1A

7

Gq/11

1B

7

Gq/11

1D

7

Gq/11

2A

7

Gi/o

2B

7

Gi/o

2C

7

Gi/o

1

7

GS

2

7

GS

3

7

GS

5-HT 1A

7

Gi/o

5-HT 1B

7

Gi/o

5-HT 1D

7

Gi/o

5-HT 1E

7

Gi/o

5-HT 2A

7

Gq/11

5-HT 2B

7

Gq/11

5-HT 2C

7

Gq/11

5-HT 3

4

Cation channel

5-HT 4

7

GS

D1

7

GS

D2

7

Gi/o

D3

7

Gi/o

D4

7

Gi/o

D5

7

GS

H1

7

Gq/11

H2

7

GS

H3

7

Gi/o

M1

7

Gq/11

M2

7

Gq/11

M3

7

Gq/11

M4

7

Gi/o

M5

7

Gq/11

Muscle

4 Multiunit

Na/K/Ca

Ganglionic

4 Multiunit

Na/K/Ca

Central nervous system

4 Multiunit

Na/K/Ca

NMDA

Multiunit ?4TM

Na/K/Ca

AMPA

Multiunit 3TM

Na/K/Ca

Kainate

?

Na/K/Ca

mGlu1

7

Gq/11

mGlu2

7

Gi/o

mGlu3

7

Gi/o

Acetylcholine Muscarinic

Nicotinic

Excitatory amino acids (glutamate) Ionotropic

Metabotropic

mGlu4

7

Gi/o

mGlu5

7

Gq/11

mGlu6

7

Gi/o

mGlu7

7

Gi/o

GABAA

Multiunit

Internal Cl

GABAB

7 Hetero-dimer gb1 gb2

Gi/o

V1A

7

Gq/11

V1B

7

Gq/11

V2

7

GS

OT

7

Gq/11

TRH

TRH

7

Gq/11

GHRH

GHRH

7

GS

LHRH

LHRH

7

Gq/11

CRH

CRH

7

GS

Somatostatin

SST1

7

Gi/o

SST2A

7

Gi/o

SST2B

7

Gi/o

SST3

7

Gi/o

SST4

7

Gi/o

SST5

7

Gi/o

7

Gi/o

7

Gi/o

7

Gi/o

MC1

7

GS

MC2 (corticotropin)

7

GS

MC4

7

GS

MC5

7

GS

7

GS

Inhibitory amino acid (-Aminobutyric Acid [GABA])

Neuropeptides Neurohypophyseal hormones Vasopressin

Oxytocin Hypophyseotropic hormones

Endogenous opioid peptides

Melanocortins

MC3

Gut-brain peptides Tachykinins Substance P

NK1

7

Gi/o

Substance K

NK2

7

Gi/o

Neurokinin B

NK3

7

Gi/o

Neurotensin

NT

7

Gq/11

VIP

VIP1

7

GS

VIP2

7

GS

PACAP

PACAP

7

GS

Galanin

G

7

Gi/o

Cholecystokinin

CCKA

7

Gq/11

CCKB (gastrin receptor)

7

Gq/11

Y1

7

Gi/o

Y2

7

Gi/o

Y5

7

Gi/o

AT1

7

Gq/11

AT2

7

cGMP

ANPA

1

cGMP

ANPB

1

cGMP

ETA

7

Gq/11

ETB

7

Gq/11

Neuropeptide Y

Vasoactive peptides Angiotensin

Atrial natriuretic peptide

Endothelin

NMDA, N-methyl-D-aspartate; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazdeproprionic acid; TRH, thyrotropin-releasing hormone; GHRH, growth hormone-releasing hormone; LHRH, luteinizing hormone-releasing hormone; CRH, corticotropin-releasing hormone; NT, neurotensin; VIP, vasoactive peptide; PACAP, pituitary adenylate cyclase activating peptide; cGMP, cyclic guanosine monophosphate. cGMP: Guanylate cyclase activity intrinsic to the receptor.

Gi/o : Receptor coupled to the G i/o family. Opens K+ channel, closes Ca2+ channel, inhibits adenylate cyclase. Gq/11 : Receptor coupled to the G q/11 family. Stimulates phosphoinositol cascade. GS : Receptor coupled to the G S family. Stimulates adenylate cyclase and increases intracellular cAMP. Some receptors have intrinsic tyrosine phosphorylase activity, others have intrinsic tyrosine hydroxylase activity. The former stimulate phosphorylation of tyrosine kinases; the latter stimulate breakdown of tyrosine kinase. The designation of functional type is oversimplified. Many examples can be cited in which receptor activation can stimulate both adenylate cyclase and phosphoinositide turnover. Adapted from Watson S, Girdlestone D. Receptor and channel nomenclature supplement. Trends Pharmacol Sci 1995; (Suppl 16):173. *Receptors cited are human or rat if human not available.

98

TABLE 7-5 -- Terms Used to Describe Cyclic Endocrine Phenomena Period:

length of the cycle

Circadian:

around a day

Diurnal:

exactly a day

Ultradian:

less than a day, i.e., minutes or hours

Infradian:

longer than a day, i.e., month or year

Mean:

arithmetic mean of all values within a cycle

Range:

difference between the highest and lowest values

Nadir:

minimal level (inferred from mathematical curve fitting calculations)

Acrophase:

time of maximal levels (inferred from curve fitting)

Zeitgeber:

"time-giver" (German), the external cue, usually the light-dark cycle that synchronizes endogenous rhythms

Entrainment:

the process by which an endogenous rhythm is regulated by a zeitgeber

Phase shift:

induced change in an endogenous rhythm

Intrinsic clock: neural structures that possess intrinsic capacity for spontaneous rhythms; for circadian rhythms these are located in the suprachiasmatic nucleus Adapted from Van Cauter E, Turek FW. Endocrine and other biological rhythms. In DeGroot LJ (ed). Endocrinology, 3rd ed. Philadelphia, WB Saunders, 1995, pp 24972548. the synchronizing time cue. Many endogenous rhythms have a period of approximately 24 hours ( circadian [around a day] or diurnal rhythms). Circadian changes follow an intrinsic program that is about 24 hours long, whereas diurnal rhythms can be either circadian or dependent on shifts in light and dark. [165] Rhythms that occur more frequently than once a day are ultradian. Infradian rhythms have a period longer than 1 day, as in the approximately 27-day human menstrual cycle and the yearly breeding patterns of some animals. Most endocrine rhythms are circadian (see Fig. 7-12) (Figure Not Available) . [164] The secretion of GH and PRL is maximal shortly after an individual has gone to sleep, and that of cortisol is maximal between 2 and 4 AM. Thyrotropin secretion is lowest in the morning between 9 AM and 12 noon and maximal between 8 PM and midnight. Gonadotropin secretion in adolescents is increased at night. [162] Superimposed on the circadian cycle are ultradian bursts of hormone secretion. Gonadotropin secretion during adolescence is characterized by rapid, high-amplitude pulsations at night, whereas in sexually mature individuals secretory episodes are lower in amplitude and occur throughout the 24 hours. [162] GH, corticotropin, [164] and PRL[166] are also secreted in brief, fairly regular pulses. The short-term fluctuations in hormonal secretion have important functional significance. In the case of gonadotropins, the normal endogenous rhythm of pituitary secretion reflects the pulsatile release of LHRH. The period of approximately 90 minutes between the peak of pulses corresponds to the optimal timing to induce maximal pituitary stimulation. Episodic secretion of GH also enhances its biopotency, but for many rhythms the function is not clear. Most homeostatic activities are also rhythmic, including body temperature, water balance, blood volume, sleep, and activity. Assessment of endocrine function must take into account the variability of hormone levels in the blood, and appropriately obtained samples at different times of day or night may provide useful dynamic indicators of hypothalamic-pituitary function. For example, the loss of diurnal rhythm of GH and corticotropin secretion may be an early sign of hypothalamic dysfunction. Furthermore, the optimal timing for the administration of glucocorticoids to suppress corticotropin secretion (as in therapy for congenital adrenal hyperplasia) must take into account the varying suppressibility of the pituitary at different times of day. The best understood neural structures responsible for circadian rhythms are the suprachiasmatic nuclei, paired structures in the anterior hypothalamus above the optic chiasm.[157] [161] Individual cells of the suprachiasmatic nuclei have an intrinsic capacity to oscillate in a circadian pattern, [167] and the nucleus is organized to permit many reciprocal neuron-neuron interactions through direct synaptic contacts. It is especially rich in neuropeptides, including somatostatin, vasoactive intestinal polypeptide (VIP), NPY, and neurotensin, and microinjections of NPY into the SCN reset the timing cycle of some circadian rhythms in hamsters. [168] The SCN also responds to the pineal hormone melatonin through melatonin receptors. [149] [169] The SCN receives neuronal input from many parts of the brain and from a direct projection from the retina that is distinct from the visual pathway, called the retinohypothalamic pathway, which is the route by which the nucleus is cued by light-dark changes. [161] Anatomic dissociation of pathways subserving subjective visual and nonvisual stimulation explains the finding that circadian hormonal rhythms in some blind individuals are entrained to the light-dark cycle. [170] Circadian rhythms during fetal life are regulated by maternal circadian rhythms. [171] Circadian changes can be detected 2 to 3 days before birth, and suprachiasmatic nuclei from fetuses of this age display spontaneous rhythmicity in vitro. Maternal regulation of fetal circadian rhythms may be mediated by circulating melatonin or by cyclic changes in the food intake of the mother. Metabolic changes in the SCN, such as increased uptake of 2-deoxyglucose and an increased level of VIP, accompany circadian rhythms. This nucleus projects to the pineal gland by way of the autonomic nervous system (see later in the section on the pineal gland) and regulates its activity. In addition to determining patterns of pituitary secretion, the circadian pacemaker influences many homeostatic functions. In humans, the alteration of sleep brought about by jet lag and by working night shifts has profound effects on the sense of well-being and efficiency [172] and may be a factor in the pathogenesis of seasonal affective disorder, a condition characterized by depression in winter when days are short and levels of illumination are low. [173] Seasonal affective disorder has been treated by manipulating the light-dark cycle. [173] The timing of the circadian pacemaker can be shifted in humans by the administration of triazolam, a short-acting benzodiazepine, earlier, or by altered patterns of intense illumination. [172]

[ 174]

or melatonin, as described



Thyrotropin-Releasing Hormone Chemistry and Evolution

TRH, the smallest known peptide releasing hormone, is the tripeptide pyroGlu-His-Pro-NH 2 . The TRH peptide sequence is repeated six times within the human TRH pre-prohormone gene (Fig. 7-13) (Figure Not Available) . The rat pro-TRH precursor contains five TRH peptide repeats flanked by dibasic residues (Lys-Arg or Arg-Arg), along with seven or more non-TRH peptides. Two prohormone convertases, PC1 and PC2, cleave the TRH tripeptides at the dibasic residues within the regulated secretory pathway. Carboxypeptidase E then removes the dibasic residues, leaving the sequence Gln-His-Pro-Gly. This peptide is then amidated at the C-terminus by peptidylglycine alpha-amidating monooxygenase, with Gly acting as the amide donor. The amino-terminal pyro-Glu residue results from cyclization of the Gln. Although the TRH tripeptide is the only established hormone encoded within its large prohormone, the rat pro-TRH yields seven additional peptides that have unique tissue distributions. [175] Several biologic activities of these peptides have

99

Figure 7-13 (Figure Not Available) Structure of human thyrotropin-releasing hormone (TRH) gene and peptide, showing six repeating codons for the TRH sequence. CPE, carboxypeptidase E; PAM, peptidylglycine alpha-amidating monooxygenase; PC1, prohormone convertase 1. (From Yamada M, Radovick S, Wondisford FE, et al. Cloning and structure of human genomic DNA and hypothalamic cDNA encoding human preprothyrotropin-releasing hormone. Mol Endocrinol 1990; 4:551556.)

been observed: pre-pro-TRH [160] [161] [162] [163] [164] [165] [166] [167] [168] [169] may be a hypophyseotropic factor because it is released from hypothalamic slices [176] and potentiates the thyrotropin-releasing effects of TRH. [177] Pro-TRH [177] [178] [179] [180] [181] [182] [183] [184] [185] [186] [187] [188] [189] [190] [191] [192] [193] [194] [195] [196] [197] [198] [199] is also released from the median eminence[176] and appears to inhibit ACTH release. [178] TRH is a phylogenetically ancient peptide, being found in primitive vertebrates, such as the lamprey, and even invertebrates such as the snail. TRH is widely expressed in both CNS and periphery in amphibians, reptiles, and fishes but does not stimulate thyrotropin release in these poikilothermic vertebrates. [179] Thus, TRH has multiple peripheral and central activities and was co-opted as a hypophyseotropic factor midway during the evolution of vertebrates, perhaps specifically as a factor needed for coordinate regulation of temperature homeostasis. Effects on the Pituitary Gland and Mechanism of Action

After intravenous injection of TRH in humans, serum thyrotropin levels rise within a few minutes (Fig. 7-14) ,[180] [181] [182] followed by a rise in serum triiodothyronine (T 3 ) levels; there is an increase in thyroxine (T 4 ) release as well, but a change in blood levels of T 4 is usually not demonstrable because the pool of circulating T 4 (most of which is bound to carrier proteins) is so large. The clinical applications of TRH testing are covered later in this chapter and in Chapter 10 . TRH action on the pituitary is blocked by previous treatment with thyroid hormone, which is a crucial element in feedback control of pituitary thyrotropin secretion. TRH is also a potent PRF (Fig. 7-15) . [180] [181] [182] The time course of response of blood PRL levels to TRH, the dose-response characteristics, and the suppression by thyroid hormone pretreatment (all of which parallel changes in thyrotropin secretion) suggest that TRH may be involved in the regulation of PRL secretion. Moreover, TRH is present in the hypophyseal-portal blood of lactating rats. [183] However, it is unlikely to be a physiologic regulator of PRL secretion [184] [185] because the PRL response to nursing in humans is unaccompanied by changes in plasma thyrotropin levels. [185] Nevertheless, TRH may occasionally cause hyperprolactinemia (with or without galactorrhea) in patients with hypothyroidism. In normal individuals TRH has no influence on the secretion of pituitary hormones other than thyrotropin and PRL, but it enhances the release of human growth hormone (hGH) in acromegaly and of corticotropin in some patients with Cushing's disease. Furthermore, prolonged stimulation of the normal pituitary with GHRH can sensitize it to the hGH-releasing effects of TRH. [186] [187] TRH also causes the release of hGH in some patients with uremia, hepatic disease, anorexia

Figure 7-14 Effect of intravenous injection of thyrotropin-releasing hormone on serum thyrotropin levels in humans. TRF, thyrotropin-releasing hormone; TSH, thyrotropin. (From Hershman JM, Pittman JA Jr. Control of thyrotropin secretion in man. N Engl J Med 1971; 285:9971006. Reprinted by permission of The New England Journal of Medicine.)

100

Figure 7-15 Prolactin (PRL) and thyrotropin (TSH) secretory responses to intravenous injection of 800 µg of thyrotropin-releasing hormone (TRH) in humans. This figure shows that TRH induces discharge of both PRL and thyrotropin, that the effect in females is greater than that in males (presumably owing to estrogen sensitization of the pituitary), and that thyrotoxicosis inhibits the response of both PRL and thyrotropin to TRH. An inhibitory effect on the TRH response is noted at the upper limit of the normal range of thyroid hormone levels and is a sensitive test of minor degrees of thyroid hormone excess. Although TRH is a potent prolactin-releasing factor (PRF), there is evidence that there is another PRF physiologically connected to PRL regulation. (Replotted from data of Bowers C, Friesen HG, Hwang P, et al. Prolactin and thyrotropin release in man by synthetic pyroglutamylhistidyl-prolinamide. Biochem Biophys Res Commun 1971; 45:10331041.)

nervosa, and psychotic depression [182] and in children with hypothyroidism. TRH inhibits sleep-induced hGH release through its actions in the CNS (see later in the section on extrapituitary actions of TRH). Stimulatory effects of TRH are initiated by binding of the peptide to specific receptors on the plasma membrane of the thyrotroph. [24] [188] Neither thyroid hormone nor somatostatin, both of which antagonize the effects of TRH, interfere with its binding. TRH was originally thought to activate membrane adenylate cyclase to stimulate formation of cAMP,[182] [189] and cAMP in turn was thought to stimulate thyrotropin secretion. However, cAMP does not increase under all conditions of TRH-induced thyrotropin release, [190] and it is now clear that TRH action is mediated mainly through hydrolysis of phosphatidylinositol, with phosphorylation of key protein kinases and an increase in intracellular free Ca 2+ as the crucial step in postreceptor activation (see Chapter 5) . [190] [191] [192] TRH effects can be mimicked by exposure to a Ca2+ ionophore and are partially abolished by a Ca 2+ -free medium. TRH stimulates the formation of mRNAs coding for thyrotropin [193] and PRL,[194] confirming that this peptide is trophic as well as a releasing factor. TRH is degraded to acid TRH and to the dipeptide histidylprolineamide, which cyclizes nonenzymatically to histidylproline diketopiperazine (cyclic His-Pro).

[195]

Acid

TRH has some behavioral effects in rats that are similar to those of TRH but no other proven actions. Cyclic His-Pro is reported to act as a PRF and to have other neural effects, including reversal of ethanol-induced sleep (TRH is also effective in this system), elevation of brain cyclic guanosine monophosphate levels, an increase in stereotypical behavior, modification of body temperature, and inhibition of eating behavior. Some of the effects of TRH may be mediated through cyclic His-Pro, but the fact that cyclic His-Pro is abundant in some areas and is not proportional to the amount of TRH suggests that the peptide may not be derived solely from TRH.[196] [197] Extrapituitary Function

TRH is present in virtually all parts of the brain: cerebral cortex, circumventricular structures, neurohypophysis, pineal gland, and spinal cord. [182] [198] [199] [200] [201] [202] TRH is also found in pancreatic islet cells and in the gastrointestinal tract. [203] Although it exists in low concentration, the total amount in extrahypothalamic tissues exceeds the amount in the hypothalamus. The extensive extrahypothalamic distribution of TRH, its localization in nerve endings, and the presence of TRH receptors in brain tissue suggest that TRH serves as a neurotransmitter or neuromodulator outside the hypothalamus. TRH is a general stimulant [182] [204] [205] [206] and induces hyperthermia on intracerebroventricular injection, suggesting a role in central thermoregulation. Clinical Applications

The use of TRH for the diagnosis of hyperthyroidism is less common since the development of ultrasensitive assays for thyroid-stimulating hormone (TSH) [182] (see Chapter 10) ; its use to discriminate between hypothalamic and pituitary causes of thyrotropin deficiency has also declined because of the test's poor specificity, [182] [207] but the application of ultrasensitive assays in conjunction with the TRH test has not been fully evaluated. [208] TRH testing is also not of value in the differential diagnosis of causes of hyperprolactinemia [209] but is useful for the demonstration of residual abnormal somatotropin-secreting cells in acromegalic patients who release hGH in response to TRH before treatment. Studies of the effect of TRH on depression have shown inconsistent results, [210] possibly because of poor blood-brain barrier penetration. [211] Intrathecal administration of TRH may improve responses in depressed patients, [211] [212] but its clinical utility is unknown. Although a role for TRH in depression is not established, many depressed patients have a blunted thyrotropin response to TRH and changes in TRH responsiveness correlate with the clinical course. [210] The mechanism by which blunting occurs is unknown. [213] TRH has been proposed as a treatment for women with threatened premature labor to stimulate the production of lung surfactant in the preterm fetus. Despite encouraging results in early studies, several large-scale trials failed to show improvement in the survival of babies so treated. [214] [215] TRH has been evaluated for the treatment of spinal muscle atrophy and amyotrophic lateral sclerosis; transient improvement in strength was reported in both disorders,[216] [217] [218] [219] but the combined experience at many centers using a variety of treatment protocols including long-term intrathecal administration failed to confirm efficacy. [220] [221] [222] TRH administration also reduces the severity of experimentally induced spinal and ischemic shock [223] [224] [225] ; preliminary studies in humans suggest that TRH treatment may improve recovery after spinal cord injury [226] and head trauma.[227] TRH has been used to treat children with neurologic disorders including West's syndrome, Lennox-Gastaut syndrome, early infantile epileptic encephalopathy, and intractable epilepsy. [228] TRH has been proposed to be an analeptic agent. Sleeping or drug-sedated animals were awakened by the administration of TRH, [229] TRH reportedly reversed sedative effects of ethanol in humans,[230] and TRH is said to have awakened a patient with a profound sleep disorder caused by a hypothalamic and midbrain eosinophilic granuloma. [231] Regulation of Thyrotropin Release

The secretion of thyrotropin is regulated by two interacting elements: negative feedback by thyroid hormone and open-loop neural control by hypothalamic hypophyseotropic factors

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Figure 7-16 Regulation of the hypothalamic-pituitary-thyroid axis. AGRP, agouti-related protein; CART, cocaine and amphetamineregulated transcript; CRH, corticotropin-releasing hormone; NPY, neuropeptide Y; POMC, proopiomelanocortin; T 3 , triiodothyronine; T 4 , thyroxine; TRH, thyrotropin-releasing hormone; TSH, thyrotropin; OB-R, leptin receptor.

(Fig. 7-16) . Thyrotropin secretion is also modified by other hormones, including estrogens, glucocorticoids, and possibly GH, and is inhibited by cytokines in the pituitary and hypothalamus. Aspects of the pituitary-thyroid axis are also considered in Chapter 10 .[182] [198] [232] [233] [234] [235] [236] Feedback Control: Pituitary-Thyroid Axis

In the context of a feedback system, the level of thyroid hormone in blood or of its unbound fraction is the controlled variable and the set-point is the normal resting level of plasma thyroid hormone. Secretion of thyrotropin is inversely regulated by the level of thyroid hormone so that deviations from the set-point of control lead to appropriate changes in the rate of thyrotropin secretion (Fig. 7-17) . Factors that determine the rate of thyrotropin secretion required to maintain a given level of thyroid hormone include the rate at which thyrotropin and thyroid hormone disappear from the blood (turnover rate) and the rate at which T 4 is converted to its more active form, T3 .

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Figure 7-17 Relationship between plasma thyrotropin levels and thyroid hormone as determined by plasma protein-bound iodine (PBI) measurements in humans and rats. These curves illustrate, in the human ( A) and the rat (B), that plasma thyrotropin levels are a curvilinear function of plasma thyroid hormone level. Human studies were carried out by giving myxedematous patients successive increments of thyrotropin T 4 at approximately 10-day intervals. Each point represents simultaneous measurements of plasma PBI and plasma thyrotropin at various times in the six patients studied. The rat studies were performed by treating thyroidectomized animals with various doses of T 4 for 2 weeks before assay of plasma thyrotropin and plasma PBI. These curves illustrate that the secretion of thyrotropin is regulated over the entire range of thyroid hormone levels. At the normal set point for T 4 , the small changes above and below the control level are followed by appropriate increases or decreases in plasma thyrotropin. TSH, thyrotropin; T 4 , thyroxine. (A from Reichlin S, Utiger RD. Regulation of the pituitary thyroid axis in man: relationship of TSH concentration to concentration of free and total thyroxine in plasma. J Clin Endocrinol Metab 1967; 27:251255, copyright by The Endocrine Society. B from Reichlin S, Martin JB, Boshans RL, et al. Measurement of TSH in plasma and pituitary of the rat by a radioimmunoassay utilizing bovine TSH: effect of thyroidectomy or thyroxine administration on plasma TSH levels. Endocrinology 1970; 87:10221031, copyright by The Endocrine Society.)

Thyroid hormones act on both the pituitary and the hypothalamus. Feedback control of the pituitary by thyroid hormone is remarkably precise. Administration of small doses of T3 and T4 inhibited the thyrotropin response to TRH, [237] and barely detectable decreases in plasma thyroid hormone levels were sufficient to sensitize the pituitary to TRH. [238] TRH stimulates thyrotropin secretion within a few minutes through its action on a membrane receptor, whereas thyroid hormone actions,

mediated by intranuclear receptors, require several hours to take effect (see Chapter 10) . The secretion of hypothalamic TRH is also regulated by thyroid hormone feedback. Systemic injections of T 3 [239] or implantations of tiny T 3 pellets in the paraventricular nucleus of hypothyroid rats [240] (Fig. 7-18) (Figure Not Available) reduced the concentration of TRH mRNA and TRH prohormone in TRH-secreting cells. Thyroid hormone also suppressed TRH secretion into hypophyseal-portal blood in sheep. [241] T4 in the blood gains access to TRH-secreting neurons in the hypothalamus by way of the CSF. The hormone is taken up by epithelial cells of the choroid plexus of the lateral ventricle of the brain, bound within the cell to locally produced transthyretin (T 4 -binding prealbumin), and then secreted across the blood-brain barrier. [242] Within the brain, T 4 is converted to T 3 by type II deiodinase, and T 3 interacts with subtypes of the thyroid hormone receptor, TR 1 , TR1 , and TR2 , in the paraventricular nucleus and other brain cells (see Chapter 10) .[243] Thereby the set-point of the pituitary-thyroid axis is determined by thyroid hormone levels within the brain. [244] T 3 in the circulation is not transported into brain in this manner but presumably gains access to the paraventricular TRH neurons across the blood-brain barrier. The brain T4 transport and deiodinase system account for the fact that higher blood levels of T 3 are required to suppress pituitary-thyroid function after administration of T 3 than after administration of T 4 . [244] Transthyretin is present in the brain of early reptiles and in addition is synthesized by the liver in warm-blooded animals. [242] During embryogenesis in mammals, transthyretin is first detected when the blood-brain barrier appears, ensuring thyroid hormone access to the developing nervous system. Neural Control

The hypothalamus determines the set-point of feedback control around which the usual feedback regulatory responses are elicited. Lesions of the thyrotropic area lower basal thyroid hormone levels and make the pituitary more sensitive to inhibition by thyroid hormone, [232] and high doses of TRH raise thyrotropin and thyroid hormone levels. [245] Synthesis of TRH in the paraventricular nuclei is regulated by feedback actions of thyroid hormones. [246] The hypothalamus can override normal feedback control through an open-loop mechanism involving neuronal inputs to the hypophyseotropic TRH neurons (see Fig. 7-16) . For example, cold exposure caused a sharp increase in thyrotropin release in animals [247] [248] and in human newborns.[249] Circadian changes in thyrotropin secretion are another example of brain-directed changes in the set-point of feedback control, but if thyroid hormone levels are sufficiently elevated, as in hyperthyroidism, TRH cannot overcome the inhibition. Hypothalamic regulation of thyrotropin secretion is also influenced by two inhibitory factors, somatostatin and dopamine. [235] [250] [251] Antisomatostatin antibodies increase basal thyrotropin levels and potentiate the response to stimuli that normally induce thyrotropin release in the rat, such as cold exposure and TRH administration. [250] [251] Thyroid hormone in turn inhibits the release of somatostatin, [252] implying coordinated, reciprocal regulation of TRH and somatostatin by thyroid hormone. GH stimulates hypothalamic somatostatin synthesis [253] and can inhibit thyrotropin secretion. The role of

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Figure 7-18 (Figure Not Available) A and B, Direct effects of triiodothyronine (T 3 ) on thyrotropin-releasing hormone (TRH) synthesis in the rat hypothalamic paraventricular nucleus (parvicellular division) were shown in this experiment by immunohistochemical detection of pre-pro TRH(2550) after implantation of a pellet of either T 3 (B) or inactive diiodotyrosine (T 2 ) as a control (A). The T 2 pellet had no effect on the concentration of pre-proTRH (A). In contrast, the TRH prohormone (B) concentrations were markedly reduced. These studies indicate that thyroid hormone regulates the hypothalamic component of the pituitary-thyroid axis as well as the pituitary thyrotrope itself. C and D, Effects of 1 hour at 4°C on TRH messenger ribonucleic acid (mRNA). E to G, Effects on TRH mRNA levels of starvation (F) and leptin replacement during starvation (G). (Photographs courtesy of Dr. R. M. Lechan. From Dyess EM, Segaerson TP, Liposits Z, et al. Triiodothyronine exerts direct cell-specific regulation of thyrotropin-releasing hormone gene expression in the hypothalamic paraventricular nucleus. Endocrinology 1988; 123:22912297, copyright by The Endocrine Society.)

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somatostatin in the regulation of thyrotropin secretion in humans is uncertain. Dopamine has modest effects on thyrotropin secretion, and blockade of dopamine receptors (in the human) stimulates thyrotropin secretion slightly. [235] Changes in the metabolism of thyroid hormone also influence T 3 homeostasis within the brain. In states of thyroid hormone deficiency, brain T 3 levels are maintained by an increase in the deiodinase that converts T 4 to T3 . [254] The pineal gland has been reported to inhibit thyroid function in some [255] but not all [256] studies. The pineal gland contains TRH, and in the frog its content changes with the season and with light and dark cycles independently of hypothalamic thyrotropin. [257] Circadian Rhythm

Plasma thyrotropin in humans is characterized by a circadian periodicity, with a maximum between 9 PM and 5 AM and a minimum between 4 PM and 7 PM. [258] Smaller ultradian thyrotropin peaks occur every 90 to 180 minutes, [259] probably because of bursts of TRH release from the hypothalamus, and are physiologically important in controlling the synthesis and glycosylation of thyrotropin. [260] Glycosylation is a determinant of thyrotropin potency. Temperature

External cold exposure activates and high ambient temperature inhibits the pituitary-thyroid axis in animals, [248] and analogous changes occur in humans under certain conditions. Exposure of infants to cold at the time of delivery causes an increase in blood thyrotropin levels, possibly because of alterations in the turnover and degradation of the thyroid hormones. [249] Blood thyroid hormone levels are higher in the winter than in the summer in individuals in cold climates [261] but not in other climates. [249] However, it is difficult to show that changes in environmental or body temperature in adults influence thyrotropin secretion. For example, exposure to cold ambient temperature or central hypothalamic cooling does not modify thyrotropin levels in young men. [262] Behavioral changes, activation of the sympathetic nervous system, and shivering appear to be more important in temperature regulation in adults than the thyroid response. [248] The autonomic nervous system and the thyroid axis work together to maintain temperature homeostasis in mammals, and TRH plays a role in both pathways. [263] Hypothalamic TRH release is rapidly (30 to 45 minutes) increased in rats exposed to cold. [247] Rapid inhibition of somatostatin release in the median eminence has also been documented, and both changes appear to play important roles in the rise in plasma TSH induced by cold exposure. TRH mRNA is elevated within an hour of cold exposure (see Fig. 7-18 C and D) (Figure Not Available) . The regulation of hypophyseotropic TRH release and expression by cold is largely mediated by catecholamines. Noradrenergic and adrenergic fibers, originating in the brain stem, are found in close proximity to TRH nerve endings in the median eminence, and a rapid rise in TRH release was seen after norepinephrine treatment of hypothalamic fragments containing mainly median eminence. [264] Brain stem adrenergic and noradrenergic fibers also make synaptic contacts with TRH neurons in the PVH (see Fig. 7-16) , [265] [266] and thus catecholamines are likely to be involved in the regulation of TRH gene expression by cold. TRH neurons in the PVH are densely innervated by NPY terminals, [267] and a portion of the NPY terminals arising from the C1, C2, C3, and A1 cell groups of the brain stem and projecting to the PVH are known to be catecholaminergic. [268] Somatostatin, dopamine, and serotonin also play a variety of roles in the regulation of TRH. Stress

Stress is another determinant of thyrotropin secretion. [232] [234] In humans physical stress inhibits thyrotropin release, as indicated by the finding that in the euthyroid sick syndrome low T 3 and T4 do not cause compensatory increases in thyrotropin secretion as would occur in normal individuals. [269] [270] [271] A number of observations demonstrate interactions between the thyroid and adrenal axes. Physiologically, the bulk of evidence suggests that glucocorticoids in humans[272] and rodents[273] act to blunt the thyroid axis through actions in the CNS. Some actions may be direct because the TRH gene (see Fig. 7-13) (Figure Not Available) contains the glucocorticoid response element consensus sequence [196] and hypophyseotropic TRH neurons appear to contain glucocorticoid receptors. [274] The diurnal rhythm of cortisol is opposite that of TSH (see Fig. 7-12) (Figure Not Available) and acute administration of glucocorticoids can block the nocturnal rise in

TSH, but disruption of cortisol synthesis with metyrapone only modestly affects the TSH circadian rhythm. [275] Several lines of evidence, however, identify conditions in which elevated glucocorticoids are associated with stimulation of the thyroid axis. Human depression is often associated with hypercortisolism and hyperthyroxinemia, [276] and TRH mRNA levels are elevated by glucocorticoids in a number of cell lines as well as in cultured fetal hypothalamic TRH neurons from the rat. [277] Thus, although glucocorticoids probably stimulate TRH production in TRH neurons, their overall inhibitory effect on the thyroid axis results from indirect glucocorticoid negative feedback on structures such as the hippocampus. Disruption of hippocampal suppression of the hypothalamic-pituitary-adrenal (HPA) axis is proposed to be involved in the hypercortisolemia commonly seen in affective illness, [278] and disruption of hippocampal inputs to the hypothalamus have been shown to produce a rise in hypophyseotropic TRH in the rat. [279] Starvation

The thyroid axis is depressed during starvation, presumably to help conserve energy by depressing metabolism (see Fig. 7-18 E to G) (Figure Not Available) . In humans, reduced T 3 , T4 , and TSH are seen during starvation or fasting. [280] There are also changes in the thyroid axis in anorexia nervosa, such as low blood levels of T 3 and low normal levels of T 4 (see Chapter 33) . Inappropriately low levels of TSH are found, suggesting defective activation of TRH production by low thyroid hormone levels. During starvation in rodents, reduced TRH release into hypophyseal portal blood [281] and reduced pro-TRH mRNA levels [282] are seen, despite lowered thyroid hormone levels. Reduced basal TSH levels are also usually present. The hypothyroidism seen in fasting or in the leptin-deficient Lepob /Lepob mouse can be reversed by administration of leptin, [283] and the evidence suggests that the mechanism involves leptin's ability to up-regulate TRH gene expression in the PVH (see Fig. 7-18 E to G) (Figure Not Available) . [284] Leptin appears to act both directly through leptin receptors on hypophyseotropic TRH neurons and indirectly through its actions on other hypothalamic cell groups, such as arcuate nucleus POMC and NPY-agouti-related peptide (AgRP) neurons. [285] TRH neurons in the PVH receive dense NPY-AgRP[267] and POMC projections [286] from the arcuate and express NPY and melanocortin-4 receptors, [287] and -MSH administration partially prevents the fasting-induced drop in thyroid hormone levels. [286] Indeed, the TRH promoter contains a signal transducer and activator of transcription (STAT) response element and a cAMP response element that have been demonstrated to mediate induction of TRH gene expression by leptin and -MSH, respectively, in a heterologous cell system (see Fig. 7-13) (Figure Not Available) . [287] The regulation of TRH by metabolic state is likely to be under redundant control,

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however, because, unlike rodents, leptin-deficient children are euthyroid, euthyroid.[290]

[ 288]

and both melanocortin-4 receptor (MC4R)deficient rodents [289] and humans are

Central TRH outside the paraventricular nucleus also plays a role in thermoregulation through the autonomic nervous system. Infection and Inflammation

The molecular basis of infection- or inflammation-induced thyrotropin suppression is now established. Sterile abscesses or the injection of interleukin-1 (IL-1; endogenous pyrogen, a secretory peptide of activated lymphocytes) [291] or of tumor necrosis factor (TNF-) inhibits thyrotropin secretion, [292] and IL-1 stimulates the secretion of somatostatin.[293] TNF- inhibits thyrotropin secretion directly and induces functional changes in the rat characteristic of the "sick euthyroid" state. [294] It is likely that the thyrotropin inhibition in animal models of the sick euthyroid syndrome is due to cytokine-induced changes in hypothalamic and pituitary function. [295] IL-6, IL-1, and TNF- contribute to the suppression of TSH in the sick euthyroid syndrome. [296]



Corticotropin-Releasing Hormone Chemistry and Evolution

The HPA axis is the humoral component of an integrated neural and endocrine system that functions to respond to internal and external challenges to homeostasis (stressors). The system comprises the neuronal pathways linked to release of catecholamines from the adrenal medulla (fight-or-flight response) and the hypothalamic-pituitary control of ACTH release in the control of glucocorticoid production by the adrenal cortex. Pituitary ACTH release is stimulated primarily by CRH and to a lesser extent by AVP (see Chapter 9) . The hypophyseotropic CRH neurons are located in the parvicellular division of the PVH and project to the median eminence (see Fig. 7-6 (Figure Not Available) Fig. 7-7 Fig. 7-8 Fig. 7-9 ). In a broader context, the CRH system in the CNS is also quite important in the behavioral response to stress. This complex system includes not only nonhypophyseotropic CRH neurons but also three CRH-like peptides (urocortin I, urocortin II or stresscopin-like peptide, and urocortin III or stresscopin), at least two cognate receptors (CRH-R1 and CRH-R2), and a high-affinity CRH-binding protein, each with distinct and complex distributions in the CNS. The Schally and Guillemin laboratories demonstrated in 1955 that extracts from the hypothalamus stimulated ACTH release from the pituitary. [297] [298] The primary active principle, CRH, was purified and characterized from the sheep in 1981 by Vale and colleagues. [299] Human CRH is an amidated 41-amino-acid peptide that is cleaved from the carboxyl terminus of a 196-amino-acid pre-prohormone precursor by PC1 and PC2 and amidated (Fig. 7-19) .[300] In general, the peptide is highly conserved; the human peptide is identical in sequence to the mouse and rat peptides but differs at seven residues from the ovine sequence. CRH and urocortin I, II, and III in mammals, fish urotensin, anuran sauvagine, [301] and the insect diuretic peptides [302] are members of an ancient family of peptides that evolved from an ancestral precursor early in the evolution of metazoans, approximately 500 million years ago. Comparison of peptide sequences in the vertebrate suggests grouping of the peptides into two families, CRH-urotensin-urocortin-sauvagine and urocortin II-urocortin III (Fig. 7-20) .[303] Urocortin and sauvagine appear to represent tetrapod orthologues of fish urotensin. Sauvagine, isolated originally from Phyllomedusa sauvagei, is an osmoregulatory peptide produced in the skin of certain frogs; urotensin is an osmoregulatory peptide produced in the caudal neurosecretory system of the fish. Whereas isolation of CRH required 250,000 ovine hypothalami, the cloning of urocortin II and III [303] [304] [305] was accomplished by computer search of the human genome data-base. The CRH peptides signal by binding to CRH-R1 [306] [307] [308] and CRH-R2 receptors [309] [310] [311] [312] that are members of the gut-brain family of G protein-coupled receptors and couple to Gs and activation of adenylyl cyclase. Two splice variants of the latter that differ in the extracellular amino-terminal domain, CRH-R2 and CRH-R2, have been found in both rodents and humans, [313] and a third N-terminal splice variant, CRH-R2, has been reported in the human. [314] CRH, urotensin, and sauvagine are all potent agonists of CRH-R1, urocortin is a potent agonist of both receptors, and urocortins II and III are specific agonists of CRH-R2. CRH-mediated activation of the HPA axis appears to be exclusively mediated through CRH-R1 expressed in the corticotroph. The PVH is the site of the majority of CRH neurons projecting to the median eminence, although some CRH neurons projecting to the median eminence are found in most hypothalamic nuclei (Fig. 7-21A) (Figure Not Available) . Some CRH fibers in the PVH also project to the brain stem, and CRH neurons are also found elsewhere, primarily in limbic structures involved in processing sensory information and in regulating the autonomic nervous system. Sites include the prefrontal, insular, and cingulate cortices; amygdala; substantia nigra; periaqueductal gray; locus coeruleus; nucleus of the solitary tract; and parabrachial nucleus. In the periphery, CRH is found in human placenta, where it is up-regulated 6-fold to 40-fold during the third trimester; lymphocytes; autonomic nerves; and gastrointestinal tract. Urocortin is found at highest levels in the Edinger-Westphal nucleus, lateral superior olive, and supraoptic nucleus of the rodent brain, with additional sites including the substantia nigra, ventral tegmental area, and dorsal raphe (Fig. 7-21 B) (Figure Not Available) . In the human, urocortin is widely distributed with highest levels in the frontal cortex, temporal cortex, and hypothalamus [315] and has also been reported in the Edinger-Westphal and olivary nuclei. [316] In the periphery, urocortin is seen in placenta, mucosal inflammatory cells in the gastrointestinal tract, lymphocytes, and cardiomyocytes. The tissue distribution of urocortins II and III is not well characterized as of this writing. In addition to its expression in pituitary corticotrophs, CRH-R1 is found in the neocortex and cerebellar cortex, subcortical limbic structures, and amygdala, with little to no expression in the hypothalamus (Fig. 7-21 C) (Figure Not Available) . CRH-R1 is also found in a variety of peripheral sites in humans, including ovary, endometrium, and skin.[317] CRH-R2 is found mainly in the brain in rodents, with high levels of expression seen in the ventromedial hypothalamic nucleus and lateral septum (see Fig. 7-21C) (Figure Not Available) . [318] CRH-R2 is seen centrally in cerebral arterioles and peripherally in gastrointestinal tract, heart, and muscle. [309] [310] [312] [319] In contrast, in humans CRH-R2 is seen in brain and periphery, and the and subtypes are primarily central. [ 313] [ 314] Little CRH-R2 message is seen in pituitary. Although CRH-R1 appears to be exclusively involved in regulation of pituitary ACTH synthesis and release, both receptors have been found to be expressed in the rodent adrenal cortex.[320] Data suggest that this intra-adrenal CRH-ACTH system may be involved in fine-tuning of adrenocortical corticosterone release. The CRH system is also regulated in both brain and periphery by a 37-kd high-affinity CRH-binding protein. [321] [322] [323] This factor was initially postulated from the observation that CRH levels rise dramatically during the second and third trimesters of pregnancy without activating the pituitary-adrenal axis. Among hypophyseotropic factors, CRH is the only one for which a specific binding protein (in addition to the receptor) exists in tissue or blood. The placenta is the principal source of pregnancy-related CRH-binding protein. Human and rat

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Figure 7-19 Structure of human corticotropin-releasing hormone (CRH) gene and protein. The sequence coding for CRH occurs at the terminus of the prohormone. Cleavage sites and the terminal Gly position are shown. PAM, peptidylglycine alpha-amidating monooxygenase; PC1/PC2, prohormone convertases 1 and 2; ERE, estrogen regulating element; GRE, glucocorticoid regulating element; CRE, cyclic AMP-responsive element; UTR, untranslated. (Redrawn from data of Shibahara S, Morimoto Y, Furutani Y, et al. Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene. EMBO J 1983; 2:775779.)

CRH-binding proteins are homologous (85% amino acid identity), but in the rat the protein is expressed only in brain. [324] The binding protein is species specific; bovine CRH, which is almost identical in sequence to rat-human CRH, has a lower affinity of binding to the human binding protein. The functional significance of the CRH-binding protein is not fully understood. [325] CRH-binding protein does not bind to the CRH receptor but does inhibit CRH action. For this reason CRH-binding protein probably acts to modulate CRH actions at the cellular level. Corticotroph cells in the anterior pituitary have membrane CRH receptors and intracellular CRH-binding protein; conceivably, the binding protein acts to sequester or terminate the action of membrane-bound CRH. CRH-binding protein is present in many regions of the CNS, including cells that synthesize CRH and cells that receive innervation

Figure 7-20 Sequence comparison of members of the corticotropin-releasing hormone (CRH) peptide family. SPP, stresscopin related peptide; SCP, stresscopin.

from CRH-containing neurons. [324] The anatomic distribution of the protein, the variability of its location in relation to the presence of CRH, and its relative sparseness in the CRH tuberohypophyseal neuronal system suggest a control system that is as yet poorly understood.

Structure-activity relationship studies have demonstrated that C-terminal amidation and an -helical secondary structure [326] are both important for biologic activity of CRH. The first CRH antagonist described was termed -helical CRH 941 .[327] A second, more potent antagonist, termed astressin, had the structure cyclo(3033)( D-Phe12 , Nle12 , Glu12 , Lys12 ,)hCRH1241 . [328] Both peptides are somewhat nonspecific, antagonizing both CRH-R1 and CRH-R2. Because of the anxiogenic activity of CRH and urocortin, a number of pharmaceutical companies have developed small molecule CRH antagonists; several of the

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Figure 7-21 (Figure Not Available) Distribution of corticotropin-releasing hormone (CRH), urocortin, and the CRH receptor 1 (CRH-R1) and CRH-R2 messenger ribonucleic acid sequences in the rat brain. A 1 , noradrenergic cell group 1; A5 , noradrenergic cell group 5; ac, anterior commissure; BST, bed nucleus of the stria terminalis; cc, corpus callosum; CeA, central nucleus amygdala; CG, central gray; DR, dorsal raphe; DVC, dorsal vagal complex; HIP, hippocampus; LC, locus coeruleus; LDT, laterodorsal tegmental nucleus; LHA, lateral hypothalamic area; ME, median eminence; MID THAL, midline thalamic nuclei; mfb, medial forebrain bundle; MPO, medial preoptic area; MR, medial raphe; MVN, medial vestibular nucleus; PB, parabrachial nucleus; POR, perioculomotor nucleus; PP, posterior pituitary; PVH, periventricular nucleus; SEPT, septal region; SI, substantia innominata; st, stria. (From Swanson LW, Sawchenko PE, Rivier J, et al. Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study. Neuroendocrinology 1983; 36:165186; Bittencourt et al. J Comp Neurol 1999; 415:285, Fig. 17; Steckler and Holsboer. Biol Psychol 1999; 46:1480, Fig. 1.)

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molecules are currently in clinical trials for anxiety and depression (discussed in more detail later). Thus far, this structurally diverse group of small molecule compounds, such as antalarmin, CP-154,526, and NBI27914, are potent antagonists of CRH-R1, with little activity at CRH-R2. [329] The efficacy of these compounds across the entire behavioral, neuroendocrine, and autonomic repertoire of response to stress has been demonstrated in a number of laboratory animal studies. For example, oral administration of antalarmin in a social stress model in the primate (introduction of strange males) reduced behavioral measures of anxiety such as lack of exploratory behavior, decreased plasma ACTH and cortisol, and reduced plasma epinephrine and norepinephrine. [330] A peptide antagonist with 100-fold selectivity for the CRH 2 receptor, ( D-Phe,11 His12 )sauvagine 1140 or antisauvagine-30, has also been described. [331] Effects on the Pituitary and Mechanism of Action

Administration of CRH to humans causes prompt release of corticotropin into the blood, followed by secretion of cortisol (Fig. 7-22) and other adrenal steroids including aldosterone. [332] [333] Most studies have used ovine CRH, which is more potent and longer acting than human CRH, but human and porcine CRHs appear to have equal diagnostic value. [332] The effect of CRH is specific to corticotropin release and is inhibited by glucocorticoids. As mentioned before, CRH acts on the pituitary corticotroph primarily by binding to CRH-R1 and activating adenylyl cyclase. The concentration of cAMP in the tissue is increased in parallel with the biologic effects and is reduced by glucocorticoids. The rate of transcription of the mRNA that encodes the corticotropin prohormone POMC is also enhanced by CRH, indicating that CRH is a trophic factor as well as a releasing hormone.

Figure 7-22 Changes in plasma levels of corticotropin and serum levels of cortisol after intravenous injection of corticotropin-releasing hormone in a group of six normal men. The initial prompt response in corticotropin is followed by a somewhat delayed secondary change in cortisol. To convert corticotropin values to picomoles per liter, multiply by 0.2202. To convert cortisol values to millimoles per liter, multiply by 27.59. ACTH, adrenocorticotropic hormone. (From Grossman A, Kruseman ACN, Perry L, et al. New hypothalamic hormone, corticotropin-releasing factor, specifically stimulates the release of adrenocorticotropic hormone and cortisol in man. Lancet 1982; 1:921922.) Extrapituitary Functions

CRH and the urocortin peptides have a wide range of biologic activities in addition to the hypophyseotropic role of CRH in regulating ACTH synthesis and release. Centrally, these peptides have behavioral activities in anxiety, mood, arousal, locomotion, reward, and feeding [334] [335] [336] and increase sympathetic activation. Many of the nonhypophyseotropic behavioral and autonomic functions of these peptides can be viewed as complementary to activation of the HPA axis in the maintenance of homeostasis under exposure to stress. In the periphery, activities have been reported in immunity, cardiac function, gastrointestinal function, and reproduction. The CRH and urocortin peptides have a repertoire of behavioral and autonomic actions after central administration that suggests a role for these pathways in mediating the behavioral-autonomic components of the stress response. Hyperactivity of the HPA axis is a common neuroendocrine finding in affective disorders (Fig. 7-23) (Figure Not Available) (for reviews see references [337] [338] [339] ). Furthermore, normalization of HPA regulation is highly predictive of successful treatment. Defective dexamethasone suppression of CRH release, implying defective corticosteroid receptor signaling, [340] is seen not only in depressed patients but also in healthy subjects with a family history of depression. [341] Depressed patients also show elevated levels of CRH in the CSF. [342] Central administration of CRH or urocortin activates neuronal cell groups involved in cardiovascular control and increases blood pressure, heart rate, and cardiac output.[343] [344] However, urocortin is expressed in cardiac myocytes, [345] and intravenous administration of CRH or urocortin decreases blood pressure and increases heart rate in most species, including humans. [346] This hypotensive effect is probably mediated peripherally because ganglion blockade did not disrupt the hypotensive effects of intravenous urocortin. [347] Furthermore, high levels of CRH-R2 have been seen in the cardiac atria and ventricles, [309] [310] [312] [319] and knockout of the CRH-R2 gene in the mouse removed the hypotensive effects of intravenous urocortin administration. [348] [349] CRH and AVP also play an important role in the regulation of inflammatory responses. As described later ("Neuroendocrine-Immune Interaction"), cytokines have an important role in extinguishing inflammatory responses through activation of CRH and AVP neurons in the paraventricular nucleus and subsequent elevation of anti-inflammatory glucocorticoids. Interestingly, CRH is generally seen to be proinflammatory in the periphery, where it is found in sympathetic efferents, sensory afferent nerves, leukocytes, and in macrophages in some species. [350] CRH is also made as a paracrine factor by the endometrium, where it may play a role in decidualization and implantation and act as a uterine vasodilator.

[351]

The relative contributions of each of the CRH-urocortin peptides and receptors to the different biologic functions reported has been the topic of considerable analysis, given the receptor-specific antagonists already described as well as the CRH, [352] CRH-R1,[353] [354] and CRH-R2[348] [349] [355] knockout mice available for study. [356] [357] Examining three potent stressorsrestraint, ether, and fastingthese studies demonstrated that other ACTH secretagogues, such as vasopressin, oxytocin, and catecholamines, could not replace CRH in its role in mounting the stress response. In contrast, augmentation of glucocorticoid secretion by a stressor after prolonged stress was not defective in the CRH knockout mouse, implicating CRH-independent mechanisms. [357] Although CRH is a potent anxiogenic peptide, [358] the CRH knockout mouse exhibits normal anxiety behaviors in, for example, conditioned fear paradigms. [359] The nonpeptide CRH-R1 specific antagonist CP-154,526 was anxiolytic in a shock-induced

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Figure 7-23 (Figure Not Available) Comparison of plasma immunoreactive adrenocortico-tropic hormone (IR-ACTH) (A) and cortisol (B) responses to ovine corticotropin-releasing hormone in control subjects, patients with depression, and patients with Cushing's disease. (From Gold PW, et al. N Engl J Med 1986; 314:1329.)

freezing paradigm in both wild-type and CRH knockout mice, [359] suggesting that the anxiogenic activity is a CRH-like peptide acting at the CRH-R1 receptor.

CRH and urocortin peptides also have potent anorexigenic activity, implicating the CRH system in stress-induced inhibition of feeding. Stress-induced inhibition of feeding remained intact, however, in the CRH knockout mouse. [360] Likewise, suppression of the proestrous LH surge by restraint was intact in the CRH knockout mouse.[361] Both CRH-R1 and CRH-R2 knockout strains had normal weight and feeding behavior but were distinctly different from wild-type mice in the anorexigenic response to centrally administered urocortin or CRH. The CRH-R1deficient mice lacked the acute anorexigenic response (0 to 1.5 hours) to urocortin seen in wild-type mice.[362] Both wild-type and CRH-R1 / mice exhibited comparable reduction in feeding 3 to 11 hours after administration. In contrast, the late phase of urocortin responsiveness appeared to depend on the presence of CRH-R2. [348] [349] [355] Thus, signaling through CRH-R1 and CRH-R2 appears to play a complex role in the acute effects of stress on feeding behavior. Clinical Applications

No useful therapeutic applications of CRH or CRH-like peptides have been reported, although the peptide has been demonstrated to have a number of activities in human and primate studies. For example, intravenous administration of CRH was found to stimulate energy expenditure and has been proposed for use in weight loss. The development of small molecule, orally available, CRH-R1 antagonists has, however, led to phase I clinical trials for anxiety and depression. An early study of 20 patients demonstrated significant reductions in scores of anxiety and depression, using ratings determined by either patient or clinician. [363] Feedback Control

The administration of glucocorticoids inhibits corticotropin secretion; removal of the adrenals (or administration of drugs that impair secretion of glucocorticoids) leads to increased corticotropin release. The set-point of pituitary feedback is determined by the hypothalamus acting through hypothalamic releasing hormones CRH and vasopressin ( see Chapter 8 and Chapter 9 ). [266] [333] [364] [365] [366] [367] [368] [369] [370] [371] [372] [373] Glucocorticoids act on both the pituitary corticotrophs and the hypothalamic neurons that secrete CRH and vasopressin. These regulatory actions are analogous to the control of the pituitary-thyroid axis. However, whereas thyrotropin becomes completely unresponsive to TRH when thyroid hormone levels are sufficiently high, severe neurogenic stress and large amounts of CRH can break through the feedback inhibition by glucocorticoids. A still higher level of feedback control is exerted by glucocorticoid-responsive neurons in the hippocampus that project to the hypothalamus; these neurons affect the activity of CRH hypophyseotropic neurons and determine the set-point of pituitary responsiveness to glucocorticoids. [368] Glucocorticoids are lipid soluble and enter the brain through the blood-brain barrier. [369] In brain and pituitary they can bind to two receptors, type I (the mineralocorticoid receptor, so named because it binds aldosterone and glucocorticoids with high affinity) and type II ( glucocorticoid receptor, which has low affinity for mineralocorticoids). [266] [368] [369] [370] [371] [372] [373] Glucocorticoid action involves binding of the steroid-receptor complex to regulator sequences in the genome. [370] Type I receptors are saturated by basal levels of glucocorticoids, whereas type II receptors are not saturated under basal conditions but approach saturation during peak phases of the circadian rhythm and during stress. These differences and differences in regional distribution within the brain suggest that type I receptors determine basal activity of the hypothalamic-pituitary axis and that type II receptors mediate stress responses. In the pituitary, glucocorticoids inhibit secretion of corticotropin and the synthesis of POMC mRNA; in the hypothalamus, the secretion of CRH and vasopressin and the synthesis of their respective mRNAs are inhibited. [365] [366] [368] [369] [370] [371] Neuron membrane excitability and ion transport properties are suppressed by changes in glucocorticoid-directed synthesis of intracellular protein. Glucocorticoids may also act directly on neuronal cell membranes to change corticotropin secretion rapidly. [374] Glucocorticoids block stress-induced corticotropin release. The latency of the inhibitory effect is so short (less than 30 minutes) [367] that it is possible that gene regulation is not the sole basis of the response. Long-term suppression (more than 1 hour) clearly acts through genomic mechanisms. Glucocorticoid receptors are also found outside the hypothalamus in the septum and amygdala, [368] [369] [373] structures that

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are involved in the emotional changes in hypercortisolism and hypocortisolism. Hippocampal neurons are damaged by prolonged elevation of glucocorticoids during prolonged stress. [368] Neural Control

Significant physiologic or psychological stressors evoke an adaptive response that commonly includes activation of both the HPA axis and the sympathoadrenal axis. The end products of these pathways then help to mobilize resources to cope with the physiologic demands in emergency situations, acutely through the fight-or-flight response and over the long term through systemic effects of glucocorticoids on functions such as gluconeogenesis and energy mobilization (see Chapter 33) . The HPA axis also has unique stress-specific homeostatic roles, the best example being the role of glucocorticoids in down-regulating immune responses after infection and other events that stimulate cytokine production by the immune system (see "Neuroendocrine-Immune Interactions"). The paraventricular nucleus is the primary hypothalamic nucleus responsible for providing the integrated whole-animal response to stress. [375] [376] [377] [378] [379] This nucleus contains three major types of effector neurons that are spatially distinct from one another within it: (1) magnocellular oxytocin and vasopressin neurons that project to the posterior pituitary and participate in the regulation of blood pressure, fluid homeostasis, lactation, and parturition; (2) neurons projecting to the brain stem and spinal cord that regulate a variety of autonomic responses including sympathoadrenal activation; and (3) parvocellular CRH neurons that project to the median eminence and regulate ACTH synthesis and release. Many CRH neurons coexpress AVP, which acts as an auxiliary ACTH secretagogue, synergistic with CRH. AVP is regulated quite differently in parvocellular versus magnocellular neurons but is also regulated somewhat differently from CRH by stressors in parvocellular cells expressing both peptides. [380] Different stressors result in different patterns of activation of the three major visceromotor cell groups within the paraventricular nucleus, as measured by the general neuronal activation marker c-fos (Fig. 7-24) . [381] For example, salt loading down-regulates CRH mRNA in parvocellular CRH cells, up-regulates CRH in a small number of magnocellular CRH cells, but only activates magnocellular cells. Hemorrhage activates every division of the paraventricular nucleus, whereas cytokine administration primarily activates parvocellular CRH cells with some minor activation of magnocellular and autonomic divisions. The synthesis and release of AVP, which regulates renal water absorption and vascular smooth muscle, are controlled mainly by the volume and tonicity of the blood. This information is relayed to the magnocellular AVP cell through the nucleus of the solitary tract and A1 noradrenergic cell group of the ventrolateral medulla and projections from a triad of CVOs lining the third ventricle, the SFO, MePO, and OVLT. Oxytocin is primarily involved in reproductive functions, such as parturition, lactation, and milk ejection, although it is cosecreted with AVP in response to osmotic and volume challenges, and oxytocin cells receive direct projections from the nucleus of the solitary tract as well as from the SFO, medial preoptic nucleus (MePO), and OVLT. In contrast to the neurosecretory neurons functionally defined by the three peptides, CRH, oxytocin, and AVP, PVH neurons projecting to brain stem and spinal cord include neurons expressing each of these peptides. In the rodent, a wide variety of stressors have been determined to activate parvocellular CRH neurons, including cytokine injection, salt loading, hemorrhage, adrenalectomy, restraint, foot shock, hypoglycemia, fasting, and ether exposure. Thus, in contrast to the simplicity of inputs to magnocellular cells (Fig. 7-25A) , it is not surprising that parvocellular CRH neurons receive a diverse and complex assortment of inputs ( Fig. 7-26 ; see Fig. 7-25B ). These may be divided into three major categories, brain stem, limbic forebrain, and hypothalamus. Because the PVH is not known to receive any direct projections from the cerebral cortex or thalamus, stressors involving emotional or cognitive processing must involve indirect relay to the PVH. Visceral sensory input to the PVH involves primarily two pathways. The nucleus of the solitary tract, the primary recipient of sensory information from the thoracic and abdominal viscera, sends dense catecholaminergic projections to the PVH, both directly and through relays in the ventrolateral medulla. [382] [383] These brain stem projections account for about half of the NPY fibers present in the PVH, [268] described in more detail subsequently. A second major input responsible for transducing signals from blood-borne substances derives from three CVOs adjacent to the third ventricle, the SFO, OVLT, and MePO. [384] [385] These pathways account for activation of CRH neurons by what are referred to as systemic or physiologic stressors. [377] By contrast, what are termed neurogenic, emotional, or psychological stressors involve, in addition, nociceptive or somatosensory pathways as well as cognitive and affective brain centers. Using elevation of c-fos as an indicator of neuronal activation, detailed studies have compared PVH-projecting neurons activated by IL-1

treatment (systemic stressor) versus foot shock (neurogenic stressor). Only catecholaminergic solitary tract nucleus and ventrolateral medulla neurons were activated by moderate doses of IL-1.[386] In contrast, foot shock activated neurons of the solitary tract nucleus and ventrolateral medulla but also cell groups in the limbic forebrain and hypothalamus. [387] Notably, pharmacologic or mechanical disruption of the ascending catecholaminergic fibers blocked IL-1mediated activation but not foot shockmediated activation of the HPA axis. [388] Data suggest that pathways activated by other neurogenic and systemic stressors may overlap significantly with those activated by foot shock and IL-1 treatment, respectively. [378] Except for the catecholaminergic neurons of the nucleus of the solitary tract and ventrolateral medulla, parts of the bed nucleus of the stria terminalis, and the dorsomedial nucleus of the hypothalamus, many inputs to the paraventricular nucleus, such as those deriving from the prefrontal cortex and lateral septum, are thought to act indirectly through local hypothalamic glutamatergic [378] and GABAergic neurons[389] with direct synapses to the CRH neurons. The bed nucleus of the stria terminalis is the only limbic region with prominent direct projections to the PVH. With substantial projections from the amygdala, hippocampus, and septal nuclei, it may thus serve as a key integrative center for transmission of limbic information to the PVH. Other Factors Influencing Secretion of Corticotropin

Circadian Rhythms

Levels of corticotropin and cortisol (in humans) peak in the early morning, fall during the day to reach a nadir at about midnight, and begin to rise between 1 AM and 4 [ AM (see Fig. 7-12) (Figure Not Available) . 164] Within the circadian cycle approximately 15 to 18 pulses of corticotropin can be discerned, their height varying with the time of day. The set-point of feedback control by glucocorticoids also varies in a circadian pattern. Pituitary-adrenal rhythms are entrained to the light-dark cycle and can be changed over several days by exposure to an altered light schedule. It has long been assumed that the rhythm of corticotropin secretion is driven by CRH rhythms, and CRH knockout mice were found to exhibit no circadian rhythm in corticosterone production. [390] Remarkably, however, a diurnal

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Figure 7-24 Regulation of neurons of the paraventricular nucleus (PVH) by diverse stressors. ADX, adrenalectomy; dp, dorsal PVH; IL-1, interleukin-1; mp, magnocellular PVH; NGFI-B, nerve growth factor I-B; pm, medial PVH. (Reprinted from Sawchentzo PE, et al. The paraventricular nucleus of the hypothalamus and the functional neuroanatomy of visceromotor responses to stress. Prog Brain Res 1996; 107:208. With permission from Elsevier Science.)

rhythm in corticosterone was restored by a constant infusion of CRH to the CRH knockout mouse, [390] suggesting that CRH is necessary to permit pituitary or adrenal responsiveness to another diurnal rhythm generator. Corticotropin ReleaseInhibiting Factor

Disconnection of the pituitary from the hypothalamus in several species leads to increased basal levels of corticotropin, and certain responses to physical stress (in contrast to psychological stress) are retained in such animals. These observations have led several investigators to postulate the existence of a corticotropin inhibitory factor analogous to dopamine in the control of PRL secretion and to somatostatin in the control of GH secretion. [391] Candidate hypothalamic peptides to inhibit corticotropin release at the level of the pituitary include atrial natriuretic peptide, activins and inhibins, and sequence 178 to 199 of the TRH prohormone. [178] [392] There is not yet a consensus on the existence of a physiologically relevant corticotropin releaseinhibiting factor or on its identity.

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Figure 7-25 Neuronal inputs to neurons of the paraventricular nucleus. AVP, arginine vasopressin; BST, bed nucleus of the stria terminalis; CG, central gray; IGL, intergeniculate leaf; LDT, laterodorsal tegmental nucleus; MePO, medial preoptic nucleus; NTS, nucleus of the tractus solitarius; OT, oxytocin; OVLT, organum vasculosum of the lamina terminalis; PB, parabrachial nucleus; PIN, posterior intralaminar nucleus; PP, peripeduncular nucleus; PPN, pedunculopontine nucleus; SFO, subfornical organ. (Reprinted from Sawchentzo PE, et al. The paraventricular nucleus of the hypothalamus and the functional neuroanatomy of visceromotor responses to stress. Prog Brain Res 1996; 107:204. With permission from Elsevier Science.)



HYPOPHYSEOTROPIC HORMONES AND NEUROENDOCRINE AXES (Continued) Growth HormoneReleasing Hormone Chemistry and Evolution

Evidence for neural control of GH secretion came from studies of its regulation in animals with lesions of the hypothalamus [393] and from the demonstration that hypothalamic extracts stimulate the release of GH from the pituitary. When it was shown that GH is released episodically, follows a circadian rhythm, responds rapidly to stress, and is blocked by pituitary stalk section, the concept of neural control of GH secretion became a certainty. However, it was only with the discovery of the paraneoplastic syndrome of ectopic GHRH secretion by pancreatic adenomas in humans that sufficient starting material became available for peptide sequencing and subsequent cloning of a complementary deoxyribonucleic acid (cDNA). [394] [395] [396] [397] Two principal molecular forms of GHRH occur in human hypothalamus: GHRH(144)-NH 2 and GHRH(140)-OH (Fig. 7-27) .[398] As with other neuropeptides, the various forms of GHRH arise from post-translational modification of a larger prohormone. [397] [399] The NH 2 -terminal tyrosine of GHRH (or histidine in rodent GHRHs) is essential for bioactivity, but a COOH-terminal NH 2 group is not. Fragments as short as (129)-NH 2 are active, but GHRH(127)-NH 2 is inactive. A circulating type IV dipeptidylpeptidase potently inactivates GHRH to its principal and more stable metabolite, GHRH(344)-NH 2 ,[400] which accounts for most of the immunoreactive peptide detected in plasma. As in the case of LHRH, there are species differences among GHRHs; the peptides from seven species range in sequence homology with the human peptide from 93% in the pig to 67% in the rat. [398] The COOH-terminal end of GHRH exhibits the most sequence diversity among species, consistent with the exon arrangement of the gene and dispensability of these residues for GHRH receptor binding. Despite its importance for the elucidation of GHRH structure, ectopic secretion of the peptide is a rare cause of acromegaly. Fewer than 1% of acromegalic patients have elevated plasma levels of GHRH (see Chapter 8) .[401] Approximately 20% of pancreatic adenomas and 5% of carcinoid tumors contain immunoreactive GHRH, but most are clinically silent. [402] [403] In addition to expression in the hypothalamus, the GHRH

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Figure 7-26 Regulation of the hypothalamic-pituitary-adrenal axis. ACTH, adrenocorticotropic hormone; AVP, arginine vasopressin; BST, bed nucleus of the stria terminalis; CNS, central nervous system; CRH, corticotropin-releasing hormone; CRIF, corticotropin releaseinhibiting factor; GABA, -aminobutyric acid; 5-HT, 5-hydroxytryptamine; IL-1, interleukin-1; MeA, medial amygdala; MePO, medial preoptic; NPY, neuropeptide Y; NTS, nucleus of the tractus solitarius; OVLT, organum vasculosum of the lamina terminalis; POMC, pro-opiomelanocortin.

gene is expressed eutopically in human ovary, uterus, and placenta, [404] although its function in these tissues is not known. Studies in rat placenta indicate that an alternative transcriptional start site 10 kilobases upstream from the hypothalamic promoter is utilized together with an alternatively spliced exon la. [405] Growth HormoneReleasing Hormone Receptor

The GHRH receptor is a member of a subfamily of G proteincoupled receptors that includes receptors for VIP, pituitary adenylyl cyclaseactivating peptide, secretin, glucagon, glucagon-like peptide 1, calcitonin, parathyroid hormone or

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Figure 7-27 Diagram illustrating the genomic organization, messenger ribonucleic acid structure, and post-translational processing of the human growth hormonereleasing hormone (GHRH) prohormone. Few details are known about the transcriptional regulation of the GHRH gene except that distinct promoter sequences and alternative 5' exons are utilized by hypothalamic neurons and extrahypothalamic tissues. All of the amino acid residues required for bioactive GHRH peptides are encoded by exon 3. An amino-terminal exopeptidase that cleaves the Tyr-Ala dipeptide is primarily responsible for the inactivation of GHRH peptides in extracellular compartments. CPE, carboxypeptidase E; PAM, peptidylglycine alpha-amidating monooxygenase; PC1/PC2, prohormone convertases 1 and 2; UTR, untranslated region. (Compiled from data of Mayo K, Cerelli GM, Lebo RV, et al. Gene encoding human growth hormonereleasing factor precursor: structure, sequence, and chromosomal assignment. Proc Natl Acad Sci USA 1985; 82:6367; Frohman LA, Downs TR, Chomczynski P, Frohman MA. Growth hormonereleasing hormone: structure, gene expression and molecular heterogeneity. Acta Paediatr Scand [Suppl] 1990; 367:8186; and González-Crespo S, Boronat A. Expression of the rat growth hormonereleasing hormone gene in placenta is directed by an alternative promoter. Proc Natl Acad Sci USA 1991; 88:87498753.)

parathyroid hormonerelated peptide, and gastric inhibitory polypeptide. [406] [407] GHRH elevates intracellular cAMP by its receptor coupling to a stimulatory G protein (Gs ), which activates adenylyl cyclase, increases intracellular free Ca 2+ , releases preformed GH, and stimulates GH mRNA transcription and new GH synthesis (see Chapter 8) .[408] GHRH also increases pituitary phosphatidylinositol turnover. Nonsense mutations in the human GHRH receptor gene are the cause of rare familial forms of GH deficiency [409] [410] and indicate that no other gene product can fully compensate for the specific receptor in pituitary. Effects on the Pituitary and Mechanism of Action

Intravenous administration of GHRH to individuals with normal pituitaries caused a prompt, dose-related increase in serum GH that peaked between 15 and 45 minutes, followed by a return to basal levels by 90 to 120 minutes (Fig. 7-28) .[411] A maximally stimulating dose of GHRH is approximately 1 µg/kg, but the response differs considerably between individuals and within the same individual tested on different occasions, presumably because of cosecretagogue and somatostatin tone that exists at the time of GHRH injection. Repeated bolus administration or sustained infusions of GHRH over several hours cause a modest decrease in the subsequent GH secretory response to acute GHRH administration. However, unlike the marked desensitization of the LHRH receptor and decline in circulating gonadotropins that occur in response to continuous LHRH exposure, pulsatile GH secretion and insulin-like growth factor I (IGF-I) production are maintained by constant GHRH in the human.[411] This response suggests the involvement of additional factors that mediate the intrinsic

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Figure 7-28 Response of normal men to growth hormonereleasing hormone (GHRH)(129) (1 µg/kg), ghrelin (1 µg/kg), or GHRH(129) and ghrelin administered by intravenous injection. Note the prompt release of GH, followed by a rather prolonged fall in hormone level in response to both secretagogues. Ghrelin alone was more efficacious than GHRH(129), and there was an additive effect from the two peptides administered simultaneously. (From Arvat E, Macario M, Di Vito L, et al. Endocrine activities of ghrelin, a natural growth hormone secretagogue (GHS), in humans: comparison and interactions with hexarelin, a nonnatural peptidyl GHS, and GH-releasing hormone. J Clin Endocrinol Metab 2001; 86:11691174.)

diurnal rhythm of GH, and these factors are addressed in the following sections. The pituitary effects of a single injection of GHRH are almost completely specific for GH secretion, and there is minimal evidence for any interaction between GHRH and the other classical hypophyseotropic releasing hormones. [411] GHRH has no effect on gut peptide hormone secretion. The GH secretory response to GHRH is enhanced by estrogen administration, glucocorticoids, and starvation. Major factors known to blunt the response to GHRH in humans are somatostatin, obesity, and advancing age. In addition to its role as a GH secretagogue, GHRH is a physiologically relevant growth factor for somatotrophs. Transgenic mice expressing a GHRH cDNA coupled to a suitable promoter developed diffuse somatotroph hyperplasia and eventually pituitary macroadenomas. [412] [413] The intracellular signal transduction pathways mediating the mitogenic action of GHRH are not known with certainty but probably involve an elevation of adenylyl cyclase activity. Several lines of evidence support this conclusion, including the association of activating mutations of the G s polypeptide in many human somatotroph adenomas.[414] Extrapituitary Functions

GHRH has few known extrapituitary functions. The most important may be its activity as a sleep regulator. The administration of nocturnal GHRH boluses to normal men significantly increased the density of slow wave sleep, as also shown in other species. [415] Furthermore, there is a striking correlation between the age-related declines in slow wave sleep and daily integrated GH secretion in healthy men. [416] These and other data suggest that central GHRH secretion is under circadian entrainment and nocturnal elevations in GHRH pulse amplitude or frequency directly mediate sleep stage and sleep-induced increases in GH secretion. GHRH has been reported to stimulate food intake in rats and sheep, and the effect is dependent on route of administration, time of administration, and macronutrient composition of the diet. [406] The neuropeptide's physiologic relevance to feeding in humans is unknown, although a study indicated that GHRH stimulated food intake in patients with anorexia nervosa but reduced it in patients with bulimia or in normal female control subjects. [417] Growth Hormone-Releasing Peptides

In studies of the opioid control of GH secretion, several peptide analogues of met-enkephalin were found to be potent GH secretagogues. These include the GH-releasing peptide GHRP-6 (Fig. 7-29) (Figure Not Available) , hexarelin (His-D2MeTrp-Ala-Trp-DPhe-Lys-NH 2 ), and other more potent analogues including cyclic peptides and modified pentapeptides. [406] [418] Subsequently, a series of nonpeptidyl GHRP mimetics were synthesized with greater oral bioavailability, including the spiropiperidine MK-0677 and the shorter acting benzylpiperidine L-163,540 (see Fig. 7-29) (Figure Not Available) . Common to all these compounds, and the basis of their differentiation from GHRH analogues in pharmacologic activity screens, is their activation of phospholipase C and inositol 1,4,5-trisphosphate. This property was exploited in a cloning strategy that led to the identification of a G proteincoupled receptor GHS-R that is highly selective for the GH secretagogue class of ligands. [419] The GHS-R is unrelated to the GHRH receptor and is highly expressed in the anterior pituitary gland and multiple brain areas, including the medial basal hypothalamus, the hippocampus, and the mesencephalic nuclei that are centers of dopamine and serotonin production. Peptidyl and nonpeptidyl GHSs are active when administered by intranasal and oral routes, are more potent on a weight basis than GHRH itself, are more effective in vivo than in vitro, synergize with coadministered GHRH and are almost ineffective in the absence of GHRH, and do not suppress somatostatin secretion. [406] [411] Prolonged infusions of GHRP amplify pulsatile GH secretion in normal men. GHRP administration, like that of GHRH, facilitates slow wave sleep. Patients with hypothalamic disease leading to GHRH deficiency have low or no response to hexarelin; similarly, pediatric patients with complete absence of the pituitary stalk have no GH secretory response to hexarelin. [420] The potent biologic effects of GHRPs and the identification of the GHS-R suggested the existence of a natural ligand for the receptor that is involved in the physiologic regulation of GH secretion. A probable candidate for this ligand is the acylated peptide ghrelin, produced and secreted into the circulation from the stomach (see Fig. 7-29) (Figure Not Available) . [28] The effects of ghrelin on GH secretion in humans are identical to or more potent than those of the non-natural GHRPs (see Fig. 7-28) .[421] In addition, ghrelin acutely increases circulating PRL, ACTH, cortisol, and aldosterone levels. [421] There is debate concerning the extent and localization of ghrelin expression in the brain that must be resolved before the implications of gastric-derived ghrelin in the regulation of pituitary hormone secretion are fully understood. A proposed role for ghrelin in appetite and regulation of food intake is discussed later in this chapter. Clinical Applications

GHRH stimulates growth in children with intact pituitaries, but the optimal dosage, route, and frequency of administration, as well as possible usefulness by the nasal route, have not been determined. The availability of recombinant hGH (which requires

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Figure 7-29 (Figure Not Available) Structure of a non-natural peptidyl (GHRP-6) and nonpeptidyl (MK-0677 and L-163,540) growth hormone secretagogues and a natural ligand (ghrelin) that all bind and activate the growth hormone secretagogue receptor. Ghrelin is an acylated 28-amino-acid peptide. The O-n-octanoylation at Ser3 is essential for biological activity and is a unique post-translational modification among the known neuropeptides. (Adapted from Smith RG, Feighner S, Prendergast K, et al. A new orphan receptor involved in pulsatile growth hormone release. Trends Endocrinol Metab 1999; 10:128135; and Kojima M, Hosoda H, Date Y, et al. Ghrelin is a growth hormonereleasing acylated peptide from stomach. Nature 1999; 402:656660.)

less frequent injections than GHRH) and the development of the more potent GHSs with improved oral bioavailability have reduced enthusiasm for the clinical use of GHRH or its analogues. GHRH is not useful for the differential diagnosis of hypothalamic and pituitary causes of GH deficiency in children. However, in adults a combined GHRH-GHRP challenge test may be ideal for the diagnosis of GH reserve. GH release in response to the combined secretagogues is not influenced by age, sex, or body mass index, and the test has a wider margin of safety than an insulin tolerance test. [422] [423] The potential clinical applications of GHSs including MK-0677 are still being explored. [406] [418] An area of intense interest is the normal decline in GH secretion with age. GH administration in healthy older individuals has been associated with increased lean body mass, increased muscle strength, and decreased fat mass, although there is a high incidence of adverse side effects. The physiologic GH profile induced by MK-0677 may be better tolerated than GH injections. However, unlike treatment with GHRH, chronic administration of GHSs leads to significant desensitization of the GHS-R and attenuation of the GH response. The release of pituitary hormones other than GH may also limit the applicability of GHS therapy. Finally, apart from actions on GH secretion, both GHRH and GHSs are being investigated for the treatment of sleep disorders commonly associated with aging. Neuroendocrine Regulation of Growth Hormone Secretion

GH secretion is regulated by hypothalamic GHRH and somatostatin interacting with circulating hormones and additional modulatory peptides at the level of both the pituitary and the hypothalamus (Fig. 7-30) .[406] [411] [424] Additional background on somatostatin and its functions other than control of GH secretion are presented in a

later section. Feedback Control

Negative feedback control of GH release is mediated by GH itself and by IGF-I, which is synthesized in the liver under control of GH. Direct GH effects on the hypothalamus are produced by short-loop feedback, whereas those involving IGF-I and other circulating factors influenced by GH, including free fatty acids and glucose, are long-loop systems analogous

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Figure 7-30 Regulation of the hypothalamic-pituitary-growth hormone (GH) axis. GH secretion by the pituitary is stimulated by GH-releasing hormone (GHRH) and is inhibited by somatostatin (SRIF). Negative feedback control of GH secretion is exerted at the pituitary level by insulin-like growth factor I (IGF-I) and by free fatty acids (FFA). GH itself exerts a short-loop negative feedback by the activation of SRIF neurons in the hypothalamic periventricular nucleus. These SRIF neurons directly synapse on arcuate GHRH neurons and project to the median eminence. Neuropeptide Y (NPY) neurons in the arcuate nucleus also indirectly modulate GH secretion by integrating peripheral GH, leptin, and ghrelin signals and projecting to periventricular SRIF neurons. Ghrelin is secreted from the stomach and is a putative natural ligand for the GH secretagogue receptor that stimulates GH secretion at both the hypothalamic and pituitary levels. On the basis of indirect pharmacologic data, it appears that release of GHRH is stimulated by galanin, -aminobutyric acid (GABA), and 2 -adrenergic and dopaminergic stimuli and inhibited by somatostatin. Secretion of somatostatin is inhibited by acetylcholine (muscarinic receptors) and 5-HT (type 1D receptors), and increased by 2 -adrenergic stimuli and corticotropin-releasing hormone (CRH). ACh, acetylcholine; CNS, central nervous system; DA, dopamine.

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to the pituitary-thyroid and pituitary-adrenal axes. Control of GH secretion thus includes two closed-loop systems (GH and IGF-I) and one open-loop regulatory system (neural). Although most of the evidence for a direct role of GH in its own negative feedback has been derived from animals, an elegant study in normal men demonstrated that GH pretreatment blocks the subsequent GH secretory response to GHRH by a mechanism that is dependent on somatostatin. [425] The mechanism responsible for GH feedback through the hypothalamus has been largely elucidated in rodent models. GH receptors are selectively expressed on somatostatin neurons in the hypothalamic periventricular nucleus and on NPY neurons in the arcuate nucleus. C-fos gene expression is acutely elevated in both populations of GH receptorpositive neurons by GH administration, indicating an activation of hypothalamic circuitry that includes these neurons. Similarly, GHRH neurons in the arcuate nucleus are acutely activated by MK-0677 because of their selective expression of the GHS-R. Zheng and colleagues [426] showed in the latter group of neurons that c-fos induction after MK-0677 administration was blocked by pretreatment of mice with GH (Fig. 7-31) . The effect must be indirect because there are no GH receptors on GHRH neurons. However, there are type 2 somatostatin receptors expressed on GHRH neurons, and the somatostatin analogue octreotide also significantly blocked c-fos activation in the arcuate

Figure 7-31 Somatostatin and the somatostatin receptor 2 subtype are involved in the short-loop inhibitory feedback of growth hormone (GH) on arcuate neurons. Activation of neurons in the arcuate nucleus was determined by the quantification of immunoreactive c-Fospositive cells after administration of the growth hormone secretagogue MK-0677 (MK). Preliminary treatment of wild-type mice (SSTR2 +/+ ) with either GH or the somatostatin analogue octreotide (Octreo) significantly attenuated the neuronal activation by MK-0677. In contrast, GH and octreotide had no effect on MK-0677 neuronal activation in somatostatin receptor 2-deficient mice (SSTR2 -/- ). (Adapted from Zheng H, Bailey A, Jian M-H, et al. Somatostatin receptor subtype 2 knockout mice are refractory to growth hormone-negative feedback on arcuate neurons. Mol Endocrinol 1997; 11:17091717.)

nucleus by MK-0677. The inhibitory effects of either GH or octreotide pretreatment were abolished in knockout mice lacking the specific somatostatin receptor (see Fig. 7-31) . Together with data from many other experiments, these results strongly support a model of GH negative feedback regulation that involves the primary activation of periventricular somatostatin neurons by GH. These tuberoinfundibular neurons then inhibit GH secretion directly by release of somatostatin in the median eminence, but they also indirectly inhibit GH secretion by way of collateral axonal projections to the arcuate nucleus that synapse on and inhibit GHRH neurons (see Fig. 7-30) . It is probable from evidence in rodents that NPY and galanin also play a part in the short-loop feedback of GH secretion, but a definitive mechanism in humans is not yet established. IGF-I has a major inhibitory action on GH secretion at the level of the pituitary gland. [406] IGF-I receptors are expressed on human somatotroph adenoma cells and inhibit both spontaneous and GHRH-stimulated GH release. In addition, gene expression of both GH and the pituitary-specific transcription factor Pit-1 is inhibited by IGF-I. Conflicting data among species suggest that circulating IGF-I may also regulate GH secretion by actions within the brain. The feedback effects of IGF-I account for the fact that in conditions in which circulating levels of IGF-I are low, such as anorexia nervosa, proteincalorie

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starvation, [427] and Laron dwarfism (the result of a defect in the GH receptor), serum GH levels are elevated. Neural Control

The predominant hypothalamic influence on GH release is stimulatory, and section of the pituitary stalk or lesions of the basal hypothalamus cause reduction of basal and induced GH release. When the somatostatinergic component is inactivated (e.g., by antisomatostatin antibody injection in rats), basal GH levels and GH responses to the usual provocative stimuli are enhanced. GHRH-containing nerve fibers that terminate adjacent to portal vessels in the external zone of the median eminence arise principally from within, above, and lateral to the infundibular nucleus in human hypothalamus, corresponding to rodent arcuate and ventromedial nuclei. [428] Perikarya of the tuberoinfundibular somatostatin neurons are located almost completely in the medial periventricular nucleus and parvocellular component of the anterior paraventricular nucleus. Neuroanatomic and functional evidence suggests a bidirectional synaptic interaction between the two peptidergic systems. [406] Multiple extrahypothalamic brain regions provide efferent connections to the hypothalamus and regulate GHRH and somatostatin neuronal activity ( Fig. 7-32 ; see Fig. 7-30 ). Somatosensory and affective information is integrated and filtered through the amygdaloid complex. The basolateral amygdala provides an excitatory input to the hypothalamus, and the central extended amygdala, which includes the central and medial nuclei of the amygdala together with the bed nucleus of the stria terminalis, provides a GABAergic inhibitory input. Many intrinsic neurons of the hypothalamus also release GABA, often with a peptide cotransmitter. Excitatory cholinergic fibers arise to a small extent from forebrain projection nuclei but mostly from hypothalamic cholinergic interneurons, which

Figure 7-32 Neural pathways involved in growth hormone (GH) regulation. This diagram illustrates the varied pathways by which impulses from the limbic system and brain stem ultimately impinge on the hypothalamic periventricular and arcuate nuclei to stimulate GH release through the mediation of somatostatin (SRIF) and growth hormonereleasing hormone (GHRH). Psychological stress modulates hypothalamic function indirectly through the bed nucleus of the stria terminalis (BNST) and amygdalar complex (Amyg). Circadian rhythms are entrained in part by projections from the suprachiasmatic nucleus (SCN). Complex reciprocal interactions between sleep stage and GHRH release involve cortex and subcortical nuclei, but the detailed mechanisms are not known. Dopaminergic and histaminergic input are from neurons located in the arcuate and mammillary nuclei, respectively, of the hypothalamus (HYP). Ascending catecholaminergic projections arise in both the nucleus of the tractus solitarius (NTS) and ventral lateral medulla (VLM). Serotoninergic (5-HT) afferents are from the raphe nuclei. In addition to these neural pathways, a variety of peripheral hormonal and metabolic signals and cytokines influence GH secretion by actions within the medial basal hypothalamus and pituitary gland.

densely innervate the external zone of the median eminence. Similarly, the origin of dopaminergic and histaminergic neurons is local with their cell bodies located in the hypothalamic arcuate and tuberomammillary bodies, respectively. Two important ascending pathways to the medial basal hypothalamus regulate GH secretion and originate from serotoninergic neurons in the raphe nuclei and adrenergic neurons in the nucleus of the tractus solitarius and ventral lateral nucleus of the medulla. Both GHRH and somatostatin neurons express presynaptic and postsynaptic receptors for multiple neurotransmitters and peptides (Table 7-6) . The 2 -adrenoreceptor agonist clonidine reliably stimulates GH release, and for this reason a clonidine test was a standard diagnostic tool in pediatric endocrinology. The stimulatory effect is blocked by the specific 2 -antagonist yohimbine and appears to involve a dual mechanism of action, inhibition of somatostatin neurons and activation of GHRH neurons. In addition, partial attenuation of the effects of clonidine by mixed 5-hydroxytryptamine type 1 and type 2 antagonists suggests that some of the relevant 2 -receptors are located presynaptically on serotoninergic nerve terminals and increase serotonin release. Both norepinephrine and epinephrine play physiologic roles in the adrenergic stimulation of GH secretion. The 1 -agonists have no effect on GH secretion in humans, but 2 -agonists such as the bronchodilator salbutamol inhibit GH secretion by stimulating the release of somatostatin from nerve terminals in the median eminence. These effects are blocked by propranolol, a nonspecific -antagonist. Dopamine generally has a net effect to stimulate GH secretion, but the mechanism is not clear because of multiple dopamine receptor subtypes and the apparent activation of both GHRH and somatostatin neurons. Serotonin's effect on GH release in humans was difficult to decipher because of the large number of receptor subtypes. However, clinical studies with the receptor-selective agonist

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Physiologic

TABLE 7-6 -- Factors That Change Growth Hormone Secretion in Humans Hormones and Neurotransmitters

Pathologic

Stimulatory Factors Episodic, spontaneous release

Insulin hypoglycemia

Exercise

2-Deoxyglucose

TRH

Stress

Amino acid infusions

LHRH

Physical Psychological

Arginine, lysine Neuropeptides

Acromegaly

Glucose Arginine

Slow wave sleep

GHRH

Interleukins 1, 2, 6

Postprandial glucose decline

Ghrelin

Protein depletion

Fasting

Galanin

Starvation

Opioids (µ-receptors)

Anorexia nervosa

Melatonin

Renal failure

Classical neurotransmitters 2

-Adrenergic agonists

Liver cirrhosis Type 1 diabetes mellitus

-Adrenergic antagonists M1-cholinergic agonists 1D-serotonin agonists H1-histamine agonists GABA (basal levels) Dopamine (? receptor) Estrogen Testosterone Glucocorticoids (acute) Inhibitory Factors* Postprandial hyperglycemia

Glucose infusion

Elevated free fatty acids

Neuropeptides

Acromegaly L-Dopa

Elevated GH levels

Somatostatin

D2R DA agonists

Elevated IGF-I (pituitary)

Calcitonin

Phentolamine

Rapid eye movement (REM) sleep

Neuropeptide Y (NPY)

Galanin

Senesence, aging

Corticotropin-releasing hormone (CRH) Classical neurotransmitters 1/2 2

-Adrenergic antagonists

Obesity Hypothyroidism Hyperthyroidism

-Adrenergic agonists

H1-histamine antagonists Serotonin antagonist Nicotinic cholinergic agonists Glucocortioids (chronic) TRH, thyrotropin-releasing hormone; LHRH, luteinizing hormonereleasing hormone; GHRH, growth hormonereleasing hormone; DA, dopamine; IGF-I, insulinlike growth factor I. *In many instances, the inhibition can be demonstrated only as a suppression of GH release induced by a pharmacologic stimulus. The inhibitory actions of NPY and CRH on GH secretion are firmly established in the rodent and are secondary to increased somatostatin tone. Contradictory evidence exists in the human for both

peptides and further studies are required.

sumatriptan clearly implicated the 5-hydroxytryptamine 1D receptor subtype in the stimulation of basal GH levels. [429] The drug also potentiates the effect of a maximal dose of GHRH, suggesting the recurring theme of GH disinhibition by inhibition of hypothalamic somatostatin neurons in its mechanism of action. Histaminergic pathways acting through H1 receptors play only a minor, conditional stimulatory role in GH secretion in humans. Acetylcholine appears to be an important physiologic regulator of GH secretion. [430] Blockade of acetylcholinergic muscarinic receptors reduces or abolishes GH secretory responses to GHRH, glucagon and arginine, morphine, and exercise. In contrast, drugs that potentiate cholinergic transmission increase basal GH levels and enhance the GH response to GHRH in normal individuals or in subjects with obesity or Cushing's disease. In vitro acetylcholine inhibits somatostatin release from hypothalamic fragments, and acetylcholine can act directly on the pituitary to inhibit GH release. There may even be a paracrine cholinergic control system within the pituitary. However, the sum of evidence suggests that the primary mechanism of action of M1 agonists is inhibition of somatostatin neuronal activity or the release of peptide from somatostatinergic terminals. Short-term cholinergic blockade with the M1 muscarinic receptor antagonist pirenzepine reduced the GH excess of patients with poorly controlled diabetes mellitus. [881] However, in the long term, cholinergic blockade did not prevent complications associated with the hypersomatotropic state. Many neuropeptides in addition to GHRH and somatostatin are involved in the modulation of GH secretion in humans (see Table 7-6) .[406] [411] Among these, the evidence is most compelling for a stimulatory role of galanin acting in the human hypothalamus by a GHRH-dependent mechanism. [431] Many GHRH neurons are immunopositive for galanin as well as neurotensin and tyrosine hydroxylase. Galanin's actions may be explained, in part, by presynaptic facilitation of catecholamine release from nerve terminals and subsequent direct adrenergic stimulation of GHRH release. [432] Opioid peptides also stimulate GH release, probably by activation of GHRH neurons, but under normal circumstances endogenous opioid tone in the hypothalamus is presumed to be low because opioid antagonists have little acute effect on GH secretion. A larger number of neuropeptides are known or suspected to inhibit GH secretion in humans, at least under certain circumstances. calcitonin, oxytocin,

[411]

The list includes NPY, CRH,

121

neurotensin, VIP, and TRH. Inhibitory actions of NPY are well established in the rat. The effect on GH secretion is secondary to stimulation of somatostatin neurons and is of particular interest because of the presumed role in GH auto-feedback (discussed earlier) and the integration of GH secretion with regulation of energy intake and expenditure (discussed in a later section). Finally, TRH has the well-established paradoxical effect of increasing GH secretion in patients with acromegaly, type 1 diabetes mellitus, hypothyroidism, or hepatic and renal failure. Factors Influencing Secretion of Growth Hormone

Human Growth Hormone Rhythms

The unraveling of rhythmic GH secretion has relied on a combination of technical innovations in sampling and GH assay, and sophisticated mathematical modeling including deconvolution analysis and the calculation of approximate entropy as a measure of orderliness or regularity in minute-to-minute secretory patterns. [411] At least three distinct categories of GH rhythms, which differ markedly in their time scales, can be considered here. The daily GH secretion rate varies over two orders of magnitude from a maximum of nearly 2.0 mg/day in late puberty to a minimum of 20 µg/day in older or obese adults. The neonatal period is characterized by markedly amplified GH secretory bursts followed by a prepubertal decade of stable, moderate GH secretion of 200 to 600 µg/day. There is a marked increase in daily GH secretion during puberty that is accompanied by a commensurate rise in plasma IGF-I to levels that constitute a state of physiologic hypersomatotropism. This pubertal increase in GH secretion is due to increased GH mass per secretory burst and not to increased pulse frequency. Although the changes are clearly related to the increases in gonadal steroid hormones and can be mimicked by administration of estrogen or testosterone to hypogonadal children, the underlying neuroendocrine mechanisms are not fully understood. One hypothesis is that decreased sensitivity of the hypothalamic-pituitary axis to negative feedback of GH and IGF-I leads to increased GHRH release and action. Young adults have a return of daily GH secretion to prepubertal levels despite continued gonadal steroid elevation. The so-called somatopause is defined by an exponential decline in GH secretory rate with a half-life of 7 years starting in the third decade of life. GH secretion in young adults exhibits a true circadian rhythm over a 24-hour period, characterized by a greater nocturnal secretory mass that is independent of sleep onset. [433] However, as discussed earlier, GH release is further facilitated when slow wave sleep coincides with the normal circadian peak. Under basal conditions GH levels are low most of the time, with an ultradian rhythm of about 10 (men) or 20 (women) secretory pulses per 24 hours as calculated by deconvolution analysis. [434] Both sexes have an increased pulse frequency during the nighttime hours, but the fraction of total daily GH secretion associated with the nocturnal pulses is much greater in men. Overall, women have more continuous GH secretion and more frequent GH pulses that are of more uniform size than men. [434] A complementary study using approximate entropy analysis concluded that the nonpulsatile regularity of GH secretion is also significantly different in men and women. [435] These sexually dimorphic patterns in the human are actually quite similar to those in the rat, although the sex differences are not as extreme in humans. [411] [435] The neuroendocrine basis for sex differences in the ultradian rhythm of GH secretion is not fully understood. Gonadal sex steroids play both an organizational role during development of the hypothalamus and an activational role in the adult, regulating expression of the genes for many of the peptides and receptors central to GH regulation. [406] [411] In the human, unlike the rat, the hypothalamic actions of testosterone appear to be predominantly due to its aromatization to 17-estradiol and interaction with estrogen receptors. Hypothalamic somatostatin appears to play a more prominent role in men than in women in the regulation of pulsatile GH secretion, and this difference is postulated to be a key factor in producing the sexual dimorphism. [434] [436] [437] External and Metabolic Signals

The various peripheral signals that modulate GH secretion in humans are summarized in Table 7-6 ( also see Fig. 7-30 and Fig. 7-32 ). Of particular importance are factors related to energy intake and metabolism because they provide a common signal between the peripheral tissues and hypothalamic centers regulating nonendocrine homeostatic pathways in addition to the classical hypophyseotropic neurons. It is also in this complex arena that species-specific regulatory responses are particularly prominent, making extrapolations between rodent experimental models and human GH regulation less reliable. [406] [411] Important triggers of GH release include the normal decrease in blood glucose level after intake of a carbohydrate-rich meal, absolute hypoglycemia, exercise, physical and emotional stress, and high intake of protein (mediated by amino acids). Some of the pathologic causes of elevated GH represent extremes of these physiologic signals and include protein-calorie starvation, anorexia nervosa, liver failure, and type 1 diabetes mellitus. A critical concept is that many of these GH triggers work through the same final common mechanism of somatostatin withdrawal and consequent disinhibition of GH secretion. In contrast, postprandial hyperglycemia, glucose infusion, elevated plasma free fatty acids, type 2 diabetes mellitus (with obesity and insulin resistance), and obesity are all associated with inhibition of GH secretion. The role of leptin in mediating either increases or decreases in GH release is complicated by its multiple sites of action and coexistent secretory environment. Similarly, other members of the cytokine family including IL-1, IL-2, IL-6, and endotoxin have been inconsistently shown to stimulate GH in humans. The actions of steroid hormones on GH secretion are complex because of their multiple loci of action within the proximal hypothalamic-pituitary components in addition to secondary effects on other neural and endocrine systems. Glucocorticoids in particular produce opposite responses that are dependent on the chronicity of administration. Moreover, glucocorticoid effects follow an inverted U-shaped dose-response curve. Both low and high glucocorticoid levels reduce GH secretion, the former because of decreased GH gene expression and somatotroph responsiveness to GHRH and the latter because of increased hypothalamic somatostatin tone and decreased GHRH. Similarly, physiologic levels of thyroid hormones are necessary to maintain GH secretion and promote GH gene expression. Excessive thyroid hormone is also inhibitory to the GH axis, and the mechanism is speculated to be a combination of increased hypothalamic somatostatin tone, GHRH deficiency, and suppressed pituitary GH production. Somatostatin

Chemistry and Evolution

A factor that potently inhibited GH release from pituitary in vitro was unexpectedly identified during early efforts to isolate GHRH from hypothalamic extracts. [438] Somatostatin, the peptide responsible for this inhibition of GH secretion and the inhibition of insulin secretion by a pancreatic islet extract, was eventually isolated from hypothalamus and sequenced by Brazeau

122

Figure 7-33 Diagram illustrating the genomic organization, messenger ribonucleic acid structure, and post-translational processing of the human somatostatin prohormone. Transcriptional regulation of the somatostatin gene, including the identification of tissue-specific elements (TSE), upstream elements (UE), and the cyclic adenosine monophosphate (cAMP) response element (CRE) that are binding sites for specific factors, has been studied extensively in pancreatic islet cell lines. It is not known whether all or some of these factors are also involved in the neural-specific expression of somatostatin. SST-28 and SST-14 are cyclic peptides containing a single covalent disulfide bond between a pair of Cys residues. A beta turn containing the tetrapeptide Phe-Trp-Lys-Thr is stabilized by hydrogen bonds to produce the core receptor binding epitope. This minimal structure has been the model for conformationally restrained analogues of somatostatin including octreotide. CPE, carboxypeptidase E; PC1/PC2, prohormone convertases 1 and 2; UTR, untranslated region. (Compiled from data by Shen LP, Rutter WJ. Sequence of the human somatostatin 1 gene. Science 1984; 224:168171; Goudet G, Delhalle S, Biemar F, et al. Functional and cooperative interactions between the homeodomain PDX1, Pbx, and Prep1 factors on the somatostatin promoter. J Biol Chem 1999; 274:40674073; and Milner-White EJ. Predicting the biologically active conformations of short polypeptides. Trends Pharmacol Sci 1989; 10:7074.)

and colleagues in 1973. [439] The term somatostatin was originally applied to a cyclic peptide containing 14 amino acids (somatostatin-14 [SST-14]; Fig. 7-33 ). Subsequently, a second form, N-extended somatostatin-28 (SST-28), was identified as a secretory product. Both forms of somatostatin are derived by independent cleavage of a common prohormone by prohormone convertases. [440] In addition, the isolation of SST-28(112) in some tissues suggests that SST-14 can be secondarily processed from SST-28. SST-14 is the predominant form in the brain (including the hypothalamus), whereas SST-28 is the 123

major form in the gastrointestinal tract, especially the duodenum and jejunum. The name somatostatin is descriptively inadequate because the molecule also inhibits thyrotropin secretion from the pituitary and has nonpituitary roles including activity as a neurotransmitter or neuromodulator in the central and peripheral nervous systems and as a regulatory peptide in gut and pancreas. As a pituitary regulator, somatostatin is a true neurohormone, that is, a neuronal secretory product that enters the blood (hypophyseal-portal circulation) to affect cell function at remote sites. In the gut, somatostatin is present in both the myenteric plexus, where it acts as a neurotransmitter, and epithelial cells, where it influences the function of adjacent cells as a paracrine secretion. Somatostatin can influence its own secretion from delta cells (an autocrine function) in addition to acting as a paracrine factor in pancreatic islets. Gut exocrine secretion can be modulated by intraluminal action, so it is also a lumone. Because of its wide distribution, broad spectrum of regulatory effects, and evolutionary history, this peptide can be regarded as an archetypical pan-system modulator. The genes that encode somatostatin in humans [441] (see Fig. 7-33) and a number of other species exhibit striking sequence homology, even in primitive fish such as the anglerfish. Furthermore, the amino acid sequence of SST-14 is identical in all vertebrates. Formerly, it was accepted that all tetrapods have a single gene encoding both SST-14 and SST-28 whereas teleost fish have two nonallelic pre-prosomatostatin genes ( PPSI and PPSII), each of which encodes only one form of the mature somatostatin peptides. This situation implied that a common ancestral gene underwent a duplication event after the split of teleosts from the descendants of tetrapods. However, both lampreys and amphibians, which predate and postdate the teleost evolutionary divergence, respectively, have now been shown to have at least two PPS genes.[442] A more distantly related gene has been identified in mammals that encodes cortistatin, a somatostatin-like peptide that is highly expressed in cortex and hippocampus.[443] [444] Cortistatin-14 differs from SST-14 by three amino acid residues but has high affinity for all known subtypes of somatostatin receptors (see later). The human gene sequence predicts a tripeptide-extended cortistatin-17 and a further N-terminally extended cortistatin-29. [445] A revised evolutionary concept of the somatostatin gene family is that a primordial gene underwent duplication at or before the advent of chordates and the two resulting genes underwent mutation at different rates to produce the distinct pre-prosomatostatin and pre-procortistatin genes in mammals. [442] A second gene duplication probably occurred in teleosts to generate PPSI and PPSII from the ancestral somatostatin gene. Apart from its expression in neurons of the periventricular and arcuate hypothalamic nuclei and involvement in GH secretion discussed earlier, somatostatin is highly expressed in the cortex, lateral septum, extended amygdala, reticular nucleus of the thalamus, hippocampus, and many brain stem nuclei. Cortistatin is present in the brain at a small fraction of the levels of somatostatin and in a more limited distribution primarily confined to cortex and hippocampus. The molecular mechanisms underlying the developmental and hormonal regulation of somatostatin gene transcription have been most extensively studied in pancreatic islet cells. [446] [447] [448] Less is known concerning the regulation of somatostatin gene expression in neurons except that activation is strongly controlled by binding of the phosphorylated transcription factor cAMP response elementbinding protein to its cognate cAMP response element contained in the promoter sequence. [449] [450] Enhancer elements in the somatostatin gene promoter that bind complexes of homeodomain-containing transcription factors (PAX6, PBX, PREP1) and up-regulate gene expression in pancreatic islets may actually represent gene silencer elements in neurons ( see Fig. 7-33 , promoter elements TSEII and UE-A).[446] Conversely, another related cis element in the somatostatin gene ( see Fig. 7-33 , promoter element TSEI ) apparently binds a homeodomain transcription factor PDX1 (also called STF1/IDX1/IPF1) that is common to developing brain, pancreas, and foregut and regulates gene expression in both the CNS and gut. [451] The function of somatostatin in GH and thyrotropin regulation was considered earlier in this chapter. Its actions in the extrahypothalamic brain and diagnostic and therapeutic roles are considered in the remainder of this section and in Chapter 8 . An additional function of somatostatin in pancreatic islet cell regulation is described in Chapter 29 , and the manifestations of somatostatin excess as in somatostatinoma are described in Chapter 35 . Somatostatin Receptors

Five somatostatin receptor subtypes (SSTR1 to SSTR5) have been identified by gene cloning techniques (Table 7-7) , and one of these (SSTR2) is expressed in two alternatively spliced forms. [452] These subtypes are encoded by separate genes located on different chromosomes, are expressed in unique or partially overlapping distributions in multiple target organs, and differ in their coupling to second messenger signaling molecules and therefore in their range and mechanism of intracellular actions. [452] [453] The subtypes also differ in their binding affinity to specific somatostatin analogues. Certain of these differences have important implications for the use of somatostatin analogues in therapy and in diagnostic imaging. All SSTR subtypes are coupled to pertussis toxinsensitive G proteins and bind SST-14 and SST-28 with high affinity in the low nanomolar range, although SST-28 has a uniquely high affinity for SSTR5. SSTR1 and SSTR2 are the two most abundant subtypes in brain and probably function as presynaptic autoreceptors in the hypothalamus and limbic forebrain, respectively, in addition to their postsynaptic actions. SSTR4 is most prominent in hippocampus. All the subtypes are expressed in pituitary, but SSTR2 and SSTR5 are the most abundant on somatotrophs. These two subtypes are also the most physiologically important in pancreatic islets, with SSTR5 responsible for inhibition of insulin secretion from beta cells and SSTR2 responsible for inhibition of glucagon from alpha cells. [454] Binding of somatostatin to its receptor leads to activation of one or more plasma membranebound inhibitory G proteins, which in turn inhibit adenylyl cyclase activity and lower intracellular cAMP. Other G proteinmediated actions common to all SSTRs are activation of a vanadate-sensitive phosphotyrosine phosphatase and modulation of mitogen-activated protein kinase (MAPK). Different subsets of SSTRs are also coupled to inwardly rectifying K + channels, voltage-dependent Ca 2+

channels, an Na+ /H+ exchanger, -amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)-kainate glutamate receptors, TABLE 7-7 -- Characteristics of the Human Somatostatin Receptors Characteristic

SSTR1

SSTR2

SSTR3

SSTR4

SSTR5

Chromosome

14q13

17q24

22q13.1 20p11.2 16p13.3

Tissue distribution

Brain

Brain

Brain

Brain

Pituitary Pituitary Pituitary Islet

Islet

Islet

Brain Pituitary

Islet

Islet

Stomach Stomach Stomach Stomach Stomach Kidney Liver

Kidney

Lung Placenta

124

phospholipase C, and phospholipase A2. [452] The lowering of intracellular cAMP and Ca 2+ is the most important mechanism for the inhibition of hormone secretion, and actions on phosphotyrosine phosphatase and MAPK are postulated to play a role in somatostatin's antiproliferative effect on tumor cells. Effects on Target Tissues and Mechanism of Action

In the pituitary, somatostatin inhibits secretion of GH and thyrotropin and, under certain conditions, of PRL and ACTH as well. It exerts inhibitory effects on virtually all endocrine and exocrine secretions of the pancreas, gut, and gallbladder (Table 7-8) . Somatostatin inhibits secretion by the salivary glands and, under some conditions, the secretion of parathyroid hormone and calcitonin. Somatostatin blocks hormone release in many endocrine-secreting tumors, including insulinomas, glucagonomas, VIPomas, carcinoid tumors, and some gastrinomas. The physiologic actions of somatostatin in extrahypothalamic brain remain the subject of investigation. In the striatum, somatostatin increases the release of dopamine from nerve terminals by a glutamate-dependent mechanism. It is widely expressed in GABAergic interneurons of limbic cortex and hippocampus, where it modulates the excitability of pyramidal neurons. Temporal lobe epilepsy is associated with a marked reduction in somatostatin-expressing neurons in the hippocampus consistent with a putative inhibitory action on seizures. [455] A wealth of correlative data has linked reduced forebrain and CSF concentrations of somatostatin with Alzheimer's disease, major depression, and other neuropsychiatric disorders, raising speculation about the role of somatostatin in modulating neural circuits underlying cognitive and affective behaviors. [456] Clinical Applications of Somatostatin Analogues

An extensive pharmaceutical discovery program has produced somatostatin analogues with receptor subtype selectivity and improved pharmacokinetics and oral bioavailability compared with the native peptide. Initial efforts focused on the rational design of constrained cyclic peptides that incorporated D-amino acid residues and included the Trp 8 -Lys9 dipeptide of somatostatin, which was shown by structure-function studies to be necessary for high-affinity binding to its receptor (see Fig. 7-33) . Many TABLE 7-8 -- Biologic Actions of Somatostatin Outside the Central Nervous System Inhibits Hormone Secretion by Pituitary gland GH, thyrotropin, ACTH, prolactin Gastrointestinal tract Gastrin Secretin Gastrointestinal polypeptide Motilin Glicentin (enteroglucagon) VIP Pancreas Insulin Glucagon Somatostatin Genitourinary tract Renin Inhibits Other Gastrointestinal Actions Gastric acid secretion Gastric and jejunal fluid secretion Gastric emptying Pancreatic bicarbonate secretion Pancreatic enzyme secretion (Stimulates intestinal absorption of water and electrolytes) Gastrointestinal blood flow AVP-stimulated water transport Bile flow Extra Gastrointestinal Actions Inhibits the function of activated immune cells Inhibition of tumor growth GH, growth hormone; ACTH, adrenocorticotropic hormone; AVP, arginine vasopressin; VIP, vasoactive intestinal peptide. such analogues have been studied in clinical trials including octreotide, lanreotide, vapreotide, and the hexapeptide MK-678. These compounds are agonists with

similarly high-affinity binding to SSTR2 and SSTR5, moderate binding to SSTR3, and no (or low) binding to SSTR1 and SSTR4. A combinatorial chemistry approach has led to a new generation of nonpeptidyl somatostatin agonists that bind selectively and with subnanomolar affinity to each of the five SSTR subtypes. [457] [458] In contrast to the marked success in development of potent and selective somatostatin agonists, there is a relative paucity of useful antagonists. [452] The actions of octreotide (SMS 201-995 or Sandostatin) illustrate the general potential of somatostatin analogues in therapy. [459] [460] It controls excess secretion of GH in acromegaly in most patients and shrinks tumor size in about one third. Octreotide is also indicated for the treatment of thyrotropin-secreting adenomas that recur after surgery. It is used to treat other functioning metastatic neuroendocrine tumors, including carcinoid, VIPoma, glucagonoma, and insulinoma, but is seldom of use for the treatment of gastrinoma. It is also useful in the management of many forms of diarrhea (acting on salt and water excretion mechanisms in the gut) and in reducing external secretions in pancreatic fistulae (thus permitting healing). A decrease in blood flow to the gastrointestinal tract is the basis for its use in bleeding esophageal varices, but it is not effective in the treatment of bleeding from a peptic ulcer. The only major undesirable side effect of octreotide is reduction of bile production and of gallbladder contractility, leading to "sludging" of bile and an increased incidence of gallstones. Other common adverse effects including nausea, abdominal cramps, diarrhea secondary to malabsorption of fat, and flatulence usually subside spontaneously within 2 weeks of continued treatment. Impaired glucose tolerance is not associated with long-term octreotide therapy, despite an inhibitory effect on insulin secretion, because of compensating reductions in carbohydrate absorption and GH and glucagon secretion that are also caused by the drug. Somatostatin analogues labeled with a radioactive tracer have been used as external imaging agents for a wide range of disorders. [459] [460] A 111 In-labeled analogue of octreotide (OctreoScan) has been approved for clinical use in the United States and several other countries (Fig. 7-34) . The majority of neuroendocrine tumors and many pituitary tumors that express somatostatin receptors are visualized by external imaging techniques after administration of this agent; a variety of nonendocrine tumors and inflammatory lesions are also visualized, all of which have in common the expression of somatostatin receptors. Such tumors include nonsmall cell cancer of the lung (100%), meningioma (100%), breast cancer (74%), and astrocytomas (67%). Because activated T cells of the immune system display somatostatin receptors, inflammatory lesions that take up the tracer include sarcoidosis, Wegener's granulomatosis, tuberculosis, and many cases of Hodgkin's disease and non-Hodgkin's lymphoma. Although the tracer lacks specificity in differential diagnosis, its ability to identify the presence of abnormality and the extent of the lesion provides important information for management, including tumor staging. The use of a small hand-held radiation detector in the operating room makes it possible to ensure the completeness of removal of medullary thyroid carcinoma metastases. [461] New developments in the synthesis of tracers chelated to octreotide for positron emission tomography have allowed the sensitive detection of meningiomas only 7 mm in diameter and located beneath osseous structures at the base of the skull. [462] The ability of somatostatin to inhibit the growth of normal and some neoplastic cell lines and to reduce the growth of experimentally induced tumors in animal models has stimulated interest in somatostatin analogues for the treatment of cancer. Somatostatin's tumoristatic effects may be a combination

125

Figure 7-34 The use of 111 In-labeled diethylenetriaminepentaacetic acid (DTPA)-octreotide (radioactive somatostatin analogue) and external imaging techniques to localize a carcinoid tumor expressing somatostatin receptors. Pictures were taken 24 hours after administration of labeled tracer. A, Anterior view of the abdomen showing nodular metastases in an enlarged liver and the primary carcinoid tumor (arrow) in the wall of the jejunum of a patient with severe flushing and diarrhea. B, Posterior view of the chest and neck showing a metastasis in a lymph node on the left side of the neck (arrow) and multiple metastases in the ribs and pleura. (Reprinted with modifications, by permission, from Lamberts SWJ, Krenning EP, Reubi J-C. The role of somatostatin and its analogs in the diagnosis and treatment of tumors. Endocrine Rev 1991; 12:450482. © 1991, The Endocrine Society.)

of direct actions on tumor cells related to inhibition of growth factor receptor expression, inhibition of MAPK, and stimulation of phosphotyrosine phosphatase. SSTR1, SSTR2, SSTR4, and SSTR5 can all promote cell cycle arrest associated with induction of the tumor suppressor retinoblastoma and p21, and SSTR3 can trigger apoptosis accompanied by induction of the tumor suppressor p53 and the proapoptotic protein Bax. [452] In addition, somatostatin has indirect effects on tumor growth by its inhibition of circulating, paracrine, and autocrine tumor growthpromoting factors and it can modulate the activity of immune cells and influence tumor blood supply. Despite this promise, the therapeutic utility of octreotide as an antineoplastic agent remains controversial. Two new treatment approaches in preclinical trials may yet effectively utilize somatostatin receptors in the arrest of cancer cells. [460] The first is receptor-targeted radionuclide therapy using octreotide chelated to a variety of gamma- or beta-emitting radioisotopes. Theoretical calculations and empirical data suggest that radiolabeled somatostatin analogues can deliver a tumoricidal radiotherapeutic dose to some tumors after receptor-mediated endocytosis. A variation on this theme is the chelation of a cytotoxic chemotherapeutic agent to a somatostatin analogue. A second approach involves somatic cell gene therapy to transfect SSTR-negative pancreatic cancer cells with an SSTR gene. [463] Therapeutic results can be obtained with the creation of autocrine or paracrine inhibitory growth effects or the addition of targeted radionuclide treatments.



Prolactin-Regulating Factors Dopamine

It is well known that PRL secretion, unlike the secretion of other pituitary hormones, is primarily under tonic inhibitory control by the hypothalamus (Fig. 7-35) . [464] Destruction of the stalk median eminence or transplantation of the pituitary gland to ectopic sites causes a marked constitutive increase in PRL secretion, in contrast to a decrease in the release of GH, TSH, ACTH, and the gonadotropins. Many lines of evidence indicate that dopamine is the principal, physiologic prolactin-inhibiting factor (PIF) released from the hypothalamus. [465] Dopamine is present in hypophyseal-portal vessel blood in sufficient concentration to inhibit PRL release, [466] [467] dopamine inhibits PRL secretion from lactotrophs both in vivo and in vitro, [468] and dopamine D2 receptors are expressed on the plasma membrane of lactotrophs. [469] [470] Mutant mice with a targeted disruption of the D2 receptor gene uniformly developed lactotroph hyperplasia, hyperprolactinemia, and eventually lactotroph adenomas, further emphasizing the importance of dopamine in the physiologic regulation of lactotroph proliferation in addition to hormone secretion. [471] [472] The intrinsic dopamine neurons of the medial-basal hypothalamus constitute a dopaminergic population with regulatory properties that are distinct from those in other areas of the brain. Notably, they lack D2 autoreceptors but express PRL receptors, which are essential for positive feedback control as discussed in detail later. In the rat, these neurons are subdivided by location into the A12 group within the arcuate nucleus and the A14 group in the anterior periventricular nucleus. The caudal A12 dopamine neurons are further classified as tuberoinfundibular (TIDA) because of their axonal projections to the external zone of the median eminence. Tuberohypophyseal (THDA) neuronal soma are located more rostrally in the arcuate nucleus and project to both the neural lobe and intermediate lobe through axon collaterals that are found in the internal zone of the median eminence. Finally, the A14 periventricular hypophyseal (PHDA) neurons send their axons only to the intermediate lobe of the pituitary gland. Although the TIDA neurons are generally considered to be the major source of dopamine to the anterior lobe through the long portal vessels originating in the median eminence, dopamine can also reach the anterior lobe from the neural and intermediate lobes by the interconnecting short portal veins. [473] Consistent with this pathway for dopamine access to the anterior lobe, surgical removal of the neurointermediate lobe in rats caused a significant increase in basal PRL levels. [474] In addition to direct actions of dopamine on lactotrophs, central dopamine can indirectly affect PRL secretion by altering the activity of inhibitory interneurons that in turn synapse on the TIDA neurons. These effects are complicated by opposing intracellular signaling pathways linked to D1 and D2 receptors located on different populations of interneurons. [475] The binding of dopamine or selective agonists such as bromocriptine to the D2 receptor has multiple effects on lactotroph function. D2 receptors are coupled to pertussis toxinsensitive G proteins and inhibit adenylyl cyclase and decrease intracellular cAMP levels. Other effects include activation of an inwardly rectifying K + channel, increase of voltage-activated K + currents, decrease of voltage-activated Ca 2+ currents, and inhibition of inositol phosphate production. Together, this spectrum of intracellular signaling events decreases free Ca 2+ concentrations and inhibits exocytosis of PRL secretory granules. [476] Dopamine also has a modest effect on thyrotrophs to inhibit the secretion of TSH. There is continuing debate concerning the mechanism by which D2 receptor activation inhibits transcription of the PRL gene. Likely pathways involve the inhibition of MAPK or protein kinase C, with a resultant reduction in the phosphorylation

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Figure 7-35 Regulation of the hypothalamic-pituitary-prolactin (PRL) axis. The predominant effect of the hypothalamus is inhibitory, an effect mediated principally by dopamine secreted by the tuberohypophyseal dopaminergic neuron system. The dopamine neurons are stimulated by acetylcholine (ACh) and glutamate and inhibited by histamine and opioid peptides. One or more prolactin releasing factors (PRFs) probably mediate acute release of PRL as in suckling and stress. There are several candidate PRFs, including thyrotropin-releasing hormone (TRH), vasoactive intestinal polypeptide (VIP), and oxytocin. PRF neurons are activated by serotonin (5-HT). Estrogen sensitizes the pituitary to release PRL, which feeds back on the pituitary to regulate its own secretion (ultrashort-loop feedback) and also influences gonadotropin secretion by suppressing the release of luteinizing hormonereleasing hormone (LHRH). Short-loop feedback is also mediated indirectly by prolactin receptor regulation of hypothalamic dopamine synthesis, secretion, and turnover. CNS, central nervous system; DA, dopamine; GABA, -aminobutyric acid.

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of Ets family transcription factors. Ets factors are important for the stimulatory responses of TRH, insulin, and epidermal growth factor on PRL expression [480] and they interact cooperatively with the pituitary-specific POU protein Pit1, which is essential for cAMP-mediated PRL gene expression. [ 481]

[477] [478] [479]

The second messenger pathways used by the D2 receptor to inhibit lactotroph cell division are also unsettled. A study using primary pituitary cultures from rats demonstrated that forskolin treatment, which activates protein kinase A and elevates intracellular cAMP, or insulin treatment, which activates a potent receptor tyrosine kinase, were both effective mitogenic stimuli for lactotrophs. Bromocriptine competitively antagonized the proliferative response caused by elevated cAMP. Furthermore, inhibition of MAPK signaling by PD98059 markedly suppressed the mitogenic action of both insulin and forskolin, suggesting an interaction of MAPK and protein kinase A signaling. [482] Another study used immortalized mammosomatotroph tumor cells that were transfected with a D2 receptor expression vector and concluded that stimulation of a phosphotyrosine phosphatase activity was an important component of dopamine's antiproliferative action. [483] Therefore, it is clear that dopamine actions on lactotrophs involve multiple different intracellular signaling pathways linked to activation of the D2 receptor, but different combinations of these pathways are relevant for the inhibitory effects on PRL secretion, PRL gene transcription, and lactotroph proliferation. The other major action of dopamine in the pituitary is the inhibition of hormone secretion from the POMC-expressing cells of the intermediate lobe, although, as noted earlier, the adult human differs from most other mammals in the rudimentary nature of this lobe. [57] THDA and PHDA axon terminals provide a dense plexus of synaptic-like contacts on melanotrophs. Dopamine release from these terminals is inversely correlated with serum MSH levels [484] and also regulates POMC gene expression and melanotroph proliferation. [485] Other hypothalamic factors probably play a role secondary to that of dopamine as additional PIFs. [464] The primary reason to conjecture the existence of these PIFs is the frequent inconsistency between portal dopamine levels and circulating PRL in different rat models. GABA is the strongest candidate and most likely acts through GABAA inotropic receptors in the anterior pituitary. Melanotrophs, like lactotrophs, are inhibited by both dopamine and GABA but with the principal involvement of G proteincoupled, metabotropic GABA B receptors.[486] Because basal dopamine tone is high, the measurable inhibitory effects of GABA on PRL release are generally small under normal circumstances. Other putative PIFs include somatostatin and calcitonin. Prolactin-Releasing Factors

Although tonic suppression of PRL release by dopamine is the dominant effect of the hypothalamus on PRL secretion, a number of stimuli promote PRL release, not

merely by disinhibition of PIF effects but by causing release of one or more neurohormonal PRFs (see Fig. 7-35) . The most important of the putative PRFs are TRH, oxytocin, and VIP, but vasopressin, angiotensin II, NPY, galanin, substance P, bombesin-like peptides, and neurotensin can also trigger PRL release under different physiologic circumstances. [464] TRH was discussed in a previous section of this chapter (see Fig. 7-15) . In humans there is an imperfect correlation between pulsatile PRL and TSH release, suggesting that TRH cannot be the sole physiologic PRF under basal conditions. [487] Like TRH, oxytocin, vasopressin, and VIP fulfill all the basic criteria for a PRF. They are produced in paraventricular hypothalamic neurons that project to the median eminence. Concentrations of the hormones in portal blood are much higher than in the peripheral circulation and are sufficient to stimulate PRL secretion in vitro. [488] [489] [490] Moreover, there are functional receptors for each of the neurohormones in the anterior pituitary gland and either pharmacologic antagonism or passive immunization against each hormone can decrease PRL secretion, at least under certain circumstances. [491] [492] [493] [494] [495] Vasopressin is released during stress and hypovolemic shock, as is PRL, suggesting a specific role for vasopressin as a PRF in these contexts. Similarly, another candidate PRF, peptide histidine isoleucine, may be specifically involved in the secretion of PRL in response to stress. Peptide histidine isoleucine and the human homologue PHM are structurally related to VIP and synthesized from the same prohormone precursor in their respective species. [496] Both peptides are coexpressed with CRH in parvocellular paraventricular neurons and presumably released by the same stimuli that cause release of CRH into the hypophyseal-portal vessels. [497] There is evidence suggesting that dopamine itself may also act as a PRF, in contrast to its predominant function as a PIF. [464] At concentrations three orders of magnitude lower than that associated with maximal inhibition of PRL secretion, dopamine was shown to be capable of stimulating secretion from primary cultures of rat pituitary cells. [498] These studies were extended to an in vivo model by Arey and colleagues, [499] who demonstrated that low-dose dopamine infusion in cannulated rats caused a further increase in circulating PRL above the already elevated baseline produced by pharmacologic blockade of endogenous dopamine biosynthesis. The physiologic relevance of these findings to humans has yet to be established. Finally, reports of "new" PRFs continue to be published. Much excitement was generated by the isolation of a peptide from bovine hypothalamus named prolactin-releasing peptide (PrRP). [500] PrRP binds with high affinity to an orphan G proteincoupled receptor (hGR3/GPR10) expressed specifically in human pituitary and selectively stimulates PRL release from rat pituitary cells with a potency similar to that of TRH. However, PrRP is expressed predominantly in a subpopulation of noradrenergic neurons in the medulla and a small population of non-neurosecretory neurons of the hypothalamus, raising the serious question of whether PrRP reaches the anterior pituitary and actually causes PRL secretion. [501] Subsequent studies found no direct evidence for release of PrRP in the arcuate nucleusmedian eminence, further suggesting that the peptide is not a hypophyseotropic neurohormone. [502] However, PrRP probably does function as a neuromodulator within the

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CNS at sites expressing its receptor and may be involved in the neural circuitry mediating satiety.

[ 503]

Intrapituitary Regulation of Prolactin Secretion

Probably more than that of any other pituitary hormone, the secretion of PRL is regulated by autocrine-paracrine factors within the anterior lobe and by neurointermediate lobe factors that gain access to venous sinusoids of the anterior lobe by way of the short portal vessels. The wealth of local regulatory mechanisms within the anterior lobe has been reviewed extensively [464] [504] [505] and is also discussed in Chapter 8 . Galanin, VIP, endothelin-like peptides, angiotensin II, epidermal growth factor, basic fibroblast growth factor, LHRH, and the cytokine IL-6 are among the most potent local stimulators of PRL secretion. Locally produced inhibitors include PRL itself, acetylcholine, transforming growth factor , and calcitonin. Although none of these stimulatory or inhibitory factors plays a dominant role in the regulation of lactotroph function and much of the research in this area has not been directly confirmed in human pituitary, it seems apparent that the local milieu of autocrine and paracrine factors plays an essential modulatory role in determining the responsiveness of lactotrophs to hypothalamic factors in different physiologic states. As noted earlier, a proportion of the inhibitory dopamine tone to the anterior lobe lactotrophs is derived from the neurointermediate lobe. It was therefore unanticipated that surgical removal of this structure in rats would block suckling-induced PRL release over the moderate basal increase attributed to partial dopamine disinhibition. [506] Further studies showed that exposure of the anterior pituitary to intermediate lobe extracts (devoid of VIP, vasopressin, and other known PRFs) stimulated PRL secretion. At least two kinds of PRF activity have been isolated from intermediate lobe tumors of the mouse, but the specific molecules involved have yet to be identified. [507] Other researchers have suggested a more passive role for the neurointermediate lobe in the regulation of PRL secretion. Melanotroph-derived N-acetylated MSH appears to act as a lactotroph responsiveness factor by recruiting nonsecretory cells to an active state and sensitizing secreting lactotrophs to the actions of other direct PRFs. [508] However, the relevance of the neurointermediate lobe for PRL regulation in primates (including humans) is not clear because of its attenuated structure in these species. Neuroendocrine Regulation of Prolactin Secretion

Secretion of PRL, like that of other anterior pituitary hormones, is regulated by hormonal feedback and neural influences from the hypothalamus. [464] [465] [509] Feedback is exerted by PRL itself at the level of the hypothalamus. PRL secretion is regulated by many physiologic states including the estrous and menstrual cycles, pregnancy, and lactation. Furthermore, PRL is stimulated by several exteroceptive stimuli including light, ultrasonic vocalization of pups, olfactory cues, and various modalities of stress. Expression and secretion of PRL are also influenced strongly by estrogens at the level of both the lactotrophs and TIDA neurons [510] (see Fig. 7-35) and by paracrine regulators within the pituitary such as galanin and VIP. Feedback Control

Negative feedback control of PRL secretion is mediated by a unique short-loop mechanism within the hypothalamus. [511] PRL activates PRL receptors, which are expressed on all three subpopulations of A12 and A14 dopamine neurons, leading to increased tyrosine hydroxylase expression and dopamine synthesis and release.[512] [513] Ames dwarf mice that secrete virtually no PRL, GH, or TSH have decreased numbers of arcuate dopamine neurons and this hypoplasia can be reversed by neonatal administration of PRL, suggesting a trophic action on the neurons. [514] However, another mouse model of isolated PRL deficiency generated by gene targeting appears to have normal numbers of hypofunctioning dopamine neurons secondary to the loss of PRL feedback. [515] Neural Control

Lactotrophs have spontaneously high secretory activity, and therefore the predominant effect of the hypothalamus on PRL secretion is tonic suppression, which is mediated by regulatory hormones synthesized by tuberohypophyseal neurons. Secretory bursts of PRL are caused by the acute withdrawal of dopamine inhibition, stimulation by PRFs, or combinations of both events. At any given moment, locally produced autocrine and paracrine regulators further modulate the responsiveness of individual lactotrophs to neurohormonal PIFs and PRFs. Multiple neurotransmitter systems impinge on the hypothalamic dopamine and PRF neurons to regulate their neurosecretion [464] (see Fig. 7-35) . Nicotinic cholinergic and glutamatergic afferents activate TIDA neurons, whereas histamine, acting predominantly through H2 receptors, inhibits these neurons. An inhibitory peptidergic input to TIDA neurons of major physiologic significance is that associated with the endogenous opioid peptides enkephalin and dynorphin and their cognate - and -receptor subtypes. [516] Opioid inhibition of dopamine release has been associated with increased PRL secretion under virtually all physiologic conditions, including the basal state, different phases of the estrous cycle, lactation, and stress. Ascending serotoninergic inputs from the dorsal raphe nucleus are the major activator of PRF neurons in the paraventricular nucleus. concerning the identity of the specific 5-hydroxytryptamine receptors involved in this activation.

[517]

There is still debate

The PRL regulatory system and its monoaminergic control have been scrutinized in detail because of the frequent occurrence of syndromes of PRL hypersecretion (see Chapter 8) . Both the pituitary and the hypothalamus have dopamine receptors, and unfortunately the response to dopamine receptor stimulation and blockade does not distinguish between central and peripheral actions of the drug. Many commonly used neuroleptic drugs influence PRL secretion. Reserpine (a catecholamine depletor) and phenothiazines such as chlorpromazine and haloperidol enhance PRL release by disinhibition of dopamine action on the pituitary, and the PRL response is an excellent predictor of the antipsychotic effects of phenothiazines because of its correlation with D2 receptor binding and activation. [518] The major

antipsychotic neuroleptic agents act on brain dopamine receptors in the mesolimbic system and in the pituitary-regulating tuberoinfundibular system. Consequently, treatment of such patients with dopamine agonists such as bromocriptine can reverse the psychiatric benefits of such drugs. A report of three patients with psychosis and concomitant prolactinomas recommended the combination of clozapine and quinagolide as the treatment of choice to manage both diseases simultaneously. [519] Factors Influencing Secretion

Circadian Rhythm

PRL is detectable in plasma at all times during the day but is secreted in discrete pulses superimposed on basal secretion and exhibits a diurnal rhythm with peak values in the early

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morning hours.[520] There is a true circadian rhythm in humans because it is maintained in a constant environment independent of the sleep rhythm. [521] The combined body of data examining TIDA neuronal activity, dopamine concentrations in the median eminence, and manipulations of the SCN suggests that endogenous diurnal alterations in dopamine tone that are entrained by light constitute the major neuroendocrine mechanism underlying the circadian rhythm of PRL secretion. External Stimuli

The suckling stimulus is the most important physiologic regulator of PRL secretion. Within 1 to 3 minutes of nipple stimulation, PRL levels rise and remain elevated for 10 to 20 minutes.[522] This reflex is distinct from the milk let-down, which involves oxytocin release from the neurohypophysis and contraction of mammary alveolar myoepithelial cells. These reflexes provide a mechanism by which the infant regulates both the production and the delivery of milk. The nocturnal rise in PRL secretion in nursing women and in non-nursing women may have evolved as a mechanism of milk maintenance during prolonged nonsuckling periods at night. [166] Pathways involved in the suckling reflex arise in nerves innervating the nipple, enter the spinal cord by way of spinal afferent neurons, ascend the spinal cord through spinothalamic tracts to the midbrain, and enter the hypothalamus by way of the median forebrain bundle (see Fig. 7-35) . In most of the pathway, neurons regulating the oxytocin-dependent milk let-down response accompany those involved in PRL regulation and then separate at the level of the paraventricular nuclei. The suckling reflex brings about an inhibition of PIF activity and a release of PRFs, although the identity of an undisputed suckling-induced PRF is unsettled. Although the significance for PRL regulation in humans is not certain, environmental stimuli from seasonal changes in

Figure 7-36 Schematic diagram of the gene for pre-pro-gonadotropin-releasing hormone (GnRH) and the GnRH peptide. Diagrams for the enhancer and promoter regions are specific to the rat gene.

light duration and auditory and olfactory cues are clearly of great importance to many mammalian species. [464] Seasonal breeders, such as the sheep, exhibit a reduction in PRL secretion in response to shortened days. The specific ultrasound vocalization of rodent pups is among the most potent stimuli for PRL secretion in lactating and virgin female rats. Olfactory stimuli from pheromones also have potent actions in rodents. A prime example is the Bruce effect or spontaneous abortion induced by exposure of a pregnant female rat to an unfamiliar male. It is mediated by a well-studied neural circuitry involving the vomeronasal nerves, corticomedial amygdala, medial preoptic area of the hypothalamus, and finally activation of TIDA neurons and a reduction in circulating PRL that is essential for maintenance of luteal function in the first half of pregnancy. Stress in many forms dramatically affects PRL secretion, although the teleologic significance is uncertain. It may be related to actions of PRL on cells of the immune system or some other aspect of homeostasis. Different stressors are associated with either a reduction or an increase in PRL secretion, depending on the local regulatory environment at the time of the stress. However, whereas well-documented changes in PRL are associated with relatively severe forms of stress in laboratory animal models, a study of academic stress in college students failed to show any significant correlation among the time periods before, during, or after final examinations and diurnal PRL levels. [523]



Gonadotropin-Releasing Hormone and Control of the Reproductive Axis Chemistry and Evolution

The hypothalamic neuropeptide that controls the function of the reproductive axis is GnRH. GnRH is a 10-amino-acid

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peptide that is synthesized as part of a larger precursor molecule and is then enzymatically cleaved to remove a signal peptide from the N-terminus and GnRH-associated peptide (GAP) from the C-terminus (Fig. 7-36) . [524] [525] [526] All forms of the decapeptide have a pyroGlu at the N-terminus and Gly-amide at the C-terminus, indicating the functional importance of the terminal regions throughout evolutionary biology. Within mammals, two genes encoding GnRH have been identified. [527] [528] The first encodes a 92-amino-acid precursor protein. This form of GnRH is now referred to as GnRH-I and is the form found in hypothalamic neurons that serves as a releasing factor to regulate pituitary gonadotroph function. [529] The second GnRH gene, GnRH-II, encodes a decapeptide that differs from the first by three amino acids. This form of GnRH is found in the midbrain region and serves as a neurotransmitter rather than as a pituitary releasing factor. Both GnRH-I and GnRH-II are found in phylogenetically diverse species, from fish to mammals, suggesting that these multiple forms of GnRH diverged from one another early in vertebrate evolution. [529] A third form of GnRH, GnRH-III, has been identified in neurons of the telencephalon in teleost fish. This form of GnRH may have been lost in higher vertebrates or simply not yet discovered in other species. [529] As discussed subsequently in more detail, GnRH-I and GnRH-III are found in cells that originate in the olfactory placode in early embryonic development. In contrast, GnRH-IIcontaining cells are derived from the midbrain ventricle. GnRH is also found in cells outside the brain. The roles of GnRH peptides produced outside the brain are not well understood but are an area of current investigation. [530] All GnRH genes have the same basic structure, with the pre-prohormone mRNA encoded in four exons. Exon 1 contains the 5' untranslated region of the gene; exon 2 contains the signal peptide, GnRH, and the N-terminus of GAP; exon 3 contains the central portion of GAP; and exon 4 contains the C-terminus of GAP and the 3' untranslated region (see Fig. 7-36) . [529] [531] Among species, the nucleotide sequences encoding the GnRH decapeptide are highly homologous. Two transcriptional start sites have been identified in GnRH genes at +1 and -579, with the +1 promoter being active in hypothalamic neurons and the other promoter active in placenta. The first 173 base pairs of the promoter are highly conserved among species. In the rat, this promoter region has been shown to contain two Oct-1 binding sites; three regions that bind the POU domain family of transcription factors, SCIP, Oct-6, and Tst-1; and three regions that can bind the progesterone receptor. [532] [533] In addition, a variety of hormones and second messengers have been shown to regulate GnRH gene expression, and the majority of the cis-acting elements thus far characterized for hormonal control of GnRH transcription have been localized to the proximal promoter region. [533] [534] The 5' untranslated region of the GnRH gene also contains a 300-base-pair enhancer region that is 1.8 kilobases upstream of the transcription start site. [535] It contains binding sites for POU homeodomain transcription factors and GATA factors. In this chapter, we focus on the hypothalamic GnRH that is derived from GnRH-1 mRNA and plays an important role in the regulation of the hypothalamic-pituitary-gonadal axis. A mutant strain of mice with a deletion of the GnRH-1 gene have hypogonadism, and the homozygous animals are infertile.

[536]

Anatomic Distribution

GnRH neurons are small, diffusely located cells that are not concentrated in a nucleus (Fig. 7-37A) . [537] [538] They are generally bipolar and fusiform in shape, with long thin axons that can exhibit spines. The location of hypothalamic GnRH neurons is species-dependent. In the rat, hypothalamic GnRH neurons are concentrated in rostral areas including the medial preoptic area, the diagonal band of Broca, the septal areas, and the anterior hypothalamus. In primates, the majority of hypothalamic GnRH neurons are located more dorsally in the medial basal hypothalamus, the infundibulum, and periventricular to the third ventricle. Throughout the hypothalamus, neuroendocrine GnRH neurons, which extend their axon terminals to the median eminence, are interspersed with non-neuroendocrine GnRH neurons, which extend their axons to other regions of the brain including other hypothalamic regions and various regions of the cortex. GnRH secreted from non-neuroendocrine neurons has been implicated in the control of sexual behavior in rodents [539] but not in higher primates. [540] Embryonic Development

GnRH neuroendocrine neurons are an unusual neuronal population in that they originate outside the CNS, from the epithelial tissue of the nasal placode. [541] [542] During embryonic development GnRH neurons migrate across the surface of the brain and into the hypothalamus, with the final hypothalamic location differing somewhat among species. Migration is dependent on a scaffolding of neurons and glial cells along which the GnRH neurons move, with neural cell adhesion molecules playing a critical role in guiding the migration process. [543] Failure of GnRH neurons to migrate properly leads to a clinical condition, Kallmann's syndrome, in which GnRH neuroendocrine neurons do not reach their final destination and thus do not stimulate pituitary gonadotropin secretion. [544] Patients with Kallmann's syndrome do not enter puberty spontaneously. X-linked Kallmann's syndrome results from a deficiency of the Kal-1 gene, which encodes a putative protein of 680 amino acids and contains four fibronectin type III repeats and a four-disulfide core motif. [545] However, this form of Kallmann's syndrome accounts for only a small percentage (about 8%) of cases, and the cause of other forms remains unknown. Administration of exogenous GnRH effectively treats this form of hypothalamic hypogonadism. Patients with Kallmann's syndrome often have other congenital midline defects, including anosmia, which results from hypoplasia of the olfactory bulb and tracts. Action at the Pituitary

Receptors

GnRH binds to a membrane receptor on pituitary gonadotrophs and stimulates both LH and FSH synthesis and secretion. The GnRH receptor is a seven-transmembrane-domain G proteincoupled receptor, but it lacks a typical intracellular C-terminal cytoplasmic domain. [546] [547] [548] Under physiologic conditions, the GnRH receptor number varies and is usually directly correlated with the gonadotropin secretory capacity of pituitary gonadotrophs. For example, across the rat estrous cycle, a rise in GnRH receptors is seen just before the surge of gonadotropins that occurs on the afternoon of proestrus. [549] [550] GnRH receptor message levels are regulated by a variety of hormones and second messengers including steroid hormones (estradiol can both suppress and stimulate, and progesterone suppresses), gonadotropins (which suppress), and calcium and protein kinase C (which stimulate). Gq/11 is the primary guanosine triphosphatebinding protein mediating GnRH responses; however, there is evidence that GnRH receptors can couple to other guanosine triphosphatebinding proteins including G s and Gi .[548] With activation, the GnRH receptor couples to a phosphoinositide-specific phospholipase C, which leads to increases in calcium transport into gonadotrophs and calcium release from internal

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stores through a diacylglycerolprotein kinase C pathway. Increased calcium entry is a critical step in GnRH-stimulated release of gonadotropin secretion. However, the MAPK cascade is also stimulated by GnRH. [548]

[548] [551]

When there is a decline in GnRH stimulation to the pituitary, as occurs in a variety of physiologic conditions including states of lactation, undernutrition, or seasonal periods of reproductive quiescence, the number of GnRH receptors on pituitary gonadotrophs declines dramatically. [549] [550] Subsequent exposure of the pituitary to pulses of GnRH restores receptor number by a Ca 2+ -dependent mechanism that requires protein synthesis. [551] [552] The effect of GnRH to induce its own receptor is termed up-regulation or self-priming. Only certain physiologic frequencies of pulsatile GnRH can augment GnRH receptor production, and these frequencies appear to differ among species. [553] Up-regulation of GnRH receptors after a period of low GnRH stimulation to the pituitary can take hours to days of exposure to pulsatile GnRH, depending on the duration and extent of the prior decrease in GnRH. The self-priming effect of GnRH to up-regulate its own receptors also plays a crucial role in the production of the gonadotropin surge that occurs at midcycle in females of spontaneously ovulating species and triggers ovulation. Just before the gonadotropin surge, two factors, the increased frequency of pulsatile GnRH release [549] [550] [554] [555] [556] and a sensitization of the pituitary gonadotrophs by rising levels of estradiol, [557] [558] make the pituitary exquisitely sensitive to GnRH and allow an output of LH that is an order of magnitude greater than the release seen during the rest of the female reproductive cycle. This surge of LH triggers the ovulatory process at the ovary. In contrast to up-regulation of GnRH receptors by pulsatile regimens of GnRH, continuous exposure to GnRH leads to down-regulation of GnRH receptors and an accompanying decrease in LH and FSH synthesis and secretion, termed desensitization. [559] Down-regulation does not require calcium mobilization or gonadotropin secretion.[559] It involves a rapid uncoupling of receptor from G proteins and sequestration of the receptors from the plasma membrane, followed by internalization and proteolytic degradation of the receptors. [548] The concept of down-regulation has a number of clinical applications. For example, the most common current therapy for precocious puberty of hypothalamic origin (i.e., precocious GnRH secretion) is to treat the child with a long-acting

Figure 7-37a Regulation of the hypothalamic-pituitary-gonadal axis. A, Gonadotropin-releasing hormone (GnRH) neurons in a coronal section of the rat hypothalamus at 4× magnification. The inset is at 20× magnification. (Micrograph provided by Patricia Williamson and Kevin Grove, Oregon National Primate Center.)

GnRH agonist, which down-regulates pituitary GnRH receptors and effectively turns off the reproductive axis. [553] [560] Children with precocious puberty can be maintained with long-acting GnRH agonists for years to suppress the premature activation of the reproductive axis, and at the normal age of puberty agonist treatment can be withdrawn, allowing a reactivation of pituitary gonadotrophs and a downstream increase in gonadal steroid hormone production. Long-acting GnRH agonists are also used in the treatment of forms of breast cancer that are estrogen-dependent as well as other gonadal steroid-dependent cancers. [553] [561] Long-acting antagonists of GnRH have been developed that can also be used for these therapies. [562] Antagonists have the advantage of not having a flare effect, that is, an acute stimulation of gonadotropin secretion that is seen during the initial treatment of individuals with superagonists. Pulsatile Gonadotropin-Releasing Hormone Stimulation

Because a single pulse of GnRH stimulates the release of both LH and FSH and chronic exposure of the pituitary to pulsatile GnRH supports the synthesis of both LH and FSH, many people believe that there is only one releasing factor regulating the synthesis and secretion of LH and FSH. [563] However, in a number of physiologic conditions there are divergent patterns of LH and FSH secretion, and thus a second FSH-releasing peptide has been proposed, but such a peptide has not been isolated to date. [564] Other mechanisms, discussed in more detail later, are likely to account for the differential regulation of LH and FSH release. The ensemble of GnRH neurons in the hypothalamus that send axons to the portal blood system in the median eminence fire in a coordinated, repetitive, episodic manner, producing distinct pulses of GnRH in the portal blood stream. [565] [566] [567] The pulsatile nature of GnRH stimulation to the pituitary leads to the release of distinct pulses of LH into the peripheral blood stream. [568] [569] In experimental animals, in which it is possible to collect blood samples simultaneously from the portal and peripheral blood stream, GnRH and LH pulses have been found to correspond in about a one-to-one ratio at most physiologic rates of secretion (Fig. 7-38) (Figure Not Available) . [566] [567] Because the portal blood stream is generally inaccessible in humans, the collection of

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Figure 7-37b B, Schematic diagram of the hypothalamic-pituitary-gonadal axis showing neural systems that regulate GnRH secretion and feedback of gonadal steroid hormones at the level of the hypothalamus and pituitary. CRH, corticotropin-releasing hormone; FSH, follicle-stimulating hormone; GABA, -aminobutyric acid; LH, luteinizing hormone; NPY, neuropeptide Y.

frequent blood samples from the peripheral blood stream is used to define the pulsatile nature of LH secretion (i.e., frequency and amplitude of LH pulses), and pulsatile LH is used as an indirect measure of the activity of the GnRH secretory system. Indirect assessment of GnRH secretion by monitoring the rate of pulsatile LH secretion is also used in many animal studies examining the factors that govern the regulation of the pulsatile activity of the reproductive neuroendocrine axis. Unlike 133

Figure 7-38 (Figure Not Available) Simultaneous detection of pulses of gonadotropin-releasing hormone (GnRH) measured in blood collected from the hypothalamic-hypophyseal portal vessels and luteinizing hormone (LH) measured in blood collected from the peripheral vasculature of an oophorectomized ewe. (Redrawn from Clarke IJ, Cummins JT. Endocrinology 1982; 111:17371739).

LH secretion, FSH secretion is not always pulsatile, and even when it is pulsatile, there is only partial concordance between LH and FSH pulses.

[ 570]

It is possible to place multiple unit recording electrodes in the medial basal hypothalamus of monkeys and other species and find spikes of electrical activity that are concordant with the pulsatile discharge of LH secretion. [571] [572] [573] It is unknown, however, whether these bursts of electrical activity reflect the activity of GnRH neurons themselves or the activity of neurons that impinge on GnRH neurons and govern their firing. With the development of mice in which the gene for green fluorescent protein has been put under the regulation of the GnRH promoter, it has been possible to identify GnRH neurons in hypothalamic tissue slices using fluorescence microscopy and record from them intracellularly. [30] These studies

Figure 7-39 The influence of gonadotropin-releasing hormone (GnRH) pulse frequency on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion in a female rhesus monkey with an arcuate nucleus lesion ablating endogenous GnRH support of the pituitary. Decreasing GnRH pulse frequency from 1 pulse/hour to 1 pulse/3 hours leads to a decrease in plasma LH concentrations but an increase in plasma FSH concentrations. (Redrawn from Wildt L, Haulser A, Marshall G, et al. Endocrinology 1981; 109:376385.)

have shown that many, but not all, GnRH neurons show a bursting pattern of electrical activity. A central, unsolved question in the field of reproductive neuroendocrinology is what causes GnRH neurons to pulse in a coordinated manner. Studies using a line of clonal GnRH neurons have shown that these neurons grown in culture can release GnRH in a pulsatile pattern, suggesting that the pulse-generating capacity of GnRH neurons may be intrinsic. [574] The term GnRH pulse generator is often used to acknowledge the fact that GnRH secretion occurs in pulses and to refer to the central mechanisms responsible for pulsatile GnRH

release.[575] A critical factor governing LH and FSH secretion and release is the rate of pulsatile GnRH stimulation of the gonadotrophs. Experimental studies in which the hypothalamus was lesioned and GnRH was replaced by pulsatile administration of exogenous GnRH showed that different frequencies of GnRH can lead to differential ratios of LH to FSH secretion from the pituitary. [576] Figure 7-39 shows that in a monkey with a hypothalamic lesion, replacement of one pulse of GnRH per hour led to a relatively low ratio of FSH to LH secretion. Subsequent institution of a slower pulse frequency of one pulse of GnRH every 3 hours led to a decrease in LH secretion but an increase in FSH secretion such that the ratio of FSH to LH secretion was greatly elevated. It is likely that this effect of pulse frequency on the ratio of FSH to LH secretion accounts, at least in part, for the clinical finding that at times when the GnRH pulse generator is just turning on, such as at the onset of puberty and during recovery from chronic undernutrition, the ratio of FSH to LH is higher than when it is measured in adults experiencing regular reproductive function. [577] [578] As discussed subsequently, steroid hormones act at both the hypothalamus and pituitary to influence strongly the rate of pulsatile GnRH release and amount of LH and FSH secreted from the pituitary. GnRH pulse frequency not only influences the rate of pulsatile gonadotropin release and the ratio of FSH to LH secretion but also plays an important role in modulating the structural makeup of the gonadotropins. LH and FSH are structurally similar glycoprotein hormones. [579] Each of these hormones is made up of an and a subunit. LH, FSH, and TSH share a common subunit, and each has a unique subunit that conveys tissue specificity to the intact hormone. Before secretion of gonadotropins, terminal sugars are attached to each gonadotropin molecule. [579] The sugars include sialic acid, galactose, N-acetylglucosamine, and mannose, but the most important is sialic acid. The extent of glycosylation of

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LH and FSH is important for the physiologic function of these hormones. Forms of gonadotropin with more sialic acid have a longer half-life because they are protected from degradation by the liver. Forms of gonadotropin with less sialic acid can have more potent effects at their biologic receptors. Both the rate of GnRH stimulation and ovarian hormone feedback at the level of the pituitary regulate the degree of LH and FSH glycosylation. [579] [580] For example, slow frequencies of GnRH, seen during follicular development, are associated with greater degrees of FSH glycosylation, which would provide sustained FSH support to growing follicles. In contrast, faster frequencies of GnRH, seen just before the midcycle gonadotropin surge, are associated with lesser degrees of FSH glycosylation, providing a more potent but shorter lasting form of FSH at the time of ovulation. [580] Regulatory Systems

Many neurotransmitter systems from the brain stem, limbic system, and other areas of the hypothalamus convey information to GnRH neurons ( see Fig. 7-37) Fig. 7-37 ). [581] These afferent systems include neurons that contain norepinephrine, dopamine, serotonin, GABA, glutamate, endogenous opiate peptides, NPY, galanin, and a number of other peptide neurotransmitters. Glutamate and norepinephrine play important roles in providing stimulatory drive to the reproductive axis, [582] [583] whereas GABA and endogenous opioid peptides provide a substantial portion of the inhibitory drive to GnRH neurons. [584] [585] Influences of specific neurotransmitter systems are discussed where appropriate in later sections on the physiologic regulation of GnRH neurons. GnRH neurons are surrounded by glial processes, and only a small percentage of their surface area is available to receive dendritic contacts from afferent neurons. Changes in the steroid hormone milieu influence the degree of glial sheathing and may play important roles in regulating afferent input to GnRH neurons by this mechanism.[586] Some glial cells also secrete substances that can modulate the activity of GnRH neurons. For example, at puberty there is an increase in the hypothalamic expression of transforming growth factor , and transforming growth factor can stimulate GnRH release by acting on astroglial cells to stimulate their release of PGE 2 , which is stimulatory to GnRH neurons. [587] Feedback Regulation

Steroid hormone receptors are abundant in the hypothalamus and in many neural systems that impinge on GnRH neurons, including noradrenergic, serotoninergic, -endorphincontaining, and NPY neurons. Early studies identifying regions of the brain that bound labeled estrogens showed that in rodents the preoptic area and ventromedial hypothalamus had the highest concentrations of estrogen receptors in the brain. [588] [589] Further localization studies, identifying estrogen receptors by immunocytochemistry or in situ hybridization, confirmed the strong presence of estrogen receptors in the hypothalamus and in brain areas with strong connections to the hypothalamus, including the amygdala, septal nuclei, bed nucleus of the stria terminalis, medial part of the nucleus of the solitary tract, and lateral portion of the parabrachial nucleus. [590] [591] In 1986 a new member of the steroid hormone receptor superfamily with high sequence homology to the classical estrogen receptor (now referred to as estrogen receptor ) was isolated from rat prostate and named estrogen receptor . [592] [593] This novel estrogen receptor was shown to bind estradiol and to activate transcription by binding to estrogen response elements. [593] In situ hybridization studies examining the localization of estrogen receptor mRNA have shown that these receptors are present throughout the rostral-caudal extent of the brain, with a high level of expression in the preoptic area, bed nucleus of the stria terminalis, paraventricular and supraoptic nuclei, amygdala, and laminae II to VI of the cerebral cortex. [594] [595] Specific receptors for progesterone are induced by estrogen in hypothalamic regions of the brain, including the preoptic area, the ventromedial and ventrolateral nuclei, and the infundibular-arcuate nucleus, although there is also evidence for constitutive expression of progesterone receptors in some regions.[596] [597] [598] [599] Androgen receptor mapping studies have shown considerable overlap in the distribution of androgen and estrogen receptors throughout the brain. The highest density of androgen receptors was found in hypothalamic nuclei known to participate in the control of reproduction and sexual behaviors, including the arcuate nucleus, paraventricular nucleus, medial preoptic nucleus, ventromedial nucleus, and brain regions with strong connections to the hypothalamus including the amygdala, nuclei of the septal region, bed nucleus of the stria terminalis, nucleus of the solitary tract, and lateral division of the parabrachial nucleus. [591] [600] [601] The anterior pituitary also contains receptors for all of the gonadal steroid hormones. Steroid hormones can dramatically alter the pattern of pulsatile release of GnRH and of the gonadotropins through actions at both the hypothalamus and the pituitary (Fig. 7-40 and Fig. 7-41 ). At the hypothalamus, estradiol, progesterone, and testosterone can all act to slow the frequency of GnRH release into the portal blood stream, an action referred to as negative feedback. [602] [603] [604] Because GnRH neurons have generally been shown to lack steroid hormone receptors, it is likely that the effects of steroid hormones on the firing rate of GnRH neurons are mediated by steroid hormone actions on other neural systems that provide afferent input to GnRH neurons. For example, progesterone-mediated negative feedback on GnRH secretion in primates appears to be regulated by -endorphincontaining neurons in the hypothalamus, acting primarily through µ-opioid receptors. [603] If a µ-receptor antagonist, such as naloxone, is administered along with progesterone, the negative feedback action of progesterone on GnRH secretion can be blocked. [603] Negative feedback of steroid hormones can also occur directly at the level of the pituitary. [602] For example, estradiol has been shown to be capable of binding to the pituitary, decreasing LH and FSH synthesis and release, and decreasing the sensitivity of pituitary gonadotrophs to the actions of GnRH such that less LH and FSH are released when a pulse of GnRH stimulates the pituitary. Evidence for such a direct pituitary action of estradiol came from studies with rhesus monkeys that had been rendered deficient in endogenous GnRH by a lesion in the arcuate nucleus and showed a decline in endogenous gonadotropin secretion. When these monkeys received a pulsatile regimen of GnRH gonadotropin secretion, subsequent estradiol infusions dramatically suppressed the responsiveness of the pituitary to GnRH and suppressed the gonadotropin secretion that was being driven by the pulsatile administration of GnRH. [606] Steroid hormones can have direct negative feedback actions at the pituitary; however, the extent of hypothalamic versus pituitary negative feedback actions is species-specific. [607] In primate species including humans, there is considerable feedback of estradiol at the pituitary, but most of the progesterone and testosterone negative feedback occurs at the level of the hypothalamus.[603] [607] Most of the time, the hypothalamic-pituitary axis is under the negative feedback influence of gonadal steroid hormones. If the gonads are removed surgically or their normal secretion of steroid hormones is suppressed pharmacologically, there is a dramatic increase (10-fold to 20-fold) in circulating levels of LH and FSH secretion.[602] [607] This type of "castration response" occurs normally at the menopause in women, when

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Figure 7-40 Pulsatile luteinizing hormone (LH) secretion in an ovariectomized rhesus monkey (A) and an ovariectomized rhesus monkey treated with estradiol (B). Estradiol causes a rapid and sustained suppression of LH secretion. (Redrawn from Yamaji T, Dierschke DJ, Knobil E. Endocrinology 70: 771777.)

ovarian follicular development and thus ovarian production of large quantities of estradiol and progesterone decrease and eventually cease.

[ 608]

In addition to negative feedback, estradiol can have a positive feedback action at the level of the hypothalamus and pituitary to lead to a massive release of LH and FSH from the pituitary. [575] [602] This massive release of gonadotropins occurs once each menstrual cycle and is referred to as the LH-FSH

Figure 7-41 Dose and duration requirements for estradiol-induced negative and positive feedback on luteinizing hormone (LH) secretion. Varying amounts of estradiol were implanted into ovariectomized rhesus monkeys. Short-term exposure to estradiol led to negative feedback on LH secretion, 36 hours of exposure led to positive feedback in 6 of 11 monkeys, and 42 hours of exposure led to robust positive feedback resulting in a surge of LH secretion in all monkeys. (Redrawn from Karsch F, Weick RF, Butler WR, et al. Endocrinology 1973; 92:17401747.)

surge. The positive feedback action of estradiol occurs as a response to the rising tide of estradiol that is produced during the process of dominant follicle development in the late follicular phase of the menstrual cycle. In women, elevated estradiol levels are generally maintained at about 500 pg/mL for about 36 hours prior to stimulation of the gonadotropin surge. Experiments have shown that both a critical concentration of plasma estradiol and a critical duration of elevated estradiol are

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necessary to achieve positive feedback and a resulting gonadotropin surge (see Fig. 7-41) . Moreover, the duration of estrogen elevation that is required to trigger a surge depends on the concentration of circulating estrogen. [609] If supraphysiologic doses of estradiol are administered, the surge can occur as early as 18 hours after their administration. Because the ovary is responsible for the production of estradiol and the time course and magnitude of estradiol release control the rate of positive feedback, the ovary has been referred to as the zeitgeber of the menstrual cycle. [575] [602] The dependence of the positive feedback system on the magnitude of estradiol production helps explain the fact that the portion of the menstrual cycle that varies most in length is the follicular phase. Production of higher levels of estradiol by a dominant follicle in one cycle would lead to a more rapid positive feedback action with earlier ovulation and thus a shorter follicular phase compared with a cycle in which the dominant follicle produced lower levels of estradiol. As with negative feedback in response to estradiol, the positive feedback actions of estradiol occur both at the hypothalamus, to increase GnRH secretion, and at the pituitary, to enhance greatly pituitary responsiveness to GnRH. At the pituitary, estradiol increases pituitary sensitivity to GnRH by increasing the synthesis of new GnRH receptors and by enhancing the responsiveness to GnRH at a postreceptor site of action. At the level of the hypothalamus in rodent species, estradiol appears to act at a "surge center" to induce the ovulatory surge of GnRH. Lesions in areas adjacent to the medial preoptic area, near the anterior commissure and septal complex, block the ability of estradiol to induce a surge in these species without blocking negative feedback effects of estradiol. [610] In primate species, there does not appear to be a separate surge center mediating the positive feedback actions of estradiol. [575] [602] The cellular mechanisms that mediate the switch from negative to positive feedback of estrogen are not fully understood, but there is support for the concept that estrogen induction of various transcription factors and receptors (notably progesterone receptors) may play an important role in mediating this switch. [611] [612] Alternatively, estrogen has been shown to have biphasic actions on hypothalamic GABAergic neurons that impinge on GnRH neurons and are strong regulators of their activity, with the switch in action dependent on the duration of estradiol exposure. [613] The molecular mechanisms by which estradiol influences GnRH gene expression are also not well understood, but it is likely that these influences occur through actions of neural systems afferent to GnRH because GnRH neurons do not appear to have estrogen receptors. Much more is known about the molecular mechanisms by which estradiol acts at the pituitary to regulate gonadotropin gene expression. Expression of LH subunit is strongly regulated (10-fold to 14-fold) by estradiol, but expression of FSH and subunits is regulated to a lesser extent (4-fold to 8-fold and 2-fold to 3-fold, respectively). [614] Although in vivo studies indicate strong negative feedback actions of estradiol on LH gene transcription, such actions have not been replicated in in vitro studies with isolated pituitaries, leading to the conclusion that estradiol negative feedback on gonadotropin synthesis occurs predominantly by extrapituitary mechanisms. [614] In contrast, estradiol can stimulate LH mRNA transcription directly at the level of the pituitary, acting by binding to an estrogen response element in the 5' promoter region of the LH gene. [614] Regulation by Inhibins and Activins

Negative feedback of pituitary FSH secretion is also exerted by a family of peptide hormones produced by the gonads, the inhibins. [615] Inhibins are produced by follicular and luteal cells of the ovary and by Sertoli cells in the testes. Inhibins are members of the transforming growth factor superfamily and comprise two subunits, an and a subunit. There are two forms of the subunit, A and B. Inhibins selectively suppress FSH secretion without simultaneous suppression of LH secretion; thus, they provide one of the mechanisms whereby the pituitary can release differential amounts of LH and FSH, even though there appears to be only a single gonadotropin-releasing factor. Interestingly, the pituitary itself produces compounds related to inhibins that are dimers of the B subunits. These are the activins. [615] [616] [617] [618]

Activins received their name from their ability to facilitate FSH release. Activins have been shown to stimulate both basal and GnRH-induced FSH release from the anterior pituitary as well as increase FSH mRNA levels by enhancing transcription. An important role of endogenous activins in stimulating FSH secretion is supported by the finding that transgenic mice deficient in activin receptor IIA have reduced serum FSH levels. [619] Activins have other actions in pituitary gonadotrophs as well, including up-regulation of GnRH receptors and enhancement of GnRH-stimulated LH release. [618] Activins and inhibins also have local actions within the ovary influencing granulosa cell growth and differentiation, the responsiveness of the ovary to gonadotropins, steroid hormone production, follicular development, and oocyte maturation.[620] Regulation of the Ovarian Cycle

Whereas in males spermatogenesis occurs continually throughout the adult years, females show a cyclic pattern of ovarian activity with intermittent maturation and release of ova from the ovaries. Cyclic activity in the ovary is controlled by an interplay between steroid hormones produced by the ovary and the hypothalamic-pituitary neuroendocrine components of the reproductive axis. [602] [621] [622] The duration of each phase of the ovarian cycle is species-dependent, but the general mechanisms controlling the cycle are similar in all species that have spontaneous ovarian cycles. In the human menstrual cycle, day 1 of the cycle is designated as the first day of menstrual bleeding. At this time, small and medium-sized follicles are present in the ovaries and only small amounts of estradiol are produced by the follicular cells. As a result, there is a low level of negative feedback to the hypothalamic-pituitary axis, LH pulse frequency is relatively fast (one pulse about every 60 minutes), and FSH concentrations are slightly elevated compared with much of the rest of the cycle (Fig. 7-42) . [621] [622] FSH acts at the level of the ovarian follicles to stimulate development and causes an increase in follicular estradiol production, which in turn provides increased negative feedback to the hypothalamic-pituitary unit. A result of the increased negative feedback is a slowing of pulsatile LH secretion over the course of the follicular phase to a rate of about one pulse every 90 minutes.[621] [622] However, as the growing follicle (or follicles, depending on the species) secretes more estradiol, a positive feedback action of estradiol is triggered that leads to an increase in GnRH release and a surge release of LH and FSH. The surge of gonadotropins acts at the fully developed follicle to stimulate the dissolution of the follicular wall and leads to ovulation of the matured ovum into the nearby fallopian tube, where fertilization takes place if sperm are present.

Ovulation results in a reorganization of the cells of the follicular wall, which undergo hypertrophy and hyperplasia and start to secrete large amounts of progesterone and some estradiol. Progesterone and estradiol have a negative feedback effect at the level of the hypothalamus and pituitary, and thus LH pulse frequency becomes very slow during the luteal phase of the menstrual cycle. [621] [622] The corpus luteum has a fixed life span, and without additional stimulation in the form of chorionic gonadotropin from a developing embryo, the corpus luteum regresses spontaneously

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Figure 7-42 Diagrammatic representation of changes in plasma levels of estradiol, progesterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and portal levels of gonadotropin-releasing hormone (GnRH) over the human menstrual cycle.

after about 14 days and progesterone and estradiol secretion diminishes. This reduces the negative feedback signals to the hypothalamus and pituitary and allows an increase in FSH and LH secretion. The fall in progesterone is also a withdrawal of steroid hormone support to the endometrial lining of the uterus, and as a result the endometrium is shed as menses and a new cycle begins. In other species, the interplay between the neuroendocrine and ovarian hormones is similar but the timing of events is different and other factors, such as circadian and seasonal regulatory factors, play a role in regulating the cycle. The rat has a 4- or 5-day ovarian cycle with no menses (the endometrial lining is absorbed rather than shed). The rat also shows strong circadian rhythmicity in the timing of the LH-FSH surge, with the surge always occurring in the afternoon of the day of proestrus. [623] Sheep are an example of a species that has a strongly seasonal pattern of ovarian cyclicity. [624] During the breeding season they have 15-day cycles, with a very short follicular phase and an extended luteal phase; during the nonbreeding season signals relaying information about day length through the visual system, pineal, and SCN cause a dramatic suppression of GnRH neuronal activity, and cyclic ovarian function is prevented by a decrease in trophic hormonal support from the pituitary. Early Development and Puberty

Neuroendocrine stimulation of the reproductive axis is initiated during fetal development, and in primates in midgestation circulating levels of LH and FSH reach values similar to those in castrated adults. [625] [626] Later in gestational development, gonadotropin levels decline, restrained by rising levels of circulating gonadal steroids. [625] [626] [627] The steroids that have this effect are probably placental in origin in that after parturition there is a rise in circulating gonadotropin levels that is apparent for variable periods of the first year of life, depending on the species. [628] [629] The decline in reproductive hormone secretion in the postnatal period appears to be due to a decrease in GnRH stimulation of the reproductive axis because it occurs even in the castrate state and gonadotropin and gonadal steroid secretion can be supported by administration of pulses of GnRH. [629] [630] Pubertal reawakening of the reproductive axis occurs in late childhood and is marked initially by nighttime elevations in gonadotropin and gonadal steroid hormone levels.[631] [632] The mechanisms controlling the pubertal reawakening of the GnRH pulse generator have been an area of intense investigation for the past two decades.[630] [633] Although the mechanisms are not fully understood, significant progress has been made in identifying central changes in the hypothalamus that appear to play a role in this process. There appear to be both a decrease in transsynaptic inhibition to the GnRH neuronal system at puberty and an increase in stimulatory input to GnRH neurons at this time. One of the major inhibitory inputs to the GnRH system is provided by GABAergic neurons. Studies in rhesus monkeys have shown that hypothalamic levels of GABA decrease during early puberty and that blocking GABAergic input before puberty, by intrahypothalamic administration of antisense oligodeoxynucleotides against the enzymes responsible for GABA synthesis, results in premature activation of the GnRH neuronal system. [584] It has been suggested, on the basis of findings that a subset of glutamate receptors (i.e., kainate receptors) increase in the hypothalamus at puberty, that the pubertal decrease in GABA

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tone may be caused by an increase in glutamatergic transmission. [633] Further evidence for a role for glutamate comes from studies showing that administration of glutamate to prepubertal rhesus monkeys can drive the reawakening of the reproductive axis. [634] Increased stimulatory drive to the GnRH neuronal system also appears to come from increases in norepinephrine and NPY at the time of puberty. [583] [635] Furthermore, as discussed earlier, there is evidence that growth factors act through release of prostaglandin from glial cells at puberty to play a role in stimulating GnRH neurons. [633] Despite an increased understanding of the neural changes occurring at puberty, the question of what signals trigger the pubertal awakening of the reproductive axis is unanswered at this time. Availability of food and nutritional status have been shown to affect the timing of puberty, but these signals appear to be only modulators of the pubertal process because puberty can be only moderately advanced by increasing food availability. [636] Determining whether there is a genetic timing mechanism that regulates the timing of puberty or whether other signals from the body or the brain are responsible for timing the reactivation of the reproductive axis awaits further research. Reproductive Function and Stress

Many forms of physical stresses, such as energy restriction, exercise, temperature stress, infection, pain, and injury, as well as psychological stresses, such as being subordinate in a dominance hierarchy or being acutely psychologically stressed, can suppress the activity of the reproductive axis. [637] [638] [639] [640] If the stress exposure is brief, there may be acute suppression of circulating gonadotropins and gonadal steroid hormones and in females disruption of normal menstrual cyclicity, but fertility is unlikely to be impaired. [639] [640] In contrast, prolonged periods of significant stress exposure can lead to complete impairment of reproductive function, also characterized by low circulating levels of gonadotropins and gonadal steroids. [637] [638] Stress appears to decrease the activity of the reproductive axis by decreasing GnRH drive to the pituitary because in all cases in which it has been examined, administration of exogenous GnRH can reverse the effects of the stress-induced decline in reproductive hormone secretion. [575] Although we do not know the neural circuits through which many forms of stress suppress GnRH neuronal activity, some forms of stress-induced suppression of reproductive function are better understood. In the case of foot shock stress in rats [641] and immune stress (i.e., injection of IL-1) in primates, [642] the suppression of gonadotropin secretion that occurs has been shown to be reversible by administration of a CRH antagonist, implying that endogenous CRH secretion mediates the effects of these stresses on GnRH neurons. In other studies, naloxone, a -opiate receptor antagonist, has been shown to be capable of reversing restraint stressinduced suppression of gonadotropin secretion in monkeys; however, naloxone is ineffective in reversing the suppression of gonadotropin secretion that occurs during insulin-induced hypoglycemia. [643] [644] In the case of metabolic stresses, multiple regulators appear to mediate changes in the neural drive to the reproductive axis. Various metabolic fuels including glucose and fatty acids can regulate the function of the reproductive axis, and blocking cellular utilization of these fuels can lead to suppression of gonadotropin secretion and decreased gonadal activity. [645] Leptin, a hormone produced by fat cells, can also modulate the activity of the reproductive axis. Transgenic mice deficient in leptin or leptin receptors are infertile, and fertility can be restored by administration of leptin. [646] Moreover, leptin administration has been shown to reverse the suppressive effects of undernutrition on the reproductive axis in some situations. [647] Leptin receptors are found in several populations that are known to have a strong influence on the reproductive axis, notably NPY neurons. [648] In summary, it appears that a number of neural circuits can mediate effects of stress on the GnRH neuronal system and that the neural systems involved are at least somewhat specific to the type of stress that is experienced.



Leptin and the Brain-Gut-Adipose Axis

Long-term energy, stored as fat in adipose tissue, is homeostatically maintained by a hypothalamic system termed the lipostat or adipostat. [649] Inputs to this system are many, including acute hormonal, nutritional, and vagal signals of hunger and satiety; the signal of long-term energy stores derived from the adipose hormone leptin; and powerful olfactory, visual, emotional, and cognitive inputs from higher brain centers. Outputs include those directed toward energy intake, primarily determined by feeding behavior, and energy expenditure, which can be broken down into basal metabolism, voluntary and involuntary activity, and diet-induced thermogenesis. As described throughout this chapter, energy homeostasis is maintained through the triad of behavioral, autonomic, and endocrine pathways. Thus, energy homeostasis is maintained by a complicated hypothalamic-brain stem-target organ axis that may be referred to as the brain-gut-adipose axis (Fig. 7-43) . The regulation of feeding behavior and metabolism is determined by both short-term and long-term control mechanisms. Short-term control involves the initiation and termination of meals. A major determinant of meal size is the perception of satiety that is produced by neural, endocrine, and nutritional inputs during ingestion of a meal. For example, gut distention and release of gastrointestinal peptides such as cholecystokinin lead to meal termination. Ghrelin, an orexigenic peptide, demonstrates a clear preprandial rise and postprandial fall in plasma levels supporting a possible physiologic role in meal initiation in humans. [650] Long-term signals that reflect the body's overall energy depots, such the adipocyte-derived hormone leptin, signal to the CNS to effect changes in feeding behavior and energy expenditure. Both these short-term and long-term factors are coordinated through the brain-gut-adipose axis to respond to changes in energy homeostasis (see Fig. 7-43) . Chemistry and Evolution of Leptin

A significant advance in understanding energy homeostasis in humans occurred with the identification of the basis for the monogenic obesity syndromes characterized in ob/ob and db/db mice.[26] [651] Parabiosis experiments in which mice were surgically fused to permit transfer of molecules from one to the other led to the hypothesis 30 years ago that ob/ob mice were deficient in a circulating signal of satiety whereas db/db mice were deficient in its cognate receptor. The ob gene was cloned in 1994 and encodes a unique member of the cytokine family now called leptin (from the Greek root leptos, meaning thin) (Fig. 7-44) . Leptin protein has been highly conserved throughout evolution, as demonstrated by mouse and human leptin being 84% homologous. Thus far, leptin has been found in birds but not in fish or amphibians. This 167-amino-acid protein has a mass of 16 kd and circulates in the blood at concentrations proportional to the amount of fat depots. Leptin circulates in the blood stream both as a free protein and bound to a soluble isoform of its receptor (Ob-Re). Its secretion is pulsatile and shows a rhythm with a nocturnal peak occurring between 1 and 2 AM and a nadir in the afternoon (Fig. 7-48) .[652] Leptin is secreted primarily from the adipocyte; however, minor levels of regulated

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Figure 7-43 Regulation of energy homeostasis by the brain-gut-adipose axis. CCK, cholecystokinin; GLP-1, glucagon-like peptide 1; PYY, peptide YY.

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Figure 7-44 Structure of the human leptin gene. cDNA, complementary deoxyribonucleic acid; UTR, untranslated region; rectangle, SP-1 site; triangle, CCAAT/enhancer binding protein (C/EBP); circle, cyclic AMP-responsive element (CRE); diamond, glucorticoid response element (GRE).

leptin expression also occur in other sites such as skeletal muscle, [653] placenta, and stomach. Effects of Leptin on the Hypothalamus and Neuroendocrine Axes

A reduction in leptin levels occurs because of loss of adipose mass such as in anorexia nervosa, weight loss induced by diet or exercise, or starvation and is crucial to metabolic adaptation to a state of negative energy balance. This metabolic adaptation includes a decrease in metabolic rate that allows extended survival periods; inhibition of the reproductive, GH, and thyroid axes [283] ; and, at least in rodents, inhibition of the activity of the sympathetic nervous system [654] [655] and activation of the HPA axis. In addition, leptin is a critical signal in the initiation of puberty. Leptin is a signal from the adipose tissue directed to the CNS that conveys readiness to proceed into puberty and is essential for fertility in the adult. Presumably, the leptin signal is a mechanism for the organism to determine whether adequate energy stores are present to maintain a pregnancy through term. For example, leptin administration restored fertility to ob/ob mice[646] and prevented the starvation-induced delay in ovulation in female mice and rats. [283] [656] In addition, leptin does not advance the onset of puberty beyond normal in animals fed a normal diet ad libitum, suggesting that leptin is not a trigger but rather a permissive signal in the complex process of initiation of puberty and reproductive competence. Furthermore, leptin exerted at least a permissive action on GnRH release from the hypothalamus and stimulated LH and FSH release from the pituitary in vitro. [657] Mechanism of Action

After secretion, leptin circulates in plasma in both free and bound forms. It is assumed that the binding protein is a soluble form of the leptin receptor, but other alternatives are being evaluated. [658] In humans, the half-life of leptin is approximately 75 minutes. [659] The precise mechanism of the transport of leptin into the CNS is unknown. Active uptake of leptin has been described in the capillary endothelium and microvasculature of brains from humans and mice, suggesting a role of short isoforms of the leptin receptor. [91] In addition, the transport of leptin into the choroid plexus is saturable. [91] After its transport through the blood-brain barrier, leptin binds to specific receptors in the hypothalamus. Leptin receptor mRNA is densely concentrated in the arcuate nucleus, and lower levels are found in the ventromedial and dorsomedial hypothalamic nuclei. [96] The leptin receptor is a member of the cytokine receptor superfamily. The leptin receptor binds Janus kinases (JAKs), tyrosine kinases involved in intracellular

cytokine signaling. Activation of JAK leads to phosphorylation of members of the

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signal transducer and activator of transcription (STAT) family of proteins. In turn, these STAT proteins activate transcription of leptin target genes. A great deal of effort has gone into characterizing the mechanism of leptin action in the hypothalamus. Leptin appears to inhibit feeding and stimulate metabolism by acting on a small number of nuclei in the hypothalamus and brain stem, including the ventromedial hypothalamus and the arcuate, dorsomedial, and paraventricular hypothalamic nuclei (Fig. 7-45) . [660] In these neurons, leptin up-regulates the expression of an assortment of anorexigenic peptides, such as -MSH, derived from the POMC pre-prohormone gene (Fig. 7-46) , cocaine and amphetamine-regulated transcript (CART), and neuromedin U, and decreases the expression of orexigenic peptides, NPY, AgRP, and melanin-concentrating hormone. Furthermore, leptin has been shown to depolarize and activate the firing rate of the anorexigenic POMC neurons and hyperpolarize the adjacent orexigenic NPY-AgRP neurons. [31]

Figure 7-45 Role of the hypothalamus as a sensory organ in the regulation of energy homeostasis. CRH, corticotropin-releasing hormone; DA, dopamine; GHRH, growth hormone-releasing hormone; GnRH, gonadotropin-releasing hormone; NPY, neuropeptide Y; POMC, pro-opiomelanocortin; SRIH, somatotropin release-inhibiting hormone; TRH, thyrotropin-releasing hormone. (Modified with permission from Cone RD, Cowley MA, Buller AA, et al. The arcuate nucleus as a conduit for diverse signals relevant to energy homeostasis. Int J Obes 2001; 25:563667.)

NPY, long known as a potent stimulator of feeding, was proved to have a role in leptin action when deletion of the NPY gene relieved a significant component of the obesity phenotype of the ob/ob mouse. [661] The role of the POMC neurons in energy homeostasis was originally discovered from studies on the agouti mouse, [289] [662] [663] one of the five naturally occurring monogenic obesity strains in the mouse. [664] Intracere-broventricular administration of -MSH agonist and antagonist analogues inhibited and stimulated feeding behavior, respectively. [663] Furthermore, deletion of the melanocortin-4 receptor (MC4R), the primary neuronal receptor for the melanocortin peptides, [665] caused an obesity syndrome identical to that seen in the obese lethal yellow agouti animal. [289] The structure and distribution of the POMC and NPY-AgRP circuits are highly conserved in humans (Fig. 7-47) (Figure Not Available) (Fig. 7-47) (Figure Not Available) . Furthermore, a null mutation in the POMC gene in humans caused an obesity syndrome similar to that seen in the lethal yellow mouse along with ACTH insufficiency and red hair [666] (see

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Figure 7-46 Organization of pro-opiomelanocortin (POMC), the precursor hormone of corticotropin (ACTH on figure), -LPH, and related peptides. The precursor protein contains a leader sequence (signal peptide), followed by a long fragment that includes sequence 5162 corresponding to -MSH. This fragment is cleaved at Lys-Arg bonds to form corticotropin 1B39, which in turn includes the sequences for -MSH (corticotropin 1B13) and corticotropin-like intermediate lobe peptide (CLIP) (corticotropin 1739), and a sequence corresponding to -LPH (191) that includes -LPH 158), and -endorphin (61B91). The -endorphin sequence also includes a sequence corresponding to met-enkephalin. The precursor molecule in the anterior lobe of the pituitary is processed predominantly to corticotropin and -LPH. In the intermediate pituitary lobe (in the rat), corticotropin and -LPH are further processed to -MSH and a -endorphinlike material. In all extrapituitary tissues, post-translational processing of the prohormone resembles that in the intermediate lobe. Hypothalamic processing is similar but not identical to that in the intermediate lobe. In the latter, -endorphin and -MSH are present predominantly in their acetylated forms. -LPH, -lipotropin; -MSH, -melanocyte-stimulating hormone; -MSH, -melanocyte-stimulating hormone; -LPH, -lipotropin.

"Neuroendocrine Disease"). Thus, it appears that the central melanocortin system subserves the same purposes in mouse and humans. Although obesity related to genetic defects in leptin or the leptin receptor is rare in humans, haploinsufficiency Figure 7-47a (Figure Not Available) A, A series of photomicrographs demonstrate that -melanocyte-stimulating hormone-immunoreactive (-MSH-IR) neurons are present in the human hypothalamus. The neurons are found in the arcuate nucleus of the hypothalamus. Arc, infundibular nucleus; 3v, third ventricle.

of the MC4R appears to be responsible for up to 3% to 5% of severe pediatric obesity.

[290] [667] [668]

Thus, the arcuate nucleus, site of the POMC-CART and NPY-AgRP neurons described earlier, is an important site

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Figure 7-47b (Figure Not Available) B, A series of photomicrographs demonstrate that agouti-related peptideimmunoreactive (AgRP-IR) neurons are present in the human hypothalamus. A and B, Two rostral to caudal low-power photomicrographs demonstrate that AgRP-IR neurons localize to the arcuate nucleus of the hypothalamus (Arc; infundibular nucleus). B, Immunoreactive fibers are also observed streaming dorsally out of the arcuate nucleus. C and D, AgRP-IR neurons are observed in the arcuate nucleus. D is a higher magnification of C (use box for orientation). 3v, third ventricle; fx, fornix; ot, optic tract. (Modified from Elias CF, Saper CB, Makatos-Flier E, et al. Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area. J Comp Neurol 1998; 402:442459.)

from which leptin exerts a subset of its actions on energy homeostasis. It is important to note that defective melanocortin signaling does not produce the severe neuroendocrine defects seen in leptin-deficient mice or people (e.g., infertility). Furthermore, as already described, the arcuate is adjacent to the median eminence, a CVO, and evidence suggests that acute signals of satiety and hunger may reach regulatory centers through the arcuate as well as vagal inputs to the brain stem. [669] The hormone ghrelin is proposed to be a hunger-initiating factor acting in part through receptor sites on arcuate NPY neurons. [670] The levels of serum ghrelin are potently decreased by food intake in humans (Fig. 7-48) . Clinical Applications

Leptin deficiency [288] and leptin receptor defects [671] in humans are rare. In fact, serum leptin levels in humans are generally proportional to adipose mass. [672] Thus, the vast majority of obese humans may be considered to manifest a leptin-resistant state rather than a deficient state. [673] [674] This concept of leptin resistance also remains poorly understood. However, it is thought that one mechanism of leptin resistance may be impaired leptin transport into the brain. Thus, suboptimal leptin transport through the blood-brain barrier may be one mechanism that underlies the development of leptin resistance in humans. [675] Furthermore, the concept of leptin resistance leads to some reservations concerning the ability of exogenously administered leptin to overcome this leptin resistance and cause effective weight reduction in obese humans. Clinical studies have now demonstrated that leptin treatment is safe and well tolerated and clearly effective in individuals with congenital leptin deficiency. [676] In this study, low doses of methionyl leptin (met-leptin) were given to subjects with congenital deficiency that resulted in leptin levels 10% of that predicted on the basis of body fat. Leptin in this study was well tolerated and resulted in dramatic declines in appetite, body weight, and food intake. [676] However, in individuals with common obesity, leptin had only modest effects on appetite and body weight. For example, studies evaluated the safety and efficacy of recombinant human met-leptin administration as well as pegylated human leptin. The first study was a double-blind, placebo-controlled, escalating-dose cohort trial in 54 lean and 73 obese subjects. [677] Higher doses of met-leptin (0.01 to 0.3 mg/kg daily) were also given for 4 to 24 weeks. Met-leptin treatment resulted in significant dose-dependent weight loss: -1.3 kg (placebo

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Figure 7-48 Average plasma ghrelin, insulin, and leptin concentrations during a 24-hour period in 10 human subjects consuming breakfast (B), lunch (L), and dinner (D) at the times indicated (0800, 1200, and 1730 hours, respectively) (Reprinted with permission from Cummings DE, et al. A preprandial rise in plasma ghrelin levels suggest a role in meal initiation in humans. Diabetes 2001; 50:17141719.).

group), -1.4 kg (0.03 mg/kg group), and -7.1 kg (0.30 mg/kg group) over a 24-week period. [677] Of note, 95% of the weight loss achieved in the two highest dose cohorts was due to loss of fat mass and not any significant changes in fat free mass. The findings have supported the idea that leptin resistance may be partially overcome by a high enough overall leptin concentration. Another study evaluated the efficacy of another long-acting leptin compound (A-200) in 200 obese subjects in a 24-week randomized, placebo-controlled pilot study with mild dietary intervention (500 calories below daily requirement). Results indicated that A-200 was safe, well tolerated, and resulted in a statistically significant decline in body weight and fat mass. [678] Most of the weight loss again was determined to be secondary to decreases in fat mass. In a randomized, double-blind trial,[679] [680] 30 patients received either 20 mg of polyethylene glycol (PEG)-leptin or placebo weekly for 12 weeks. At the end of the study, patients receiving placebo had increased appetite and hunger levels in the fasting state, compared with reduced appetite and hunger in the treatment group. However, the treatment group did not experience reductions in daily food intake or body mass or changes in body composition compared with the control group. These findings led researchers to conclude that PEG-leptin has central rather than peripheral biologic activity in obese men. [680] Studies in rodents have demonstrated that leptin is highly efficacious as an antidiabetic agent in lipodystrophy, in which leptin deficiency is directly responsible for a hyperinsulinemic diabetic syndrome. [681] The National Institute of Diabetes and Digestive and Kidney Diseases is currently studying the long-term efficacy of leptin replacement in patients with lipodystrophy ( www.clinicaltrials.gov, National Institutes of Health protocols 02-DK-0146 and 02-DK-0022). Results from these studies are not yet available. Feedback Control

Little is known about the cellular pathway involving leptin secretion. However, the rapid effects of -adrenergic stimulation on leptin release from adipose tissue suggest that leptin secretion is regulated by cAMP. As well, leptin secretion is upregulated by the hormones insulin and cortisol working synergistically and down-regulated by catecholamines, norepinephrine, and epinephrine. A report also suggests that cholecystokinin may regulate leptin secretion directly. [682] Finally, TNF may be an important paracrine regulator of leptin secretion. [683] The interesting phenomenon of leptin resistance in obesity was initially suggested on the basis of the elevation of plasma leptin levels in obese humans. It turns out that, as with other cytokine receptors, activation of the leptin receptor induces expression of a protein called suppressor of cytokine signaling-3 (SOCS-3), which may inhibit further leptin signal transduction. The contribution of SOCS-3 to acquisition of leptin resistance and obesity remains an active area of investigation. As well, leptin receptors are expressed in the endothelial cells of the blood-brain barrier and it is plausible that dysfunction of this process may also lead to a state of obesity and leptin resistance.



Neuroendocrine-Immune Interactions

Stimulation of the immune system by foreign pathogens leads to a stereotyped set of responses orchestrated by the CNS. These responses are the result of the complex interaction of the immune system and the CNS and are often referred to as the cerebral component of the acute phase reaction. [84] This constellation of stereotyped responses is adaptive, is mediated in large part by the hypothalamus, and includes coordinated autonomic, endocrine, and behavioral components. These responses include fever, alterations in the activity of nearly every neuroendocrine axis, changes in the sleep-wake cycle, anorexia, and inactivity. It is now clear that cytokines produced by white blood cells of the immune system mediate the CNS responses. Early evidence supporting this hypothesis was provided by the seminal observations that cytokines such as IL-1 can activate the HPA axis. [684] [685] [686] In fact, these and other observations provided the framework for a new area of research in neuroscience and neuroendocrinology. This discipline is often referred to as neuroimmunology. Thus, the term neuroimmunomodulation has been used to describe the study of the interactions of immune system cues and nervous system function. [687] [688]

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Although it is established that cytokines modulate hypothalamic activity, it is also important to note that the immune system is modulated by the nervous system. This modulation occurs largely by two routes, endocrine mechanisms and direct innervation. The innervation includes lymphoid organs such as the thymus and spleen, which receive direct inputs from the autonomic nervous system. [689] [690] As noted earlier in the section on CRH, the hallmark of cytokine action on the hypothalamus is the activation of the HPA axis. The resultant glucocorticoid secretion acts as a classical negative feedback to the immune system to damp the immune response (Fig. 7-49) . In general, glucocorticoids inhibit most limbs of the immune response, including lymphocyte proliferation, production of immunoglobulins, cytokines, and cytotoxicity. These inhibitory reactions form the basis of the anti-inflammatory actions of glucocorticoids. Glucocorticoid feedback on immune responses is regulatory and beneficial because loss of this function makes animals with adrenal insufficiency vulnerable to inflammation. Moreover,

Figure 7-49 A, Effect of injection of Escherichia coli endotoxin (lipopolysaccharide [LPS]) on circulating levels of corticotropin, vasopressin, and cortisol. B, Effect of injection of LPS on circulating levels of IL-1; IL-1RA; and TNF-. AVP, vasopressin; ACTH, corticotropin; IL-1RA, interleukin-1 receptor antagonist; IL-1, interleukin-1; TNF-, tumor necrosis factor . (A redrawn from Michie HR, Majzoub JA, O'Dwyer ST, et al. Both cyclooxygenase-dependent and cyclooxygenase-independent pathways mediate the neuroendocrine response in humans. Surgery 1990; 108:254259. B redrawn from Granowitz EV, Santos AA, Poutsiaka DD, et al. Production of interleukin-1-receptor antagonist during experimental endotoxaemia. Lancet 1991; 338:14231424, copyright by the Lancet Ltd. 1991; and Michie HR, Manoque KR, Spriggs DR, et al. Detection of circulating tumor necrosis factor after endotoxin injection. N Engl J Med 1988; 318:14811486. Copyright 1988. Massachusetts Medical Society. All rights reserved.)

this feedback response can have pathophysiologic consequences, as chronic activation of the HPA axis can certainly be detrimental. [687] [688] Indeed, it is now established that chronic stress can lead to immunosuppression. The fact that products of inflammation such as IL-1 can activate the HPA axis suggests the operation of a negative feedback control loop to regulate the intensity of inflammation. The role of the hypothalamus in regulating pituitary-adrenal function is an excellent example of neuroimmunomodulation. This section addresses several of the hypothesized mechanisms by which cytokines engage neural pathways to mediate neuroendocrine and autonomic effects. In addition, some of the autonomic and endocrine pathways that are engaged by immune system cues are briefly discussed. It is important to note that many nonlymphocytic cells including endocrine and adipose cells and neurons also synthesize cytokines that exert effects independent of immunomodulation. Examples of cytokines secreted by adipocytes include leptin, adipsin, and TNF-, which have profound effects on metabolism. [691] Cytokines Signal the Central Nervous System

Cytokines made outside the CNS can alter the activity and function of populations of hypothalamic neurons. Although the interactions of cytokines with the nervous system have been studied extensively, the mechanisms by which immune signals influence the CNS remain unsettled. LPS (or endotoxin) is a cell wall component of all gram-negative bacteria that is a potent immune system stimulant. LPS administration is widely used as an experimental model and induces the secretion of several pyrogenic cytokines including IL-1, TNF-, and IL-6 that mimic the patterns of cytokine production seen in natural infections. [688] [692] [693] [694] Many other studies have used systemic injections of cytokines such as IL-1 and TNF- to stimulate the CNS. Using methodologies such as these, at least four models have been proposed to explain how immune system signals might act upon the CNS (Fig. 7-50) . Interaction of Cytokines with the Circumventricular Organs

The CVOs, described in detail earlier, are specialized regions along the margins of the ventricular system that have fenestrated capillaries and therefore no blood-brain barrier. [695] Many circulating hormones such as angiotensin II act on neurons in the CVOs, converting blood-borne signals into CNS responses. [84] Several models of fever production have hypothesized that cytokines may enter the CNS through the CVOs, particularly at the OVLT ( Fig. 7-51A ; see Fig. 7-50 ). [101] [696] [697] [698] However, definitive evidence establishing this model as a predominant mechanism is still lacking. Large lesions of the preoptic area of the hypothalamus including the OVLT block fever, but they inevitably damage nearby regions that are critical for thermoregulation. [101] Small lesions of the OVLT do not block fever or corticotropin responses. [699] However, an inherent limitation of this type of study is that the lesion itself breaches the blood-brain barrier, allowing entry of cytokines. Moreover, knife cuts just caudal to the OVLT, interrupting connections from the OVLT to the PVH, did not block activation of the HPA axis by IL-1. [386] [700] Other studies have focused on the area postrema, a CVO located in the medulla oblongata lying along the surface of the nucleus of the solitary tract at the caudal end of the fourth ventricle ( see Fig. 7-50 and Fig. 7-51D ). Lesions of the area postrema can block the IL-1induced activation of the HPA axis and the induction of c-fos mRNA in the PVH. [701] However,

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Figure 7-50 A model of the central nervous system circuitry mediating the activation of the paraventricular hypothalamic nucleus (PVN or PVH) and the hypothalamic-pituitary-adrenal (HPA) axis by immune system stimulation. The immune system probably uses several pathways and sites of entry to communicate with the brain. This model predicts that circumventricular organs (organs devoid of blood-brain barrier; CVOs) and the blood vessels (bv) are crucial target sites of cytokines of systemic origin produced during the acute-phase response, whereas activated regions of the brain stem and deep limbic system might play a determinate role in the integration of information received from the periphery. Among these integrative structures, the PVN is critical in coordinating autonomic and endocrine responses including the activity of the HPA axis. For example, corticotropin-releasing factor (CRF) neurons of the parvicellular PVN expressed c-fos messenger ribonucleic acid, and that transcription of the gene coding CRF is activated essentially in this hypothalamic nucleus indicates the importance and the specificity of this neuroendocrine

nucleus in endotoxin-treated animals. The mechanisms and the circuitry controlling the CRF release and the activity of the HPA axis might also be different from those involved in the biosynthetic machinery of CRF during immune challenge. ACTH, adrenocorticotropic hormone; AP, area postrema; ARC, arcuate nucleus; BnST, bed nucleus of the stria terminalis; bv, blood vessels; chp, choroid plexus; CeA, central nucleus of the amygdala; COX-2, cyclooxygenase-2; DMH, dorsomedial nucleus of the hypothalamus; EP, prostaglandin E receptor; IL-1, interleukin-1; IL-1R1, IL-1 type 1 receptor; IL-6, interleukin-6; IB, NF-B inhibitor; LC, locus coeruleus; LDT, laterodorsal tegmental nucleus; LPS, lipopolysaccharide; LRNm, lateral reticular nucleus medial; ME, median eminence; MPOA, medial preoptic area; NF-B, nuclear factor B; NTS, nucleus of the solitary tract; OVLT, organum vasculosum of the lamina terminalis; PGE 2 , prostaglandin E2 ; PB, parabrachial nucleus; PP, posterior pituitary; PVN, paraventricular nucleus of the hypothalamus (parvicellular [pc] and magnocellular [mc] divisions); SFO, subfornical organ; SON, supraoptic nucleus; TNF-, tumor necrosis factor ; VLM, ventrolateral medulla. (Modified from Rivest S, Lacroix S, Vallieves L, et al. How the blood talks to the brain parenchyma and the paraventricular nucleus of the hypothalamus during systemic inflammatory and infectious stimuli. Proc Soc Exp Biol Med 2000; 223:2238.)

others have found that more circumscribed lesions, which do not injure the nucleus of the solitary tract, do not prevent CNS responses to intravenous IL-1

[ 702]

Interactions of Cytokines at the Barriers of the Brain: The Requirement for Prostaglandins

One of the hallmarks of CNS responses to inflammation is that many of the components, including fever and activation of the HPA axis, can be prevented by blocking the production of prostaglandins. This is typically done by administration of nonsteroidal anti-inflammatory drugs such as aspirin and indomethacin. [703] [704] [705] Indeed, decades ago the work of Milton and Wendlandt [706] [707] demonstrated that central injections of prostaglandins increase body temperature. However, the site of action in the CNS of drugs such as aspirin that inhibit cyclooxygenase (COX), the enzyme that produces prostaglandins from arachidonic acid, has never been fully established. Two isoforms of COX exist. COX 1 is the constitutive form of the enzyme and is not thought to be regulated by inflammatory stimuli. COX 2 is an inducible isoform and is increased in several cell types in response to immunologic stimuli. [708] [709] In the normal brain, COX 2 mRNA and protein are found exclusively in neurons.[694] [710] [711] [712] In contrast, immune stimulation by LPS or cytokines induces COX 2 mRNA and protein throughout the brain in non-neuronal cells associated with blood vessels, the meninges, and the choroid plexus. In addition, systemic administration of IL-1 induces the expression of prostaglandin E synthase mRNA. [713] The cells probably include endothelial cells, perivascular microglial cells, and meningeal

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Figure 7-51 Immune stimulation activates key brain regions. A series of photomicrographs demonstrating the distribution of Fos-like immunoreactivity (Fos-IR) in the rat brain 2 hours after intravenous injections of lipopolysaccharide (LPS; 125 µg/kg). LPS administration is a commonly used model of immune stimulation, and Fos-IR is a widely used marker of neuronal activation. LPS activates (induces Fos-IR) in the ventral medial preoptic area and organum vasculosum of the lamina terminalis (VMPO and OVLT; A), in the subfornical organ (SFO; B), in the paraventricular nucleus of the hypothalamus (PVH; C), and in the area postrema and nucleus of the solitary tract in the brain stem (AP, NTS; D). Note that prominent Fos-IR is seen throughout the subdivisions of the PVH including the dorsal (dp), ventral (vp), and medial (mp) parvicellular and posterior magnocellular (pm) divisions. Also note that LPS activates neurons in the circumventricular organs (OVLT, SFO, AP). 3v, third ventricle.

macrophages (Fig. 7-62).[714] [715] [716] Regardless of the cell type, it seems clear that circulating LPS or cytokines induce COX 2 in cells in the perivascular space, which in turn may produce prostaglandins to stimulate nearby brain regions inside the blood-brain barrier. PGE2 , the predominant endogenous isoform of PGE in the brain, is thought to be an essential mediator of cytokine modulation of hypothalamic function. [717] This claim is supported by the finding that microinjections of PGE receptor agonists into the brain of rats [698] [718] [719] and other species [720] [721] produce fever. The preoptic area of the hypothalamus surrounding the OVLT is thought to be critical in the response to PGE (see Fig. 7-51A) . For example, microinjections of as little as 1 ng of PGE 2 into the anteroventral preoptic area of rats reliably produced fevers. [718] Conversely, the COX-2 inhibitor ketorolac attenuated LPS-induced fever with injections placed in the same region. [722] This PGE-sensitive zone is the same as the region containing the highest concentrations of PGE 2 binding sites. [723] [724] The cloning of the prostaglandin E (EP) receptors has allowed more definitive analysis of the receptors in the hypothalamus that mediate the effects of PGEs (see Fig. 7-50) . Four EP receptors have been identified, EP 1 , EP2 , EP3 , and EP4 . [693] [725] [726] All four subtypes are expressed in the preoptic area of the hypothalamus. [102] [693] [727] [728] [729] [730] Despite the established role of PGE in producing fever and activating the HPA axis, the EP receptor subtypes that are crucial in the febrile response are not 2 [ 102] [ 727] [ 731] [732] yet established. Pharmacologic evidence suggests that EP 1 and EP3 receptor agonist administration has an effect mimicking PGE 2 -induced fever.[731] [732] Moreover, an EP1 receptor antagonist blocked PGE 2 fever.[732] In contrast, targeted deletion of the EP 3 gene resulted in mice that did not show an early phase of fever after intracerebroventricular injection of LPS or PGE 2 . [726] Interestingly, there is up-regulation of EP 4 receptor expression in several areas of the brain, including the CRH neurons of the paraventricular nucleus, after immune challenge. [102] [729] In addition, paraventricular neurons that express Fos after intracerebroventricular PGE 2 also express EP 4 receptors.[729] Thus, production of PGE2 is certainly an obligate step in the pathogenesis of the febrile response; the identity of the EP receptor subtypes required for distinct components of the response remains to be established. Entry of Cytokines into the Brain

Circulating cytokines are proteins that cannot easily penetrate the blood-brain barrier. The kinetics of entry of cytokines into the brain have been examined, and evidence suggests

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that there is saturable transport of IL-1, IL-1, IL-6, and TNF- into the brain. [733] [734] [735] However, it is not clear whether sufficient levels of cytokines are detectable in brain after acute intravenous administration to account for CNS responses to acute infection. Thus, the physiologic setting and significance of this mechanism remain to be established. Moreover, it is noteworthy that levels of circulating IL-1 do not rise significantly during immune challenges. [736] [737] In contrast, large increases in circulating and brain IL-6 are found during fever. Although it is not completely understood, it appears that synthesis of IL-6 within the blood-brain barrier and not peripheral IL-6 crossing the barrier is critical in the production of fever. [738] Moreover, several studies have demonstrated that cells located at the blood-brain barrier and cells within the meninges respond to LPS stimulation with induction of IL-1 and TNF-, the nuclear factor B inhibitor IB, and the LPS receptor CD14 (Fig. 7-62). [693] [739] [740] [741] Cells with a similar morphology lining the blood vessels that penetrate the CNS and the meninges that cover it also have IL-1 receptors, suggesting that they may respond to cytokines as well. [742] Hence, endothelial and perivascular cells at the blood-brain interface may have the ability to elaborate cytokines after an LPS or cytokine signal. The physiologic role of centrally produced cytokines in the response to peripheral immune cues has been reviewed in detail. [693] [737] Interactions of Cytokines with Peripheral Nerves

Another proposed model by which cytokines may alter the activity of CNS neurons involves stimulation of peripheral sensory nerves, the prototypical example being the vagus nerve (see Fig. 7-50) .[743] [744] Several pieces of experimental evidence support the idea that the vagus may provide a conduit for cytokines to activate CNS pathways. For example, IL-1 receptor antagonist binds to vagal paraganglia. [743] [745] In addition, neurons in the nodose ganglia (vagal sensory neurons) expressed type 1 IL-1 receptor mRNA.[746] Peripheral administration of LPS induced Fos expression, a marker of neuronal activation, in the vagal sensory nodose ganglia, and this could be blocked by prior vagotomy. [745] Similarly, IL-1 administration induced the expression of Fos in neurons in the nodose ganglia and increased the firing rate of vagal afferent nerve fibers. [746] The IL-1induced response was blocked by pretreatment with a COX inhibitor, demonstrating the need for prostaglandin production in this model as well. Severing the vagus nerve below the diaphragm blocked fever, sickness behavior, and induction of IL-1 mRNA and Fos protein in the brain after intraperitoneal LPS or IL-1.[743] [747] [748] [749] [750] [751] In contrast, the abdominal vagus nerve did not seem to be necessary for the CNS response to intravenous administration of LPS or IL-1 in rats.[747] [751] These observations suggest that, although vagal sensory mechanisms may contribute to CNS responses to immune stimuli, particularly with local

infections in the abdominal or thoracic cavities, blood-borne immune challenge may activate the CNS by other routes. In the end, it is likely that redundant mechanisms exist by which the CNS is made aware of inflammatory signals in the periphery. The relative contribution of distinct mechanisms may depend upon the route of administration and dose of inflammatory mediators, and future studies should increase our understanding of these mechanisms. Cell Groups Throughout the Brain Responsive to Cytokines

Many studies have used the expression of immediate early genes such as c- fos or its protein product, Fos, [752] as a marker of neuronal activity. In this way, investigators have assessed the involvement of extended neuronal systems during the complex physiologic responses after immune challenge. Mapping the patterns of activation in the CNS after either IL-1 or LPS administration has yielded new insights into the functional neuroanatomy underlying the coordinated autonomic, endocrine, and behavioral responses during the febrile response. [381] [386] [703] [747] [753] [754] [755] [756] [757] [758] [759] [760] [761] [762] Immune activation using moderate to high doses of LPS and IL-1 activates central autonomic and endocrine structures at nearly every level of the neuraxis including several neuroendocrine regulator sites such as the central nucleus of the amygdala, paraventricular hypothalamic nucleus, arcuate nucleus of the hypothalamus, SFO, OVLT, and ventral medial preoptic area (see Fig. 7-51) . Brain stem sites engaged include the parabrachial nucleus, nucleus of the solitary tract, area postrema, and the rostral and caudal levels of the ventrolateral medulla. [381] [386] [703] [747] [753] [754] [755] [756] [757] [758] [759] [760] [761] [762] In the paraventricular nucleus, LPS and cytokines activate parvicellular CRH neurons. Although it is established that the hypothalamus is responsible for inducing a febrile response, it is also important to note that there are hypothalamic systems that act to attenuate rises in body temperature. These include arcuate POMC neurons ( see Fig. 7-45 and Fig. 7-47 (Figure Not Available) (Fig. 7-47) (Figure Not Available) ) and AVP neurons, both of which are thought to be endogenous antipyretic neuromodulators. [763] [764] [765] [766] Thus, neuronal activation patterns elicited by LPS or cytokines probably include neurons engaged to limit rises in body temperature. Indeed, Tatro and colleagues [767] have found that exogenous -MSH administration can block LPS-induced fever. Moreover, the central melanocortin system has been shown to contribute to anorexia during systemic illness. [768] [769] It is possible, therefore, that central melanocortin pathways may act in parallel to inhibit food intake and to reduce fever during immune challenge. Experimental work has coupled neuroanatomic tract tracing with methods assessing immediate early gene expression to investigate the circuitry that is activated by peripheral immune signals. For example, intravenous administration of IL-1 induced Fos in C1 adrenergic neurons in the ventrolateral medulla that project to the PVH. The C1 adrenergic cell group targets the medial parvicellular subdivision of the PVH, the site of the CRH neurons ( see Fig. 7-25 and Fig. 7-26 ). [386] Lesions interrupting the input from the C1 cells to the PVH prevent the HPA response to IL-1. These studies suggest that the activation of C1 cells by locally produced prostaglandins [702] may play a critical role in activating the HPA axis in response to IL-1. Sympathetic preganglionic neurons in the intermediolateral cell column (IML), extending from the first thoracic through the upper lumbar segments of the spinal cord, also show Fos expression in response to LPS. [761] Preganglionic neurons in the upper thoracic (T1 to T4) levels mediate thermogenesis by brown adipose tissue, [770] [771] which is a key mechanism used by rats to control heat production and body temperature. [772] Sympathetic preganglionic neurons in the T2 to T5 levels are important for control of the heart, [773] [774] which is important because there are changes in cardiac output in the febrile state. Another important concept is that sympathetic preganglionic neurons receive direct, monosynaptic input from a series of well-defined nuclei in the brain stem and the hypothalamus (see Fig. 7-1) . These cells provide another way in which the hypothalamus can contribute to the coordinated autonomic response to inflammatory signals. The major input to the sympathetic preganglionic column arises from neurons in the hypothalamus. [128] This innervation includes the paraventricular nucleus (dorsal, ventral, and lateral parvicellular subnuclei), the lateral hypothalamic area, and the arcuate nucleus and retrochiasmatic area. [50] [775] Direct projections to the intermediolateral cell column also arise in the brain stem from the A5

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noradrenergic cell group in the ventral pons, the caudal part of the nucleus of the solitary tract, the ventromedial medulla including the medullary raphe nuclei, and the rostral ventrolateral medulla, including the C1 adrenergic cell group. [50] [774] [776] Fos expression in hypothalamic and brain stem neurons projecting to the IML after LPS administration has been examined. [776] LPS-activated cells that innervate the IML are found in the rostral ventrolateral medulla (C1 adrenergic cell group) and the A5 noradrenergic cell group in the brain stem. Moreover, a prominent population of cells was found in the dorsal parvicellular division of the paraventricular nucleus in the hypothalamus. These results suggest that neurons in the parvicellular PVH specifically innervate sympathetic preganglionic neurons in the spinal cord that regulate LPS-induced fever. Furthermore, as noted earlier, activation of CRH neurons in the PVH is a signature of the CNS response to immune stimulation. Thus, the paraventricular hypothalamic nucleus is a key site for mediating both neuroendocrine and autonomic responses to immune stimulation.



NEUROENDOCRINE DISEASE Disease of the hypothalamus can cause pituitary dysfunction, neuropsychiatric and behavioral disorders, and disturbances of autonomic and metabolic regulation (Fig. 7-52) . In the diagnosis and treatment of suspected hypothalamic or pituitary disease, four issues must be kept in mind: the extent of the lesion, the physiologic impact, the specific cause, and the psychosocial setting. The etiology of hypothalamic neuroendocrine disorders categorized by age and syndrome is summarized in Table 7-9 and Table 7-10 . Manifestations of pituitary insufficiency secondary to hypothalamic or pituitary stalk damage are not identical to those of primary pituitary insufficiency. Hypothalamic injury causes decreased secretion of most pituitary hormones but can cause hypersecretion of hormones normally under inhibitory control by the hypothalamus, as in hypersecretion of PRL after damage to the pituitary stalk and precocious puberty caused by loss of the normal restraint over gonadotropin maturation. [777] Impairment of inhibitory control of the neurohypophysis can lead to the syndrome of inappropriate vasopressin secretion (SIADH) (see Chapter 9) . More subtle abnormalities in secretion can result from impairment of the control system. For example, loss of the normal circadian rhythm of corticotropin

Figure 7-52 Typical pituitary response to thyrotropin-releasing hormone (TRH) administration in patients with hypothalamic-pituitary disease that has caused hypothyroidism. If there is intrinsic pituitary damage, the response is abnormally low. If there is hypothalamic damage, the response is normal or exaggerated. It must be emphasized that some patients with hypothalamic disease may not respond to TRH and that some patients with pituitary disease may respond to TRH. T 4 , thyroxine; TSH, thyrotropin. (From Jackson IMD. Diagnostic tests for the evaluation of pituitary tumors. In Jackson IMD, Reichlin S [eds]. The Pituitary Adenoma. New York, Plenum, 1980, pp 219238.)

secretion may occur before loss of pituitary-adrenal secretory reserve, [778] and responses to physiologic stimuli may be paradoxical. Because hypophyseotropic hormone levels cannot be measured directly and pituitary hormone secretion is regulated by complex, multilayered controls, assay of pituitary hormones in blood does not necessarily give a meaningful picture of events at hypothalamic and higher levels. Rarely, tumors secrete excessive amounts of releasing peptides and cause hypersecretion of hormones from the pituitary. Disorders of the hypothalamic-pituitary unit can result from lesions at several levels (Fig. 7-53) . Defects can arise from destruction of the pituitary (as by tumor, infarct, inflammation, or autoimmune disease) or from a hereditary deficiency of a particular hormone as in rare cases of isolated FSH, GH, or POMC deficiency (Fig. 7-54) . Selective loss of thyroid hormone receptors in the pituitary can give rise to increased thyrotropin secretion and thyrotoxicosis. Furthermore, disorders can arise through disruption of the stalkmedian eminence contact zone, the stalk itself, or the nerve terminals of the tuberohypophyseal system; such disruption occurs after surgical stalk section, with tumors involving the stalk, and in some inflammatory diseases. At a higher level, tonic inhibitory and excitatory inputs can be lost as manifested by absence of circadian rhythms or the development of precocious puberty. Physical stress, cytokine products of inflammatory cells, toxins, and reflex inputs from peripheral homeostatic monitors also impinge on the tuberoinfundibular system. At the highest level of control, emotional stress and psychological disorders can activate the pituitary-adrenal stress response and suppress gonadotropin secretion (e.g., psychogenic amenorrhea) or inhibit GH secretion (e.g., psychosocial dwarfism) (see Chapter 23) . Intrinsic disease of the anterior pituitary is reviewed in Chapter 8 , and disturbances in neurohypophyseal function are discussed in Chapter 9 . This chapter considers diseases of the hypothalamic-pituitary unit. Pituitary Isolation Syndrome

Destructive lesions of the pituitary stalk, as occur with head injury, surgical transection, tumor, or granuloma, produce a characteristic pattern of pituitary dysfunction.[777] [779] [780] Central diabetes inspidus (DI) develops in a large percentage of patients, depending on the level at which the stalk has been sectioned. If the cut is close to the hypothalamus, DI is almost always produced, whereas if the section is low on the stalk, the incidence is lower. The extent to which nerve terminals in the upper stalk are preserved determines the clinical course. The

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TABLE 7-9 -- Etiology of Hypothalamic Disease by Age Premature Infants and Neonates Intraventricular hemorrhage Meningitis: bacterial Tumors: glioma, hemangioma Trauma Hydrocephalus, kernicterus 1 mo2 yr Tumors Glioma, especially optic glioma Histiocytosis X Hemangioma Hydrocephalus Meningitis Familial disorders Laurence-Moon-Biedl syndrome Prader-Labhart-Willi syndrome 210 yr Neoplasms Craniopharyngioma Glioma, dysgerminoma, hamartoma Histiocytosis X, leukemia Ganglioneuroma, ependymoma Medulloblastoma Meningitis

Bacterial Tuberculous Encephalitis Viral Exanthematous demeyelinating Familial Diabetes insipidus Radiation therapy Diabetic ketoacidosis Moyamoya disease, circle of Willis 1025 yr Tumors Craniopharyngioma Glioma, hamartoma, dysgerminoma Histiocytosis X, leukemia Dermoid, lipoma, neuroblastoma Trauma Vascular Subarachnoid hemorrhage Aneurysm Arteriovenous malformation Inflammatory disease Meningitis Encephalitis Sarcoidosis Tuberculosis Structural brain defect Chronic hydrocephalus Increased intracranial pressure 2550 yr Nutritional: Wernicke's disease Tumors Glioma, lymphoma, meningioma Craniopharyngioma, pituitary tumors Angioma, plasmacytoma, colloid cysts Ependymoma, sarcoma, histiocytosis X Inflammatory disease Sarcoidosis Tuberculosis, viral encephalitis Vascular Aneurysm, subarachnoid hemorrhage Arteriovenous malformation Damage from pituitary radiation therapy 50 yr and Older Nutritional: Wernicke's disease Tumors: Pituitary tumors, sarcoma, glioblastoma, ependymoma, meningioma, colloid cysts, lymphoma Vascular disease Infarct, subarachnoid hemorrhage Pituitary apoplexy Inflammation: encephalitis, sarcoidosis, meningitis Damage from radiation therapy for ear-nose-throat carcinoma, pituitary tumors Adapted from Plum F, Van Uitert R. Nonendocrine diseases and disorders of the hypothalamus. In Reichlin S, Baldessarini RJ, Martin JB (eds). The Hypothalamus, vol 56. New York, Raven Press, 1978, pp 415473. classical triphasic syndrome of initial polyuria followed by normal water control and then by vasopressin deficiency over a period of 1 week to 10 days is seen in less than half of the patients. The sequence is attributed to an initial loss of neurogenic control of the neural lobe, followed by autolysis of the neural lobe with release of vasopressin into the circulation and finally by complete loss of vasopressin. However, full expression of polyuria requires adequate cortisol levels; if cortisol is deficient, vasopressin deficiency may be present with only minimal polyuria. DI can also develop after stalk injury without an overt transitional phase. When DI occurs after head injury or operative trauma, varying degrees of recovery can be seen even after months or years. Sprouting of nerve terminals in the stump of the pituitary stalk may give rise to sufficient functioning tissue to maintain water balance. In contrast to the effects of stalk section, nondestructive injury to the neurophypophysis or stalk, as during surgical resection of optic chiasmatic astrocytomas, can sometimes give rise to transient SIADH. [781] Although head injury, granulomas, and tumors are the most common causes of acquired DI, other cases develop in the absence of a clear-cut cause. [782] Some cases may be due to autoimmune disease of the hypothalamus as suggested by the finding of autoantibodies to neurohypophyseal cells in a third of cases of "idiopathic" DI in one series. [783] However, autoantibodies were also frequently found in association with histiocytosis-X. Later reports suggest the importance of continued vigilance in cases of idiopathic DI because a definite cause is frequently uncovered in time, including a high proportion of occult germinomas whose detection by magnetic

resonance imaging may be preceded by elevated levels of human chorionic gonadotropin (hCG) in CSF.

[784] [785]

TABLE 7-10 -- Etiology of Endocrine Syndromes of Hypothalamic Origin Hypophyseotropic Hormone Deficiency Surgical pituitary stalk section Basilar meningitis and granuloma, sarcoidosis, tuberculosis, sphenoid osteomyelitis, eosinophilic granuloma Craniopharyngioma Hypothalamic tumor Infundibuloma Teratoma (ectopic pinealoma) Neuroglial tumor, particularly astrocytoma Maternal deprivation syndrome, psychosocial dwarfism Isolated growth hormonereleasing hormone (GHRH) deficiency Hypothalamic hypothyroidism Panhypophyseotropic failure Disorders of Regulation of Gonadotropin-Releasing Hormone Secretion Female Precocious puberty GnRH-secreting hamartoma hCG-secreting germinoma Delayed puberty Neurogenic amenorrhea Pseudocyesis Anorexia nervosa "Functional" amenorrhea "Functional" oligomenorrhea Drug-induced amenorrhea Male Precocious puberty Fröhlich's syndrome Olfactory-genital dysplasia (Kallmann's syndrome) Disorders of Regulation of Prolactin-Regulating Factors Tumor Sarcoidosis Drug-induced Reflex Herpes zoster of chest wall Post-thoracotomy Nipple manipulation Spinal cord tumor "Psychogenic" Hypothyroidism Carbon dioxide narcosis Disorders of Regulation of Corticotropin-Releasing Hormone Paroxysmal corticotropin discharge (Wolff's syndrome) Loss of circadian variation Depression CRH-secreting gangliocytoma CRH, corticotropin-releasing hormone; GnRH, gonadotropin-releasing hormone; hCG, human chorionic gonadotropin.

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Figure 7-53 Effect of hypothalamic-pituitary disconnection on the growth hormone (GH) secretory responses to GH-releasing hormone (GHRH) (1 µg/kg) and hexarelin (2 µg/kg) administered intravenously to children with GH deficiency. Top, mean responses in a group of 24 prepubertal children with short stature secondary to familial short stature or constitutional growth delay. Children with GH deficiency and an intact vascular pituitary stalk as visualized by dynamic magnetic resonance imaging exhibited a clear, but blunted, GH response to both secretagogues (middle). In contrast, children with pituitary stalk agenesis (both vascular and neural components) had no or a markedly attenuated response to both peptides (bottom). (From Maghnie M, Spica-Russotto V, Cappa M, et al. The growth hormone response to hexarelin in patients with different hypothalamic-pituitary abnormalities. J Clin Endocrinol Metab 1998; 83:38863889.)

Congenital DI can be part of a hereditary disease. DI in the Brattleboro rat is due to an autosomal recessive genetic defect that impairs production of vasopressin but not of oxytocin.[786] Inherited forms of DI in humans have been attributed to mutations in the vasopressin V2 receptor gene or less frequently in the aquaporin or

vasopressin genes. [787] [788] [789] [790] Menstrual cycles cease after stalk section although urinary gonadotropins may still be detectable, unlike the situation after hypophysectomy. Plasma glucocorticoid levels and urinary excretion of cortisol and 17-hydroxycorticoids decline after hypophysectomy and stalk section, but the change is slower after stalk section. A transient increase in cortisol secretion after stalk section is believed to be due to release of ACTH from preformed stores. The ACTH response to the lowering of blood cortisol is markedly reduced but ACTH release after stress may be normal, possibly because of CRH-independent mechanisms. Reduction in thyroid function after stalk section is similar to that seen with hypophysectomy. The fall in GH secretion is said to be the most sensitive indication of damage to the stalk; however, the insidious nature of this endocrinologic change in adults who have suffered traumatic brain injuries may cause it to be overlooked and therefore contribute to delayed rehabilitation. [791] Humans with stalk sections or with tumors of the stalk region have widely varying levels of hyperprolactinemia and may have galactorrhea. [792] PRL responses to hypoglycemia and to TRH are blunted, in part because of loss of neural connections with the hypothalamus. PRL responses to dopamine agonists and antagonists in the pituitary isolation syndrome are similar to those in patients with prolactinomas. Interestingly, PRL secretion continues to show a diurnal variation in patients with either hypothalamopituitary disconnection or microprolactinoma. [520] Both forms of hyperprolactinemia are characterized by a similarly increased frequency of PRL pulses and a marked rise in nonpulsatile or basal PRL secretion, although the disruption is greater in the tumoral hyperprolactinemia. An incomplete pituitary isolation syndrome may occur with the empty sella syndrome, intrasellar cysts, or pituitary adenomas. [793] [794] [795] Anterior pituitary failure after stalk section is in part due to loss of specific neural and vascular links to the hypothalamus and in part due to pituitary infarction.



Hypophyseotropic Hormone Deficiency

Selective pituitary failure can be due to a deficiency of specific pituitary cell types or a deficiency of one or more hypothalamic hormones. Isolated GnRH deficiency is the most common hypophyseotropic hormone deficiency. In Kallmann's syndrome (gonadotropin deficiency commonly associated with

Figure 7-54 A neuroendocrine syndrome of adrenocorticotropic hormone insufficiency, obesity, and red hair resulting from a null mutation in the pro-opiomelanocortin gene. (Photo kindly provided by Dr. A. Gruters, Berlin.)

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hyposmia),[796] hereditary agenesis of the olfactory lobe may be demonstrable by magnetic resonance imaging. [797] Abnormal development of the GnRH system is due to defective migration of the GnRH-containing neurons from the olfactory nasal epithelium in early embryologic life (see the earlier section on GnRH). Other malformations of the cranial midline structures, such as absence of the septum pellucidum in septo-optic dysplasia (De Morsier's syndrome), can cause hypogonadotropic hypogonadism (HH) or, less commonly, precocious puberty. A surprisingly large percentage of children with septo-optic dysplasia who otherwise have multiple hypothalamic-pituitary abnormalities actually retain normal gonadotropin function and enter puberty spontaneously. [798] The genetic basis of HH has now been established in approximately 10% of patients. [799] [800] Mutations in the KAL (Kallmann's syndrome) gene and the AHC-DAX1 (adrenal hypoplasia congenitaHH) gene cause X-linked recessive disease. Autosomal recessive HH has been associated with mutations in the GnRH receptor, leptin, leptin receptor, FSH, LH, PROP-1 (combined pituitary deficiency), and HESX (septo-optic dysplasia) genes. The GnRH response test is of little value in the differential diagnosis of hypothalamic hypogonadism. Most patients with GnRH deficiency show little or no response to an initial test dose, but normal responses are seen after repeated injection. This slow response has been attributed to down-regulation of GnRH receptors in response to prolonged GnRH deficiency. Furthermore, with intrinsic pituitary disease the response to GnRH may be absent or normal. Consequently, it is not possible to distinguish between hypothalamic and pituitary disease with a single injection of GnRH. Prolonged infusions or repeated administration of GnRH agonists after hormone replacement therapy priming may aid in the diagnosis or provide therapeutic options for women with Kallmann's syndrome wishing to become pregnant. [801] [802]

Deficiency of TRH secretion gives rise to hypothalamic hypothyroidism, also called tertiary hypothyroidism, which can occur in hypothalamic disease or more rarely as an isolated defect. [803] Molecular genetic analyses have revealed infrequent autosomal recessive mutations in the TRH and TRH receptor genes in the etiology of central hypothyroidism. [804] Hypothalamic and pituitary causes of TSH deficiency are most readily distinguished by imaging methods. Although theoretically reasonable, the TRH stimulation test for the differentiation of hypothalamic disease from pituitary disease is of limited value. The typical pituitary response to TRH administration in patients with TRH deficiency is an enhanced and somewhat delayed peak, whereas the response with pituitary failure is subnormal or absent. The hypothalamic type of response has been attributed to an associated GH deficiency that sensitizes the pituitary to TRH (possibly through suppression of somatostatin secretion), but GH also affects T 4 metabolism and may alter pituitary responses as well. [805] In practice, the responses to TRH in hypothalamic and pituitary disease overlap so much that they cannot be used reliably for a differential diagnosis. Persistent failure to demonstrate responses to TRH is good evidence for the presence of intrinsic pituitary disease, but the presence of a response does not mean that the pituitary is normal. Deficient TRH secretion leads to altered TSH biosynthesis by the pituitary, including impaired glycosylation. Poorly glycosylated TSH has low biologic activity, and dissociation of bioactive and immunoreactive TSH can lead to the paradox of normal or elevated levels of TSH in hypothalamic hypothyroidism. [803] [806] GHRH deficiency appears to be the principal cause of hGH deficiency in children with idiopathic dwarfism. [807] This condition is frequently associated with abnormal electroencephalograms, a history of birth trauma, and breech delivery. Furthermore, magnetic resonance imaging scans show that a substantial proportion of children with idiopathic hGH deficiency have evidence of a torn pituitary stalk, [808] [809] which is presumed evidence for birth trauma as the cause. Human GH is the most vulnerable of the anterior pituitary hormones when the pituitary stalk is damaged. It can be difficult to differentiate between primary pituitary disease and GHRH deficiency by standard tests of GH reserve. However, a substantial GH secretory response to a single administration of hexarelin occurs only in the presence of at least a partially intact vascular stalk. [420] In many children with dwarfism, the anatomic abnormalities of the intrasellar contents and pituitary stalk together with the frequent occurrence of other midline defects, such as those in septo-optic dysplasia, are consistent with the alternative hypothesis of a developmental defect occurring in embryogenesis. [808] There has been a remarkable advance in our understanding of the molecular ontogeny of the hypothalamic-pituitary unit, much of it based on mutant mouse models. [810] [811] Parallel genetic analyses have been conducted in children with isolated GH deficiency or combined pituitary hormone deficiencies. These studies have identified autosomal recessive mutations in both structural and regulatory genes including the GHRH receptor, PIT1, PROP1, and HESX1 that are responsible for a sizable proportion of congenital hypothalamic-pituitary disorders once considered idiopathic. [409] [807] [812] [813] Adrenal insufficiency is another manifestation of hypothalamic disease and can be due to CRH deficiency. [814] [815] Isolated ACTH deficiency is uncommon, but there is suggestive evidence in at least one family of genetic linkage to the CRH gene locus. [816] Later investigations have revealed mutations in the TPIT gene, a T box transcription factor expressed only in pituitary corticotrophs and melanotrophs, associated with cases of isolated ACTH deficiency. [817] The CRH stimulation test does not distinguish hypothalamic from pituitary failure as a cause of corticotropin deficiency. [818] [819] Apart from intrinsic diseases of the hypothalamus such as tumors and granulomas, two environmental causes of central hypophyseotropic deficiencies are of increasing clinical importance. These are trauma to the brain, [777] [779] [791] particularly from motor vehicle accidents, and the sequelae of chemotherapy and radiation therapy for intracranial lesions in children and adults. [806] [820] [821] Improved short-term survival from head injuries associated with coma and CNS malignancies has greatly increased the prevalence of long-term neuroendocrine consequences.



Hypophyseotropic Hormone Hypersecretion

Pituitary hypersecretion is occasionally caused by tumors of the hypothalamus. [822] GnRH-secreting hamartomas can cause precocious puberty. [822] CRH-secreting gangliocytomas can cause Cushing's syndrome, [823] and GHRH-secreting gangliocytomas of the hypothalamus can cause acromegaly. [824] Although they do not arise from the hypothalamus, paraneoplastic syndromes can also cause pituitary hypersecretion, as with CRH-secreting tumors and GHRH-secreting tumors of the bronchi and pancreas. Bronchial carcinoids and pituitary islet cell tumors are the usual causes of this phenomenon.



Neuroendocrine Disorders of Gonadotropin Regulation Precocious Puberty

The term precocious puberty is used when physiologically normal pituitary-gonadal function appears at an early age. [825] [826] By convention, the onset of androgen secretion and spermatogenesis must occur before the age of 9 or 10 in boys and the onset of estrogen secretion and cyclic ovarian activity before

153

age 8 in girls. [827] [828] Central precocious puberty is due to disturbed CNS function, which may or may not have an identifiable structural basis. Pseudoprecocious puberty refers to premature sexual development resulting from excessive secretion of androgens, estrogens, or hCG caused by tumors (both gonadal and extragonadal), administration of exogenous gonadal steroids, or genetically determined activation of gonadotropin receptors (see Chapter 15) . Central precocious puberty with neurogenic causes and pineal gland disease is discussed in this chapter. Idiopathic Sexual Precocity

Familial occurrence is uncommon, but there is a hereditary form of idiopathic sexual precocity that is largely confined to males. Abnormal electroencephalograms and behavioral disturbances, suggesting the presence of brain damage, have been reported occasionally in girls with idiopathic precocious puberty. The pathogenesis may be related to the rate of hypothalamic development or other as yet undetermined nutritional, environmental, or psychosocial factors. Many cases previously thought to be idiopathic are due to small hypothalamic hamartomas discussed in more detail in the following. It has been argued that localized activation of discrete cellular subsets connected to GnRH neurons may be sufficient to initiate puberty. [829] Neurogenic Precocious Puberty

Approximately two thirds of hypothalamic lesions that influence the timing of human puberty are located in the posterior hypothalamus, but in the subset of patients who come to autopsy, damage is extensive. Specific lesions known to cause precocity include craniopharyngioma (although delayed puberty is more common), astrocytoma, pineal tumors, subarachnoid cysts, encephalitis, miliary tuberculosis, tuberous sclerosis or neurofibromatosis type 1, the Sturge-Weber syndrome, porencephaly, craniostenosis, microcephaly, hydrocephalus, empty sella syndrome, and Tay-Sachs disease. [830] [831] Hamartoma of the hypothalamus is an exception to the generalization that tumors of the brain cause precocious puberty by impairment of gonadotropin secretion (although hamartomas on occasion cause hypothalamic damage). A hamartoma is a tumor-like collection of normal-appearing nerve tissue lodged in an abnormal location. The parahypothalamic type consists of an encapsulated nodule of nerve tissue attached to the floor of the third ventricle or suspended from the floor by a peduncle and typically less than 1 cm in diameter. The intrahypothalamic or sessile type is enveloped by the posterior hypothalamus and can distort the third ventricle. These tend to be larger than the pedunculated variety, grow in the interpeduncular cistern, and are frequently accompanied by seizures, mental retardation, developmental delays, and roughly half the incidence of precocious puberty associated with the parahypothalamic lesions. [832] [833] Before the development of high-resolution scanning techniques, this tumor was considered rare, but small ones can now be visualized. Miniature hamartomas of the tuber cinereum are common at autopsy. Precocious puberty occurs when the hamartoma makes connections with the median eminence and thus serves as an accessory hypothalamus. Peptidergic nerve terminals containing GnRH have been found in the tumors. [834] Early pubertal development is presumably due to unrestrained GnRH secretion, although the hamartomas almost certainly have an intrinsic pulse generator of GnRH secretion because pulsatility is required for stimulation of gonadotropin secretion (see earlier section on GnRH). Manifestations of premature puberty in patients with hamartomas are similar to those associated with other central causes of precocity. Hamartomas occur in both sexes and may be present as early as age 3 months. In the past most cases were thought to be fatal by age 20, but many hamartomas cause no brain damage and need not be excised.[833] The interpeduncular fossa of the brain is difficult to approach, and surgical experience is somewhat limited. Early in the course of illness, epilepsy manifested as "brief, repetitive, stereotyped attacks of laughter" [835] may provide a clue to the disease. Late in the course, hypothalamic damage can cause severe neurologic defects and intractable seizures. Hypothyroidism

Hypothyroidism can cause precocious puberty in girls that is reversible with thyroid therapy. Hyperprolactinemia and galactorrhea may be present. One possibility is that elevated thyrotropin levels (in children with thyroid failure) cross-react with the FSH receptor. [836] Alternatively, low levels of thyroid hormone might simultaneously activate release of LH, FSH, and TSH. A third possibility is that hypothyroidism causes hypothalamic encephalopathy that impairs the normal tonic suppression of gonadotropin release by the hypothalamus. The high PRL levels that sometimes accompany this disorder may be due to a deficiency in PIF secretion, increased secretion of TRH, or increased sensitivity of the lactotrophs to TRH secretion. Tumors of the Pineal Gland

Pineal gland tumors account for only a small percentage of intracranial neoplasms. They occur as a central midline mass with an enhancing lesion on magnetic resonance imaging frequently accompanied by hydrocephalus. Pinealomas cause a variety of neurologic abnormalities (Table 7-11) . Parinaud's syndrome, which consists of paralysis of upward gaze, pupillary areflexia (to light), paralysis of convergence, and a wide-based gait, occurs with about half of pinealomas. Gait disturbances can also occur because of brain stem or cerebellar compression. Several discrete cytopathologic entities account for mass lesions in the pineal region (Table 7-12) . [836] The most common non-neoplastic conditions are degenerative pineal cysts, arachnoid cysts, and cavernous hemangioma. Pinealocytes give rise to primitive neuroectodermal tumors, the so-called small blue cell tumors that are immunopositive for the neuronal marker synaptophysin and negative for the lymphocyte marker CD45. True pinealomas can be relatively well-differentiated pineocytomas, intermediate mixed forms, or the less differentiated pineoblastomas, [837] [838] which are basically the same as medulloblastomas, neuroblastomas, and oat cell carcinomas of the lung. The most common tumors of the pineal gland are actually germinomas (a form of teratoma), so designated because of their presumed origin in germ cells. Germinomas may also occur in the anterior hypothalamus or the floor of the third ventricle, where they are often associated with the clinical triad of DI, pituitary insufficiency, and visual abnormalities. [830] Identical tumors can be found in the testis and anterior mediastinum. Intracranial germinomas have a tendency to spread locally, infiltrate the hypothalamus, and metastasize to the spinal cord and CSF. Extracranial metastases (to the skin, lung, or liver) are rare. Teratomas derived from two or more germ cell layers also occur in the pineal region. Chorionic tissue in teratomas and germinomas may secrete hCG in sufficient amounts to cause gonadal maturation, and some of these tumors have histologic and functional characteristics of choriocarcinomas. Diagnosis is confirmed by the combination of a mass lesion, cytologic analysis of CSF, and radioimmunoassay detection of hCG in the CSF. Precocious puberty is a relatively unusual manifestation of

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TABLE 7-11 -- Classification of Tumors of the Pineal Region

A. Germ Cell Tumors 1. Germinoma a. Posterior third ventricle and pineal lesions b. Anterior third ventricle, suprasellar or intrasellar lesions c. Combined lesions in anterior and posterior third ventricle, apparently noncontiguous, with or without foci of cystic or solid teratoma 2. Teratoma a. Evidencing growth along two or three germ lines in varying degrees of differentiation b. Dermoid and epidermoid cysts with or without solid foci of teratoma c. Histologically malignant forms with or without differentiated foci of benign, solid, or cystic teratoma-teratocarcinoma, chorioepithelioma, embryonal carcinoma (endodermal-sinus tumor or yolk-sac carcinoma), combinations of these with or without foci of germinoma, chemodectoma B. Pineal Parenchymal Tumors 1. Pinealocytes a. Pineocytoma b. Pineoblastoma c. Ganglioglioma and chemodectoma d. Mixed forms exhibiting transitions between these 2. Glia a. Astrocytoma b. Ependymoma c. Mixed forms and other less frequent gliomas (e.g., glioblastoma, oligodendroglioma) C. Tumors of Supporting or Adjacent Structures 1. Meningioma 2. Hemangiopericytoma D. Non-neoplastic Conditions of Neurosurgical Importance 1. "Degenerative" cysts of pineal lined by fibrillary astrocytes 2. Arachnoid cysts 3. Cavernous hemangioma From DeGirolami U. Pathology of tumors of the pineal region. In Schmidek HH (ed). Pineal Tumors. New York, Masson, 1977, pp 119. pineal gland disease. When it occurs, neuroanatomic studies suggest that the cause is secondary to pressure or destructive effects of the pineal tumor on the function of the adjacent hypothalamus or to the secretion of hCG. Most patients have other evidence of hypothalamic involvement such as DI, polyphagia, somnolence, obesity, or behavioral disturbance. Choriocarcinoma of the pineal gland is associated with high plasma levels of hCG. The hCG can stimulate testosterone secretion from the testis but not estrogen secretion by the ovary and TABLE 7-12 -- Pinealomas: Frequency (%) of Presenting Symptoms and Signs Increased intracranial pressure 85 Spasticity

35

Ataxia

30

Parinaud's syndrome

25

Cerebellar-type nystagmus

25

Syncope

20

Vertigo

20

Cranial nerve palsy (other than cranial nerves VI, VIII)

20

Intention tremor

15

Scotoma

10

Tinnitus

10

Other

10

From Brady WL. The role of radiation therapy. In Schmidek HH (ed). Pineal Tumors. New York, Masson, 1977, pp. 99113. hence causes premature puberty almost exclusively in boys. The prevalence of elevated hCG levels in children with premature puberty related to tumors in the pineal region is unknown, but the fact that this phenomenon occurs further challenges the theory that nonparenchymal tumors cause precocious puberty by damaging the normal pineal gland. Rarely, pinealomas cause delayed puberty, raising speculation about a role of melatonin in inhibiting gonadotropin secretion in these cases. Management of tumors in the pineal region is not straight-forward. [837] [839] Operative mortality rates can be high, but the rationale for an aggressive approach to the pineal region is based on the need to make a histologic diagnosis, the variety of lesions found in this region, the possibility of cure of an encapsulated lesion, and the effectiveness of chemotherapeutic agents for germinomas and choriocarcinoma. Stereotaxic biopsy of the pineal region provided diagnosis in 33 of 34 cases in one series, suggesting that this is a useful alternative to open surgical exploration for diagnostic purposes. [840] Long-term palliation or cure of many pineal region tumors is possible by combinations of surgery, radiation, gamma knife, or chemotherapy, depending on the nature of the lesion. [841] Approach to the Patient with Precocious Puberty

Several groups have reviewed the diagnostic approach to suspected central precocious puberty. [842] [843] [844] Although guidelines differ, the index of suspicion is clearly inversely proportional to the age of the patient. A GnRH stimulation test to assess gonadotropin release and thereby differentiate between primed and inactive gonadotrophs is probably the single most important endocrinologic measure. If LH and FSH levels are not stimulated and there is no evidence of gonadal germ cell maturation, the cause of precocious puberty lies outside the hypothalamic-pituitary axis and the diagnostic process should focus on the adrenal glands and gonads ( see Chapter 13 and Chapter 15 ). Magnetic resonance imaging studies are central to the work-up for exclusion or characterization of organic lesions in the areas of the sella, optic chiasm, suprasellar hypothalamus, and interpeduncular cistern. [826] Management of Sexual Precocity

Structural lesions of the hypothalamus are treated by surgery, radiation, chemotherapy, or combinations of these as indicated by the pathologic diagnosis and extent of disease. Endocrinologic manifestations of precocious puberty are best treated by GnRH agonists with the therapeutic goals of delaying sexual maturation to a more appropriate age and achieving optimal linear growth and bone mass, possibly with the combined use of GH treatment. [845] [846] [847] Other approaches include the use of cyproterone acetate, testolactone, or spironolactone to antagonize or inhibit gonadal steroid biosynthesis. [848] [849] Precocious puberty is stressful to both the child and

the parents, and it is essential that psychological support be provided. Psychogenic Amenorrhea

Menstrual cycles can cease in young nonpregnant women with no demonstrable abnormalities of the brain, pituitary, or ovary in several situations, [850] [851] including pseudocyesis (false pregnancy), anorexia nervosa, excessive exercise, psychogenic disorders, and hyperprolactinemic states (see Chapter 13) . Psychogenic amenorrhea, the most common cause of secondary amenorrhea except for pregnancy, can occur with major psychopathology or minor psychic stress and is often temporary. Psychogenic

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amenorrhea is probably mediated by excessive endogenous opioid activity because naloxone or naltrexone (opiate receptor blockers) can induce ovulation in some patients with this disorder. [850] [852] Exercise-induced amenorrhea may be a variant of psychogenic amenorrhea or may result from loss of body fat. [851] [853] The syndrome is associated with intense and prolonged physical exertion such as running, swimming, or ballet dancing. Such women are always below ideal body weight and have low stores of fat. If the activity is begun before puberty, normal sexual maturation can be delayed for many years. The mass of fat may be a regulator of gonadotropin secretion with adipocyte-derived leptin as the principal mediator between peripheral energy stores and hypothalamic regulatory centers. [854] Studies in nonhuman primates showed a direct role of caloric intake in the pathogenesis of amenorrhea associated with long-distance running. [855] Exercise and psychogenic amenorrhea can have adverse effects because of the associated estrogen deficiency and accompanying osteopenia (also see Chapter 23) .[856] Neurogenic Hypogonadism in Males

A discussion of neurogenic hypogonadism in males should begin with an account of Fröhlich's syndrome (adiposogenital dystrophy), originally characterized as delayed puberty, hypogonadism, and obesity associated with a tumor that impinges on the hypothalamus. It was subsequently recognized that either hypothalamic or pituitary dysfunction can induce hypogonadism and the presence of obesity indicates that the appetite-regulating regions of the hypothalamus have been damaged. Several organic lesions of the hypothalamus can cause this syndrome, including tumors, encephalitis, microcephaly, Friedreich's ataxia, and demyelinating diseases. Other important causes of hypogonadotropic hypogonadism are Kallmann's syndrome, a disorder caused by failure of GnRH-containing neurons to migrate normally (see earlier in the section on GnRH and hypophyseotropic hormone deficiency), and a subset of the Prader-Willi syndrome. [857] However, most males with delayed sexual development do not have serious neurologic conditions. Furthermore, most obese boys with delayed sexual development have no structural damage to the hypothalamus but have constitutional delayed puberty, which is commonly associated with obesity. It is not known whether there is a functional disorder of the hypothalamus in this condition. It is generally believed that psychosexual development of brain maturation depends on the presence of androgens and that hypogonadism in boys (regardless of cause) should be treated by the middle teen years (15 years at the latest). In adult men, hypogonadism (including reduced spermatogenesis) can be induced by emotional stress or severe exercise, [858] but this abnormality is seldom diagnosed because the symptoms are more subtle than menstrual cycle changes in similarly stressed women. Prolonged physical stress and sleep and energy deficiency can also decrease testosterone and gonadotropin levels. [859] Chronic intrathecal administration of opiates for the control of intractable pain syndromes is strongly associated with hypogonadotropic hypogonadism, and to a lesser extent hypocorticism and GH deficiency, in both men and women. [860] Finally, critical illness with multiple causes is well known to be associated with hypogonadism and ineffectual altered pulsation of GnRH. [861]



Neurogenic Disorders of Prolactin Regulation

Neurogenic causes of hyperprolactinemia include irritative lesions of the chest wall (herpes zoster, thoracotomy), excessive tactile stimulation of the nipple, and lesions within the spinal cord such as ependymoma. [862] [863] Prolonged mechanical stimulation of the nipples by suckling or the use of a breast pump can initiate lactation in some women who are not pregnant, and neurologic lesions that interrupt the hypothalamic-pituitary connection can cause hyperprolactinemia, as discussed earlier. Hyperprolactinemia also occurs after certain forms of epileptic seizures. In one series, six of eight patients with temporal lobe seizures had a marked increase in PRL, whereas only one in eight frontal lobe seizures led to hyperprolactinemia. [864] Agents that block dopamine receptors (such as the phenothiazines) or prevent dopamine release (e.g., reserpine and methyldopa) must be excluded in all cases. Because the nervous system exerts such profound effects on PRL secretion, patients with hyperprolactinemia (including those with adenomas) may have a deficit of PIF or an excess of PRF activity. In studies of PRL secretion in patients apparently cured of hyperprolactinemia by removal of a pituitary microadenoma, regulatory abnormalities persisted in some but not all patients. Persistence of regulatory abnormalities may be due to incomplete removal of tumor, abnormal function of the remaining part of the gland, or underlying hypothalamic abnormalities. [865]



Neurogenic Disorders of Growth Hormone Secretion Hypothalamic Growth Failure

Loss of the normal nocturnal increase in GH secretion and loss of GH secretory responses to provocative stimuli occur early in the course of hypothalamic disease and may be the most sensitive endocrine indicator of hypothalamic dysfunction. As noted earlier, anatomic malformations of midline cerebral structures are associated with abnormal GH secretion, presumably related to failure of the development of normal GH regulatory mechanisms. Such disorders include optic nerve dysplasia and midline prosencephalic malformations (absence of the septum pellucidum, abnormal third ventricle, and abnormal lamina terminalis). Certain complex genetic disorders including Prader-Willi syndrome also commonly involve reduced GH secretory capacity. [866] Idiopathic hypopituitarism with GH deficiency was considered earlier in this chapter. Maternal Deprivation Syndrome and Psychosocial Dwarfism

Infant neglect or abuse can impair growth and cause failure to thrive (the maternal deprivation syndrome). Malnutrition interacts with psychological factors to cause growth failure in children with the maternal deprivation syndrome, and each case should be carefully evaluated from this point of view. Older children with growth failure in a setting of abuse or severe emotional disturbance (termed psychosocial dwarfism) may also have abnormal circadian rhythms and deficient hGH release after insulin-induced hypoglycemia or arginine infusion (see Chapter 8) .[867] [868] Deficient release of corticotropin and gonadotropins may also be present. A new variant termed hyperphagic short stature has been identified. [869] These disorders are reversible by placing the child in a supportive milieu where growth and neuroendocrine hGH responses rapidly return to normal. [870] The pathogenesis of altered GH secretion in children in response to deprivation is unknown. In the adult human, furthermore, physical or emotional stress usually causes an increase in hGH secretion, as noted earlier.

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Neuroregulatory Growth Hormone Deficiency

The availability of biosynthetic hGH for treatment of short stature has brought into focus a group of patients who grow at low rates (below the third percentile) and have low levels of serum IGF-I but a normal hGH secretory reserve. Studies of 24-hour hGH secretion profiles indicate that many of these children do not have normal spontaneous hGH secretion (abnormal ultradian and circadian rhythms and decreased number or amplitude of secretory bursts, or both). These children with idiopathic short stature may have a functional regulatory disturbance of the hypothalamus and appear to grow normally when given exogenous hGH. [871] There is considerable uncertainty about the criteria for the diagnosis of neuroregulatory hGH deficiency. Many normally growing children have profiles of hGH secretion that are indistinguishable from those in children with the postulated syndrome. [872] Patterns of hGH secretion do not predict which child will benefit from therapy, and there is a poor correlation between hGH secretion and growth. Furthermore, the results of repeated tests in children show considerable variability. It has been suggested that specific genetic defects may underlie the pathogenesis of a subset of children with this heterogeneous syndrome of growth failure. [873] The prevalence of an hGH neuroregulatory deficiency syndrome is thus unclear, and the decision to treat short children with hGH should be made cautiously. [874] [875] Neurogenic Hypersecretion of Growth Hormone

Diencephalic Cachexia

Children and infants with tumors in and around the third ventricle frequently become cachectic, which is often associated with elevated hGH levels and paradoxical GH secretory responses to glucose and insulin. [876] [877] GH hypersecretion may be due to a hypothalamic abnormality [878] or to malnutrition. Deficits of pituitary-adrenal regulation are less common. A TABLE 7-13 -- Clinical Features of Diencephalic Syndrome (Pooled Data of 67 Anatomically Defined Tumors) Clinical Feature

%

Emaciation

100

Alert appearance

87

Increased vigor or hyperkinesis, or both

72

Vomiting

68

Euphoria

59

Pallor

55

Nystagmus

55

Irritability

32

Hydrocephalus*

33

Optic atrophy

24

Tremor

23

Sweating

15

Large hands, feet

5

Large genitalia

5

Polyuria

5

Papilledema

5

Positive pneumoencephalogram results

98

Endocrine anomalies

90

Cerebrospinal fluid protein

64

Cerebrospinal fluid abnormal cells

23

Modified from Burr IM, Slonim AE, Danish RK, et al. Diencephalic syndrome revisited. J Pediatr 1976; 88:439444. *Hydrocephalus includes clinical plus radiologic findings. Positive in 9 of 10 cases with adequately recorded investigation. (Occasionally, patients had electrolyte and blood pressure anomalies and eosinophilia.)

TABLE 7-14 -- Tumors Producing Diencephalic Syndrome Tumor Gliomas

No. of Patients 56

Astrocytoma

37

Not subclassified

10

Spongioblastoma

5

Astroblastoma

1

Oligodendroglioma

1

Mixed astrocytoma-spongioblastoma

1

Mixed astrocytoma-oligodendroglioma

1

Ependymoma

2

Ganglioglioma

1

Dysgerminoma

1

No histology

10

From Burr IM, Slonim AE, Danish RK, et al. Diencephalic syndrome revisited. J Pediatr 1976; 88:439444. striking feature is an alert appearance and seeming euphoria despite the wasted state. A variety of associated neurologic abnormalities may be present (Table 7-13) ; the tumors that produce this syndrome are summarized in Table 7-14 and include a high proportion of chiasmatic-hypothalamic gliomas. [879] Syndrome of Inappropriate Growth Hormone Hypersecretion

Apparently inappropriate hGH hypersecretion (the syndrome of inappropriate somatotropin secretion) occurs with uncontrolled diabetes mellitus, hepatic failure, uremia, anorexia nervosa, and protein-calorie malnutrition. Nutritional factors are probably important in this response because in normal persons obesity inhibits and fasting stimulates episodic GH hypersecretion. [880] In diabetes mellitus cholinergic blockers reverse the abnormality, [881] possibly by inhibiting hypothalamic somatostatin secretion (see earlier in the section on neurotransmitter regulation of GH). Loss of inhibition of GH secretion by IGF-I may also play a role because most disorders in which this syndrome occurs are associated with low IGF-I levels.



Neurogenic Disorders of Corticotropin Regulation

Hypothalamic CRH hypersecretion is the likely cause of sustained pituitary-adrenal hyperfunction in at least two situations: Cushing's syndrome caused by the rare CRH-secreting gangliocytomas of the hypothalamus [882] and severe depression. Severe depression is associated with pituitary-adrenal abnormalities, including inappropriately elevated corticotropin levels, abnormal cortisol circadian rhythms, and resistance to dexamethasone suppression. [883] [884] The dexamethasone suppression test has, in fact, been used as an aid to the diagnosis of depressive illness. Patients with depression also have diminished responses to CRH, suggesting that depressed individuals hypersecrete CRH (see earlier section on CRH). Another possible example of disordered neurogenic control of CRH associated with stress is the metabolic syndrome. [885] [886] [887] This syndrome is characterized by mild hypercortisolism, blunted dexamethasone suppression of the HPA axis, visceral obesity, and hypertension and may be strongly associated with greater risks for cardiovascular disease and stroke. A unique syndrome of corticotropin hypersecretion termed periodic hypothalamic discharge (Wolff's syndrome) has been described in one young man. The patient had a recurring cyclic disorder characterized by high fever, paroxysms of glucocorticoid

157

hypersecretion, and electroencephalographic abnormalities. [888]



Genetic Obesity Disorders Involving Hypothalamic Circuits

In the past 5 years there have been a number of important discoveries related to genetic mutations underlying certain human obesity disorders. [668] [889] These clinical advances have closely paralleled the advances of basic research in the neuro-endocrine control of energy homeostasis discussed in an earlier section. Linkage studies and quantitative trait loci analyses have strongly implicated the POMC gene locus as an important determinant of weight homeostasis in humans of many, but not all, different ethnic populations, although specific alleles associated with obesity have not yet been demonstrated. [890] [891] [892] [893] Because no mutations within the coding region of the POMC gene that alter peptide activity have been identified in these populations, a current hypothesis is that mutations in regulatory regions of the gene decrease the level of POMC expression in the brain. However, a small number of children from consanguineous parents have been found to have null mutations in the POMC gene resulting in absence of detectable circulating ACTH. [666] [894] These children presented with a syndrome of red hair, adrenal insufficiency, and severe, early-onset obesity (see Fig. 7-54) . In addition, both dominant and recessive mutations in the MC4R gene have been found in the human population, and MC4R mutations have been proposed to play a role in as many as 5% of pediatric obesity cases. [290] [667] [895] [896] [897] The genetic mirror image may also be true; an association between a polymorphism linked to the gene encoding the MC4R antagonist agouti-related protein and anorexia nervosa has been reported. Taking all these data into account, it is safe to say that obesity in a subpopulation of humans can be considered a genetic disorder of the hypothalamus. [668] [898]



Nonendocrine Manifestations of Hypothalamic Disease

The hypothalamus is involved in the regulation of diverse functions and behaviors (Table 7-15) . Psychological abnormalities in hypothalamic disease include antisocial behavior; attacks of rage, laughing, and crying; disturbed sleep patterns; excessive sexuality; and hallucinations. Both somnolence (with posterior lesions) and pathologic wakefulness (with anterior lesions) occur, as do bulimia and profound anorexia. The abnormal eating patterns are analogous to the syndromes of hyperphagia produced in rats by destruction of the ventromedial nucleus or of connections to the paraventricular nucleus. Lateral hypothalamic damage causes profound anorexia. Patients with hypothalamic damage may experience hyperthermia, hypothermia, unexplained fluctuations in body temperature, and poikilothermy. Disturbances of sweating, acrocyanosis, loss of sphincter control, and diencephalic epilepsy are occasional manifestations. Hypothalamic damage also causes loss of recent memory, believed to be due to damage of the mammillothalamic pathways. Severe memory loss, obesity, and personality changes (apathy, loss of ability to concentrate, aggressive antisocial behavior, severe food craving, inability to work or attend school) may occur with suprasellar extension of pituitary tumors, hypothalamic radiation, or damage incurred from surgical removal of parasellar tumors. Hypothalamic tumors grow slowly and may reach a large size while producing minimal disturbance of behavior or visceral homeostasis, whereas surgery of limited extent can produce striking functional TABLE 7-15 -- Neurologic Manifestations of Nonendocrine Hypothalamic Disease Disorders of Temperature Regulation Hyperthermia Hypothermia Poikilothermia Disorders of Food Intake Hyperphagia (bulimia) Anorexia, aphagia Disorders of Water Intake Compulsive water drinking Adipsia Essential hypernatremia Disorders of Sleep and Consciousness Narcolepsy Somnolence Sleep rhythm reversal Akinetic mutism Coma Delirium Periodic Disease of Hypothalamic Origin Diencephalic epilepsy Kleine-Levin syndrome Periodic discharge syndrome of Wolff Hereditary Hypothalamic Disease Laurence-Moon-Biedl syndrome Prader-Willi syndrome Disorders of Psychic Function Rage behavior Hallucinations Hypersexuality Disorders of Autonomic Nervous System Pulmonary edema Cardiac arrhythmias Sphincter disturbance Miscellaneous Diencephalic syndrome of infancy Cerebral gigantism abnormalities. Presumably, this is because slowly growing lesions permit compensatory responses to develop. These potential consequences should be weighed carefully with the neurosurgeon, patient, and patient's family in planning the therapeutic approach. Adverse effects of treatment have led to more conservative surgical guidelines for the treatment of craniopharyngioma. A convergence of functional genomics from two animal species, the dog and mouse, has refocused attention on neuropeptide circuits of the hypothalamus in the control of sleep. Positional cloning was used to identify mutations in the hypocretin-orexin receptor 2 as the cause of canine narcolepsy. [899] Knockout of the gene encoding the hypocretin-orexin peptide precursor produced an equivalent narcoleptic syndrome in mice, [900] further establishing this neuropeptide system as a major component of sleep-modulating neural circuits. Histaminergic neurons of the tuberomammillary nucleus express both forms of the orexin receptor and make reciprocal synaptic connections with orexin neurons in the lateral hypothalamus. Furthermore, orexin is an excitatory transmitter for the histamine neurons, suggesting that the two populations cooperate in the regulation of rapid eye movement sleep. [901] Targeted ablation of orexin neurons in the lateral hypothalamus of rats by means of a hypocretin receptor 2saporin conjugate produced narcoleptic-like sleep behavior, [902] closely paralleling the clinical findings and selected loss of hypocretin-orexin neurons in the lateral hypothalamus of humans with narcolepsy. [903] These new discoveries add to the list of other neuropeptides including GHRH, somatostatin, and cortistatin with established function in modulation of the sleep cycle.



ACKNOWLEDGEMENT

The authors are highly indebted to Dr. Seymour Reichlin, not only for text and figures he shared from the ninth edition of this text, but also for the inspiration and mentorship he provided to the current generation of neuroendocrinologists.

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Chapter 8 - Anterior Pituitary Shlomo Melmed David Kleinberg

DEVELOPMENT, ANATOMY, AND OVERVIEW OF CONTROL OF HORMONE SECRETION The pituitary gland situated within the sella turcica derives its name from the Greek ptuo and Latin pituita, phlegm, reflecting its nasopharyngeal origin. Galen hypothesized that nasal phlegm originated from the brain and drained through the pituitary gland. It is now clear that together with the hypothalamus the pituitary orchestrates the structural integrity and function of endocrine glands, including the thyroid gland, adrenal gland, and gonads, in addition to target tissues including cartilage and breast. The pituitary stalk serves as an anatomic and functional connection to the hypothalamus. Preservation of the hypothalamic-pituitary unit is critical for integration of anterior pituitary control of sexual function and fertility, linear and organ growth, lactation, stress responses, energy, appetite, and temperature regulation and secondarily for carbohydrate and mineral metabolism. Integration of vital body functions by the brain was first proposed by Descartes in the 17th century. In 1733, Morgagni recorded the absence of adrenal glands in an anencephalic neonate, providing early evidence for a developmental and functional connection between the brain and the adrenal glands. In 1849 Claude Bernard set the stage for the subsequent advances in neuroendocrinology by demonstrating that central lesions in the area of the fourth ventricle resulted in polyuria. [1] Subsequent studies led to the identification and chemical isolation of pituitary hormones, and astute clinical observations led to the realization that pituitary tumors were associated with functional hypersecretory syndromes, including acromegaly and Cushing's disease. [2] [3] [4] In 1948 Geoffrey Harris, the founder of modern neuroendocrinology, in reviewing anterior pituitary gland hormone control, proposed their hypothalamic regulation, predicting the subsequent discovery of specific hypothalamic regulating hormones. [5] Anatomy

The pituitary gland comprises the predominant anterior lobe, the posterior lobe, and a vestigial intermediate lobe (Fig. 8-1) (Figure Not Available) . The gland is situated within the bony sella turcica and is overlain by the dural diaphragma sella, through which the stalk connects to the median eminence of the hypothalamus. The adult pituitary weighs approximately 600 mg (range, 400 to 900 mg) and measures about 13 mm in the longest transverse diameter, 6 to 9 mm in vertical height, and about 9 mm anteroposteriorly. Structural variation may occur in multiparous women, and gland volume also changes during the menstrual cycle. During pregnancy these measurements may be increased in either dimension, with pituitary weight increasing up to 1 g. Normal pituitary hypertrophy without evidence for the presence of an adenoma was described in seven eugonadal women with pituitary height greater than 9 mm and a convex upper gland boundary observed on magnetic resonance imaging (MRI). [6] The sella turcica located at the base of the skull forms the thin bony roof of the sphenoid sinus. The lateral walls comprising either bone or dural tissue abut the cavernous sinuses, which are traversed by the third, fourth, and six cranial nerves and internal carotid arteries (Fig. 8-2) (Figure Not Available) . Thus, the cavernous sinus contents are vulnerable to increased intrasellar expansion. The dural roofing protects the gland from compression by fluctuant cerebrospinal fluid (CSF) pressure. The optic chiasm, located anterior to the pituitary stalk, is directly above the diaphragma sella. The optic tracts and central structures are therefore vulnerable to pressure effects by an expanding pituitary mass, which is likely to follow the path of least tissue resistance by lifting the diaphragma sella (Fig. 8-3) (Figure Not Available) . The intimate relationship of the pituitary and chiasm is borne out in optic chiasmal hypoplasia associated with developmental pituitary dysfunction seen in patients with septo-optic dysplasia. The posterior pituitary gland, in contrast to the anterior pituitary, is directly innervated by supraopticohypophyseal and tuberohypophyseal nerve tracts of the posterior stalk. Hypothalamic neuronal lesions, stalk disruption, or direct systemically derived metastases are therefore often associated with attenuated vasopressin (diabetes insipidus) or oxytocin secretion, or both. The hypothalamus contains nerve cell bodies that synthesize hypophysiotropic releasing and inhibiting hormones as well as the neurohypophyseal hormones of the posterior pituitary (arginine vasopressin and oxytocin). Five distinct hormone-secreting

178

Figure 8-1 (Figure Not Available) Schematic representation of the blood supply of the hypothalamus and pituitary. (From Scheithauer BW. The hypothalamus and neurohypophysis. In Kovacs K, Asa SL [eds]. Functional Endocrine Pathology. Boston, Blackwell Scientific, 1991, pp 170244.)

cell types are present in the mature anterior pituitary gland. Corticotroph cells express pro-opiomelanocortin (POMC) peptides including adrenocorticotropic hormone (ACTH); somatotroph cells express growth hormone (GH); thyrotroph cells express the common glycoprotein subunit and the specific thyrotropin (thyroid-stimulating hormone, TSH) subunit; gonadotrophs express the and subunits for both follicle-stimulating hormone (FSH) and luteinizing hormone (LH); Figure 8-2 (Figure Not Available) Coronal section of the sellar structures and cavernous sinus showing the relationship of the oculomotor (III), trochlear (IV), trigeminal ophthalmic and maxillary divisions (V 1 and V2 ), and abducent (VI) cranial nerves to the pituitary gland. (From Stiver SI, Sharpe JA. Neuro-ophthalmologic evaluation of pituitary tumors. In Thapar K, Kovacs K, Schithauer BW, Lloyd RV [eds]. Diagnosis and Management of Pituitary Tumors. Totowa, NJ, Humana Press, 2001, pp 173200.)

the lactotroph expresses prolactin (PRL). Each cell type is under highly specific signal controls that regulate their respective differentiated gene expression.



Pituitary Development

The pituitary gland arises from within the rostral neural plate. Rathke's pouch, a primitive ectodermal invagination anterior

179

Figure 8-3 (Figure Not Available) Relationship of the pituitary gland to the optic chiasm. A, The intracranial optic nerve/chiasmal complex lies up to 10 mm above the diaphragma sellae. C, Anterior clinoid process; D, dorsum of the sella turcica. B, Coronal section from magnetic resonance scan of patient with large pituitary adenoma tumor; suprasellar extension has elevated and distorted the chiasm. (From Miller NR, Newman NJ [eds]. Walsh and Hoyt's Clinical Neuro-Ophthalmology, vol 1, 4th ed. Baltimore, Williams & Wilkins, 1985, pp 6069.)

to the roof of the oral cavity, is formed by the fourth to fifth week of gestation and gives rise to the anterior pituitary gland (Fig. 8-4) (Figure Not Available) . [7] [8] The pouch is directly connected to the stalk and hypothalamic infundibulum and ultimately becomes distinct from the oral cavity and nasopharynx. Rathke's pouch proliferates toward the third ventricle, where it fuses with the diverticulum and subsequently obliterates its lumen, which may persist as Rathke's cleft. The anterior lobe is formed from Rathke's pouch, and the diverticulum gives rise to the adjacent posterior lobe. Remnants of pituitary tissue may persist in the nasopharyngeal midline and rarely give rise to functional ectopic hormone-secreting tumors in the nasopharynx. The neurohypophysis arises from neural ectoderm associated with third-ventricle development. [9] Functional development of the anterior pituitary cell types involves complex spatiotemporal regulation of cell lineagespecific transcription factors expressed in pluripotential pituitary stem cells as well as dynamic gradients of locally acting soluble factors. [10] [11] [12] Critical neuroectodermal signals for organizing the dorsal gradient of pituitary morphogenesis include infundibular bone morphogenetic protein 4 (BMP4) required for the initial pouch invagination, [8] fibroblast growth factor 8 (FGF-8), Wnt 5, and Wnt 4. Subsequent ventral developmental patterning and transcription factor expression are determined by spatial and graded expression of BMP2 and sonic hedgehog protein (shh), which appears critical for directing early patterns of cell proliferation. [13] The human fetal Rathke pouch is evident at 3 weeks, and the pituitary grows rapidly in utero. By 7 weeks, the anterior pituitary vasculature begins to develop, and by 20 weeks the entire hypophyseal-portal system is already established. The anterior pituitary undergoes major cellular differentiation during the first 12 weeks, by which time all the major secretory cell compartments are structurally and functionally intact, except for lactotrophs. Totipotential pituitary stem cells give rise to acidophilic (mammosomatotroph, somatotroph, and lactotroph) and basophilic (corticotroph, thyrotroph, and gonadotroph) differentiated pituitary cell types, which appear at clearly demarcated developmental stages. Corticotroph cells are morphologically identifiable at 6 weeks, and immunoreactive ACTH is detectable by 7 weeks. At 8 weeks, somatotroph cells are evident with abundant immunoreactive cytoplasmic GH expression. Glycoprotein hormonesecreting cells express a common subunit, and at 12 weeks differentiated thyrotrophs and gonadotrophs express immunoreactive subunits for TSH, LH, and FSH. Interestingly, gonadotrophs expressing LH and FSH are equally distributed in females, whereas in the male fetus, LH-expressing gonadotrophs predominate. [14] Fully differentiated PRL-expressing lactotrophs are evident only late in gestation (after 24 weeks). Prior to that time, immunoreactive PRL is detectable only in mixed mammosomatotrophs, also expressing GH, reflecting the common genetic origin of these two hormones. [15]



Pituitary Transcription Factors

Determination of anterior pituitary cell type lineages results from a temporally regulated cascade of homeodomain transcription factors. Although most pituitary developmental information has been acquired from murine models, [16] histologic and pathogenetic observations in human subjects have largely corroborated these developmental mechanisms (see Fig. 8-4) (Figure Not Available) . Early cell differentiation requires intracellular Rpx and Ptx expression. Rathke's pouch expresses several transcription factors of the LIM homeodomain family, including Lhx3, Lhx4, and IsI-1, [17] which are early determinants of functional pituitary development. Pitx1 is expressed in the oral ectoderm and subsequently in all pituitary cell types, particularly those arising ventrally. [18] Rieger's syndrome, characterized by defective eye, tooth, umbilical cord, and pituitary development, is caused by defective related Pitx2. [19] [20] Ptx behaves as a universal pituitary regulator and activates transcription of -GSU (the -subunit of gonadotroph hormones), POMC and LH (Ptx1), and GH (Ptx2). Lhx3 determines GH-PRL and TSH cell differentiation, and Prop-1 behaves as a prerequisite for Pit-1, which activates GH, PRL, TSH, and growth hormone-releasing hormone (GHRH) receptor transcription. TSH and gonadotropin-expressing cells share a common subunit (GSU) expression under developmental control of GATA-2. [11] These specific anterior pituitary transcription factors participate in a highly orchestrated cascade leading to the commitment of the five differentiated cell types (see Fig. 8-4) (Figure Not Available) . The major proximal determinant of pituitary cell lineage derived from a totipotential stem cell is thus Prop-1 expression, which determines subsequent development of Pit-1dependent and gonadotroph cell lineages. [21] POU1F1, the renamed Pit-1, is a POU-homeodomain transcription factor that determines development and appropriate temporal and spatial expression of cells committed to GH, PRL, TSH, and GHRH receptor expression. POU1F1 binds to specific deoxyribonucleic acid (DNA) motifs and activates and regulates somatotroph, lactotroph, and thyrotroph development and mature secretory function. Signal-dependent coactivating factors also cooperate with Pit-1 to determine specific hormone expression. Thus, in POU1F1-containing cells, high estrogen receptor levels induce a commitment to express PRL, whereas thyrotroph embryonic factor (TEF) favors TSH expression. Selective pituitary cell type specificity is also perpetuated by binding of POU1F1 to its own DNA regulatory elements as well as those contained within the GH, PRL, and TSH genes. Steroidogenic factor-1 (SF-1) and dosage-sensitive sex reversal, adrenal hypoplasia congenita, X-chromosome factor (DAX-1) determine subsequent gonadotroph development. [22] [23] Corticotroph cell commitment, although occurring earliest during fetal development, is independent of POU1F1-determined

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Figure 8-4 (Figure Not Available) Model for development of the human anterior pituitary gland and cell lineage determination by a cascade of transcription factors. Trophic cells are depicted with transcription factors known to determine cell-specific human or murine gene expression. (Adapted from Shimon I, Melmed S. In Conn P, Melmed S [eds]. Scientific Basis of Endocrinology. Totowa, NJ, Humana Press, 1996, pp 3047; Amselem S. Perspectives on the molecular basis of developmental defects in the human pituitary region. In Rappaport R, Amselem S [eds]. Hypothalamic-Pituitary Development: Genetic and Clinical Aspects. Basel, Karger, 2001; and Dasen JS, Rosenfeld MG. Curr Opin Cell Biol 1999; 11:669677. [849] )

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Figure 8-5 Model for regulation of anterior pituitary hormone secretion by three tiers of control. Hypothalamic hormones traverse the portal system and impinge directly upon their respective target cells. Intrapituitary cytokines and growth factors regulate tropic cell function by paracrine (and autocrine) control. Peripheral hormones exert negative feedback inhibition of respective pituitary trophic hormone synthesis and secretion. (From Ray D, Melmed S. Pituitary cytokine and growth factor expression and action. Endocr Rev 1997; 18:206228.)

lineages, and Tpit protein appears to be a prerequisite for POMC expression. [24] Hereditary mutations arising within these transcription factors may result in isolated or combined pituitary hormone failure syndromes (see later).



Pituitary Blood Supply

The pituitary gland enjoys an abundant blood supply derived from several sources (see Fig. 8-1) (Figure Not Available) . The superior hypophyseal arteries branch from the internal carotid arteries to supply the hypothalamus, where they form a capillary network in the median eminence, external to the blood-brain barrier. Long and short hypophyseal portal vessels originate from infundibular plexuses and the stalk, respectively. These vessels form the hypothalamic-portal circulation, the predominant blood supply to the anterior pituitary gland. They deliver hypothalamic releasing and inhibiting hormones to the trophic hormone-producing cells of the adenohypophysis without significant systemic dilution, allowing the pituitary cells to be sensitively regulated by timed hypothalamic hormone secretion. Vascular transport of hypothalamic hormones is also locally regulated by a contractile internal capillary plexus (gomitoli) derived from stalk branches of the superior hypophyseal arteries. [25] Retrograde blood flow toward the median eminence also occurs, facilitating bidirectional functional hypothalamic-pituitary interactions. [26] Systemic arterial blood supply is maintained by inferior hypophyseal arterial branches, which predominantly supply the posterior pituitary. Disruption of stalk

Figure 8-6 Control of hypothalamic-pituitarytarget organ axes. (Reproduced from Melmed S. Disorders of anterior pituitary and hypothalamus. In Braunwald E, et al (eds). Harrison's Textbook of Medicine, 15th ed. New York, McGraw-Hill, 2001, p 2030.)

integrity may lead to compromised pituitary portal blood flow, depriving the anterior pituitary cells of hypothalamic hormone access.



Pituitary Control

Three levels of control subserve the regulation of anterior pituitary hormone secretion (Fig. 8-5) . Hypothalamic control is mediated by adenohypophysiotropic hormones secreted into the portal system and impinging directly upon anterior pituitary cell surface receptors. G proteinlinked cell surface membrane binding sites are highly selective and specific for each of the hypothalamic hormones and elicit positive or negative signals mediating pituitary hormone gene transcription and secretion. Peripheral hormones also participate in mediating pituitary cell function, predominantly by negative feedback regulation of trophic hormones by their respective target hormones. Intrapituitary paracrine and autocrine soluble growth factors and cytokines act locally to regulate neighboring cell development and function. The net result of these three tiers of complex intracellular signals is the controlled pulsatile secretion of the six pituitary trophic hormones, ACTH, GH, PRL, TSH, FSH, and LH, through the cavernous sinus, petrosal veins, and ultimately the systemic circulation through the superior vena cava (Fig. 8-6) . The temporal and quantitative control of pituitary hormone secretion is critical for physiologic integration of peripheral hormonal systems such as the menstrual cycle, which relies on complex and precisely regulated pulse control.



PITUITARY MASSES Pituitary Mass Effects

An expanding pituitary mass may inexorably alter the sellar size and shape by bone erosion and remodeling (Fig. 8-7) . Although the exact time course of this process is unknown, it appears to be slowly progressive over years or decades. The tumor may invade soft tissue, and the dorsal sellar roof presents the least resistance to expansion from within the confines of the bony sella. Nevertheless, both suprasellar and parasellar compression and invasion may occur with an enlarging mass, with resultant clinical manifestations (Table 8-1) . As tumors impinge upon the optic chiasm, they interfere with vision. Because of the anatomy of the chiasm, pressure from below affects temporal visual fields, starting superiorly and ultimately extending to the entire temporal field. Loss of nasal fields also occurs and may result in blindness. Long-standing optic chiasmal pressure results in optic disc pallor. Lateral invasion of pituitary lesions may invade the dural

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Figure 8-7 Magnetic resonance coronal section of a normal pituitary gland (top). A large pituitary adenoma is seen lifting and distorting the optic chiasm (arrow) and is also invading the sphenoid sinus (middle). A sagittal section of a large macroadenoma with bone invasion and impinging brain structures is shown (bottom).

wall of the cavernous sinus affecting the third, fourth, and sixth cranial nerves as well as the ophthalmic and maxillary branches of the fifth cranial nerve and surround the internal carotid artery. Varying degrees of diplopia, ptosis, ophthalmoplegia, and decreased facial sensation may infrequently occur, depending on the extent of the neural involvement by the cavernous sinus mass. Downward extension into the sphenoid sinus indicates that the parasellar mass has eroded the bony sellar floor. Aggressive tumors may invade the roof of the palate and cause nasopharyngeal obstruction, infection, and CSF leakage. Infrequently, temporal or frontal lobes may be invaded, causing uncinate seizures, personality disorders, and anosmia. In addition to the anatomic lesions caused by the expanding mass, direct hypothalamic involvement of the encroaching mass may lead to important metabolic sequelae discussed in Chapter 7 . Patients with intrasellar tumors commonly present with headaches, even in the absence of demonstrable suprasellar extension. Small changes in intrasellar pressure caused by a microadenoma within the confined sella are sufficient to stretch the dural plate with resultant headache. Headache severity does not correlate with the size of the adenoma or the presence of suprasellar extension. [27] Relatively minor diaphragmatic distortions or dural impingement may be associated with persistent headache. Successful medical management of small functional pituitary tumors with dopamine agonists or somatostatin analogues is often accompanied by a remarkable improvement in or disappearance of headache. Regardless of their etiology or size, pituitary masses, including adenomas, may be associated with compression of surrounding healthy tissue and resultant hypopituitarism. In 49 patients undergoing transsphenoidal resection of pituitary adenomas, mean intrasellar pressure was elevated twofold to threefold in patients with pituitary failure. Furthermore, prevalence of headache and elevated PRL levels correlated positively with intrasellar pressure levels, [28] suggesting interrupted portal delivery of hypothalamic hormones. Thus, surgical decompression of a sellar mass may lead to recovery of compromised anterior pituitary function. In the patients who do not recover pituitary function postoperatively, ischemic necrosis is likely to have occurred. Stalk compression may result in pituitary failure caused by encroachment of the portal vessels that normally provide pituitary access to the hypothalamic hormones. Stalk compression also usually leads to hyperprolactinemia and concomitant failure of other pituitary trophic hormones.



Pituitary Adenomas Pathogenesis

Pituitary tumors account for about 15% of all intracranial neoplasms and are commonly encountered at autopsy. The Brain Tumor Registry of Japan reported that 15.8% of 28,424

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TABLE 8-1 -- Local Effects of an Expanding Pituitary or Hypothalamic Mass Clinical Effect

Affected Structure Pituitary

Growth failure Adult hyposomatotrophism Hypogonadism Hypothyroidism Hypoadrenalism

Optic tract

Loss of red perception, bitemporal hemianopsia, superior or bitemporal field defect, scotoma, blindness

Hypothalamus

Temperature dysregulation, obesity, diabetes insipidus, thirst, sleep, appetite, behavioral and autonomic nervous system dysfunctions

Cavernous sinus

Ptosis, diplopia, ophthalmoplegia, facial numbness

Temporal lobe

Uncinate seizures

Frontal lobe

Personality disorder, anosmia

Central

Headache, hydrocephalus, psychosis, dementia, laughing seizures

Neuro-ophthalmologic tract Field defects Bitemporal hemianopia (50%) Amaurosis with hemianopia (12%) Contralateral or monocular hemianopia (7%) Scotomas Junctional; monocular central, arcuate, altitudinal; hemianopic Homonymous hemianopia Acuity loss Snellen Contrast sensitivity Color vision Visual evoked potential Pupillary abnormality Impaired light reactivity Afferent defect Optic atrophy Papilledema Cranial nerve palsyoculomotor, trachlear, abducens, sensory trigeminal Nystagmus Visual hallucinations Postfixation blindness Adapted from Melmed S. In DeGroot LJ, Jameson JL (eds). Endocrinology. Philadelphia, WB Saunders, 2001; Arnold AC. Neuro-ophthalmologic evaluation of pituitary disorders. In Melmed S (ed) The Pituitary, 2nd ed. Malden, Mass, Blackwell Science, 2002, pp 687708.

TABLE 8-2 -- Factors Involved in Pituitary Tumor Pathogenesis Hereditary MEN-1 Transcription factor defect (e.g., Prop-1 excess) Carney complex Hypothalamic Excess GHRH or CRH production Receptor activation? Dopamine deprivation? Pituitary Signal transduction mutations (e.g., gsp, CREB) Disrupted paracrine growth factor or cytokine action (e.g., FGF2, FGF4, LIF, EGF, NGF) Activated oncogene or cell cycle disruption (e.g., PTTG; ras; p27) Intrapituitary paracrine hypothalamic hormone action (e.g., GHRH, TRH)

Loss of tumor suppressor gene function (11q13; 13) Environmental Estrogens Irradiation Peripheral Target failure (ovary, thyroid, adrenal) CREB, cyclic adenosine monophosphate response elementbinding protein; CRH, corticotropin-releasing hormone; EGF, epidermal growth factor; FGF, fibroblast growth factor; GHRH, growth hormonereleasing hormone; LIF, leukemia inhibitory factor; NGF, nerve growth factor; PTTG, primary tumor transforming gene; TRH, thyrotropin-releasing hormone. Compiled from Heaney AP, Melmed S, PTTG: a novel factor in pituitary tumor formation. Baillieres Clin Endocrinol Metab 1999, 12:367. cases were histologically confirmed pituitary adenomas. [29] [30] They are benign monoclonal adenomas that may express and secrete hormones autonomously, leading to hyperprolactinemia, acromegaly, and Cushing's disease, or may be functionally silent and initially diagnosed as a sellar mass. Although these adenomas are invariably benign, their neoplastic features represent a unique tumor biology that is reflected in their important local and systemic manifestations. These monoclonal neoplasms have a slow doubling time and, if small, may rarely resolve spontaneously. Nevertheless, they can be aggressive and locally invasive or compressive to vital central structures. They usually express a single gene product, but polyhormonal expression may reflect a primitive stem cell or mature bimorphous cellular origin. Hypothalamic factors may have a specific role in the pathogenesis of pituitary tumors, in addition to regulating pituitary hormone gene expression and secretion (Table 8-2) . Ectopic GHRH-secreting tumors (bronchial carcinoids, pancreatic islet cell tumors, or small cell lung carcinomas) result in GH hypersecretion, acromegaly, somatotroph hyperplasia, and occasionally somatotroph adenoma formation. [31] [32] In transgenic mice overexpressing a GHRH transgene, the pituitary size increased dramatically because of somatotroph hyperplasia, and older mice developed GH-secreting adenomas. [33] However, adenomatous hormonal secretion is usually independent of physiologic hypothalamic control, and the surgical resection of small well-defined adenomas usually results in definitive cure of hormonal hypersecretion. These observations imply that these tumors do not arise because of excessive polyclonal pituitary cell proliferation related to generalized hypothalamic stimulation. However,

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Gene Gsp

Protein

Tumor Type

GNAS 40% GH-secreting tumors

TABLE 8-3 -- Candidate Genes in Pituitary Tumorigenesis Mechanism of Overexpression or Inactivation

Function, Defect

Point mutation

Signal transduction, elevated cAMP

McCune-Albright syndrome Minority other types PTTG1

PTTG All pituitary tumors

Unknown Estrogen?

Chromatid separation, regulates bFGF secretion, disrupted cell cycle, chromsomal instability, bFGF-mediated mitogenesis and angiogenesis

Hst

FGF4

Unknown

Angiogenesis, overexpression

Large prolactinomas

Enhanced PRL transcription CREB H-ras

Ras

GH-secreting

Increased Ser-phosphorylated CREB promoted by Dimerizes with cAMP response elements gsp overexpression

Metastatic pituitary carcinoma only

Point mutation, amplification

Signal transduction, stimulates tyrosine kinase pathway

Prolactinomas in familial MEN-1

11q13 loss of heterozygosity

Nuclear tumor suppressor, loss-of-function mutations

Inactivating Men 1

Menin

13q14

RB?

Highly invasive tumors

13q14 loss of heterozygosity

Inconsistent Rb protein loss, disrupted cell cycle regulation

CDKN2A

p16

All tumor types examined

Gene methylation leading to absent p16, allowing Rb phosphorylation and cell cycle progression

Cell cycle regulation, absent p16 protein leading to disrupted cell cycle regulation

CIP1/KIP1

p27

Transgenic mouse models

Gene methylation leading to absent p27

Regulate multiple CDK enzymes including CDK4/6-cyclin Ds, absent p27 protein

Adapted from Heaney AP, Melmed S. Molecular pathogenesis of pituitary tumors. In Wass J (ed). Oxford Textbook of Endocrinology: Endocrine-Related Career. New York, Oxford University Press, 2002. hypothalamic factors may promote and maintain growth of already transformed pituitary adenomatous cells. Normal and hyperplastic pituitary tissues are polyclonal, and pituitary adenomas arise as the result of monoclonal pituitary cell proliferation. Using X-chromosomal inactivation analysis, the monoclonal origin of adenomas secreting GH, PRL, [34] and ACTH[35] [36] and nonfunctioning pituitary tumors was confirmed in female patients heterozygous for variant alleles of the X-linked genes hypoxanthine phosphoribosyltransferase ( HPRT) and phosphoglycerate kinase ( PGK). Thus, an intrinsic somatic pituitary cell genetic alteration probably gives rise to clonal expansion of a single cell, resulting in adenoma formation (Table 8-3) . Activating gsp mutations are present in up to 40% of human GH-secreting adenomas. [37] [38] [39] These somatic heterozygous activating point mutations of the G protein subunit (Gs) gene involving either arginine 201 (replaced by cysteine or histidine) or glutamine 227 (replaced with arginine or leucine) constitutively activate the Gs protein and convert it into an oncogene (gsp). This G protein activation increases cyclic adenosine monophosphate (cAMP) levels and activates protein kinase A, which in turn phosphorylates the cAMP response elementbinding protein (CREB) and leads to sustained constitutive GH hypersecretion and cell proliferation. The gsp-bearing adenomas are smaller, have mildly lower GH levels and enhanced intratumoral cAMP, do not respond briskly to GHRH, and are extremely sensitive to the inhibitory effect of somatostatin. [39] These gsp activating mutations do not occur in PRL-secreting or in TSH-producing adenomas and are rarely present in nonfunctioning pituitary tumors or ACTH-secreting tumors (1 mg/ day) over prolonged periods (>10 days), especially near term. Maternal amiodarone therapy causes thyroidal dysfunction in up to 20% of newborns. [135] It is not known whether iodide goiter in newborns results from an inherent hypersensitivity of the fetal thyroid or from the fact that the placenta concentrates iodide several-fold or both. [129] [136] As discussed earlier ( see Chapter 10 , "Regulation of Thyroid Function"), large doses of iodine cause an acute inhibition of organic binding that abates in the normal individual, despite continued iodine administration (acute Wolff-Chaikoff effect and escape). [137] Iodide goiter appears to result from a more pronounced inhibition of organic binding and the failure of the escape phenomenon. As a consequence of decreased hormone synthesis and the consequent increase in TSH, iodide transport is enhanced. Because inhibition of organic binding is a function of the intrathyroidal concentration of iodide, a vicious circle, augmented by this increase in serum TSH, is set in motion. The disorder usually appears as a goiter with or without

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hypothyroidism, although in rare instances iodine may produce hypothyroidism unaccompanied by goiter. Usually the thyroid gland is firm and diffusely enlarged, often greatly so. Histopathologic examination reveals intense hyperplasia. The FT 4 I concentration is low, TSH concentration is increased, and the 24-hour urinary iodine excretion and the serum inorganic iodide concentration are increased. The disorder regresses after iodine is withdrawn. Thyroid hormone may also be given to relieve severe symptoms. Drugs Blocking Thyroid Hormone Synthesis or Release

Ingestion of compounds that block thyroid hormone synthesis or release may cause goiter with or without hypothyroidism. Apart from the agents used in the treatment of hyperthyroidism, antithyroid agents may be encountered either as drugs for the treatment of disorders unrelated to the thyroid gland or as natural agents in foodstuffs. [132] Goiter with or without hypothyroidism can occur in patients given lithium, usually for bipolar manic-depressive psychosis. [138] Like iodide, lithium inhibits thyroid hormone release, and in high concentrations can inhibit organic binding reactions. At least acutely, iodide and lithium act synergistically in the latter respect. The mechanisms underlying the several effects of lithium are uncertain; what differentiates patients who develop goiter during lithium therapy from those who do not is also unclear. Underlying autoimmune thyroiditis may be at least one factor because many patients with this combination have autoimmune thyroid disease. Other drugs that occasionally produce goitrous hypothyroidism include para-aminosalicylic acid, phenylbutazone, aminoglutethimide, and ethionamide. [139] [140] Like the thionamides, these drugs interfere with both the organic binding of iodine and the later steps in hormone biosynthesis. Although soybean flour is not an antithyroid agent, soybean products in feeding formulas formerly resulted in goiter in infants by enhancing fecal loss of hormone, which, together with the low iodine content of soybean products, produced a state of iodine deficiency. Feeding formulas containing soybean products are now enriched with iodine. Cigarette smoking increases the risk of hypothyroidism in patients with underlying autoimmune thyroid disease. Although the mechanism is unclear, certain components of cigarette smoke, including thiocyanate, hydroxyperidine, and benzopyrene derivatives, may be responsible. [141] [142] Both the goiter and the hypothyroidism usually subside after the antithyroid agent is withdrawn. If continued administration of pharmacologic goitrogens is required, however, replacement therapy with thyroid hormone causes the goiter to regress. Goitrogens in Foodstuffs or as Endemic Substances or Pollutants

Antithyroid agents also occur naturally in foods. These are widely distributed in the family Cruciferae or Brassicaceae, particularly in the genus Brassica, including cabbages, turnips, kale, kohlrabi, rutabaga, mustard, and various plants that are not eaten by humans but that serve as animal fodder. It is likely that some thiocyanate is present in such plants (particularly cabbage). [143] Cassava meal, a dietary staple in many regions of the world, contains linamarin, a cyanogenic glycoside, the metabolism of which leads to the formation of thiocyanate. Ingestion of cassava can accentuate goiter formation in areas of endemic iodine deficiency. Except for thiocyanate, dietary goitrogens influence thyroid iodine metabolism in the same manner as do the thionamides, which they resemble chemically; their role in the induction of disease in humans is uncertain. Waterborne, sulfur-containing goitrogens of mineral origin are believed to contribute to the development of endemic goiter in certain areas of Colombia. [121] A number of synthetic chemical pollutants have been implicated in causing goitrous hypothyroidism, including polychlorinated biphenyls and resorcinol derivatives. Perchlorate has also been noted in high concentrations in geographic regions in which explosives were made. It is not clear whether the concentrations are significant enough to produce hypothyroidism. [144] [145] [146] Cytokines

Patients with chronic hepatitis C or various malignancies may be given interferon or interleukin-2. Such patients may experience hypothyroidism, which is usually transient but may persist. These agents activate the immune system and can induce a clinical picture suggesting an exacerbation of underlying autoimmune disease such as occurs during postpartum thyroiditis ( see Chapter 11 and Chapter 37 ). [100] [101] [147] Graves' disease with hyperthyroidism may also develop, and ablative therapy may be required to treat this condition. Patients with preexisting evidence of autoimmune thyroid disease who have positive TPO antibodies are probably at higher risk for this complication and should be monitored carefully during and after a course of treatment with either of these cytokines. Autoimmune hypothyroidism may also develop after successful treatment of Cushing's disease, presumably as a result of the release of the glucocorticoid-induced immunosuppression. [148] Congenital Causes

Inherited defects in hormone biosynthesis are rare causes of goitrous hypothyroidism and account for only about 10% to 15% of the 1 in 3500 newborns with congenital hypothyroidism. [4] In most instances, the defect appears to be transmitted as an autosomal recessive trait. Individuals with goitrous hypothyroidism are believed to be homozygous for the abnormal gene, whereas euthyroid relatives with slightly enlarged thyroids are presumably heterozygous. In the latter group, appropriate functional testing may disclose a mild abnormality of the same biosynthetic step that is defective in the homozygous individual. In contrast with nontoxic

goiter, which is more common in females than in males, these defects, as a group, affect females only slightly more commonly than males. Although goiter may be present at birth, it usually does not appear until several years later. Therefore, the absence of goiter in a child with functioning thyroid tissue does not exclude the presence of hypothyroidism. The goiter is initially diffusely hyperplastic, often intensely so, suggesting papillary carcinoma, but eventually becomes nodular. In general, the more severe the biosynthetic defect, the earlier the goiter appears, the larger it is, and the greater the likelihood of early development of hypothyroidism or even cretinism. Five specific defects in the pathways of hormone synthesis have been identified. Iodide Transport Defect

Iodide transport defect is rare, a result of impaired iodide transport by the sodium-iodide symporter (NIS) protein mechanism and is reflected by defective iodide transport in the thyroid, salivary gland, and gastric mucosa. [149] [150] [151] Administration of iodide, by raising the plasma concentration, increases the intrathyroidal concentration of iodide sufficiently to permit the synthesis of normal quantities of hormone, demonstrating that this is the cause of the deficiency. [152]

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Defects in Expression or Function of Thyroid Peroxidase

TPO is a protein that is required for normal synthesis of iodothyronines. Quantitative or qualitative abnormalities of TPO have been identified in 1 in 66,000 infants in the Netherlands. [153] The most common of the 16 mutations identified in 35 families was a GGCC insertion in exon 8, leading to premature termination of TPO synthesis. Pendred's Syndrome

The most common presentation in patients with Pendred's syndrome is a defect in iodine organification accompanied by sensory nerve deafness. The abnormality is in the PDS gene encoding pendrin, which is involved in the apical secretion of iodide into the follicular lumen ( see Fig. 10-2 and Chapter 10 , "Iodine Metabolism"). [154] [155] [156] Thyroid function is only mildly impaired in this disorder. [157] Defects in Thyroglobulin Synthesis

Defects in the synthesis of thyroglobulin due to genetic causes are rare, having been identified only in a small number of families with congenital hypothyroidism. [158] [159] [160] [ 161] Some defects lead to premature termination of translation, whereas another defect causes deficiency in endoplasmic reticulum processing of the thyroglobulin molecule. [160] The complex regulation and huge size of this gene makes screening for mutations a difficult task, and considerable work is still required to unravel the extent of the defects in this gene. Iodotyrosine Dehalogenase Defect

The pathogenesis of goiter and hypothyroidism in the iodotyrosine dehalogenase defect is complex. The major abnormality is an impairment of both intrathyroidal and peripheral deiodination of iodotyrosines, presumably because of a lack (or dysfunction) of the iodotyrosine dehalogenase. The gene encoding this enzyme has yet to be identified. As a consequence of intense thyroid stimulation and lack of intrathyroidal recycling of iodide derived from dehalogenation, iodide is rapidly accumulated by the thyroid gland and is rapidly released; monoiodotyrosine (MIT) and diiodotyrosine (DIT) are elevated in plasma and, together with their deaminated derivatives, in the urine. Hypothyroidism is presumed to result from the loss of large quantities of MIT and DIT in the urine and to secondary iodine deficiency. The goiter and hypothyroidism are relieved by administration of large doses of iodine. Thyroid Infiltration

A number of infiltrative or fibrosing conditions may cause hypothyroidism. Some are often associated with goiter, such as Riedel's struma (see later). [162] [163] [164] Others, such as amyloidosis, [165] [166] hemochromatosis,[167] [168] or scleroderma[169] may not be. Although the other manifestations of these conditions are usually obvious and hypothyroidism is only a complication, the presence of significant hypothyroidism without evidence of autoimmune thyroiditis should lead to a consideration of these rare causes of this condition. Atrophic Hypothyroidism

In some patients, manifestations of hypothyroidism are apparent but there is no obvious thyroid enlargement (atrophic hypothyroidism). This may be due to either acquired or congenital abnormalities, prominent among the former being Autoimmune Thyroiditis Type 2B (Table 12-3) . The pathophysiology and thyroid function tests are similar to those found when goiter is present. Acquired Causes

Nongoitrous Hypothyroidism

Hypothyroidism in the absence of a classic Hashimoto's goiter has often been termed primary hypothyroidism (or myxedema); this condition is more common in women than in men and occurs most often between the ages of 40 and 60 years. Many years ago, the presence of circulating thyroid autoantibodies in almost all patients and the clinical and immunologic overlap with autoimmune diseases indicated that this represented the end stage of an autoimmune thyroiditis in which goiter either did not develop or went unnoticed (Autoimmune Thyroiditis 2B). Although most cases are due to autoimmune-induced apoptosis of the thyroid epithelial cells ( see Fig. 12-7 and Fig. 12-8 ), some cases of nongoitrous hypothyroidism are also associated with TSH receptor antibodies that block the response of thyroid cells to endogenous TSH (see Chapter 11) .[170] [171] [172] In primary thyroid failure, the thyroid gland is not usually palpable but may be normal in size or even somewhat enlarged on sonography and of firm consistency. Circulating TPOAbs or TgAbs are detectable in most patients but may be absent in long-standing disease. Postablative Hypothyroidism

Postablative hypothyroidism is a common cause of thyroid failure in adults. One type follows total thyroidectomy usually performed for thyroid carcinoma. Although functioning remnants may be present, as indicated by foci of radioiodine accumulation, hypothyroidism invariably develops. Another etiologic mechanism is subtotal resection of the diffuse goiter of Graves' disease or multinodular goiter. Its frequency depends on the amount of tissue remaining, but continued autoimmune destruction of the thyroid remnant in patients with Graves' disease may be a factor because some studies suggest a correlation between the presence of circulating thyroid autoantibodies in thyrotoxicosis and the development of hypothyroidism after surgery. Hypothyroidism can be manifested during the first year after surgery, but, as with postradioiodine hypothyroidism, the incidence increases with time to approach 100%. In some patients, mild hypothyroidism appears during the early postoperative period and then may occasionally remit, as also occurs after radioiodine treatment. [173] Hypothyroidism after destruction of thyroid tissue with radioiodine is common and is the one established disadvantage of this form of treatment for hyperthyroidism in adults. Its frequency is determined, in large part, by the dose of radioiodine but is also influenced by variations in individual susceptibility, including autoimmune factors. [76] The incidence of post-radioiodine hypothyroidism increases with time, approaching 100%. Although the FT 4 I is low in patients with postablative hypothyroidism, serum TSH levels may be anomalously low for several months after either surgical or 131 I-induced hypothyroidism if TSH synthesis has been suppressed for a long period prior to treatment. [173] [174] [175] Primary atrophic thyroid failure may also develop in patients with Hodgkin's disease after treatment with mantle irradiation [176] or after high-dose neck irradiation for other forms of lymphoma or carcinoma. [176] [177] [178] Surgical, radioiodine, or external beam therapy may also lead to a state of subclinical hypothyroidism, which usually

represents an interim phase in the evolution of thyroid failure. During this phase, the patient is eumetabolic but has a modest increase in the serum TSH level

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(5 to 15 mU/L), low-to-normal FT 4 I, and a normal serum T3 concentration (see Table 12-1) . Congenital Causes

Thyroid Agenesis or Dysplasia

Developmental defects of the thyroid are often responsible for the hypothyroidism that occurs in 1 in 3500 newborns. [4] These defects may take the form of complete absence of thyroid tissue or failure of the thyroid to descend properly during embryologic development. Thyroid tissue may then be found anywhere along its normal route of descent from the foramen caecum at the junction of the anterior two thirds and posterior third of the tongue (lingual thyroid) to the normal site or below. Absence of thyroid tissue or its ectopic location can be ascertained by scintiscanning. As indicated, a number of proteins are known to be crucial for normal thyroid gland development. These include the thyroid-specific transcription factor PAX8 as well as thyroid transcription factors 1 and 2 (TTF1 and 2). It might be anticipated that defects in one or more of these proteins may explain abnormalities in thyroidal development. These have been identified in several patients with PAX8 mutations,[179] and a mutation in the human TTF2 gene was associated with thyroid agenesis, cleft palate, and choanal atresia. [180] Despite a specific search, no mutations have been found in the TTF1 gene in infants with congenital hypothyroidism. [181] [182] Thyroid Aplasia Due to Thyrotropin Receptor Unresponsiveness

Several families exist in which thyroid hypoplasia, high TSH concentrations, and a low free T 4 level are associated with loss-of-function mutations in the TSH receptor. [183] [184] [185] The thyroid glands were in the normal location but did not trap pertechnetate (TCO 4 - ). Somewhat surprisingly, thyroglobulin levels were still detectable. The molecular details of these patients are still under study. A second type of abnormality that may cause TSH unre-sponsiveness is a mutation in the Gs protein that occurs in pseudohypoparathyroidism type 1A. These patients have inactivating mutations in the -subunit of the Gs protein and, consequently, mild hypothyroidism. [186] Other as yet unexplained patients with elevated TSH levels and hypothyroidism in which the molecular nature of the defect has not been defined have been reported. [187] Transient Hypothyroidism

Transient hypothyroidism is defined as a period of reduced FT 4 I with suppressed, normal, or elevated TSH levels that are eventually followed by a euthyroid state. This unusual form of hypothyroidism usually occurs in the clinical context of a patient with subacute (postviral), lymphocytic (painless), autoimmune, or postpartum thyroiditis. These conditions are reviewed in detail in Chapter 11 . The patient reports mild to moderate symptoms of hypothyroidism of short duration, and serum TSH concentrations are typically elevated, although not greatly so. The patient often has a preceding episode of symptoms consistent with mild or moderate thyrotoxicosis. If these symptoms cannot be elucidated from the history, it may be difficult to distinguish such patients from those with a permanent form of hypothyroidism. In the early phases of post-thyroiditis hypothyroidism, TSH concentrations may still be suppressed even though the FT 4 I is low because of the delayed recovery of pituitary TSH synthesis, such as in patients with Graves' disease or with toxic nodules who have undergone surgery and who have experienced rapid relief of hypothyroidism (see Table 12-1) . In that situation, the TSH response to hypothyroidism may be suppressed for many months; in post-thyroiditis hypothyroidism, this period is rarely longer than a few weeks. A significant fraction (33%) of women with autoimmune thyroiditis but normal thyroid function have episodes of hypothyroidism during the postpartum period. [93] [117] [188] In some, the preceding hyperthyroidism is relatively asymptomatic, which can make an accurate clinical diagnosis difficult. Patients who have had an episode of typical subacute postviral thyroiditis with pain, tenderness, and hyperthyroidism are not difficult to recognize. Diagnostic evaluation should include a determination of TSH, FT 4 I, and TPOAbs. Negative or low antibodies argue strongly for a nonautoimmune cause. This is significant, in that it may be possible for the patient not to be treated only temporarily for hypothyroidism. In such patients, a trial of a lower levothyroxine dosage after 3 to 6 months may reveal that thyroid function has recovered (see Fig. 12-6) . This may also occur in patients with hypothyroidism that follows acute autoimmune thyroiditis (e.g., in the postpartum period), but it is somewhat less likely to occur because of the underlying progressive nature of the autoimmune thyroiditis. In patients with hypothyroidism due to postviral thyroiditis, the thyroid gland is usually relatively small and atrophic. In patients with hypothyroidism that follows an episode of acute lymphocytic thyroiditis, the gland is usually slightly enlarged and somewhat firm, reflecting the underlying scarring and infiltration associated with that condition. Consumptive Hypothyroidism

Consumptive hypothyroidism is the term given to an unusual cause of hypothyroidism that has been identified in infants with visceral hemangiomas or related tumors.[67] [189] The first patient reported with this syndrome presented with abdominal distention caused by a large hepatic hemangioma with respiratory compromise secondary to upward displacement of the diaphragm. However, clinical signs suggested hypothyroidism, which was confirmed by finding a markedly elevated TSH level and undetectable T 4 and T3 levels. The infant's response to an initial IV infusion of liothyronine was transient, leading to the decision to use parenteral thyroid hormone replacement to relieve the clinical hypothyroidism. The accelerated degradation of thyroid hormone was apparent from the fact that it required 96 µg of liothyronine plus 50 µg of levothyroxine to normalize the TSH level. The equivalent dosage as levothyroxine alone is roughly nine times that ordinarily required for treatment of infants with congenital hypothyroidism. The infant succumbed to complications of the hemangioma, and a postmortem tumor biopsy showed type 3 iodothyronine deiodinase (D3) activity in the tumor at levels eightfold higher than those normally present in term placenta. The serum reverse T 3 was extremely elevated (400 ng/dL), and the serum thyroglobulin was higher than 1000 ng/mL. Retrospective search revealed two other patients with similar pathophysiology in whom the cause of the hypothyroidism had not been recognized. Significant D3 expression has subsequently been noted in all proliferating cutaneous hemangiomas studied to date. The cutaneous hemangiomas of infancy, although they express D3, are not associated with hypothyroidism owing to their small size. Because a significant fraction of hemangiomas remit with glucocorticoid and interferon therapy, it is important to treat such patients with adequate doses of thyroid hormone to prevent the permanent neurologic complications associated with untreated hypothyroidism during the critical phase of neurologic development. A recent report described a similar syndrome in a 21-year-old with epithelioid hemangioendothelioma.[189A]

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Central Hypothyroidism

Central hypothyroidism is due to TSH deficiency caused by either acquired or congenital hypothalamic or pituitary gland disorders ( see Chapter 7 and Chapter 8 ). The causes of TSH deficiency may be classified as those of pituitary ( secondary hypothyroidism) and hypothalamic ( tertiary hypothyroidism) origins, but this distinction is not necessary in the initial separation of primary from central hypothyroidism. In many cases, hyposecretion of TSH is accompanied by decreased secretion of other pituitary hormones, with the result that evidence of somatotroph, gonadotroph, and corticotroph failure is also present. Hyposecretion of TSH as the sole demonstrable abnormality (monotropic deficiency) is less common but does occur in both acquired and congenital forms. Hypothyroidism due to pituitary insufficiency varies in severity from instances in which it is mild and overshadowed by features of

gonadal and adrenocortical failure to those in which the features of the hypothyroid state are predominant. Because a small but significant fraction of thyroid gland function is independent of TSH (10% to 15%), hypothyroidism due to central causes is less severe than primary hypothyroidism. The causes of central hypothyroidism are both acquired and congenital. The general subject has been discussed in Chapter 7 and Chapter 8 , and those causes with relatively specific thyroid-related deficiencies are mentioned here for completeness. In addition to pituitary tumors, hypothalamic disorders, and the like, an unusual cause of secondary hypothyroidism occurs in individuals given bexarotene (a retinoid X [RXR] receptor agonist) for T-cell lymphoma. [190] This drug suppresses the activity of the human TSH -subunit promoter in vitro. Serum T 4 concentrations are reduced about 50%, and patients experience clinical benefit from thyroid hormone replacement. Dopamine, dobutamine, high-dose glucocorticoids, or severe illness may suppress TSH release transiently, leading to a pattern of thyroid hormone abnormalities suggesting central hypothyroidism. As discussed earlier ( see Chapter 10 , "Changes in Thyroid Function During Severe Illness"), this severe state of hypothalamic-pituitary-thyroid suppression is a manifestation of stage 3 illness (see Table 10-10) . Although these agents might be expected to have similar effects when given chronically, they do not; nor does somatostatin have a similar effect when given for acromegaly, although it does block the response of TSH to TRH and it has been administered to patients with thyrotropin-secreting pituitary adenomas. [191] [192] Congenital defects in either the stimulation or the synthesis of TSH or in its structure have been identified as rare causes of congenital hypothyroidism. These include the consequences of defects in several of the homeobox genes, including POU1F1 (formerly termed Pit-1), PROP1, and HESX1. The latter factor is necessary for the development of the hypothalamus, pituitary, and olfactory portions of the brain, and its targeted deficiency in the mouse produces a condition resembling septo-optic dysplasia in humans. [193] Defects in POU1F1 and PROP1 cause hereditary hypothyroidism, usually accompanied by deficiencies in growth hormone and prolactin. [194] [195] [196] [ 197] [ 198] One patient has been identified with a familial defect in the TRH receptor gene. [199] All of these conditions are associated with the typical pattern of reduced FT 4 I and TSH. Structural defects in TSH have also been described. These include those with a mutation in the CAGYC peptide sequence of the -subunit, thought to be necessary for its association with the -subunit [200] or defects that produce premature termination of the TSH -subunit gene. [201] [202] As mentioned, some of these abnormalities may be associated with elevations in TSH, suggesting the diagnosis of primary hypothyroidism, but the TSH molecule is immunologically, but not biologically, intact. Resistance to Thyroid Hormone

Patients with resistance to thyroid hormone (RTH) may have features of hypothyroidism if the resistance is severe and affects all tissues. Alternatively, patients with RTH may have hyperthyroidism if the resistance is more severe in the hypothalamic-pituitary axis than in the remainder of the tissues. In clinical terms, patients in the former group are said to have generalized resistance to thyroid hormone, whereas patients in the latter group are said to have pituitary resistance to thyroid hormone.[203] [204] [205] [206] Patients with both forms almost always have mutations in one allele of the TR-beta (TR) gene that interfere with the capacity of that receptor to respond normally to T 3 , usually by reducing its binding affinity (see Fig. 10-9) . The mutations in the TR gene causing RTH cluster in three areas of the thyroid hormone receptor, which have been recognized to have important contacts with the hydrophobic ligand-binding domain cavity of TR as recognized from its crystal structure. [207] [208] [209] The mutations do not interfere with the function of the DNA-binding domain, its co-repressor binding domain, or its region of heterodimerization with RXR. Some mutations affect the activation domain in the carboxy-terminus of the TR receptor. RTH is probably produced by the heterodimerization of the mutant TR with RXR or homodimerization with a normal TR or TR. These mutant TR-containing dimers compete with wild-type TR-containing dimers for binding to the thyroid hormone response elements (TREs) of thyroid hormone-dependent genes (see Fig. 10-8) . Because these complexes bind co-repressor molecules that cannot be released in the absence of T 3 binding, genes containing these TREs are more repressed than they would be normally at the prevailing concentrations of circulating thyroid hormones. Receptors that contain mutations in the activation domain may have a combination of both decreased affinity for T 3 as well as impaired activating potential. Thus, the mutant TR complex can interfere with the function of the three normal TR-expressing genes, producing a pattern termed dominant negative inhibition with an autosomal dominant pattern of inheritance. At least 400 families have been identified with this condition, and there are probably many more unreported cases. The gene frequency estimate is about 1 : 50,000, and the study of the function of the mutant receptors in this disorder has provided valuable insights into the mechanism of thyroid hormone action. [205] [208] [209] [210] [211] [212] Patients with RTH usually are recognized because of thyroid enlargement, which is present in about two thirds of these individuals. Despite one's expectations, patients usually report a peculiar mixture of symptoms of hyperthyroidism and hypothyroidism. With respect to the heart, palpitations and tachycardia are more common than a reduced heart rate; however, patients may also demonstrate growth retardation and retarded skeletal maturation. This has been attributed to the fact that thyroid hormone effects in the heart appear to be primarily dependent on TR rather than TR, whereas the hypothalamic-pituitary axis is primarily regulated through TR, particularly TR2. [205] Abnormalities in neuropsychological development exist, with an increased prevalence of attention deficient hyperactivity disorder, which is found in approximately 10% of such individuals. Other neuropsychological abnormalities have also been described. [213] [214] [215] [216] Deafness in patients with RTH reflects the important role of TR and thyroid hormone in the normal development of auditory function. [214] The mixture of symptoms, some suggesting hypothyroidism and others suggesting hyperthyroidism, may even differ in individuals within the same family, despite the identical mutation, thus confusing the clinical picture.

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Because patients may present with symptoms suggesting hyperthyroidism, it is important to keep this diagnosis in mind in a patient with tachycardia, goiter, and elevated thyroid hormones. RTH is discussed here because a reduced response to thyroid hormone is the biochemical basis for the condition. However, the laboratory results may be the first clear evidence that a patient otherwise thought to have hyperthyroidism has RTH. These tests show the unusual combination of an increased FT 4 I accompanied by normal or slightly increased TSH levels. Thus, the principal differential diagnosis is between a TSH-secreting pituitary tumor and RTH. [214] [217] Factors that may assist in the differential diagnosis are as follows: 1. Absence of a family history in patients with TSH-producing tumors. 2. Normal thyroid hormone levels in family members of individuals with TSH-induced hyperthyroidism due to pituitary tumor. 3. Presence of an elevated glycoprotein -subunit in patients with pituitary tumor but not in those with thyroid hormone resistance. A definitive diagnosis requires sequencing of the TR gene demonstrating the abnormality. Although virtually all patients with RTH have such abnormalities, in a few individuals this is not the case, suggesting that there may be mutations in coactivator proteins or one of the RXR receptors, which can also present in a similar fashion.[218] Treatment is difficult because thyroid hormone analogues designed to suppress TSH, thereby relieving the hyperthyroxinemia, may lead to worsening of the cardiovascular manifestations of the condition. [203] [219] Therapy with 3,5,3'-triiodothy-roacetic acid (TRIAC) has been used in several patients. [220] [221] [222] The development of analogues of thyroid hormone with TR, as opposed to mixed or TR preferential effects, may eventually prove useful in treatment. [223]



Treatment

Hypothyroidism, either primary or central, is gratifying to treat because of the ease and completeness with which it responds to thyroid hormone. Treatment is nearly always with levothyroxine, and the proper use of this medication has been reviewed extensively. [122] [224] [225] A primary advantage of levothyroxine therapy is that the peripheral deiodination mechanisms can continue to produce the amount of T 3 required under physiologic control. If one accepts the principle that replicating the natural state is the goal of hormone replacement, it is logical to provide the "prohormone" and allow the peripheral tissues to activate it by physiologically regulated mechanisms. Pharmacologic and Physiologic Considerations

Levothyroxine has a 7-day half-life; about 80% of the hormone is absorbed relatively slowly and equilibrates rapidly in its distribution volume, therefore avoiding large postabsorptive perturbations in FT 4 I levels. [226] With its long half-life, omission of a single day's tablet has no significant effect and the patient may safely take an omitted tablet the following day. In fact, the levothyroxine dosage can be calculated almost as satisfactorily on a weekly, as on a daily, basis. According to the U.S. Pharmacopeia, the levothyroxine content of replacement tablets must be between 90% and 110% of the stated amount, although narrower restrictions are being introduced in the United States. The availability in many countries of a multiplicity of tablet strengths with content ranging from 25 to 300 µg allows precise titration of the daily levothyroxine dosage for most patients with a single tablet, improving compliance significantly. The typical dose of levothyroxine, approximately 1.6 to 1.8 µg/kg ideal body weight per day (0.7 to 0.8 µg/pound), generally results in the prescription of between 75 and 112 µg/day for women and 125 to 200 µg/day for men. Replacement doses need not be adjusted upward in obese patients. This dosage is about 20% greater than the T4 production rate owing to incomplete absorption of the levothyroxine. In patients with primary hypothyroidism, these amounts usually result in serum TSH concentrations that are within the normal range. Because of the 7-day half-life, approximately 6 weeks is required before there is complete equilibration of the FT 4 I and the biologic effects of levothyroxine. Accordingly, assessments of the adequacy of a given dose or the effects of a change in dosage should not be made until this interval has passed. By and large, levothyroxine products are clinically equivalent, although problems do occur. [227] [228] However, the variation permitted by the U.S. Food and Drug Administration in tablet content can result in slight variations in serum TSH in patients with primary hypothyroidism even when the same brand is used. Although the serum TSH level is an indirect reflection of the levothyroxine effect in patients with primary hypothy-roidism, it is superior to any other readily available method of assessing the adequacy of therapy. Return of the serum TSH level to normal is therefore the goal of levothyroxine therapy in the patient with primary hypothyroidism. Some patients may require slightly higher or lower doses than generally used, owing to individual variations in absorption, and a number of conditions or associated medications may change levothyroxine requirements in patients with established hypothyroidism (see later). In decades past, desiccated thyroid was successfully employed for the treatment of hypothyroidism and still accounts for a small fraction of the prescriptions written for thyroid replacement in the United States. Although this approach was successful, desiccated thyroid preparations contain thyroid hormone derived from animal thyroid glands that have significantly higher ratios of T 3 to T4 than the 1:11 value in normal human thyroid gland. [229] [230] Accordingly, such preparations may lead to supraphysiologic levels of T 3 in the immediate postab-sorptive period (2 to 4 hours) owing to the rapid release of T 3 from thyroglobulin, its immediate and nearly complete absorption, and the 1-day period required for T 3 to equilibrate with its 40-L volume of distribution (see Table 10-5) .[231] Mixtures of liothyronine and levothyroxine ( liotrix) contain in a 1-grain (64-mg) equivalent tablet (Thyrolar in the United States), the amounts of T 3 (12.5 µg) and T 4 (50 µg) present in the most popular desiccated thyroid tablet. [232] The levothyroxine equivalency of a 1-grain desiccated thyroid tablet or its liotrix equivalent can be estimated as follows. The 12.5 µg of liothyronine (T 3 ) is completely absorbed from desiccated thyroid or from liotrix tablets. [231] Levothyroxine is approximately 80% absorbed, [233] [234] and about 36% of the 40 µg of levothyroxine absorbed is converted to T 3 , with the molecular weight of T 3 (651) being 84% that of T 4 (777). Accordingly, a 1-grain tablet should provide about 25 µg of T 3 (12.5 + 12.1), which would be approximately equivalent to that obtained from 100 µg of levothyroxine. This equivalency ratio can be used as an initial guide in switching patients from desiccated thyroid or liotrix to levothyroxine. As indicated earlier, the use of levothyroxine as thyroid hormone replacement is a compromise with the normal pathway of T 3 production, in which about 80% of T 3 is derived from T 4 5-monodeiodination and approximately 20% (6 µg) is secreted directly from the thyroid gland. [235] Studies in thyroidectomized rats, for example, show that it is not possible to

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normalize T 3 simultaneously in all tissues by an IV infusion of T 4 . [236] However, it should be recalled from the earlier discussion of T 4 deiodination that the ratio of T 3 /T 4 in the human thyroid gland is about 0.09 but is 0.17 in the rat thyroid gland. [237] Thus, about 40% of the rat's daily T 3 production is derived from the thyroid versus about 20% in humans. [67] Accordingly, the demonstration that T 4 alone cannot provide normal levels of T 3 in all tissues in the rat is of interest but is not strictly applicable to thyroid hormone replacement in humans. [236] [238] Nonetheless, the ratio of T 3 /T4 in the serum of a patient receiving levothyroxine as the only source of T 3 must be about 20% lower than is that in a normal individual. Similarly, the quantity of levothyroxine required to normalize TSH in an athyreotic patient results in a slightly higher serum T 4 concentration than is present in normal individuals. [226] Although this may, to some extent, compensate for the lack of T 3 secretion, the fact that T 4 has an independent mechanism for TSH suppression owing to the intracellular generation of T 3 in the hypothalamic-pituitary-thyroid axis results in a portion of the feedback regulation being independent of the plasma T 3 concentration. Does this slightly lower T 3 concentration in patients receiving levothyroxine make any difference physiologically? Probably not, although the question is difficult to answer definitively because the most readily measurable end point, TSH, cannot be used. In one study, patients who received 12.5 µg of T 3 as a substitution for 50 µg of their levothyroxine preparation scored, on average, somewhat higher on tests of mood than when they were taking levothyroxine alone. [239] The dosage of thyroid hormone used in these studies was excessive, as judged by the fact that 20% of the group had serum TSH values below normal on either regimen and the test period was only a few months, making it difficult to extrapolate to the chronic replacement setting. On the other hand, another study showed that the FT 4 I correlated as closely with the resting energy expenditure, as did TSH levels in a group of patients in whom small supplements or decrements in their ideal replacement levothyroxine dosage were made. [240] The correlation with serum T 3 was not statistically significant, suggesting that in humans, perhaps as a result of differences in the peripheral metabolism of T 4 from that in rodents, the FT 4 I may be as accurate as the TSH value as an index of satisfactory thyroid hormone replacement. The practical difficulty with the design of tablets providing combinations of T 3 and T4 is that the approximate dose of 6 µg of T 3 provided would need to be released in a sustained fashion over 24 hours, which is quite different from the rapid absorption of T 3 with a peak at 2 to 4 hours when given in its conventional form. [231] Thus, for the present, it appears that the current approach to thyroid replacement using levothyroxine, although not a perfect replication of the normal physiology, is satisfactory for most patients. Institution of Replacement Therapy

The initial dose of levothyroxine prescribed depends on the degree of hypothyroidism and the age and general health of the patient. Patients who are young or middle-aged and otherwise healthy with no associated cardiovascular or other abnormalities and mild to moderate hypothyroidism (TSH concentrations 5 to 50 mU/L)

can be given a complete replacement dose of about 1.7 µg/kg of ideal body weight. The resulting increase in serum T 4 concentration to normal requires 5 to 6 weeks, and the biologic effects of T 3 are sufficiently delayed that these patients do not experience adverse effects. At the other extreme, the older patient with heart disease, particularly angina pectoris, without reversible coronary lesions, should be given small initial doses of levothyroxine (25 or even 12.5 µg/day), and the dosage should be increased in 12.5 µg increments at 2- to 3-month intervals with careful clinical and laboratory evaluation. [241] The goal in the patient with primary hypothyroidism is to return serum TSH concentrations to normal, reflecting normalization of that patient's thyroid hormone supply. This usually results in a mid to high-normal serum FT 4 I. The serum TSH should be evaluated 6 weeks after a theoretically complete replacement dose has been instituted to allow minor adjustments to optimize the individual dose. In patients with central hypothyroidism, serum TSH is not a reliable index of adequate replacement and the serum FT4 I should be restored to a concentration in the upper half of the normal range. Such patients should also be evaluated and treated for glucocorticoid deficiency before institution of thyroid replacement (see Chapter 8) . Although the adverse effects of the rapid institution of therapy are unusual, pseudotumor cerebri has been reported in profoundly hypothyroid juveniles between ages 8 and 12 years who were given even modest initial levothyroxine replacement. [242] This complication appears 1 to 10 months after initiation of treatment and responds to acetazolamide and dexamethasone. The interval between the initiation of treatment and the first evidence of improvement depends on the strength of dose given and the degree of the deficit. An early clinical response in moderate to severe hypothyroidism is a diuresis of 2 to 4 kg. The serum sodium (Na + ) level increases even sooner if hyponatremia was present initially. Thereafter, pulse rate and pulse pressure increase, appetite improves, and constipation may disappear. Later, psychomotor activity increases and the delay in the deep tendon reflex disappears. Hoarseness abates slowly, and changes in skin and hair do not disappear for several months. In individuals started on a complete replacement dose, the serum FT 4 I level should return to normal after 6 weeks; a somewhat longer period may be necessary for serum TSH levels to return to normal, perhaps up to 3 months. In addition to myxedema coma (see later), it is sometimes clinically appropriate to alleviate hypothyroidism rapidly. For example, patients with severe hypothyroidism withstand acute infections or other serious illnesses poorly and myxedema coma may develop as a complication. In such circumstances, rapid repletion of the peripheral hormone pool in the average adult can be accomplished by a single IV dose of 500 µg of levothyroxine. Alternatively, by virtue of its rapid onset of action, liothyronine (25 µg orally every 12 hours) can be administered if the patient can take medication by mouth. With both approaches, an initial effect is achieved within 24 hours. Parenteral therapy with levothyroxine is then continued with a dose that is 80% of the appropriate oral dose but not in excess of 1.4 µg/kg of ideal body weight. Because of the possibility that rapid increases in metabolic rate will overtax the existing pituitary-adrenocortical reserve, supplemental glucocorticoid (IV hydrocortisone 5 mg/hour) should also be given to patients with severe hypothyroidism receiving high initial doses of thyroid hormones. Finally, in view of the tendency of hypothyroid patients to retain free water, IV fluids containing only dextrose should not be given. When replacement therapy is withdrawn for short periods (4 to 6 weeks) for purposes of evaluating therapy for thyroid cancer, rapid reinstitution of levothyroxine using a loading dose of three times the daily replacement dose for 3 days can usually be given unless there are other complicating medical illnesses. When hypothyroidism results from administration of iodine-containing or antithyroid drugs, withdrawal of the offending agent usually relieves both the hypothyroidism and the accompanying goiter, although it is appropriate to provide interim

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replacement until the gland recovers its function. This is especially true for amiodarone, which may remain in tissues for up to a year. Infants and Children

In infants with congenital hypothyroidism, the determining factor for eventual intellectual attainment is the age at which adequate treatment with thyroid hormone is begun. The therapy for infants with congenital hypothyroidism should consist initially of raising the serum T 4 level to more than 130 nmol/L (10 µg/dL) as rapidly as possible and maintaining it at that level for the first 3 to 4 years of life. This is usually accomplished by administering an initial levothyroxine dose of 50 µg/day, [4] which is higher than the adult dose on a weight basis and in keeping with the higher metabolic clearance of the hormone in the infant. [243] The serum TSH concentration may not return to normal even with this high dose because of residual reset of the pituitary feedback mechanism. After 2 years of age, however, a TSH level in the normal range is an index of optimal therapy as it is in adults. [244] [245] Monitoring Replacement Therapy

Monitoring the adequacy of, and compliance with, thyroid hormone therapy in patients with primary hypothyroidism is easily done by measurement of serum TSH. This value should be within the normal range for an assay sufficiently sensitive to measure, with confidence, the lower limit of the normal range. The normal serum TSH concentration varies between 0.5 and 4.0 mU/L in most second-generation and third-generation assays, and results within this range are associated with the elimination of all clinical and biochemical manifestations of primary hypothyroidism, except in patients with RTH. After the first 6 months of therapy, the dose should be reassessed because restoration of euthyroidism increases the metabolic clearance of T 4 . A dose that was adequate during the early phases of therapy may not be adequate when the same patient is euthyroid owing to an acceleration in the clearance of thyroid hormone. Under normal circumstances, the finding of a normal serum TSH level on an annual basis is adequate to ensure that the proper dose is prescribed and is being taken by the patient. If the serum TSH level is above the normal range and noncompliance is not the explanation, small adjustments, usually in 12-µg increments, can be made with reassessment of TSH concentrations after full equilibration (6 weeks) with the new dose to confirm that such adjustments are appropriate. [241] In North America, this strategy is simplified by the availability of multiple tablet strengths, many of which differ by only 12 µg. Most patients can receive the same dose until they reach the 7th or 8th decade, at which point a downward adjustment of 20% to 30% is indicated because thyroid hormone clearance decreases in the elderly. [69] [246]

Thyroid hormone requirements may be altered in several situations (Table 12-4) . A reduction in replacement dosage may be required in women who are receiving androgen therapy for adjuvant treatment of breast carcinoma. [247] Most other conditions or medications increase the levothyroxine requirement in patients receiving maintenance therapy. During pregnancy, the levothyroxine requirement is increased by 25% to 50%. [248] [249] Hypothyroid patients who are planning a pregnancy should be advised to increase the dose by up to 50% as soon as the diagnosis is confirmed because the change in requirement appears soon after implantation. The increased requirement is probably due to a combination of factors, including increases in thyroxine-binding globulin and the volume of distribution of T 4 , an increase in body mass, and an increase in D3 in placenta and perhaps the uterus. [250] [251] [252] The increased requirement TABLE 12-4 -- Conditions That Alter Levothyroxine Requirements Increased Levothyroxine Requirements Pregnancy Gastrointestinal Disorders Mucosal diseases of the small bowel (e.g., sprue) After jejunoileal bypass and small-bowel resection Diabetic diarrhea Therapy with Certain Pharmacologic Agents Drugs That Interfere with Levothyroxine Absorption Cholestyramine

Sucralfate Aluminum hydroxide Calcium carbonate Ferrous sulfate Drugs That Increase the Cytochrome P450 Enzyme (CYP3A4) Rifampin Carbamazepine Estrogen Phenytoin Sertraline ? Statins Drugs That Block T 4 to T 3 Conversion Amiodarone Conditions That May Block Deiodinase Synthesis Selenium deficiency Cirrhosis Decreased Levothyroxine Requirements Aging (65 years and older) Androgen therapy in women T4 , thyroxine; T3 , triiodothyronine. persists throughout pregnancy but returns to normal within a few weeks after delivery. Therefore, the dose should be reduced to the original pre-pregnancy level at the time of delivery. Maternal T 4 is critically important to the athyreotic fetus, and pregnant patients should be monitored carefully. [47] [243] [253] [254] Other conditions in which levothyroxine requirements are increased (Table 12-4) [132] include malabsorption due to either bowel disease or adsorption of levothyroxine to coadministered medications such as sucralfate [255] aluminum hydroxide and perhaps calcium carbonate, [256] ferrous sulfate,[257] lovastatin, [258] or various resins.[259] [260] Certain medications, notably rifampin, [261] carbamazepine, [262] phenytoin,[263] and sertraline, [264] increase the clearance of levothyroxine by inducing CYP3A4 in the liver. Estrogen given to postmenopausal women may act in the same way, although the changes in thyroglobulin and distribution volume make the exact resolution of the cause of the increased levothyroxine requirement uncertain. [265] Amiodarone increases levothyroxine requirements by blocking conversion of T 4 to T3 and perhaps by interfering with T 3 -thyroid hormone receptor binding. [266] Selenium deficiency is rare, but because it is rate-limiting in the synthesis of D1 (see Fig. 10-6) , a deficiency, such as may occur in patients receiving diets restricted in protein, may increase levothyroxine requirements. [267] [268] [269] Occasionally, in patients who have been treated with radioactive iodine for Graves' disease or toxic nodular goiter, some degree of thyroid hormone secretion persists and, although inadequate, is autonomous. Such patients have a suppressed TSH on what otherwise would be considered a replacement dose of levothyroxine. The levothyroxine dose in these individuals should be reduced until TSH levels rise to normal, keeping in mind that several months may be required before TSH secretion recovers after its prolonged suppression. Because of

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either the delayed effects of radioiodine or the natural history of Graves' disease, per se, this autonomous T 4 secretion may decrease with time, leading to an increase in levothyroxine requirements in subsequent years. Rarely, the opposite occurs; that is, a patient treated with radioiodine develops an increased TSH level, but, after several months of therapy, the requirement for such replacement is either reduced or eliminated. This may reflect transient impairment of thyroid function by a combination of preirradiation antithyroid drug therapy and immediate effects of radiation on the thyroid. In such patients, frequent monitoring of levothyroxine replacement is required to avoid over-replacement. In North America, clinical experience with the most commonly used levothyroxine preparations suggests that these products are equally effective, and this is supported by small clinical trials. [227] Nonetheless, the possibility of a problem with tablet levothyroxine content should be considered if a new preparation changes the biologic effects of the same dosage. [228] Adverse Effects of Levothyroxine Therapy

Although the administration of excessive doses of levothyroxine causes osteoporosis in postmenopausal patients, most authorities believe that returning thyroid status to normal does not have adverse effects on bone density. [34] [270] [271] Administration of excessive doses also increases cardiac wall thickness and contractility and, in elderly patients, increases the risk of atrial fibrillation. [7] [272] [273] In some patients, TSH levels remain elevated despite the prescription of adequate replacement doses. This is most often a consequence of poor compliance. The combination of normal or even elevated serum FT 4I values and elevated TSH levels can occur if the patient does not take levothyroxine regularly but ingests several pills the day before testing. The integrated dose of levothyroxine over prior weeks is best reflected in the serum TSH level, and noncompliant patients require careful education as to the rationale for treatment. Subtle changes in dietary habits, such as increasing the ingestion of bran-containing products, may decrease levothyroxine absorption, and their recognition requires a careful history. Patients with Hypothyroid Symptoms Despite Restitution of Normal Thyroid Function

In rare circumstances, symptoms consistent with hypothyroidism persist despite appropriate treatment of the hypothyroid state. Such patients should be educated as to the relationship between symptoms of hypothyroidism and the role of thyroid hormone in relieving these, and other causes should be sought for the symptomatology. In rare cases, hypothyroid symptoms are associated with hypometabolism despite normal levels of serum thyroid hormones and TSH. [274] Such patients may have RTH in peripheral but not central tissues, a situation that has been documented only rarely. [203]



Special Aspects of Hypothyroidism Subclinical Hypothyroidism

The term subclinical hypothyroidism designates a situation in which an asymptomatic patient has a low-normal FT 4 I but a slightly elevated serum TSH level. Other terms for this condition are mild hypothyroidism, preclinical hypothyroidism, biochemical hypothyroidism, and decreased thyroid reserve (see Table 12-1) . The TSH elevation in such patients is modest, with values typically between 5 and 15 mU/L. This syndrome is most often seen in patients with early Hashimoto's disease and is a common phenomenon, occurring in 7% to 10% of older women.[1] [275] A number of studies on the effects of thyroid hormone treatment in such patients have used physiologic end points (e.g., measurements of various serum enzymes, systolic time intervals, serum lipids, psychometric testing), and results have been variable. In the most carefully controlled studies, one or another of the parameters has returned to normal in about 25% to 50% of patients. [7] [12] [58] [276] [277] In general, FT 4 I and TSH levels normalize, but FT 3 I, usually normal at the outset, does not change. In one study that employed a double-blind, crossover approach, the 4 of 17 women who improved could be differentiated from the remainder only by a somewhat lower serum free T3 at the start of the study.[277] Modest improvements in cardiac indices have been noted in some [278] [279] but not all [276] reports, and the same is true for lipids [12] [58] . Thus, when one is confronted with this clinical situation, there is no clearly correct approach. [280] One factor favoring a decision to recommend levothyroxine therapy is the presence of antibodies to TPO or thyroglobulin or the presence of a goiter. There is a risk of progression of thyroid dysfunction in patients with Hashimoto's disease, and this premonitory sign of thyroid failure is, to many, a justification for initiating therapy. To be weighed against this are the expense and bother of daily medication, not acceptable to some patients, and the possibility that overdosage with levothyroxine may exacerbate osteoporosis or cause cardiac arrhythmias. If a therapeutic trial is performed, the TSH concentration should be monitored carefully and should not be reduced below normal. If no therapy is given, such patients should be monitored at intervals of 6 to 12 months both clinically and by measurements of serum TSH. Metabolic Insufficiency

Nonspecific symptoms of true hypothyroidism include mild lassitude, fatigue, slight anemia, constipation, apathy, cold intolerance, menstrual irregularities, loss of hair, and weight gain (see Fig. 12-5) . For this reason, some patients with such complaints but with normal laboratory results have been considered candidates for levothyroxine therapy. The response to thyroid hormone therapy is sometimes gratifying, at least initially, but symptomatic improvement usually disappears after a time unless the dose is increased. Eventually, even larger doses fail to alleviate the symptoms, confirming that they do not arise from a deficiency of thyroid hormone. Thus, thyroid hormone therapy should be avoided in patients with no biochemical documentation of impaired thyroid function. Furthermore, even in patients with subclinical hypothyroidism, symptoms may be out of proportion to FT 4 I abnormalities. It is unwise to raise the patient's expectations that such symptoms will be relieved by correction of mild biochemical abnormalities. Thyroid Function Testing in Patients Receiving Replacement Therapy

Physicians are frequently confronted with patients receiving levothyroxine in whom the historical diagnosis of hypothyroidism has been made on what appear to be questionable grounds. In this circumstance, it may be impossible to determine, from retrospective clinical or laboratory findings, whether thyroid hormone replacement is indicated. If serum TSH is in the normal range and primary hypothyroidism is suspected, a simple way of assessing the need for levothyroxine therapy is to switch levothyroxine to every-other-day dosage or to reduce the daily dose by 50% and to reevaluate TSH and FT 4 I after 4 weeks. If there has been no significant increase in

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TSH concentration and FT 4 I remains constant during that period, levothyroxine is withdrawn and blood tests are repeated 4 and 8 weeks later. If the initial TSH level is suppressed, indicating overreplacement, the dose should be reduced until TSH becomes detectable before this trial is instituted. If central hypothyroidism is suspected, the FT 4 I must be monitored during these procedures. Emergent Surgery in the Hypothyroid Patient

The perioperative course of patients with untreated hypothyroidism has been evaluated in several studies. In general, such patients were not recognized to be hypothyroid or did not require surgery despite the presence of significant hypothyroidism. Complications were uncommon. Perioperative hypotension, ileus, and central nervous system disturbances were more common in hypothyroid patients, and patients with major infections had fewer episodes of fever than did euthyroid control subjects. [281] [282] [283] Other complications were delayed recovery from anesthesia and abnormal hemostasis, possibly owing to an acquired form of von Willebrand's disease. [39] From these studies, one may conclude that emergent surgery should not be postponed in hypothyroid patients but that such patients should be rigorously monitored for evidence of carbon dioxide retention, bleeding, infection, and hyponatremia. These findings are also relevant to the treatment of hypothyroid individuals with symptomatic coronary artery disease. Considering the lack of significant increase in perioperative complications in the hypothyroid patient, the option of surgery for remediable coronary artery lesions is open to hypothyroid individuals without the risk of a myocardial infarction in association with restitution of the euthyroid state (see later). [284]



Heart Disease and Thyroid Hormone Therapy Coexisting Coronary Artery Disease and Hypothyroidism

In many patients with coronary artery disease and primary hypothyroidism, cardiac function is corrected during institution of levothyroxine therapy because of a decrease in peripheral vascular resistance and improvement in myocardial function. [7] [10] [17] However, patients with preexisting angina pectoris should be evaluated for correctable lesions of the coronary arteries and treated appropriately before levothyroxine is administered. [8] [285] [286] Retrospective studies indicate that this approach is safer than the institution of replacement therapy prior to angiography and angioplasty or even coronary artery bypass grafting. [284] [285] In a few patients, lesions may not be remediable or small-vessel disease is severe even after bypass grafting, so that complete replacement cannot be instituted. Such patients must receive optimal antianginal therapy combined with -adrenergic receptor blockers in judicious quantities, and complete restitution of the euthyroid state may not be possible. [287] Thyroid Hormone for Compromised Cardiovascular Function

In addition to the issues raised in patients with combined hypothyroidism and coronary artery disease, there is interest in the potential therapeutic use of thyroid hormone in the treatment of patients with either cardiomyopathy or status postcoronary artery bypass grafting (CABG) or other cardiac procedures. [288] [289] [290] [291] [292] As expected, T3 levels are reduced in patients with advanced congestive heart failure, as with any illness. [293] In one report, 23 patients with advanced heart failure (mean ejection fraction, 22%) were given up to 2.7 µg/kg of liothyronine over 6 hours with an increase in cardiac output and decrease in systemic vascular resistance but without increase in heart or metabolic rate. [288] Similar effects were seen with a dose of liothyronine, 110 µg, over 6 hours after CABG. [286] In addition, decreases in the frequency of atrial fibrillation following surgery were found in liothyronine-treated patients, [290] although this was not confirmed in a second study. [294] However, there were no adverse effects associated with the infusion of T 3 in either study. Liothyronine has also been given postoperatively for congenital heart disease and, again, an improvement in cardiac output and decrease in vascular resistance occurred without adverse side effects. [292] These results suggest that, in certain selected circumstances, liothyronine may be useful as adjunctive therapy in patients with congestive heart failure because of its effect of relaxing vascular smooth muscle. It is conceivable that more selective thyroid hormone analogues may be on the horizon that might produce this effect but not increase myocardial oxygen demands.



Screening for Primary Hypothyroidism

The high incidence of primary hypothyroidism in women, particularly if the 7% to 10% prevalence of subclinical hypothyroidism is included, raises the issue of whether the cost of systematic periodic screening of an asymptomatic population is justified. [1] [275] A number of studies have addressed this complex issue. The conclusions depend, to a great extent, on assumptions regarding the effectiveness and economic value of therapy in asymptomatic patients with TSH elevation alone. [275] [295] [296] [297] One study concluded that the cost of an every-5-year TSH determination for women and men would be approximately $9000 per quality-adjusted life-year in women.[295] Other studies have indicated somewhat lower financial benefits. [297] An assessment of TSH levels at 5-year intervals in women older than age 50 years seems justified, but further analyses of more extensive screening programs are in order. A second complex issue involves whether women planning pregnancy should be screened for the presence of hypothyroidism as a routine part of a prenatal visit. This question is raised because of the results of a study indicating that mild to modest hypothyroidism is associated with an impairment of mental development in infants of mothers with elevated TSH during the first trimester. [254] The prevalence of hypothyroidism during pregnancy has been found to be approximately 2%, [298] and screening of all patients has been advocated by several professional organizations. Maternal FT4 I concentrations in the lowest 10% of the normal range, even with normal TSH levels, have also been suggested as a risk factor for impaired neuropsychological development of the fetus. [299] It is not clear why this is a risk factor for impaired fetal neuropsychological development, because such patients are not hypothyroid. For the moment, it appears that any patient with a family history of autoimmune thyroid disease, with symptoms suggesting hypothyroidism, or with thyroid enlargement should be screened for thyroid dysfunction prior to pregnancy or as soon after conception as is feasible.



Myxedema Coma

Myxedema coma is the ultimate stage of severe long-standing hypothyroidism. This state, which almost invariably affects

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older patients, occurs most commonly during the winter months and is associated with a high mortality rate. It is usually, but not always, accompanied by a subnormal temperature. Values as low as 23°C having been recorded. The external manifestations of severe myxedema, bradycardia, and severe hypotension are invariably present. The characteristic delay in deep tendon reflexes may be lacking if the patient is areflexic. Seizures may accompany the comatose state. Although the pathogenesis of myxedema coma is not clear, factors that predispose to its development include exposure to cold, infection, trauma, and central nervous system depressants or anesthetics. Alveolar hypoventilation, leading to carbon dioxide retention and narcosis, and dilutional hyponatremia resembling that seen with inappropriate secretion of arginine vasopressin (AVP) may also contribute to the clinical state. From the foregoing, it appears that myxedema coma should be readily recognized from its clinical signs, but this is not the case. After a brain stem infarction, elderly patients with features suggestive of hypothyroidism may be both comatose and hypothermic. In addition, hypothermia of any cause, due for example to exposure to cold, may cause changes suggestive of myxedema, including delayed relaxation of deep tendon reflexes. The importance of the difficulty in diagnosing myxedema coma is that a delay in therapy worsens the prognosis. Consequently, the diagnosis should be made on clinical grounds, and, after sending blood for thyroid function tests, therapy should be initiated without awaiting the results of confirmatory tests because mortality may be 20% or higher. [300] [301] Treatment consists of administration of thyroid hormone and correction of the associated physiologic disturbances. [300] [302] Because of the sluggish circulation and severe hypometabolism, absorption of therapeutic agents from the gut or from subcutaneous or intramuscular sites is unpredictable, and medications should be administered intravenously if possible. Administration of levothyroxine as a single IV dose of 500 to 800 µg serves to replete the peripheral hormone pool and may cause improvement within hours. Daily doses of IV levothyroxine, 100 µg, are given thereafter. Hydrocortisone (5 to 10 mg/hour) should also be given because of the possibility of relative adrenocortical insufficiency as the metabolic rate increases. Alternatively, IV liothyronine may be given at a dose of 25 µg every 12 hours. [303] [304] Others have used a combination of 200 to 300 µg T 4 and 25 µg T3 intravenously as a single dose, followed by 25 µg T 3 and 100 µg T 4 24 hours later, and then 50 µg T 4 daily until the patient regains consciousness. [305] Hypotonic fluids should not be given because of the danger of water intoxication owing to the reduced free water clearance of the hypothyroid patient. Hypertonic saline and glucose may be required to alleviate severe dilutional hyponatremia and the occasional hypoglycemia. A critical element in therapy is support of respiratory function by means of assisted ventilation and controlled oxygen administration. Internal warming by gastric perfusion may be useful, but external warming should be avoided because it may lead to vascular collapse due to peripheral vasodilatation. Further heat loss can be prevented with blankets. An increase in temperature may be seen within 24 hours in response to levothyroxine. General measures applicable to the comatose patient should be undertaken, such as frequent turning, prevention of aspiration, and attention to fecal impaction and urinary retention. Finally, the physician should assess the patient for the presence of coexisting disease, such as infection and cardiac or cerebrovascular disease. In particular, the myxedematous patient may be afebrile despite a significant infection. As soon as the patient is able to take medication by mouth, treatment with oral levothyroxine should be instituted.



THYROIDITIS Overview

Thyroiditis is a term indicating the presence of thyroid inflammation, and thus comprises a large group of diverse inflammatory conditions. These include the following: 1. 2. 3. 4. 5.

Autoimmune or quasi-autoimmune causes. Viral or postviral conditions. Infections, including those of bacterial and fungal origins. A chronic sclerosing form of thyroiditis, termed Riedel's thyroiditis (or struma). Miscellaneous causes of various types, including radiation-induced and granulomatous causes, such as sarcoidosis.

Not only are the causes of thyroiditis extremely varied; their clinical presentations may also be diverse and are difficult to categorize in a simple fashion (Table 12-5) . Thus, as already discussed, autoimmune thyroiditis may present with hypothyroidism but often patients remain euthyroid for long periods after the disease is initiated. On the other hand, in a euthyroid patient with Hashimoto's disease who becomes pregnant, the postpartum period is often complicated by an acute form of hyperthyroidism due to the transient exacerbation of thyroiditis, often followed by a period of hypothyroidism. A similar syndrome has been observed in nonpregnant patients, called silent or painless thyroiditis. It is manifested primarily as thyrotoxicosis of sudden onset without localized pain and often without evidence of autoimmune disease. This condition may be viral in origin in some patients; however, the most classic presentation of viral thyroiditis is as subacute, nonsuppurative thyroiditis, also known as de Quervain's thyroiditis, pseudotuberculous thyroiditis, or migratory or creeping thyroiditis. Unlike typical autoimmune thyroiditis, this condition is characterized by extreme thyroid tenderness, with pain radiating to the oropharynx and ears, and must be differentiated from acute suppurative thyroiditis caused by bacterial or fungal infection. [306] Thus, inflammatory conditions of the thyroid present a dilemma because one must decide whether to discuss these entities as a group with the common denominator of inflammation or to categorize them according to their principal clinical effects, namely thyrotoxicosis or thyroid hormone deficiency. We have chosen the latter approach and have already discussed autoimmune thyroiditis, the major cause of thyroid gland failure (see Table 12-2) . However, patients with acute autoimmune thyroiditis may also develop thyrotoxicosis, such as in postpartum silent or painless thyroiditis ( see Chapter 11 on autoimmune thyroiditis). These patients must be differentiated from those with Graves' disease. In addition, some patients with viral thyroiditis have thyrotoxicosis as a major manifestation with varying degrees of neck discomfort ranging from none to full-blown subacute, nonsuppurative (granulomatous) thyroiditis. For that reason, this thyroiditis syndrome is also discussed in Chapter 11 , even though the pain associated with the typical form of this condition makes the principal differential TABLE 12-5 -- Causes of Thyroiditis Autoimmune thyroiditis (see Table 12-3) Postpartum, silent, or painless thyroiditis (see Chapter 11) Subacute (nonsuppurative) thyroiditis (see Chapter 11) Acute infectious thyroiditis Riedel's thyroiditis Miscellaneous Postirradiation ( 131 I or external-beam therapy) Sarcoidosis

448

diagnosis lie between that and pyogenic thyroiditis. In that context, subacute thyroiditis is also mentioned later.



Acute Infectious Thyroiditis

Although the thyroid gland is remarkably resistant to infection, congenital abnormalities of the piriform sinus, underlying autoimmune disease, or immunocompromise of the host may lead to the development of an infectious disease of the thyroid gland. [307] [308] The etiology may be any bacterium, including Staphylococcus, Pneumococcus, Salmonella, or Mycobacterium tuberculosis. [309] [310] [311] In addition, infections with certain fungi, including Coccidioides immitis, Candida, or Aspergillus and Histoplasma have been reported.[312] The most common cause of repeated childhood pyogenic thyroiditis, particularly in the left lobe, is a consequence of an internal fistula extending from the piriform sinus to the thyroid. [313] [314] This sinus is the residual connection following the path of migration of the ultimobranchial body from the fifth pharyngeal pouch to the thyroid gland. The predominance of thyroiditis of the left lobe is explained by the fact that the right ultimobranchial body is often atrophic, whereas this is not the case for the left side. Nonetheless, a patient with a completely normal thyroid gland may develop bacterial thyroiditis. This is an extremely rare disease even as a complication of direct puncture of the thyroid gland, such as in fine-needle aspiration. In individuals with midline infections, persistence of the thyroglossal duct should be considered. Incidence

Infectious thyroiditis is extremely rare, with no more than a few cases being seen in large tertiary care centers.

TABLE 12-6 -- Features Useful in Differentiating Acute Suppurative Thyroiditis and Subacute Thyroiditis Characteristic Acute Thyroiditis History

Physical examination of the thyroid

Laboratory

Needle aspiration

Radiologic

Clinical course

Subacute Thyroiditis

Preceding upper respiratory infection

88%

17%

Fever

100%

54%

Symptoms of thyrotoxicosis

Uncommon

47%

Sore throat

90%

36%

Painful thyroid swelling

100%

77%

Left side affected

85%

Not specific

Migrating thyroid tenderness

Possible

27%

Erythema of overlying skin

83%

Not usually

Elevated white blood cell count

57%

2550%

Elevated erythrocyte sedimentation rate (>30 mm/hr)

100%

85%

Abnormal thyroid hormone levels (elevated or depressed)

510%

60%

Alkaline phosphatase, transaminases increased

Rare

Common

Purulent, bacteria or fungi present

100%

0

Lymphocytes, macrophages, some polyps, giant cells

0

100%

123

Uncommon

100%

I uptake low

Abnormal thyroid scan

92%

Thyroid scan or ultrasound helpful in diagnosis

75%

Gallium scan positive

100%

100%

Barium swallow showing fistula

Common

0

CT scan useful

Rarely

Not indicated

Clinical response to glucocorticoid treatment

Transient

100%

Incision and drainage required

85%

No

Recurrence following operative drainage

16%

No

Piriform sinus fistula discovered

96%

No

From DeGroot LJ, Larsen PR, Hennemann G. Acute and subacute thyroiditis. In The Thyroid and Its Diseases, 6th ed. New York, Churchill Livingstone, 1996, p 700.

Clinical Manifestations

The clinical manifestations of infectious thyroiditis are dominated by local pain and tenderness in the affected lobe or entire gland. This is accompanied by painful swallowing and difficulty on swallowing. Because of the tendency for referral of pain to the pharynx or ear, the patient may not recognize the tenderness in the anterior neck. Depending on the virulence of the organism and the presence of septicemia, symptoms such as fever and chills may also accompany the condition. The major differential diagnosis lies between an infectious form of thyroiditis and subacute, nonsuppurative thyroiditis. It is instructive to compare the principal features of these two diseases to arrive at an accurate diagnosis (Table 12-6) . By and large, patients with acute thyroiditis caused by a bacterium are much sicker than patients with subacute thyroiditis; they have more severe and localized tenderness and are less likely to have laboratory evidence of hyperthyroidism, which is present in approximately 60% of patients with subacute thyroiditis. Ultrasonographic examination often reveals the abscess in the thyroid gland or evidence of swelling, and needle aspiration may help pinpoint the responsible organism. [310] [315] A gallium scan will be positive as a result of the diffuseness of the inflammation and, particularly in children with thyroiditis of the left lobe, a barium swallow showing a fistula connecting the piriform sinus and left lobe of the thyroid is diagnostic.

[316]

[317]

Occasionally, pertechnetate scanning is useful in showing normal function of one lobe of the thyroid gland, which is much less common in subacute thyroiditis (which more often affects the entire gland). Needle aspiration should be used to drain the affected lobe, although occasionally surgical drainage may be required. If a piriform sinus fistula can be demonstrated, it must be removed to prevent recurrence of the problem.

449

Antibiotics should be administered appropriate to the offending organism. Fungal infections should be treated appropriately, especially because many of these individuals are immunocompromised. Endemic organisms should be kept in mind as a cause, since both Echinococcus and Trypanosomiasis infections of the thyroid

gland have been reported. The prognosis is excellent with preservation of thyroid function in general, although post-thyroiditis thyroid function tests should be monitored to ascertain that thyroid failure has not occurred.



Riedel's Thyroiditis

Riedel's chronic sclerosing thyroiditis is rare and dramatic and occurs chiefly in middle-aged women. [318] [319] The etiologic mechanism is uncertain, although some cases are considered to be an advanced state of Hashimoto's disease. [320] [321] This condition is characterized by fibrosis of the thyroid gland and adjacent structures and may be associated with fibrosis elsewhere, especially in the retroperitoneal area. [318] The presence of eosinophils has been demonstrated histologically, suggesting a unique autoimmune response to fibrous tissue. [322] Symptoms develop insidiously and are related chiefly to compression of adjacent structures, including the trachea, esophagus, and recurrent laryngeal nerves. Constitutional evidence of inflammation is uncommon. The thyroid gland is moderately enlarged, stony hard, and usually asymmetrical. The consistency of the gland and the invasion of adjacent structures suggest carcinoma, but there is no enlargement of regional lymph nodes. Temperature, pulse, and leukocyte count are normal. Severe hypothyroidism is unusual but does occur, as does loss of parathyroid function. The RAIU may be normal or low. Circulating thyroid autoantibodies are less common and are found in lower titers than in Hashimoto's disease. Surgery may be required to preserve tracheal and esophageal function. If extensive involvement of perithyroid tissues is present, resection of the isthmus may relieve some symptoms. Treatment with thyroid hormone relieves the hypothyroidism but has no effect on the primary process, which may progress inexorably. Immunosuppressive treatment and even chemotherapy has been tried in individual cases.



Miscellaneous Causes

Only a few causes of generalized inflammation of the thyroid gland have been reported. These include inflammation arising after 131 I treatment of individuals for Graves' disease, a residual thyroid lobe in a patient with thyroid cancer of the contralateral lobe, and thyroiditis arising from external-beam therapy for conditions such as Hodgkin's or non-Hodgkin's lymphoma, breast carcinoma, or other lesions of the oropharynx. In general, only radioiodine-induced thyroiditis is associated with pain and glucocorticoid treatment may be useful in symptomatic therapy.



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B, Palmieri E, Fazio S, et al. Endogenous subclinical hyperthyroidism affects quality of life and cardiac morphology and function in young and middle-aged patients. J Clin Endocrinol Metab 2000; 85:47014705. 274. Kaplan

MM, Swartz SL, Larsen PR. Partial peripheral resistance to thyroid hormone. Am J Med 1981; 70:11151121.

275. Weetman 276. Cooper

AP. Hypothyroidism: screening and subclinical disease. BMJ 1997; 314:11751178.

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283. Ladenson

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284. Hay

ID, Duick DX, Vlietstra RE, et al. Thyroxine therapy in hypothyroid patients undergoing coronary revascularization: a retrospective analysis. Ann Intern Med 1981; 95:456457.

285. Sherman

SI, Ladenson PW. Percutaneous transluminal coronary angioplasty in hypothyroidism. Am J Med 1991; 90:367370.

286. Klemperer 287. Rossant

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288. Hamilton 289. Moruzzi

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MA, Stevenson LW, Fonarow GC, et al. Safety and hemodynamic effects of intravenous triiodothyronine in advanced congestive heart failure. Am J Cardiol 1998; 81:443447.

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JD, Klein IL, Ojamaa K, et al. Triiodothyronine therapy lowers the incidence of atrial fibrillation after cardiac operations. Ann Thorac Surg 1996; 61:13231327; discussion 13281329.

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SL, Argenziano M, Corwin S, et al. A randomized double-blind study of the effect of triiodothyronine on cardiac function and morbidity after coronary bypass surgery. J Thorac Cardiovasc Surg 1999; 117:11281134. 292. Chowdhury

D, Parnell VA, Ojamaa K, et al. Usefulness of triiodothyronine (T3) treatment after surgery for complex congenital heart disease in infants and children. Am J Cardiol 1999; 84:11071109, A10. 293. Hamilton

MA, Stevenson LW, Luu M, et al. Altered thyroid hormone metabolism in advanced heart failure. J Am Coll Cardiol 1990; 16:9195.

294. Bennett-Guerrero

E, Jimenez JL, White WD, et al. Cardiovascular effects of intravenous triiodothyronine in patients undergoing coronary artery bypass graft surgery. A randomized, double-blind, placebo-controlled trial. Duke T3 Study Group. JAMA 1996; 275:687692. 295. Danese

D, Sciacchitano S, Farsetti A, et al. Diagnostic accuracy of conventional versus sonography-guided fine-needle aspiration biopsy of thyroid nodules. Thyroid 1998; 8:1521.

296. Helfand

M, Redfern CC. Clinical guideline: II. Screening for thyroid disease: an update. American College of Physicians. Ann Intern Med 1998; 129:144158.

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M, Santini F, Rivolta G, et al. Cost effectiveness of screening for subclinical hypothyroidism in the elderly: a decision-analytical model. Pharmacoeconomics 1998; 14:209216.

298. Klein

RZ, Haddow JE, Faix JD, et al. Prevalence of thyroid deficiency in pregnant women. Clin Endocrinol (Oxf) 1991; 35:4146.

299. Pop

VJ, Kuijpens JL, van Baar AL, et al. Low maternal free thyroxine concentrations during early pregnancy are associated with impaired psychomotor development in infancy. Clin Endocrinol (Oxf) 1999; 50:149155. 300. Jordan

RM. Myxedema coma: the prognosis is improving. Endocrinologist 1993; 3:149153.

301. Reinhardt 302. Nicoloff

W, Mann K. Incidence, clinical picture, and treatment of hypothyroid coma: results of a survey. Med Klin 1997; 92:521524.

JT, LoPresti JS. Myxedema coma: a form of decompensated hypothyroidism. Endocrinol Metab Clin North Am 1993; 22:279290.

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CM, McConahey WM, Keating RF, et al. Calorigenic effects of single intravenous doses of

304. MacKerrow 305. Hylander

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SD, Osborn LA, Levy H, et al. Myxedema-associated cardiogenic shock treated with intravenous triiodothyronine. Ann Intern Med 1992; 117:10141015.

B, Rosenquist U. Treatment of myxedema coma: factors associated with fatal outcome. Acta Endocrinol (Copenh) 1985; 108:6571.

306. Szabo

SM, Allen DB. Thyroiditisdifferentiation of acute suppurative and subacute: case report and review of the literature. Clin Pediatr 1989; 28:171174.

307. Singer

PA. Thyroiditis: acute, subacute, and chronic. Med Clin North Am 1991; 75:6177.

308. Fernandez

JF, Anaissie EJ, Vassilopoulou-Sellin R, et al. Acute fungal thyroiditis in a patient with acute myelogenous leukemia. J Intern Med 1991; 230:539541.

309. Nieuwland

Y, Tan KY, Elte JW. Miliary tuberculosis presenting with thyrotoxicosis. Postgrad Med J 1992; 68:677679.

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DK, Pant CS, Chachra KL, et al. Fine-needle aspiration cytology diagnosis of tuberculous thyroiditis: a report of eight cases. Acta Cytol 1992; 36:517522.

311. Chiovato 312. Goldani

L, Canale G, Maccherini D, et al. Salmonella brandenburg: a novel cause of acute suppurative thyroiditis. Acta Endocrinol (Copenh) 1993; 128:439442.

LZ, Klock C, Diehl A, et al. Histoplasmosis of the thyroid. J Clin Microbiol 2000; 38:38903891.

313. Miyauchi

A, Matsuzuka F, Kuma K, et al. Piriform sinus fistula: an underlying abnormality common in patients with acute suppurative thyroiditis. World J Surg 1990; 14:400405.

314. Lucaya

J, Berdon WE, Enriquez G, et al. Congenital pyriform sinus fustula: a cause of acute left-sided suppurative thyroiditis and neck abscess in children. Pediatr Radiol 1990; 21:2729.

315. Gandhi

RT, Tollin SR, Seely EW. Diagnosis of Candida thyroiditis by fine-needle aspiration. J Infect 1994; 28:7781.

316. Bernard 317. Hatabu

PJ, Som PM, Urken ML, et al. The CT findings of acute thyroiditis and acute suppurative thyroiditis. Otolaryngol Head Neck Surg 1988; 99:489493.

H, Kasagi K, Yamamoto K, et al. Acute suppurative thyroiditis associated with piriform sinus fistula: sonographic findings. Am J Med 1990; 155:845847.

318. Bartholomew 319. Chopra

LG, Cain JC, Woolner LB, et al. Sclerosing cholangitis: its possible association with Riedel's struma and fibrous retroperitonitisreport of two cases. N Engl J Med 1963; 269:813.

D, Wool MS, Grossen A, et al. Riedel's struma associated with subacute thyroiditis, hypothyroidism, and hypoparathyroidism. J Clin Endocrinol Metab 1978; 46:869871.

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F, Zelmanovitz T, Beck M, et al. Riedel's thyroiditis associated with high titers of antimicrosomal and antithyroglobulin antibodies and hypothyroidism. J Endocrinol Invest 1994;

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C, Vieillefond A, Desligneres S, et al. Hashimoto's thyroiditis associated with Riedel's thyroiditis and retroperitoneal fibrosis. Pathol Res Pract 1997; 193:573577.

322. Heufelder

AE, Goellner JR, Bahn RS, et al. Tissue eosinophilia and eosinophil degranulation in Riedel's invasive fibrous thyroiditis. J Clin Endocrinol Metab 1996; 81:977984.

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Chapter 13 - Nontoxic Goiter and Thyroid Neoplasia Martin-Jean Schlumberger Sebastiano Filetti Ian D. Hay

After thyroid dysfunction and neck pain, the discovery of an apparent structural abnormality of the thyroid gland is the most common reason for a patient to seek the expertise of a clinical thyroidologist. In this chapter, we review the imaging techniques available for evaluating thyroid structural abnormalities; the units of measurement used in evaluation of the radiation dose and radioactivity are defined in Table 13-1 . Goiter resulting in thyrotoxicosis and other thyroid conditions arising from autoimmune thyroid disease are considered in Chapter 11 and Chapter 12 . This chapter describes simple or nontoxic goiter in addition to the increasingly recognized problem of nodular thyroid disease. Moreover, thyroid neoplasia, both benign and malignant, is discussed authoritatively. We consider an appropriate histologic classification and staging of thyroid cancer and present a management program for the most common thyroid cancer types.



EVALUATION OF STRUCTURAL ABNORMALITIES BY IMAGING TECHNIQUES External Scintiscanning

Localization of functioning or nonfunctioning thyroid tissue in the area of the thyroid gland or elsewhere is made possible by techniques of external scintiscanning. The underlying principle is that isotopes that are selectively accumulated by thyroid tissue can be detected and quantified in situ and the data transformed into a visual display. Two types of apparatus are available. The rectilinear scanner is a device that moves a highly collimated (focused) scintillation detector back and forth across the area of study in a series of parallel tracks. A printing device records a mark whenever a predetermined number of counts has been received to provide a visual representation of the localization of radioactivity. The stationary scintillation camera has now replaced the rectilinear scanner in most centers. It is equipped with a pinhole collimator that views the entire field of interest and translates the counting rates from specific areas of the field into images. Radioactivity in specific areas can be quantified. These cameras provide better resolution than rectilinear scanners, but anatomic localization may be more difficult. [1] Several radioisotopes are employed in thyroid imaging. Technetium 99m ( 99m Tc) pertechnetate is a monovalent anion that is actively concentrated by the thyroid gland but undergoes negligible organic binding and diffuses out of the thyroid gland as its concentration in the blood decreases. The short physical half-life of 99m Tc (6 hours), its low fractional uptake, and its transient stay within the thyroid make the radiation delivered to the thyroid gland by a standard dose very low. Consequently, the intravenous administration of large doses (>37 MBq [1 mCi]) permits, about 30 minutes later, adequate imaging of the thyroid. Two radioactive isotopes of iodine have been used in thyroid imaging. Iodine 131 ( 131 I) was commonly used in the past and is still useful when functioning metastases of thyroid carcinoma are being sought; however, 131 I is a beta emitter, its physical half-life is 8.1 days, and the energy of its main gamma ray is high and thus poorly adapted for its detection. 123 I is, in many respects, ideal but is expensive. The energy of its main gamma ray is adapted for its detection by gamma cameras. Its short half-life (0.55 day) and the absence of beta radiation result in a radiation dose to the thyroid that is about 1% of that delivered by a comparable activity of 131 I.[2] [3] It is the isotope of choice for thyroid scintigraphy in pediatric practice.

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TABLE 13-1 -- Radiation Nomenclature: Traditional and International System (SI) Units Radiation dose

Abbreviation

1 Gy = 100 rad = absorption of 1 joule/kg

Gy = gray

1 rad = 0.01 Gy = 1 cGy

rad = radiation absorbed dose

1 Sv = 100 rem

rem = roentgen-equivalent-man

Radioactivity 1 Bq = 1 disintegration per second

mCi = millicurie Bq = becquerel

1 mCi = 37 MBq

kBq = kilobecquerel

1 GBq = 103 MBq = 106 kBq = 109 Bq

MBq = megabecquerel GBq = gigabecquerel

The most important use of scintigraphic imaging of thyroid tissue is to define areas of increased or decreased function ("hot" or "cold" areas, respectively) relative to function of the remainder of the gland, provided that they are 1 cm in diameter or larger. Almost all malignant nodules are hypofunctioning, but more than 80% of benign nodules are also nonfunctioning. Conversely, functioning nodules (hot nodules), particularly if they are either more active than surrounding tissue or the sole functioning tissue, are rarely malignant. In the past, several nuclear medical tests were used to evaluate thyroid disorders. In patients with a single area of thyroid uptake, scintiscans after administration of exogenous thyrotropin (TSH) may demonstrate the presence of hemiagenesis of the thyroid or document the functional capability of suppressed thyroid tissue. Conversely, scans performed after a period of exogenous thyroid hormone administration (suppression scans) can reveal areas of autonomous function that may not be detectable in baseline studies. These tests should no longer be used because the use of sensitive TSH assays and of scanning with a gamma camera permits the diagnosis of most of these hot nodules. Scintiscanning with radioactive iodine can also be used to demonstrate that intrathoracic masses represent thyroid tissue, to detect ectopic thyroid tissue in the neck, and to detect functioning metastases of thyroid carcinoma. The choice of the scanning agent depends on many factors. 99m Tc pertechnetate delivers a small dose of radiation to the thyroid gland, is readily available, and is inexpensive. Because imaging is performed soon after administration of the scanning agent, the entire procedure requires only a single visit to the laboratory. However, 5% to 10% of thyroid tumors appear to be functioning when examined with 99m Tc pertechnetate but not with radioiodine. Because 99m Tc pertechnetate imaging is done early, the intravascular activity and the activity in salivary tissue may obscure or confuse the findings. For the same reason, 99m Tc pertechnetate is inappropriate for scanning substernal or intrathoracic goiter or for detecting ectopic tissue in the neck. In these cases, radioactive iodine should be used. Total-body scanning is performed with 131 I in the follow-up of patients with papillary and follicular thyroid carcinoma. As detailed subsequently, radioiodine uptake by neoplastic tissue may be found only after TSH stimulation and is always lower than in normal thyroid tissue. For this reason, sufficiently high doses of 131 I should be given, and scanning should be performed 2 to 3 days after the dose (or even later), when background blood activity is low and when the contrast is optimal. Scanning conditions should be optimized, preferably by use of a gamma camera with two opposed heads equipped with thick crystals and high-energy collimators. Scanning at low speed with spot images on regions of interest is performed. There are two aims: (1) to verify the completeness of ablation and to detect and localize foci of uptake, and (2) to quantify any uptake. This quantification permits a dosimetric evaluation that indicates the usefulness of 131 I treatment.



Fluorescent Scan

Fluorescent scanning provides information concerning the content of stable iodine within the gland. [4] In this technique, discrete zones of the thyroid gland are subjected to radiation from radioactive americium ( 241 Am) or from an x-ray tube. When incident radiation encounters 127 I, a fluorescent x-ray, registered by a suitable detector, is emitted. Nonfunctioning nodules generally have a low iodine content and are therefore cold on fluorescent scan; thyroid iodine is depleted during subacute thyroiditis [5] and increased during chronic iodine overload, such as with amiodarone treatment. The technique has limited clinical utility.



Ultrasonography

Sonography is noninvasive, is less expensive than computed tomography (CT) or magnetic resonance imaging (MRI), and produces no known tissue damage. No special preparation of the patient is necessary, and the technique requires only portable equipment, allowing it to be performed in the physician's examining room. A major limitation of ultrasonography is a high degree of observer dependence. High-frequency sound waves are emitted by a transducer and reflected as they pass through the body, whereupon the returning echoes are received by the transducer, which also acts as a receiver. The amplitude of the reflections of the sound waves is influenced by differences in the acoustic impedance of the tissues encountered by the sound; for example, fluid-filled structures reflect few echoes and therefore have no or few internal echoes and well-defined margins; solid structures reflect varying amounts of sound and thus have varying degrees of internal echoes and less well-defined margins; and calcified structures reflect virtually all incoming sound and yield pronounced echoes with an acoustic "shadow" posteriorly. High-frequency sound waves, such as those used in current thyroid sonography, are attenuated rapidly in the body tissues. Therefore, they cannot be used to image structures deeper than about 5 cm from the skin. Fortunately, the thyroid gland is usually well within this limit and can be completely imaged. [6] High-frequency (7 to 13 MHz), small-parts instruments have become widely available since the middle 1980s and provide good spatial resolution and image quality. The theoretical axial resolution of these systems is about 1 mm; no other thyroid imaging method can achieve this degree of resolution. [6] Intrathyroidal nodules as small as 3 mm in diameter and cystic nodules as small as 2 mm can be readily detected. [8] Color flow Doppler ultrasonography allows visualization of very small vessels, so that vascularity of thyroid nodules can be assessed, but its diagnostic performance for malignancy is lower, as compared with fine-needle aspiration biopsy (FNAB).

[7]

Thyroid sonography is typically performed with the patient supine. The patient's neck is hyperextended by a pad centered under the scapulae to provide optimal exposure. The examiner usually sits at the head of the examining table and can steady the transducer by resting an elbow or a forearm on the table next to the patient's head. The thyroid gland must be examined thoroughly in transverse and longitudinal planes. Imaging of the lower poles can be enhanced by swallowing, which momentarily raises the thyroid gland in the neck. The examination should cover the entire gland, including the isthmus. Imaging should also include the region of the carotid artery and jugular vein to identify enlarged cervical lymph nodes. [6] The normal thyroid parenchyma has a characteristic homogeneous medium-level echogenicity, with little identifiable internal

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Figure 13-1 Transverse composite sonogram (A) and corresponding anatomic map (B) of the normal thyroid gland. C, common carotid artery; CVII, seventh cervical vertebra; LC, longus colli muscle; SM, strap muscles; SCM, sternocleidomastoid muscle; T, thyroid; TR, trachea. (From Rifkin MD, Charboneau JW, Laing FC. Special course: ultrasound 1991. In Reading CC [ed]. Syllabus: Thyroid, Parathyroid, and Cervical Lymph Nodes. Oak Brook, Ill, Radiological Society of North America, 1991, pp 363377.)

architecture (Fig. 13-1) . The surrounding muscles have the appearance of hypoechoic structures. The air-filled trachea in the midline gives a characteristic curvilinear reflecting surface with an associated reverberation artifact. The esophagus is usually hidden from sonographic visualization by the tracheal air shadow. A portion of the esophagus, however, may swing laterally, usually toward the left, where it may lie adjacent to the posteromedial surface of the thyroid. Neck ultrasonography may confirm the presence of a thyroid nodule when the findings on physical examination are equivocal. A diagrammatic representation of the neck showing the location or locations of any abnormal finding is a useful supplement to the routine film images recorded during an ultrasound examination. [6] Such a cervical map (Fig. 13-2) can help communicate the anatomic relationships of the pathology more clearly to the referring clinician and serves as a reference for the sonographer on follow-up examinations. In patients with known thyroid cancer, sonography can be useful in evaluating the extent of disease, both preoperatively and postoperatively. In most instances, sonography is not performed routinely before thyroidectomy but can be useful in patients with large cervical masses for evaluation of nearby structures (e.g., the carotid artery and internal jugular vein) to exclude the possibility of direct invasion or encasement by the tumor. Alternatively, in patients who present with cervical lymphadenopathy caused by papillary thyroid carcinoma (PTC) but in whom the gland is palpably normal, sonography may be used preoperatively to detect an occult, primary intrathyroid focus. Some surgeons do regularly obtain a preoperative sonogram in patients with PTC or medullary thyroid carcinoma (MTC) in order to identify prior to surgery the anatomic locations of any sonographically suspicious regional lymph nodes and thereby to permit planning of the extent of nodal dissection. Occasionally, a hand-held ultrasound probe can be used intraoperatively to identify impalpable residual cancer that has been identified by preoperative ultrasonography and proved to be cytologically positive by ultrasound-guided FNAB. After surgery for thyroid cancer, sonography is the preferred method for detecting residual, recurrent, or metastatic disease in the neck. [9] In patients who have undergone less than a near-total thyroidectomy, the sonographic appearance of the remaining thyroid tissue may be an important factor in the decision whether to recommend completion thyroidectomy. Also, it is more sensitive than neck palpation in detecting recurrent disease within the thyroid bed and metastatic disease in cervical lymph nodes. [10] [11] The location at the lower part of the neck and the sonographic appearance (hypoechoic, without a central echogenic line), the size (>1 cm in diameter), the

Figure 13-2 Cervical map, derived from sonographic images, helps to communicate anatomic relationships of pathology to clinicians and serves as a reference for follow-up examinations. SMG, submandibular gland. (From James EM, Charboneau JW, Hay ID. The thyroid. In Rumack CM, Wilson SR, Charboneau JW [eds]. Diagnostic Ultrasound, vol 1. St. Louis, MosbyYear Book, 1991, pp 507528.)

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shape (round), the presence of fine microcalcifications or a cystic component, and the use of color Doppler ultrasonography (hypervascularization) may aid in recognition of lymph node metastases. Sonography may also be useful to guide fine-needle biopsy of thyroid bed masses and lymph nodes, especially when these abnormalities are not palpable. [10] [11] [12]



Computed Tomography

The CT appearance of the anatomic structures depends on the attenuation of the tissue examined. The thyroid gland, because of its high concentration of iodine, has higher attenuation than do the surrounding soft tissues. [13] The diagnostic utility of CT in the evaluation of nodular thyroid disease is limited because thyroid masses, whether benign or malignant, may be hypodense, hyperdense, or isodense compared with adjacent normal thyroid tissue. [14] In aggressive pathologic processes, such as anaplastic thyroid carcinoma, CT can define the extension of the tumor to the mediastinum and its relationships to surrounding structures, such as the carotid artery, jugular vein, and trachea, before attempted surgical excision. In patients with known thyroid cancer, CT is less useful in evaluating recurrence in the neck because of the difficulty in detecting small masses in the indistinct tissue planes in the postoperative neck. [15] CT imaging can, however, improve the detection of lymph node metastases in the neck, although there is considerable overlap in the appearance of malignant and inflammatory nodes and CT lacks the ability to guide fine-needle biopsy of minimally enlarged nodes. [7] In patients with thyroid cancer, CT is used most frequently to search for lymph node metastases in the mediastinum and for distant metastases in the chest and abdomen. CT scanning can provide useful information regarding the presence and extent of intrathoracic (substernal) goiters. The CT findings of an intrathoracic mass in continuity with the thyroid gland, with high attenuation on noncontrast-enhanced images and marked enhancement after intravenous contrast material injection, all suggest intrathoracic goiter. [16] Radionuclide scanning can also be performed in this clinical setting, but false-negative results can occur when little or no functional tissue is present in the intrathoracic goiter. Because of the necessity of infusing iodine-containing contrast agents, CT should be performed at least 4 weeks before any radioiodine therapy. [15]



Magnetic Resonance Imaging

Because the hydrogen atoms of different tissues have different relaxation times (termed T1 and T2), a computer-assisted analysis of T1-weighted and T2-weighted signals is used to differentiate the thyroid gland from skeletal muscles, blood vessels, or regional lymph nodes. Normal thyroid tissue tends to be slightly more intense than muscle on a T1-weighted image, and tumors often appear more intense than normal thyroid tissue. MRI is rapidly evolving, with improvements in spatial resolution, reduction of artifacts, and development of new contrast agents. Currently obtained MR images have superior tissue contrast resolution but poorer spatial resolution than comparable CT images. Like CT, MRI does not distinguish benign from malignant nodules and does not assess functional status [17] ; however, it can define the anatomic extent of large goiters with great clarity. [18] Coronal and sagittal images provide a simultaneous view of the cervical and thoracic components of substernal goiters. The relation of the goiter to surrounding vessels in the mediastinum is also well visualized. [19] Recurrent neoplasms in the thyroid bed or regional lymph nodes can be detected with MRI; MRI is more accurate than palpation and comparable in accuracy to CT. [20] Recurrence is characterized by a mass with low to medium intensity on T1-weighted images and medium to high signal intensity on T2-weighted images. Conversely, scar tissue or fibrous tissue has low signal intensity on both T1-weighted and T2-weighted images. Tumor invasion of adjacent skeletal muscle has high signal intensity on T2-weighted images. [21] Edema or inflammation in the muscle can cause a similar appearance and can be difficult to differentiate from recurrent tumor. [17]



Positron Emission Tomography

Positron emission tomography (PET) is a special nuclear medical imaging technique that is both quantitative and tomographic. The radionuclide used emits a positron that is converted into a pair of photons after a short path of a few millimeters in the tissue. The coincidence detection of the two photons, which travel on a line in opposite directions, permits the localization of the site of the radionuclide decay. The agent most widely used with PET is [ 18 F]fluorodeoxyglucose (18 FDG). This agent is transported and phosphorylated as a glucose substitute but remains metabolically trapped inside tumor cells because of its inability to undergo glycolysis. PET scanners with a large field of view permit in vivo images related to regional glucose metabolism, with high sensitivity and a spatial resolution less than 5 mm. Superimposition of CT and PET images greatly improves both the sensitivity and specificity of the technique and the anatomic localization of any focus of abnormal uptake. Elevated glucose metabolism is present in most malignant tumor tissues, and PET scanning has been shown to be particularly useful for the detection of lymph node metastases in the neck or mediastinum in patients with papillary and follicular thyroid carcinoma who have no tumoral radioiodine uptake. [22] [23] [24] High uptake has also been observed in several thyroid diseases, such as thyroiditis, but PET cannot be used to differentiate benign from malignant thyroid nodules.



SIMPLE (NONTOXIC) GOITER: DIFFUSE AND MULTINODULAR Simple, or nontoxic, goiter may be defined as any thyroid enlargement that is not associated with hyperthyroidism or hypothyroidism and that does not result from inflammation or neoplasia. The term is usually restricted to the form that occurs sporadically or in regions that are not the locus of endemic goiter (i.e., where more than 10% of the children in the population have a thyroid enlargement) as a result of iodine deficiency. Although the term simple goiter is useful in indicating the presence of the characteristics just noted, the condition can be a result of different underlying abnormalities. [25] Pathogenesis and Pathophysiology

Goiter has been traditionally regarded as the adaptive response of the thyroid follicular cell to any factor that impairs thyroid hormone synthesis. This classic concept no longer appears to encompass the many aspects of goiters. Indeed, goiter is characterized by a variety of clinical, functional, and morphologic presentations, and whether this heterogeneity represents different entities remains to be clarified. Also, iodine deficiency as the sole factor responsible for goiter appears to be an oversimplification. Thus, not all inhabitants in an iodine-deficient region develop goiter; moreover, endemic goiter

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has been observed in countries with no iodine deficiency, and even with iodine excess, and has not been observed in some regions with severe iodine deficiency. These findings suggest that other factors, both genetic and environmental, may play a role in the genesis of simple and nodular goiter, and some of these factors may act synergistically. The role of genetic factors is suggested by several lines of evidence, 1. 2. 3. 4.

[26] [27]

such as

The clustering of goiters within families. The higher concordance rate for goiters in monozygotic than in dizygotic twins. The female/male ratio (1:1 in endemic versus 7:1 to 9:1 in sporadic goiters). The persistence of goiters in areas where a widespread iodine prophylaxis program has been properly implemented.

By studying families affected by goiter, researchers have been able to detect several gene abnormalities involving proteins related to thyroid hormone synthesis, such as mutations in thyroglobulin ( Tg), [28] sodium/iodide symporter (NIS), [29] thyroid peroxidase ( TPO), [30] pendrin syndrome (PDS), [31] and TSH receptor (TSHR) [32] genes. In addition, two loci for this disorder have been identified. The first locus, identified on chromosome 14q, was designated MNG1 (Online Mendelian Inheritance in Man [OMIM] 138800) for multinodular goiter 1 [33] ; the other, MNG2 (OMIM 300273), maps to chromosome Xp22.[34] Although an autosomal dominant inheritance has been demonstrated in several families, multiple genes may be involved in other families. This may explain why predisposing gene alterations remain unidentified in most patients with simple goiter. Such genetic predispositions are believed to cause abnormalities in thyroid hormone synthesis. Thus, in some cases, defects can be detected by abnormalities of perchlorate discharge (see Chapter 10) ; more often, however, no abnormality can be demonstrated. [35] Goiter should thus be regarded as a complex trait in which both genetic susceptibility and environmental factors probably contribute to the development of disease. Whereas iodine deficiency represents the main environmental factor in the genesis of endemic goiter, other factors, such as cigarette smoking, infections, drugs, and goitrogens, may play a role in the genesis of goitrous disease together with a genetic background of susceptibility. Interestingly, in a population-based twin study, a critical role of the genetic background in the etiology of goiter was demonstrated in females. [27] TSH has long been considered the major agent determining thyroid growth in response to any factor that impairs thyroid hormone synthesis. When such factors are operative, hypersecretion of TSH stimulates thyroid growth and increases the aspects of hormone biosynthesis that are capable of response. As a consequence of the increase in thyroid mass and functional activity, a normal rate of hormone secretion is restored and the patient is goitrous but eumetabolic. Indeed, in the rare clinical setting of functioning TSH-secreting pituitary tumor, the increased blood TSH levels typically cause an enlargement of the thyroid gland. [36] It is interesting that goiter is also a typical part of the clinical picture of Graves' disease, in which a stimulatory growth effect on thyroid tissue is induced by thyroid-stimulating antibody through TSHR activation. [37] Moreover, thyroid enlargement may appear during the course of Graves' disease when increased TSH levels result from overtreatment with antithyroid drugs. In addition, toxic thyroid hyperplasia is usually present in non-autoimmune autosomal dominant hyperthyroidism, a disorder related to germ lineactivating mutations of the TSHR gene.[32] This clinical condition further emphasizes the role of TSH-TSHR system activation in the genesis of thyroid hyperplasia in diffuse nontoxic or toxic goiter. This concept of the pathogenesis of nontoxic goiter is inconsistent with the fact that the serum TSH concentration is normal in most patients with nontoxic goiter. [38] [39] Nonetheless, a participatory role of TSH in the maintenance of goiter is indicated by the regression of goiter that sometimes follows administration of suppressive doses of thyroid hormone. Several possible mechanisms may accommodate these apparently divergent findings. The mechanism with experimental support in rats is that iodine depletion enhances the promotion of thyroid growth by TSH. [39] Hence, any factor that impairs intrathyroidal iodine levels may lead to gradual development of goiter in response to normal concentrations of TSH. A second possibility is that the increase in serum TSH concentration is significant but too small to be detected by immunoassay methods. Finally, a goitrogenic stimulus may have been present in the past but may no longer be detectable at the time of study. Thus, the residual normal TSH concentration can maintainbut not initiatethe goiter. However, this primary, if not exclusive, role for TSH in determining thyroid growth and hyperplasia has been challenged. [40] [41] [42] Indeed, a complex network of both TSH-dependent and TSH-independent pathways directs thyroid follicular cell growth and function and plays a role in the goitrogenic process. In particular, a variety of growth factors, derived either from the blood stream or through autocrine or paracrine secretion, may serve to regulate thyroid cell proliferation and differentiation processes. [43] Among these factors, epidermal growth factor (EGF) and insulin-like growth factor (IGF) have been recognized as thyroid growthpromoting substances in different species. IGF-I stimulates cell proliferation and differentiation (i.e., thyroglobulin expression) in thyroid tissue both in vitro and in vivo. Indeed, enhanced IGF-I expression may play a role in the goitrogenic process. In this regard, it is worth emphasizing that acromegalic patients with elevated levels of serum growth hormone and IGF-I and normal TSH levels have an increased prevalence of goiter. [44] Similarly, fibroblast growth factor (FGF) stimulates thyroid function, and its expression has been associated with thyroid hyperplasia. Interestingly, the proliferation effect of these growth factors also occurs through stimulation of their respective receptors in thyrocytes. Other factors, including IGF-binding proteins (IGF-BPs), transforming growth factor and , cytokines, prostaglandins, norepinephrine, acetylcholine, and vasoactive intestinal peptide, may also participate in the regulation of thyroid cell proliferation. However, the relative contribution of these factors to the goitrogenic process has not yet been clarified. In the propylthiouracil-induced goiter in the rat, Wollman and colleagues [45] recognized the importance of the development of new blood vessels in goiter formation and demonstrated that growth of perifollicular blood vessels was induced by angiogenic factors produced by follicular cells. Indeed, many molecules involved in promoting or inhibiting thyroid angiogenesis, including vascular endothelial growth factor, angiopoietins 1 and 2, hepatocyte growth factor, endothelin, angiogenin or thrombospondin, angiostatin, and endostatin, have now been identified. [46] Goitrogenesis, therefore, appears to be a complex process in which TSH, growth factors, and angiogenic substances either play a distinct and separate role or act

synergistically through complex interaction mechanisms. Another pathogenetic concept is based on autoradiographic and clinical studies of normal thyroid tissue and nontoxic and toxic multinodular goiters. [47] Early in the course of goiter formation, areas of microheterogeneity of structure and function are intermixed and include areas of functional autonomy and small areas of focal hemorrhage. Indeed, as judged from the presence of scattered foci of persistent radioiodine uptake in the thyroid glands of patients given suppressive doses of thyroid

462

Figure 13-3 Outer and cut surfaces of a nontoxic nodular goiter of 15 years' duration. Note variations in size and structure of the nodules; there are thick areas of fibrous tissue, flecks of calcium, scattered areas of thyroid tissue, cysts, and small hemorrhages.

hormone before surgery, some cells with functional autonomy are present in the normal thyroid gland; this is in accordance with the heterogeneous staining for NIS observed in normal thyroid and goitrous tissues. [48] Thus, in addition to the variability in thyroid microcirculation, heterogeneity may result from clonal differences among cells that give rise to thyroid follicles, some being more and some less responsive to external stimulation factors, including TSH, and others being autonomous from the outset. This concept implies that the anatomic and functional heterogeneity observed within the thyroid at the outset of the disease is exaggerated by prolonged stimulation. Further insights into the pathogenesis of sporadic multinodular goiter have been gained by assessment of the clonality of individual thyroid nodules. Polyclonality implies a multicellular origin related to the proliferation of a group of cells, whereas a monoclonal tumor is thought to be formed by expansion of a single cell. [49] Studies involving X-chromosome inactivation analysis have produced variable results in multinodular goiters. Some dominant nodules are monoclonal, especially if they showed evidence of recent rapid growth. [50] Other researchers have found a monoclonal pattern in only a minority of large nodules. [51] Two groups reported that in multinodular glands more than one nodule can be monoclonal, and both monoclonal and polyclonal nodules can coexist within the same gland. [52] [53] Analysis of hyperplastic nodules by rigid criteria [54] also indicated that morphologically indistinguishable hyperplastic thyroid nodules may be either monoclonal or polyclonal. Monoclonal adenomas within hyperplastic thyroid glands may reflect a stage in progression along the hyperplasia-neoplasia spectrum; accumulation of multiple somatic mutations may subsequently confer a selective growth advantage to this single-cell clone. [54] Cytogenetic [55] and in situ hybridization follicular adenomas. [57]

[56]

studies also support the idea of a biologic continuum and karyotypic evolution between hyperplastic nodules and true

Eventually, the amount of functionally autonomous tissue in a multinodular goiter may be sufficient to suppress TSH secretion. [38] [58] Ultimately, autonomous hyperfunction may be sufficient to produce subclinical or overt thyrotoxicosis, or thyrotoxicosis may supervene when the patient is exposed to an iodine load. For this reason, patients with nontoxic multinodular goiter should not be given medications that contain iodine and should be observed after radiologic procedures that involve administration of iodinated contrast media. Some investigators administer antithyroid agents to patients with nodular goiter who are to receive agents containing iodine. This is a reasonable suggestion, especially in areas of iodine deficiency where jodbasedow is likely to occur. Nontoxic goiter has a female preponderance (7:1 to 9:1) and seems to be common during adolescence or pregnancy. There appears to be no physiologic increase in thyroid volume during normal adolescence, and development of a goiter during adolescence is a pathologic rather than a physiologic process. [59] However, as evidenced by sonographic measurement of thyroid volume in women living in an area of moderate iodine intake, normal pregnancy is goitrogenic, especially in women with preexisting thyroid disorders. [60] The increased thyroid volume during pregnancy is associated with biochemical features of thyroid stimulation (i.e., an increased triiodothyronine/thyroxine [T 3 /T4 ] ratio) owing to slightly elevated serum TSH levels at delivery or a high human chorionic gonadotropin (hCG) concentration during the first trimester. [60] [61] [62] Repeated pregnancies may play a role in the development of later thyroid disorders, a relation that might explain the high prevalence of thyroid disorders in women.[63]



Pathology

Simple goiter is a noninflammatory, non-neoplastic, diffuse or nodular enlargement of the thyroid gland without hyperthyroidism. [64] The gland is usually large and may have a distorted shape (Fig. 13-3) . The cut surface shows areas of nodularity, fibrosis, hemorrhage, and calcification. The nodules vary in size, number, and appearance, the last according to their colloid or cellular content. Single or multiple cystic areas may contain colloid or brown fluid, representing previous hemorrhage. Histologically, nodules contain irregularly enlarged, involuted follicles distended with colloid or clusters of smaller follicles lined by taller epithelium and containing small colloid droplets. These microfollicles may be surrounded by an edematous or a fibrous stroma. Large nodules tend to compress the surrounding parenchyma and may have a partially developed fibrous capsule. Markedly distended follicles may coalesce to form colloid cysts several millimeters in diameter. The nodules tend to be incompletely encapsulated and are poorly demarcated from and merge with the internodular tissue, which also has an altered architecture. However, the nodules in some glands appear to be localized, with areas of apparently normal architecture elsewhere. Here, the distinction from a follicular adenoma may be difficult, and some pathologists apply terms such as colloid or adenomatous nodules to such lesions. Studies of clonality may be helpful in distinguishing between focal or nodular hyperplasia and true adenomas. [65] [66] Whereas nodular goiters are polyclonal in origin, solitary thyroid nodules are monoclonal and therefore true benign neoplasms. [51]



Clinical Picture

Diffuse or nodular goiters are usually not associated with abnormal thyroid hormone secretion. Therefore, affected patients

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do not exhibit clinical symptoms or signs of thyroid dysfunction. The only clinical features of nontoxic goiter are those of thyroid enlargement. Nearly 70% of patients with sporadic nontoxic goiter complain of neck discomfort; the remainder have cosmetic concerns or a fear of possible malignancy. [67] Large goiter, which may displace or compress the trachea, esophagus, and neck vessels, can be associated with symptoms and signs including inspiratory stridor, dysphagia, and a choking sensation. These obstructive symptoms may be accentuated by the so-called Pemberton maneuver. This maneuver, which consists of "elevating both arms until they touch the sides of the head," is considered positive if, after a minute or so, congestion of the face, some cyanosis, and lastly distress become apparent. [68] Compression of the recurrent laryngeal nerve, with hoarseness, suggests carcinoma rather than nontoxic goiter, but vocal cord paralysis can occasionally result from benign nodular goiters. [69] Hemorrhage into a nodule or cyst produces acute, painful enlargement locally and may enhance or induce obstructive symptoms. Endogenous subclinical thyrotoxicosis caused by autonomously functioning nodules should be carefully investigated. It is particularly relevant in elderly patients, whose cardiac morphology and function may be affected, thereby increasing their risk of developing cardiac arrhythmias. [70]



Laboratory Tests

Serum TSH, measured in a highly sensitive immunometric assay, combined with a single measurement of free thyroid hormone concentrations may be used as a first-line screening test. Serum free thyroid hormones and TSH are, by definition, within the normal range. However, the T 3 /T4 ratio may be increased, perhaps reflecting defective iodination of Tg. Patients with sporadic nontoxic goiter tend to have high-normal free T 4 and T3 concentrations and low TSH levels. [67] The prevalence of so-called subclinical hyperthyroidism is higher when patients with nodular goiter have clear-cut autonomous areas on scintigraphy. [71] An undetectable serum TSH, even associated with normal free thyroid hormone levels, should suggest the possibility of toxic, autonomously functioning nodular areas in the goiter. Such a finding should prompt further cardiac investigation, especially in elderly patients, whose risk of atrial fibrillation may be increased as much as threefold when serum TSH levels are less than 0.1 mU/L. [72] Moreover, it has been demonstrated that this condition, by affecting cardiac morphology and function, has a relevant clinical impact even in young patients and that many patients are, in fact, symptomatic. Therefore, this disorder should be considered a mild form of tissue thyrotoxicosis that may necessitate treatment. [73] In a cross-sectional study of 102 patients with sporadic nontoxic goiter, the serum TSH level correlated negatively with the thyroid volume, which in turn correlated positively with both the age of the patient and the duration of the goiter. [67] In a prospective study of 242 patients with nodular goiter, no correlation was found between thyroid volume and any thyroid biochemical parameters, but there were significant negative correlations between the number of nodules identified by ultrasonography and the levels of basal TSH and the TSH response to thyrotropin-releasing hormone (TRH) stimulation. [71]



Imaging in Goiter Evaluation

A diagnosis of goiter usually does not warrant the use of imaging procedures. When a nodular goiter is present, however, both scintigraphy and sonography provide useful information for disease management and treatment. Indeed, the former should be used to detect hot or warm nodules in the thyroid tissue. This finding affects the therapeutic approach. Sonography should be used to assess both morphology and size of the goiter. [74] Thus, sonography in patients with a nodular goiter may allow a determination of the number and the individual features of the nodules and serve as guidance for FNAB. [75] Sonography also permits an accurate, objective measure of goiter growth over time or after treatment. Conventional radiography of the neck and the upper mediastinum should be used to determine the presence of tracheal compression. CT and MRI are indicated in the presence of intrathoracic goiter to define the relationships with surrounding structures.



Differential Diagnosis

The differential diagnosis of nontoxic goiter can be considered in functional and anatomic terms. As indicated, the same factors that lead to goitrous hypothyroidism can, if they are less severe, cause nontoxic goiter. Consequently, some patients with putative nontoxic goiter are slightly hypothyroid. On the other hand, foci of autonomous function may develop in multinodular goiters in which the spectrum of function can range from clinical euthyroidism with intact regulatory control to euthyroidism with some degree of functional autonomy to thyrotoxicosis. [76] Anatomically, the diffuse stage of nontoxic goiter can resemble the thyroid of either Graves' or Hashimoto's disease. If Graves' disease is not in an actively thyrotoxic phase, and if the ocular manifestations are lacking, there is no way to distinguish between the two except to demonstrate the presence of TSHR antibody in the serum. In one study of 108 patients with diffuse nontoxic goiter observed for more than 5 years, 33% had a family history of autoimmune thyroid disease and five patients developed Graves' disease during follow-up. [77] Diffuse nontoxic goiter is sometimes also difficult to differentiate from Hashimoto's disease, although the thyroid of Hashimoto's disease is usually firmer and more irregular. Demonstration of high titers of antithyroid antibodies should indicate autoimmune disease. In its multinodular stage, nontoxic goiter may suggest thyroid carcinoma. The approach to distinguishing between the two is discussed in the following section on thyroid neoplasms.



Treatment

Patients with small, asymptomatic goiters can be monitored by clinical examination and evaluated periodically with ultrasound measurements. In fact, goiter growth can be variable, and some patients have stable goiters for many years. For more than a century, thyroid "feeding" has been employed to reduce the size of nontoxic goiters. [78] The 1953 report of Greer and Astwood, in which two thirds of patients' goiters regressed with thyroid therapy, led to widespread acceptance of suppressive therapy [79] despite some doubts about the value of such therapy. [80] [81] An overview of studies performed from 1960 to 1992 suggested that 60% or more of sporadic nontoxic goiters respond to suppressive therapy. [80] In a prospective placebo-controlled, double-blind randomized clinical trial, 58% of the thyroxine-treated group had a significant response at 9 months, as measured by ultrasonography, in contrast with 5% after placebo. [82] However, ultrasonographic measurement of goiter size demonstrated a return to pretherapy values within 3 months of treatment discontinuation. [83] Therefore, maintenance of the size reduction may require continuous long-term treatment. Nodular goiters appear to be less responsive than diffuse goiters, and the therapeutic efficacy of thyroxine treatment is increased in younger patients and in those with small or recently diagnosed goiters. [80] It has been proposed that a basal serum TSH greater than

464

1 mU/L in a patient with sporadic nontoxic goiter is an indication to administer levothyroxine to lower the serum TSH level to the low-normal range (0.5 to 1.0 mU/L). Others[84] have suggested that TSH levels on treatment should be subnormal but not profoundly suppressed (0.1 to 0.3 mU/L). The validity of this approach remains to be ascertained. If the goiter size decreases or remains stable, treatment should be continued indefinitely, with periodic monitoring of serum TSH levels to detect possible development of functional autonomy. [85] A major concern in relation to long-term thyroxine suppression therapy is the possibility of detrimental effects on the skeleton and heart. [86] It has been reported that TSH suppression therapy is associated with variable degrees of bone loss, particularly in postmenopausal women. [87] [88] However, other studies did not demonstrate significant change in bone mass after long-term thyroxine therapy. [89] [90] Furthermore, although marginal cardiac changes may occur with levothyroxine therapy, there is no evidence that levothyroxine per se is detrimental to the heart. [86] It is now generally accepted that TSH should be suppressed with the lowest effective dose of levothyroxine, [80] [85] [86] [89] usually between 1.5 and 2.0 µg/kg body weight per day; the risk of deleterious effects may be minimized by monitoring serum TSH and free T3 concentrations.[86] Surgery for simple nontoxic goiter is physiologically unsound because it further restricts the ability of the thyroid to meet hormone requirements. Nevertheless, surgery may become necessary because of persistence of obstructive manifestations despite a trial of levothyroxine. Surgery, which should consist of a near-total or total thyroidectomy, rapidly and effectively removes the goiter, but recurrence is seen in about 10% to 20% within 10 years. [91] Surgical complications have been reported in 7% to 10% of cases and are more common with large goiters and with reoperation. [92] Prophylactic treatment with levothyroxine after goiter resection probably does not prevent recurrence of goiter. [93] [94] Traditionally, the role of 131 I therapy for nontoxic goiter was to reduce the size of a massive goiter in elderly patients who were poor candidates for surgery [95] [96] or to treat goiter that recurs after resection. [97] However, several studies have demonstrated that primary treatment of multinodular goiter with 131 I is followed by a reduction in thyroid volume (Fig. 13-4) .[98] [99] In

Figure 13-4 Median changes in thyroid volume alterations after iodine 131 treatment in 39 patients with nontoxic multinodular goiter who remained euthyroid after a single dose. Bars represent quartiles. (From Nygaard B, Hegedus L, Gervil M, et al. Radioiodine treatment of multinodular nontoxic goiter. BMJ 1993; 307:828832.)

one study, thyroid volume (assessed by ultrasonography) was reduced by 40% after 1 year and 55% after 2 years with no further reduction thereafter, the total reduction occurred within the first 3 months.

[98]

and 60% of

A randomized trial comparing levothyroxine at suppressive doses with radioactive iodine treatment (120 µCi/g corrected for 24-hour thyroid uptake) showed impressive differences in outcome. After 131 I therapy, 97% of patients responded, with a mean decrease in goiter size of 39% at 1 year and 46% at 2 years; the initial side effects were neck tenderness and slight thyrotoxic symptoms in 12% of patients, and at 2 years 35% of patients were hypothyroid and 10% had subclinical thyrotoxicosis. In contrast, with levothyroxine therapy 43% of patients responded with a mean decrease of 23% at 1 year and 22% at 2 years; the initial side effect was a mild thyrotoxicosis in 30% of patients and at 2 years a significant decrement in spine bone density. [88] It was formerly argued that treatment of large goiters or goiters with substernal extension with 131 I should be avoided because of the risks of acute swelling of the gland and consequent tracheal compression. [100] Ultrasonographic studies of thyroid volume after 131 I have failed to demonstrate significant early volume increase. [101] Moreover, decreased tracheal deviation and increased tracheal lumen size were demonstrable by MRI in patients who had compression by nontoxic goiters with substernal extension. [96] Therefore, it appears that 131 I treatment of nontoxic multinodular goiter is effective and safe, [81] but hypothyroidism may occur in 22% to 40% within 5 years after 131 I therapy. [98] [99] Regular follow-up, preferably by a systematic annual recall scheme, is necessary. [102] Although reassuring data are available on the long-term thyroid and nonthyroidal cancer risk after 131 I treatment in hyperthyroidism,[103] [104] the follow-up of patients with 131 I-treated nontoxic goiters is short-term and involves small numbers of patients. Children and adolescents should not be treated with 131 I. Stimulation with low doses of recombinant human TSH (rhTSH) (0.01 to 0.03 mg) increases the thyroid 131 I uptake and therefore may allow the administration of a lower dosage of 131 I. [105] Long-term randomized studies comparing the effects, side effects, and costs and benefits of surgery and 131 I treatment need to be performed.[81]



THYROID NEOPLASIA In an era when patients are advised on self-examination to detect cancer at an early stage, the finding of a palpable mass in such a superficial location as the thyroid gland can be disconcerting. The affected patient is likely to seek medical evaluation. At the end of an appropriate investigation, the clinician can usually reassure the patient that the nodule is benign. Alternatively, if the evaluation does suggest malignancy, the patient can be advised that the management of typical thyroid cancer is effective and usually consists of surgical resection, [106] followed by medical therapy [107] and regular surveillance. [108] The major challenge in this circumstance is to determine whether the discovered thyroid nodule is malignant. Some degree of consensus has been achieved with regard to both the initial evaluation of nodular thyroid disease [109] [110] [111] and the management of differentiated thyroid cancer,[112] [113] [114] but important clinical and biologic questions remain unanswered. [115] [116] [117] In the following discussion, we describe a clinical approach to nodular thyroid disease and present a widely used scheme for classifying and staging tumors of the thyroid gland. We also review the features of the principal types of benign and malignant thyroid neoplasms and the controversies in the management of differentiated thyroid carcinoma.

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Initial Investigation

Thyroid tumors are the most common endocrine neoplasms. They usually arise as anterior neck nodules that usually can be localized to the thyroid gland by palpation. Most of these nodules are benign hyperplastic (or colloid) nodules or benign follicular adenomas, but about 5% to 10% of nodules coming to medical attention are carcinomas. Differentiating true neoplasms from hyperplastic nodules and distinguishing between benign and malignant tumors are major challenges. Moreover, with the widespread practice of medical checkups in healthy individuals and the increasing use of imaging technology, this problem is likely to become more common. High-resolution ultrasound studies suggest that the prevalence of nodular thyroid disease in healthy adults is above 60%. [118] However, during 2001 in the United States, only about 19,500 new cases of thyroid cancer were likely to be diagnosed. [119] Therefore, most of these so-called thyroid incidentalomas are obviously benign and do not progress to clinical tumors. [120] In identifying the nodules that are likely to be malignant, a thorough history and a careful physical examination should be supplemented with laboratory testing, imaging procedures, and, most important, FNAB of the nodule in question. With the use of this approach, it is possible to assess the likelihood of malignancy and to advise appropriate treatment in the majority of patients. History and Physical Examination

Historical features that favor benign disease include the following: 1. A family history of Hashimoto's thyroiditis, benign thyroid nodule, or goiter. 2. Symptoms of hypothyroidism or hyperthyroidism. 3. A sudden increase in size of the nodule with pain or tenderness, suggesting a cyst or localized subacute thyroiditis. Historical features that suggest malignancy include the following: 1. 2. 3. 4. 5.

Young (70 years old) age; Male sex; A history of external neck radiation during childhood or adolescence; Recent changes in speaking, breathing, or swallowing; A family history of thyroid cancer or multiple endocrine neoplasia (MEN) type 2.

On physical examination, manifestations of thyroid malignancy should be sought, including firm consistency of the nodule, irregular shape, fixation to underlying or overlying tissues, and suspicious regional lymphadenopathy. In both prospective [121] and retrospective [122] [123] studies, the sensitivity and specificity rates for detecting thyroid malignancy by history and physical examination were about 60% and 80%, respectively. In these historical series, only about 20% of patients with later confirmed malignancy had, when initially seen, neither suspicious historical features nor evidence of potential malignancy on neck examination. Further testing may include assessment of thyroid function, measurement of tumor markers, genetic screening, thyroid imaging, and the only decisive parameter, FNAB. Laboratory Evaluation

The serum TSH level is measured to exclude thyroid dysfunction. Patients with thyroid cancer rarely have abnormalities in serum TSH levels. A low (suppressed) serum TSH level may indicate a toxic nodule and should lead to thyroid scintigraphy. Measurement of serum anti-TPO antibody and anti-Tg antibody levels may be helpful in diagnosis of chronic autoimmune thyroiditis, especially if the serum TSH level is elevated. In chronic autoimmune thyroiditis, the thyroid gland's size and consistency may simulate either a solitary nodule or bilateral nodules. Evidence of autoimmune thyroiditis, however, does not preclude the presence of cancer in the gland. Follicular cellderived thyroid cancers (FCTCs) may release increased amounts of Tg into the blood stream. Unfortunately, there is overlap of serum Tg levels in FCTCs and in a number of benign conditions, and measurement of serum Tg levels is not useful in the initial work-up of nodular thyroid disease. [111] Similarly, some investigators [124] routinely measure calcitonin (Ct) levels in all patients with nodular thyroid disease to identify cases of MTC. In fact, the calcitonin level is increased in virtually all patients with clinical MTC. However, because of the rarity of unsuspected MTC, the high frequency of false-positive results that may prompt a thyroidectomy despite a reassuring cytologic result, and the unknown clinical relevance of medullary microcarcinomas, it is neither cost-effective nor necessary to measure calcitonin levels in patients with nodular thyroid disease in the absence of clinical suspicion of MTC or abnormal cytologic findings. The molecular abnormality in more than 95% of familial MTC cases is a germline mutation of the RET proto-oncogene that is located on the long arm of chromosome 10.[125] Many investigators advocate RET mutation testing in all patients with MTC, including apparently sporadic cases, because 4% to 6% of such patients have germline mutations of the gene (see Chapter 36) .[126] Such tests are highly accurate, reproducible, and reliable. If a mutation is found, family members at risk are then tested to identify affected individuals. A negative result obviates the need for any further testing, and individuals who harbor such mutations should undergo prophylactic total thyroidectomy to prevent later development of the multicentric MTC that occurs in this disorder. [127] Thyroid Imaging

The traditional imaging procedure is thyroid scintigraphy using 131 I, 123 I, or 99m Tc. Most thyroid carcinomas are inefficient in trapping and organifying iodine and appear on scans as areas of diminished isotope uptake, so-called cool or cold nodules. Unfortunately, most benign nodules also do not concentrate iodine and therefore are cold nodules. Furthermore, not all nodules with normal or slightly increased 99m Tc uptake are benign and may appear cold on a thyroid scan with radioactive iodine. The only situation in which an iodine scan can exclude malignancy with reasonable certainty is in the case of a toxic adenoma, which is characterized by significantly

increased uptake within the nodule and markedly suppressed or absent uptake in the remainder of the gland. These lesions account for fewer than 10% of thyroid nodules and are almost invariably benign. [128] When isotopic thyroid scanning is compared with history and physical examination, most authors have found scanning to be of negligible or no value for the diagnosis of malignancy. [129] [130] In an attempt to improve the performance of isotopic scanning, a number of radioisotopes other than iodine-related compounds have been tried, such as thallium 201 (201 Tl)[131] [132] and 99m Tc-labeled methoxyisobutyl isonitrile (MIBI). In the hands of dedicated experts, these techniques may be valuable, but they are expensive and their widespread use must await more extensive evaluation. Ultrasonography is capable of detecting even minute thyroid nodules and increases the sensitivity of carcinoma detection but does little to enhance specificity. In fact, of 1000 normal control subjects, 65% had detectable nodularity on high-resolution scanning. [118] Attempts have been made to develop criteria

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TABLE 13-2 -- Probability of Malignancy at Histology Based on Fine-Needle Aspiration Biopsy Cytology (Summary of the Literature) Cytology Percent of Results (%), Mean (Range) Probability of Malignancy (%), Range Inadequate or nondiagnostic

16 (1520)

1020

Benign

70 (5390)

12

Suspicious*

10 (523)

1020

4 (110)

>95

Malignant

*The suspicious category includes follicular neoplasms (hyperplastic nodules, follicular adenomas, and follicular carcinomas) and some Hürthle cell tumors.

for distinguishing benign and malignant nodules. Echo-free (cystic) and homogeneously hyperechoic lesions are reputed to carry a low risk of malignancy. [133] [134] Positive predictive criteria of malignancy include solid hypoechoic nodules, presence of calcifications, irregular shape, absence of halo, and absence of cystic elements; however, in one study only 64% of malignant nodules displayed patterns typical of malignancy. [135] In addition, nodules that can be clearly identified as benign by sonography are uncommon, limiting the usefulness of ultrasound scanning. Ultrasonography is useful in identifying hypoechoic nodules that should be submitted to FNAB and also in examining the rest of the thyroid gland and lymph node areas. [75] It may also be used in case of nonpalpable nodules to guide FNAB, especially when the diameter of the nodule is 1 cm or more. [12] Cystic lesions may be treated by aspiration of the fluid and ethanol injection to avoid recurrence; this is optimally performed under ultrasonographic guidance. [136] CT scanning and MRI in the initial diagnosis of thyroid malignancy do not provide higher-quality images of the thyroid and cervical nodes than those of ultrasonography. CT examination of the lower central neck is preferable when tracheal or mediastinal invasion is suspected.

Figure 13-5 Sonographically guided thyroid nodule fine-needle aspiration. Transverse sonogram of the right thyroid lobe ( A, left panel) shows a 1.5-cm solid thyroid nodule (arrows) containing a central cystic component. C, common carotid artery; J, jugular vein. Palpation-guided aspiration biopsy obtained nondiagnostic fluid only. B, right panel, shows sonographically guided needle aspiration biopsy (curved arrow) of the solid portion of the nodule, which proved that this was a benign adenomatous nodule. (From Rifkin MD, Charboneau JW, Laing FC. Special course: ultrasound 1991. In Reading CC [ed]. Syllabus: Thyroid, Parathyroid, and Cervical Lymph Nodes. Oak Brook, Ill, Radiological Society of North America, 1991, pp 363377.) Fine-Needle Aspiration Biopsy

FNAB of thyroid nodules has eclipsed all other techniques for diagnosing thyroid cancer, with reported overall rates of sensitivity and specificity exceeding 90% in iodine-sufficient areas. [137] [138] The technique is easy to perform and safe, with only a handful of complications having been reported in the literature, [139] [140] and causes little discomfort. However, care must be taken to obtain an adequate specimen; most authors recommend between three and six aspirations. [137] [138] A satisfactory specimen contains at least five or six groups of 10 to 15 well-preserved cells. The cells are categorized by their cytologic appearances into benign, indeterminate or suspicious, and malignant (Table 13-2) . The diagnosis of PTC by FNAB on the basis of characteristic nuclear changes is particularly reliable and accurate, with sensitivity and specificity both approaching 100%. For follicular neoplasms, however, the performance of FNAB is inferior. If strict criteria for malignancy are used, sensitivity may be as low as 8%. [123] If any follicular neoplasm that is not clearly benign on cytologic examination is classified as cancerous, sensitivity rises to about 90% or more. Unfortunately, this increase is associated with a considerable drop in specificity to less than 50% (i.e., a large number of false-positive results). This seriously limits the usefulness of FNAB in iodine-deficient regions, where the incidence of follicular thyroid carcinoma (FTC) approaches that of PTC and where both follicular adenomas and hyperplastic adenomatous nodules are prevalent. TPO immunochemistry with a monoclonal antibody (MoAb 47) shows promise in improving the accuracy of FNAB for follicular lesions. [141] For 100% sensitivity, a specificity of almost 70% has been achieved with this technique. Pending independent confirmation of these results, TPO immunocytochemistry may be a valuable adjunct to the standard cytologic techniques. The use of large-needle biopsy in addition to standard FNAB has improved diagnostic accuracy in difficult FNA cases, [143] but the technique is more exacting than FNAB alone and is associated with increased morbidity and, possibly, increased complication rates.

[ 142]

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Particularly for cystic thyroid nodules, sampling from the margin of the nodule, rather than from the cystic fluid and debris in the center, increases accuracy. [137] Ultrasonographically guided FNA can be used for this purpose (Fig. 13-5) . Although such guided biopsies are sometimes helpful, routine use of ultrasound-guided biopsy for clinically palpable nodules is not any better than "freehand" aspiration. [12] [144] However, some centers are evaluating this approach to allow recognition and FNA of nonpalpable nodules 1 cm or smaller in size and to reduce the number of passes to three. [75] In some European centers, both preoperative FNAB and intraoperative frozen section are combined in endemic goitrous regions with high rates of follicular tumors. In the hands of experienced surgeon-pathologist teams, this approach results in less than 5% misdiagnoses, as evidenced by subsequent review of paraffin-embedded specimens. The approach avoids unnecessarily extensive surgery in patients with benign tumors, achieves resection of nearly all malignant tumors, and rarely necessitates a second operation for completion thyroidectomy. [145] Such an approach is employed at the Mayo Clinic and at the Institut Gustave Roussy, where intraoperative frozen section is routine.

[145]

Apart from its limited utility in the evaluation of follicular neoplasms, the only other limitation of FNAB is nondiagnostic specimens, which may be obtained in up to 20% of cases.[138] Although repeated aspiration increases both the accuracy and the rate of diagnostic aspirations, even repeated attempts may sometimes fail. Many persistently nondiagnostic FNAB specimens may be neoplastic, possibly 50%. [146] Hence, either close observation or surgical removal of the nodule is probably the best option. Some authorities recommend a trial of TSH suppression, which can sometimes shrink benign nodules. [137] However, a significant proportion of benign nodules do not shrink, and some carcinomas do shrink; consequently, the diagnostic value of TSH suppression is doubtful. Whether ultrasound-guided FNAB can help overcome this problem is unclear, but confirmation is required. [12] [75] [144] Figure 13-6 is an algorithm for the management of nodular thyroid disease in which FNAB is the first diagnostic test and subsequent management is based on cytologic results.

The most expeditious way to diagnose thyroid malignancy is to obtain a thorough history and physical examination, followed by FNAB and evaluation of the sample by an experienced cytologist. In some cases, FNAB should be performed

Figure 13-6 Management of nodular goiter based on fine-needle aspiration (FNA) biopsy as the first diagnostic test. Subsequent management is based on cytologic results. Percentages in parentheses indicate satisfactory or unsatisfactory biopsy results. (From Gharib H. Fineneedle aspiration biopsy of thyroid nodules: advantages, limitations, and effect. Mayo Clin Proc 1994; 69:4449.)

under ultrasound guidance. Imaging procedures, in addition to ultrasonography, and other tests may occasionally be helpful, but diagnostic thyroid scintiscanning, as traditionally practiced, is of little or no value and should be abandoned. In iodine-sufficient areas with a high relative prevalence of PTC, the combination of history and physical examination and FNAB is usually sufficient to confirm malignancy. Conversely, if history and physical examination, FNAB, and ultrasonography do not suggest malignancy, the chances of missing PTC are probably less than 1%.[123] In areas where the prevalence of follicular tumors is higher, more patients may require neck exploration because FNAB may not be conclusive; in experienced hands, however, intraoperative frozen sections can limit the number of unnecessarily extensive, bilateral procedures. Surgery should also be considered for large tumors (>4 cm), especially in young subjects, in order to avoid repeated evaluations; in addition, because these tumors may be composed of various cell populations, results of FNAB may be less reliable. Finally, micronodules less than 1 cm in diameter, found incidentally during imaging, do not need to be tested any further, unless there are sonographic features suggestive of PTC or MTC. The usual advice is to repeat ultrasonography of such lesions after an interval of 6 to 12 months. [120]



Classification of Thyroid Tumors Histologic Classification

Two monographs have had a major impact on the histologic classification of thyroid tumors. One is from the World Health Organization (WHO), [147] the other, from the Armed Forces Institute of Pathology (AFIP). [148] The classification described in Table 13-3 is modified from the guidelines described by these organizations. [147] [148] Lesions of follicular cell origin constitute more than 95% of the cases, and the remainder are largely made up of tumors exhibiting C cell differentiation. [149] Mixed medullary and follicular carcinomas, made up of cells with both C-cell and follicular differentiation, are rare and of uncertain histogenesis. [147] Nonepithelial thyroid tumors mainly include malignant lymphomas, which may involve the thyroid gland as the only manifestation of the disease or as part of a systemic disease. True sarcomas and malignant hemangioendotheliomas are exceptional. Blood-borne metastases to the thyroid are not uncommon at autopsy in patients with widespread malignancy but rarely cause clinically detectable thyroid enlargement. Staging of Thyroid Carcinoma

In addition to the histologic classification of thyroid tumors developed by WHO and AFIP groups, the International Union Against Cancer (UICC) and the American Joint Committee TABLE 13-3 -- Classification of Thyroid Neoplasms Primary Epithelial Tumors Tumors of Follicular Cells Benign: follicular adenoma Malignant: carcinoma Differentiated Papillary Follicular Poorly differentiated Insular Others Undifferentiated (anaplastic) Tumors of C Cells Medullary carcinoma Tumors of Follicular and C Cells Mixed medullary-follicular carcinoma Primary Nonepithelial Tumors Malignant Lymphomas Sarcomas Others Secondary Tumors

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TABLE 13-4 -- American Joint Committee on Cancer Stage Groupings for Thyroid Carcinoma * Papillary or Follicular Stage

(Age 14 cm; T3, >4 cm; T4, extrathyroid invasion); N, regional nodal metastases (0, absent; 1, present); M, distant metastases (0, absent; 1, present).

on Cancer (AJCC) have agreed on a staging system in thyroid cancer. [150] As stated by the AJCC, "the principal purpose served by international agreement on the classification of cancer cases by extent of disease was to provide a method of conveying clinical experience to others without ambiguity." [150] The AJCC based its system of classification on the TNM system, which relies on assessing three components: (1) extent of the primary tumor (T), (2) absence or presence of regional lymph node metastases (N), and (3) absence or presence of distant metastases (M). The TNM system allows a reasonably precise description and recording of the anatomic extent of disease. The classification may be either clinical (cTNM), based on evidence (including biopsy) acquired before treatment, or pathologic (pTNM), by which intraoperative and surgical pathology data are available. Obviously, pTNM classification is preferable because a precise size can be assigned to the primary tumor, the histo-type is identified, and extrathyroid invasion is demonstrated unequivocally. Typically, the primary thyroid tumor (T) status is defined according to the size of the primary lesion: T1, greatest diameter 1 cm or smaller

T2, larger than 1 cm but not larger than 4 cm T3, larger than 4 cm T4, direct (extrathyroidal) extension or invasion through the thyroid capsule A thyroid tumor with four degrees of T, two degrees of N, and two degrees of M can have 16 different TNM categories. For purposes of tabulation and analysis, these categories have been condensed into a convenient number of TNM stage-groupings (Table 13-4) . Whereas head and neck cancer is usually staged entirely on the basis of anatomic extent, in thyroid cancer staging both the histologic diagnosis and the age of the patient for PTC and FTC are included because of their importance in predicting the behavior and prognosis of thyroid cancer. According to this staging scheme, all patients younger than age 45 years with PTC or FTC are in stage I, unless they have distant metastases (DM), in which case they would be in stage II. In young patients and especially in children, the risk of recurrence is high [151] and may be underestimated by the TNM staging system. Older patients (aged 45 years or more) with node-negative papillary or follicular microcarcinoma (T1 N0 M0) are in stage I. Tumors between 1.1 and 4.0 cm are classified as stage II, and those with either nodal spread (N1) or extrathyroidal invasion (T4), stage III. For MTC, the scheme is similar, in that microcarcinoma is stage I and a node-positive tumor is stage III. There is no age distinction for MTC, although age is a significant independent prognostic indicator in most multivariate analyses, [152] [153] [154] [155] and local (extrathyroidal) invasion is grouped within stage II. For patients with MTC and older patients with PTC or FTC, stage IV denotes the presence of DMs. Independent of age or tumor extent, all patients with undifferentiated (anaplastic) cancer are considered to be in stage IV.



Follicular Adenoma

Follicular adenoma is a benign, encapsulated tumor with evidence of follicular cell differentiation. [147] [148] It is the most common thyroid neoplasm and may be found in 4% to 20% of glands examined at autopsy. [156] [157] The tumor has a well-defined fibrous capsule that is grossly and microscopically complete. There is a sharp demarcation and distinct structural difference from the surrounding parenchyma. These adenomas vary in size, but most have a diameter of 1 to 3 cm at the time of excision. Degenerative changes, including necrosis, hemorrhage, edema, fibrosis, or calcification, are common features, particularly in larger tumors. Follicular adenomas can be classified into subtypes (Table 13-5) according to the size or presence of follicles and degree of cellularity. Each adenoma tends to have a consistent architectural pattern. Microfollicular, normofollicular, and macrofollicular adenomas owe their names to the size of their follicles compared with follicles in the neighboring, non-neoplastic areas of the gland. Trabecular adenomas are cellular and consist of columns of cells arranged in compact cords. They show little follicle formation and rarely contain colloid. A variant, the hyalinizing trabecular adenoma, has unusually elongated cells and prominent hyaline changes in the extracellular space. [158] The histologic differences between these subtypes are striking but of no clinical importance. The only practical value of the classification is that the more cellular a follicular nodule is, the more one should search for evidence of malignancy in the form of invasion of blood vessels and capsule, either singly or in combination. [148] Atypical adenomas are hypercellular or heterogeneous, or both, with gross and histologic appearances TABLE 13-5 -- Subtypes of Follicular Adenoma Conventional Trabecular/solid (embryonal) adenoma Microfollicular (fetal) adenoma Normofollicular (simple) adenoma Macrofollicular (colloid) adenoma Variants Hyalinizing trabecular adenoma Oncocytic (oxyphilic or Hürthle cell) tumor Adenomas with papillary hyperplasia Hyperfunctioning ("toxic") adenoma Atypical (hypercellular) adenoma

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Figure 13-7 Genetic events in thyroid tumorigenesis. Activating point mutations of the RAS genes are found with a similar frequency in follicular adenomas and follicular carcinomas and are considered an early event in follicular tumorigenesis. The PPAR-PAX8 rearrangement was found only in follicular carcinomas. Rearrangements of transmembrane receptors with tyrosine kinase activity (RET, TRK genes) are found only in papillary thyroid carcinomas. Inactivating point mutations of the P53 gene are found only in poorly differentiated and anaplastic thyroid carcinomas. Activation of the cyclic adenosine monophosphate pathway, by point mutation of the thyrotropin receptor (TSH-R) or the subunit of the G protein genes, leads to the appearance of hyperfunctioning thyroid nodules. G s stimulatory guanyl nucleotide protein.

that suggest the possibility of malignancy but not invasion. They account for fewer than 3% of all follicular adenomas. Follow-up indicates that this lesion behaves in a benign fashion. The fact that the tumor does not recur or produce metastases after removal does not prove that it is actually benign; removal may have interrupted a natural history that would have culminated in invasion and metastases. [64] The most important cytologic variant is the oxyphilic or oncocytic (Hürthle cell) adenoma, which is composed predominantly (at least 75%) or entirely of large cells with granular, eosinophilic cytoplasm. [159] Ultrastructurally, the cells are rich in mitochondria and may exhibit nuclear pleomorphism with distinct nucleoli. Although all such neoplasms are thought by some to be potentially malignant, [160] the biologic behavior and clinical course of oncocytic tumors correlate closely with the histology and the size of the initial lesion. The absence of invasion predicts a benign outcome, [161] [162] but larger tumors may rarely be associated with later recurrence or metastases, even in the absence of obvious microscopic evidence of invasion; fortunately, such an occurrence is extremely rare, and generally a diagnosis of benign Hürthle cell adenoma can be reliable. [163] [164] Some normofollicular adenomas may contain pseudopapillary structures that can be confused with the papillae of papillary carcinoma. These structures are probably an expression of localized hyperactivity and are most common in adenomas that show autonomous function. In the majority of hyperfunctioning follicular adenomas, activating point mutations have been identified in the TSHR [32] [165] or in the subunit of the stimulatory guanyl nucleotide protein (G s ) (Fig. 13-7) . Such mutations may impair guanosine triphosphatase (GTP) activity, trapping the G protein in a state of constitutive activation, resulting in enhanced cyclic adenosine monophosphate (cAMP) production and constitutive hyperstimulation of the cells. [166] [167]



Papillary Thyroid Carcinoma

PTC has been defined as "a malignant epithelial tumor showing evidence of follicular cell differentiation, and characterized by the formation of papillae and/or a set of distinctive nuclear changes." [168] The most common thyroid malignancy, PTC constitutes 50% to 90% of differentiated FCTCs worldwide. [169] Papillary thyroid microcarcinoma (PTM) is defined by the WHO as a PTC 1.0 cm in diameter or smaller. [147] [170] [171] The incidence rates for clinically diagnosed PTC in the United States are approximately 5 per 100,000 for tumors larger than 1 cm in diameter and 1 per 100,000 for PTM. By contrast, the incidence of PTM in autopsy material from various continents ranges from 4% to 36%. [168] Typically, PTC shows a predominance of papillary structures, consisting of a fibrovascular core lined by a single layer of epithelial cells, but the papillae are usually admixed with neoplastic follicles having characteristic nuclear features. The nuclei of PTC cells have a distinctive appearance that has a diagnostic significance comparable to that of the papillae. Indeed, the preoperative diagnosis of PTC can often be made on the basis of the characteristic nuclear changes seen in FNA material: Nuclei are larger than in normal follicular cells and overlap, they may be fissured like coffee beans, chromatin is hypodense (ground glass nuclei), limits are irregular, and they frequently contain an inclusion corresponding to a cytoplasmic invagination. Several subtypes exist: 1. The tumor is designated a follicular variant of PTC when the lining cells of the neoplastic follicles have the same nuclear features as seen in typical PTC and the follicular predominance over the papillae is complete. [147] [148] [149] 2. The diffuse sclerosing variant is characterized by diffuse involvement of one or both thyroid lobes, widespread lymphatic permeation, prominent fibrosis, and lymphoid infiltration. 3. The tall cell variant is characterized by well-formed papillae that are covered by cells twice as tall as they are wide. 4. The columnar cell variant differs from other forms of PTC because of the presence of prominent nuclear stratification. The tall cell and columnar cell variants are more aggressive, [168] but controversy exists regarding outcome for the diffuse sclerosing variant. [172] Molecular Pathogenesis

The thyroid follicular cell may give rise to both benign and malignant tumors, and the malignancy can be of either papillary or follicular histotype. There is no evidence that benign tumors ever undergo malignant transformation into classic PTC. Structural abnormalities of the chromosomes may occur in about 50% of PTCs, frequently involving the long arm of chromosome 10.[173] The RET proto-oncogene is located on chromosome 10q11-2. It encodes a transmembrane receptor with a tyrosine kinase domain. Its ligands are the glial cell linederived neutrophilic factor (GDNF) and the neurturin, both of which induce protein dimerization. RET activation was first demonstrated in transfection experiments and has been found only in PTC tumors. [174] [175] [176] [177] [178] [179] It was therefore called RET/PTC. All activated forms of the RET proto-oncogene are the consequence of oncogenic rearrangements fusing the tyrosine kinase

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domain of the RET gene with the 5' domain of different genes. The foreign gene is constitutively expressed, and its 5' domain acts as a promoter, resulting in permanent expression of the RET gene. Furthermore, these genes have domains that induce RET activation by permanent dimerization; because of this fusion, the chimeric protein is localized in the cytoplasm and not in the plasma cell membrane. Three major classes of RET/PTC have been identified: 1. RET/PTC1 is formed by an intrachromosomal rearrangement fusing the RET tyrosine kinase domain to a gene designated H4 (D10S170), whose function is still unknown. 2. RET/PTC2 is formed by an interchromosomal rearrangement fusing the RET tyrosine kinase domain to a gene located on chromosome 17 encoding the RI regulatory subunit of protein kinase A. 3. RET/PTC3 is formed by an intrachromosomal rearrangement fusing the RET tyrosine kinase domain to a gene designated ELE1, whose function is still unknown. Several variants of RET/PTC have been observed in post-Chernobyl thyroid tumors, including rearrangements formed by fusing the tyrosine kinase domain of the RET gene at other breakpoint sites or with other partners. The frequency of RET/PTC rearrangements occurring in PTC patients without prior childhood neck irradiation varies between 2.5% and 35%. In these tumors, the frequencies of RET/PTC1 and RET/PTC3 were similar and that of RET/PTC2 was lower. The RET/PTC rearrangements were more frequently found (in 60% to 80% of cases) in PTC cases occurring either after external irradiation in childhood or in children after the Chernobyl accident. [174] [175] [176] RET/PTC3 was more frequently found in aggressive tumors that occurred early after the accident and RET/PTC1 in less aggressive tumors that occurred later. The finding of RET/PTC rearrangement in micropapillary thyroid carcinomas suggests that it constitutes an early event in thyroid carcinogenesis. [177] On the other hand, RET/PTC-positive tumors lack evidence of progression to poorly or undifferentiated tumor phenotypes. [176] Several additional oncogenes may occasionally be involved in PTC, including NTRK1 (also named TRKA), which codes for a neural growth factor receptor with a tyrosine kinase domain and which is activated by rearrangement in about 10% of PTCs. [174] The receptor for hepatocyte growth factor is a transmembrane tyrosine kinase encoded by the MET oncogene; it is overexpressed in some patients with PTC, and low expression has been associated with the occurrence of DM. [180] [181] A high incidence of PTC has been reported in patients with adenomatous polyposis coli and Cowden's disease (the multiple hamartoma syndrome), suggesting that the predisposing genes may play a role in the occurrence of papillary carcinoma. About 3% of cases of PTC are familial; their behavior is similar to or slightly more aggressive than that of nonfamilial cases. [182] The gene predisposing to familial thyroid tumors with cellular oxyphilia has been mapped to chromosome 19q13.2, and in a family with PTC and renal carcinoma a separate gene was mapped to chromosome 1q21. [183] [184] The expression of thyroid-specific genes has been studied at the messenger ribonucleic acid (mRNA) and protein levels in a large series of human thyroid tumors. Expression of NIS was profoundly decreased in both benign and malignant thyroid hypofunctioning nodules; moreover, in malignant nodules, low expression of TPO, PDS, and Tg was also found. [185] These abnormalities clearly explain many of the metabolic defects typically observed in thyroid cancer tissues: a low iodine concentration, a low rate of iodine organification, low hormonal synthesis, and a short intrathyroidal half-life of iodine. [186] However, Tg is expressed in all FCTCs and can be shown by immunohistochemistry, which can prove useful in cases with atypical histology.

Figure 13-8 Distribution of pathologic-Tumor-Node-Metastases (pTNM) stages in 2284 patients with papillary thyroid carcinoma ( upper left), 218 patients with medullary thyroid cancer (lower left), 141 patients with follicular thyroid cancer ( upper right), and 125 patients with Hürthle cell cancer (lower right) undergoing primary surgical treatment at the Mayo Clinic from 1940 to 1997. Presenting Features

Although PTCs can occur at any age, most occur in patients between 30 and 50 years of age (mean age, 45 years). Women are affected more frequently (female predominance, 60% to 80%). Most primary tumors are 1 to 4 cm in size; they average about 2 to 3 cm in greatest diameter. [169] [187] Extrathyroidal invasion of adjacent soft tissues is present in about 15% (range 5% to 34%) at primary surgery, and about one third of PTC patients have clinically evident lymphadenopathy at presentation. [187] About 35% to 50% of excised neck nodes have histologic evidence of involvement, and in patients 17 years of age or younger nodal involvement may be present in up to 90%. [151] [188] Only 1% to 7% of PTC patients have DM at diagnosis. [169] [187] Spread to superior mediastinal nodes is usually associated with extensive neck nodal involvement. The TNM classification is a widely used system for tumor staging. [189] Most PTC patients present with either stage I (60%) or stage II (22%). Patients aged 45 years or older with either nodal metastases or extrathyroidal extension (stage III) account for fewer than 20% of cases. [169] As already noted, few (1% to 7%) of PTC patients present with DM and have stage IV disease (age 45 years or older with any T, any N, M1). Figure 13-8 (upper left) illustrates the distribution of TNM stages in 2284 PTC cases seen at the Mayo Clinic, and Figure 13-9 demonstrates survival by TNM stage in this cohort of PTC patients treated from 1940 to 1997. Recurrence and Mortality

Three types of tumor recurrence may occur with PTC: Postoperative nodal metastases (NM) Local recurrence (LR) Postoperative distant metastases (DM) LR may be defined as "histologically confirmed tumor occurring in the resected thyroid bed, thyroid remnant, or other adjacent tissues of the neck (excluding lymph nodes)" after

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Figure 13-9 Cause-specific survival according to pathologic-Tumor-Node-Metastases (pTNM) stage in a cohort of 2284 patients with papillary thyroid carcinoma treated at the Mayo Clinic from 1940 to 1997. The numbers in parentheses represent the percentages of patients in each pTNM stage grouping.

complete surgical removal of the primary tumor. [190] Nodal or distant spread may be considered postoperative if the metastases are discovered within 180 or 30 days, respectively.[169] Ideally, tumor recurrence should be considered only as it occurs in patients without initial DM who had complete surgical resection of the primary tumors. Figure 13-10 illustrates rates of PTC recurrence at local, nodal, and distant sites in 2150 patients with PTC treated at one institution from 1940 to 1997. After 20 years of follow-up, postoperative NM had been discovered in 9%, and LR and DM occurred in 5% and 4%, respectively. Both LR and DM are less common in PTC than in FTC (Fig. 13-11) . However, postoperative cases of NM were more frequent in PTC than in FTC. Cause-specific mortality (CSM) rates for differentiated thyroid cancer are shown in Figure 13-12 . CSM rates for PTC were 2% at 5 years, 4% at 10 years, and 5% at 20 years. Among those with lethal PTC, 20% of deaths occurred in the first year after diagnosis, and 80% of the deaths occurred within 10 years. The 25-year cause-specific survival rate of 95% for PTC was significantly higher than the 79%, 71%, and 66% rates seen with MTC, Hürthle cell cancer (HCC), and FTC, respectively.

Figure 13-10 Development of neck nodal metastases, local recurrences, and distant metastases in the first 20 years after definitive surgery for papillary thyroid cancer (PTC) or medullary thyroid cancer (MTC) performed at the Mayo Clinic from 1940 to 1997. Based on 2150 consecutive PTC ( left) and 194 MTC (right) patients who had complete surgical resection (i.e., had no gross residual disease) and were without distant metastases on initial examination. Postop, postoperative.

Figure 13-11 Development of neck nodal metastases (NM), local recurrences (LR), and distant metastases (DM) in the first 20 years after definitive surgery for follicular thyroid cancer (FTC) or Hürthle cell cancer (HCC) performed at the Mayo Clinic from 1940 to 1997. Based on 110 consecutive FTC patients ( left) and 115 HCC patients (right) who had complete surgical resection and were without distant metastases on initial examination. Outcome Prediction

Only a fraction (15%) of patients with PTC are likely to experience relapse of disease, and even fewer (5%) have a lethal outcome. Exceptional patients, who have an aggressive course, tend to experience relapse early (Fig. 13-13) , and the rare fatalities usually occur within 5 to 10 years of diagnosis. [169] [170] [187] [188] Multivariate analyses have been used to identify variables predictive of CSM. [191] [192] [193] [194] Increasing age of the patient and the presence of extrathyroidal invasion are independent prognostic factors in all studies. [191] [192] [193] [194] The presence of initial DM and large size of the primary tumor are also significant variables in most studies, [191] [193] [194] and some groups[169] [191] [192] [195] have reported that histopathologic grade (degree of differentiation) is an independent variable. The completeness of initial tumor resection (postoperative status) is also a predictor of mortality.[169] [193] [196] The presence of initial neck NM, although relevant to future nodal recurrence, does not influence CSM (Fig. 13-14) .[169] [187] [196] Several scoring systems based on these significant prognostic indicators have been devised. Each system allows one to assign the majority of PTC patients (80% or more) to a low-risk

Figure 13-12 Cumulative cause-specific mortality rates for patients with differentiated thyroid carcinoma in the first 25 years after treatment with initial surgery performed at the Mayo Clinic from 1940 to 1997. Based on 2768 consecutively treated patients (2284 with papillary thyroid carcinoma [PTC], 141 with follicular thyroid cancer [FTC], 125 with Hürthle cell cancer [HCC], and 218 with medullary thyroid cancer [MTC]).

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Figure 13-13 Survival to death from all causes and to death from thyroid cancer (cause-specific mortality) in 2284 consecutive patients with papillary thyroid carcinoma undergoing initial management at the Mayo Clinic from 1940 to 1997. Also plotted is the expected survival (all causes) of persons of the same age and sex and with the same date of treatment but living under mortality conditions of the northwest central United States.

group, in which the CSM at 25 years is less than 2%, and the others (a small minority) to a high-risk group, in which almost all cancer-related deaths are observed. In general, these systems provide prediction of postoperative events comparable to that of the internationally accepted TNM staging system. [197] A scoring index devised to assign PTC patients to prognostic risk groups [191] was named the AGES scheme after the four independent variables: patient's age, tumor grade, tumor extent (local invasion, DM), and tumor size. With the use of such a scoring system, 86% of patients were in the minimal risk group (AGES score < 4) and they experienced a 20-year CSM rate of only 1%. [169] By contrast, patients with AGES scores of 4+ (high-risk; 14% of the total) had a 20-year CSM of 36%. Figure 13-15 compares the AGES scores with TNM stage and with two other subsequently introduced schemes designed to stratify PTC patients into groups at either minimal risk or high risk of cancer-related death. Such a prognostic scoring system makes it possible to counsel patients and to aid in the planning of individualized postoperative management programs in PTC.[191] [196] Although the AGES scheme had the potential for universal application, some academic centers could not include the differentiation (G) variable because their surgical pathologists did not recognize higher-grade PTC tumors. [198] Accordingly, a

Figure 13-14 Lack of influence of nodal metastases at initial operation on cumulative mortality from papillary thyroid carcinoma in 1941 patients with pT1-3 intrathyroidal tumors (completely confined to the thyroid gland) and 209 pT4 patients with extrathyroidal (locally invasive) tumors. All patients had initial surgical treatment at the Mayo Clinic from 1940 to 1997. DM, distant metastases.

Figure 13-15 Cumulative mortality from papillary thyroid carcinoma in patients at either minimal risk or higher risk of cancer-related death as defined by International Union Against Cancer (UICC) pathologic-Tumor-Node-Metastases (pTNM) stages (upper left), AGES scores (upper right), AMES risk groups (lower left), and MACIS scores (lower right). The minimal risk group constitutes 81% of the 2284 patients when defined by pTNM stages I and II, 86% as defined by AGES scores less than 4, 88% as defined by AMES low-risk, and 83% when defined by a MACIS score less than 6. The cause-specific mortality (CSM) rates at 20 years were 25% for stages III and IV, 36% for AGES scores of 4+, 39% for AMES high-risk, and 32% for patients with MACIS scores of 6+. The CSM ratios between the high-risk and low-risk groups at 20 years were 19 for pTNM, 36 for AGES, 35 for AMES, and 40 for MACIS.

prognostic scoring system for predicting PTC mortality rates was devised with the use of candidate variables that included completeness of primary tumor resection but excluded histologic grade. [196] Cox model analysis and stepwise variable selection led to a final prognostic model that included five variables: metastasis, age, completeness of resection, invasion, and size (MACIS). The final score was defined as 3.1 (age 39 years or younger) or 0.08 × age (age 40 years or older) +0.3 × tumor size (in centimeters) +1 (if tumor not completely resected) +1 (if locally invasive) +3 (if DM present) As illustrated by Figure 13-16 , the MACIS scoring system

Figure 13-16 Cause-specific survival according to MACIS ( metastases, age, completeness of resection, invasion, and size) scores of less than 6, 6 to 6.99, 7 to 7.99, and 8+ in a cohort of 2284 consecutive patients with papillary thyroid carcinoma (PTC) undergoing initial treatment at the Mayo Clinic from 1940 to 1997. The numbers in parentheses represent the numbers and percentages of PTC patients in each of the four risk groups.

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permits identification of groups of patients with a broad range of risk of death from PTC. Twenty-year cause-specific survival rates for patients with MACIS scores of less than 6, 6 to 6.99, 7 to 7.99, and 8+ were 99%, 89%, 56%, and 27%, respectively ( P < .0001). When cumulative mortality from all causes of death was considered, approximately 85% of PTC patients with AGES scores below 4 or MACIS scores below 6 had no excess mortality over rates predicted for control subjects. [191] [196] It should be emphasized that the five variables in MACIS scoring are easy to define after primary operation; consequently, the system can be applied in any clinical setting. The MACIS system can be used for counseling individual PTC patients and can help guide decision making concerning the intensity of the postoperative tumor surveillance and the appropriateness of adjunctive radioiodine therapy. Because the CIS ( completeness of resection, invasion, and size) variables require information obtained at surgery, the system probably should not be used to decide the extent of primary surgery. [198]



Follicular Thyroid Carcinoma

FTC is "a malignant epithelial tumor showing evidence of follicular cell differentiation but lacking the diagnostic features of papillary carcinoma." [147] Such a definition excludes the follicular variant of PTC, and it is also customary to exclude both the poorly differentiated insular carcinoma [199] and the rare mixed medullary and follicular carcinoma. [200] The correct classification of tumors with predominant oncocytic features (Hürthle cell carcinomas) is controversial. [159] The WHO committee has taken the stance that this tumor is an oxyphilic variant of FTC. [147] The AFIP monograph, by contrast, states that "the tumors made up of this cell type have gross, microscopic, behavioral, cytogenetic (and conceivably etiopathogenic) features that set them apart from all others and justify discussing them in a separate section." [148] Thus categorized, FTC is a relatively rare neoplasm whose identification requires invasion of the capsule, blood vessel, or adjacent thyroid. In epidemiologic surveys, FTC constituted from 5% to 50% of differentiated thyroid cancers and tended to be more common in areas with iodine deficiency. [201] Owing to a combination of changing diagnostic criteria and an increase in the incidence of PTC associated with dietary iodine supplementation, the diagnosis of FTC has decreased in frequency; in one North American experience, minimally invasive nonoxyphilic FTC made up fewer than 2% of thyroid malignancies. [202] The microscopic appearance of FTC varies from well-formed follicles to a predominantly solid growth pattern. [147] [148] [149] Poorly formed follicles and atypical patterns (e.g., cribriform) may occur, and multiple architectural types may coexist. Mitotic activity is not a useful indicator of malignancy. FTC is best divided into two categories on the basis of degree of invasiveness: Minimally invasive or encapsulated Widely invasive There is little overlap between these two types. Minimally invasive FTC is an encapsulated tumor whose growth pattern resembles that of a trabecular or solid, microfollicular, or atypical adenoma. The diagnosis of malignancy depends on the demonstration of blood vessel or capsular invasion, or both. The criteria for invasion must therefore be strict. [149] Blood vessel invasion is almost never seen grossly. Microscopically, the vessels "should be of venous caliber, be located in or immediately outside of the capsule and contain one or more clusters of tumor cells attached to the wall and protruding into the lumen." [149] Interruption of the capsule must involve the full thickness to qualify as capsular invasion. Penetration of only the inner half or the presence of tumor cells embedded in the capsule does not qualify for the diagnosis of FTC. Foci of capsular invasion must be distinguished from the capsular rupture that can result from FNA. The acronym WHAFFT (worrisome histologic alterations following FNA of the thyroid) is applied to such changes. [203] In contrast, the rare, widely invasive form of FTC can be distinguished easily from benign lesions. Although the tumor may be partially encapsulated, the margins are infiltrative even on gross examination and vascular invasion is often extensive. The structural features are variable, with solid and trabecular areas, but a follicular element is always present. When follicular differentiation is poor or absent, the tumor may be classified as a poorly differentiated (insular) carcinoma. [64] [149] Focal or extensive clear-cell changes can occur. A rare clear cell variant of FTC has been described in which glycogen accumulation or dilatation of the granular endoplasmic reticulum is responsible for the clear cells. [204] When more than 75% of cells in an FTC exhibit Hürthle cell (or oncocytic) features, the tumor is classified as a Hürthle cell or an oncocytic carcinoma [148] [205] or an oxyphilic variant FTC. [64] [147] Molecular Pathogenesis

There is still no accepted paradigm for the pathogenesis of follicular thyroid cancer. A multistep adenoma-to-carcinoma pathogenesis, similar to that for colon cancer and other adenocarcinomas, [176] [206] is not universally accepted because pathologists do not recognize follicular carcinoma in situ and documentation of the evolution of adenoma to carcinoma is rare. Nevertheless, several facts about the pathogenesis of FTC are firmly established. First, most follicular adenomas and all FTCs are probably of monoclonal origin. [51] [65] [66] Second, oncogene activation, particularly by point mutation of the RAS oncogene, is common both in follicular adenomas and in FTCs (40%), supporting a role in early tumorigenesis. [167] [206] Such RAS mutations are not specific for follicular tumors and also occur in PTC. The RET oncogene does not appear to be significantly involved in follicular tumors. [207] Third, cytogenetic abnormalities and evidence of genetic loss are more common in FTC than in PTC and also occur in follicular adenomas. [208] [209] [210] [211] Losses in FTC are particularly associated with chromosomes 3, 10, 11, and 17.[209] [212] Of the cytogenetic abnormalities described in FTC, [213] the most common are deletions, partial deletions, and deletion-rearrangements involving the p arm of chromosome 3.[211] [214] Loss of heterozygosity (LOH) on chromosome 3p appears to be limited to FTC because no evidence for 3p LOH has been found in follicular adenomas or PTC.[209] [210] A translocation, t(2;3)(q13;p25), resulting in the fusion of the deoxyribonucleic acid (DNA) binding domains of the thyroid transcription factor PAX-8 to domains of the peroxisome proliferatoractivated receptor (PPAR) 1, was detected in five of eight FTCs but not in follicular adenomas, PTCs, or multinodular hyperplasia. The chimeric protein may retard growth inhibition and follicular differentiation normally induced by PPAR 1. [215] Presenting Features

FTC tends to occur in older people, with the mean age in most studies being more than 50 years, about 10 years older than that for typical PTC. [201] [216] The average median age of patients with oxyphilic FTC (HCC) is about 60 years. [201] [205] As in most thyroid malignancies, women outnumber men by more than 2 to 1. Most patients with FTC present with a painless thyroid nodule, with or without background thyroid nodularity, and they rarely (4% to 6%) have clinically evident lymphadenopathy at presentation. [201] Lymph node metastases to the neck in FTC are so exceptional that "wherever they are observed, the alternative possibilities of follicular variant papillary

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Figure 13-17 Postoperative recurrence (any site) in the first 20 years after definitive surgery for differentiated thyroid carcinoma performed at the Mayo Clinic from 1940 to 1997. Based on 2569 consecutive patients (2150 papillary thyroid carcinoma, 110 follicular thyroid carcinoma, 115 Hürthle cell carcinoma, and 194 medullary thyroid carcinoma) who had complete tumor resection and had no distant metastases at presentation. The ages in parentheses represent the median age at diagnosis for each of the four histologic subtypes.

carcinoma, oncocytic carcinoma, and poorly differentiated (insular) carcinoma should be considered."

[148]

In most series in which tumor sizes were reported, the average tumor in FTC (oxyphilic or nonoxyphilic) was larger than those seen with PTC. [201] [205] [216] Direct extrathyroidal extension, by definition, does not occur with minimally invasive FTC but is not uncommon in the rare patient with invasive FTC. Between 5% and 20% of patients may have DM at presentation. [201] [216] The most common sites for DM in FTC are lung and bone.[148] [216] The bones most often involved are long bones (e.g., femur), flat bones (particularly the pelvis, sternum, and skull), and vertebrae. When a DM is the first manifestation of the disease, definitive proof of its thyroid origin

should be obtained, usually by a biopsy of a metastasis, before performing any thyroid surgery. It is unusual for patients with FTC to have thyrotoxicosis caused by massive tumor burden.[217] Most patients (53% to 69%) with FTC or HCC have pTNM stage II disease. Patients aged 45 years or older with nodal metastases or extrathyroidal extension (stage III) account for only 4% of FTCs and 9% of HCCs (see Fig. 13-8) . About 5% of HCCs and 17% or more of nonoxyphilic FTCs have DM at the time of diagnosis (stage IV). Recurrence and Mortality

Nodal metastases are rare in typical FTC, and the nodal recurrence rate at 20 postoperative years is the lowest in differentiated thyroid carcinoma, being around 2% (see Fig. 13-11) . About 6% of patients with HCC have node involvement at presentation, [218] but within 25 years after primary surgery about 17% of HCC patients have nodal recurrence. [201] When recurrences at either neck or distant sites are taken into consideration, patients with HCC (Fig. 13-17) have the highest numbers of tumor recurrences after 10 to 20 years. As illustrated by Figure 13-11 , local recurrences at 20 years have occurred in 20% of FTCs and 30% of HCCs. Comparable DM rates are 23% and 28%, respectively. CSM rates vary with the presenting TNM stage in both FTC (Fig. 13-18) and HCC. The death rates tend to parallel the curves for development of DM (see Fig. 13-11) . In more than five decades of experience at the Mayo Clinic, the mortality rate for FTC initially exceeds that of HCC, but by 20 to 30 postoperative years there are no significant differences in cause-specific survival rates between FTC and HCC (Fig. 13-19) , both being around 80% at 20 and 70% at 30 postoperative

Figure 13-18 Cause-specific survival according to pathologic-Tumor-Node-Metastases (pTNM) stages in a cohort of 141 patients with follicular thyroid carcinoma ( left panel) and 125 patients with Hürthle cell carcinoma ( right panel) treated at the Mayo Clinic from 1940 to 1997. Numbers in parentheses represent the number of patients in each pTNM stage grouping.

years. [201] Curves representing mortality from all causes differ in FTC and HCC. On average, patients with FTC are about 5 years younger, tend to die within the first 10 postoperative years, and have high all-cause mortality for 10 to 30 postoperative years (Fig. 13-20) . Deaths related to HCC occur gradually over the first 15 years; however, by 25 years, the average survivor of HCC is 84 years old, and by that time, almost 50% of the treated cohort would be predicted by the actuarial curve to have died from all causes. Outcome Prediction

The risk factors that predict outcome in FTC are largely the same as in PTC [201] : DM at presentation, increasing age of the patient, large tumor size, and the presence of local (extrathyroidal) invasion. To a lesser degree, increased mortality is associated with male sex and higher grade (less well-differentiated) tumors. In addition, vascular invasiveness, lymphatic involvement at presentation, DNA aneuploidy, and oxyphilic histology are potential prognostic variables unique to FTC. [201] The importance of vascular invasion is underscored by a study showing that FTC patients with minimal capsular invasion and no evidence of vascular invasion had 0% CSM at 10-year follow-up. [219] Prognostic scoring systems for FTC [216] [220] allow stratification of patients into high-risk and low-risk categories. A multivariate analysis at the Mayo Clinic found that DM at presentation, patient's age greater than 50 years, and marked vascular invasion predict a poor outcome. [216] As illustrated by Figure 13-21 , if two or more of these factors are present, the 5-year survival rate is only 47%, and 20-year survival is 8%. By contrast, if only one of these factors is present, 5-year survival is 99%, and 20-year survival is 86%. [216] Systems developed to predict outcome in either PTC or FTC have been applied to FTC patients. Specifically, the pTNM as well as the AMES risk group categorization (age, metastasis, extent, size) is useful in FTC. [221] From a multivariate analysis of 228 patients with FTC treated at the Memorial Sloan Kettering Cancer Center, the independent adverse prognostic factors were identified as age older than 45 years, Hürthle cell histotype, extrathyroidal extension, tumor size exceeding 4 cm, and the presence of DM.[222] The prognostic importance in FTC of histologic grade was also confirmed, [222] [223] and this factor was included in assignment of risk groups to low, intermediate, or high categories (Fig. 13-22) . The AGES scheme, originally developed for PTC, has also

475

Figure 13-19 Comparison of cause-specific survival in 1472 papillary thyroid carcinoma (PTC) and 250 follicular thyroid carcinoma (FTC) patients treated at the Mayo Clinic from 1940 to 1990. 138 of the PTCs were "pure" papillary in histotype (no follicular elements). 97 of the FTC patients had predominantly oxyphilic tumors. There is a significant difference ( P = .0001) between the PTC and the FTC survival curves. However, within either the PTC or FTC groups, the two survival curves are insignificantly different. (From Grebe SKG, Hay ID. Follicular thyroid cancer. Endocrinol Metab Clin North Am 1995; 24:761801.)

been successfully applied to FTC. [224] [225] It thus appears that scoring systems used in PTC may be cautiously applied in FTC as long as some of the unique features of this tumor, such as vascular invasiveness and the remarkable significance of DNA aneuploidy in HCC, are kept in mind. [201]



Poorly Differentiated (Insular, Solid, or Trabecular) Carcinoma

Poorly differentiated thyroid carcinoma has been defined as "a tumor of follicular cell origin with morphological and biologic attributes intermediate between differentiated and anaplastic carcinomas of the thyroid." [64] The most distinctive histologic feature is the presence of small cells with round nuclei and scant cytoplasm with a diffuse solid pattern or organized in round or oval nests (insulae) or in trabeculae. The predominant

Figure 13-20 Survival to death from all causes in 141 consecutive patients with follicular thyroid carcinoma (left) and 125 patients with Hürthle cell cancer (right) undergoing initial management at the Mayo Clinic from 1940 to 1997. Also plotted is the expected survival (all causes) of persons of the same age and sex and with the same date of treatment but living under mortality conditions of the northwest central United States.

pattern of growth is solid, but microfollicles are also seen, some of which contain dense colloid. Extrathyroidal extension and blood vessel invasion are common. Most such tumors exhibit foci of necrosis, are larger than 5 cm in diameter at diagnosis, and have an invasive margin on gross examination. The mean age at diagnosis is about 55 years, and the female/male ratio is about 2:1. [64] Poorly differentiated carcinoma is aggressive and often lethal. Metastases are common in regional nodes and distant sites (lung, bone, brain). In one series,

Figure 13-21 Cumulative cause-specific survival among 100 patients with nonoxyphilic follicular thyroid carcinoma treated at the Mayo Clinic from 1946 to 1970, plotted by high-risk and low-risk categories. High risk means that two or more of the following factors were present: age older than 50 years, marked vascular invasion, and metastatic disease at time of initial diagnosis. (From Brennan MD, Bergstralh EJ, van Heerden JA, et al. Follicular thyroid cancer treated at the Mayo Clinic, 1946 through 1970: initial manifestations, pathologic findings, therapy, and outcome. Mayo Clin Proc 1991; 66:1122.)

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Figure 13-22 Survival differences in low-risk, intermediate-risk, and high-risk groups for 228 consecutive patients with follicular thyroid carcinoma who were seen and treated at the Memorial Sloan-Kettering Cancer Center during a period of 55 years from 1930 to 1985. (From Shaha AR, Loree TR, Shah JP. Prognostic factors and risk group analyses in follicular carcinoma of the thyroid. Surgery 1995; 118:11311138.)

56% of patients died from their tumor within 8 years of initial therapy. [199] The tumor is viewed by the WHO committee [147] as a morphologic variant of FTC, but others view it as a poorly differentiated variant of either PTC or FTC. [64] [149] Some tumors formerly classified as the compact form of undifferentiated small cell carcinoma probably belonged to this category. [64] The AFIP group also considers that a large proportion of "low-risk" young patients with aggressive PTC or FTC belong to this category of high-grade poorly differentiated FTC. [148]



Undifferentiated (Anaplastic) Carcinoma

Anaplastic carcinoma constitutes about 5% of all thyroid carcinomas, usually occurs after the age of 60 years, and is slightly more common in women (1.3:1 to 1.5:1).[226] This carcinoma is highly malignant, nonencapsulated, and extends widely. Evidence of invasion of adjacent structures, such as the skin, muscles, nerves, blood vessels, larynx, and esophagus, is common. DM occur early in the course of the disease in lungs, liver, bones, and brain. On histopathologic examination, the lesion is composed of atypical cells that exhibit numerous mitoses and form a variety of patterns. Spindle-shaped cells, multinucleate giant cells, and squamoid cells usually predominate. Areas of necrosis and polymorphonuclear infiltration are common, and the presence of PTC or FTC suggests that they may be the precursors of anaplastic carcinoma. Mutations of the p53 gene are present in many undifferentiated carcinomas but may not be found in the residual well-differentiated component, [227] [228] suggesting that these mutations occurred after the development of the original tumor and may have played a key role in tumor progression. The usual clinical complaint is of a rapid, often painful enlargement of a mass that may have been present in the thyroid gland for many years. The tumor invades adjacent structures, causing hoarseness, inspiratory stridor, and difficulty in swallowing. On examination, the overlying skin is often warm and discolored. The mass is tender and is often fixed to adjacent structures. It is stony hard in consistency, but some areas may be soft or fluctuant. The regional lymph nodes are enlarged, and there may be evidence of DM. Anaplastic carcinomas do not accumulate iodine and do not typically produce thyroglobulin. Treatment should be initiated rapidly to avoid death from locally infiltrative disease and possible suffocation. It consists of surgical resection of the tumor tissue present in the neck, when this is feasible, followed by a combination of external irradiation and chemotherapy. When the extent of disease is limited and when these protocols can be applied, local control may be obtained in about two thirds of the patients and long-term survival in about 20%. [229]



Medullary Thyroid Carcinoma

MTC accounts for less than 10% of thyroid malignancies (see Chapter 36) . It arises from the parafollicular or C cells of the thyroid gland, and the tumor cells typically produce an early biochemical signal (hypersecretion of calcitonin). [230] MTC readily invades the intraglandular lymphatics and spreads to other parts of the gland, in addition to the pericapsular and regional lymph nodes. It also regularly spreads through the blood stream to the lungs, bone, and liver. [152] [153] [154] [155] MTC tumors are firm and usually unencapsulated. On histopathologic examination, the tumor is composed of cells that vary in morphologic features and arrangement. Round, polyhedral, and spindle-shaped cells form a variety of patterns, which may vary from solid, trabecular to endocrine or glandular-like structures. An amyloid stroma is commonly present.[231] Gross or microscopic foci of carcinoma may be present in other parts of the gland, and blood vessels may be invaded. The histopathologic appearance of the metastases resembles that of the primary lesion. In all cases, the diagnosis can be confirmed by positive immunostaining of tumor tissue for calcitonin and carcinoembryonic antigen (CEA). MTC first appears either as a hard nodule or mass in the thyroid gland or as an enlargement of the regional lymph nodes. Occasionally, a metastatic lesion in a distant site is found first. The neck masses are frequently painful; they are sometimes bilateral and are often localized to the upper two thirds of each lobe of the gland, which reflects the anatomic location of the parafollicular cells. The tumor occurs in both sporadic and hereditary forms, the latter making up about 20% of the total. The hereditary variety can be transmitted as a single entity, familial MTC, or it can arise as part of MEN syndrome type 2A or 2B. The hereditary form is typically bilateral [232] and is usually preceded by a premalignant C cell hyperplasia. Total thyroidectomy at this premalignant stage can cure the disease in more than 90% of cases. [126] [127] [233] [234] RET proto-oncogene testing should be performed in all MTC patients. The finding of a germline mutation in this gene indicates a hereditary disease; the mutation should then be sought in all first-degree family members. Early series of MTC mainly described sporadic cases, in which 80% of patients presented with TNM stage II or III. [231] As more patients with familial MTC[235] or MEN 2A have been diagnosed, more patients have curable (stage I) disease, and the survival rate has improved, a trend that should continue with widespread application of RET proto-oncogene testing. [236] Patients with MTC now have outcomes similar to or better than those of patients with nonpapillary FCTC (see Fig. 13-10) . The cause-specific survival curves for 218 consecutive MTC cases treated from 1940 to 1997 at the Mayo Clinic, according to TNM stage, are presented in Figure 13-23 . Prognostic factors relevant to outcome in MTC include (1) age at diagnosis, (2) male gender, (3) initial extent of the disease, such as NM and DM, (4) tumor size, (5) extrathyroidal invasion, (6) vascular invasion, (7) calcitonin immunoreactivity and amyloid staining in tumor tissue, (8) postoperative gross residual disease, and (9) postoperative plasma calcitonin levels. [231] In multivariate analysis, only the age of the patient at initial treatment and the stage of the disease remain significantly independent indicators of survival. This suggests that, in routine

477

Figure 13-23 Cause-specific survival according to pathologic-Tumor-Node-Metastases (pTNM) stage in a cohort of 218 patients with medullary thyroid carcinoma treated at the Mayo Clinic from 1940 to 1997. Numbers in parentheses represent the percentages of patients in each pTNM stage grouping.

practice, clinicians attempting to predict outcome in MTC should take into account not only the presenting disease stage, as assessed by the pTNM system (see Fig. 13-23) , but also the age of the patient at diagnosis. [153] [154] [155] In patients with MTC, Cushing's syndrome may occur because of secretion of corticotropin by the tumor. Prostaglandins, serotonin, kinins, and vasoactive intestinal peptide may also be secreted and are variously responsible for flushing and for the attacks of watery diarrhea that about one third of patients experience, usually at an advanced stage of the disease. [154] In MEN-2A, hyperparathyroidism occurs late and is usually due to parathyroid hyperplasia rather than adenoma. Pheochromocytomas invariably occur later than MTC; they are often bilateral and may be clinically silent, and patients at risk should be screened with measurements of urinary metanephrine excretion. In MEN-2B, MTC and pheochromocytomas are associated with multiple mucosal neuromas (bumpy lip syndrome), a marfanoid habitus, and typical facies, but such patients do not regularly have hyperparathyroidism. [234] Differentiation of sporadic MTC from other types of thyroid nodule on clinical grounds alone may be difficult. In patients with a family history of thyroid cancer associated with hypertension or hyperparathyroidism, the MEN-2A syndrome should be suspected. FNAB has made it possible to diagnose MTC before surgery. In some patients, however, cytologic findings may be misleading because the type of carcinoma is difficult to determine and HCC may occasionally be confused with MTC. Positive immunocytochemical staining for calcitonin allows confirmation of the diagnosis. Basal plasma calcitonin levels are elevated in virtually all patients with clinical MTC. Infusions of pentagastrin or calcium elicit secretion of calcitonin, and the response may be exaggerated in patients with either MTC or the antecedent C-cell hyperplasia; its use should be restricted to patients with an undetectable or borderline plasma calcitonin level (see Chapter 36) . When the diagnosis of MTC is made from calcitonin measurements or FNAB, patients should be evaluated for hyperparathyroidism and for pheochromocytoma. If these diagnoses are satisfactorily excluded, a total thyroidectomy with removal of regional nodes can safely be performed. [230] In patients with MEN, surgery should be performed for pheochromocytomas before surgery for MTC is performed. First-degree relatives of patients with MEN or familial MTC should undergo DNA testing for the presence of the mutant RET gene (see Chapter 36) . Gene carriers should undergo a prophylactic total thyroidectomy between 5 and 7 years of age. [232] [233]



Primary Malignant Lymphoma

Primary lymphomas of the thyroid are uncommon tumors, constituting fewer than 2% of all thyroid malignancies. The peak incidence is in the seventh decade, and the male/female ratio is 1:3. [237] [238] [239] [240] Thyroid lymphomas are almost invariably seen as a rapidly enlarging, painless neck mass, fixed to surrounding tissues; they cause compressive symptoms and should be differentiated from anaplastic carcinoma. Unilateral or bilateral lymph node enlargement is present in about 50% of affected patients. Clinically evident distant disease is uncommon. The palpated mass is solid and, if studied by imaging, would be hypoechoic on ultrasonography and nonfunctioning on thyroid scintiscan. Most primary thyroid lymphomas arise in patients who have chronic autoimmune thyroiditis. Nonetheless, the disease is a rare complication of Hashimoto's thyroiditis. [241] Primary thyroid lymphomas should be distinguished from generalized lymphomas with thyroid involvement. FNAB can be useful in distinguishing lymphoid proliferation from epithelial tumors. However, differentiating lymphoma from chronic autoimmune thyroiditis by thyroid cytology may be difficult. [242] Therefore, surgical specimens are needed for diagnosis. Immunohistochemical studies identify lymphoid proliferation if findings are positive for leukocyte common antigen. Because chronic autoimmune thyroiditis reproduces the exact features of a mucosa-associated lymphoid tissue (MALT), most cases of thyroid lymphoma are considered MALT lymphomas.[243] Those small cell lymphomas are characterized by a low grade of malignancy, slow growth, and a tendency for recurrence in other MALT sites, such as the gastrointestinal or respiratory tract, the thymus, or the salivary glands. A large proportion of clinical cases are large cell lymphomas and have an aggressive course. With immunohistochemistry, nearly all of them show B-cell markers. Monoclonality for light chain immunoglobulin is considered a strong indication of malignant lymphoma. Usually, immunohistochemistry is positive for BCL2 in small cell and negative in large cell lymphomas. Although accurate staging is very important for planning treatment, patients are often elderly, in poor condition, or may require urgent therapy to relieve symptoms, thus making a full staging investigation before treatment impractical. Staging includes physical examination; complete blood count; serum lactate dehydrogenase and 2- microglobulin measurements; liver function tests; bone marrow biopsy; CT scanning of the neck, thorax, abdomen, and pelvis; and appropriate biopsies at sites where tumor is suspected. Involvement of Waldeyer's ring and of the gastrointestinal tract has been associated with thyroid lymphomas, and for this reason upper gastrointestinal radiography or endoscopy should be performed. Disseminated disease necessitates chemotherapy. In patients with disease apparently confined to the neck, therapy is guided by the histologic features of the lymphoma. Chemotherapy with an anthracycline-based regimen and involved-field radiotherapy should be given to all patients with large cell thyroid lymphoma and in some series has provided long-term survival rates of nearly 100%. For small cell MALT lymphomas, radiation alone may be adequate if the disease is determined to be localized after accurate staging. [238] [239] [240]



SURGICAL TREATMENT OF THYROID CARCINOMA The extent of surgery appropriate for thyroid malignancy is a matter of controversy. [190] [191] Factors that influence this decision

478

include the histologic diagnosis, the size of the original lesion, the presence of DM, the patient's age, and the risk group category. [221] [223] Obviously, the surgeon must be appropriately skilled in thyroid surgery, and the goal of surgery should be to remove all the malignant neoplastic tissue present in the neck. Therefore, the thyroid gland and affected neck lymph nodes should all be carefully identified and adequately resected. In the case of PTC and FTC, although some debate still exists regarding the extent of thyroid surgery, many favor a near-total (leaving no more than 2 to 3 g of thyroid tissue) thyroidectomy for all patients. [169] [187] Near-total thyroidectomy reduces the recurrence rate, compared with more limited surgery, because many PTCs are both multifocal and bilateral. Removal of most, if not all, of the thyroid gland facilitates postoperative remnant ablation with 131 I. For extremely low-risk patients (i.e., those with unifocal intrathyroidal PTM and possibly small [10%) should lead to completion surgery. 131 I therapy can be administered to the other patients, usually with 24-hour uptakes considerably less than 10%. A total body scan is performed 4 to 7 days after the treatment dose, and levothyroxine suppressive therapy is initiated. Total ablation (defined as no visible uptake) may be verified by an 131 I total-body scan 6 to 12 months later, typically with 2 to 5 mCi (74 to 185 MBq). [253] Total ablation is achieved after administration of either 100 TABLE 13-6 -- Indications for No indication

131

I Treatment in Patients with Papillary, Follicular, or Hürthle Cell Thyroid Carcinoma after Initial Definitive Near-Total Thyroidectomy

Patients at low risk of cause-specific mortality or of relapse (e.g., PTC patients with MACIS scores < 6 and pTNM stage I FTC or HCC patients) Indications Definite Distant metastasis at diagnosis Incomplete tumor resection Patients at high risk for mortality or recurrence (e.g., PTC with MACIS 6+ and pTNM stage II/III FTC or HCC) Probable PTC or FTC in children younger than 16 years Tall cell or columnar cell variant of PTC Possible Diffuse sclerosing variant PTC Bulky bilateral nodal metastases Elevated Tg at 3+ months postoperatively FTC, follicular thyroid carcinoma; HCC, Hürthle cell carcinoma; MACIS, metastasis, age, completeness of resection, invasion, and size; PTC, papillary thyroid carcinoma; pTNM, pathologic-Tumor-Node-Metastasis; Tg, thyroglobulin. mCi (3700 MBq) or 30 mCi (1100 MBq) in more than 80% of patients who had at least a near-total thyroidectomy. After less extensive surgery, ablation is achieved in only two thirds of patients with 30 mCi (1100 MBq). Therefore, a near-total thyroidectomy should be performed in all patients who are to be treated with 131 I. Total ablation requires that a dose of at least 300 Gy (30,000 rad) is delivered to thyroid remnants, and a dosimetric study can allow a more precise estimate of the 131 I dose to be administered. [253] Obviously, the only patients eligible for such a protocol would be those selected high-risk patients with either PTC or FTC. 131 I ablation therapy does not play a regular role in the management of patients with anaplastic thyroid cancer, MTC, or thyroid lymphoma.



External Radiotherapy

External radiotherapy to the neck and mediastinum is indicated only for older patients with extensive PTC in whom complete surgical excision is impossible and in whom the tumor tissue does not take up 131 I. Retrospective studies have shown that in these selected patients, external radiotherapy decreases the risk of neck recurrence.[254] [255] [256] The target volume encompasses the thyroid bed, bilateral neck lymph node areas, and the upper part of the mediastinum. Typically, 50 Gy (5000 rad) would be delivered in 25 fractions over 5 weeks. In patients with MTC, this protocol may be applied after incomplete resection of the tumor and also after apparently complete surgery, when plasma calcitonin remains detectable in the absence of DM. In these patients, it may decrease the risk of neck recurrence by a factor of 2 to 4. [153] [254] In patients with anaplastic thyroid carcinoma, when the extent of disease is limited and surgery is feasible, accelerated external radiotherapy in combination with chemotherapy permits local control of the disease in two thirds of the patients and long-term survival in about 20%. [229]



Levothyroxine Treatment

The growth of thyroid tumor cells is controlled by TSH, and inhibition of TSH secretion with levothyroxine is thought to improve the recurrence and survival rates. Therefore, levothyroxine should be given to all patients with FCTC, whatever the extent of thyroid surgery and other treatment. The initial effective dose is about 2.5 µg/kg body weight in adults; children require a higher dose. The adequacy of therapy is monitored by measuring serum TSH 3 months after it is begun, the initial goal being a serum TSH concentration of 0.1 mU/L or less. In some centers, the serum free T 3 concentration is also documented to be within the normal range. [257] When these guidelines are followed, levothyroxine therapy does not have deleterious effects on the heart or bone. [89] In patients with anaplastic thyroid carcinoma, MTC, or thyroid lymphoma, a replacement dose of levothyroxine is given with the aim of obtaining a serum TSH level in the normal range.



FOLLOW-UP In patients with PTC or FTC, the goals of follow-up after initial therapy are to maintain adequate levothyroxine suppressive therapy and to detect persistent or recurrent thyroid carcinoma. Most recurrences occur during the first years of follow-up, but some occur late. Therefore, follow-up is necessary throughout the patient's life.

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Early Detection of Recurrent Disease Clinical and Ultrasonographic Examinations

Palpation of the thyroid bed and lymph node areas should be routinely performed at all follow-up visits in patients with thyroid cancer. Ultrasonography should be performed in patients at high risk of recurrence and in any patient with clinically suspicious findings. Palpable lymph nodes that are small, thin, or oval; in the posterior neck chains; and especially if they decrease in size after an interval of 3 months are considered benign. By contrast, round shape, hypoechogenicity and absence of a central echogenic line, microcalcifications, a cystic component, and hypervascularization on color Doppler ultrasonography are suspicious findings. Serum Tg is undetectable in 20% of patients receiving levothyroxine treatment who have isolated lymph node metastases, and undetectable values do not exclude metastatic lymph node disease. If in doubt, ultrasound-guided node biopsy for cytology and Tg measurement in the fluid aspirate may be performed. [258] Sensitive reverse transcriptasepolymerase chain reaction (RT-PCR) to amplify Tg mRNA appears to be even more sensitive but is not yet being used by commercial laboratories. [259] Radiographs

Bone and chest radiographs are no longer routinely obtained for patients with undetectable serum Tg concentrations. The reason is that virtually all patients with abnormal radiographs have readily detectable serum Tg concentrations. Serum Thyroglobulin Determinations

Tg is a glycoprotein that is produced only by normal or neoplastic thyroid follicular cells. Methods used for serum Tg determination and serum interferences are detailed in Chapter 10 . It should not be detectable in patients who have had total thyroid ablation, and its detection in that setting probably signifies the presence of persistent or recurrent disease (Table 13-7) . In patients who are in complete remission after total thyroid ablation, serum Tg antibodies decline gradually to low or undetectable levels. Their persistence or their reappearance during follow-up should be considered suspicious for persistent or recurrent disease. [260]

TABLE 13-7 -- Percentages of Patients with Detectable (>1 ng/mL) Serum Thyroglobulin Concentrations during Thyroxine Treatment and after Discontinuation of Thyroxine According to the Presence or Absence of Normal Thyroid Tissue * Thyroid Tumor Status Total Ablation Total Thyroidectomy Thyroxine treatment

On

Off

On

Off

Complete remission

25 to 30 mU/L) in patients treated in this way; if it is not, 131 I administration should be delayed until it is. Intramuscular injections of rhTSH (0.9 mg for 2 consecutive days) are an alternative because levothyroxine treatment need not be discontinued and side effects are minimal. When combining serum Tg measurement and 131 I total-body scanning, its efficiency is comparable to that of levothyroxine withdrawal in most patients. [261] [262] [263]

TABLE 13-8 -- Nonthyroidal Conditions Associated with Contamination

131

I Accumulation

Skin, hair, clothes Physiologic accumulations Salivary glands (mouth, nose) Stomach, esophagus, colon Bladder Breast Diffuse hepatic uptake ( 131 I-labeled iodoproteins) Inflammatory processes Lung or bronchial, cutaneous, dental, sinusoidal Various conditions Nonthyroidal neoplasms: salivary glands, stomach, lung, meningioma, struma ovarii Cysts: renal, pleuropericardial, hepatic, salivary, mammary, testicular hydrocele Thymus: normal or hyperplastic Ectasia of the common carotid artery with stasis Esophagus: dilatation, hiatal hernia Pericardial effusion, cardiac insufficiency

481

Figure 13-24 Total-body scan performed 5 days after administration of 100 mCi (3700 MBq) of radioactive iodine ( 131 I). The chest radiograph of this asymptomatic 34-year-old patient, who was being monitored for a papillary thyroid carcinoma, was normal; the only abnormality was an elevated serum thyroglobulin level, at 45 ng/mL, during levothyroxine suppressive treatment. Note the presence of diffuse uptake in the lungs (L) and in the left iliac bone (I). After four treatments with 100 mCi of 131 I, metastatic uptake disappeared and the thyroglobulin level became undetectable during levothyroxine therapy. B, bladder; M, mouth; N, nose; S, stomach.

When 131 I scanning is planned, patients should be instructed to avoid iodine-containing medications and iodine-rich foods, and urinary iodine should be measured in doubtful cases. Pregnancy must be excluded in women of childbearing age. For routine diagnostic scans, from 2 to 5 mCi (74 to 185 MBq) of 131 I is given; higher doses may reduce the uptake of a subsequent therapeutic dose of 131 I.[268] The scan is done and uptake, if any, is measured 48 to 72 hours after the dose, preferably using a double-head gamma camera equipped with thick crystals and high-energy collimators. False-positive results are rare and are usually easily recognized (Table 13-8) . PostIodine 131 Therapy Total-Body Scans

Assuming equivalent fractional uptake after administration of either a diagnostic or a therapeutic dose of 131 I, uptake too low to be detected with 2 to 5 mCi (74 to 185 MBq) may be detectable after the administration of 100 mCi (3700 MBq). Thus, a total-body scan should be routinely performed 4 to 7 days after a high dose (Fig. 13-24) . This is also the rationale for administering a large dose of 131 I in patients with elevated Tg levels (>10 ng/mL in the absence of levothyroxine treatment), even if the diagnostic scan is negative. [264] Other Tests

These should be performed only in selected cases and may include spiral CT or MR imaging of the neck and chest, bone scintigraphy, PET scanning using 18 FDG, and scintigraphy using a less specific tracer (e.g., thallium, MIBI, tetrofosmin). The FDG PET scan is more frequently positive in patients with no detectable 131 I uptake in the metastases and is particularly sensitive for the discovery of neck lymph nodes (Fig. 13-25) ; a spiral CT scan is more sensitive than a FDG PET scan for the discovery of small lung metastases. [22] [23] [24] Follow-up Strategy

If the total-body scan performed after administration of 131 I to destroy the thyroid remnants does not show any uptake outside the thyroid bed, physical examination is performed and serum TSH and Tg are measured during levothyroxine treatment 3 months later (Fig. 13-26) . In most centers, the serum Tg level is measured and a diagnostic 131 I total-body scan is done after thyroid hormone withdrawal or rhTSH stimulation 6 to 12 months later. Visible uptake in the thyroid bed that is too low to be quantified should not be considered evidence of disease in the absence of any other abnormality. If any significant uptake is detected outside the thyroid bed, a therapeutic dose of 100 mCi (3700 MBq) of 131 I is given. Serum Tg determination after TSH stimulation, obtained after either thyroid hormone withdrawal or injections of rhTSH, may help to select for scanning with a large amount of 131 I those patients with negative diagnostic 131 I total-body scans who have detectable serum Tg levels. [269] In low-risk patients considered cured, the dose of levothyroxine is decreased to maintain a low but detectable serum TSH concentration (0.1 to 0.5 mU/L). In high-risk patients, higher doses of levothyroxine are given, the goal being a serum TSH concentration less than 0.1 mU/L. [270] Clinical and biochemical evaluations are performed annually; neck ultrasonography is frequently performed in case of doubt or in high-risk patients, but any other testing is unnecessary as long as the patient's serum Tg concentration is undetectable and the patient does not produce an interfering anti-Tg autoantibody. In patients receiving levothyroxine in whom serum Tg becomes detectable, neck ultrasonography is performed and serum Tg may be measured again after levothyroxine is discontinued or after rhTSH stimulation. If residual neck disease is found on sonography, the diagnosis should be confirmed by guided biopsy and consideration given to surgical reexploration

482

Figure 13-25 The patient was being monitored for a papillary thyroid carcinoma. The serum thyroglobulin level was 22 ng/mL during levothyroxine suppressive treatment, and local imaging modalities were not interpretable because of three previous extensive neck operations. Left, Total-body scan performed 4 days after administration of 100 mCi (3.7 GBq); there is no visible uptake in the neck. Right, Positron emission tomography scan using [ 18 F]fluorodeoxyglucose ( 18 FDG) demonstrating significant uptake in a paratracheal lymph node (arrow) that measured 12 mm in diameter at surgery.

of the neck. If there is no demonstrable neck disease but the stimulated serum Tg concentration increases above 10 ng/mL, even if no uptake is seen on a diagnostic 131 I total-body scan performed with 2 to 5 mCi, consideration should be given to the administration of a therapeutic dose of 100 mCi of 131 I. In the absence of 131 I uptake, spiral CT of the neck and lungs, bone scintigraphy, and FDG PET scanning can be useful in

Figure 13-26 Follow-up of high-risk patients with papillary or follicular thyroid carcinoma after near-total thyroidectomy based on serum thyroglobulin (Tg) measurements and I ablation, total-body scanning. LT 4 , levothyroxine; TBS, total-body scan; TSH, thyrotropin. Thyroglobulin values are method specific, and the normal range should be determined in each assay. For the total-body scan, above 0 is positive, with 131 I uptake indicative of neoplastic foci; below 0 is negative. 131

localizing hitherto unrecognized sites of recurrent disease. In patients whose serum Tg levels are initially undetectable during levothyroxine treatment but later become detectable (levels 50%) of circulating androgens in premenopausal females. [129] In males this contribution is much smaller because of the testicular production of androgens, but adrenal androgen excess even in males may be of clinical significance, notably in patients with CAH. The adult adrenal secretes DHEA at approximately 4 mg/day, DHEAS at 7 to 15 mg/day, androstenedione at 1.5 mg/day, and testosterone at 0.05 mg/day. DHEA is a weak sex steroid but can be converted to androgens and estrogens through the activities of 3-HSD, a superfamily of 17-HSD isozymes, and aromatase, expressed in peripheral target tissues, and this is of clinical importance in many diseases. [130] ACTH stimulates androgen secretion; DHEA (but not DHEAS because of its increased plasma half-life) and androstenedione demonstrate a similar circadian rhythm to cortisol.[131] However, there are many discrepancies between adrenal androgen and glucocorticoid secretion, which has led to the suggestion of an additional androgen-stimulating hormone (Table 14-4) . Many putative androgen-stimulating hormones have been proposed including POMC derivatives such as joining peptide, prolactin, and insulin-like growth factor-I (IGF-I), but conclusive proof is lacking. Adrenal androgen steroidogenesis is dependent upon the relative activities of 3-HSD and 17-hydroxylase and in particular upon the 17,20-lyase activity of 17-hydroxylase. Factors that determine whether 17-hydroxylated substrates, 17-hydroxypregnenolone and 17-OHP, undergo 21-hydroxylation to form glucocorticoid or side-chain

500

TABLE 14-4 -- Dissociation of Adrenal Androgen and Glucocorticoid Secretion: Evidence for an Adrenal-Stimulating Hormone Dexamethasone studies: Complete cortisol suppression with chronic high-dose dexamethasone. DHEA falls by only 20%. Greater sensitivity of DHEA to acute low-dose dexamethasone administration. Adrenarche: Rise in circulating DHEA at 68 years of age. Cortisol production unaltered. Aging: Reduction in DHEA production, no change in cortisol. Anorexia nervosa and illness: Fall in DHEA, no change (or increase) in cortisol. DHEA, dehydroepiandrosterone. cleavage by 17-hydroxylase to form DHEA and androstenedione are unresolved and seem likely to be important in defining the activity of any putative androgen-stimulating hormone. [131]



CORTICOSTEROID HORMONE ACTION Receptors and Gene Transcription

Both cortisol and aldosterone exert their effects after uptake of free hormone from the circulation and binding to intracellular receptors, termed the glucocorticoid and mineralocorticoid receptors (GR and MR). [132] [133] [134] These are both members of the thyroid/steroid hormone receptor superfamily of transcription factors comprising a C-terminal ligand-binding domain, a central deoxyribonucleic acid (DNA) binding domain interacting with specific DNA sequences on target genes, and an N-terminal hypervariable region. In both cases, although there is only a single gene encoding the GR and MR, splice variants have been described resulting in and variants [135] [136] (Fig. 14-9) . Glucocorticoid hormone action has been studied in more depth than mineralocorticoid action. The binding of steroid to

Figure 14-9 Schematic structure of the human genes encoding the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). In both cases splice variants have been described; in the case of the GR, there is evidence that the GR isoform can act as a dominant negative inhibitor of GR action. mRNA, messenger ribonucleic acid.

the GR in the cytosol results in activation of the steroid-receptor complex through a process that involves the dissociation of heat shock proteins (HSP 90 and HSP 70). [137] After translocation to the nucleus, gene transcription is stimulated or repressed following binding of dimerized GR-ligand complexes to specific DNA sequences in the promoter regions of target genes. [138] [139] This glucocorticoid response element is invariably a palindromic CGTACAnnnTGTACT sequence that binds with high affinity to two loops of DNA within the DNA binding domain of the GR (zinc fingers). This stabilizes the RNA polymerase II complex, facilitating gene transcription. The GR variant may act as a dominant negative regulator of GR transactivation. [140] Naturally occurring mutations in the GR (as seen in patients with glucocorticoid resistance, discussed later) and GR mutants generated in vitro have highlighted critical regions of the receptor responsible for binding and transactivation, [141] [142] [143] but numerous others factors are also required (coactivators, corepressors [144] ) that may confer tissue specificity of response. This is a rapidly evolving field and is reviewed in Chapter 4 . However, the interactions between GR and two particular transcription factors are important in mediating the anti-inflammatory effects of glucocorticoids and explain the effect of glucocorticoids on genes that do not contain obvious glucocorticoid response elements in their promoter regions. Activator protein-1 (AP-1) comprises Fos and Jun subunits and is a proinflammatory transcription factor induced by a series of cytokines and phorbol ester. The GR-ligand complex can bind to c-jun and prevent interaction with the AP-1 site to repress AP-1 and GR trans-activation functions. [145] [146] Similarly, functional antagonism exists between the GR and nuclear factor B (NF-B). NF-B is a ubiquitously expressed transcription factor that activates a series of genes involved in lymphocyte development, inflammatory response, host defense, and apoptosis [146] (Fig. 14-10) . In keeping with the diverse array of actions of cortisol, many hundred glucocorticoid-responsive genes have been identified. Some glucocorticoid-induced genes and repressed genes are given in Table 14-5 . In contrast to the diverse actions of glucocorticoids, mineralocorticoids have a more restricted role, principally to stimulate

501

Figure 14-10 The anti-inflammatory action of glucocorticoids. Cortisol binds to the cytoplasmic glucocorticoid receptor (GR). Conformational changes in the receptor-ligand complex result in dissociation from heat shock proteins (HSPs) 70 and 90 and migration to the nucleus. Binding occurs to specific DNA motifsglucocorticoid response elements in association with the activator protein-1 (AP-1) comprising c-fos and c-jun. Glucocorticoids mediate their anti-inflammatory effects through several mechanisms: (1) The inhibitory protein IB, which binds and inactivates nuclear factor B (NFB), is induced. (2) The GR-cortisol complex is able to bind NFB and thus prevent initiation of an inflammatory process. (3) Both GR and NFB compete for the limited availability of coactivators that include cyclic adenosine monophosphate response element binding protein (CREB) binding protein and steroid receptor coactivator-1.

epithelial sodium transport in the distal nephron, distal colon, and salivary glands. [147] This stimulation is mediated through the induction of the apical sodium channel (comprising three subunits, , and ) [148] and the 1 and 1 subunits of the basolateral Na + ,K+ -adenosine triphosphatase [149] through transcriptional regulation of an aldosterone-induced gene, serum and glucocorticoid-induced kinase ( SGK). [150] Aldosterone binds to the MR, principally in the cytosol (although there is evidence for expression of the unliganded MR in the nucleus), followed by translocation of the hormone-receptor complex to the nucleus (Fig. 14-11) . The MR and GR share considerable homology: 57% in the steroid binding domain and 94% in the DNA binding domain. It is perhaps not surprising, therefore, that there is promiscuity of ligand binding with aldosterone (and the synthetic mineralocorticoid fludrocortisone) binding to the GR and cortisol binding to the MR. For the MR this is particularly impressivein vitro the MR has the same inherent affinity for aldosterone, corticosterone, and cortisol. [133] [151] Specificity upon the MR is conferred through the "prereceptor" metabolism of cortisol through the enzyme 11-HSD, which inactivates cortisol and corticosterone to inactive 11-keto metabolites, enabling aldosterone to bind to the MR. [152] [153] For both glucocorticoids and mineralocorticoids there is accumulating evidence for so-called nongenomic effects involving hormone responses obviating the genomic GR or MR. A series of responses have been reported within seconds or minutes of exposure to corticosteroids and are thought to be mediated by as yet uncharacterized membrane-coupled receptors. [154] [155]



Cortisol-Binding Globulin and Corticosteroid Hormone Metabolism

Over 90% of circulating cortisol is bound, predominantly to the 2 -globulin cortisol-binding globulin (CBG). [156] This 383-amino-acid protein is synthesized in the liver and binds cortisol with high affinity. Affinity for synthetic corticosteroids (except prednisolone, which has an affinity for CBG about 50% of that of cortisol) is negligible. Circulating CBG concentrations are approximately 26 mg/dL (700 nmol/L); levels are increased by estrogens and in some patients with chronic active hepatitis but reduced by glucocorticoids and in patients with cirrhosis, nephrosis, and hyperthyroidism. [157] The estrogen effect can be marked, with levels increasing twofold to threefold during pregnancy, and this should also be taken into account when measuring plasma "total" cortisol in pregnancy and in women taking estrogens.

502

Site of Action Immune system

TABLE 14-5 -- Some of the Genes Regulated by Glucocorticoids or Glucocorticoid Receptors Induced Genes

Repressed Genes

IB (NFB inhibitor)

Interleukins

Haptoglobin

TNF-

TCR

IFN-

p21, p27, and p57

E-selectin

Lipocortin

ICAM-1 Cyclooxygenase 2 iNOS

Metabolic

PPAR-

Tryptophan hydroxylase

Tyrosine aminotransferase

Metalloprotease

Glutamine synthase Glycogen synthase Glucose-6-phosphatase PEPCK Leptin -Fibrinogen Cholesterol 7-hydroxylase C/EBP/ Bone

Androgen receptor

Osteocalcin

Calcitonin receptor

Collagenase

Alkaline phosphatase IGF-BP-6 Channels and transporters

Epithelial sodium channel (ENaC) , , Serum and glucocorticoidinduced kinase (SGK) Aquaporin 1

Endocrine

bFGF

GR

VIP

PRL

Endothelin

POMC/CRH

RXR

PTHrP

GHRH receptor

Vasopressin

Natriuretic peptide receptors Growth and development

Surfactant protein A, B, C

Fibronectin -Fetoprotein NGF Erythropoietin G1 cyclins Cyclin-dependent kinases

Modified from McKay LI, Cidlowski JA. Molecular control of immune/inflammatory responses: interactions between nuclear factorB and steroid receptorsignalling pathways. Endocr Rev 1999; 20:435459. bFGF, basic fibroblast growth factor; CRH, corticotropin-releasing hormone; C/EBP/, CAAT-enhancer binding protein-beta; GR, glucocorticoid receptor; GHRH, growth hormonereleasing hormone; ICAM, intercellular adhesion molecule; IFN, interferon; IGF-BP, insulin-like growth factorbinding protein; IB, inhibitory kappa B; iNOS, inducible nitric oxide synthase; NFB, nuclear factor B; NGF, nerve growth factor; PEPCK, phosphoenolpyruvate carboxykinase; POMC, proopiomelanocortin; PPAR, peroxisome proliferatoractivated receptor; PTHrP, parathyroid hormonerelated protein; RXR, retinoid X receptor; SGK, serum and glucocorticoid-induced kinase; TCR, T-cell receptor; TNF-, tumor necrosis factor-alpha; VIP, vasoactive intestinal peptide.

Inherited abnormalities in CBG synthesis are much rarer than those described for thyroxine-binding globulin but include elevated CBG, partial and complete deficiency of CBG, or CBG variants with reduced affinity for cortisol. [158] [159] In each case, alterations in CBG concentrations change total circulating cortisol concentrations accordingly but free cortisol concentrations are normal. Only this free circulating fraction is available for transport into tissues for biologic activity. The free cortisol excreted through the kidneys is termed urinary free cortisol and represents only 1% of the total cortisol secretion rate. The circulating half-life of cortisol varies between 70 and 120 minutes. [161] The major steps for cortisol metabolism are depicted in Figure 14-12 . [162] [163] These

[160]

comprise: 1. The interconversion of the 11-hydroxyl (cortisol, Kendall's compound F) to the 11-oxo group (cortisone, compound E) through the activity of 11-HSD (EC 1.1.1.146). [164] [165] The metabolism of cortisol and cortisone then follow similar pathways. 2. Reduction of the C4-C5 double bond to form dihydrocortisol or dihydrocortisone followed by hydroxylation of the 3-oxo group to form tetrahydrocortisol (THF) and tetrahydrocortisone (THE). The reduction of the C4-C5 double bond can be carried out by either 5-reductase [166] or 5-reductase [167] to yield, respectively, 5-THF (THF) or 5-THF (allo-THF). In normal subjects the 5 metabolites predominate (5:5-THF 2:1). THF, allo-THF, and THE are rapidly conjugated with glucuronic acid and excreted in the urine. [168] 3. Further reduction of the 20-oxo group by either 20HSD or 20-HSD to yield - and -cortols and cortolones from cortisol and cortisone, respectively. Reduction of the C20 position may also occur without A ring reduction, giving rise to 20-hydroxycortisol and 20-hydroxycortisol. [163] 4. Hydroxylation at C6 to form 6-hydroxycortisol. [169] 5. Cleavage of THF and THE to the C 19 steroids 11-hydroxy and 11-oxo androsterone or etiocholanolone. 6. Oxidation of the C21 position of cortols and cortolones to form the extremely polar metabolites cortolic and cortolonic acids. [170]

503

Figure 14-11 Mineralocorticoid hormone action. An epithelial cell is depicted in the distal nephron or distal colon, or both. The much higher concentrations of cortisol are inactivated by the type 2 isozyme of 11-hydroxysteroid dehydrogenase (11-HSD2) to cortisone, permitting the endogenous ligand, aldosterone, to bind to the mineralocorticoid receptor (MR). Relatively few mineralocorticoid target genes have been identified, but these include serum and glucocorticoid-induced kinase (SGK), subunits of the epithelial sodium channel (ENaC), and basolateral Na+ ,K+ -adenosine triphosphatase.

Approximately 50% of secreted cortisol appears in the urine as THF, allo-THF, and THE; 25% as cortols and cortolones; 10% as C 19 steroids; and 10% as cortolic and cortolonic acids. The remaining metabolites are free, unconjugated steroids (cortisol, cortisone, 6- and 20/20-metabolites of THF and THE). [162] [163] The principal site of cortisol metabolism has been considered to be the liver, but many of the preceding enzymes have been described in the mammalian kidney, notably in the inactivation of cortisol to cortisone by 11-HSD. Quantitatively, the interconversion of cortisol to cortisone by 11-HSD is also the most important pathway. Furthermore, the bioactivity of glucocorticoids is in part related to the hydroxyl group at C11; cortisone with a C11 oxo group is an inactive steroid so that 11-HSD expressed in peripheral tissues plays a crucial role in regulating corticosteroid hormone action. Two distinct 11-HSD isozymes have been reported: a type 1 oxoreductase dependent on reduced nicotinamide adenine dinucleotide phosphate and expressed principally in the liver, which confers bioactivity upon orally administered cortisone by converting it to cortisol, and a type 2, nicotinamide adenine dinucleotidedependent dehydrogenase. It is 11-HSD2, coexpressed with the MR in the kidney, colon, and salivary gland, that inactivates cortisol to cortisone and permits aldosterone to bind to the MR in vivo. If this enzyme-protective mechanism is impaired, cortisol is able to act as a mineralocorticoid; this explains some forms of endocrine hypertension (apparent mineralocorticoid excess, licorice ingestion; see Chapter 15 ) and the mineralocorticoid excess state that characterizes the ectopic ACTH syndrome (see later). [164] [165] Hyperthyroidism results in increased cortisol metabolism and clearance and hypothyroidism the converse, principally because of an effect of thyroid hormone on hepatic 11-HSD and 5/5-reductases. [164] [171] [172] IGF-I increases cortisol clearance by inhibiting hepatic 11-HSD (conversion of cortisone to cortisol). [173] [174] 6-Hydroxylation is normally a minor pathway, but cortisol itself induces 6-hydroxylase so that 6-hydroxycortisol excretion is markedly increased in patients with Cushing's syndrome.[169] Furthermore, some drugs, notably rifampicin [175] and phenytoin, [176] increase cortisol clearance through this pathway. Patients with renal disease have impaired cortisol clearance because of reduced renal conversion of cortisol to cortisone. [177] These observations have clinical implications for patients with thyroid disease, acromegaly, and renal disease and for patients taking cortisol replacement therapy. Adrenal crisis has been reported in steroid-replaced addisonian patients given rifampicin, [178] and hydrocortisone replacement therapy may need to be increased in treated patients in whom hyperthyroidism develops. Aldosterone is also metabolized in the liver and kidneys. In the liver it undergoes tetrahydro reduction and is excreted in the urine as a 3-glucuronide tetrahydroaldosterone derivative; however, glucuronide conjugation at the 18 position occurs directly in the kidney, as does 3 and 5/5 metabolism of the free steroid. [179] Because of the aldehyde group at the C18 position, aldosterone is not metabolized by 11-HSD. Hepatic aldosterone clearance is reduced in patients with cirrhosis, ascites, and severe congestive heart failure



Effects of Glucocorticoids (Fig. 14-13) Carbohydrate, Protein, and Lipid Metabolism

Glucocorticoids increase blood glucose concentrations through their action on glycogen, protein, and lipid metabolism. In the liver, cortisol stimulates glycogen deposition by increasing glycogen synthase and inhibiting the glycogen-mobilizing enzyme glycogen phosphorylase. [180] Hepatic glucose output increases through the activation of key enzymes involved in gluconeogenesis, principally glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. [181] [182] In peripheral tissues (muscle, fat), cortisol inhibits glucose uptake and utilization. [183] In adipose tissue lipolysis is activated, resulting in the release of free fatty acids into the circulation. [184] An increase in total circulating cholesterol and triglycerides is observed, but HDL cholesterol levels fall. Glucocorticoids also have a permissive effect on other hormones including catecholamines and glucagon. The resultant effect is to cause insulin resistance and an increase in blood glucose concentrations at the expense of protein and lipid catabolism. Glucocorticoids stimulate adipocyte differentiation, promoting adipogenesis through the transcriptional activation of key differentiation genes including lipoprotein lipase, glycerol-3-phosphate dehydrogenase, and leptin. [185] Chronically, the effects of glucocorticoid excess on adipose tissue are more complex, at least in humans, in whom the deposition of visceral or central adipose tissue is stimulated, [186] providing a useful discriminatory sign for the diagnosis of Cushing's syndrome. The explanation for the predilection for visceral obesity may be related to the increased expression of both the GR [187] and type 1 isozyme of 11-HSD (generating cortisol from cortisone) in omental compared with subcutaneous adipose tissue. [188] Skin, Muscle, and Connective Tissue

In addition to inducing insulin resistance in muscle tissue, glucocorticoids cause catabolic changes in muscle, skin, and connective tissue. In the skin and connective tissue, glucocorticoids inhibit epidermal cell division and DNA synthesis and reduce collagen synthesis and production. [189] In muscle, glucocorticoids cause atrophy (but not necrosis), and this seems to be specific for type II or "phasic" muscle fibers. Muscle protein synthesis is reduced.

504

Figure 14-12 The principal pathways of cortisol metabolism. Interconversion of hormonally active cortisol to inactive cortisone is catalyzed by two isozymes of 11-hydroxysteroid dehydrogenase (11-HSD), 11-HSD1 principally converting cortisone to cortisol and 11-HSD2 the reverse. Cortisol can be hydroxylated at the C6 and C20 positions. A ring reduction is undertaken by 5-reductase or 5-reductase and 3-hydroxysteroid dehydrogenase.

505

Figure 14-13 The principal sites of action of glucocorticoids in humans highlighting some of the consequences of glucocorticoid excess. CNS, central nervous system; GI, gastrointestinal; FSH, follicle-stimulating hormone; GH, growth hormone; LH, luteinizing hormone; TSH, thyroid-stimulating hormone. Bone and Calcium Metabolism

The effects of glucocorticoids on osteoclast function are debated, but osteoblast function is inhibited by glucocorticoids, and this is thought to explain the osteopenia and osteoporosis that characterize glucocorticoid excess. [190] [191] With 0.25% to 0.5% of Western populations receiving chronic glucocorticoid therapy, [192] glucocorticoid-induced osteoporosis is becoming a prevalent health concern. [193] Glucocorticoids also induce a negative calcium balance by inhibiting intestinal calcium absorption [194] and increasing renal calcium excretion. [195] As a consequence, parathyroid hormone secretion is usually increased. Salt and Water Homeostasis and Blood Pressure Control

Glucocorticoids increase blood pressure by a variety of mechanisms involving actions on the kidney and vasculature. [196] In vascular smooth muscle they increase sensitivity to pressor agents such as catecholamines and angiotensin II while reducing nitric oxidemediated endothelial dilatation. [197] [198] Angiotensinogen synthesis is increased by glucocorticoids. [198] [199] In the kidney, depending on the activity of the type 2 isozyme of 11-HSD, cortisol can act on the distal nephron to cause sodium retention and potassium loss (mediated by the MR). [164] Elsewhere across the nephron, glucocorticoids increase glomerular filtration rate, proximal tubular epithelial sodium transport, and free water clearance. [200] The last effect involves antagonism of the action of vasopressin and explains the dilutional hyponatremia seen in patients with glucocorticoid deficiency. [201] [202] Anti-Inflammatory Actions and the Immune System

Glucocorticoids suppress immunologic responses, and this has been the stimulus to develop a series of highly potent pharmacologic glucocorticoids to treat a variety of autoimmune

506

and inflammatory conditions. The inhibitory effects are mediated at many levels. In the peripheral blood, glucocorticoids reduce lymphocyte counts acutely (T lymphocytes > B lymphocytes) by redistributing lymphocytes from the intravascular compartment to spleen, lymph nodes, and bone marrow. [203] Conversely, neutrophil counts increase after glucocorticoid administration. Eosinophil counts fall rapidly, an effect that was used historically as a bioassay for glucocorticoids. The immunologic actions of glucocorticoids involve direct actions on both T and B lymphocytes that include inhibition of immunoglobulin synthesis and stimulation of lymphocyte apoptosis.[204] Inhibition of cytokine production from lymphocytes is mediated through inhibition of the action of NF-B. NF-B plays a crucial and generalized role in inducing cytokine gene transcription; glucocorticoids can bind directly to NF-B to prevent nuclear translocation and can induce NF-B inhibitor, which sequesters NF-B in the cytoplasm, thereby inactivating its effect. [146] [205] [206] [207] Additional anti-inflammatory effects involve inhibition of monocyte differentiation into macrophages and macrophage phagocytosis and cytotoxic activity.

Glucocorticoids reduce the local inflammatory response by preventing the action of histamine and plasminogen activators. Prostaglandin synthesis is impaired through the induction of lipocortins, which inhibit phospholipase A 2 activity. [208] [209] Central Nervous System and Mood

Clinical observations of patients with glucocorticoid excess and deficiency reveal that the brain is an important target tissue for glucocorticoids, with depression, euphoria, psychosis, apathy, and lethargy being important manifestations (see the following). Both glucocorticoid and mineralocorticoid receptors are expressed in discrete regions of the rodent brain including hippocampus, hypothalamus, cerebellum, and cortex. [210] Glucocorticoids cause neuronal death notably in the hippocampus, [211] and this may underlie the interest in glucocorticoids and cognitive function, memory, and neurodegenerative diseases such as Alzheimer's. In the eye, glucocorticoids act to raise intraocular pressure through an increase in aqueous humor production and deposition of matrix within the trabecular meshwork, which inhibits aqueous drainage. Steroid-induced glaucoma appears to have a genetic predisposition, but the underlying mechanisms are unknown. [212] [213] Gut

Chronic but not acute administration of glucocorticoids increases the risk of developing peptic ulcer disease. with glucocorticoid

[ 214]

Pancreatitis with fat necrosis is reported in patients

TABLE 14-6 -- Therapeutic Use of Corticosteroids Endocrine: Replacement therapy (Addison's disease, pituitary disease, congenital adrenal hyperplasia), Graves' ophthalmopathy Skin: Dermatitis, pemphigus Hematology: Leukemia, lymphoma, hemolytic anemia, idiopathic thrombocytopenic purpura Gastrointestinal: Inflammatory bowel disease (Ulcerative colitis, Crohn's disease) Liver: Chronic active hepatitis, transplantation, organ rejection Renal: Nephrotic syndrome, vasculitides, transplantation, rejection Central nervous system: Cerebral edema, raised intracranial pressure Respiratory: Angioedema, anaphylaxis, asthma, sarcoidosis, tuberculosis, obstructive airway disease Rheumatology: Systemic lupus erythematosus, polyarteritis, temporal arteritis, rheumatoid arthritis Muscle: polymyalgia rheumatica, myasthenia gravis excess. The GR is expressed throughout the gastrointestinal tract and the MR in the distal colon, and these mediate the corticosteroid control of epithelial ion transport. [215] Growth and Development

Although glucocorticoids stimulate growth hormone (GH) gene transcription in vitro, [216] glucocorticoids in excess inhibit linear skeletal growth, [217] [218] probably as a result of catabolic effects on connective tissue, muscle, and bone and inhibition of the effects of IGF-I. Experiments on mice lacking the GR gene [111] emphasize the role of glucocorticoids in normal fetal development. In particular, glucocorticoids stimulate lung maturation through the synthesis of surfactant proteins (SP-A, SP-B, SP-C),[219] [220] and mice lacking the GR die shortly after birth as a result of hypoxia from lung atelectasis. Glucocorticoids also stimulate the enzyme phenylethanolamine N-methyltransferase, [221] which converts norepinephrine to epinephrine in adrenal medulla and chromaffin tissue. Mice lacking the GR do not develop an adrenal medulla. Endocrine Effects

Glucocorticoids suppress the thyroid axis, probably through a direct action on thyroid-stimulating hormone (TSH) secretion. In addition, they inhibit 5' deiodinase activity, mediating the conversion of thyroxine to active triiodothyronine. Glucocorticoids also act centrally to inhibit gonadotropin-releasing hormone pulsatility and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (see later).



THERAPEUTIC CORTICOSTEROIDS Since the dramatic anti-inflammatory effect of cortisone was first demonstrated in the 1950s, a series of synthetic corticosteroids have been developed for therapeutic purposes. These are used to treat a diverse variety of human diseases and rely principally on their anti-inflammatory and immunologic actions (Table 14-6) . The main corticosteroids used in clinical practice, together with their relative glucocorticoid and mineralocorticoid potencies, are listed in Table 14-7 . The structures of common synthetic steroids are depicted in Figure 14-14 . Biologic activity of a corticosteroid is dependent upon a 4-3-keto, 11-hydroxy, 17,21-trihydroxyl configuration. [222] Conversion of the C11 hydroxyl group to a C11 keto group (cortisol to cortisone) inactivates the steroid. The addition of a 1,2 unsaturated bond to cortisol results in prednisolone, which is four times more potent than cortisol in classical glucocorticoid bioassays such as hepatic glycogen deposition, suppression of eosinophils, and anti-inflammatory actions. Prednisone is the "cortisone equivalent" of prednisolone and relies upon conversion by 11-HSD type 1 in the liver for bioactivity. [223] Potency is further increased by the addition of a 6-methyl group to prednisolone (methylprednisolone). Fludrocortisone is a synthetic mineralocorticoid that has 125-fold greater potency than cortisol in stimulating sodium reabsorption. [224] This is achieved through the addition of a 9-fluoro group to cortisol. Interestingly, fludrocortisone also has glucocorticoid potency (12-fold greater than cortisol), and the addition of a 16-methyl group and 1,2 saturated bond to fludrocortisone results in dexamethasone, a highly potent glucocorticoid (25-fold greater than cortisol) but with negligible mineralocorticoid activity. [225] Betamethasone has the same structure but with a 16-methyl group and is widely used in respiratory and nasal aerosol sprays.

507

Steroid

TABLE 14-7 -- Relative Biologic Potencies of Synthetic Steroids in Bioassay Systems Anti-inflammatory Action Hypothalamic-Pituitary-Adrenal Suppression

Salt Retention

Cortisol

1

1

1

Prednisolone

3

4

0.75

Methylprednisolone

6.2

4

0.5

Fludrocortisone

12

12

1

14

Fludrocortisone

Triamcinolone Dexamethasone

125 225

5

4

0

26

17

0

Figure 14-14 Structures of the natural glucocorticoid cortisol, some of the more commonly prescribed synthetic glucocorticoids, and the mineralocorticoid fludrocortisone. Note that triamcinolone is identical to dexamethasone except that a 16-hydroxyl group is substituted for the 16-methyl group. Betamethasone, another widely used glucocorticoid, has a 16-methyl group.

Corticosteroids are given orally, parenterally, and by numerous topical routes (e.g., eyes, skin, nose, inhalation, rectal suppositories). [226] Unlike hydrocortisone, which has a high affinity for CBG, most synthetic steroids have low affinity for this binding protein and circulate as free steroid (30%) or bound to albumin (70%). Circulating half-lives vary depending upon individual variability and underlying disease, particularly renal and hepatic impairment. Cortisone acetate should not be used parenterally as it requires metabolism by the liver to active cortisol. It is beyond the consideration of this chapter to describe which steroid should be given and by which route for the nonendocrine conditions listed in Table 14-6 . The acute and chronic administration of corticosteroid therapy in patients with hypoadrenalism and CAH is discussed in these sections. In addition to the undoubted benefit that corticosteroids provide, there is increasingly a misuse of corticosteroid therapy, particularly in patients with respiratory or rheumatologic disease, to such an extent that up to 0.5% of the population is now prescribed chronic corticosteroid therapy. Because of their established euphoric effect, corticosteroids often make patients feel better but without any objective measures of improvements in underlying disease parameters. In view of the long-term sequelae of chronic glucocorticoid excess, decisions regarding treatment should be evidence-based and subject to constant review for efficacy and side effects. The endocrinologic consequences, notably suppression of the HPA axis, are an important aspect of modern clinical practice. Endocrinologists need to be aware of the effects of chronic therapy and advise on steroid withdrawal. Chronic Corticosteroid Therapy, Hypothalamo-Pituitary-Adrenal Axis Suppression, and Steroid Withdrawal

The negative feedback control of the HPA axis by endogenous cortisol has already been detailed. Synthetic corticosteroids similarly suppress the function of the HPA axis through a process that is dependent on both dose and duration of treatment. As a result, sudden cessation of corticosteroid therapy may result in adrenal failure. [226] This may also occur after treatment with high doses of the synthetic progestagen medroxyprogesterone acetate, which possesses glucocorticoid agonist activity.[227] In patients taking any steroid dose for less than 3 weeks, clinically significant suppression of the HPA axis is rarely a problem and patients can withdraw from steroids suddenly with no ill effect. [228] The possible exception to this is the patient who receives frequent short courses of corticosteroid therapy, for example, patients with recurrent episodes of severe asthma. Conversely, suppression of the HPA axis is invariable in patients taking the equivalent of 15 mg or more of prednisolone

508

Dose (mg pred/day) 7.5 mg

3 wk Can stop

*

TABLE 14-8 -- Duration of Glucocorticoid Treatment >3 wk Reduce rapidly e.g., 2.5 mg every 34 days THEN

57.5 mg

Can stop

Reduce by 1 mg every 24 wk

OR

THEN

20 pmol/L [90 pg/mL]) but nevertheless overlap values seen in Cushing's disease in 30% of cases [362] and cannot therefore be used to differentiate these two conditions (Fig. 14-20) . The most discriminatory time of day to measure ACTH is actually between 11 PM and 1 AM, when ACTH-cortisol secretion is at a nadir, and in our practice ACTH is usually measured with cortisol in the circadian rhythm studies. A midnight ACTH result greater than 5 pmol/L (23 pg/mL) in a patient with biochemical hypercortisolism confirms that the underlying disease is ACTH-dependent. The measurement of ACTH precursors (pro-ACTH, POMC) is not routinely available but may be more useful in detecting an ectopic source of ACTH; more data are required on patients with occult tumors causing the syndrome. In patients with adrenal tumors, plasma ACTH is invariably undetectable (5000 nmol/L

25005000 nmol/L

5002500 nmol/L (ACTH stimulation)

Testosterone

Increased

Increased

Variable, increased

Growth

-23 SD

-12 SD

Probably normal

21-Hydroxylase activity (% of wild type)

0%

1%

20%50%

Typical CYP21A2 mutations

Deletions, conversions, nt656g

I172N

V281L

G1108nt, R356W

nt656g

P30L

Hormones

I236N, V237E, M239K, Q318X ACTH, adrenocorticotropic hormone; SD, standard deviation. mutations in the human CYP21A2 gene, whereas in the carrier, heterozygote state, only one allele is mutated. The clinical significance of the heterozygote state is uncertain; it does not appear to affect reproductive capability but may cause signs of hyperandrogenism in women. [471] Two CYP21A2 genes are located within 50 kb of the short arm of chromosome 6 within the major histocompatibility locus, a 3' CYP21A2B gene encoding the functional enzyme and a pseudogene, CYP21A2A (Fig. 14-33) . These two genes are closely homologous, and at least 25% of cases of 21-hydroxylase deficiency arise because of unequal crossover and genetic recombination of these two genes at meiosis. Although mutations have been identified within the CYP21A2B gene in affected kindreds (point mutations, gene conversions, and deletions [471] [478] ), the relationship between genotype and phenotype is complex. [479] Severe mutations within the gene do not correlate with a severe phenotype either within families or in individual

Figure 14-33 Map of the short arm of human chromosome 6 (upper bar), showing the relative positions of the genes encoding the major histocompatibility proteins A, C, B, DR, DQ, and DP. The detail (lower bar) shows the approximately 120-kilobase region containing the genes for complement component C2, properdin factor B (Bf), and the duplicated complement C4 gene (C4A and C4B). The pseudogene CYP21A1 and the functional gene CYP21A2 are in tandem array with the two C4 genes. HLA, human leukocyte antigen.

cases. Phenotypic variability (e.g., salt wasting, age of onset) seems likely to depend on other interacting genes rather than CYP21A2B itself. The condition is inherited as an autosomal recessive trait, and the higher incidence of the condition in some ethnic communities is almost certainly related to consanguinity. A diagnosis of 21-hydroxylase deficiency should be considered in any newborn infant with genital ambiguity, salt wasting, or hypotension. Hyponatremia and hyperkalemia with raised plasma renin activity are found in salt wasters. In later life, adrenal androgen excess (DHEAS, androstenedione) is found in patients presenting with sexual precocity or a PCOS-like phenotype. 17-Hydroxyprogesterone (17-OHP) is invariably elevated, and clinically useful nomograms have been developed comparing circulating concentrations of 17-OHP before and 60 minutes after exogenous ACTH. [480] This separates patients with classical and nonclassical 21-hydroxylase deficiency from heterozygote carriers and normal subjects, but there is some overlap between values seen in heterozygotes and normal people. 17-OHP is measured basally and then 60 minutes after 250 µg of Synacthen. Stimulated values are invariably grossly elevated in patients with classical and nonclassical varieties (in excess of 35 nmol/L [11 µg/dL]). Heterozygote patients usually have stimulated values between 10 and 30 nmol/L (3 to 9 µg/dL) (Fig. 14-34) . Stimulation tests are not always required to make a diagnosis; for example, a basal 17-OHP concentration less than 5 nmol/L in the follicular phase of the menstrual cycle effectively excludes late-onset 21-hydroxylase deficiency. [476] Increasingly, genotyping programs will form a useful adjunct to hormonal measurements. Androgen excess in 21-hydroxylase deficiency is readily suppressed after glucocorticoid administration. Prenatal diagnosis of 21-hydroxylase deficiency has been advocated because treatment of an affected female may prevent masculinization in utero. [481] 17-OHP can be assayed in amniotic fluid, but the most robust approach is the rapid genotyping of fetal cells obtained by chorionic villus sampling in early gestation. Unlike hydrocortisone, which is inactivated by placental 11-HSD, maternally administered dexamethasone can cross the placenta to suppress the fetal HPA axis. One approach is to advocate dexamethasone therapy as soon as pregnancy is confirmed in high-risk cases and to continue this until the diagnosis is excluded in the fetus. If the fetus is affected, only those of female sex require dexamethasone therapy during

535

Figure 14-34 Basal and stimulated plasma 17-hydroxyprogesterone (17OHP) concentrations in patients with CYP21A2 (21-hydroxylase) deficiency. To convert values to nmol/L, multiply by 0.0303. The mean for each group is indicated by a large cross and the adjacent letter: c, patients with classical CYP21A2 deficiency; v, patients with nonclassical (acquired and cryptic) CYP21A2 deficiency; h, heterozygotes for all forms of CYP21A2 deficiency; p, general population; u, known unaffected persons (e.g., siblings of patients with CYP21A2 deficiency who carry neither affected parental haplotype as determined by human leukocyte antigen typing). (From White PC, New MI, Dupont B. Congenital adrenal hyperplasia: part 1. Reprinted by permission of The New England Journal of Medicine 1987; 316:15191524.)

gestation. Therapy must be initiated before 8 to 10 weeks of gestation to be effective. However, because only one in eight cases treated in this way have an affected female fetus, the use of steroid therapy in this setting has been questioned. [482] Dexamethasone can lead to maternal cushingoid effects in pregnancy [483] and may in turn have long-term, deleterious effects on the fetus. In patients with known cases requesting fertility (be they male or female), determination of 17-OHP levels through a Synacthen test in the partner before conception uncovers lateonset or heterozygote cases and provides the endocrinologist or geneticist with some assignment of risk before pregnancy. Treatment

The objectives in treating 21-hydroxylase deficiency differ with age, but at all ages treatment and overall management can be fraught with difficulties. In childhood the overall goal is to replace glucocorticoid and mineralocorticoid, thereby preventing further salt-wasting crises, but also to suppress adrenal androgen secretion so that normal growth and skeletal maturation can proceed. [473] Accurate replacement is essential; glucocorticoids in excess suppress growth, and inadequate replacement results initially in accelerated linear growth but ultimately in short stature because of premature epiphyseal closure. [471] Response is best monitored through growth velocity and bone age, with biochemical markers (17-OHP, DHEAS, testosterone) being useful adjuncts. In difficult cases, a day curve study as described for patients with primary adrenal failure, but measuring the ACTH and 17-OHP response before and after corticosteroid replacement, may confirm overreplacement or underreplacement. Corrective surgery is frequently required (clitoral reduction, vaginoplasty) during childhood. In late childhood and adolescence, appropriate replacement therapy is equally important. Overtreatment may result in obesity and delayed menarche or puberty with sexual infantilism, whereas underreplacement results in sexual precocity. Compliance with regular medication is often an issue through adolescence. Although much has been written about adequate control in childhood, adults with CAH often provide an ongoing dilemma for the endocrinologist. The follow-up of such patients should involve multidisciplinary clinics, initially with transition adolescence clinics to facilitate transfer from pediatric to adult

536

care. Problems in adulthood are related to fertility concerns, hirsutism and menstrual irregularity in women, obesity and impact of short stature, sexual dysfunction, and psychological problems [471] [484] [485] ; counseling is often required in addition to endocrine support. In the absence of any evidence-based data, there are no prescriptive steroid regimens to treat patients with CAH at any age, and as a result many individualized regimens are used in clinical practice. Usual starting doses of hydrocortisone in childhood are 10 to 25 mg/m 2 per day in divided doses. Reverse-phase therapy may be appropriate, giving the largest dose of hydrocortisone at night to suppress early morning ACTH secretion. Long-acting steroids such as dexamethasone are more effective in this regard, but care should be taken to avoid oversuppression and reduction in linear growth. Fludrocortisone is required for patients with salt wasting (although this may improve spontaneously with age); doses of 0.1 to 0.2 mg/day should be given and blood pressure, electrolytes, and supine-erect plasma renin activity monitored to assess response. Fludrocortisone may improve linear growth in patients with simple virilizing CAH even if they are not salt wasters. [486] In women with hyperandrogenism and untreated late-onset CAH, there is no evidence that final height is affected. In this setting, glucocorticoid suppression in isolation rarely controls hirsutism and additional antiandrogen therapy is often required (cyproterone acetate, spironolactone, flutamide together with an oral estrogen contraceptive pill). However, ovulation induction rates with gonadotropin therapy are improved after suppression of nocturnal ACTH levels with 0.25 to 0.5 mg of dexamethasone. Once final height is achieved in adult males, strict control is required only for patients with adrenal rests within the testes or to ensure fertility; inadequate replacement therapy may result in adrenal androgen excess suppressing pituitary FSH secretion and lowering sperm counts. [487] 11-Hydroxylase Deficiency

11-Hydroxylase deficiency accounts for 7% of all cases of CAH with an incidence of 1 per 100,000 live births. [488] The incidence is higher in Israel (1 per 30,000). The condition arises because of mutations in the CYP11B1 gene that result in loss of enzyme activity and a block in the conversion of 11-deoxycortisol to cortisol. As

reported for 21-hydroxylase deficiency, there remains a poor correlation between genotype and phenotype. There is loss of negative cortisol feedback and enhanced ACTH-mediated adrenal androgen excess (Fig. 14-35) . Clinical features are therefore similar to those reported in the simple virilizing form of CAH (virilized female fetus, sexual ambiguity), and again milder cases can present later in childhood or even young adulthood. The principal difference compared with 21-hydroxylase deficiency is hypertension, and this is thought to be secondary to the mineralocorticoid effect of DOC excess (see Table 14-23) . However, there is a poor correlation between DOC secretion and the presence of hypertension; furthermore, unexplained salt wasting has been reported in a few cases. [489] [490] On this clinical background, the diagnosis can be made by demonstrating a plasma ACTH-stimulated 11-deoxycortisol value that is more than three times the 95th percentile for an age-matched normal group. Although established heterozygotes do not demonstrate a rise in 11-deoxycortisol above normal after Synacthen [491] (unlike the 17-OHP response observed in heterozygote 21-hydroxylase patients), exaggerated ACTH-stimulated responses have been observed in patients with hirsutism[492] and in patients with essential hypertension, [493] suggesting partial defects in 11-hydroxylase activity. As reported for 21-hydroxylase deficiency, treatment is with replacement glucocorticoid therapy; with suppression of DOC secretion,

Figure 14-35 Congenital adrenal hyperplasia related to 11-hydroxylase deficiency. The normal synthesis of cortisol is impaired, and adrenocorticotropic hormone (ACTH) levels increase because of loss of normal negative feedback inhibition resulting in an increase in adrenal steroid precursors proximal to the block. The results are cortisol deficiency, mineralocorticoid excess related to excessive deoxycorticosterone (DOC) secretion, and excessive secretion of adrenal androgens. DHEA, dehydroepiandrosterone; StAR, steroidogenic acute regulatory protein.

plasma renin activity (suppressed at baseline) increases into the normal range. 17-Hydroxylase Deficiency

Fewer than 150 cases of 17-hydroxylase deficiency have been reported. [494] [495] Mutations within the CYP17 gene result in the failure to synthesize cortisol (17-hydroxylase activity), adrenal androgens (17,20-lyase activity), and gonadal steroids [496] (Fig. 14-36) . Thus, in contrast to 21-hydroxylase and 11-hydroxylase deficiencies, 17-hydroxylase deficiency results in adrenal and gonadal insufficiency. A single enzyme is expressed in adrenal and gonad and possesses both 17-hydroxylation and 17,20-lyase activities, [41] but patients with isolated deficiency in the hydroxylation of 17-OHP or 17,20-lyase deficiency have rarely been reported.[497] Loss of negative feedback results in increased secretion of steroids proximal to the block, and mineralocorticoid synthesis is enhanced. However, aldosterone levels are variable and the mineralocorticoid excess state that characterizes this condition is thought to be induced by DOC excess in over 80% of cases.[498] The genetic basis for the disease has been established in many cases, involving point mutations, gene deletions, and conversions in the CYP17 gene.[499] [500] Relative hydroxylase and lyase activities of mutant CYP17 complementary DNAs vary in in vitro transfection assays, but correlations with clinical phenotype are lacking. Thus, patients with clinically pure 17,20-lyase deficiency may have mutant CYP17 complementary DNAs that exhibit compromised 17-hydroxylase activity.[494] The diagnosis is usually made at the time of puberty when patients present with hypertension, hypokalemia, and hypogonadism, the latter occurring because of lack of CYP17 expression within the gonad and impaired gonadal steroidogenesis. As

537

Figure 14-36 Congenital adrenal hyperplasia related to 17-hydroxylase deficiency. The normal synthesis of cortisol is impaired, and adrenocorticotropic hormone (ACTH) levels increase because of loss of normal negative feedback inhibition resulting in an increase in adrenal steroid precursors proximal to the block. The result is cortisol deficiency and mineralocorticoid excess usually related to deoxycorticosterone (DOC) excess. Because gonadal 17-hydroxylase activity is also absent, sex steroid secretion in addition to adrenal androgen secretion is severely impaired, resulting in hypogonadism. DHEA, dehydroepiandrosterone; StAR, steroidogenic acute regulatory protein.

a result, LH and FSH levels are elevated. Female patients (XX) have primary amenorrhea with absent sexual characteristics, and males (46,XY) have complete pseudohermaphroditism with female external genitalia but absent uterus and fallopian tubes. The intra-abdominal testes should be removed, and such patients are usually reared as female. Glucocorticoid replacement reverses the DOC-induced suppression of the renin-angiotensin system and lowers blood pressure. Additional sex steroid replacement is required from puberty onward. 3-Hydroxysteroid Dehydrogenase Deficiency

In this rare form of CAH, the secretion of all classes of adrenal and ovarian steroids is impaired because of mutations within the HSD3B2 gene encoding 3-HSDII. [501] [502] Patients usually present in early infancy with adrenal insufficiency. Loss of mineralocorticoid secretion results in salt wasting, although this is absent in 30% to 40% of cases (Fig. 14-37) . As with 21-hydroxylase deficiency, absence of salt wasting may delay the presentation into childhood or puberty. [503] The correlation between genotype and phenotype is once again poor; identical mutations have been found in the HSD3B2 gene in both salt wasters and nonsalt wasters. [501] The spectrum of genital development is variable in both sexes. In males, because the 3-HSDII enzyme is also expressed within the gonad, male pseudohermaphroditism may occur with female external genitalia. In milder cases, hypospadias may be found or even normal male genitalia. In females, genital development can be normal but there is usually evidence of mild virilization, presumably because of enhanced adrenal secretion of DHEA, which is converted peripherally to testosterone. A late-onset form has been described in patients with premature pubarche [504] and a PCOS-like phenotype (hirsutism, oligomenorrhea, amenorrhea). [505] Because activity of the 3-HSDI enzyme present in skin and other peripheral tissues is intact, circulating 4 steroid levels (progesterone, 17-hydroxyprogesterone, androstenedione) may be normal (or even increased). However, a diagnosis is established by demonstrating an increased ratio of 5 steroids (pregnenolone, 17-hydroxypregnenolone, DHEA) to 4 steroids in plasma or urine. ACTH stimulation may be required to detect a late-onset presentation. Treatment is with replacement glucocorticoids, fludrocortisone (if indicated), and sex steroids from puberty onward. Steroidogenic Acute Regulatory Protein Deficiency

Mutations in the gene encoding StAR result in a failure of transport of cholesterol from the outer to the inner mitochondrial

Figure 14-37 Congenital adrenal hyperplasia related to 3-hydroxysteroid dehydrogenase (3-HSD) deficiency resulting in cortisol deficiency and variable mineralocorticoid deficiency. Gonadal 3-HSD activity is also absent, resulting in male pseudohermaphroditism and hypogonadism or primary amenorrhea in females. ACTH, adrenocorticotropic hormone; DOC,

deoxycorticosterone; DHEA, dehydroepiandrosterone; StAR, steroidogenic acute regulatory protein.

538

membrane in steroidogenic tissues; as a result, there is deficiency of all adrenal and gonadal steroid hormones. [28] [506] Presentation is with acute adrenal insufficiency in the neonatal period, and males exhibit pseudohermaphroditism because of absent gonadal steroids. The condition is fatal in infancy in two thirds of all cases. The adrenal glands are often massively enlarged and full of lipid; prior to the characterization of StAR, the condition was termed congenital "lipoid" hyperplasia and the candidate gene was thought to be cholesterol side-chain cleavage ( CYP11A1).[506] In fact, to date, no mutations have been reported in the CYP11A1 gene; such mutations are thought to be lethal in utero. This clinical phenotype is endorsed by recombinant mouse models lacking the StAR gene. [507] Apparent Cortisone Reductase Deficiency

In this condition, adrenal glands become hyperplastic because of ACTH stimulation resulting from a defect in cortisol metabolism rather than an inherent defect within the gland itself. Patients with apparent cortisone reductase deficiency have a defect in the conversion of cortisone to cortisol, suggesting inhibition of 11-oxoreductase activity and, by implication, inhibition of the type 1 isozyme of 11-hydroxysteroid dehydrogenase (11-HSD1) (see Fig. 14-12) . Eight cases have TABLE 14-25 -- Clinical and Biochemical Characteristics of Reported Cases of Apparent Cortisone Reductase Deficiency Age Sex

28

F

Clinical Features

Hirsuitism

Serum Androgens

THF + allo THF: THE ratio

Testosterone

Comments

Reference

Marked fall in serum androgens on treatment with dexamethasone

[510]

Fall in androgens with dexamethasone treatment although developed cushingoid side effects.

[511]

Sibling of preceding patient. Fall in androgens with treatment.

[511]

Fall in testosterone with treatment.

[512]

Sibling of preceding patient. No mutations on genetic sequence analysis of HSD11B1

[508]

No mutations on genetic sequence analysis of HSD11B1

[514]

17-OHP levels suppressed completely with prednisolone, indicative of an inability to activate cortisone acetate. No mutations on genetic sequence analysis of HSD11B1

[513]

No mutations on genetic sequence analysis of HSD11B1

[509]

DHEAS Androstenedione 17

F

Oligomenorrhea, hirsuitism, acne, obesity

Testosterone 0.039 DHEAS

18

F

Oligomenorrhea, hirsuitism, acne 0.045

30

37

F

Oligomenorrhea, hirsuitism, infertility

M

Excess body hair (sibling of preceding patient)

F

Obesity, oligomenorrhea, hirsuitism

Testosterone

Testosterone 0.03 DHEAS

(0.51.15)

Androstenedione

55

F

Congenital adrenal hyperplasia diagnosed shortly after birth (21-hydroxylase deficiency). 17-Hydroxyprogesterone levels unresponsive to cortisone acetate

F

Androgenetic alopecia, mild hirsutism

Testosterone 0.04 (0.50.8)

DHEAS, dehydroepiandrosterone sulfate; allo-THF, 5-tetrahydrocortisol; THE, tetrahydrocortisone; THF, 5-tetrahydrocortisol. been described; with one exception, all are female (Table 14-25) . Cortisol clearance is increased, and ACTH secretion is elevated to maintain normal circulating cortisol concentrations but at the expense of adrenal androgen excess. As a consequence, patients described are usually women who present with hirsutism, menstrual irregularity, or androgenic alopecia. Dexamethasone treatment to suppress ACTH has been used with some success to control the hyperandrogenism in these cases. Urinary tetrahydro metabolites of cortisol and cortisone show almost exclusively THE with little or no detectable THF or allo-THF (ratio of THF + allo-THF to THE less than 0.05, reference range 0.8 to 1.3). Further studies have also shown impaired plasma cortisol concentrations after an oral dose of cortisone acetate. Despite this biochemical evidence implicating a defect in 11-HSD1, investigations have revealed no mutations to date in the HSD11B1 gene in affected cases. [508] [509] [510] [511] [512] [513] [514] Patients with PCOS share many of the same clinical characteristics as those with apparent cortisone reductase deficiency. Although there is evidence to support increased cortisol secretion rates in PCOS, perhaps indicative of a defect in conversion of cortisone to cortisol, there remains to be a consensus with respect to THF + allo-THF/THE ratios. Both normal and reduced ratios have been reported in the literature.



539

Mineralocorticoid Deficiency

These syndromes are listed in Table 14-26 . They can be divided into those that are congenital and others that are acquired. Mineralocorticoid deficiency may occur in some forms of CAH and with other causes of adrenal insufficiency (e.g., Addison's disease and congenital adrenal hypoplasia) (see preceding). Primary Defects in Aldosterone Biosynthesis: Aldosterone Synthase Deficiency

Failure of conversion of corticosterone to 18-hydroxycorticosterone or of 18-hydroxycorticosterone to aldosterone usually results in a salt-wasting crisis in neonatal life. Hyperkalemia, metabolic acidosis, dehydration, and hyponatremia are found. The condition has been called corticosterone methyl oxidase (CMO) deficiency, but this was before the final enzyme or enzymes involved in the conversion of DOC to aldosterone were characterized and cloned. [515] [516] In fact, a single enzyme, aldosterone synthase, carries a multistep reaction involving 11-hydroxylation of DOC to corticosterone and 18-hydroxylation of corticosterone to 18-hydroxycorticosterone followed by 18-dehydrogenation to aldosterone (see Fig. 14-3) . Two variants of CMO deficiency are described; CMO I is characterized by low 18-hydroxycorticosterone and aldosterone levels, whereas patients with CMO II deficiency have hypoaldosteronism but high 18-hydroxycorticosterone levels. In both cases, mutations in the gene encoding aldosterone synthase have been described and the discrepant 18-hydroxycorticosterone levels seem likely to be explained on the basis of variable 18-hydroxylase activity of the related CYP45011-hydroxylase enzyme. [488] [517] [518] CMO II is much more common in Iranian Jews than the white population. Defects in Aldosterone Action: Pseudohypoaldosteronism

Pseudohypoaldosteronism type I may occur in neonatal life with respiratory difficulties but is usually found in infancy with severe salt wasting and failure to thrive, with very high plasma aldosterone and plasma renin activity levels and inappropriate urinary sodium loss. [519] [520] The MR appears to be defective, as judged by studies evaluating the binding of aldosterone to monocytes, but molecular studies have failed to show any abnormality in the MR itself. [521] Rather, inactivating mutations in the , , and subunits of the epithelial sodium channel have been shown to explain the condition. [522] Acquired forms of pseudohypoaldosteronism can occur in patients after renal transplantation, following obstructive uropathy, and in premature infants. Pseudohypoaldosteronism type II or Gordon's syndrome is an autosomal dominant disorder characterized by hyperkalemia TABLE 14-26 -- Causes of Mineralocorticoid Deficiency Addison's disease Adrenal hypoplasia Congenital adrenal hyperplasia (21-hydroxylase and 3-hydroxysteroid dehydrogenase deficiencies) Pseudohypoaldosteronism types I and II Hyporeninmic hypoaldosteronism Aldosterone biosynthetic defects Drug induced but not salt wasting, in contrast to the type I condition. Patients have resistance to the mineralocorticoid effects of aldosterone on tubular potassium transport but not to those of sodium and chloride transport. As a result, affected individuals have hyperchloremia, hypertension, and suppression of plasma renin activity. [523] Recently, deletions in the WNK4 gene (a member of the WNK family of serine-threonine kinases) have been described in affected cases. [524] Hyporeninemic Hypoaldosteronism

Angiotensin II is a key stimulus to aldosterone secretion, and damage or blockade of the renin-angiotensin system may result in mineralocorticoid deficiency. Various renal conditions have been associated with damage to the juxtaglomerular apparatus and hence renin deficiency. These include systemic lupus erythematosus, myeloma, amyloid, AIDS, and use of nonsteroidal anti-inflammatory drugs, but the most common (greater than 75% cases) is diabetic nephropathy. [525] [526] [527] The usual picture is of an elderly patient with hyperkalemia, acidosis, and mild to moderate impairment of renal function. Plasma renin activity and aldosterone are low and fail to respond to sodium depletion, the erect posture, or furosemide administration. In contrast to those with adrenal insufficiency, patients have normal or elevated blood pressure and no postural hypotension. Muscle weakness and cardiac arrhythmias may also occur. Other factors may contribute to the hyperkalemia, including the use of potassium-sparing diuretics, potassium supplementation, insulin deficiency, and -adrenoceptor blocking drugs and prostaglandin synthetase inhibitors, which inhibit renin release. Treatment of primary renin deficiency is with fludrocortisone in the first instance together with dietary potassium restriction. However, these patients are not salt depleted and may become hypertensive with fludrocortisone. In such a scenario, the addition of a loop-acting diuretic such as furosemide is appropriate. This increases acid excretion and improves the metabolic acidosis.



Adrenal Adenomas, Incidentalomas, and Carcinomas Etiology of Adrenal Tumors

The underlying basis for adrenal tumorigenesis is unknown. Clonal analysis suggests progression from a normal to adenomatous to carcinomatous lesion, but the molecular pathways involved remain obscure. Several factors have been associated with malignant transformation including genes encoding p53, p57 cyclin-dependent kinase, menin, IGF-II, MC2R, and inhibin-. [528] Mice lacking the inhibin- gene develop adrenal tumors through a process that is also gonadotropin-dependent. [529] Adenomas

Cortisol-secreting adrenal adenomas have been discussed in detail ("Cortisol-Secreting Adrenal Adenoma and Carcinoma"), and aldosterone-secreting adenomas (Conn's syndrome) are discussed in Chapter 15 . Pure virilizing benign adrenal adenomas are rare, with approximately 50 cases reported in the literature. The majority of cases occur in women; male cases are restricted to childhood, when presentation is with sexual precocity and accelerated bone age. In females, the majority of cases arise before the menopause with marked hirsutism, deepening of the voice, and amenorrhea. Clitorimegaly is found in 80% of cases. Testosterone is usually strikingly elevated, but gonadotropin levels may not be suppressed. By definition, urinary free cortisol is

540

Figure 14-38 A, Adrenal incidentaloma discovered in a woman undergoing investigation for abdominal pain. B, Incidentally discovered right adrenal myelolipoma.

normal. Tumors vary in size and should be treated surgically. Postoperatively, clinical features invariably improve and normal menses return.

[530]

Incidentalomas

Autopsy series had defined the prevalence of adrenal adenomas more than 1 cm in diameter to be between 1.5% and 7%. It is perhaps not surprising, therefore, that with the advent of high-resolution imaging procedures (CT, MRI), incidentally discovered adrenal masses have become a common clinical problem. An adrenal mass is uncovered in up to 4% of patients imaged for nonadrenal pathology. [531] Incidentalomas are uncommon in patients younger than 30 years but increase in frequency with age; they occur equally in males and females. In more than 85% of cases these lesions are nonfunctioning, benign adenomas. Occasionally they may represent myelolipomas, hamartomas, or granulomatous infiltrations of the adrenal and result in a characteristic CT or MRI appearance (Fig. 14-38) . Functioning tumors (pheochromocytomas or those secreting cortisol, aldosterone, or sex steroids) and carcinomas make up the remainder. In addition, it is established that some incidentalomas may cause abnormal hormone secretion without obvious clinical manifestations of a hormone excess state; the best example of this is "preclinical" Cushing's syndrome, which may occur in up to 20% of all cases. [532] This may explain why incidentalomas appear to be commoner in patients with obesity and diabetes mellitus. As a result, all patients with incidentally discovered adrenal masses should undergo appropriate endocrine screening tests. These should comprise 24-hour urinary catecholamine collection, 24-hour urinary free cortisol, and overnight dexamethasone suppression tests. Because of the reported poor sensitivity of serum potassium measurements in detecting primary aldosteronism, our practice has been to measure supine circulating plasma renin activity and aldosterone levels. DHEAS should be measured as a marker of adrenal androgen secretion. Low levels may occur in patients with suppressed ACTH concentrations related to autonomous cortisol secretion from the adenoma, [533] and it is important that DHEAS not be measured during the overnight dexamethasone study. Some studies have also documented high levels of 17-OHP after ACTH stimulation tests, suggesting partial defects in 21-hydroxylase in some tumors. [534] The possibility of malignancy should be considered in each case. In patients with a known extra-adrenal primary, the incidence of malignancy is obviously much higher (up to 20% of patients with lung cancer, for example, have adrenal metastases on CT scanning). In those with no evidence of malignancy, adrenal carcinoma is rare; in one study, only 26 of 630 incidentalomas were found to be adrenal carcinomas. [531] In true incidentalomas, size appears to be predictive of malignancy; a lesion less than 5 cm in diameter is most unlikely to be malignant. The majority of nonfunctioning lesions less than 5 cm can therefore be treated conservatively and patients followed up with annual imaging. Even incidentalomas larger than 5 cm are more likely to be benign than malignant, but because of an increased risk of malignancy many centers recommend removal of tumors more than 5 cm in diameter, preferably by laparoscopic adrenalectomy. Additional characteristic MRI appearances or scintigraphy studies may aid in differentiating malignant from nonmalignant lesions. CT-guided biopsy is useful in differentiating adrenal from nonadrenal tissue in the case of a suspected metastasis but is poor in differentiating benign adenomas from malignant adrenal lesions. Carcinomas

Primary adrenal carcinoma is rare, with an incidence of 1 per million population per year. Women are more commonly affected than men (2.5:1); mean age of onset is 40 to 50 years, although men tend to be older at presentation. Eighty percent of tumors are functional, most commonly secreting glucocorticoids alone (45%), glucocorticoids and androgens (45%), or androgens alone (10%). Less than 1% of all cases secrete aldosterone. Patients present with features of the hormone excess state (glucocorticoid or androgen excess, or both), but abdominal pain, weight loss, anorexia, and fever occur in 25% of cases. An abdominal mass may be palpable.[310] [311] [312] Current treatments for what is often an aggressive tumor are poor. Surgery offers the only chance of cure for patients with local disease, but metastatic spread is evident in 75% of cases at presentation. Radiotherapy is ineffective, as are most chemotherapeutic regimens. Mitotane in high doses offers transient benefit in reducing tumor growth in 25% to 30% of cases and controlling hormonal hypersecretion in 75% of cases. [311] Overall, the prognosis is poor, with 5-year survival rates of less than 20%.[535]



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Chapter 15 - Endocrine Hypertension Robert G. Dluhy Jennifer E. Lawrence Gordon H. Williams

Hypertension is a common disorder, occurring in approximately 20% of the United States population. The great majority of hypertensive subjects have the diagnosis of essential or primary hypertension. [1] Essential hypertension is a heritable syndrome reflecting a variety of pathophysiologic abnormalities that can lead, independently or together, to an elevated arterial blood pressure. [2] Although secondary causes exist in a smaller percentage (10%) of hypertensive subjects, they still represent a large number of patients. [3] Broadly speaking, the secondary causes of hypertension can be divided into renal causes (e.g., parenchymal or renovascular disease) and endocrine causes. In some disorders, many cases can be diagnosed by an astute clinician because the signs and symptoms are often distinct (e.g., pheochromocytoma and Cushing's syndrome). In addition, hypertension refractory to antihypertensive treatment may prompt the physician to screen for secondary causes. The age and sex of the hypertensive patient may also be helpful in the diagnosis of disorders with the secondary etiologies. For example, fibromuscular hyperplasia and Cushing's syndrome are more commonly seen in younger females, whereas primary hypothyroidism most commonly occurs in older female patients. Finally, making a diagnosis of a secondary disorder is gratifying because it may lead to significant amelioration or in some instances cure of the elevated blood pressure.



PHYSIOLOGY OF THE SYMPATHOADRENAL SYSTEM AND PHEOCHROMOCYTOMA The autonomic nervous system consists of the parasympathetic nervous system and the sympathoadrenal system. The neurotransmitter in the parasympathetic nervous system is primarily acetylcholine, and the neurotransmitters in the sympathoadrenal system include norepinephrine at sympathetic nerve endings in the periphery and central nervous system and epinephrine, which is secreted by the adrenal medulla into the systemic circulation. Another catecholamine, dopamine, acts primarily as a neurotransmitter in the central nervous system but is also secreted from peripheral sympathetic nerve endings. The sympathetic nervous system is under direct control of the central nervous system, allowing rapid onset of actions of short duration as a result of the abbreviated half-lives of catecholamines. Structure and Organization of the Sympathoadrenal System

The sympathoadrenal system, which is composed of the ganglia of the sympathetic nervous system and the adrenal medulla, is embryologically derived from neural crest tissue.[4] The precursor sympathogonia differentiate into neuroblasts, ultimately giving rise to the paravertebral and preaortic ganglion cells. Sympathetic preganglionic axons arise in large part from cells located in the thoracolumbar spinal cord. [5] [6] These preganglionic sympathetic neurons in turn have synapses with descending tracks from neurons in the pons, medulla, and hypothalamus, allowing regulation of sympathetic activity by the brain ( Fig. 15-1 ; see also Fig. 15-16 ). Thus, the limbic system and cortex can also regulate sympathetic activity by connections with central nuclei in the hypothalamus and medulla. In turn, these central nervous system neurons that influence sympathetic activity are regulated by a variety of factors including substrates (glucose) and hormones (corticotropin-releasing hormone). The axons of the preganglionic neurons synapse with postganglionic cell bodies located in the paravertebral and preaortic

553

Figure 15-1 Organization of the sympathoadrenal system. Sympathetic preganglionic axons arise in large part from cells located in the thoracolumbar spinal cord. These preganglionic sympathetic neurons in turn have synapses with descending tracks from neurons in the pons, medulla, and hypothalamus, allowing regulation of sympathetic activity by the brain. In turn, these central nervous system neurons that influence sympathetic activity are regulated by a variety of factors, including substrates (glucose) and hormones (corticotropin-releasing hormone). (From Landsberg L, Young JB. Catecholamines and the adrenal medula. In Bondy PK, Rosenberg JE (eds). Metabolic Control and Disease, 8th ed. Philadelphia: WB Saunders, 1980:16211693.)

ganglia as well as neurons in the celiac and superior and inferior mesenteric ganglia (Fig. 15-2) . Postganglionic axons from the cell bodies located in these ganglia in turn innervate the visceral organs. The splanchnic outflow of the lower thoracic and lumbar preganglionic axons also directly innervates the cells of the adrenal medulla (see Fig. 15-2) . Acetylcholine is the neurotransmitter at the ganglionic synapses and at the adrenal medullary neurons. In different tissues, the postganglionic sympathetic innervation can be cholinergic or noradrenergic, or both. For example, at arteriolar synaptic clefts norepinephrine is released from postganglionic nerve terminals, and sweat glands have sympathetic cholinergic innervation. Adrenergic nerves also contain other mediators including the peptide substance P, neuropeptide Y, somatostatin, and chromogranin A. [7] [8] [9] [10] These substances, which are released in peripheral and central adrenergic nerves, may have synergistic or direct actions with norepinephrine on effector cells. On the other hand, the adrenal medullary cells that secrete epinephrine into the systemic circulation are innervated by the splanchnic outflow of cholinergic preganglionic neurons. The arterial blood supply to the adrenal gland is derived from the aorta (middle adrenal artery), the inferior phrenic artery, and the renal artery. Adrenal blood flow drains centrally toward the medulla, eventually forming a single adrenal vein, which drains into the renal vein on the left and into the vena cava on the right. Although the existence of a corticomedullary portal system remains controversial, it is likely that local high concentrations of glucocorticoids influence the biosynthesis of epinephrine by induction of the enzyme phenylethanolamine N-methyltransferase (PNMT).[11] [12]



Catecholamines

All naturally occurring catecholamines contain a catechol nucleus (Fig. 15-3) . Epinephrine is synthesized and stored in the adrenal medulla and released into the systemic circulation. Norepinephrine is synthesized and stored at peripheral nerve endings. Dopamine acts primarily as a neurotransmitter in the central nervous system, although epinephrine and norepinephrine also act as central nervous system neurotransmitters. Catecholamines act widely in the body and affect many cardiovascular and metabolic processes. Specific catecholamine receptors mediate the biologic actions of these compounds.[13] [14] The amount of endogenous catecholamines released at peripheral nerve endings and the plasma concentration of epinephrine are the main determinants of the physiologic responses to activation of the sympathetic nervous system. Identification of -adrenergic and -adrenergic receptors and their receptor subtypes ( 1 , 2 , 1 , and 2 ) in target tissue has led to an understanding of the physiologic responses to exogenous and endogenous administration of catecholamines. [15] [16] [17] Moreover, the pharmacologic development of selective - and -adrenergic antagonists has added a wide range of treatments for a variety of clinical disorders. For example, 2 -agonists (terbutaline and albuterol), among their actions, can cause bronchial smooth muscle relaxation and are commonly prescribed in aerosol formulation for the treatment of bronchial asthma. [18] [19] On the other hand, 1 -antagonists (such as atenolol and metoprolol) are considered standard therapies for angina pectoris, hypertension, and cardiac arrhythmias. The - and -adrenergic receptors on cell surfaces reciprocally increase or decrease in response to the receptor-specific agonist concentration. Inhibition or stimulation of the intracellular adenylate cyclase (cyclic adenosine monophosphate [cAMP]) system mediates the majority of responses to receptor subtypespecific agonists. Catecholamine Synthesis

Catecholamines are formed from the amino acid tyrosine by a process of hydroxylation and decarboxylation (see Fig. 15-3) . This process of amine precursor uptake and decarboxylation (APUD) is a feature of neuroendocrine tissues that have a common origin. Most reactions occur in the cytoplasm except for hydroxylation of dopamine into norepinephrine, which occurs in the secretory vesicle. The rate-limiting step in catecholamine biosynthesis is the conversion of tyrosine to 3,4-dihydroxyphenylalanine (dopa) by the enzyme tyrosine hydroxylase (TH). [20] [21] The reaction requires tyrosine as substrate and oxygen, iron (Fe 2+ ), and tetrahydrobiopterin as cofactors. Tyrosine hydroxylase is expressed only in neuronal tissues that synthesize catecholamines, and several factors regulate its activity. The intraneuronal or intracellular transport of tyrosine may be affected by other amino acids or drugs that compete for transport or act as competitive inhibitors of the transport system, such as -methylparatyrosine. Increased intracellular levels of catechols down-regulate the activity of the enzyme. As catechols are released from secretory granules in response to a stimulus, cytoplasmic catecholamines are depleted and the feedback inhibition of tyrosine hydroxylase is released. Four isoforms of tyrosine hydroxylase exist. [22] Transcription is stimulated by glucocorticoids, cAMP-dependent protein kinases, Ca 2+ /phospholipid-dependent protein kinase, and Ca 2+ /calmodulin-dependent protein kinase. [23] [24] Aromatic L-amino acid decarboxylase (AADC) catalyzes the decarboxylation of dopa to dopamine, a process that can occur in any APUD tissue in which dopa is present. AADC is not specific for dopa. For example, decarboxylation of 5-hydroxytryptophan produces serotonin. Pyridoxal 5-phosphate is the cofactor required.

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Figure 15-2 Sympathoadrenomedullary efferent autonomic pathways. The axons of the preganglionic neurons synapse with postganglionic cell bodies located in the paravertebral and preaortic ganglia as well as neurons in the celiac and superior and inferior mesenteric ganglia. Postganglionic axons from the cell bodies located in these ganglia in turn innervate the visceral organs. The splanchnic outflow of the lower thoracic and lumbar preganglionic axons also directly innervates the cells of the adrenal medulla.

Dopamine is actively transported into granulated vesicles to be hydroxylated to norepinephrine by the copper-containing enzyme dopamine -hydroxylase (DBH). Oxygen is required, as is ascorbic acid, which acts as a cofactor and hydrogen donor. The enzyme is structurally similar to tyrosine hydroxylase and may share similar transcriptional regulatory elements, and both are stimulated by glucocorticoids and cAMP-dependent kinases. [25] These reactions occur in the synaptic vesicle of adrenergic neurons in the central nervous system, the peripheral nervous system, or the chromaffin cells of the adrenal

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Figure 15-3 Biosynthetic pathway for catecholamines (left to right). All catecholamines contain the catechol nucleus. L-Tyrosine is converted to L-3,4-dihydroxyphenylalanine (L-dopa) in the rate-limiting step by tyrosine hydroxylase (TH). Aromatic L-amino acid decarboxylase (AADC) converts L-dopa to dopamine. Dopamine is hydroxylated to L-norepinephrine by dopamine -hydroxylase (DBH). L-Norepinephrine is converted to L-epinephrine by phenylethanolamine N-methyltransferase (PNMT).

medulla. The major constituents of the granulated vesicle are dopamine -hydroxylase (in either a membrane-bound or soluble form), ascorbic acid, chromogranin A, and adenosine triphosphate (ATP). [26] In the adrenal medulla, norepinephrine is released from the granule into the cytoplasm, where it combines with the cytosolic enzyme PNMT to produce epinephrine (see Fig. 15-3) . Epinephrine is then transported back into another storage vesicle. The N-methylation reaction by PNMT involves S-adenosylmethionine as the methyl donor as well as oxygen and magnesium. PNMT expression is regulated by the presence of glucocorticoids, which are in high concentration in the medulla through the corticomedullary portal system. Cholinergic stimulation through both nicotinic and muscarinic components affects transcriptional regulation of PNMT. [27] [28] Epinephrine produces a noncompetitive negative feedback inhibition of PNMT activity. PNMT expression occurs in other tissues, including the lung, pancreas, and kidney. In spite of peripheral conversion, the plasma concentration ratio of norepinephrine to epinephrine is almost 9:1. In normal adrenal medullary tissue, approximately 80% of the catecholamine released is epinephrine. In patients with Addison's disease who have diminished cortisol production, adrenal medullary epinephrine production is decreased. [29] Small adrenal pheochromocytomas tend to secrete predominantly epinephrine, whereas larger tumors often secrete predominantly norepinephrine. [30] Larger tumors may outgrow the corticomedullary blood supply, thus losing the exposure to high local concentrations of glucocorticoids that regulate the activity of PNMT. Catecholamine Uptake and Release

Catecholamines are taken up into storage vesicles by active transport using an H + +-ATPdriven proton pump and carrier proteins, vesicular monoamine transporters (VMATs). [31] The ATP-driven pump maintains a steep electrical gradient. For every monoamine transported, ATP is hydrolyzed and two hydrogen ions are transported from the vesicle into the cytosol. Calcium is also maintained in high concentration within the vesicle. [32] [33] [34] Iodine 131labeled metaiodobenzylguanidine (MIBG) appears to be imported by VMATs into the storage vesicles in the adrenal medulla, which makes imaging with MIBG useful for evaluation of pheochromocytomas. [35]

Catecholamine uptake, as well as MIBG, is inhibited by reserpine. [36] Acetylcholine originating from preganglionic sympathetic fibers stimulates nicotinic cholinergic receptors and causes depolarization of the adrenomedullary chromaffin cell. Depolarization leads to activation of voltage-gated Ca 2+ channels resulting in exocytosis of secretory vesicle contents. [37] A Ca2+ -sensing receptor appears to be involved in the process of exocytosis. [38] During exocytosis, all of the granular contents are released into the extracellular space. Catecholamine Metabolism

Metabolism of catecholamines occurs through two enzyme pathways (Fig. 15-4) . Catechol-O-methyltransferase (COMT) is found primarily outside neuronal tissue and converts epinephrine to metanephrine and norepinephrine to normetanephrine by meta- O-methylation. S-Adenosylmethionine is used as the methyl donor, and Ca2+ is required. Metanephrine and normetanephrine are oxidized by monoamine oxidase (MAO) to vanillylmandelic acid (VMA) by oxidative deamination. MAO may also oxidize epinephrine and norepinephrine to 3,4-dihydroxymandelic acid, which is then converted by COMT to VMA. MAO is located on the outer membrane of mitochondria. In the storage vesicle, norepinephrine is protected from metabolism by MAO. MAO action may play an important role in regulating the metabolism of norepinephrine and dopamine. Intravesical stores of norepinephrine increase when MAO is inhibited. [39]



Pheochromocytoma Incidence and Importance

Pheochromocytomas are tumors of neuroectodermal origin arising from chromaffin cells. They are named for the dark staining reaction that is caused by the oxidation of intracellular catecholamine stores on exposure to dichromate salts. Although pheochromocytomas are a rare cause of hypertension, failure to recognize and treat a pheochromocytoma could prove a fatal oversight. Reportedly, less than 1% of patients who are evaluated for hypertension have pheochromocytomas, but this may be an underestimate. In one series, the incidence rate was calculated to be 0.8 per 100,000 person-years. In an autopsy series, approximately half of the pheochromocytomas were diagnosed at postmortem examination, demonstrating that this disorder is frequently not recognized. [40] [41] Tumors that arise from chromaffin cells of the adrenal medulla are referred to as pheochromocytomas, and those that arise in paraganglia are termed paragangliomas or extra-adrenal pheochromocytomas. The paraganglia are collections of specialized neural crest cells that have migrated to their final

556

Figure 15-4 Catecholamine metabolism. Metabolism of catecholamines occurs through two enzyme pathways. Catechol- O-methyltransferase (COMT) converts epinephrine to metanephrine and converts norepinephrine to normetanephrine by meta- O-methylation. Metanephrine and normetanephrine are oxidized by monoamine oxidase (MAO) to vanillylmandelic acid (VMA) by oxidative deamination. MAO also may oxidize epinephrine and norepinephrine to dihydroxymandelic acid (DOMA), which is then converted by COMT to VMA.

destination throughout the body. [42] Tumors can arise from sympathetic ganglia located from the neck to the bladder as well as the carotid body, vagal body, mediastinum, aorta, organs of Zuckerkandl, and pelvis (the most common site) (see Fig. 15-2) . It is commonly believed that the malignant potential is higher for extra-adrenal tumors than intra-adrenal tumors. However, some studies suggest that disease-free survival is similar for patients with extra-adrenal and intra-adrenal tumors.[43] Clinical Manifestations

Hypertension is the most common clinical manifestation of pheochromocytoma and is present in 90% to 100% of patients. Sustained hypertension is seen in approximately half, paroxysmal hypertension in a third, and normal blood pressure in less than a fifth of patients. [44] In children, sustained hypertension occurs most frequently. Patients with pheochromocytoma frequently present with paroxysmal episodes or spells that include the classic triad of severe headaches, palpitations, and diaphoresis.[45] These episodes may occur daily or as frequently as every few months. More than 90% of patients present with at least two of the three symptoms in the classic triad. The headaches are typically abrupt in onset, throbbing, and bilateral and diminish within an hour. The headaches may be associated with pallor or nausea, may be brief, or may persist over a week. The presence of palpitations, anxiety, or tremulousness may suggest the predominant secretion of epinephrine. [41] [46] [47] Less common symptoms include tremor, angina, nausea, Raynaud's phenomenon, livedo reticularis, and mass effect from the tumor. Increased total peripheral resistance causes the hypertension in patients with pheochromocytoma, as in patients with essential hypertension. Heart rate is variably increased in patients with pheochromocytoma. [48] [49] [50] Normal cardiac output is maintained by a decreased stroke volume resulting from intravascular volume depletion. [51] Lability of blood pressure is caused by a combination of the following: (1) episodic catecholamine release, (2) impaired sympathetic reflexes, and (3) unrecognized chronic volume depletion. [51] Altered sympathetic vascular regulation may underlie the orthostatic hypotension often seen in pheochromocytoma. [52] In rare cases of pheochromocytoma with predominant secretion of epinephrine, dopa, or dopamine, orthostatic hypotension may be the presenting symptom. [45] Patients with pheochromocytoma who are asymptomatic despite high circulating levels of catecholamines may have adrenergic receptor desensitization related to chronic stimulation.[53] Other cardiovascular manifestations of pheochromocytomas include dilated cardiomyopathy [54] resulting from catecholamine excess or hypertrophic cardiomyopathy.[55] Both forms have been reported to be reversible with tumor resection. Myocarditis has also been described in patients with pheochromocytoma with a pathology characterized by infiltration of inflammatory cells, specifically perivascularly, and focal contraction band necrosis. [56] Patients may also present with features of acute myocardial infarction including chest pain or electrocardiographic abnormalities including ST segment elevation or depression or inversion of T waves, or both. [46] [57] Other electrocardiographic manifestations of pheochromocytoma include left ventricular hypertrophy, sinus tachycardia, T-wave inversion, and rhythm disturbances such as supraventricular tachycardia or supraventricular ectopic beats. [51] Hereditary Pheochromocytoma

The majority of pheochromocytomas are sporadic. However, approximately 10% or more occur in association with a familial disorder such as multiple endocrine neoplasia type 2A or 2B (MEN-2A or MEN-2B), von HippelLindau (VHL) disease, or neurofibromatosis (see Chapter 36) . The MEN-2 syndromes and VHL disease are inherited in an autosomal dominant pattern with age-related penetrance. MEN-2A (Sipple's syndrome) is characterized by pheochromocytoma, medullary carcinoma of the thyroid, and hyperparathyroidism. MEN-2B is characterized by pheochromocytoma, medullary carcinoma of the thyroid, and multiple mucosal neuromas, often in association with a marfanoid habitus. Germ line mutations of the RET proto-oncogene have been described in the MEN-2 syndromes, and the MEN-2A gene has been localized to chromosome 10q11.2. [58] [59] The mutation confers constitutive activation of the tyrosine kinase receptor leading to unregulated hyperplasia and increased susceptibility to malignant transformation. The VHL disease phenotype includes pheochromocytoma,

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cerebellar and retinal hemangioblastomas, renal carcinoma, and renal and pancreatic cysts. The VHL disease suppressor gene has been cloned to chromosome 3p25-p26.[60] A loss-of-function mutation leads to tumor formation with different kindreds demonstrating varied clinical manifestations related to different types of mutations. Missense mutations are thought to be more commonly associated with pheochromocytoma.[61] [62] [62A] A low percentage (0.1% to 5.7%) of patients with von Recklinghausen's neurofibromatosis have pheochromocytoma. [63] However, a much higher percentage (50%) of pheochromocytoma is seen in such patients who have hypertension. [64] The majority of patients with von Recklinghausen's disease have solitary pheochromocytomas, whereas bilateral disease is often seen in other hereditary syndromes. Inactivating mutations in the neurofibromatosis F1 ( NF1) gene, a tumor suppressor gene on

chromosome 17 that encodes neurofibromin, lead to this disorder. [65] Hereditary pheochromocytomas are typically intra-adrenal and bilateral. In one series, up to 83% of the patients with familial pheochromocytoma had bilateral tumors.[66] Patients with hereditary pheochromocytoma typically present at younger ages than those with sporadic pheochromocytoma. The mean age of diagnosis of familial pheochromocytoma was 38 ± 11 years, compared with 47 ± 16 years for patients with sporadic tumors. [66] [66A] Sporadic pheochromocytoma cases usually present with hypertension; in contrast, many cases of the familial syndrome are diagnosed earlier as a result of biochemical surveillance or genetic testing, often before hypertension is detected. [67] The MEN-2 and VHL tumors make up most of the hereditary pheochromocytomas, but about 25% apparently sporadic patients may have germline mutations. [66A] It has been shown that MEN-2 tumors typically produce metanephrine, the metabolite of epinephrine, whereas tumors in patients with VHL disease produce normetanephrine, the metabolite of norepinephrine. These specific biochemical phenotypes demonstrate that unique mutation-dependent differential gene expression is probably involved in catecholamine synthesis. PNMT has been reported to be overexpressed in the MEN-2 tumors providing the epinephrine metabolism profile, whereas PNMT is under-expressed in VHL tumors providing the norepinephrine metabolism profile. [68] MEN-2 pheochromocytomas also appear to have increased tyrosine hydroxylase activity, which accounts for the greater concentration of catecholamine metabolites measured and clinical symptoms seen in MEN-2 patients compared with VHL patients. Patients with MEN-2 typically demonstrate episodic symptoms of hypertension. On the other hand, a pattern of sustained hypertension is seen in VHL patients. Thus, the biochemical phenotypes in these syndromes appear to be associated with particular patterns of catecholamine synthesis and release. Measurement of plasma-free metanephrines has been used to distinguish between MEN-2 and VHL disease and to reveal the presence of pheochromocytoma prior to clinical symptoms with greater sensitivity and specificity than urine testing [68] [69] (see later). Hereditary paragangliomas of the neck (glomus tumors) are associated with germ line mutations in a mitochondrial complex II gene, succinyl dehydrogenase subunit D (SDHD),[70] which encodes an enzyme that is involved in oxidative phosphorylation. Somatic and germ line mutations of the SDHD gene may also be associated with nonsyndromic, sporadic, [66A] [71] and familial pheochromocytoma.[72] Diagnosis

Differential Diagnosis

Pheochromocytoma may be suspected when a crisis, the physiologic consequence of abrupt catecholamine release, is precipitated by factors such as exertion, trauma, certain drugs, anesthesia, surgery, or surgical manipulation of the tumor. Tricyclic antidepressants, droperidol, glucagon, metoclopramide, phenothiazines, and naloxone have all been reported to induce hypertensive episodes. [45] [46] Foods or beverages, such as certain aged cheeses or red wine that contain tyramine, may precipitate a crisis. The -blockers may cause a paradoxical rise in blood pressure. Several disorders may mimic the symptoms of pheochromocytoma and also cause elevations in catecholamines. Abrupt withdrawal from medications such as clonidine or from alcohol may produce such a picture. Cerebral events such as cerebral vasculitis, preeclampsia, subarachnoid hemorrhage, migraine, and intracranial lesions associated with increased intracranial pressure may mimic pheochromocytoma. Agents such as amphetamines, ephedrine, pseudoephedrine, isoproterenol, phenylpropanolamine, cocaine, phencyclidine (PCP), and lysergic acid diethylamide (LSD) also lead to excess catecholamine levels. On the other hand, the symptoms of pheochromocytoma may be mistaken for those of panic attacks, hypoglycemic episodes, or accelerated hypertension of other etiologies. Lastly, disorders such as mastocytosis and the carcinoid syndrome, which are characterized by spells and episodic symptoms, may also mimic pheochromocytoma. [30] [47] [73] However, hypertensive crises, which often occur with pheochromocytoma, are notably absent in these disorders. In fact, episodic hypotension may occur with mastocytosis or the carcinoid syndrome as a result of peripheral vasodilation. Indication for Screening

Because pheochromocytomas do not occur frequently, physicians must appreciate when screening for the disorder is appropriate. The following are reasonable indications for screening: 1. 2. 3. 4. 5. 6. 7. 8.

Hypertension with episodic features suggesting pheochromocytoma (the classic triad of headaches, palpitations, and diaphoresis) Refractory hypertension Prominent lability of blood pressure Severe pressor response during anesthesia, surgery, or angiography Unexplained hypotension during anesthesia, surgery, or pregnancy Family history of pheochromocytoma or a familial disorder such as MEN-2, VHL disease, neurofibromatosis, or glomus tumors Incidentally discovered adrenal masses Idiopathic dilated cardiomyopathy

Biochemical Assessment

In pheochromocytoma, enzyme activity involved in synthesis of catecholamines is augmented and enzyme activity involved in catabolism is decreased. Because the catecholamine excess cannot be effectively stored, the hormones spill into the peripheral circulation. Biochemical measurement of excessive catecholamine production by the tumor confirms the diagnosis of pheochromocytoma (see later). However, because catecholamines are normally constitutively produced by the sympathoadrenal system, it is the magnitude of the elevation that is diagnostic of pheochromocytoma. Basal Measurements

The diagnosis is made with the demonstration of elevated circulating or urinary catecholamines or metabolites (see Fig. 15-3) . Screening

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Figure 15-5 Algorithm for diagnosis of pheochromocytoma. CT, computed tomography; MRI, magnetic resonance imaging.

methods include (1) 24-hour urine collection for excretion of unmetabolized or so-called free catecholamines (epinephrine and norepinephrine) or catecholamine metabolites (metanephrine, normetanephrine, and vanillylmandelic acid) and (2) determination of plasma metanephrines and catecholamines (Fig. 15-5) . Pheochromocytomas are heterogeneous in hormone metabolism and secretion. Therefore, there is no one optimal test for

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screening and there is disagreement about the preferred test for diagnosis. For example, as catecholamines have short half-lives and are secreted episodically, a

random plasma measurement may miss the peak catecholamine levels. On the other hand, plasma levels are particularly helpful when samples are collected during a paroxysm. Although the 24-hour urine collection has the advantage of integration of the catecholamine secretion over time, it is more cumbersome for patients and may yield a false-negative result if the collection is performed in absence of symptoms or hypertension in patients with episodic catecholamine secretion. The urinary metanephrine-to-creatinine ratio can be useful in compensating for overcollection (false-positives) or undercollection (false-negatives). [74] Overnight measurements of urine catecholamines have also been used to diagnose pheochromocytoma but involve an increased risk of both false-positive and false-negative results. [75] In the initial evaluation of a patient suspected of having a pheochromocytoma, we recommend a 24-hour urine collection for free or unmetabolized catecholamines (epinephrine and norepinephrine), total metanephrines, and creatinine (see Fig. 15-5) . Of the different metabolites that can be detected in a 24-hour urine collection, metanephrines are the most sensitive and specific. [74] There are less data concerning the appropriate use of plasma measurements. However, more experience with the measurement of plasma metanephrines is accruing, and it is considered by some to be a highly sensitive method for biochemical diagnosis, especially for hereditary pheochromocytoma (sensitivity 97%, specificity 96%). [76] Because pheochromocytomas secrete primarily metabolized catecholamines, plasma metanephrines may be more useful than plasma catecholamines. Measurement of plasma-free metanephrines as opposed to the conjugated or sulfated forms is particularly helpful because free fraction levels result from the actions of COMT on tumor catecholamine production. As a result, plasma-free metanephrines may show larger increases above normal than plasma catecholamines in pheochromocytoma. On the other hand, although plasma metanephrines are highly sensitive in detecting pheochromocytoma, a high number of false-positives may occur, particularly in older patients. Accordingly, the sensitivities and specificities of urine and plasma biochemical tests remain under investigation. As a result, some centers advocate urine testing as the initial screening test whereas others suggest plasma-free metanephrine levels. It is important to remember that the current technology for measuring plasma metanephrines requires that the patient abstain from acetaminophen for 3 to 5 days before testing [77] (see Fig. 15-5) . Typically, a measurement of urinary catecholamines or metabolites that is two or three times above the upper limit of normal is considered diagnostic of pheochromocytoma. For example, the upper limit of normal for total urinary catecholamines is approximately 100 µg per 24 hours, and a measurement above 250 µg per 24 hours is obtained in most patients with pheochromocytoma. Urine collections should include measurement of urinary creatinine to verify the adequacy of collection. A strong acid (such as 6 N HCl) is added to a sealed container. The optimal system for plasma catecholamine determination includes having the patient fast overnight and lie comfortably in a supine position with a heparin lock inserted 20 to 30 minutes before collection for withdrawing the blood. Certain precautions should be taken in interpreting catecholamine or metanephrine values. For example, iodinated contrast dyes can interfere with some biochemical measurements. Labetalol can give falsely elevated results when the following assays are employed: fluorometric methods of analysis used for catecholamine measurements, spectrophotometric methods used for metanephrine measurements, or radioenzymatic assays used for urinary-free catecholamine measurements. [78] Tricyclic antidepressants, prochlorperazine (Compazine), reserpine, clonidine, and clofibrate may interfere with urinary catecholamines and metabolite measurements. [79] [80] Such medications should be discontinued, preferably 2 weeks before collection. Blood pressure should be controlled with agents such as dihydropyridine calcium channel blockers that do not interfere with the assays. To provide better resolution against interfering substances, many laboratories use a reverse-phase high-performance liquid chromatography method with electrochemical detection. Measurement of these compounds by mass spectroscopy, which eliminates the problem of interfering substances, and use of immunoassay techniques are future directions. Stresses associated with serious illnesses, such as myocardial infarction, cerebral vascular accidents, or congestive heart failure, cause elevation in catecholamine levels. In renal insufficiency, plasma and urinary levels may be falsely elevated and urinary collections should be expressed in milligrams of creatinine. [81] In these circumstances, other diagnostic tests including imaging modalities are required for evaluation. Chromogranin A is a soluble protein stored and secreted with catecholamines in chromaffin tissue. This biochemical marker is not specific for pheochromocytoma, and elevations may be seen with other neuroendocrine tumors. Plasma chromogranin A levels are elevated in more than 80% of patients with pheochromocytomas. [83] This assay is most often utilized in the postoperative surveillance of patients after resection of catecholamine-secreting tumors (see "Medical and Surgical Management"). Chronic renal failure is also associated with elevated chromogranin A levels.

[ 82]

Stimulation and Suppression Tests

The clonidine and glucagon tests are dynamic tests that are not routinely performed but are usually used when the suspicion of pheochromocytoma is high but the basal catecholamine levels are not diagnostic or are equivocal. Clonidine is a centrally acting 2 -adrenoceptor agonist that normally suppresses the release of catecholamines from neurons but does not affect the autonomous release of catecholamines from a neoplasm. [46] [84] In patients without pheochromocytoma, a decrease in basal plasma catecholamines by 50% or less than 3 nmol/L (500 pg/mL) is expected 2 to 3 hours after 0.3 mg of clonidine is administered. [46] Provocative testing is utilized when clinical suspicion of pheochromocytoma is not supported by the biochemical testing. Patients with pheochromocytomas typically demonstrate a threefold increase in plasma catecholamine levels or a concentration greater than 12 nmol/L (2000 pg/mL) 2 minutes after administration of 1.0 mg of intravenous glucagon. The glucagon provocative test is considered highly specific but poorly sensitive, whereas the clonidine test is considered highly sensitive with poor specificity. If both tests are negative, the diagnosis of pheochromocytoma may be reasonably excluded. [46] [85] Imaging Techniques

After the diagnosis of pheochromocytoma is confirmed by biochemical testing, imaging techniques are employed for tumor location. Localization techniques include magnetic resonance imaging (MRI), computed tomography (CT), and MIBG or octreotide scintigraphy. Pheochromocytomas are typically large tumors (2 to 5 cm in diameter) and may contain areas of hemorrhage or necrosis. Pheochromocytomas in hereditary syndromes tend to be bilateral and smaller. [86] The latter feature is probably related to early detection as the result of periodic surveillance. Approximately 98% of pheochromocytomas are intra-abdominal, and 90% originate within the adrenal gland. However,

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Figure 15-6 A, Axial in-phase gradient-echo image demonstrates left adrenal mass (arrow). B, Axial image from the out-of-phase gradient-echo sequence demonstrates the left adrenal mass. In comparison to the in-phase image, no suppression of the adrenal mass is present. Suppression is the rule in lipid-containing cortical adenomas.

pheochromocytomas may occur anywhere in the autonomic nervous system including the posterior mediastinum, pericardium, or bladder (see Fig. 15-2) . Because of their size, intra-adrenal tumors are usually easily imaged by either CT or MRI (Fig. 15-6) . Although MRI may have greater specificity, CT has a sensitivity between 93% and 100%. Because of the tumor size, contrast agents are not required for visualization and, in fact, may precipitate a hypertensive crisis. With T2-weighted MRI, adrenal pheochromocytoma is usually three times as intense as liver. On T1-weighted images, the tumor is usually isointense with the liver. With gadolinium-diethylenetriaminepentaacetic acid (DTPA), the tumor appears hypervascular. [86] These general characteristics are not uniformly met, and rarely a pheochromocytoma is indistinguishable from other adrenal tumors. MRI is preferred for localization of paragangliomas, especially those located outside the abdomen, such as a posterior mediastinal or intracardiac tumor. MIBG has chemical similarities to norepinephrine and is concentrated within intracellular storage granules of catecholamine-secreting tissues. [87] Radioiodinated MIBG is especially valuable for localization of pheochromocytomas of extra-adrenal origin and for confirmation of tumor resection in postoperative surveillance. [88] The sensitivity of MIBG is reported to be greater than 90% with a specificity of 100%. [87] [89] MIBG is labeled with 123 I or 131 I; the former isotope with gamma ray flux has fewer particulate emissions and may provide greater sensitivity. Thyroid uptake should be blocked by administration of iodide preparations 3 days before and 1 week after iodine-labeled MIBG is given. Medicines that could interfere with catecholamine metabolism, uptake, or release should be discontinued at least 72 hours before MIBG evaluation. For example, tricyclic antidepressants and phenylpropanolamine inhibit MIBG uptake; sympathomimetics (cocaine, labetalol, and reserpine) deplete the storage vesicle contents. Atypical antidepressants, phenothiazines and butyrophenones, which block adrenergic receptors, may also produce false-negative

results. [90] Somatostatin receptor scintigraphy is another localization technique because somatostatin receptors are normally expressed in adrenomedullary and paraganglionic tissues.[91] The receptor density is increased on pheochromocytoma tissue, which enables imaging with the somatostatin analogue octreotide. Octreotide, which binds to somatostatin receptor subtypes 2 and 5, is labeled with 111 In-DTPA. As with MIBG, octreotide scanning is best employed for detection of pheochromocytomas of extra-adrenal origin and of metastases from malignant pheochromocytoma. [92] Some malignant tumors down-regulate the expression of somastatin receptors. As a result, lack of uptake by octreotide may be a poor prognostic indicator in such patients. In contrast to MIBG or octreotide scintigraphy, which requires 24 to 48 hours for optimal visualization, a promising new technology that can immediately image a pheochromocytoma is 6-[18 F]fluorodopamine positron emission tomography. [93] The uptake and retention of this radiopharmaceutical in chromaffin cells with subsequent imaging by emission scanning may provide a useful diagnostic test in patients with pheochromocytoma. Finally, venous sampling has been utilized in the past to confirm or rule out the diagnosis of pheochromocytoma. The adrenal sampling effluent has a norepinephrine/epinephrine ratio less than 1. Higher ratios suggest the presence of pheochromocytoma. [94] [95] Medical and Surgical Management

Preoperative Management

Surgical excision of a pheochromocytoma is the treatment of choice, but it involves a risk of morbidity as high as 40% and a risk of mortality of 2% to 4%. [73] Surgical outcomes have improved with preoperative treatment such as -receptor blockade and volume expansion. [96] Volume expansion is initially achieved with a high-sodium diet (150 to 200 mEq/L [150 to 200 mmol/day]) unless contraindicated by congestive heart failure or renal insufficiency. Phenoxybenzamine, a noncompetitive -blocker, has traditionally been used for preoperative preparation. Phenoxybenzamine is titrated to reduce blood pressure to normal levels or orthostasis, or both. The starting dose, 10 mg/day by mouth, is titrated upward every 2 days. Most

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patients require 80 to 100 mg daily given in divided doses. When -blockade is established, -blockade may be initiated if the patient is tachycardic or has arrhythmias. Without prior -blockade, -blockade alone can lead to unopposed -receptor stimulation and further elevation of blood pressure. Because phenoxybenzamine blocks catecholamine binding to receptors, it minimizes the risk of a hypertensive crisis during intubation, during induction with anesthesia, or during exploration and tumor manipulation. A noncompetitive -blocker is theoretically preferred to a competitive inhibitor because catecholamine levels, which can increase 500-fold, may overcome a competitive inhibitor. [96] However, complete -blockade can mask the dramatic fall in blood pressure seen after tumor resection that signals to the surgeon that the pheochromocytoma is resected. Phenoxybenzamine may also lead to postoperative hypotension because of its prolonged half-life of 24 hours. [97] Calcium channel blockers, in particular nicardipine from the dihydropyridine class, are increasingly popular. These agents improve intraoperative systemic vascular resistance by blunting catecholamine-mediated arterial vasoconstriction during tumor manipulation, and they have few side effects. [98] [99] The selective 1 inhibitor doxazosin has been effective in preoperative management without causing tachycardia or other serious side effects. [100] The oral formulation of labetalol, with an /-blocking ratio of 1:3, may not be ideal for preparation for surgery because the -blockade is weaker than that of phenoxybenzamine. As a result, additional vasodilators may be required intraoperatively in labetalol-pretreated subjects. [101] Finally, metyrosine, which inhibits catecholamine synthesis, has been used in combination with -blockade for preoperative management. [102] Tradition and experience have guided the length of preoperative therapy with volume expansion and /-blockade. Patients are typically treated for 10 to 14 days, although this time course has not been consistently associated with better operative and postoperative outcomes. [96] Unfortunately, there are no reliable features that predict a smooth surgical course. Thus, each patient must be evaluated on an individual basis when selecting antihypertensive medicines. Surgical Management

The surgical approach is dictated by the clinical situation. In patients with familial pheochromocytoma, a transabdominal incision allows adequate visualization and bilateral adrenalectomy if required. The flank approach offers better exposure and reduced blood loss for the patient with a solitary tumor. Surgeons are gaining experience with laparoscopic adrenalectomy performed when the tumor is smaller than 6 cm. [103] [104] The patient with pheochromocytoma should be referred to a surgeon who has experience in the management of pheochromocytoma and who collaborates with an experienced anesthesiologist to establish a smooth team effort. Intraoperative hypotension is managed initially with volume expansion and then with intravenous pressor agents if necessary. Postoperative hypoglycemia, which may be due to reactive hyperinsulinemia, should be anticipated and warrants routine screening of glucose monitoring in the early postoperative hours. [105] Surgical outcomes have been improved by intraoperative hemodynamic monitoring, the combination of fast-acting intravenous vasodilators and -blockers (sodium nitroprusside and esmolol, respectively), and the use of intravenous vasoconstrictors (norepinephrine or epinephrine). The following is an approach followed in our institution to prepare the patient for surgery. As soon as the diagnosis of pheochromocytoma is established, -blockade is started and titrated upward (see earlier). Five days before surgery, if not earlier, a high-sodium (150 to 200 mEq/L [150 to 200 mmol]) diet is initiated; therapy with -blockade is continued, and daily weights and vital signs are monitored. Admission for volume expansion with intravenous saline is considered, depending on the patient's status. One day before surgery, the patient is transferred to a monitored intensive care setting with intravenous arterial and Swan-Ganz catheters. Isotonic saline is administered to achieve a pulmonary capillary wedge pressure greater than 10 mm Hg. If systemic vascular resistance is elevated (>1000 dyne second/cm 5 /m2 ), intravenous sodium nitroprusside at 0.5 to 2 µg/kg per minute is initiated and increased as needed (500 to 1000 mg/minute may be required). On the day of surgery, the patient is given two separate intravenous lines, one for administration of pressors and the other for administration of vasodilators. Sodium nitroprusside, esmolol, epinephrine, and norepinephrine infusions are on standby in the event that they are required. When surgery is performed, the catecholamine levels usually return to normal in approximately 2 weeks. If hypertension persists despite normal catecholamine levels and surgical etiologies such as inadvertent ligation of the renal artery are excluded, essential hypertension or hypertension secondary to renal damage may be the cause. In a series from the Mayo Clinic, 20% of patients who had postoperative hypertension were found to have essential hypertension. [45] Alternatively, the patient could harbor another pheochromocytoma or have metastatic disease. As a result, patients should be monitored indefinitely after surgical resection with annual biochemical screening and chromogranin A levels. Factors that predict recurrent pheochromocytoma include a hereditary pheochromocytoma syndrome, a low ratio of epinephrine to total catecholamines, and the presence of large, extra-adrenal or bilateral tumors. Pregnancy

Management of pheochromocytoma in pregnancy is especially challenging. The mortality rate for mother and fetus is reported to be approximately 50%. Diagnosis before term improves these rates considerably. [106] Clinical symptoms are similar to those in nonpregnant individuals, but unique features can occur. For example, the gravid uterus may compress the pheochromocytoma, causing paroxysms in the supine position with normal blood pressure in the erect posture. Pheochromocytomas may also be easily misdiagnosed as preeclampsia, especially later in pregnancy. The diagnosis is typically made by evaluation of the urinary collection of catecholamines and metanephrines. Methyldopa should be discontinued before collection because of interference in catecholamine measurements. MRI is the preferred imaging modality because there is no ionizing radiation, and MIBG is contraindicated. Surgery is typically performed before 20 to 24 weeks of gestation. Thereafter, medical therapy is attempted, depending on the maternal status, and cesarean section is planned followed by tumor resection. [107] [108] [109] Phenoxybenzamine has been used during pregnancy, but it does cross the placental barrier; as a result, calcium channel blockers may be preferable to control blood pressure.[110]

Malignant Pheochromocytoma

Malignant pheochromocytoma occurs in 3% to 13% of all cases. The 5-year survival rate is 23% to 44%, compared with 97% 5-year survival in benign pheochromocytoma.[45] [51] [111] These tumors typically grow slowly, and evidence of malignancy may not be seen for several years. Malignancy is defined by direct local invasion of sites that do not typically have chromaffin tissue. Malignant tumors most commonly metastasize to the lungs, bone, liver, or lymph nodes or may recur

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locally. Surgical removal or debulking is the treatment of choice. After surgery, treatment goals are palliative to control the symptoms related to excess catecholamines. To achieve this end, -blockade followed by -blockade has most often been used (as discussed earlier). However, use of other antihypertensive treatments, such as dihydropyridine calcium channel blockers, is increasing. Unfortunately, the response to chemotherapeutic agents has been disappointing, but they may be tried in combination with antihypertensive treatment. Chemotherapeutic agents such as vincristine, cyclophosphamide, and dacarbazine have been used, often in combination. [112] Although the response rate is suboptimal, the experience with high-specific-activity 131 I-labeled MIBG is increasing, sometimes in combination with chemotherapy [113] External beam radiation has been attempted for palliation of bone metastases. Tumor embolization is another approach when surgery is not possible. [114]



RENIN-ANGIOTENSIN-ALDOSTERONE AXIS Several different mechanisms can lead to an increase in blood pressure in patients with essential hypertension and are similar to the mechanisms that cause an increase in blood pressure in individuals with secondary forms. A leading candidate for one of these mechanisms is a derangement in the renin-angiotensin system.[115] Components

Components of the renin-angiotensin system are shown in Figure 15-7 .[116] Renin

Renin is an enzyme produced in a number of cells in the body, principally in the juxtaglomerular apparatus of the kidney. [117] In tissues that produce renin, it is stored in granules and released in response to specific secretagogues. It is a member of the aspartyl proteinase family of enzymes and is synthesized

Figure 15-7 Components of the renin-angiotensin system. (Redrawn from Williams GH, Chao J, Chao L. Kidney hormones. In Conn PM, Melmed S [eds]. Endocrinology: Basic and Clinical Principles. Totowa, NJ, Humana Press, 1997, pp 393404.)

as a pre-proprotein. In humans, the gene that encodes renin is located on the short arm of chromosome 1 (1q321q42). In the rat the gene is located on chromosome 13, and in the mouse it is located on chromosome 1 [118] [119] (the mouse has two renin genes). In each species, the nucleotide sequence is approximately 12 kb, with 10 exons and 9 introns. The transcription product is a 1.5-kb messenger ribonucleic acid, and the initial protein consists of 340 amino acids, of which the first 43 are a prosegment cleaved to produce the active enzyme. Renin is termed a double-domain enzyme because the N-terminal and C-terminal halves are similar. [118] Each domain contains a single aspartic acid residue critical for its catalytic activity. The three-dimensional structure of the enzyme has been characterized. A number of factors can regulate the transcription of the renin gene; consensus elements are present in the 5-flanking region of the gene, including those for cAMP and a number of steroid receptors (estrogen, progesterone, and glucocorticoids). [118] [119] Angiotensinogen

Angiotensinogen is the only known substrate for renin and is catabolized to angiotensin peptides. The interaction between enzyme and substrate appears to be species specific because minor structural variations in the substrate render it relatively inactive in different species. [120] Human angiotensinogen belongs to the serpin superfamily of proteins and is encoded by a gene on chromosome 1q42.3 near the renin gene. [121] The angiotensinogen gene consists of five exons and four introns and is approximately 13 kb long. The transcript encodes a protein of 485 amino acids, 33 of which constitute a presegment that is cleaved after secretion. Angiotensin I is composed of the first 10-amino-acid sequence following the presegment. The 5 promoter region has consensus sequences for control by glucocorticoids, estrogens, and cytokines. [122] [123] Angiotensin-Converting Enzyme

Angiotensin-converting enzyme (ACE), a second enzyme involved in the final production of angiotensin II (see Fig. 15-7) , is a dipeptidyl carboxyl zinc metallopeptidase usually found bound to cell membranes. [124] It is also present in intracellular

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AI

TABLE 15-1 -- Amino Acid Composition of Angiotensin Peptides Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu

AII

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe

A1-7

Asp-Arg-Val-Tyr-Ile-His-Pro

AIII

Arg-Val-Tyr-Ile-His-Pro-Phe

AIV

Val-Tyr-Ile-His-Pro-Phe

A, angiotensin. granules in certain tissues that produce angiotensin II. Its molecular weight is considerably greater than that of renin and it consists of two homologous domains, suggesting that there are two active sites in each molecule. In humans, the ACE gene is located on chromosome 17q23 and consists of 26 exons and 25 introns. Two molecular forms of ACE are products of a single gene but have separate promoter regions. One product is a somatic, or endothelial, ACE that consists of 1306 amino acids, and the second is a germinal ACE with a promoter region upstream from the 13th exon. [125] Angiotensin Receptors

In humans, the two primary forms of the angiotensin receptor are termed AT 1 and AT2 . [126] A single gene on chromosome 3 encodes the angiotensin receptor in humans; rats have two genes. The 5'-flanking region contains three putative glucocorticoid response elements. The receptor has seven transmembrane regions, with a disulfide bridge linking the first and fourth extracellular segments. The principal signaling mechanism involved in the AT 1 receptor operates through a G q protein-mediated activation of phospholipase C. [127] However, some data suggest a linkage to protein tyrosine kinase. [128] [129] [130] The AT2 receptor gene has three exons and two introns and a seven-transmembrane-domain structure. [129] [130] Angiotensin Peptides

At least four angiotensin-like peptides have biologic activity (Table 15-1) .[131] [132] The action of renin on angiotensinogen produces angiotensin I, a decapeptide that does not appear to have biologic activity. Angiotensin II is formed by cleavage of the two carboxyl-terminal peptides by ACE [117] and has full biologic activity. Amino peptidase A can remove the aminoterminal aspartic acid to produce the heptapeptide, angiotensin III. Angiotensin II and angiotensin III have equivalent efficacy in promoting aldosterone secretion and modifying renal blood flow. However, angiotensin III has less pressor activity. Amino peptidase B can cleave an additional amino acid from angiotensin III to form angiotensin IV (angiotensin 38). [132] The function of this peptide is not clear, but it may be involved in the regulation of cerebral circulation and may produce vasodilation rather than vasoconstriction. A fourth biologically active compound is produced from angiotensin I by the action of a propyl

endopeptidase to form angiotensin 17, [133] whose function is unclear.



Functions of Angiotensin II

The effects of the renin-angiotensin system can be mediated by local paracrine effects or through endocrine action. [117] [134] The endocrine system primarily involves renin from the juxtaglomerular apparatus of the kidney and angiotensinogen from the liver. In the circulation, the concentrations of each are such that variations in the angiotensinogen levels can modify angiotensin I generation. The half-life in the circulation of angiotensin II is short (probably less than a minute). Although circulating levels of angiotensin II are in the picomolar range, its affinity for its receptor is in the nanomolar range, suggesting that some angiotensin II effects may actually be mediated not by the circulating peptide but by its local generation. Elements of the renin-angiotensin system are present in the adrenal, the kidneys, the heart, and the brain. [134] For example, the adrenal glomerulosa cells contain the proteins needed to produce and secrete angiotensin II. [135] Other tissues contain one or more components of the renin-angiotensin system and require other cells or circulating components, or both, to generate angiotensin II. For example, fat cells synthesize angiotensinogen but not renin or ACE, but they can generate angiotensin II locally.[136] An increasing body of evidence suggests that many of the functions of angiotensin II are mediated by these paracrine effects. In some tissues, such as the heart, the angiotensin II may be generated by a nonrenin systemthe chymase system. Angiotensin II functions through the AT 1 receptor to maintain normal extracellular volume and blood pressure in five ways [117] : (1) constriction of vascular smooth muscle, thereby increasing blood pressure and reducing renal blood flow; (2) release of norepinephrine and epinephrine from the adrenal medulla; (3) enhancement of the activity of the sympathetic nervous system by increasing central sympathetic outflow, thereby increasing norepinephrine discharge from sympathetic nerve terminals; (4) promotion of the release of vasopressin; and (5) increasing aldosterone secretion. Other functions of angiotensin II mediated through the AT 1 receptor include (1) central nervous system effects, including modification of thirst or the sense of well-being, or both; (2) modification of the release of corticotropin from the pituitary gland; (3) possible effects on placental and ovarian function; (4) activation of plasminogen activator inhibitor type 1, thereby contributing to the coagulation cascade [137] [138] ; and (5) modification of growth of the heart, kidneys, and vascular smooth muscle. [139] [140] In many respects, the action of angiotensin II through the AT 2 receptor antagonizes its effects through the AT 1 receptor. Thus, AT2 -mediated effects include vasodilatation, renal sodium loss, and apoptosis (thereby antagonizing the growth-promoting effects of AT 1 receptor activation). AT 2 receptors are highly expressed in fetal compared with adult tissue unless the adult tissue is damaged. [141]



Functions of Aldosterone

Aldosterone's classical functions are twofold: regulation of extracellular volume and control of potassium homeostasis. [142] These effects are mediated by binding to the mineralocorticoid receptor in the cytosol of epithelial cells, principally in the renal collecting duct. Transport to the nucleus and binding to specific binding domains on targeted genes lead to their increased expression. Although not all the genes have been identified, serum and glucocorticoid-induced kinase appears to be a key intermediary. [143] [144] [145] Its increased expression leads to modification of the apical sodium channel and the basal lateral Na + ,K+ -adenosine triphosphatase (ATPase), resulting in increased sodium ion transport across the cell membrane (see Chapter 14) . Glucocorticoids and mineralocorticoids bind equally to the mineralocorticoid receptor. Specificity of action is provided in many tissues by the presence of a glucocorticoid-degrading enzyme, 11-hydroxysteroid dehydrogenase, which prevents glucocorticoids from interacting with the receptor (see Chapter 14) . A second protective mechanism for untoward mineralocorticoid action is "escape" from its renal sodium-retaining effect. This usual occurs within 3 to 5 days of continued administration. Several mechanisms contribute to this escape, including

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Figure 15-8 (Figure Not Available) Renin-angiotensin-aldosterone and potassium-aldosterone negative feedback loops. Aldosterone production is determined by input from each loop. (Redrawn from Williams GH, Dluhy RG. Diseases of the adrenal cortex. In Braunwald E, Fauci AD, Kasper D, et al [eds]. Harrison's Principles of Internal Medicine, 15th ed. New York, McGraw-Hill, 2001, p 2087.)

renal hemodynamic factors and an increase in atrial natriuretic peptide. In addition to these classical genomic actions, mediated by aldosterone binding to cytosolic receptors, an increasing body of data suggests that mineralocorticoids have acute, nongenomic actions secondary to activation of an unidentified cell surface receptor. This action involves a G protein signaling pathway and probably a modification of the sodium-hydrogen exchange activity. In both epithelial and nonepithelial cells (e.g., myocytes and leukocytes) this effect has been demonstrated.

[146]

[147] [148] [149]

There are additional nonclassical effects of aldosterone primarily on nonepithelial cells. These actions, although probably genomic and therefore mediated by activation of the cytosolic mineralocorticoid receptor, do not include modification of sodium-potassium balance. Aldosterone-mediated actions include the expression of several collagen genes; genes controlling tissue growth factors, such as transforming growth factor , and plasminogen activator inhibitor type 1; or genes mediating inflammation. The resultant actions lead to microangiopathy, necrosis (acutely), and fibrosis in a variety of tissues, such as heart, the vasculature, and kidney. Increased levels of aldosterone are not necessary to cause this damage. Rather, an imbalance between the volume or sodium balance state and the level of aldosterone appears to be the critical factor. [150] [151] [152] [153] [154] [155] [156] [157] [158]



Regulation Renin

The release of renin into the circulation from the kidneys is controlled by four factors: (1) the macula densa, a specialized group of distal convoluted tubular cells that function as chemoreceptors for monitoring the sodium and chloride loads present in the distal tubule; (2) juxtaglomerular cells acting as miniature pressure transducers that sense renal perfusion pressure; (3) the sympathetic nervous system, which modifies the release of renin, particularly in response to upright posture in humans; and (4) humoral factors including potassium, angiotensin II, and atrial natriuretic peptides. The tissue renin-angiotensin systems are not necessarily regulated in the same manner as the circulating renin-angiotensin system. [119] [159] [160] For example, a high potassium intake reduces renal renin release and increases adrenal renin secretion. Aldosterone

The action of angiotensin II on aldosterone involves a negative feedback loop that also includes extracellular fluid volume (Fig. 15-8) (Figure Not Available) . The major function of this feedback loop is to modify sodium homeostasis and, secondarily, to regulate arterial pressure. [161] [162] Thus, sodium restriction activates the renin-angiotensin-aldosterone axis. The effects of angiotensin II on both the adrenal cortex and the renal vasculature promote renal sodium conservation. Conversely, with suppression of renin release and suppression of the level of circulating angiotensin, aldosterone secretion is reduced and renal blood flow is increased, thereby promoting sodium loss. In addition to the usual internal regulation of this negative feedback loop, a secondary fine-tuning component is related to the level of dietary sodium intake. [161] [162] Most endocrine negative feedback loops are not particularly sensitive to environmental factors. In contrast, the renin-angiotensin-aldosterone loop is exquisitely sensitive to dietary sodium intake. Sodium excess enhances the renal and peripheral vasculature responsiveness and reduces the adrenal responsiveness to angiotensin II (Fig. 15-9) . Sodium restriction has the opposite effect. Thus, sodium intake modifies, or modulates, target tissue responsiveness to angiotensin II, a fine tuning that appears to be critical to maintaining normal sodium homeostasis without modifying blood pressure, particularly chronically. The mechanism or mechanisms by which dietary sodium intake induces these changes in the adrenal is unclear, but in the vascular system the effects are a consequence of angiotensin II down-regulation of the target tissue responsiveness to its agonists.



ESSENTIAL HYPERTENSION The renin-angiotensin system has a powerful influence on both vasoconstrictor activity and volume regulation. Thus, defects in its regulation could lead to a rise in blood pressure by either or both of these mechanisms. Two other hormonal systems are implicated in the pathogenesis of essential hypertension: insulin (either directly or mediated by selective insulin resistance) and the calcium regulating systems. Role of the Renin-Angiotensin System in the Pathogenesis of Essential Hypertension

In the late 1960s and early 1970s, Laragh and colleagues [163] developed a classification of hypertension based on the level of circulating renin activity. By controlling dietary sodium and potassium intake, they classified patients into those whose values were low, normal, or high and used this information to define whether an individual case of hypertension was more volume-dependent or vasoconstrictor-dependent (Fig. 15-10) . The model predicted that individuals with low plasma renin activity (PRA) levels would have a volume-sensitive form of hypertension and those with high plasma levels would have a vasoconstrictor form of hypertension. It was presumed that classifying patients in this manner would lead to a more rational treatment program. However, several concerns have been raised. First, age modifies

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Figure 15-9 Modification of vascular and aldosterone response to angiotensin II by dietary salt intake. Sodium intake has a reciprocal influence on vascular and adrenal responses to angiotensin II. On a high salt intake the vascular response is enhanced while the adrenal response is suppressed. Sodium restriction has the opposite effect. (Redrawn from Williams GH, Hollenberg NK. "Sodium-sensitive" essential hypertension: emerging insights into pathogenesis and therapeutic implications. In Klahr S, Massry SG [eds]. Contemporary Nephrology. Vol 3. New York, Plenum Press, 1985, p 303, with permission.)

the level of renin activity, older subjects having lower renin levels regardless of volume status. Second, race modifies the level of renin activity (whites, in general, have higher levels than blacks). Finally, in individuals who consume a relatively large amount of sodium (more than 175 mmol/day) low, normal, and high PRA levels are difficult to distinguish from each

Figure 15-10 Relationship between the activity of the renin-angiotensin system and the mechanisms underlying the hypertension. (Redrawn from Laragh JH, Sealey JE, Niarchos AP, et al. The vasoconstrictor volume spectrum in normotension and in the pathogenesis of hypertension. Fed Proc 41:24152423, 1982.)

other. However, the concept of subclassification of hypertensive patients on the basis of the level of renin activity was useful in the development of better approaches to subclassifying patients.



Pathophysiologic Mechanisms in Low-Renin Essential Hypertension

Several mechanisms are thought to cause volume expansion and suppress renin activity in some patients with essential hypertension. [164] Adrenal mechanisms may be involved in some subjects with low-renin hypertension because spironolactone (a mineralocorticoid antagonist) and aminoglutethimide (an inhibitor of steroid hormone biosynthesis) substantially reduce their blood pressure. [165] [166] Wisgerhof and Brown [167] reported that the adrenal response to angiotensin II is enhanced in some patients with low-renin essential hypertension and that the enhanced responsiveness alters the renin-angiotensin-aldosterone negative feedback loop, allowing restoration of normal sodium homeostasis with decreased PRA and angiotensin II levels. [168] However, with normal to high sodium intake, this enhanced adrenal response could result in a scenario in which aldosterone secretion would not be suppressed adequately, promoting sodium retention and an increase in blood pressure. The frequency of this abnormality is unclear because no population-based studies have been reported. However, even some patients with so-called normal renin essential hypertension appear to have a similar defect. [169]



Nonmodulating Hypertension: Salt Sensitivity and Normal to High Renin Levels

Some patients with normal or high renin levels have a peculiar form of salt-sensitive hypertension in which increased sodium intake fails to change the vascular and the adrenal response to angiotensin II. [170] These patients, termed nonmodulators, appear to be a subset of the essential hypertensive population, as documented by a bimodal distribution of several of their biochemical features. [171] Patients with these features have been reported from Argentina, Brazil, Japan, The Netherlands, France, Italy, and the United States. [172] [173] [174] In whites, between 25% and 30% of hypertensive subjects are nonmodulators, and in black hypertensives the frequency is likely to be greater. Nonmodulators share several features with low-renin essential hypertensive patients: (1) they both have salt-sensitive hypertension,

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and (2) they tend to be older than the rest of the hypertensive population. However, nonmodulators have several features that are not similar to those of low-renin hypertension, including (1) fasting hyperinsulinemia, (2) a positive family history of hypertension and myocardial disease, (3) elevated levels of cholesterol and triglycerides, and (4) a decreased adrenal response to angiotensin II as assessed with a sodium-restricted intake. Finally, and perhaps most important, the characteristics associated with nonmodulation distribute in a bimodal fashion in the hypertensive population, suggesting a discrete subgroup. [171] [174] [175] [176] In nonmodulators, target tissue responsiveness to angiotensin II does not change when sodium intake is modified. Two functional tests have been used to distinguish them from the rest of the hypertensive population. One measures the aldosterone response to an angiotensin II infusion of 3 ng/kg per minute with a low-salt (10 mEq) diet.[170] [171] [177] The other approach is to measure the renal blood flow response to the same dose of angiotensin II with a high-salt (200 mEq) diet. [170] [178] Unless the dietary sodium intake is precisely controlled, a hypertensive subject can be misclassified. The correlation between these two criteria is 70% to 80%. [171] [173] Thus, if feasible, the best approach is to require both criteria to be positive in defining a nonmodulator. Other characteristics of this subset include failure of renal blood flow to increase when dietary sodium intake is changed from low to high and an enhanced response of atrial natriuretic peptide to infused angiotensin II. [173] [179] Nonmodulators appear to have an inherited form of hypertension, as evidenced by (1) bimodality of the distribution of the nonmodulating characteristic in the hypertensive population, [171] (2) the presence of the nonmodulating characteristic in normotensive subjects, [172] (3) a strong family history of hypertension in nonmodulators (approximately 80%, compared with about 30% for the rest of the hypertensive population), [176] [179] (4) familial aggregation of nonmodulating characteristics with hypertension, [180] and (5) the association of the nonmodulating phenotype with individuals who are homozygous for the angiotensinogen 235T genotype (Fig. 15-11) . [181] Data also support the involvement of the ACE and aldosterone synthase (CYP11B2) genes. Nonmodulators are twice as likely as the rest of the hypertensive population to be homozygous for the angiotensinogen 235T genotype, four times as likely if they have both the angiotensinogen and ACE dd genotypes, and nearly six times as likely if they have the angiotensinogen, ACE, and CYP11B2-344T genotypes. A defect in the renin-angiotensin system is likely to underlie nonmodulating hypertension. It is probably a defect in the local renal and adrenal renin-angiotensin systems, as evidenced by the following: (1) the previously cited genetic data involving genes of the renin-angiotensin-aldosterone system; (2) low renal blood flow with a high-sodium diet and a reduced renal vascular response to infused angiotensin II, suggesting inappropriately high local renal angiotensin II levels; (3) correction of the renal blood flow defect by administration of a converting enzyme inhibitor; and (4) correction of the nonmodulating adrenal defect by a converting enzyme inhibitor. [177] [178] [179] The effect of sodium intake on blood pressure in nonmodulators has been extensively evaluated. Either short-term (3 days) or chronic (2 weeks) salt loading increases blood pressure in nonmodulators but not in other normal or high-renin hypertensive patients. [176] [179] The salt sensitivity of the hypertension is due to the tendency for nonmodulators to retain more of a salt load both acutely and chronically. [179] The abnormality in sodium handling is probably due to the alteration in renal hemodynamics with salt loading described previously [170] [178] and secondary to an inappropriately high local angiotensin II level. Support for this conclusion comes from correction of salt-sensitive

Figure 15-11 Effect of angiotensinogen genotype on renal blood flow responses to angiotensin II infusions. Subjects were classified according to their alleles at the 235 codon of the angiotensinogen gene as to whether they were homozygous for the wild type (MM), heterozygous (MT), or homozygous for the hypertensive-link (TT) alleles. The subjects with the TT235 genotype had a renal blood flow response to angiotensin II similar to that of nonmodulators. (Redrawn from Hopkins P, Lifton RP, Hollenberg NK, et al. Blunted renal vascular response to angiotensin II is associated with a common variant of the angiotensinogen gene and obesity. J Hypertens 1996; 14:199207. Copyright 1996, Rapid Science Publishers.)

hypertension in nonmodulators by converting enzyme inhibitors. In summary, nonmodulators are a distinct subgroup of the essential hypertensive population and may constitute as much as 30% of that population. They have a sodium-sensitive form of hypertension, probably owing to a derangement of the local renin-angiotensin system in the kidney and the adrenal (Fig. 15-12) . These patients also have insulin resistance, hypercholesterolemia, a family history positive for myocardial infarction,

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Figure 15-12 Pathogenesis of nonmodulating hypertension. ANG II, angiotensin II.

Figure 15-13 Relationship of arterial pressure to insulin resistance in normotensive ethnic subgroups. An index of insulin sensitivity, the glucose disposal rate, was determined with an insulin clamp and fasting insulin levels were measured in normotensive blacks, whites, and Pima Indians. There was a significant negative correlation between arterial blood pressure and glucose disposal rates in whites but not in the other subgroups. (From Saad MF, Lillioja S, Nyomba BL, et al. Racial differences in the relationship between arterial pressure and insulin resistance. N

Engl J Med 1991; 324:733739. Copyright 1991, Massachusetts Medical Society.)

and an association with a specific allelic variant of the angiotensinogen gene. Finally, the defect appears to be correctable by the administration of converting enzyme inhibitors.



Insulin Resistance and Hypertension

Noninsulin-dependent diabetes mellitus (NIDDM), hypertension, and obesity are commonly associated, and the frequency of this association may be greater than their occurrence in the general population (see the review by Hopkins and colleagues [182] ), suggesting a common etiology. In support of this possibility is the fact that insulin resistance and hypertension can coexist without obesity or other stigmata of NIDDM. [183] [184] There may be a genetic component of this interaction. For example, in whites of European descent there is a strong relation between insulin resistance and blood pressure, whereas in normotensive blacks or Pima Indians there is no such relationship (Fig. 15-13) . [185] However, most hypertensive blacks are insulin resistant. Causative Role of Insulin in Hypertension

Several mechanisms have been proposed to explain the insulin-resistant state, including abnormalities in insulin binding to its receptor, defects in glucose transport, changes in the signal transduction pathway within insulin-sensitive cells, and metabolic abnormalities in glycolysis, glucose oxidation, or glucagon

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Figure 15-14 Mechanisms by which insulin resistance may produce hypertension.

synthesis. Yet little is known concerning the cause of insulin resistance in essential hypertension

[182]

(Fig. 15-14) .

Several features are relevant. First, insulin resistance is common in essential hypertension whether defined by fasting or postglucose load insulin levels or by euglycemic, hyperinsulinemic clamps. [186] [187] [188] Second, obesity cannot explain all cases of insulin resistance. Third, insulin directly stimulates the calcium pump in insulin-sensitive tissues and promotes calcium loss from the cell, [189] and raising cytosolic calcium levels in an adipocyte can induce insulin resistance. [190] [191] If a cell is resistant to insulin, the insulin-induced calcium loss from cells would be decreased, and in vascular smooth muscle cells the resultant increase in intracellular calcium would enhance responsiveness to vasoconstrictors and increase blood pressure. Two other mechanisms have been proposed to explain the linkage between insulin resistance and hypertension: increased activity of the adrenergic nervous system[192] and increased renal sodium retention. [193] Underlying both these hypotheses is the assumption that insulin resistance in a hypertensive subject may be selective. Accordingly, insulin resistance in the skeletal muscle or liver, or both, would induce a rise in circulating insulin levels. However, there would be little, if any, resistance at the renal tubule or adrenergic nervous system. Finally, for the vasoconstrictor hypothesis to be correct, there would have to be an imbalance between insulin's direct vasodilator effect and the vasoconstriction induced by activation of the adrenergic nervous system. A hyperinsulinemic response to glucose loading [194] and insulin resistance [195] has been described in salt-sensitive but not in salt-resistant normotensive subjects. This salt sensitivity also extends to metabolic abnormalities that are often associated with insulin resistance, such as increased levels of circulating low-density lipoprotein cholesterol in salt-sensitive hypertensives compared with salt-resistant hypertensives. [196] Thus, salt sensitivity of blood pressure is associated with lipid and glucose metabolic abnormalities and increased cardiovascular risk. Impaired insulin sensitivity [197] and hyperlipidemia [198] have been described in healthy volunteers with normal to high plasma renin levels compared with those with low renin levels. Thus, the data derived from these sources are inconsistent. Individuals who are salt-sensitive, as noted earlier, are likely to have low PRA. Yet salt-sensitive subjects as a group also have an increased risk of carbohydrate and lipid abnormalities. In summary, insulin resistance occurs in some patients with essential hypertension who do not have obesity or NIDDM. Several lines of evidence suggest that insulin resistance per se or hyperinsulinemia, or both, could result in increased sodium reabsorption, enhanced vascular tone, and activation of the adrenergic nervous system. Alternatively, this state could be associated with abnormal regulation of the renin-angiotensin system. Environmental factors can exacerbate this defect. For example, if patients with insulin resistance gain weight or receive drugs that increase insulin resistance, such as diuretics or beta-blockers, the hypertension may be worsened. However, it is still unclear whether the insulin resistance is a marker for some other abnormality or a primary defect in these patients.



Calcium and Hypertension

In 1982 McCarron and co-workers [199] reported that dietary calcium intake in humans with hypertension was lower than in normotensive control subjects, and the inverse relation between blood pressure and calcium intake has been confirmed in epidemiologic studies. [200] [201] These studies also suggest an association between the level of calcium intake and the degree of sensitivity of the blood pressure to sodium intake. In part, this may not be surprising given the known relationship between the reabsorption of calcium and that of sodium by the proximal tubule of the kidney. Blood pressure, in part, may also correlate with magnesium intake, at least in women.[200] Indeed, the relative risk of developing hypertension was 0.65 when both magnesium (50% per donor oocytein vitro fertilization cycle). Patients with premature ovarian failure are at increased risk for having an abnormal complement of chromosomes. [485] The risk of having an abnormal karyotype increases with decreasing age of onset of the ovarian failure. A chromosomal analysis is recommended for some of these patients because of increased risk of a gonadal tumor associated with the presence of a Y chromosome. [501] The arbitrarily chosen age group for chromosomal analysis includes women 30 years of age or younger because it is extraordinarily rare to encounter a gonadal tumor in patients with premature ovarian failure after the age of 30. [502] [503] The presence of mosaicism including a Y chromosome has been associated with a high incidence of gonadal tumors. [501] These malignant tumors arise from germ cells and include gonadoblastomas, dysgerminomas, yolk sac tumors, and choriocarcinoma. In particular, the presence of secondary virilization in these patients with karyotypic abnormalities and premature ovarian failure significantly increases the risk of a dysontogenetic gonadal tumor. The precise risk of a tumor in various subsets of these patients is not well known because a significant number of women carrying a Y chromosome do not have symptoms of virilization. The frequency of Y-chromosome material determined by polymerase chain reaction is high in Turner's syndrome (12.2%), but the occurrence of a gonadal tumor among these Y-positive patients seems to be as low as 7% to 10%. [504]

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TABLE 16-9 -- Laboratory Evaluation of Premature Ovarian Failure FSH (to establish the diagnosis of premature ovarian failure) Karyotype (< 30 yr of age or sexual infantilism) Cortisol after ACTH stimulation (adrenal insufficiency) TSH (hypothyroidism) Glucose (fasting and 2 hr after 75-g glucose load, diabetes mellitus)

Calcium and phosphorus (hypoparathyroidism) Sedimentation rate, complete blood count with differential, antinuclear antibody, rheumatoid factor (autoimmune disease) Pregnenolone (to evaluate 17-hydroxylase deficiency in sexually infantile women) Galactose-1-phosphate (galactosemia) ACTH, adrenocorticotropic hormone; FSH, follicle-stimulating hormone; TSH, thyroid-stimulating hormone.

Premature ovarian failure may also occur as an isolated autoimmune disorder or in association with hypothyroidism, diabetes mellitus, hypoadrenalism, hypoparathyroidism, or systemic lupus erythematosus. [505] Therefore, the tests listed in Table 16-9 should be performed every few years because premature ovarian failure can be part of an autoimmune polyendocrine syndrome. [487] Thyroid and adrenal insufficiency and diabetes mellitus are the endocrine disorders most frequently associated with premature ovarian failure. It should be noted, however, that overall it is fairly rare to encounter any endocrine disorder associated with premature ovarian failure. [506] Treatment of premature ovarian failure should be directed toward its specific cause, if this is possible. In most cases, however, it is not possible to identify a specific etiology if there are no karyotypic anomalies. If the patient desires pregnancy, she should be offered ovum donation and in vitro fertilization using her partner's sperm (see Fig. 16-29) . If pregnancy is not desired, she should be treated with an oral contraceptive or with estrogen and progestin replacement. Estrogen therapy promotes and maintains secondary sexual characteristics and prevents premature osteoporosis.



Differential Diagnosis and Management of Anovulatory Uterine Bleeding

Acyclic production of estrogen during anovulatory cycles gives rise to irregular shedding of the endometrium. These bleeding manifestations of anovulatory cycles in the absence of uterine pathology or systemic illness are commonly referred to as dysfunctional uterine bleeding. Anovulatory uterine bleeding is the most common cause of chronic menstrual irregularities and is a diagnosis of exclusion. Pregnancy, uterine leiomyomas, endometrial polyps, and adenomyosis should be ruled out as anatomic causes of irregular uterine bleeding. Malignancies of the vagina, cervix, endometrium, myometrium, fallopian tubes, and ovaries should also be ruled out before a diagnosis of anovulatory uterine bleeding is made. Finally, coagulation abnormalities should be excluded. Anovulatory uterine bleeding can be managed without surgical intervention by either restoring ovulation or mimicking the ovulatory hormonal profile by providing exogenous steroids. The rationale for using exogenous steroids is based on the knowledge of predictable responses of the endometrium to estrogen and progesterone. Physiologic responses of the endometrium to natural ovarian steroids have been uncovered by observing the gross and microscopic changes in the endometrium during thousands of normal ovulatory cycles in humans and other primates. [198] [201] [202] [203] [204] The pharmacologic application of exogenous estrogens and progestins in women with anovulatory bleeding aims to correct the production of local tissue factors, which mediate physiologic steroid action, and thus reverse the excessive and prolonged flow typical of anovulatory cycles. Clinical management of irregular uterine bleeding with exogenous hormones is a time-honored method and is also of diagnostic value. Failure to control vaginal bleeding with hormonal therapy, despite appropriate application and utilization, makes the diagnosis of anovulatory uterine bleeding considerably less likely. In this case, attention is directed to an anatomic pathologic entity within the reproductive axis as the cause of abnormal bleeding. Heavy but regular menstrual bleeding (hypermenorrhea) can be encountered in ovulatory women. It may be due to anatomic causes such as a leiomyoma impinging on the endometrial cavity or the diffuse and pathologic presence of benign endometrial glands in the myometrium (adenomyosis). In the absence of a specific pathologic cause, however, it is presumed that hypermenorrhea reflects subtle disturbances in the endometrial tissue mechanism. In essentially all cases, evaluation and treatment are identical to the approach detailed in this section. Characteristics of Normal Menses

Normal menstruation takes place about 14 days after each ovulation episode as a consequence of postovulatory estrogen-progesterone withdrawal. The quantity and duration of bleeding are quite reproducible. This predictability leads many women to expect a certain characteristic flow pattern. Any slight deviations, such as plus or minus 1 day in duration or minor deviation from expected tampon utilization, are causes for major concern in the patient. Most women of reproductive age can predict the timing of their flows so accurately that even some instances of minor variability may require reassurance by the clinician. Although variability of menstrual cycles is a common feature during teenage years and the perimenopausal transition, the characteristics of menstrual bleeding do not undergo appreciable change between ages 20 and 40. [507] For ovulatory women, the changes in the length of menstrual cycles over the period of reproductive age are predictable. Between menarche and age 20, the cycle length for most ovulatory women is relatively longer. Between 20 and 40, there is increased regularity as cycles shorten. In the 40s, cycles begin to lengthen again. The highest incidence of anovulatory cycles occurs before age 20 and after age 40. [508] [509] In this age group, the average length of a cycle is between 25 and 28 days. Among ovulatory women, the frequency of a cycle less than 21 days long or a cycle greater than 35 days is extremely rare (less than 2%). [510] Overall, most women have cycles that last from 24 to 35 days (Fig. 16-39) . [507] Between ages 40 and 50, menstrual cycle length increases and anovulation becomes more prevalent. [511] The average postovulatory bleeding lasts from 4 to 6 days. The normal volume of menstrual blood loss is 30 mL. More than 80 mL is considered abnormal. Most of the blood loss occurs during the first 3 days of a period, so excessive flow may exist without prolongation of flow. [512] [513] During an ovulatory cycle, the duration from the ovulation to menses is relatively constant and averages 14 days (see Fig. 16-22) . Greater variability in the length of proliferative phase, however, produces a distribution in the duration of a menstrual cycle. Menstrual bleeding more often than every 24 days or less often than every 35 days requires evaluation. [507] [511] Flow that lasts 7 or more days also requires evaluation. A flow that totals more than 80 mL per month usually leads to anemia and should be treated. [514] [515] In clinical practice, however, it is quite difficult to quantify menstrual flow because evaluation and treatment are based solely on the patient's perceptions regarding the duration, amount, and timing of her menstrual bleeding. Despite

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Figure 16-39 Variation of the duration of the menstrual cycle in women with regular cycles. (From Cunningham FG, MacDonald PC, Gant NF, et al. The endometrium and decidua: menstruation and pregnancy. In Williams Obstetrics, 19th ed. Stamford, Conn, Appleton & Lange, 1993, pp 81109.)

this difficulty in quantifying menstrual blood loss, the clinician should evaluate the cause of excessive uterine bleeding. Anemia should be ruled out by a complete blood count. [516] A low hemoglobin value accompanied by microcytic and hypochromic red blood cells suggests excessive blood loss during menses. These patients should be provided with iron supplementation. The likely presence of coagulation defects, uterine leiomyomas, or adenomyosis underlying prolonged menses should also be evaluated in anemic patients through a meticulous history and physical examination followed by relevant laboratory tests. Terminology Describing Abnormal Uterine Bleeding

Oligomenorrhea is defined as intervals between episodes of uterine bleeding greater than 35 days, and the term polymenorrhea is used to describe intervals less than 24 days. Hypermenorrhea refers to regular intervals (24 to 35 days) but excessive flow or duration of bleeding, or both. Hypomenorrhea refers to diminution of the flow or shortening of the duration of regular menses, or both. Uterine Bleeding in Response to Steroid Hormones

Estrogen Withdrawal Bleeding.

Uterine bleeding follows acute cessation of estrogen support to the endometrium. Thus, this type of uterine bleeding can occur after bilateral oophorectomy, radiation of mature follicles, or administration of estrogen to a castrate and then discontinuation of therapy. Similarly, the bleeding that occurs after castration can be delayed by concomitant estrogen therapy. Flow occurs on discontinuation of exogenous estrogen. Thus, estrogen withdrawal by itself (in the absence of progesterone) almost invariably causes uterine bleeding. Estrogen Breakthrough Bleeding.

Chronic exposure to varying quantities of estrogen stimulates the growth of endometrium continuously in the absence of progesterone, as in the case of excessive

extragonadal estrogen production in PCOS. After a certain point, the amount of estrogen produced in extraovarian tissue remains insufficient to maintain structural support for the endometrium. This gives rise to unpredictable episodes of shedding of the surface endometrium. Relatively low doses of estrogen yield intermittent spotting that may be prolonged but is generally light in quantity of flow. On the other hand, high levels of estrogen and sustained availability lead to prolonged periods of amenorrhea followed by acute, often profuse episodes of bleeding with excessive loss of blood. Progesterone Withdrawal Bleeding.

The typical progesterone withdrawal bleeding occurs after ovulation in the absence of pregnancy. Removal of the corpus luteum is another example that leads to endometrial desquamation. Pharmacologically, a similar event can be achieved by administration and discontinuation of progesterone or a synthetic progestin. Progesterone withdrawal bleeding occurs only if the endometrium is initially primed by endogenous or exogenous estrogen. If estrogen therapy is continued as progesterone is withdrawn, the progesterone withdrawal bleeding still occurs. Only if estrogen levels are increased markedly is progesterone withdrawal bleeding delayed. [517] Thus, progesterone withdrawal bleeding is quite predictable in the presence of previous or concomitant estrogen exposure. Progestin Breakthrough Bleeding.

This is a pharmacologic phenomenon that occurs in the presence of an unfavorably high ratio of progestin to estrogen. In the absence of sufficient estrogen, continuous progestin therapy leads to intermittent bleeding of variable duration, similar to the low-dose estrogen breakthrough bleeding noted previously. This type of bleeding is associated with the combination oral contraceptives that contain low-dose estrogen and the long-acting progestin-only contraceptive methods such as Norplant and Depo-Provera. [518] Progestin breakthrough bleeding is highly unpredictable and characterized by extensive variability between women. Causes of Irregular Uterine Bleeding

Pregnancy and its complications represent one of the most common causes of irregular uterine bleeding (Table 16-10) . Pregnancy should be ruled out by a urine test in any woman of reproductive age presenting with irregular bleeding (Table 16-11) . As pointed out earlier, anovulatory uterine bleeding arising from responses of the endometrium to inappropriate production of ovarian steroids has also been called dysfunctional uterine bleeding because treatments that restore ovulatory function potentially reverse the irregular bleeding pattern. Common examples of anovulatory bleeding include those associated with exercise-related anovulation, hyperprolactinemia, hypothyroidism, or PCOS. [519] In these cases, either restoring ovulatory menses by correction of the underlying disorder or use of exogenous hormones can achieve predictable uterine bleeding. On the other hand, various pathologic entities of the genital tract (ovaries, uterus, vagina, or vulva) or a coagulation abnormality may also cause deviation from normal menses (see Table 16-10) . Anovulatory uterine bleeding is a diagnosis of exclusion for the following reasons. Vulvar, vaginal, or uterine malignancies can give rise to irregular bleeding. Moreover, an estrogen- or androgen-secreting ovarian tumor may cause abnormal uterine bleeding (see Table 16-10) . Pregnancy and pregnancy-related problems such as ectopic pregnancy or spontaneous miscarriage are extremely common causes of abnormal uterine bleeding. In fact, the most common cause of disruption of a normal menstrual pattern is pregnancy or a complication of pregnancy. Another

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TABLE 16-10 -- Causes of Irregular Uterine Bleeding Complications of Pregnancy Threatened miscarriage Incomplete miscarriage Ectopic pregnancy Anovulation Physiologic Uncomplicated pregnancy (amenorrhea) Pubertal (postmenarchal) anovulation Premenopausal anovulation Medications (e.g., oral contraceptives, LHRH agonists, danazol) Hypothalamic (frequently presents as amenorrhea) Functional (e.g., diet, exercise, stress) Anatomic (e.g., tumor, granulomatous disease, infection) Medications Other Hyperprolactinemia, other pituitary disorders Prolactinoma Other pituitary tumors, granulomatous disease Hypothyroidism Medications Other Androgen excess PCOS, hyperthecosis Ovarian tumor (e.g., Sertoli-Leydig cell tumor) Nonclassical adrenal hyperplasia Cushing's syndrome Glucocorticoid resistance Adrenal tumor (e.g., adenoma, carcinoma) Medications (e.g., testosterone, danazol) Other Premature ovarian failure (frequently presents as amenorrhea) Chronic illness Liver failure

Renal failure AIDS Other Anatomic Defects Affecting the Uterus Uterine leiomyomas Endometrial polyps Adenomyosis (usually presents as hypermenorrhea) Intrauterine adhesions (usually presents as amenorrhea) Endometritis Endometrial hyperplasia, cancer Chronic estrogen exposure (e.g., PCOS, medication, liver failure) Estrogen-secreting ovarian tumor (e.g., granulosa cell tumor) Advanced cervical cancer Other Coagulation Defects (Usually Present as Hypermenorrhea) Von Willebrand's disease Factor XI deficiency Other Extrauterine Genital Bleeding (May Mimic Uterine Bleeding) Vaginitis Genital trauma Foreign body Vaginal neoplasia Vulvar neoplasia Other AIDS, acquired immunodeficiency syndrome; LHRH, luteinizing hormonereleasing hormone; PCOS, polycystic ovary syndrome. common cause of irregular uterine bleeding is observed in oral contraceptive users in the form of progestin breakthrough bleeding. Progestin breakthrough bleeding during postmenopausal hormone replacement is also common (see later). Patients may be using other hormonal medications unknowingly with an impact on the endometrium. For example, the use of ginseng, an herbal root, has been associated with TABLE 16-11 -- Diagnostic Tests to Evaluate Irregular Uterine Bleeding Commonly Used Tests Urine hCG test Serum hCG level (incomplete miscarriage, ectopic pregnancy) Transvaginal pelvic ultrasonography (intrauterine or ectopic pregnancy, uterine leiomyoma, endometrial polyp or neoplasia, ovarian tumor) Serum FSH, LH (anovulation; ovarian failure) Serum prolactin, TSH (anovulation; hyperprolactinemia) Complete blood count, PT, PTT (coagulation defect) Liver and renal functions, HIV (anovulation; chronic disease) Endometrial biopsy (endometrial disease; polyp, neoplasia, endometritis) Less Commonly Used Tests Evaluation for PCOS, ovarian or adrenal tumor, nonclassical adrenal hyperplasia, Cushing's syndrome and glucocorticoid resistance (androgen excess) Head CT or MRI scan (hypothalamic anovulation, hyperprolactinemia) Pelvic MRI scan (adenomyosis, uterine leiomyoma) Hysterosonography with intrauterine saline installation (endometrial polyp, uterine leiomyoma) Hysteroscopy (endometrial polyp, uterine leiomyoma) Dilatation and curettage (endometrial disease not diagnosed by ultrasonography or biopsy) CT, computed tomography; FSH, follicle-stimulating hormone; hCG, human chorionic gonadotropin; HIV, human immunodeficiency virus; LH, luteinizing hormone; MRI, magnetic resonance imaging; PCOS, polycystic ovary syndrome; PT, prothrombin time; PTT, partial thromboplastin time; TSH, thyroid-stimulating hormone. estrogenic activity and abnormal bleeding. [520] Although uterine bleeding is a common benign side effect of various long-term hormonal treatments, the clinician should always be convinced that no other pathology is present. Anatomically demonstrable pathologies of the menstrual outflow tract include endometrial hyperplasia and cancer, endometrial polyps, leiomyomata uteri, adenomyosis, and endometritis. Irregular, serious bleeding may also be associated with chronic illness, such as renal failure, liver failure, and acquired immunodeficiency syndrome. Finally, careful examination is worthwhile to discover genital injury or a foreign object (see Table 16-10) . At puberty, the most common cause of irregular uterine bleeding is anovulation. Approximately 20% of these adolescents with excessive irregular uterine bleeding, however, have a coagulation defect. [521] [522] Among all women of reproductive age with hypermenorrhea, the prevalence of a coagulation disorder was reported to be 17%. Von Willebrand's disease was the most common defect, and factor XI deficiency was the second common diagnosis. Bleeding secondary to a coagulation defect is usually a heavy flow with regular, cyclic menses (hypermenorrhea), and the same pattern can be seen in patients being treated with anticoagulants. [523] Bleeding disorders are usually associated with hypermenorrhea since menarche and a history of bleeding with surgery or trauma. Hypermenorrhea may be the only sign of an inherited bleeding disorder. [524] Early pregnancy or its complications should always be ruled out first by a sensitive urine hCG measurement in any reproductive-age women presenting with irregular bleeding (see Table 16-11) . Threatened or incomplete miscarriage and ectopic pregnancy are extremely common causes of irregular uterine bleeding. Other tests should be ordered if necessary on the basis of the initial clinical evaluation. These include tests to evaluate anovulatory disorders of various etiologies (see Table 16-11) . In patients with a history of prolonged heavy menses (hypermenorrhea) of pubertal origin, coagulation studies (e.g., prothrombin time, partial thromboplastin time, and bleeding time) and a complete blood count should be obtained.

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Pelvic ultrasonography through a vaginal probe is an extremely useful test for the evaluation of normal or abnormal pregnancy, uterine leiomyomas, endometrial neoplasia, and ovarian tumors (see Table 16-11) . Other imaging studies may be used judiciously to rule out pathologies of the hypothalamus, pituitary, or adrenal (see earlier). Of note is the use of pelvic MRI to rule out adenomyosis, a uterine disorder characterized by the abnormal presence of diffuse endometrial tissue in the myometrial layer (see Table 16-11) . Advanced adenomyosis is associated with diffuse enlargement of the uterus, hypermenorrhea, and anemia. Endometrial histology should be determined by an endometrial biopsy performed in the physician's office in patients at risk for the development of endometrial hyperplasia or cancer (e.g., PCOS, liver failure, obesity, diabetes mellitus, hormone replacement). A benign endometrial polyp or a uterine leiomyoma protruding into the uterine cavity can be diagnosed by hysterosonography using intrauterine saline installation or hysteroscopy. Hysterosonography and hysteroscopy are not appropriate tests to evaluate endometrial hyperplasia or cancer because these procedures may cause dissemination of malignant cells. If malignancy is suspected, it should be ruled out by an office endometrial biopsy (see Table 16-11) . Occasionally, an office endometrial biopsy cannot be performed or is not diagnostic of endometrial neoplasia. In these rare instances, endometrial curettage under anesthesia is performed for a reliable tissue diagnosis. We should underscore again that a careful history and physical examination eliminate the need for most of these diagnostic tests. A useful question to ask oneself before ordering a certain diagnostic study is whether that particular test will alter the ultimate clinical management. Management of Anovulatory Uterine Bleeding

The terms dysfunctional uterine bleeding and anovulatory bleeding are used interchangeably and denote inappropriate stimulation of the endometrium during dysfunctional states of the reproductive system. If ovulatory function can be restored, anovulatory bleeding usually gives way to cyclic predictable periods. Because restoring ovulatory function may not be possible or practical in a large number of these women, exogenous estrogen and progestin are administered for a number of purposes. The indications for hormonal treatment of uterine bleeding include the need to stop acute uterine bleeding, to maintain predictable bleeding episodes, or to prevent endometrial hyperplasia. A number of hormonal treatments are used to stop anovulatory uterine bleeding and to induce predictable bleeding episodes. We should reemphasize that anovulatory uterine bleeding is a diagnosis of exclusion. Various anatomically demonstrable pathologies of the genital tract as listed in Table 16-10 should be ruled out before administration of the following regimens. Oral Contraceptives

Use of combination oral contraceptives in an acute or chronic fashion is the most common treatment for irregular uterine bleeding. The estrogen component of the combination pill stabilizes the endometrial tissue and stops shedding within hours and decreases ovarian secretion of sex steroids by suppression of gonadotropins within several days. The progestin component of the pill directly affects endometrial tissue to decrease shedding over days and potentiates ovarian suppression induced by estrogen. The progestin (in the presence of estrogen) induces differentiation of the endometrial tissue into a stable form termed pseudodecidua. Typically, a monophasic oral contraceptive preparation that contains 30 or 35 µg of ethinyl estradiol is preferred. Triphasic oral contraceptives or those with less than 30 µg of ethinyl estradiol are not suitable for the treatment of excessive anovulatory uterine bleeding. A combination oral contraceptive in high doses (two or three pills a day) can be used for short intervals (weeks) to treat an acute episode of excessive uterine bleeding. A usual dose (one pill per day) may be administered for years to manage chronic anovulatory bleeding associated with PCOS or hyperprolactinemia. Oral Contraceptives and Acute Excessive Uterine Bleeding Associated with Anemia

Unopposed estrogen exposure in women with anovulatory uterine bleeding is commonly associated with chronic endometrial buildup and heavy bleeding episodes. Therapy is administered as one pill twice a day for 1 week. In obese women, the oral contraceptive may be given three times a day. This therapy is maintained despite cessation of flow within 2 days. If flow does not abate, other diagnostic possibilities (polyps, incomplete abortion, and neoplasia) should be reevaluated. In case of anovulatory bleeding, the flow does diminish rapidly within 2 days after the beginning of high-dose (one pill two or three times a day) oral contraceptive treatment. Specific causes of anovulation and possible coagulation disorders are evaluated during the following few days. At this time, the physician also considers whether blood replacement or initiation of iron therapy is necessary. The high-dose estrogen-progestin combination has produced the structural rigidity intrinsic to the compact pseudodecidual reaction for the moment. Continued random breakdown of formerly fragile tissue is avoided and blood loss stopped. A large quantity of tissue, however, remains to react to estrogen-progestin withdrawal. The patient must be warned to anticipate a heavy flow with severely cramping flow a few days after stopping this therapy. The patient should also be warned of possible nausea that may be caused by high-dose oral contraceptive treatment. At the end of a week of high-dose oral contraceptive treatment, the pill is stopped temporarily. A heavy flow usually starts within a few days. On the third day of this withdrawal bleeding, a regular dose of combination oral contraceptive medication (one pill a day) is started. This is repeated for several 3-week treatments interrupted by 1-week withdrawal intervals. A decrease in volume with each successive cycle is expected. Oral contraceptives reduce menstrual flow by more than half in most women.[525] Early application of the estrogen-progestin combination limits growth and allows orderly regression of excessive endometrial height to normal levels. Because oral contraceptives do not treat the underlying cause of anovulation but provide symptomatic relief by directly affecting the endometrium, cessation of oral contraceptives results in the return of erratic uterine bleeding. Regardless of the requirement for contraception, oral contraceptives represent the best choice for hormonal management of heavy anovulatory bleeding and should be offered as long-term management. Oral Contraceptives and Chronic Irregular Uterine Bleeding

PCOS is a common form of anovulation associated with chronic steady-state levels of unopposed estrogen that may give rise to endometrial hyperplasia and cancer (see earlier). Hypothalamic anovulation and hyperprolactinemia, on the other hand, are associated with low estrogen levels, insufficient to prevent bone loss. A combination oral contraceptive is a suitable long-term treatment for both forms of chronic anovulation.

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Oral contraceptives represent the most suitable long-term symptomatic management option for any kind of anovulatory uterine bleeding, including oligomenorrhea. Before the administration of an oral contraceptive, pregnancy should be ruled out. For this purpose, one pill per day is ordinarily administered for 3-week periods interrupted by 1-week hormone-free intervals. Withdrawal bleeding is expected during the hormone-free interval. The progestin component serves to prevent endometrial hyperplasia associated with steady-state unopposed estrogen exposure in PCOS (see earlier). In cases of anovulation associated with hypoestrogenism (e.g., hypothalamic anovulation, hyperprolactinemia), on the other hand, the estrogen component of the pill provides sufficient replacement to prevent bone loss. The risk of thromboembolism, stroke, or myocardial infarction associated with long-term administration is extremely low in current nonsmokers and in the absence of a history of thromboembolism. Provided that an oral contraceptive controls the abnormal uterine bleeding effectively, a chronically anovulatory woman can continue this regimen until the menopause. Synthetic Progestins

Synthetic progestins enhance endometrial differentiation and antagonize proliferative effects of estrogen on the endometrium (see Fig. 16-28) .[213] [215] [526] The effects of progestins or natural progesterone include limitation of estrogen-induced endometrial growth and prevention of endometrial hyperplasia. The absence of naturally synthesized progesterone in anovulatory states is the rationale for administering a progestin. The most common indication for long-term cyclic progestin administration is to prevent endometrial malignancy in a patient with PCOS and unopposed chronic estrogen exposure of the endometrium. A combination oral contraceptive is the treatment of choice in these cases. If the patient cannot use an oral contraceptive for some reason (e.g., history of thromboembolism), a progestin can be administered in a cyclic fashion to prevent endometrial hyperplasia. Before the administration of a progestin (or oral contraceptive), pregnancy should be ruled out. In the treatment of oligomenorrhea associated with PCOS, orderly limited withdrawal bleeding can be accomplished by administration of a progestin such as medroxyprogesterone acetate, 10 mg/day for at least 10 days every 2 months. Alternatively, norethindrone

acetate at 5 mg/day or megestrol acetate at 20 mg/day may be administered for 10 days every 2 months. Absence of withdrawal bleeding requires further work-up. In the treatment of excessive uterine bleeding (hypermenorrhea or polymenorrhea), these progestins at higher daily doses (medroxyprogesterone acetate 20 mg/day, norethindrone acetate 10 mg/day, or megestrol acetate 40 mg/day) are prescribed for 2 weeks to induce predecidual stromal changes in the endometrium. A heavy progestin withdrawal flow usually follows within 3 days after the last dose. Thereafter, repeated progestin treatment (medroxyprogesterone acetate 10 mg/day, norethindrone acetate 5 mg/day, or megestrol acetate 20 mg/day) is offered cyclically for at least the first 10 days of every other month to ensure therapeutic effect. Failure of progestin to correct irregular bleeding requires diagnostic reevaluation such as endometrial biopsy. On the other hand, predictable withdrawal bleeding within several days after each cycle of progestin administration suggests the absence of endometrial malignancy. High-Dose Estrogen for Acute Excessive Uterine Bleeding

As already outlined, an oral contraceptive given two or three times a day is the treatment of choice to stop heavy anovulatory bleeding. A high-dose oral contraceptive regimen should be offered to women with heavy uterine bleeding plus or minus asymptomatic anemia after an anatomically demonstrable pathology of the genital tract has been ruled out (see Table 16-10) . On the other hand, a patient with acute and severe anovulatory bleeding accompanied by symptomatic anemia represents a medical emergency. These patients should be hospitalized immediately and offered a blood transfusion. When genital tract pathology has been ruled by history, physical examination, and pelvic ultrasonography, intravenously administered high-dose estrogen is the treatment of choice to stop life-threatening bleeding. A well-established regimen is 25 mg of conjugated estrogen administered intravenously every 4 hours until bleeding markedly slows down or for at least 24 hours. [527] Estrogen most likely acts on the capillaries to induce clotting. [528] Before intravenous estrogen treatment is discontinued, an oral contraceptive pill is started three times a day. Oral contraceptive treatment is continued as described previously. Because high-dose estrogen is a risk factor for thromboembolism, taking two or three oral contraceptives per day for a week or large doses of intravenous conjugated estrogens for 24 hours should also be regarded as significant risks. There are no data available, however, to evaluate any risk associated with this type of acute use of hormonal therapy for such short intervals. The physician and patient should make a decision regarding high-dose hormone therapy after considering its risks and benefits. Alternative treatment options may be offered to patients with significant risk factors. In women with a past episode of idiopathic venous thromboembolism or a strong family history, exposure to high doses of estrogen should be avoided. High-dose hormone treatment should also be avoided in women with severe chronic illness such as liver failure or renal failure. One alternative for these patients is dilatation and curettage, followed by an oral contraceptive at one pill per day until the uterine bleeding is under control. Luteinizing Hormone Releasing Hormone Analogues for Excessive Anovulatory Uterine Bleeding

LHRH analogues (see Table 16-1) may be given to women with excessive anovulatory bleeding or hypermenorrhea related to severe chronic illness such as liver failure or coagulation disorders. It should be pointed out that monthly depot injections of LHRH agonists are not effective for acute excessive uterine bleeding and may increase uterine bleeding for the first 2 weeks. LHRH antagonists, on the other hand, down-regulate FSH and LH without a delay and achieve amenorrhea more rapidly. Depot formulations of LHRH antagonists, however, are not available at present. Long-term side effects of LHRH analogues including osteoporosis make this an undesirable choice for long-term therapy. If long-term treatment with LHRH analogues is chosen, a combination of 0.625 mg of conjugated estrogens and 2.5 mg of medroxyprogesterone acetate daily should be added back when excessive anovulatory bleeding is controlled. This add-back regimen is usually sufficient to prevent osteoporosis and does not ordinarily worsen the uterine bleeding.



Hormone-Dependent Benign Gynecologic Disorders Endometriosis

Endometriosis is defined as the presence of endometrium-like tissue outside the uterine cavity, most often on the peritoneal surfaces of the pelvis and the ovaries. It is one of the most common causes of infertility and chronic pelvic pain and affects 1 in 10 women in the reproductive age group. [529] The

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incidence increases to 30% in patients with infertility and to 45% in patients with chronic pelvic pain.

[530]

Reliable diagnosis of endometriosis can be made only by direct visualization of these peritoneal lesions by laparoscopy or laparotomy. As in other common chronic diseases such as diabetes mellitus and asthma, endometriosis is inherited in a polygenic manner. [529] Relatives of women with this disease have a sevenfold increase in the incidence of endometriosis compared with relatives of control subjects. [529] Sampson proposed the most widely accepted mechanism for the development of endometriosis on pelvic peritoneal surfaces as the implantation of endometrial tissue on the peritoneum through retrograde menstruation. [529] Because retrograde menstruation occurs in more than 90% of all women, endometriosis may be caused by genetic defects that favor survival and establishment of endometrial tissue in menstrual debris on the peritoneum. [401] Endometriosis and normal endometrial tissues respond to estrogen and progesterone with similar histologic changes. [401] Estrogen favors the growth of endometriosis, whereas progesterone may limit this mitogenic action of estrogen. Some endometriotic implants undergo atrophy in response to prolonged oral contraceptive therapy just as the normal endometrium does, the so-called pregnancy state. Yet, endometriotic tissue does not respond to progestins or native progesterone as predictably as normal endometrium does.[401] Endometriotic tissue in ectopic locations such as the peritoneum or ovary is strikingly different from the eutopic endometrium within the uterus with respect to production of cytokines and prostaglandins, steroid biosynthesis and metabolism, steroid receptor content, and clinical response to progestins.[195] [401] [530] Although current hormonal therapy for infertility associated with endometriosis is not of proven value, it is somewhat successful for pelvic pain associated with endometriosis.[529] The duration of relief provided by medical (hormonal) treatment, however, is relatively short. [531] Various agents used are comparable in terms of efficacy. Most current medical treatments were designed to decrease estrogen secretion by the ovaries (e.g., LHRH agonists, oral contraceptives, danazol and progestins) or to antagonize the effects of estrogen on endometriotic implants (e.g., oral contraceptives, danazol and progestins). A possible alternative mechanism of action of the androgenic steroid danazol or a progestin is a direct antiproliferative effect on endometriotic tissue. Many patients and physicians do not favor danazol because of its anabolic and androgenic side effects of weight gain and muscle cramps and occasional irreversible virilization (e.g., clitoromegaly and voice changes). [532] In fact, up to 50% of patients with endometriosis fail to complete 6 months of treatment with danazol. [533] The rest of the hormonal agentsoral contraceptives, progestins, and LHRH agonistsshow comparable efficacy for the control of endometriosis-associated pain. [530] [534] [535] A 6-month course using any one of these agents results in a significant reduction of pain in more than 50% of patients. [530] [534] [535] Induction of pain relief with a continuously administered oral contraceptive or progestin takes longer than with an LHRH agonist. There is, however, a high incidence of persistence of the disease after all of these medical therapies. [531] Six months after completion of a 6-month course of treatment with a progestin, oral contraceptive, or LHRH agonist, moderate to severe pain symptoms recurred in 50% of initial responders. [535] The recurrence rate of pain in the rest of the patients was approximately 5% to 20% per year during a 5-year follow-up.[531] A 6-month course of LHRH agonist treatment is currently the most popular regimen. The most serious side effect of the LHRH agonist treatment for endometriosis is bone loss related to estrogen deficiency, and oral estrogen-progestin preparations or bisphosphonates are usually added back to minimize bone loss.[32] We are still far from the cure of endometriosis, and current treatments are not satisfactory for effective control of pain. The radical treatment is the removal of both ovaries, and even this was not found to be effective in a number of cases of postmenopausal endometriosis. [536] New strategies are needed to offer women with endometriosis a reasonable chance to live without suffering from chronic pelvic pain for decades. There are two important caveats, which are not addressed by the LHRH agonist treatment. First, large quantities of estrogen can be produced locally within the endometriotic cells. This represents an intracrine mechanism of estrogen action, in contrast to ovarian secretion, which is an endocrine means of supplying this steroid to target tissues (see Fig. 16-23) .[536] [401] Second, estradiol produced in peripheral tissue sites (e.g., adipose tissue and skin fibroblasts) may give rise to pathologically significant circulating levels of estradiol in a subset of women.[401] LHRH agonists do not inhibit peripheral estrogen formation or local estrogen production within the estrogen-responsive lesion. As a further twist, endometriosis is resistant to selective effects of progesterone and currently used progestins. [401] Thus, aromatase inhibitors and selective progesterone response modulators are candidate therapeutic agents for endometriosis. Preliminary evidence suggests that unusually aggressive endometriotic lesions resistant to other therapy can be treated successfully with aromatase inhibitors. [401] [536] There are a number of ongoing trials investigating the use of aromatase inhibitors and selective progesterone receptor modulators in the treatment of endometriosis. Uterine Leiomyomas

Uterine leiomyomas originate from the myometrium and are the most common solid tumor of the pelvis. [537] Leiomyomas are responsible for over 200,000 hysterectomies per year in the United States. They are almost invariably benign and represent clonal expansion of individual myometrial cells. Leiomyomas can cause a variety of symptoms including irregular and excessive uterine bleeding, pressure sensation in the lower abdomen, pain during intercourse, pelvic pain, recurrent pregnancy loss, infertility, and compression of adjacent pelvic organs, or they may be totally asymptomatic. The prevalence rate of uterine leiomyomas is estimated to be 25% to 30%. [537] Leiomyomas are more common in black women and have a polygenic inheritance pattern. Diagnosis can be made by abdominal and transvaginal ultrasonography. Transvaginal ultrasonography is a sensitive method for determining the size, number, and location of uterine leiomyomas. Uterine leiomyomas appear during the reproductive years and regress after menopause, indicating their ovarian steroiddependent growth potential. The role of steroids or other growth factors in the initiation and growth of these tumors, however, is not well understood. The neoplastic transformation of myometrium to leiomyoma probably involves somatic mutations of normal myometrium and the complex interactions of sex steroids and growth factors. [538] Traditionally, estrogen has been considered the major promoter of myoma growth. More recent biochemical, histologic, and clinical evidence suggests an important role for progesterone in the growth of uterine leiomyomas. Biochemical and clinical studies suggest that progesterone and progestins, acting through the PR, might enhance proliferative activity in leiomyomas.[538] The therapeutic choices depend on the goals of therapy, with hysterectomy most often used for definitive treatment and myomectomy when preservation of childbearing is desired. [539] Intracavitary and submucous leiomyomas can be removed by hysteroscopic resection. Laparoscopic myomectomy is now technically possible but apparently involves an increased risk of uterine rupture during pregnancy. The overall recurrence rate after myomectomy varies widely (10% to 50%). Although LHRH agonistinduced hypogonadism can reduce the overall

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volume of the uterus containing leiomyomas and tumor vascularity, the severe side effects and prompt recurrences make LHRH agonists useful only for short-term goals such as reducing anemia related to uterine bleeding or decreased tumor vascularity before hysteroscopic resection. [539]



MANAGEMENT OF THE MENOPAUSE Consequences of the Menopause The Climacteric

The menopause is the permanent cessation of menses as a result of the irreversible loss of a number of ovarian functions, including ovulation and estrogen production. The climacteric is a critical period of life during which striking endocrinologic, somatic, and psychologic alterations occur in the transition to the menopause. The climacteric is also referred to as the perimenopause. The climacteric encompasses the change from ovulatory cycles to cessation of menses and is marked by irregularity of menstrual bleeding. The most sensitive clinical indication of the climacteric is the progressively increasing occurrence of menstrual irregularities. The menstrual cycle for most ovulatory women lasts from 24 to 35 days, whereas approximately 20% of all reproductive-age women experience irregular cycles. [507] When women are in their 40s, anovulation becomes more prevalent; prior to anovulation, the menstrual cycle length increases, beginning several years before menopause. [511] The median age for the onset of the climacteric transition is 47.5 years. [540] Regardless of the age of its onset, the menopause (cessation of menses) is consistently preceded by a period of prolonged cycle intervals. [541] Elevated circulating FSH marks this menstrual cycle change before menopause and is accompanied by decreased inhibin levels, normal levels of LH, and slightly elevated levels of estradiol. [542] [543] [544] [545] [546] These changes in serum hormone levels reflect a decreasing ovarian follicular reserve and can be detected more reliably on day 2 or 3 of the menstrual cycle. During the climacteric, serum estradiol levels do not begin to decline until less than a year before menopause. [546] The average circulating estradiol levels in perimenopausal women are estimated to be somewhat higher than those in younger women because of an increased follicular response to elevated FSH. [547] The decline in inhibin production by the follicle, allowing a rise in FSH, in the later reproductive years reflects diminishing follicular reserve and competence. [543] [544] Ovarian follicular output of inhibin begins to decrease after 30 years of age, and this decline becomes much more pronounced after age 40. These hormonal changes are parallel to a significant decrease in fecundity, which starts at age 35. The climacteric is a transitional period during which postmenopausal levels of FSH can be observed despite continued menses, whereas LH levels still remain in the normal range. The perimenopausal woman is not beyond the realm of an unexpected pregnancy because there is occasional ovulation and functional corpus luteum formation. Thus, until complete cessation of menses is observed or FSH levels higher than 40 IU/L are measured on two separate occasions, some form of contraception should be recommended to prevent unwanted pregnancies. [545] The climacteric represents an optimal period to evaluate the general health of the mature woman and introduce the measures to prepare her for striking physiologic changes that come with the menopause. The patient and her clinician should attempt to achieve several important aims during the climacteric. The long-term goal is to maintain an optimal quality of physical and social life. Another immediate objective is the detection of any major chronic disorders that occur with aging. Finally, the clinician should counsel the perimenopausal woman about the symptoms and long-term consequences of menopause. The benefits and risks of lifelong hormone replacement should be discussed at great length at this time. The Menopause

The median age of the menopause is approximately 51. [548] The age of menopause is probably determined in part by genetic factors because mothers and daughters tend to experience menopause at the same age. [549] [550] [551] A number of environmental factors may modify the age of menopause. For example, current smoking is associated with an earlier menopause, whereas alcohol consumption delays menopause. [548] Oral contraceptive use does not affect the age of menopause. The symptoms frequently seen and related to decreased estrogen production in menopause include irregular frequency of menses followed by amenorrhea, vasomotor instability manifest as hot flashes and sweats, urogenital atrophy giving rise to pain during intercourse and a variety of urinary symptoms, and consequences of osteoporosis and cardiovascular disease. The combination and the extent of these symptoms differ widely for each patient. Some patients experience multiple severe symptoms that may be disabling, whereas others have no symptoms or mild discomfort associated with the climacteric. Biosynthesis of Estrogen and Other Steroids in the Postmenopausal Woman

No follicular units can be detected histologically in the ovaries after the menopause. [552] In reproductive-age women, the granulosa cell of the ovulatory follicle is the major source of inhibin and estradiol. In the absence of these factors that inhibit gonadotropin secretion, both FSH and LH levels increase sharply after menopause. These levels peak a few years after menopause and decrease gradually and slightly thereafter. [553] [554] The postmenopausal serum level of either gonadotropin may be more than 100 IU/L. FSH levels are usually higher than LH levels because LH is cleared from the blood strikingly more quickly and possibly because the low levels of inhibin in the menopause selectively lead to increased FSH secretion. Nevertheless, increased LH is a major factor that maintains significant quantities of androstenedione and testosterone secretion from the ovary, although the total production rates of both steroids decline after menopause. The primary steroid products of the postmenopausal ovary are androstenedione and testosterone. [555] The average premenopausal rate of production of androstenedione of 3 mg/day is decreased by half to approximately 1.5 mg/day. [555] This decrease is primarily due to a substantial reduction in the ovarian contribution to the circulating androstenedione pool. Adrenal secretion accounts for most of the androstenedione production in the postmenopausal woman, with only a small amount secreted from the ovary.[556] Both DHEA and DHEAS originate almost exclusively from the adrenal and decline steadily with advancing age independent of the menopause. The serum levels of both DHEA and DHEAS after menopause are about one fourth of those in young adult women. [557] Testosterone production is decreased by approximately one third after menopause. [555] Total testosterone production can be approximated by the sum of ovarian secretion and peripheral formation from androstenedione (see Fig. 16-32) . In the premenopausal woman, significant amounts of testosterone are produced by reduction of androstenedione in extraovarian tissues. Because ovarian androstenedione secretion is substantially decreased after the menopause, the decrease in postmenopausal

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Figure 16-40 Tissue sources of estrogen in postmenopausal breast cancer. This figure exemplifies the important pathologic roles of extraovarian (peripheral) and local estrogen biosynthesis in an estrogen-dependent disease in postmenopausal women. The estrogen precursor androstenedione (A) originates primarily from the adrenal in the postmenopausal woman. Aromatase expression and enzyme activity in extraovarian tissues such as fat increase with advancing age. The aromatase activity in skin and subcutaneous adipose fibroblasts gives rise to formation of systemically available estrone (E 1 ) and to a smaller extent estradiol (E 2 ). The conversion of circulating A to E 1 in undifferentiated breast adipose fibroblasts compacted around malignant epithelial cells and subsequent conversion of E 1 to E 2 in malignant epithelial cells provide high tissue concentrations of E 2 for tumor growth. The clinical relevance of these findings is exemplified by the successful use of aromatase inhibitors to treat breast cancer.

testosterone production is accounted for, in large measure, by a decrease in the relative contribution of extraovarian sources. [555] With the disappearance of follicles and decreased estrogen, the elevated gonadotropins drive the remaining stromal tissue in the ovary to maintain testosterone secretion at levels observed during the

premenopausal years. Thus, the contribution of the postmenopausal ovary to the total testosterone production is increased in the presence of seemingly unaltered ovarian secretion. [555] The most dramatic endocrine alteration of the climacteric involves the decline in the circulating level and production rate of estradiol. The average menopausal level of circulating estradiol is less than 20 pg/mL. Both estradiol and estrone levels in postmenopausal women are usually slightly less than those in adult men. Circulating estradiol in postmenopausal women (and men) is derived from the peripheral conversion of androstenedione to estrone, which is, in turn, converted peripherally to estradiol (see Fig. 16-23) .[403] [558] [559] The mean circulating level of estrone in postmenopausal women (37 pg/mL) is higher than that of estradiol. The average postmenopausal production rate of estrone is approximately 42 µg per 24 hours. After menopause, almost all estrone and estradiol are derived from the peripheral aromatization of androstenedione. Thus, there is a drastic change in the androgen-to-estrogen ratio because of the sharp decrease in estradiol levels and slightly reduced testosterone. The frequent onset of a mild hirsutism after menopause reflects this striking shift in the hormone ratio. During the postmenopausal years, DHEAS and DHEA levels continue to decline steadily with advancing age, whereas serum androstenedione, testosterone, estrone, and estradiol levels do not change significantly. [554] [558] The aromatization of androstenedione to estrone in extraovarian tissues correlates positively with weight and advancing age ( see Fig. 16-23 and Fig. 16-37 ). [168] Body weight correlates positively with the circulating levels of estrone and estradiol. [558] Because aromatase enzyme activity is present in significant quantities in adipose tissue, increased aromatization of androstenedione in overweight individuals may reflect the increased bulk of tissue containing the enzyme. [166] In addition, there is a twofold to fourfold increase in the specific activity of aromatase per cell with advancing age. [169] An increased overall number of adipose fibroblasts with aromatase activity and a decrease in the levels of TeBG cause an increased free estradiol level and contribute to the increased risk of endometrial cancer in obese women.[169] The production rate and circulating levels of estradiol after menopause are clearly insufficient to provide support for urogenital tissues and bone. Thus, osteoporosis and urogenital atrophy are some of the most dramatic and unwanted consequences of estradiol deficiency during the menopause. Estrogen is also produced locally in pathologic tissues such as breast cancer through aromatase and reductive 17-HSD (Fig. 16-40) .[169] [196] In postmenopausal women, androstenedione of adrenal origin is the most important substrate for aromatase in tumor tissue. [169] [196] Estrone is produced primarily by aromatase that resides in undifferentiated adipose fibroblasts surrounding malignant epithelial cells (see Fig. 16-40) .[169] [196] Estrone then diffuses into malignant epithelial cells that contain reductive 17-HSD activity and is converted to biologically active estradiol (see Fig. 16-40) .[194] Thus, paracrine interactions in breast tumor tissue serve to produce estradiol in malignant epithelial cells to give rise to an effect such as proliferation. The clinical relevance of these findings was exemplified by the successful use of aromatase inhibitors as both first-line and second-line endocrine treatments for postmenopausal breast cancer. [560] Management of Postmenopausal Uterine Bleeding

Perimenopausal or postmenopausal bleeding can be due to hormone administration or excessive extraovarian estrogen formation. Irregular uterine bleeding is commonly observed during the perimenopausal transition as anovulatory cycles alternate with ovulatory cycles. Uterine bleeding after the menopause is less common if the patient is not receiving hormone replacement treatment (HRT). Obese patients are more likely to experience postmenopausal bleeding because of increased peripheral aromatization of adrenal androstenedione. Patients receiving a continuous combination regimen of HRT may experience unpredictable uterine bleeding (see later). The major objective in these circumstances is to rule out endometrial malignancy. This can be best achieved by tissue diagnosis through an office endometrial biopsy using a plastic cannula. Transvaginal ultrasonographic measurement of endometrial thickness may be used in postmenopausal women to avoid unnecessary biopsies. [226] A biopsy is required if an endometrial thickness greater than or equal to 5 mm is observed. Before employing ultrasonography and endometrial biopsy to explore the etiology of bleeding that is assumed to arise from the intrauterine cavity, the clinician should rule out diseases of the vulva, vagina, and cervix as other potential causes of vaginal bleeding. Careful inspection of these organs and a normal cervical Pap smear within the past year are sufficient to rule out the vulva, vagina, and cervix as potential sources of bleeding. Postmenopausal uterine bleeding is the most common initial event that alerts the patient and her physician to the possibility of endometrial cancer. On the other hand, the causes of postmenopausal uterine bleeding are benign most of the time. Endometrial malignancy is encountered in patients with bleeding in only about 1% to 2% of postmenopausal endometrial biopsies. [561] [562] Approximately three quarters of

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these biopsies reveal either no pathology or an atrophic endometrium. Other histologic findings include hyperplasia (15%) and endometrial polyps (3%). Persistent unexplained uterine bleeding requires repeated evaluation, biopsy, hysteroscopy, or dilatation and curettage. Unpredictable irregular uterine bleeding is observed in approximately 20% of postmenopausal women receiving a long-term (>1 year) continuous estrogen-progestin combination. This should also be evaluated appropriately with ultrasonography or biopsy, or both (see later). [226] Hot Flash

The most frequent and striking symptom in the climacteric is the hot flash. The hot flash typically occurs at the time of transition from perimenopause to postmenopause, that is, the climacteric. The flash is also a major symptom of the postmenopause and can last up to 5 years after menopause. [563] More than four fifths of postmenopausal women experience hot flashes within 3 months after the cessation of ovarian function, whether natural or surgical in origin. Of these women, more than three fourths have them for more than 1 year and approximately half for up to 5 years. [563] Hot flashes lessen in frequency and intensity with advancing age, unlike other sequelae of the menopause, which progress with time. A hot flash is a subjective sensation of intense warmth of the upper body, which typically lasts for 4 minutes but may range in duration from 30 seconds to 5 minutes. It may follow a prodrome of palpitations or headache and is frequently accompanied by weakness, faintness, or vertigo. This episode usually ends in profuse sweating and a cold sensation. The frequency may vary from extremely rare to recurring every few minutes. At night, flashes are more frequent and severe enough to awaken a woman from sleep. They are also more intense during times of stress. In a cool environment, hot flashes are fewer, less intense, and shorter in duration than in a warm environment.[564] The hot flash results from a sudden reduction of estrogen levels rather than from hypoestrogenism itself. Therefore, regardless of the cause of menopause, natural, surgical, or estrogen withdrawal caused by a long-acting LHRH agonist, hot flashes are associated with an acute and significant drop in estrogen level. The consistent association between the onset of flashes and acute estrogen withdrawal is also supported by the effectiveness of estrogen therapy and the absence of flashes in prolonged hypoestrogenic states, such as gonadal dysgenesis. Hypogonadal women experience hot flashes only after estrogen is administered and withdrawn.[565] Not all hot flashes, however, are due to estrogen deficiency. Sudden episodes of sweating and flash may be due to catecholamine- or histamine-secreting tumors (e.g., pheochromocytoma, carcinoid), hyperthyroidism, or chronic infection (e.g., tuberculosis). The hot flash may also be psychosomatic in origin and not due to estrogen withdrawal. Under these circumstances of doubt, the clinician should obtain a serum FSH level to confirm the climacteric or menopause before initiating hormone replacement. Obese women tend to be less troubled by hot flashes. Asymptomatic women are found to have significantly increased weight compared with severely symptomatic women, even when matched for age, ovarian status, and years since menopause. [566] The lower frequency and intensity of hot flashes in obese women may result from the elevated circulating free estradiol concentrations. [567] Urogenital Atrophy

The urogenital sinus gives rise to the development of the lower vagina, vulva, and urethra during embryonic development, and these tissues are estrogen-dependent. The decrease in estrogen at menopause causes the vaginal walls to become pale because of diminished vascularity and to thin down to only three to four cell layers. The vaginal epithelial cells in postmenopausal women contain less glycogen, which prior to menopause was metabolized by lactobacilli to create an acidic pH, thereby protecting the vagina from bacterial overgrowth. Loss of this protective mechanism leaves the thin, friable tissue vulnerable to infection and ulceration. The vagina also loses its rugae and becomes shorter and inelastic. Postmenopausal women may complain of symptoms secondary to vaginal dryness, such as pain during intercourse, vaginal discharge, burning, itching, or bleeding. Genitourinary atrophy leads to a variety of symptoms that affect the ease and quality of living. Urethritis with dysuria, stress urinary incontinence, and urinary frequency are further results of mucosal thinning of the urethra and bladder. Intravaginal estrogen

treatment can effectively alleviate recurrent urinary tract infections and vaginal symptoms in the postmenopausal patient. reverses vaginal atrophy and urethral symptoms caused by estrogen deficiency.

[ 568]

Oral estrogen replacement also rapidly

Cognitive Function and Estrogen

Although Alzheimer's disease affects both men and women, studies in many different populations show that 1.5 to 3 times as many women as men suffer from this disease. [569] For women with this disease, one needs to consider behavioral and cognitive problems, therapeutic issues, and other gender-related risks. One obvious consideration is a possible link between estrogen deficiency and the increased incidence of Alzheimer's disease in postmenopausal women. [570] A number of studies have been published regarding the association of estrogen with cognition in women with or without Alzheimer's disease. Treatment of women free of Alzheimer's disease with hormone replacement for signs and symptoms of menopause led to improvements in verbal memory, vigilance, reasoning, and motor speed but no enhancement of other cognitive functions. [571] A meta-analysis of observational studies suggested that HRT was associated with a decreased incidence of dementia.[571] However, possible biases and lack of control for potential confounders limit interpretation of these studies. [571] Two small but randomized studies regarding the effects of short-term estrogen treatment on cognition in women with Alzheimer's disease produced conflicting data. [572] [573] Thus, future large randomized trials should target the potential benefits of HRT in improving cognition in women with menopausal symptoms as well as in the prevention and treatment of Alzheimer's disease. The ongoing large trial called the Women's Health Initiative may provide some answers in several years. Cardiovascular Disease

It has long been suggested that estrogen protects against atherosclerosis because the incidence of cardiovascular disease is lower in women than in men in all age groups. The gender gap is widest during the premenopausal years. [574] [575] [576] For example, myocardial infarction is six times less common in premenopausal women than in men of the same age. [577] [578] This discrepancy has been attributed, in part, to the effects of estrogen. During reproductive life, women have lower LDL cholesterol than men, although these levels gradually increase with advancing age and rise rapidly after menopause.[574] [575] [576] In contrast, despite consistently higher high-density lipoprotein (HDL) cholesterol levels in women than men throughout adulthood, this discrepancy persists after the menopause. HDL cholesterol becomes higher after puberty in girls but does not change greatly at menopause. The increases in LDL and total

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cholesterol levels at menopause are partially reversible with estrogen treatment. [579] Therefore, the increased myocardial infarction rate after menopause may be a function of rising LDL cholesterol levels that seem to be related to decreased estrogen levels. [574] [575] [576] Although HDL cholesterol levels do not decrease significantly in postmenopausal women, replacement with oral estrogen increases HDL cholesterol levels significantly; this may contribute to the cardioprotective effect of HRT. [580] A strong correlation suggestive of a cause-and-effect relationship has been established between cholesterol levels and coronary heart disease in postmenopausal women.[581] [582] Postmenopausal women with elevated total cholesterol levels have a significantly increased risk of coronary heart disease compared with women with low levels. [581] [582] This risk is more pronounced in the first two decades after the menopause. The association becomes less pronounced with advancing age. [583] In both men and women, an HDL cholesterol level is the most specific determinant of coronary heart disease. [581] [582] [584] High levels of HDL cholesterol are protective, whereas a low level is a strong predictor of increased cardiovascular risk. Therefore, monitoring HDL cholesterol as well as total and LDL cholesterol levels is important in determining cardiovascular risk in postmenopausal women. [585] Replacement with estrogen decreases LDL and increases HDL cholesterol levels in postmenopausal women. [580] Although estrogen also increases triglyceride levels, the impact of this effect on the cardiovascular system is unknown. If a progestin is added, the beneficial effects of estrogen may diminish. Overall, postmenopausal estrogen replacement with or without added progestin produces a favorable lipid profile. [586] These favorable biochemical effects may provide some protection against cardiovascular risk. Many trials including a large, randomized study demonstrated a favorable impact on cardiovascular risk factors in women taking estrogen as well as a combination of estrogen and progestin. [587] Moreover, the great majority of studies investigating coronary or cerebrovascular disease as an outcome concluded that postmenopausal use of estrogens protected against cardiovascular disease, although these studies have been observational rather than randomized and blinded trials.[533] Thus, the opinion of the medical community in general was that exogenous estrogen given in postmenopausal replacement doses decreased cardiovascular risk for all women. A later randomized study of estrogen-progestin users with preexisting coronary disease (Heart and Estrogen/progestin Replacement Study, or HERS) showed an early increase in mortality. [588] Consequently, in 1999 the American College of Cardiology and American Heart Association revised their guidelines for providing HRT to patients with a history of acute myocardial infarction. [588] [589] The authors of HERS concluded that, over an average follow-up of 4 years, treatment with oral conjugated equine estrogen plus medroxyprogesterone acetate did not reduce the overall rate of coronary heart disease events in postmenopausal women with established coronary disease. [588] [589] HRT, however, was associated with small but statistically significant increases in the risks of deep venous thrombosis and pulmonary embolism.[590] No significant effect of HRT on the risk of stroke was detected in these postmenopausal women with coronary disease. [591] HRT in HERS also resulted in a marginally significant increase in the risk of symptomatic gallbladder disease and biliary tract surgery. [592] It was recommended that hormone replacement therapy should not be initiated solely for prevention of cardiovascular disease in postmenopausal women with preexisting coronary heart disease but can be continued in patients with cardiovascular disease already receiving HRT for other reasons. [588] [589] Another randomized trial showed that estradiol does not reduce mortality or the recurrence of stroke in postmenopausal women with preexisting cerebrovascular disease. [593] Thus, HRT should not be prescribed for the secondary prevention of cerebrovascular disease. [593] Recent findings from the Women's Health Initiative reinforce and expand the concerns raised by these randomized trials. In the Women's Health Initiative, more than 16,000 women with intact uteri were randomized to receive either placebo or conjugated estrogens 0.625 mg and medroxyprogesterone 2.5 mg daily. The study was terminated early, after an average of 5.2 years of therapy, because of a small but definite increase in the number of cases of invasive breast cancer and a negative global index of risks and benefits. [593A] Absolute excess risks per 10,000 person-years attributable to HRT were 7 more coronary heart disease events, 8 more strokes, 8 more pulmonary embolism events, and 8 more invasive breast cancers, while risk reductions were 6 fewer colon cancers and 5 fewer hip fractures. A significant decrease in vertebral fractures was also reported. It should also be noted that this study was not designed to investigate some potentially important benefits of HRT, including prevention of urogenital atrophy and improved sexual and cognitive functions. In summary, both the HERS trial and the Women's Health Initiative have failed to find any of the cardiovascular benefits predicted by earlier observational studies. Postmenopausal Osteoporosis

Osteopenia and osteoporosis are extremely common in elderly postmenopausal women. Osteopenia indicates low bone mass measured by densitometry, whereas the term osteoporosis implies severely decreased bone mass associated with a significantly increased risk for fractures. The most frequent sites of fracture are the vertebral bodies, distal radius, and femoral neck. Osteoporosis has become a global health issue. It is currently at epidemic proportions in the United States, affecting over 20 million people. [594] The majority of osteoporotic patients are postmenopausal women. Osteoporosis in postmenopausal women is a function of both advancing age and estrogen deficiency. Seventy-five percent or more of the bone loss in women during the first 15 years after menopause is attributed to estrogen deficiency rather than to aging itself. [595] [596] For the first 20 years after the cessation of ovarian estrogen secretion, postmenopausal osteoporosis accounts for a 50% reduction in trabecular bone and 30% loss of cortical bone. [595] [596] Vertebral bone is especially vulnerable because the trabecular bone of the vertebral bodies is metabolically very active and decreases dramatically in response to estrogen deficiency. Vertebral bone mass is already significantly decreased in perimenopausal and early postmenopausal women who have rising FSH and decreasing estrogen levels, whereas bone loss from the radius is not detected until at least a year after the menopause. [597] The risk of fracture depends on two factors: the peak bone mass achieved at maturity (at approximately age 30) and the subsequent rate of bone loss. An accelerated rate of bone loss after menopause strongly predicts an increased risk of fracture. The combination of low premenopausal bone mass and accelerated loss of bone after menopause is additive, and these individuals are at the highest risk of fracture. An increased rate of average bone loss during menopause is an indicator of lower endogenous estrogen levels because postmenopausal bone loss is considerably slower in women with increased adipose tissue mass and thus elevated

circulating estrogen. [169] It has been shown conclusively by numerous studies that hormone replacement started at the climacteric prevents postmenopausal bone loss. [598] Hormone replacement started at any age in a postmenopausal woman has potential beneficial effects by at least preventing additional bone loss. It should be noted, however, that the incidence of fractures or rate of height loss was not reduced during a 4-year follow-up in women starting

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HRT at a mean age of 66.7 (± 6.7) years.[257] More recently, in the part of the Women's Health Initiative discussed above, a decreased number of hip and vertebral fractures were noted in the group of postmenopausal women receiving estrogen/progestin [593A] ; this important evidence was the first from randomized trials suggesting that estrogen prevents fractures.



Postmenopausal Hormone Replacement

The most common current practice is to treat all women disturbed by the symptoms of hormone deprivation (hot flashes and urogenital atrophy) with estrogen and to use long-term hormonal prophylaxis against osteoporosis and cardiovascular disease on the basis of an informed decision by the patient. Patients who have undergone a hysterectomy can be given estrogen therapy alone. A progestin is added to estrogen in the postmenopausal woman with a uterus in order to prevent endometrial hyperplasia or cancer. The recent findings from the Women's Health Initiative trial [593A] are too new to allow prediction of how the negative findings from this trial will affect practice. Modest increases in breast cancer, coronary heart disease, and stroke were balanced by modest decreases in fractures and colon cancer. How individual patients and physicians will balance the immediate and common benefits in the treatment of hot flashes and urogenital atrophy against the predominantly negative long-term consequences that affect a modest fraction of patients (at least over 5 years) remains to be determined. It should be noted that the part of the Women's Health Initiative involving the use of estrogen without progestin to treat women after hysterectomy has not been halted; those results will become available in 2005. The decision for using long-term hormone replacement should be made by the patient based on accurate information. The primary role of the physician is to provide scientific information to a patient, using understandable language. [598] This is not an easy task, given the complexity of the existing data and occasionally opposite opinions. [598] Estrogens and progestins used for postmenopausal hormone replacement are among the most commonly prescribed medications in the United States. Currently, 46% of women who have experienced a natural menopause and 71% of women who have had bilateral oophorectomy report having used postmenopausal HRT. [599] The average duration of use in the United States as of 1992 was 6.6 years, but only 20% of users had maintained treatment for at least 5 years. Emphasizing the education of the patient and primary care physician on the basis of appropriate interpretation of epidemiologic studies and clinical trials will ensure appropriate use of long-term postmenopausal HRT. Target Groups for Hormone Replacement

In women with gonadal dysgenesis and surgical menopause, the duration of estrogen deprivation is prolonged. Estrogen replacement is recommended for these patients for the reduction of hot flashes and for long-term prophylaxis against cardiovascular disease, osteoporosis, and target organ atrophy. A low-dose contraceptive may be offered to nonsmoking women until the age of 45. After this age, doses of estrogen equivalent to 0.625 mg of conjugated estrogens may be more appropriate because of a sharp age-related increase in risk for thromboembolic events. The physician should recommend a continuous estrogen-progestin combination to those with a uterus and an estrogen-only regimen to women without a uterus. During the climacteric, hot flashes can be suppressed with an estrogen-progestin combination. Because bone loss related to estrogen deprivation also begins during this period, starting hormone replacement therapy during the climacteric is of paramount importance for minimizing osteoporosis. [600] In climacteric women, unexplained uterine bleeding should be evaluated with an endometrial biopsy before the start of hormone replacement. The lifelong use of hormone therapy after menopause is dependent on the informed decision of the woman based on balanced and evidence-based advice provided by her physician. The benefits of hormone replacement with respect to bone metabolism, cognitive function, urogenital health, and sexual function are substantial and for many women outweigh the increased breast cancer and cardiovascular disease risk. [601] [602] Estrogen Preparations and Beneficial Dose of Estrogen

The amount of estrogen that is optimally effective in maintaining the spine and femoral neck bone mass is equivalent to 0.625 mg/day of conjugated estrogens. [601] The effective doses of oral estrogen that reduce the incidence of fracture are 0.625 mg/day of conjugated estrogens and 1.25 mg/day of estrone sulfate. [601] [603] [604] Also, transdermal estradiol delivered at a rate of 0.1 mg/day was reported to reduce fracture risk. [605] [606] Transdermal estradiol at a dose of 0.05 mg/day is also presumed to lower fracture risk based on equivalent doses of various preparations that provide similar average circulating estradiol levels. [607] [608] Oral intake of 0.625 mg of conjugated estrogen, 1.25 mg of estrone sulfate, or 1 mg of micronized estradiol results in similar average serum levels of estrogens: estradiol, 30 to 40 pg/mL, and estrone, 150 to 250 pg/mL. [609] [610] Transdermal administration of estradiol with patches releasing 0.05 mg/day gave rise to similar average serum estradiol (30 to 40 pg/mL) but much lower estrone (40 pg/mL).[611] [612] [613] Short-term cardiovascular or hemodynamic effects of estrogen vary according to blood estrogen levels. Improvements in left ventricular contraction and function are associated with levels achieved by 0.625 mg of oral conjugated estrogens. [614] [615] [616] Extremely high estradiol levels achieved with large doses of estrogen, on the other hand, decrease left ventricular function and aortic blood flow. [617] The beneficial effect of postmenopausal estrogen in preventing the hyperinsulinemia associated with aging is present with a dose of 0.625 mg of conjugated estrogens but lost with a dose of 1.25 mg. [618] The effect of estrogen on arterial thrombosis is also dose-related. For example, oral contraceptives with high doses of estrogen significantly increase the risks of myocardial infarction and stroke, especially in smokers. Numerous studies suggest that doses of conjugated estrogens greater than 0.625 mg are, in fact, not as beneficial in terms of cardiovascular disease and mortality. However, the number of patients receiving these high doses was not large enough to achieve statistical significance. [619] [620] [621] Thus, it is imperative to achieve and maintain the lowest beneficial levels of circulating estradiol and avoid higher levels associated with unfavorable hemodynamic effects or thrombosis. Estrogen-Progestin Regimens

The addition of a progestin, either cyclically or continuously, to concomitant estrogen replacement reduces the risk of estrogen-induced endometrial hyperplasia or carcinoma but poses additional problems. [618] These problems include regular withdrawal bleeding in up to 90% of women treated with cyclic therapy and irregular spotting in 20% of women treated with continuous estrogen plus progestin. Furthermore, progestins appear to reduce the beneficial effects of estrogen on HDL and LDL cholesterol and possibly cardiovascular risk. [618] A time-honored sequential regimen involves oral administration

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Figure 16-41 Regimens of hormone replacement therapy (HRT). Estrogen (E) is replaced in a postmenopausal woman to prevent osteoporosis, urogenital atrophy, and hot flashes. In the postmenopausal woman with a uterus, a progestin (P) is added to estrogen to prevent endometrial hyperplasia and cancer. E and P can be administered in a number of ways. A and B, The postmenopausal women receiving hormone replacement have predictable withdrawal bleeding episodes after each P course. C, These women take E and P together continuously. After a year of continuous combination therapy, the rate of unpredictable breakthrough spotting is 20%.

of 0.625 mg of conjugated estrogens or the equivalent doses of a variety of available products from day 1 to 25 of each month (Fig. 16-41) . A daily dose of 10 mg of medroxyprogesterone acetate is added from day 12 to 25 or from day 16 to 25. Withdrawal bleeding is expected on or after day 26 of each month. Another common cyclic regimen involves continuous oral administration of 0.625 mg of conjugated estrogens or the equivalent daily dose (see Fig. 16-41) . A daily dose of 5 to 10 mg of medroxyprogesterone acetate is added for the first 10 to 14 days of every month. One-year randomized trial data indicate that the 5-mg dose protects the endometrium as well as the 10-mg dose.[622] Progestin withdrawal bleeding occurs in 90% of women with a sequential or cyclic regimen. [587] [623] These regimens can also cause adverse symptoms related to the relatively high daily doses of progestin, such as breast tenderness, bloating, fluid retention, and depression. Thus, the

lowest possible dose of a progestin is recommended. The continuous combined method of treatment, on the other hand, has the potential benefit of reduced bleeding and amenorrhea but is occasionally complicated by breakthrough bleeding (see Fig. 16-41) .[587] [623] In this regimen, a combination of 0.625 mg of conjugated estrogens and 2.5 mg of medroxyprogesterone acetate is given orally every day. The continuous combination regimen is simple, convenient, and associated with a higher incidence of amenorrhea in 80% of patients after at least 6 months of use. The rest of the patients continue to experience some degree of unpredictable spotting. Thus, overall compliance is much better in users of the continuous combination regimen. Moreover, the lower daily dose of medroxyprogesterone acetate is associated with a lower incidence of breast tenderness with this regimen. Other estrogen-progestin combinations are also available for similar continuous use. Most postmenopausal women stop HRT within several years after the initiation of therapy because hot flashes decrease significantly or disappear at this time. Upon the cessation of HRT that had lasted several years, hot flashes usually do not return or are less severe than during the climacteric. Consequently, long-term compliance with HRT remains poor. [624] [625] The fear of malignancy and irregular uterine bleeding are two major factors that stop women from continuing HRT indefinitely. [626] The current data on breast cancer are indicative of a slightly increased risk for both estrogen-only and estrogen plus progestin regimens. [624] [625] The addition of a progestin to the estrogen-only regimen, on the other hand, has effectively prevented endometrial cancer. [622] Irregular uterine bleeding is another major reason for discontinuation of HRT. The incidence of persistent uterine bleeding with the traditional sequential regimen can be as high as 90%, which deters women from taking lifelong hormone replacement. By switching to the continuous combination regimen, this bleeding can be reduced to a 20% incidence of spotting with long-term (>1 year) use. Thus, more clinicians start women with the continuous combination treatment with the hope of improving compliance for long-term use (see Fig. 16-41) . Selective Estrogen Receptor Modulators as Hormone Replacement

Selective estrogen receptor modulators are compounds that act like estrogen in some target tissues but antagonize estrogenic effects in others. [627] One of the first selective estrogen receptor modulators was tamoxifen, for which estrogen-like agonist activity on bone was observed to occur simultaneously with estrogen antagonist activity on the breast. [628] An unwanted effect of tamoxifen is its estrogen-like action on the endometrium. Second-generation compounds have since been developed, most notably raloxifene, which has estrogen-like actions on bone, lipids, and the coagulation system; estrogen antagonist effects on the breast; and no detectable action in the endometrium. [629] In randomized placebo-controlled studies involving postmenopausal women or patients with osteoporosis, raloxifene at 60 to 150 mg/day was effective in increasing bone mineral density over 12 to 36 months. [629] Raloxifene also decreased the risk of vertebral fractures. Raloxifene is similar to placebo in its endometrial effects and similar to estrogen in causing a twofold to threefold increase in the risk of venous thromboembolism. [630] Raloxifene lowers total and LDL cholesterol. HDL cholesterol and triglycerides are virtually unaffected. [84] The propensity of raloxifene to cause hot flashes precludes its use in women with vasomotor symptoms. On the other hand, the lack of stimulatory effects on the endometrium and the reduction in the incidence of invasive breast cancer indicate that raloxifene is an alternative to estrogen-progestin preparations for the management of postmenopausal osteoporosis, especially for patients reluctant to use estrogen. It should be emphasized that, at this time, postmenopausal hormone replacement with estrogen remains the "gold standard" for health benefits with respect to bone, cardiovascular system, urogenital organs, and possibly the central nervous system. Management of Breakthrough Bleeding during Postmenopausal Hormone Replacement

Approximately 90% of women receiving estrogen plus cyclic administration of a progestin have monthly progestin withdrawal bleeding in a predictable fashion, whereas continuous combined estrogen-progestin therapy causes breakthrough bleeding in approximately 40% of women during the first 6 months. (The rest of the women with a continuous combination regimen are amenorrheic.) The pattern of vaginal bleeding in the continuous regimen is unpredictable and causes anxiety in most patients. Fortunately, the incidence of breakthrough bleeding with the continuous combined regimen decreases to 20% after 1 year of treatment. [587] [623] [631] Breakthrough bleeding with the combined continuous regimen remains the most important reason for discontinuance of this therapy. Most patients find it unacceptable and prefer to

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switch to a cyclic progestin regimen or discontinue hormone replacement altogether. There is no effective pharmacologic method to manage the breakthrough bleeding associated with continuous combined estrogen-progestin regimens. One can only reassure the patient that the bleeding is likely to subside within a year from the start of HRT. If breakthrough bleeding continues beyond a year, the regimen should be changed to daily estrogen plus cyclic progestin monthly for 10 days. HRT can be started in the amenorrheic postmenopausal patient at any time. Perimenopausal women with oligomenorrhea, hot flashes, or other associated symptoms should also be given HRT. In the oligomenorrheic patient, a hormone replacement regimen can be initiated on day 3 of one of the infrequent menses. If the candidate for hormone replacement does not have irregular uterine bleeding, it is not essential to perform endometrial biopsies routinely before beginning treatment. Studies indicate that asymptomatic postmenopausal women rarely have endometrial abnormalities. [631] [632] [633] Pretreatment biopsies using a thin plastic biopsy cannula in the office may be limited to patients at higher risk for endometrial hyperplasia (e.g., unpredictable uterine bleeding, history of PCOS or chronic anovulation, obesity, liver disease, and diabetes mellitus). Giving a woman a combined estrogen-progestin regimen does not preclude the development of endometrial cancer. [634] It is, therefore, necessary to rule out endometrial malignancy in women receiving HRT who are experiencing irregular uterine bleeding. The important task is to differentiate breakthrough bleeding from bleeding induced by hyperplasia or cancer. Because breakthrough bleeding is extremely common, a large number of biopsies would have to be performed to detect a rare case of endometrial abnormality during HRT. In order to decrease the number of endometrial biopsies, a screening method using transvaginal ultrasonography has been introduced. [226] The thickness of the postmenopausal endometrium as measured by transvaginal ultrasonography in postmenopausal women correlates with the presence or absence of pathology. [226] Patients receiving either a cyclic or daily combination hormone replacement regimen who have an endometrial thickness less than 5 mm can be managed conservatively.[635] [636] [637] An endometrial thickness equal to or greater than 5 mm requires biopsy. Following this algorithm, it is estimated that 50% to 75% of bleeding patients receiving HRT and evaluated by ultrasonography require biopsy. [226] Side Effects of Postmenopausal Hormone Replacement

Deep Venous Thrombosis.

It has been debated whether continuous combination hormone replacement using conjugated estrogens (0.625 mg/day) plus medroxyprogesterone acetate (2.5 mg/day) increases the risk of venous thromboembolism. [638] [639] [640] [641] [642] [643] A recent randomized clinical trial has shown that this regimen of HRT significantly increases the risks of deep venous thrombosis and pulmonary embolism, although these increases were modest. [593A] Even if the relative risk is significantly increased, the actual risk remains extremely low because of the low frequency of this event in the general population. Hyperlipidemia.

This rare side effect is observed in patients with severe familial hypertriglyceridemia. An oral estrogen regimen can hasten severe hypertriglyceridemia or pancreatitis in women with severely elevated triglyceride levels. [644] Therefore, estrogen replacement is a relative contraindication in women with substantially increased triglyceride levels. Gallbladder Disease.

There is a minimally increased risk of gallbladder disease with estrogen use during the menopause. [645] There is a marginally significant increase in the risk of cholecystectomy in past and current users of HRT. [646] [647] Preexisting gallbladder disease is a relative contraindication for estrogen replacement. Breast Cancer.

Breast tissue is a major target for estrogen, and most breast tumors are estrogen-responsive. A number of case-control and cohort studies concluded that 5 or more years of current use of postmenopausal HRT is associated with a slight increase in the risk of breast cancer, a risk that is less than that associated with postmenopausal obesity or daily alcohol consumption. [648] [649] Many observational studies, however, have failed to develop evidence that long-term postmenopausal HRT increases the risk of breast cancer. [648] [649] Moreover, none of the epidemiologic studies found an increased risk of breast cancer associated with less than 5 years of use or past use of postmenopausal HRT. [649] The addition of a progestin to the treatment regimen slightly increased the risk observed in estrogen-only users. [649] In this context, the recent findings from the Women's Health Initiative support and extend the previous work, in that the combined use of estrogen and progestin led to a statistically significant increase in invasive breast cancer over the 5.2 years of the study. [593A] Because breast cancer was uncommon in this group of postmenopausal women, this increase represented an absolute increase of 8 cases per 10,000 women receiving hormone replacement. Epidemiologic data indicate that a positive family history of breast cancer should not be a contraindication to postmenopausal estrogen use. Moreover, postmenopausal women in whom the cancer develops during HRT have a reduced risk of dying from breast cancer. [649] This reduced risk may be due to an increased rate of early detection and development of less aggressive tumors in association with HRT. In conclusion, there may be a minimally increased real risk of developing breast cancer in long-term HRT users. This risk, however, is extremely small compared with the clear-cut benefits of estrogen such as osteoporosis prevention and urogenital tissue support. Hormone Replacement Therapy after a Diagnosis of Breast Cancer.

HRT is typically withheld from women with breast cancer because of concerns that estrogen may stimulate recurrence. Surprisingly, a number of relatively small studies showed either unaltered or lower risks of recurrence and mortality in women who used HRT after a diagnosis of breast cancer compared with nonusers. [650] [651] HRT in most of these small studies was started after at least a 5-year disease-free interval. [ 650] [ 651] On the basis of these insufficient but encouraging data, a decision to provide HRT is dependent on the choice of the individual patient. In these patients, tamoxifen or raloxifene represents a viable alternative to estrogen replacement for long-term prophylaxis against osteoporosis.



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A, Fisman E, Shapira I, et al. Exercise echocardiography in postmenopausal hormone users with mild hypertension. Am J Cardiol 1996; 78:13851389.

617. Pines

A, Fisman E, Drory Y, et al. The effects of sublingual estradiol on left ventricular action at rest and exercise in postmenopausal women: an echocardiographic assessment. Menopause 1998;

5:7985. 618. Lindheim

S, Presser S, Kitkoff E, et al. A possible bimodal effect of estrogen on insulin sensitivity in postmenopausal women and the attenuating effect of added progestin. Fertil Steril 1993;

60:664667. 619. Stampfer

M, Colditz G, Willett W, et al. Postmenopausal estrogen therapy and cardiovascular disease: ten-year follow-up from the Nurse's Health Study. N Engl J Med 1991; 325:756762.

620. Henderson 621. Ettinger

B, Paganini-Hill A, Ross R. Decreased mortality in users of estrogen replacement therapy. Arch Intern Med 1991; 151:7578.

B, Friedman G, Bush T, et al. Reduced mortality associated with long-term postmenopausal estrogen therapy. Obstet Gynecol 1996; 87:612.

622. Woodruff

J, Pickar J. Incidence of endometrial hyperplasia in postmenopausal women taking conjugated estrogens (Premarin) with medroxyprogesterone acetate or conjugated estrogens alone. The Menopause Study Group. Am J Obstet Gynecol 1994; 170:12131223. 623. Archer

D, Pickar J, Bottiglioni F. Bleeding patterns in postmenopausal women taking continuous combined or sequential regimens of conjugated estrogens with medroxyprogesterone acetate. Menopause Study Group. Obstet Gynecol 1994; 83:686692. 624. Speroff

T, Dawson N, Speroff L, et al. A risk-benefit analysis of elective bilateral oophorectomy: effect of change in compliance with estrogen therapy on outcome. Am J Obstet Gynecol 1991; 164:165174. 625. Berman

R, Epstein R, Lydick E. Compliance of women in taking estrogen replacement therapy. J Womens Health 1996; 213.

626. Ravnikar 627. Dardes 628. Diez

V. Compliance with hormonal therapy. Am J Obstet Gynecol 1987; 156:13321334.

R, Jordan V. Novel agents to modulate oestrogen action. Br Med Bull 2000; 56:773786.

J. Skeletal effects of selective oestrogen receptor modulators (SERMs). Hum Reprod Update 2000; 6:255258.

629. Clemett 630. Burger 631. Nand

D, Spencer C. Raloxifene: a review of its use in postmenopausal osteoporosis. Drugs 2000; 60:379411.

H. Selective oestrogen receptor modulators. Horm Res 2000; 53(suppl):2529.

S, Webster M, Baber R, et al. Bleeding pattern and endometrial changes during continuous combined hormone replacement. Obstet Gynecol 1998; 91:678684.

632. Archer

D, McIntyre-Seltman K, Wilborn W, et al. Endometrial morphology in asymptomatic postmenopausal women. Am J Obstet Gynecol 1991; 165:317320.

633. Korhonen

M, Symons J, Hyde B, et al. Histologic classification and pathologic findings for endometrial biopsy specimens obtained from 2964 perimenopausal and postmenopausal women undergoing screening for continuous hormones as replacement therapy (CHART 2 Study). Am J Obstet Gynecol 1997; 176:377380. 634. McGonigle

K, Karlan B, Barabuto D, et al. Development of endometrial cancer in women on estrogen and progestin hormone replacement therapy. Gynecol Oncol 1994; 55:126132.

635. Karlsson

B, Granberg S, Wikland M, et al. Transvaginal ultrasonography of the endometrium in women with postmenopausal bleeding: a Nordic multicenter study. Am J Obstet Gynecol 1995; 172:14881494. 636. Bakos

O, Smith P, Heimer G. Transvaginal ultrasonography for identifying endometrial pathology in postmenopausal women. Maturitas 1995; 20:181189.

664

637. Granberg

S, Ylosstalo P, Wikland M, et al. Endometrial sonographic and histologic findings in women with and without hormonal replacement therapy suffering from postmenopausal bleeding. Maturitas 1997; 27:3540. 638. Grodstein 639. Daly 640. Jick

F, Stampfer M, Goldhaber S, et al. Prospective study of exogenous hormones and risk of pulmonary embolism in women. Lancet 1996; 348:983987.

E, Vesey M, Hawkins M, et al. Risk of venous thromboembolism in users of hormone replacement therapy. Lancet 1996; 348:977980.

H, Derby L, Myers M, et al. Risk of hospital admission for idiopathic venous thromboembolism among users of postmenopausal oestrogens. Lancet 1996; 348:981983.

641. Varas-Lorenzo

C, Garcia-Rodriguez L, Cattaruzzi C, et al. Hormone replacement therapy and the risk of hospitalization for venous thromboembolism: a population-based study in Southern Europe. Am J Epidemiol 1998; 147:387390. 642. Gutthann

S, Rodriguez L, Castellsague J, et al. Hormone replacement therapy and risk of venous thromboembolism: population based case-control study. Br Med J 1997; 314:796800.

643. Cummings

S, Norton L, Eckert S, et al. Raloxifene reduces the risk of breast cancer and may decrease the risk of endometrial cancer in postmenopausal women: two-year findings from the Multiple Outcomes of Raloxifene Evaluation (MORE) trial. http:/www.asco.org/, 1998. 644. Glueck

C, Lang J, Hamer T, et al. Severe hypertriglyceridemia and pancreatitis when estrogen replacement therapy is given to hypertriglyceridemic women. J Lab Clin Med 1994; 123:5964.

645. Grodstein 646. Petitti

F, Colditz G, Stampfer M. Postmenopausal hormone use and cholecystectomy in a large prospective study. Obstet Gynecol 1994; 83:511.

D, Sidney S, Perlamn J. Increased risk of cholecystectomy in users of supplemental estrogen. Gastroenterology 1988; 94:9195.

647. LaVecchia 648. Koukoulis 649. Santen 650. Col

C, Negri E, D'Avanzo B, et al. Oral contraceptives and noncontraceptive oestrogens in the risk of gallstone disease requiring surgery. J Epidemiol Community Health 1992; 46:234236.

G. Hormone replacement therapy and breast cancer risk. Ann NY Acad Sci 2000; 900:422428.

R, Pinkerton J, McCartney C, Petroni G. Risk of breast cancer with progestins in combination with estrogen as hormone replacement therapy. J Clin Endocrinol Metab 2001; 86:1623.

N, Hirota L, Orr R, et al. Hormone replacement therapy after breast cancer: a systematic review and quantitative assessment of risk. J Clin Oncol 2001; 19:23572363.

651. O'Meara

E, Rossing M, Daling J, et al. Hormone replacement therapy with a diagnosis of breast cancer in relation to occurrence and mortality. J Natl Cancer Inst 2001; 93:754762.



665

Chapter 17 - Fertility Control: Current Approaches and Global Aspects Michael Kafrissen Eli Adashi

GLOBAL ASPECTS OF FERTILITY CONTROL Regulation of human fertility encompasses social, legal, and health care measures, including the employment of medical technologies and procedures that result in limiting the number of offspring. This can be achieved by spacing the birth of individual children in the sense of family planning or by terminating procreation when the desired family size has been achieved. The aim of fertility control is to achieve a family or population size that is compatible with a reasonable quality of life and is culturally and economically supported. Growth of the Human Population History and Current Status

The explosion of the world population is a relatively recent phenomenon (Fig. 17-1) . By AD 1, 200 million people lived on this planet. By 1650, more than 1600 years later, this number had increased to 500 million. Thereafter, the population-doubling time became progressively shorter. By 1850, the human population numbered 1 billion, and within 80 years, in 1930, the population reached 2 billion. In 2000 the world population crossed the 6 billion milestone, and the growth continues. [1] [2] Consequences of Overpopulation: From Malthus to Ehrlich

During the industrial revolution, Thomas Malthus, an English political economist, noticed the soaring population of the British Isles, a geographic area with limited arable land, and concluded that the human population would outrun the available food supplies. Between 1798 and 1816, Malthus published a series of essays in which he predicted that a collision of population growth and lack of world food supplies would result in worldwide disasters such as famines, epidemics, and wars. Malthus believed that strict limits on reproduction were essential to the betterment of humankind. [3] In 1968, at a time when humankind experienced the most prominent population increase (see Fig. 17-1) , Paul Ehrlich, a biologist at Stanford University, brought the issue of overpopulation to the public consciousness in his disquisition The Population Bomb. Ehrlich pointed out the negative effects of population growth on the environment, nonrenewable natural resources, and general economic progress. [4] [5]

666

Figure 17-1 The growth of the world population (billion) and increments by decades (million). () Publication of Malthus' essay on population. Since then, the world population has increased six times. () Publication of Paul Ehrlich's The Population Bomb. Since then, the world population has increased from 4 billion to 6 billion. (Modified from Raleigh VS. Trends in world population: how will the millennium compare with the past? Hum Reprod Update 1999; 5:500.) Coping with the Population Growth

Human ingenuity has largely disproved the predictions of both Malthus and Ehrlich. The extraordinary demographic history of the 20th century that occurred without global malthusian disasters can be explained mainly by two phenomena: innovations in agriculture and advances in medicine. [6] [7] [8] Agriculture: From the Neolithic to the Green Revolution

Historically, the phases in which the growth of the world population took an upward trend have coincided with improvement in agricultural techniques. In the Neolithic era, humans started to grow food supplies and this transition from hunting and gathering resulted in the first substantial population growth. However, further agricultural progress was slow, and so was the population growth. Not until the 18th and 19th centuries were more advanced agricultural methods applied. The industrialization of agriculture dates only from the beginning of the 20th century, when the farm tractor, introduced in Iowa in 1901, led to mechanization of the farmer's work. Further improvements, known collectively as the green revolution, followed. They included the use of chemical fertilizers, herbicides, and insecticides; the development of hybrid grains; and, more recently, the genetic manipulation of rice and grains. Farmers in the United States planted hybrid seeds and increased yields of grains by one third in only one decade, between 1930 and 1940.[6] [8] During the first 35 years of the green revolution, global grain production doubled. Famine, which historically has been a worldwide and perennial problem, became much less of a threat in the 20th century. Although the last 100 years have seen devastating famines in numerous areas of the world and millions of undernourished people have died, the demographic impact of these famines was relatively local and short-term. Famines are endemic on the Indian subcontinent, for example, but its population increased from 300,000 in the year 1900 to 1.3 billion in 2001. Despite predictions that the world population would outstrip food production, food production has risen a full 16% above population growth. [9] Today, there are fewer hungry people than ever before in history. In 1996, the number of hungry people of the world was 17%, whereas in 1970, 25 years earlier, 35% of the world's people fit in the "hungry" classification. [9] The increased productivity of industrialized agriculture has demographic consequences. Currently, 2% of the world's farmers, most of them in developed and rapidly developing countries, produce one fourth of the world's food. The reduced need for workers has liberated the industrial farmer from the pressure to have a large family. By contrast, traditional farmers, mostly in the underdeveloped countries, still feel the need to secure the necessary agricultural workforce through having more children. [6] [8] The success of industrial farming, however, came at a price. The expansion of arable land disturbed the balance of the ecosystem. For example, deforestation and reduction of natural pastures led to droughts and floods in certain areas. During the next 50 years, the growing global population will require further advances in agricultural production to ensure a sufficient, secure, and equitable food supply. To controland possibly

667

Figure 17-2 The population growth in individual continents. (From United Nations Population Division. World Population Prospects: The 2000 Revision. New York, United Nations, 2000.)

reversethe environmental impacts of agricultural expansion, intensive scientific efforts must be implemented along with regulatory, technologic, and policy changes.

[10]

[11]

Medicine: From Art to Science

During the 20th century, major advances in the practice of medicine and the application of preventive medicine increased worldwide population survival rates. A number of factors contributed to enhanced survival: an increase in the number of infants born alive, a decrease in infant and child mortality, and the containment of the spread of major epidemics. In the developed countries, the perinatal mortality rate fell from 225 per 1000 live births to under 20 per 1000. In the less developed countries, the perinatal mortality rate also decreased, but the disparity between underdeveloped and some industrialized nations was staggering. In 1988, the perinatal mortality was 5 per 1000 for Japan but it was 118 per 1000 for Bangladesh. [8] Globally, life expectancy increased from approximately 30 years at the beginning of the 20th century to 47 years in the middle of the century and 65 years by the end of the century. It is projected that by the year 2050, life expectancy will be 76 years. The current 180,000 living centenariansmost of them in Europe, Japan, China, and the United Statesbest attest to the improved health conditions of humankind. The kinds of epidemics that decimated populations in the past have become less of a peril. Historians estimate that in the 1350s, in medieval England, the epidemic of plagueknown as the "black death"reduced the total English population by 20% and decreased life expectancy to under 18 years. [7] The acquired immunodeficiency syndrome (AIDS), a modern-day parallel to a medieval plague, has taken its toll mainly in Africa, where 28 million of the 36 million AIDS-afflicted people live. Nevertheless, even in the sub-Saharan nations, where the incidence of AIDS is as high as 30% and life expectancy has been reduced to 37.2 years, the population continues to expand, although at a slower rate than before the outbreak of the epidemic. However, in some countries of sub-Saharan Africa, notably in South Africa and Zimbabwe, a negative growth rate is expected. [1] In summary, malthusian predictions of global catastrophes caused by overpopulation did not materialize. However, the balance of a steadily growing population and potential technologic limitations on resources remains precarious.



Population Projections

Current projections for the growth of the world population by the middle of the 21st century vary from 7.9 billion to 10.9 billion (see Fig. 17-1) . [1] [2] With respect to individual continents, Asia will remain the most populous. The most significant growth is expected to occur in Africa, with the population rising from the current 900 million to 2 billion. Notable population growth is also projected for countries in both North America and Latin America. The United States will probably be the only industrialized nation with a population increase. The population of Europe is expected to decrease (Fig. 17-2) . [1] However, there is a broader question related to the world population growth, namely what is the optimal number of people the world can support? In 1994, a serious attempt to answer this question was made at the International Conference on Population and Development, now known as the Cairo Conference. The conference forecasted a world population of 10 billion in 2050 and recommended holding the population at that level. [12] [13]



The Future of the World Population

Medical progress has affected human demographics by another innovation: the development of effective means of preventing pregnancy. Introduced in the 1960s when the population was increasing, modern contraception was immediately recognized as a potential instrument for large-scale family planning. [6] Indeed, available demographic data show that since the 1980s, population growth rates, along with fertility rates, have

668

Figure 17-3 Birth rates from 1950 to 2000 and projections for the first half of the 21st century. (From United Nations Population Division. World Population Prospects: The 2000 Revision. New York, United Nations, 2000.)

been falling ( Fig. 17-3 ; see Fig. 17-1 ). This signifies a tapering off of the most rapid phase of world population growth. However, the data also show that the momentum of growth will persist into the middle of this century, when the world population is projected to be 10 billion. The goal of holding the world population at 10 billion can be accomplished if the fertility rate is limited to an average of 2.1 children per woman, the essential replacement rate; this can be achieved by effective fertility control. [14]



The Role of Family Planning

Historically, most societies rewarded families for having children by reducing their tax burden and giving awards to mothers of numerous children. In the second half of the last century, however, certain societies, pressured by overpopulation, installed strong disincentives for families that had more than a prescribed number of children. In China, for example, a family that has more than the allowable one child is castigated. In Singapore, a highly developed but crowded country, the acceptable number of children for a family used to be determined, among other things, by the parents' educational level. In other cultural environments, the government resorted to positive incentives by providing gifts to individuals who underwent voluntary sterilization. Coercive methods of family planning have been criticized and are globally unacceptable. [15] Modern family planning must respect the freedom of reproductive choices, free access to family planning facilities, and availability of up-to-date methods of fertility control. Individual families as well as entire nations must be educated to comprehend that effective family planning makes good social and economic sense. Currently available data show that the decrease of birth rates is inversely proportional to the percentage of the population practicing contraception. The data also show that nations enjoying the highest living standardsor those striving to overcome economic hurdleshave the lowest birth rates. Examples of the latter are the countries of the former Eastern European bloc (Fig. 17-4) . Effective fertility control can be achieved only when the society supports it. Initially, after their introduction in the 1960s, modern methods of human fertility control were accessible only to the affluent. Today, in the milieu of globalization, contraception has become a matter of governmental policy in many nations. It is also of primary concern to global organizations such as the World Health Organization (WHO) and the United Nations Educational, Scientific, and Cultural Organization as well as private institutions such as the Population Council and the Alan Guttmacher Institute of Family Planning.



The Role of the Physician and Other Health Care Givers in Fertility Control

Health care givers in nearly all specialties have become increasingly involved in issues of fertility control. Practitioners ranging from pediatricians who care for teenage girls to internists who care for premenopausal women are frequently asked to provide contraceptive advice. The role of medical specialists in reproductive health care is also undergoing a transition. Two major challenges have arisen. The first is a result of the existing and widening cultural diversification in developed countries, where physicians frequently face problems that not only challenge their medical skills but also test their ability to deal with the family planning needs of communities with diverse ethnic, cultural, and religious backgrounds. The second challenge has to be met by all those working in reproductive health care and family planning: the increasing emphasis on state-of-the-art medical practice. There is a global need for the rapid incorporation of appropriate technologies into daily clinical practice, substantially increasing the quality of rational medical care, worldwide. [16] The individual methods and technologies of fertility control are discussed in the following sections of this chapter.



Methods of Fertility Control: Efficacy, Continuation of Use, Changing Trends

When making a recommendation to a candidate for contraception, three aspects of each method have to be examined: efficacy, tolerability, and whether the method is suitable for temporary or permanent cessation of procreation. Table 17-1 lists the principal methods of fertility control currently available and indicates their contraceptive efficacy as

669

Figure 17-4 The decrease of the birth rate is inversely proportional to the percentage of the population practicing contraception and positively related to the standard of living. (Modified from Potts M. The unmet need for family planning. Sci Am 2000; 282[1]:69.)

well as the average time patients adhere to an individual method. [17] [18] [19] The estimates of contraceptive failure during "perfect" use are derived from studies conducted for research and registration purposes; they include volunteersa self-selected groupwho are highly motivated to adhere to the study protocol. Therefore, the results are more favorable than those obtained during "typical" use, that is, use in the general population. Data on the efficacy for typical use are generated by the National Surveys of Family Growth, among others. [20] Efficacy and Continuation

If the outcome was left to chancethat is, no method of fertility control was practiced85 of 100 women of reproductive age would become pregnant within a year. Surgical sterilization, for both men and women, remains one of the most effective methods of contraception, although it is not failure proof. The small proportion of early failures may be associated with suboptimal surgical techniques, such as mistaking other anatomic structures for the fallopian tube or vas deferens; insufficient electrocoagulation when this method is used in women; or unprotected early intercourse after vasectomy while live spermatozoa are still present in the part of vas deferens that is distal to the interruption. Late failures, between the 3rd and 10th years after surgery, also occur and are discussed in the section on sterilization. The major advantage of surgical sterilization is that it is permanent and can be a welcome solution to parents with large families. Chemical sterilization of women aims at producing tubal occlusion by injection of a substance (e.g., quinacrine) into the junction between the tube and the uterus. Data from a large study conducted in Vietnam show that this method is inferior to surgical sterilization. [21] [22] Among the hormonal contraceptive methods, long-acting approaches such as implants, injectables, and copper- or hormone-containing intrauterine devices (IUDs) achieve a higher level of efficacy than oral contraceptives (OCs) that require a daily conscientious action by the user. According to revised data of 1995, [20] the failure rates of OC formulations are higher than previously thought. Emergency postcoital contraception, when properly used, is also effective; however, it is recommended only as an emergency provision. Hormonal methods of fertility control also encompass non-surgical termination of pregnancy by pharmacologic means ("contragestion"). Mifepristone and prostaglandins administered not later than the seventh week of pregnancy achieve complete abortion in a high proportion of cases. The procedure is less effective when employed after the seventh week of pregnancy. The efficacy of IUDs has been improved by incorporating either progesterone or a synthetic progestogen, levonorgestrel. The copper IUD has staged an impressive revival, principally outside the United States. Its efficacy and safety have been improved and are now comparable to those of hormonal contraception. Copper- and progestin-bearing IUDs are likely to continue to grow in popularity. Other methods of fertility control are less effective. For the barrier methods, there is a large discrepancy between the efficacy of ideal use and that of typical use. With the cervical cap and the vaginal sponge, failure rates are high even with perfect use. The same applies to the "natural" methods of fertility control. They rely, one way or another, on prediction or detection of ovulation and require continuous watchfulness and motivation of both partners. Therefore, their efficacy does not compare favorably with that of the hormonal or intrauterine

670

TABLE 17-1 -- Fertility Control Methods: Failure Rates and Continuation of Use (United States Data) Percent Pregnant during First Year of Use Estimates 1987 and 1990, * All Methods Perfect use

Typical

Estimates 1995, Reversible Methods

Continuation after First Year of Use * (%)

85

85

85

?

Male

0.1

0.2

Female

0.2

0.4

Method Chance Sterilization

100 ND

100

Surgical Chemical (quinacrine) Women 7 wk)

ND

ND

IUD-progesterone T

1.5

2.0

IUD-levonorgestrel 20

0.1

0.1

IUD-T 380 (copper)

0.6

0.8

Male

3.0

14.0

14.0

63

Female

5.0

21.0

ND

56

6.0

20.0

18.0

58

Parous women

26.0

40.0

12.0

42

Nulliparous women

9.0

20.0

56

Parous women

20.0

40.0

42

Nulliparous women

9.0

20.0

ND

56

Spermicides

6.0

26.0

26.0

40

Withdrawal

4.0

19.0

24.0

?

Intrauterine devices (IUDs) 80 ND

81 78

Barrier methods Condom

Diaphragm Cervical cap

Sponge

Periodic abstinence §

63

Calendar

9.0

?

Ovulation method

3.0

?

Postovulation

1.0

?

Symptothermald

2.0

?

21.0

Lactational amenorrhea provides an effective but temporary method of contraception ND, no data. *Data from references [ 17] [ 18] [ 19] . New estimates of contraceptive failure according to correction for abortion underreporting, from 1995 National Survey of Family Growth. [20] Data on quinacrine sterilization from reference [21] . §Periodic abstinence methods. Calendar: The woman records the length of 6 to 12 cycles and determines the beginning of the fertile period by subtracting 18 days from the shortest cycle. The end of the fertile period is estimated by subtracting 11 days from the longest cycle. Ovulation method: Women are taught to recognize the character of the cervical mucus during the fertile period. Sexual abstinence begins on the day when the mucus becomes clear, slippery, and stretchy (usually a few days before ovulation). At the peak of the fertile period, the mucus stretches between the finger and thumb to the maximum (spinnbarkeit). Intercourse is resumed on the third or fourth day after the peak mucus. Symptothermal method: Cervical mucus (ovulation) method supplemented by calendar in the preovulatory phase and basal body temperature in the postovulatory phase. Postovulatory method: Ovulation is estimated by the basal body temperature or cervical mucus methods; unprotected intercourse is allowed 3 to 4 days thereafter. Note: Sophisticated electronic devices are currently available to estimate ovulation more precisely, some of them measuring the estrogen and luteinizing hormone concentrations in urine.

methods. Lactational amenorrhea affords protection against pregnancy; however, breakthrough ovulations do occur, particularly when breast-feeding is not exclusive and is interrupted by other types of baby nourishment. Women frequently discontinue the use of contraception. Overall, 31% of women discontinue use of reversible contraceptives for a method-related reason within 6 months of use; 44% do so within 12 months. However, 68% of women overall resume use of a method within 1 month and 76% do so within 3 months. High rates of method-related discontinuations reflect dissatisfaction with available methods or management, or both. [23] Trends in Fertility Control in the United States

In the third quarter of the last century, the use of fertility control methods in the United States underwent a major change (Table 17-2) .[24] [25] Among women, hormonal contraception has remained the most popular method. Two events positively influenced the employment of hormonal methods: the advent of hormonal implants and the approval of medroxyprogesterone acetate (MPA) depot injections for contraception. As physicians became versatile in laparoscopic surgeries, the

671

TABLE 17-2 -- Trend in Contraceptive Use by Women 15 to 44 Years of Age in the United States, 1973 to 1995 (Percent Users) Method 1973 * 1982 1988 1995 Sterilization

23.5

34.1 39.2

38.6

Female

12.3

23.2 27.5

27.7

Male

11.2

10.9 11.7

10.9

36.1

28.0 30.7

31.2

Pill

36.1

28.0 30.7

26.9

Implant

NA

NA

NA

1.3

Injectable

NA

NA

NA

3.0

Intrauterine device

9.6

7.1

2.0

0.8

Diaphragm

3.4

8.1

5.7

1.9

Male condom

13.5

12.0 14.6

20.4

Foam

5.0

2.4

1.1

0.4

Periodic abstinence

4.0

3.9

2.3

2.3

Hormonal methods

Withdrawal

2.1

2.0

2.2

3.0

Other

2.7

2.5

2.1

1.3

NA, not applicable. *Data from Ford K. Contraceptive utilization among currently married women 1544 years of age: United States, 1973. Mon Vital Stat 1976; 25/7 (suppl): 115. Data from Piccino LJ, Mosher WD. Trends in contraceptive use in the United States: 19821995. Fam Plann Perspect 1998; 30:410, 46. Other includes douche, sponge, jelly or cream alone, and other methods.

frequency of female sterilization jumped from 12% in 1973 to 23% in 1982; 1995 brought a further increase to nearly 30%. The proportion of male sterilization remained stable at 11%. The condom now ranks as the third method of choice. Between 1982 and 1995, the frequency of its use rose from 12% to 27%, most markedly among unmarried women in the age group 15 to 29 years. Fear of human immunodeficiency virus (HIV) and other sexually transmitted diseases (STDs) impelled this shift. Possibly the most dramatic change in the use of contraceptive methods was the virtual abandonment of IUDs in the United States. This was triggered by the Dalkon Shield episode ending in 1973the occurrence of excess pelvic infection in women using the device. Another negative factor was the recall of the "copper 7" IUD from the market in 1986 because of putative infertility of women who had discontinued its use. A newly designed copper IUD has surfaced, although U.S. physicians are still more hesitant to prescribe it than their colleagues outside the United States. Uptake of the hormone-bearing IUD is similarly slow. Contraceptive foams alone are used rarely; currently, manufacturers and physicians recommend that they be used simultaneously with barrier methods.

TABLE 17-3 -- Fertility Control in the Developing World (Percent Users), Trend 19801993 Method

1980 1993

Female sterilization

24

39

Intrauterine device

32

26

Pill

13

11

Male sterilization

13

8

Injectables

0

4

Vaginal

0

0.3

Condom

5

4

Traditional

12

9

From Bongaarts J, Johansson E. Future trends in contraception in the developing world: prevalence and method mix. Stud Fam Plan 2002; 32:24.

Figure 17-5 Intended and unintended pregnancies in the United States, 1994. (Data adapted from Henshaw SK. Unintended pregnancy in the United States. Fam Plann Perspect 1998; 30:2429, 46.) Trends in Fertility Control Methods in the Developing World

Surgical female sterilization is the most frequently used method of effective fertility control in the developing countries (Table 17-3) .[26] The biggest difference between the United States and developing countries is the high use of IUDs; use by approximately 30% of women in developing countries makes IUD insertion the second most frequent method of contraception. Hormonal contraception ranks third, possibly because of its high cost and problems with access. In Sri Lanka, for instance, some women buy only five pills at a time because their financial situation does not allow them to acquire a full month's supply. [14] Contraceptive Failures

Given the length of time that most women practice a reversible method of fertility control, experiencing at least one contraceptive failure is likely. By sheer statistical probability, failure can happen even if the most effective methods are used perfectly. Between the ages of 16 to 30, a hormonal contraception user has a more than 50% chance of becoming pregnant. During a lifetime of use of reversible methods, the typical woman experiences 1.8 contraceptive failures. This applies particularly when the health status of a woman prohibits the use of a highly effective method, such as the pill, and the couple has to resort to methods that have fewer adverse effects but may also be less effective. [27] [28] A National Survey of Family Growth analyzed the outcome of unplanned pregnancies in the United States that occurred despite contraception between 1994 and 1995.[28] [29] The analysis took into account the total number of births and therapeutic abortions in the year 1994. Miscarriages were excluded from analysis because it was difficult to calculate the proportion of women who did not plan the pregnancy. For purposes of the analysis, the authors assumed that all therapeutic abortions resulted from unintended pregnancies. In 1994, there were 5.38 million pregnancies in the United States; of those, 2.73 million (51%) were intended and resulted in births of live children (Fig. 17-5) . A full 2.65 million (49%) pregnancies were unintended; of those, 1.22 million or 46% resulted in births and 1.43 million or 54% ended as therapeutic abortions. Alternatively, of the total 3.95 million births (100%), the majority of 2.75 million or 70% were intended and 1.22 million or 30% were unintended. The survey further revealed that 48% of women from 15 to

672

TABLE 17-4 -- Contraceptive Antecedents to Medical Abortion, 19941995: Percentage Distribution of Abortion Patients by Contraceptive Method Used at Time of Conception Method Long-acting methods

All Abortion Patients, Total 9985 (100%)

Patients Who Were Using Contraception at Time of Conception (58% of Total)

Sterilization

0.2

Implant

0.1

Intrauterine device

0.1

Injectable

0.5

0.9

1.5

Pill

11.7

20.3

Male condom

32.4

56.6

Withdrawal

5.9

10.3

Other*

6.4

11.3

42.5

NA

No contraception Total

100.0

100.0

NA, not applicable. From Henshaw SK, Kost K. Abortion patients in 19941995: characteristics and contraceptive use. Fam Plann Perspect 1998; 28:140147, 158. *Female condom, diaphragm, sponge, foam, suppository, periodic abstinence.

44 years of age had had one or more unintended pregnancies, either an unplanned birth or an abortion, or both. The percentage increased with age to a high of 60% among women in the age group 35 to 39 years. The information on the use of contraceptive methods at the time when an unplanned pregnancy was conceived and later resulted in therapeutic abortion is based on a representative subset of 9985 patients who had abortions (Table 17-4) . Of these, 42.5% did not use any contraception at the time of conception and 57.5% practiced some method of fertility control. The male condom was most frequently associated with contraceptive failure. More than half of the women who resorted to abortion reported the use of condoms. [29] Among women who experienced contraceptive failure, 76% of those younger than 18 years preferred the condom. However, only 46% of women older than 30 years used this method. Pill use peaked at 25% among women aged 20 to 29 and decreased to 16% after the age of 30. On the other hand, the use of methods classified as other (see Table 17-4) rose from 1% in the age group younger than 18 years to 24% in the age group 30 years or older. The Need for Future Improvements

The high discontinuation rate, the number of unintended pregnancies, and the number of abortions testify to the fact that present reproductive health care is still deficient in providing adequate fertility control options to all women. The health care system alone cannot be blamed for the large number of unplanned pregnancies. Patients frequently choose methods of low efficacy, the primary example being the use of the male condom. When effective methods are recommended, they are frequently applied incorrectly or their use is interrupted, even if it is resumed later on. These problems can be partially remedied by appropriate information about the available contraceptive choices and by intensive counseling adjusted to the level of the individual patient. Informing the general public about new findings and advances in reproductive health care has been largely facilitated by access to electronic sources of information; the medical profession should take full advantage of this progress. The proportion of women who did not use any contraception at the time of an unplanned conception is disturbing. This problem could be partially addressed by the development of a highly effective postcoital method. An alternative solution would be implementation of an effective system that would make the presently available postcoital contraceptives promptly accessible. Finally, there must be renewed interest in improving present methods and designing new effective and safe approaches to fertility control.



TECHNOLOGIES OF FERTILITY CONTROL Hormonal Contraception Development of Hormonal Contraceptives

The knowledge that estrogens and progestagens can inhibit ovulation has been the foundation of the modern hormonal methods of contraception. In 1940, for the first time, Sturgis and Albright [30] achieved inhibition of ovulation in women by estrogens in order to relieve dysmenorrhea. The foundation of modern contraception was laid in Manhattan in 1950 at a lengthy conference. Among the participants were Gregory Pincus, director of the Worcester Foundation, and a small group of fertility control advocates including Margaret Sanger, the founder of the Planned Parenthood movement. The conference ended by granting Pincus seed money of $2100 to initiate his research on hormonal contraception. [31] In 1953, working with a Boston gynecologist, John Rock, Pincus started to test oral progesterone for ovulation inhibition. In 1956, Pincus' group of scientists and physicians published results of the first successful contraceptive clinical trial, which took place in Puerto Rico using a synthetic progestagen, norethynodrel, provided by the G.D. Searle company. [32] Norethynodrel, however, was not the first orally active progestagen. This priority belongs to norethindrone, synthesized in 1951, which became the lead compound in the development of clinically important oral progestagens. [33] [34] Starting in the 1930s, chemists searched for orally active steroids. In 1938, a group at the Schering Corporation in Berlin, Germany, developed the first orally active steroidal estrogen by attaching the ethinyl group (···CCH) to be 17t carbon of the estradiol molecule ( Fig. 17-6 and Fig. 17-8 ). [35] Ethinylestradiol still constitutes the estrogenic component of nearly all combined OCs. The development of orally active progestagens was triggered by the discovery that when the C-19 methyl group of testosterone is split off, the resulting molecule loses androgenicity and acquires progestagenic properties. In 1951, Carl Djerassi, leading a team of chemists in Mexico City, reasoned that attaching the ethinyl group (···CCH) to the 17th carbon of the nortestosterone molecule would greatly enhance its progestagenic activity and make the compound orally active ( Fig. 17-7 and Fig. 17-8 ). Djerassi and colleagues produced 17-ethinyl-19-nortestosterone. [36] The compound, known by its generic names norethindrone and in Europe norethisterone (commonly abbreviated NET), had 10 times the activity of natural progesterone when orally administered. To this day, norethindrone has remained the progestagenic component of many combined OCs (Table 17-6) . In a parallel development, manipulation of the progesterone molecule produced a group of orally highly active progestagens, such as medroxyprogesterone acetate (MPA) (Provera) ( see Fig. 17-10 later). Originally, hormonal contraception included only the pill, that is, an orally active combination of an estrogen and a progestagen. The pill offered several advantages over other available contraceptive methods. For the first time, its high

673

Figure 17-6 Attachment of the ethinyl group to C-17 of estradiol creates an orally highly active estrogen, ethinylestradiol (EE). Mestranol has a methyl group on C-3 of EE. Mestranol is a prohormone because it must be metabolically converted to EE to be able to bind to estrogen receptors. Modification of the estradiol molecule on C-17 provides long-acting 17-cypionate, used in injectable preparations. For numbering of the steroid molecule, see Figure 17-8 .

efficacy brought confidence in a contraceptive method and freedom from the fear of pregnancy. It produced only temporary and reversible infertility; therefore, it was ideal for family planning. Because the method was not linked to coitus, the spontaneity of the sexual act could be preserved. The pill has become popular among women in both developed and developing countries, and today 10 million women in the United States and 60 million women worldwide adhere to this method.

Figure 17-7 Development of norethindrone from testosterone. Splitting off the C-19 radical from the testosterone molecule changes this androgen to a progestagen. Attachment of the ethinyl group to C-17 enhances the progestagenic activity of the compound and makes it orally active. For numbering of the steroid molecule, see Figure 17-8 .

However, use of the pill requires a conscious daily action on the part of the user, and it has been postulated that missing pills could be the reason for the discrepancy between pregnancy rates during perfect and typical use (see Table 17-1) . To close this gap, long-term methods of hormonal contraception have been invented that require only one or a limited number of actions on the part of the user. Hormonal implants, depot injections, and hormonal IUDs are examples of such long-term methods with high efficacy during typical use. Unintended pregnancies can result from failure of contraceptives or from exposure to an unplanned sexual contact. Such events necessitate short-term preventive steps. Two important developments took place in this direction: emergency post-coital contraception and the use of antiprogestagenic steroids for contragestion. Hormonal Contraceptives in Clinical Use

The hormonal contraceptives available today can be categorized by the way they are administered and by the duration of their action (see Table 17-5) .



Oral Contraception Steroidal Components

In the following paragraphs we describe the pharmacologic and biologic properties of the two hormonal components of OCs, the estrogens and the progestagens. Contraceptive Estrogens

Structure and Function

In the combined estrogen-progestagen OCs, one type of estrogen prevails, 17-ethinylestradiol. A limited number of OC preparations contain a derivative of ethinylestradiol, mestranol. The ethinyl group protects ethinylestradiol and mestranol (MEE) from oxidation and conversion into less active estrogens such as estriol. Mestranol is a prodrug that does not bind

674

Figure 17-8 Contraceptive progestagens are derived from three skeleton structures, pregnane, estrane, and gonane (see details in the text). The pregnane molecule shows the numbering system of contraceptive steroids.

to estrogen receptors and has to be converted into ethinylestradiol in order to become biologically active. MEE has been replaced by ethinylestradiol in virtually all OCs worldwide. Pharmacokinetics and Metabolism

After ingestion, ethinylestradiol is rapidly absorbed from the gastrointestinal tract. The time to the maximum ethinylestradiol concentration in plasma (T max ) is 1 to 2 hours and the elimination half-life is wide, ranging from about 9 to 27 hours. In the intestine and in the liver, ethinylestradiol is readily conjugated with sulfuric and glucuronic acids and undergoes enterohepatic circulation. Intestinal bacteria possessing the appropriate enzymes can hydrolyze ethinylestradiol sulfates, and some of the deconjugated estrogen is reabsorbed. One could speculate that the use of oral antibiotics that affect intestinal flora may influence blood levels of ethinylestradiol; so far, clinical proof of this assumption is lacking. The pharmacokinetics and metabolism of mestranol are similar to those of ethinylestradiol except that Tmax is longer than might have been expected because mestranol has to be converted to ethinylestradiol. [37] Individual subjects vary considerably in the amount of absorbed ethinylestradiol and the circulating concentrations of ethinylestradiol as well as in the elimination time. Intrasubject variations can also be prominent. These variations may explain why adverse effects and contraceptive failures occur in only certain individuals and why the same woman can experience side effects during some treatment cycles but remains symptom-free during others. The basis of the intersubject and intrasubject variations has not been explained satisfactorily. As we are witnessing another wave of reduction of the ethinylestradiol content in the combination pills to 20 µg, bioavailability becomes an issue. As these and ever lower doses of ethinylestradiol become more common, we must be certain to provide OCs in formulations that deliver the digested amounts of steroids at appropriate concentrations for the individual user. Contraceptive Progestagens

In contrast to estrogen synthesis, the synthesis of progestagens has been prolific. The reasons for this dichotomy are several. Because ethinylestradiol exhibited potent oral activity and was not protected by patents, there was little incentive to search for other OC estrogens. New progestagens were synthesized in order to secure proprietary rights for the developers of OCs. Also, synthesis of new compounds was prompted by the desire to produce a highly effective progestagen that would minimize the dose of the progestagen in the OC combination and thus reduce the incidence of progestagen-related adverse events. Currently, about a dozen progestagens are being used clinically in established contraceptive preparations and several compounds are in various stages of preclinical and clinical testing. According to the classical definition, progestagens transform the estrogen-primed endometrium into a secretory one and support the development and maintenance of pregnancy. The advent of molecular biology has defined progestagens as compounds that bind to and activate progesterone receptors within the target cells. However, binding to progesterone receptors does not preclude the progestagen molecule binding with other receptors or expressing effects other than progestagenic effects, or both. For example, norethindrone, under certain circumstances, can stimulate the proliferation of the atrophic endometrium. [38] [39] Besides being a potent progestagen, cyproterone

675

TABLE 17-5 -- Hormonal Contraceptives in Clinical Use Oral contraception (see details in Table 17-6 ) Cyclic estrogen-progestagen combinations Continuous progestagen-only oral contraception Long-acting preparations Injectable preparations Progestagen-only preparations Depotmedroxyprogesterone acetate, 150 mg, q3mo, IM Norethindrone enanthate, 200 mg, q2mo, IM (not used in the United States) Estrogen-progestagen combination injectables Medroxyprogesterone acetate, 25 mg + estradiol cypionate, 5 mg, q28 ± 5d, IM Hormonal subdermal implants (see Table 17-12)

Norplant: 6 capsules with total amount of 216 mg of levonorgestrel Norplant II (Jadelle): 2 rods with total amount of 150 mg of levonorgestrel Hormonal intrauterine systems Progestasert: T-shaped; vertical arm releases progesterone, 65 µg/d Mirena (levonorgestrel-20): T-shaped; vertical arm releases levonorgestrel, 15 µg/d Vaginal rings releasing contraceptive hormonesNuvaRing approved in 2001 Transdermal patch releasing contraceptive hormones Ortho EVRA: 20 cm2 patch, delivers 150 µg norelgestromin, 20 µg/d ethinylestradiol Emergency methods of fertility control Postcoital hormonal contraception a. 1 mg norgestrel and 100 µg ethinylestradiol within 72 h of unprotected intercourse, repeated after 12 h b. 0.75 mg levonorgestrel within 72 h of unprotected intercourse, repeated after 12 h Contragestion Mifepristone, 200600 mg, orally, followed by 400800 µg misopristol orally

Figure 17-9 Classification of contraceptive steroids. Hybrid progestagens are in gray boxes. In parentheses are the active progestagenic metabolites of norgestimate and desogestrel.

acetate is a recognized antiandrogen in both men and women. [40] Classification of Contraceptive Progestagens

Contraceptive progestagens have been classified in various ways, for example, according to the amount of ethinylestradiol with which they are combined, the type of progestagen they contain, and the time when they became available for clinical use. According to the chemical structure of the steroids, the classification of contraceptive progestagens presented recognizes three basic groups, pregnanes, estranes, and gonanes (Fig. 17-9 ; see Fig. 17-8 ). Certain compoundsnorpregnanes, drospirenone, and dienogestcombine structural elements of other progestagens or chemical groups. They are designated as hybrid progestagens. Their structural and biologic characteristics are discussed in the section "Hybrid Progestagens." The structures of pregnane, estrane, and gonane and the numbering of the steroid molecule are given in Figure 17-8 . Structure and Function of Contraceptive Progestagens

Pregnanes

Pregnanes can be divided into three subgroups. The first consists of derivatives of 17-hydroxyprogesterone acetate, which has been developed by an intricate manipulation of the progesterone molecule (Fig. 17-10) . The C-6 and C-17 positions are of key importance for progestagenic activity. Chemical manipulations at C-17 profoundly change the function of the molecule. [41] Thus, progesterone loses its biologic activity with the introduction of an -hydroxyl group at C-17. Esterification of this group not only restores the progestational activity but also renders the resulting substance17-hydroxyprogesterone

676

Figure 17-10 Progestagens derived from progesterone. Progesterone loses its activity when a hydroxyl group is attached to C-17. Formation of an acetate restores the progestational activity, which is further enhanced by manipulations at C-6. Derivatives of hydroxyprogesterone acetate are potent progestagens as well as antiandrogens.

acetateorally active. This became the starting point for the synthesis of a number of potent pregnanes, made so mainly by modifications at C-6. Examples are MPA and acetates of megestrol, chlormadinone, and cyproterone. The last two are not available in the United States (see Fig. 17-10) . The pregnane group also includes the hybrid progestagens norpregnanes and drospirenone. They are discussed in a later section. Estranes

The estrane structure lacks the C-19 angular methyl radical between rings A and B. Removal of a radical from a certain position in the molecule is abbreviated in chemical shorthand as NOR, that is, "no radical." Removal of the C-19 methyl radical from the testosterone molecule changed this androgen into a progestagen; moreover, attachment of the ethinyl group (···CCH) to the 17th carbon made the compound more potent and orally active ( see Fig. 17-7 and Fig. 17-8 ) These chemical reactions led to the synthesis of norethindrone, the first orally highly active progestagen, which enabled the development of oral contraception. All estranes are 19-norsteroids. [41] Figure 17-11 shows the structural formulas of norethindrone and its four derivatives, which must be metabolically converted into norethindrone in order to become biologically active. [37] Gonanes

The gonane structure lacks both the C-18 and the C-19 angular methyl radicals. However, all gonane progestagens bear an ethyl group between rings C and D at C-13 ( Fig. 17-12 and Fig. 17-13 ; see Fig. 17-8 ). This chemical modification makes them more active progestational agents than estranes. Early Gonanes.

dl-Norgestrel and levonorgestrel are derivatives of 13-ethylgonane. Because they were developed in the 1960s, they are categorized as early gonanes. Only the

l-isomer of dl-norgestrel, levonorgestrel, is biologically active. As expected, levonorgestrel exhibited twice the potency of dl-norgestrel. The successful separation of levonorgestrel enabled the development of OC regimens with an extremely low amount of progestagen, and levonorgestrel soon replaced all dl-norgestrelcontaining OC preparations. dl-Norgestrel and levonorgestrel were synthesized in order to acquire a more potent progestagen than norethindrone, but they also increased the compound's androgenic properties with undesired clinical and metabolic effects. Thus, the next task of the steroid chemist was to produce highly active progestagens without the androgenic effects. The synthesis of the later gonanes met this challenge. Later Gonanes.

Three progestagens belong to this family, norgestimate, desogestrel, and gestodene (see Fig. 17-13) . Norgestimate.

The key difference between norgestimate and other progestagens is the oxime group (-C=N-OH) in position 3 of the molecule instead of the keto group (C=O). Norgestimate is solely the biologically active levo form. Because the C-3 keto group is typical of androgenic compounds, its replacement by the oxime may contribute to the reduced androgenicity of norgestimate as compared with levonorgestrel. Desogestrel.

This advanced gonane is interesting in that its progestagenic activity has been increased by substitution on C-11. Manipulations

677

Figure 17-11 Norethindrone (NET) and its derivatives. In order to become biologically active, the individual derivatives must be converted into NET.

Figure 17-12 Early gonanes: levonorgestrel and norgestrel.

at C-11 give the steroid molecule the ability to bind to the progesterone, glucocorticoid, and mineralocorticoid receptors. The intensity of the binding depends on the structure of the substituting groups. Gestodene.

Gestodene differs from norgestrel in a single feature, namely the double bond between C-15 and C-16. This seemingly simple change has a profound effect on the configuration of the molecule, principally on the spatial arrangement of the D ring and C-17. One can speculate that these changes affect the conformation of the molecule in a way that affects its binding to hormone receptors. In vitro studies have shown that gestodene binds to the heme of the P450 enzymes that inactivate estrogens, with consequent increased concentrations of these hormones. [42] The clinical relevance of this finding is unknown. Some Aspects of the Clinical Pharmacology of Contraceptive Progestagens

The orally active contraceptive progestagens are rapidly absorbed from the digestive tract and are transported to the liver

678

Figure 17-13 Progestagens of the advanced gonane group. Norgestimate and desogestrel are prohormones, metabolically converted into the active progestagenic substances, norelgestromin and etonogestrel, respectively. Gestodene is active without metabolic conversion.

through the portal circulation. Thereafter, they enter the general circulation, where they form a hormonal pool. In the general circulation, progestagens are present either in the free form or bound to albumins or to the sex hormonebinding globulin (SHBG), or both. Only the free form reaches receptors in the respective target tissues and becomes biologically active. Progestagens can easily be released from the steroid-albumin complex; the bond with SHBG is firmer. During first-pass liver metabolism, progestagens can be structurally modified with consequent changes of their biologic activity. Glucuronidation and sulfuration of progestagens facilitate their excretion by the kidneys. A variable fraction of progestagens is excreted in the feces. During first-pass liver metabolism, progestagens act on hepatocytes in ways that can alter their metabolism (see later). The pharmacologic properties of progestagens determine their clinical use. Progestagens of the pregnane series can bind strongly to progestagenic as well as to androgenic receptors and can act as antiestrogens. Oral MPA is used for treatment of various gynecologic disorders, for example, for the management of dysfunctional uterine bleeding, and in the menopause. The compound has been developed as the first injectable long-term contraceptive. In the past, medroxyprogesterone and other pregnane derivatives were proscribed for contraceptive use in the United States because preclinical toxicology had shown an accelerated development of benign and malignant breast nodules in beagle dogsa breed that suffers spontaneously from a high incidence of breast tumors, including carcinoma. Extensive clinical trials conducted by the WHO have lifted the cloud of potential carcinogenicity hanging over MPA, and it is used as an injectable contraceptive globally. [43] The accelerated growth of breast nodules in beagle dogs was not observed in toxicologic studies with progestagens of the estrane and gonane series. The reasons for this difference in animal carcinogenic potential of pregnanes versus estranes and gonanes have not been adequately elucidated. It is noteworthy that long-term toxicologic studies in monkeys have not shown the formation of any type of breast nodules. In monkeys, spontaneous breast carcinoma is unknown, and the induction

of hormone-associated breast pathology would have been particularly important. Of the other pregnanes, acetates of chlormadinone, cyproterone, and megestrol are used in OCs in some countries outside the United States. They are also used for management of breast, endometrial, and prostatic carcinomas. Because they are also highly effective antiandrogens, they have been part of the management of benign prostatic hypertrophy, prostatic carcinoma, precocious puberty, and certain hyperandrogenic symptoms in women. In the estrane series, the four derivatives of norethindrone are rapidly converted to norethindrone. Within 30 minutes after ingestion of any of the derivatives, only norethindrone can be detected in the general circulation. The earliest OCs contained norethindrone and norethynodrel, originally combined with up to 150 µg of ethinylestradiol. Today, OCs containing more than 50 µg of ethinylestradiol are considered "high-dose" estrogen preparations and have been removed from the OC market in the United States. "Mid-dose"

679

Figure 17-14 Hybrid progestagens: norpregnanes nomegestrol and nestorone, dienogest, and drospirenone. Drospirenone is a spironolactone derivative.

estrogen preparations contain 50 µg of ethinylestradiol, and "low-dose" preparations contain less than 50 µg of ethinylestradiol. Currently, the low-dose OCs are recommended; the mid-dose preparations are recommended only exceptionally. The early gonanes, norgestrel and levonorgestrel, display certain unique features. [44] [45] The time required for the circulating levels of levonorgestrel to decline by 50% is about 15 hours, and for norethindrone it is about 7 hours. The difference in the elimination time is one of the reasons that contraceptive doses of levonorgestrel can be lower than those of norethindrone. Norgestrel and levonorgestrel are strong progestagens with antiestrogenic properties; however, they also show some androgenicity. Levonorgestrel also decreases the plasma concentration of SHBG by 50% and, in combined OCs, suppresses the estrogen-induced formation of SHBG. Consequently, less SHBG is available for binding testosterone. A combined OC composed of 150 µg of levonorgestrel and 30 µg of ethinylestradiol increases the levels of SHBG only slightlyabout 20% from baseline. In contrast, women using norgestimate or desogestrel-ethinylestradiol OCs have a three-fold increase in circulating levels of SHBG, which results in a 50% decrease of free testosterone. Compounds of the late gonane series, norgestimate and desogestrel, are metabolized extensively (see Fig. 17-13) . After ingestion, norgestimate is rapidly converted into norelgestromin and desogestrel is converted into etonogestrel. These metabolites account for the biologic activity of the parent compounds. Norelgestromin has been synthesized as a specific hormone for the contraceptive patch, and etonogestrel is used in silastic implants for contraception. [46] Gestodene is the only compound of the gonane series that is not rapidly metabolized. It is a highly potent progestagen; however, at the time of this writing the compound has not been approved for clinical use in the United States. Hybrid Progestagens

19-Norpregnanes

The 19-norpregnanes are a cross between the pregnanes and estranes. These compounds are derived from 17- hydroxyprogesterone acetate but lack the C-19 methyl radical and in that respect are related to estranes (Fig. 17-14) . Nomegestrol, an important member of this series, is currently being investigated as a contraceptive implant.[47] Nestorone is another 19-norpregnane of the 17-acetoxyprogesterone series, which has a methylene group on C-16. In receptor assays, nestorone had progestational effects equal to or better than those of levonorgestrel without estrogenic, androgenic, and anabolic activities. [48] However, nestorone binds to glucocorticoid receptors. Nestorone has low oral but high parenteral progestational activity. Therefore, the Population Council is studying nestorone as a contraceptive in subdermal implants, vaginal rings, and transdermal formulations. The compound may be suitable for nursing mothers because of its low oral bioavailability. Other Hybrid Progestagens

Dienogest is a new addition to the estranes. In this compound, the cyanomethyl group (···CH 2 CN) has replaced the

680

C-17 ethinyl group (···CCH) (see Fig. 17-14) . Dienogest is 100% orally available. Some evidence suggests that the compound lacks androgenic activity and produces less glucocorticoid antagonism than mifepristone. The compound suppresses endometrial growth and is being tested in the management of endometriosis. The antigonadotropic activity of dienogest is relatively low, mandating the use of 2 mg in combination with 30 µg of ethinylestradiol in a 21-day cyclic regimen. [49] Drospirenone.

An addition to contraceptive progestagens has been drospirenone. It is a progestagen derived from spironolactone, a potent steroid with antimineralocorticoid activity, which also has progestagenic properties. Drospirenone is a relatively weak progestagen; a daily dose for a contraceptive regimen is 3 mg combined with 30 µg of ethinylestradiol. The compound has antiandrogenic and antimineralocorticoid properties and causes potassium retention. Therefore, it should not be taken by patients with kidney, liver, or adrenal gland disease or with other drugs that increase potassium concentrations in the circulation. Such drugs include nonsteroidal anti-inflammatory drugs, potassium-sparing diuretics (spironolactone and others), potassium supplementation, angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists, and heparin. [50] Progestagenic Potency, Androgenicity, and Comparative Metabolic Effects of Oral Contraception

Multiple Steroid Actions

Significant determinants of the biologic activity of a steroid include its bioavailability to the target tissues and its affinity for relevant receptors. The multifaceted nature of the steroid molecule is illustrated by its capacity to bind to several different receptors and activate them to various degrees. [51] [52] The final biologic activity depends on the proportion of activated receptorsprogestagenic, estrogenic, androgenic, and glucocorticoidin the target tissue. Most contraceptive progestagens, besides binding strongly to progesterone receptors, also bind to a lesser extent to androgen receptors. Moreover, some progestagens bind to glucocorticoid receptors. Progestagenic and Androgenic Activity

It is difficult to establish the relative progestagenic and androgenic potency of progestagens because systematic testing of all progestagens by one method has not been performed. Different progestagens have been tested by different methods. The relative binding affinities of progesterone and gonanes for rabbit uterine

progestagen receptors are shown in Figure 17-15 . The binding affinity of norgestimate and its 17-deacetylated metabolite (norelgestromin) was similar to that of progesterone; levonorgestrel was about five times and gestodene and 3-ketodesogestrel were about nine times more active than progesterone. The relative binding activities of progesterone and gonane progestagens for rat prostatic androgen receptors are depicted in Figure 17-16 . Norgestimate and norelgestromin, which have the same progestagenic activity as progesterone, display low androgenic activity. Levonorgestrel, a 5 times more potent progestagen than progesterone, has 44 times higher androgenic activity than progesterone. Although 3-ketodesogestrel and gestodene are more active progestagens than levonorgestrel, they have less androgenic activity than levonorgestrel. In this assay, the androgenic activity of dihydrotestosterone is 200-fold greater than that of progesterone (not shown in Fig. 17-16 ). The androgenic activity of levonorgestrel is one fourth the activity of dihydrotestosterone. Biologic tests of androgenicity were conducted on castrated rats, the end point being the weight increase of the prostatic gland. In this assay, the androgenic potency of testosterone equals 100%; that of levonorgestrel is 15% and that of norethindrone is only 1.6%. Medroxyprogesterone and chlormadinone acetates displayed no androgenic action in this test. [53] NonReceptor-Mediated Action

In skin and some other tissues, testosterone is a prohormone and becomes biologically active only after conversion into dihydrotestosterone by 5-reductase. In vitro experiments have demonstrated that norgestimate and desogestrel inhibit the action of 5-reductase. [54] This may partially explain the beneficial effects of these progestagens in the management of androgenic skin lesions such as acne. Metabolic Actions of Progestagens

Lipid Metabolism

Important differences exist among progestagens in their effect on lipid metabolism, particularly on the cardioprotective lipoproteins. Estrogens increase total cholesterol, but they also increase the high-density lipoprotein (HDL) fraction and decrease the low-density lipoprotein (LDL) fraction of cholesterol. Progestagens exert an antagonistic effect on these positive actions of estrogens by various mechanisms, including an increase in the activity of hepatic lipase, which degrades HDL. There are quantitative differences among individual progestagens, however, and the net metabolic effect depends on an intricate interplay between the two components of the combined contraceptives, the type and dose of the progestagen, and the treatment regimen, whether it is monophasic or triphasic. In general, the higher the androgenic properties of a progestagen, the more pronounced are the negative effects on cardioprotective lipoproteins. Because norethindrone and estrane progestagens are derived from testosterone, and testosterone is part of the chemical name of the compounds, it is sometimes assumed that norethindrone and its analogues have androgenic properties. In doses and combinations used in current clinical practice, norethindrone and its analogues do not exhibit substantial clinical or metabolic androgenic effects. A monophasic combination OC (0.5 µg of norethindrone plus 35 µg of ethinylestradiol per day) has been associated with a 10% increase of HDL and a 10% decrease of LDL. [55] With respect to gonanes, levonorgestrel increases LDL only slightly but reduces HDL significantly. Norgestimate, a later gonane, significantly elevates HDL with a nonsignificant effect on LDL (Fig. 17-17) .[55] [56] Protein Binding

Natural and synthetic sex steroid hormones enter the target cells by passive diffusion. The capacity of sex steroids to reach receptors in these target cells is modulated by SHBG and other proteins, such as albumins. Natural sex steroids and some synthetic progestagens bind to SHBG with higher affinity and specificity than they bind to albumin. [57] [58] As long as a sex steroid is bound to SHBG, it cannot affect its biologic action, which is accomplished by the free or nonSHBG-bound fraction of the steroid. The binding of sex steroids to albumin is less tight, and albumin-bound steroids are more readily available to target cells, ensuring a rapid pharmacologic response. Estrogens stimulate hepatocytes to produce SHBG, and androgens and some progestagens interfere with this action. Depending on their composition, OCs are associated with increased formation of SHBG and reduced levels of free

681

Figure 17-15 Relative binding affinities of contraceptive progestagens for progesterone receptors. The assay measures displacement of 3 H-labeled R5020 from progestagen receptors isolated from the rabbit uterus. The [ 3 H]R5020 is a radiolabeled synthetic progestagen used in in vitro studies. (Data from Phillips A, Demarest K, Hahn DW, et al. Progestational and androgenic receptor binding affinities and in vivo activities of norgestimate and other progestins. Contraception 1990; 41:399.)

testosterone. For example, OCs with desogestrel and norgestimate do not interfere with the estrogen-stimulated formation of SHBG, and free testosterone levels are reduced. These changes are thought to confer clinical benefits in certain hyperandrogenic conditions in women. [59] Progestagens may also compete for SHBG binding sites and indirectly increase the concentration of free estradiol and testosterone in plasma. [60] The binding affinity of norgestrel for SHBG is relatively high. Desogestrel binding to SHBG is much less tight, and the affinity of norgestimate and its main metabolite, norelgestromin, for SHBG is practically nil. Insulin and Carbohydrate Metabolism

The original high-dose OCs were associated with insulin resistance and glucose intolerance. Glucose tolerance tests showed a significant increase of blood glucose and insulin after 1 year of OC use. [61] It was initially thought that these changes in glucose tolerance were related to the estrogenic component of the pill. However, after tests of estrogens alone, even in

Figure 17-16 Relative binding affinity of contraceptive progestagens for androgen receptors. The assay measures displacement of 3 H-labeled dihydrotestosterone from rat prostatic androgen receptors. (Data from Phillips A, Demarest K, Hahn DW, et al. Progestational and androgenic receptor binding affinities and in vivo activities of norgestimate and other progestins. Contraception 1990; 41:399.)

high doses, demonstrated no such negative effects, it was found that high doses of many progestagens can impair carbohydrate metabolism. With the advent of low-dose OCs, the effect of contraceptive hormones on carbohydrate metabolism has been minimized. [55] [56] [61] [62] [63] [64] Mechanism of Contraceptive Action

Originally, it was assumed that the contraceptive action of combined steroid hormones primarily involved inhibition of the pituitary gonadotropins with consequent blocking of ovulation. This is certainly the case with the combined estrogen-progestagen OC. [65] However, blood levels of progesterone and pituitary gonadotropins indicated that during progestagen-only contraception, ovulatory function has been preserved during many cycles. Therefore, other mechanisms of contraceptive action were explored. Among these were changes in the consistency and increases in the thickness of the cervical mucus that impair the ability of the sperm to penetrate it. Low doses

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Figure 17-17 Contrasting effects of oral contraceptives containing levonorgestrel (LNG) and norgestimate (NGM) on high-density and low-density lipoproteins (HDL and LDL). (Modified from Henzl M. Norgestimate: from the laboratory to three clinical indications. J Reprod Med 2001; 46:647661.)

of progestagens also alter the function of the endometrium, leading to an intrauterine milieu hostile to pregnancy.

[66]

Oral Contraceptive Treatment Regimens

Combined Oral Contraception: Cyclic Estrogen-Progestagen Combinations

In this method both hormonal components are given in a cyclic fashion, usually from the 5th through the 25th day of the menstrual cycle. With most of the currently used preparations, a 7-day placebo period follows the 21-day hormonal treatment so that women do not need to keep track of when to start a new cycle of contraceptive pills. There is growing interest in developing combined oral contraception (COC) for continuous use to avoid monthly withdrawal bleeding. Since the inception of COC, the developmental trend aimed at reduction of the amounts of hormonal components in the combination to a level that would make the pill safer and still provide high contraceptive protection and cycle control. The current COC preparations can be classified according to the amount of ethinylestradiol in the daily dose: (1) 20 µg of estrogen, the lowest dose used in the United States; (2) more than 20 µg to less than 50 µg, the dose most frequently recommended today; and (3) 50 µg, a dose that is rarely recommended. Preparations with an estrogen content greater than 50 µg/day have been removed from the market in the United States. A further distinction is made according to whether the dosage regimen is monophasic, biphasic, or triphasic. The monophasic dosage regimens consist of contraceptive steroids given in a fixed estrogen-progestagen combination from the 1st through the 21st day of treatment. With the biphasic preparations, the daily dose of ethinylestradiol is usually constant throughout the entire 21 days of use but the initially low progestagen dose increases at the middle of the cycle, from day 11 on. A special case of biphasic dosage is a preparation (Mircette) employing a constant dose of 20 µg of ethinylestradiol combined with 150 µg of desogestrel given from day 1 through 21. This is followed by 2 days of placebo and then 5 days of ethinylestradiol at 10 µg/day. In the triphasic treatment regimens, the daily doses of one or both steroidal components are modified three times during the treatment period. The individual dosage regimens for OC are listed in Table 17-6 . The development of the biphasic and triphasic treatment regimens was motivated by the desire to mimic the hormonal events of the normal menstrual cycle and to decrease the total load of contraceptive steroids per month. These dosage modifications have contributed to the variety of OC choices. Rigorous comparative studies have not been conducted to show whether the phasic regimens offer any clinically meaningful advantages over the monophasic schedules. However, similar performance at lower hormonal doses has theoretical appeal. The Sequential Method

In this method, estrogens were given in a fixed dose throughout the 21-day treatment period; however, progestagens were given only during the last 5 to 9 days. The use of this regimen was discontinued in the United States in the 1970s because of concerns that repeated exposure of the endometrium to unopposed estrogens may induce atypical hyperplasia. The method is mentioned here because it is described in earlier literature. Continuous Progestagen-Only Oral Contraception

The continuous progestagen-only method, known as the minipill, was developed to eliminate the estrogens entirely from OC preparations. The preparations currently available in the United States contain norethindrone, 350 µg/day; norgestrel, 75 µg/day; and levonorgestrel, 30 µg/day. Outside the United States, preparations with other progestagens are available: lynestrenol, 500 µg/day; ethynodiol diacetate, 500 µg/day; and desogestrel, 75 µg/day. The pregnancy rate for a typical user is higher than with the estrogen-progestagen OC, and the cycle control is less satisfactory. The method is well suited for breast-feeding mothers. The amounts of progestagen that are excreted into the milk of breast-feeding mothers are negligibly low and do not affect the quantity and the composition of the milk. The combination of the ovulation-suppressive effect of prolactin and the contraceptive effects of the progestagen-only pill offers excellent protection from pregnancy. Another group of women who could benefit from this method are women around the age of 40 with naturally decreased fecundity. Good candidates for progestagen-only contraception are women who do not tolerate estrogens and who reject the use of an IUD. Some preparations using the continuous progestagen-only method (minipill) have been associated with ectopic pregnancy. For this reason, when pregnancy occurs, all efforts must be made to rule out ectopic pregnancy. The physician must be attentive to complaints of pelvic pain by users of the progestin-only method; sometimes the diagnosis of ectopic pregnancy is delayed because symptoms of ectopic pregnancy such as irregular uterine bleeding, prolonged cycles, and amenorrhea resemble typical side effects. The decreased efficacy of the minipill compared with combined OCs is probably due to its mechanism of action. The minipill does not consistently inhibit ovulation (about 40% of cycles are ovulatory); thus, other mechanisms become operative. Prevention of fertilization is largely due to changes in viscosity of the cervical mucus in addition to other changes that are inhospitable to impregnation of the ovum. Benefits of Oral Contraception

Physicians, patients, and the general public have been made well aware of adverse effects of hormonal contraception. Data obtained over the last two decades brought evidence that the

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TABLE 17-6 -- Oral Contraceptives Used in the United States Manufacturer Brand Name Number/Doses Product Type

Progestagen

Estrogen* (µg)

Berlex Levlen

Combination

Tri-Levlen

Combination, triphasic

6

0.15 mg levonorgestrel

30


30

5


40

10


30

Combination

3.0 mg drospirenone

30

Ovcon-35

Combination

0.4 mg norethindrone

35

Ovcon-50

Combination


50

Desogen

Combination

0.15 mg desogestrel

35

Mircette

Biphasic, special combination 0.15 mg desogestrel

20

Yasmin Bristol-Myers Squibb

Organon

21 5 Cyclessa

10 Combination, triphasic

7

0.1 mg desogestrel

25

7

0.125 mg desogestrel

25

7

0.15 mg desogestrel

25

Ortho-MacNeil Pharmaceutical Micronor

Progestagen-only


Modicon

Combination


35

Ortho-Cept

Combination

0.15 mg desogestrel

30

Ortho-Cyclen

Combination

0.25 mg norgestimate

35

Ortho-Novum 1/35

Combination


35

Ortho-Novum 1/50

Combination


50

Ortho-Novum


7


35

7


35

7


35

10


35

11


35

7


35

7


35

7


35

5

1.0 mg norethindrone acetate

20

7


30

9


35

Ortho-Novum

Ortho-Tricyclen

Combination, biphasic


Parke-Davis Estrostep


Loestrin 1/20

Combination


20

Loestrin 1.5/30

Combination


30

Norlestrin 1/50

Combination


50

Norlestrin 2.5/50

Combination


50

Brevicon

Combination


35

Norinyl 1 + 35

Combination


35

Norinyl 1 + 50

Combination


50

Nor-Q.D.

Progestagen-only


Tri-Norinyl


Watson Laboratories

7


35

9


35

5


35

Searle Demulen 1/35

Combination

1.0 mg ethynodiol diacetate

35

Demulen 1/50

Combination

1.0 mg ethynodiol diacetate

50

Alesse

Combination


20

Lo/Ovral

Combination


30

Nordette

Combination


30

Ovral

Combination

0.500 mg norgestrel

50

Ovrette

Progestagen-only

0.075 mg norgestrel

Triphasil


Wyeth-Ayerst

6


30

5


40

10


30

Adapted and updated from Mishell DR. Contraception. In Yen SSC, Jaffe RB, Barbieri RT (eds). Reproductive Endocrinology, 4th ed. Philadelphia, WB Saunders, 1999, pp 676708. *Ethinylestradiol unless noted otherwise. Mestranol.

use of hormonal contraception has also been associated with numerous and not insignificant beneficial effects in addition to contraception. These noncontraceptive benefits can be conveniently divided into those affecting the reproductive tract and nonreproductive benefits (Table 17-7) . Noncontraceptive Reproductive Benefits of Oral Contraception

Endometrial and Ovarian Carcinoma

The most impressive noncontraceptive benefit of hormonal contraception is the decreased incidence of endometrial and ovarian carcinomas. In the United States, endometrial carcinoma is the most common pelvic cancer in women. Current estimates indicate that 36,000 new cases are diagnosed yearly with a death rate of 6500. The 5-year survival rate is 83% when it is diagnosed early; more advanced cases have a survival rate of 65%. For the most part endometrial carcinoma affects TABLE 17-7 -- Noncontraceptive Health Benefits of Hormonal Contraception Condition Relative Risk (Risk for Nonusers = 1) A. Reduced Risk of Morbidity Endometrial carcinoma Years of use 1

0.8

2

0.6

4

0.4

Overall

0.5

Ovarian carcinoma* Years of use >3

0.5

7

0.20.4

Overall

0.3

Ovarian cysts

0.4

Pelvic inflammatory disease

0.1

Ectopic pregnancy

0.1

Benign breast tumors

0.5

B. Reduced Risk and Improvement of Quality of Life Dysmenorrhea

0.4

Menorrhagia

0.5

Anemia

0.6

Premenstrual syndrome

0.7

Irregular menses

0.7

*Residual protective effect lasts for 10 to 15 years after termination of use.

women past reproductive age, although younger women are not immune to the disease. The decreased risk is related to the length of use of OCs. A decrease is evident after only 1 year of use; women who have been practicing hormonal contraception for 4 years or more have risk reduced by more than 50%. It is important to note that hormonal contraception offers long-term protection and that the residual protective effects persist for 20 years or longer. It is also important that the protective effect has been associated with the use of all OCs for which data have been gathered. Data on progestagen-only contraception and preparations with 20 µg of ethinylestradiol are not yet available, but it is likely that they would offer the same protection as the other preparations. The mechanism of the protective effect on the endometrium most likely involves the direct antiestrogenic effects of the progestagen component of the OCs. Estrogens stimulate synthesis of both estrogen and progestagen receptors, and progestagens inhibit this synthesis. Part of the antiestrogenic effect of progestagens may also involve the stimulation of estradiol 17-hydrolases in the endometrial cell and accelerated conversion of estradiol to estrone, a less potent estrogen than estradiol. [67] Consequently, the proliferation of the endometrial epithelium, both endometrial glands and stroma, proceeds at a reduced rate with less mitotic activity. With an estimated 26,700 new cases per year, ovarian carcinoma occurs less frequently than endometrial carcinoma; however, it is more deadly. With 14,800 deaths per year, ovarian cancer is the fourth leading cause of death from cancer, after lung, breast, and colon and rectal carcinomas. The 5-year survival rate is less than 45% after the diagnosis has been established. The protective effect of hormonal contraception is evident with only 3 to 6 months of use. It becomes highly significant after 7 years of use (relative risk 0.2 to 0.4), and the residual protective effect extends at least 10 to 15 years after termination of use. [68] Hormonal contraception reduces the risk of functional ovarian cysts by 60%. This can be important because functional ovarian cysts are the fourth leading reason for hospitalization of women in the United States, with 160,000 admissions per year. We can speculate that the decreased relative risk of both ovarian carcinoma and functional ovarian cysts is probably due to inhibition of monthly proliferation of the graafian follicles. [69] Ovarian suppression associated with low-dose OCs is less pronounced than with higher dose OCs. [70] Each year, more than 1 million women in the United States experience an episode of pelvic inflammatory disease (PID). Epidemiologic data suggest that women taking OCs have a reduced risk of being hospitalized for PID, but further work is needed to assess the effects of OCs on the incidence of PID. [71] [72] With respect to ectopic pregnancy, studies from the early

685

1990s showed that the incidence rate of ectopic pregnancy per 1000 woman-years is 3.0 for women who do not practice contraception at all. The incidence rate is reduced to 0.005 in women using combination OCs. In women using a Cu-T IUD or the levonorgestrel-containing implant Norplant and in women who have undergone tubal sterilization, the incidence rate is still low (0.2 to 0.3). [73] [74] Uterine Leiomyomas

High doses of norethindrone only, or norethindrone given simultaneously with gonadotropin-releasing hormone (GnRH) agonists, can achieve reduction of the size of leiomyomas. Because leiomyoma is the most frequently encountered tumor of the female genital tract, there has been great interest in determining whether OCs protect against the occurrence of this tumor. The question has been addressed by several studies that provided opposing results, and the problem remains unresolved. At least, there does not appear to be an increased risk. [75] Endometriosis

OCs have been recommended for mild forms of endometriosis. However, well-designed studies proving a substantive effect are lacking. OCs may protect against the occurrence of endometriosis, [76] and some clinicians use OCs as a follow-up for the prevention of recurrence of endometriosis after a completed course of GnRH agonists. Other Reproductive Noncontraceptive Benefits

Table 17-7 also shows the beneficial effect of OCs on conditions that are not always serious but negatively affect the quality of life. Such conditions include dysmenorrhea, mittelschmerz, menorrhagia and irregular menses, premenstrual syndrome, and iron deficiency anemia. The beneficial effects are achieved by suppression of ovulation and by influencing the endometrium. Nonreproductive Benefits of Oral Contraception

Management of Hyperandrogenism

OCs have been used in the management of hyperandrogenic conditions such as acne, seborrhea, and hirsutism. Triphasic norgestimate-ethinylestradiol regimens and three other OC combinations of ethinylestradiol with norethindrone acetate, levonorgestrel, or desogestrel [77] [78] [79] [80] have been shown in randomized, blinded, placebo-controlled trials to be effective in the treatment of acne. The efficacy of other OCs has also been supported by studies conducted under less rigorous protocols.[81] [82] For example, a single-blind randomized study demonstrated alleviation of acne by treatment with a pregnane type of progestagen, chlormadinone acetate, combined with ethinylestradiol. [83] Several modes of action have been considered to explain the efficacy of OCs in hyperandrogenism. OCs can suppress production of ovarian androgens by inhibiting pituitary gonadotropins. An increase of circulating levels of SHBG is associated with a decrease of bioavailable testosterone, and inhibition of 5-reductase in the skin tissues can also contribute to this antiandrogenic effect. Medical treatment of hirsutism with OCs is more difficult. In addition to a number of supportive case series reports, two well-controlled clinical trials using OCs with or without GnRH agonists have been reported. [84] [85] The first one employed a randomized, double-blind, placebo-controlled study design. Neither the OC Norinyl 1/35 (1 mg of norethindrone plus 35 µg of ethinylestradiol) nor placebo had a beneficial effect on hirsutism. In the second study, which was investigator-blind but not placebo-controlled, the contraceptive Demulen (1 mg of ethynodiol diacetate plus 35 µg of ethinylestradiol) had no effect on hirsutism. However, OC complements GnRH agonist analogues in the management of hirsutism. In these two studies, only the combination of OC and a GnRH agonist had a clinically and statistically significant beneficial effect on hirsutism. Bone Mineral Density

Well-designed studies demonstrate that the use of OCs increases bone mineral density so that users enter menopause with higher bone mass than nonusers, by 12% on the average. This beneficial effect depends on the duration of OC use; the greatest protection is afforded to women who use OCs for 10 years or more. The ultimate question, whether previous OC users suffer fewer bone fractures during menopause than nonusers, has been addressed by a large case-control study. The results have shown a 25% reduction in hip fractures in previous users of OCs. [86] [87] Rheumatoid Arthritis

The relationship between OC use and rheumatoid arthritis has been of interest in countries where this disease affects larger segments of the population. A case-control study from Holland reported 60% protection in ever-users of OCs. [88] Other studies and meta-analyses of various clinical trials have not provided an unequivocal conclusion. [89] Colorectal Cancer

Several studies employing epidemiologic methodology have provided evidence that OCs afford about 50% protection from colorectal cancer and that this effect is directly proportional to the duration of OC use. The subject remains controversial because other clinical observations failed to confirm the protective effect. The mechanism by which OCs would exert a protective effect against colorectal cancer is not clear. [90] [91] Adverse Events Associated with the Use of Oral Contraception

The frequency of adverse events associated with oral contraception has decreased continuously since hormonal contraception was first introduced for general clinical use. There are several reasons for this favorable development. The amount of estrogens in the pill has been gradually reduced since estrogens were identified as the culprit in the most severe adverse events, principally cardiovascular and cerebrovascular complications. This reduction was paralleled by decreases in the daily doses of the progestagenic component of OC. In addition, candidates for OCs are being selected more carefully as risk factors for potential complications have been identified, notably smoking among women older than 35. Researchers realized that more frequent follow-up of new OC users could detect early signs and symptoms of complications. For example, measurements of blood pressure before therapy and during the first 3 months of OC use identify individuals predisposed to hypertension. Finally, physicians defined appropriate contraceptive options for patients with medical problems. Most Commonly Reported Adverse Events

The use of hormonal contraception is associated with a number of less serious adverse events that are nonetheless important because they constitute reasons for discontinuation.

686

Breakthrough Bleeding.

This event can be expected in 10% to 30% of women during the first 3 months of use of hormonal contraception; thereafter, it is much less frequent. Depending on its intensity and duration, it can be handled by reassuring the patient or by short-term administration of a supplementary estrogen such as micronized estradiol.

Amenorrhea.

Amenorrhea, that is, lack of bleeding during the active pill-free period, is always a cause for anxiety because of the possibility of pregnancy. Pregnancy should be ruled out by a sensitive urine or blood pregnancy test. If pregnancy is ruled out and the patient is comfortable with amenorrhea, she may resume the same OC. Alternatively, she may be switched to another OC, usually with more estrogen dominance. Sometimes supplementation by estrogens is recommended. Repeated episodes of amenorrhea can be bothersome and irritating, and it is sometimes best to recommend another method of contraception. A positive pregnancy test should alert the physician to the unlikely but critical possibility of an ectopic pregnancy. Other Adverse Effects.

One study compared two groups of patients, randomly assigned to receive either a tricyclic combination of ethinylestradiol and norgestimate (Ortho Tri-Cyclen) or a placebo. Symptoms that are usually attributed to the use of OCs, such as common headaches, nausea, breast tension and tenderness, weight gain, and mood change, were assessed before and during treatment. The difference in the incidence of side effects between the two groups was not statistically significant. Only the higher incidence of breast tenderness and mood change in the OC group approached significance ( P = .07).[92] Women who discontinue OC use conceive later than women practicing nonhormonal methods of contraception. "Postpill amenorrhea" develops in 1% of users. Caution.

Despite the reassuring reports with respect to adverse events, the prescribing physician must be aware that certain conditions warrant caution or constitute a frank contraindication to hormonal contraception. These conditions are discussed in the sections "Contraindications" and "Contraception for Women with Health Problems." Cardiovascular and Cerebrovascular Adverse Events

From the inception of their clinical use in the 1960s, OCs have been associated with cardiovascular and cerebrovascular complications, namely idiopathic venous thromboembolism (VTE), stroke, and myocardial infarction (MI). These adverse events merit reevaluation in the light of epidemiologic studies that were prompted by the introduction of low-dose OCs into clinical practice. [93] Venous Thromboembolism

The Role of Estrogens.

Since the first reports of VTE in OC users, clinicians suspected that the noxious agent is the estrogenic component of the combination pill. A dose-response relationship between the estrogen dose and VTE was demonstrated in an epidemiologic study of 234,218 women between 1980 and 1986, when both high-dose and low-dose estrogen pills were being prescribed. The highest incidence of VTE, 10 per 10,000 woman-years, occurred among women who used OCs with an ethinylestradiol content of more than 50 µg/day. With preparations containing the medium dose of ethinylestradiol, 50 µg/day, the incidence of VTE decreased to 7 per 10,000 woman-years; with pills having an ethinylestradiol content less than 50 µg/day, the rate of VTE decreased to 4.2 events per 10,000 woman-years. The difference between the TABLE 17-8 -- Relative Risk of Idiopathic Venous Thromboembolism in Pregnancy and during the Use of Contraceptive Hormones Population Relative Risk Estimates 95% Confidence Interval Unexposed

1

Emergency contraception*

0

Hormone replacement therapy

2.3

0.415.0

Progestagen-only contraception

2.4

0.86.5

Levonorgestrel-containing oral contraceptives (second generation)

3.4

0.813.7

Third generation oral contraceptives (gestodene, desogestrel)

8.0

2.129.9

Progestogens for menstrual disorders

5.3

1.518.7

Pregnant, postpartum

12.3

4.636.4

Modified from Vasilakis C, Jick H, del Mar Melero-Montez M. Risk of idiopathic venous thromboembolism in users of progestagens alone. Lancet 1999; 354: 16101611. *Vasilakis C, Jick SS, Jick H. The risk of venous thromboembolism in users of post-coital contraceptive pills. Contraception. 1999; 59:7983.

incidence of VTE at 50 µg/day ethinylestradiol and the incidence at less than 50 µg/day ethinylestradiol was statistically significant.

[93]

A subsequent study analyzed idiopathic VTE in a cohort of 74,086 women during the 5-year period from 1993 to 1997. [94] In this epidemiologic study, the relative risk of VTE was defined for pregnancy, various forms of oral contraception including combination OC and the progestagen-only method, therapeutic use of progestagens, and hormonal replacement therapy. The results are summarized in Table 17-8 , to which we have added an analysis of the relative risk of VTE associated with emergency contraception. [95] The highest risk of VTE is associated with pregnancy13 times higher than in nonpregnant women. Emergency contraception is not associated with a substantive risk. There is a slight but nonsignificant association between progestagen-only contraception and VTE, the relative risk being 2.4 with a 95% confidence interval (CI) of 0.8 to 6.5. A similar association has been reported for women using hormonal replacement therapy, the relative risk being 2.3 (CI 0.4 to 15.0). With the therapeutic use of progestagens for menstrual disorders, the relative risk for development of VTE rose to 5.3 (CI 1.5 to 18.7). The daily doses in progestagen-only contraception are normally less than 0.5 mg, whereas the therapeutic doses range from 5.0 to 30.0 mg/day. An ongoing controversy involves reports that the risk of VTE associated with combination OCs containing desogestrel and gestodene is more than twice that for OCs with norgestrel and norethindrone (3.4 versus 8.0). This finding merits a discussion of the role of progestagens in the genesis of VTE. The Role of Progestagens.

In 1995, several independent clinical epidemiologic studies presented evidence that OCs containing desogestrel and gestodene are associated with double the risk of nonfatal VTE compared with earlier OCs containing norethindrone or levonorgestrel. [96] [97] [98] The results were surprising because thrombotic phenomena have not been conventionally associated with the progestagenic component of OCs. The data were immediately questioned, and the relationship of the various progestagens to VTE became controversial. However, a study analyzing OC use for the 7-year period between 1993 and 1999 confirmed that the risk for development of

687

VTE of women using OCs containing desogestrel and gestodene is twice that of women using OCs with levonorgestrel. [99] The controversy concerning the third-generation pills containing gestodene and desogestrel continued into 2001, when a meta-analysis established a 1.7-fold risk for third-generation versus second-generation pills. [100] Norgestimate has not been included in these analyses. An analysis based on postmarketing surveillance of adverse events associated with the use of COCs

containing desogestrel and norgestimate showed a threefold to fourfold excess of reported cases of deep vein thrombosis in desogestrel users compared with norgestimate users. Both compounds were launched at approximately the same time, but the estimated number of distributed pill cycles was markedly higher for norgestimate.[101] It is important to note that all low-dose OCs are safer than older high-dose products and that all current products are much safer than pregnancy vis-à-vis VTE. Venous Thromboembolism and Coagulation Factors.

Studies of the effects of hormonal contraceptive agents on blood coagulation factors reveal that OCs increase the synthesis of globulins in the liver, including many clotting factors. Consequently, circulating concentrations of many clotting factors are affected but not to a clinically significant level. Most frequently affected are fibrinogen and factors dependent on vitamin K (prothrombin and factors VII, IX, and X) and factor XII. At the same time, a decrease in the levels of antithrombin III, an anticoagulation factor, was noted. Despite the fact that these changes had occurred in virtually all OC users tested, VTE remains a rare event. In discussing these findings, several issues have to be taken into account. Even under physiologic conditions, concentrations of the clotting factors are excessive in the circulation of healthy women, in some cases reaching 200% of the "normal" values. For hemostasis, however, only a fraction of this activity is needed. The coagulation factors are proenzymes that are present in the circulation in their inactive form. Damage to the blood vessel must occur to activate the coagulation cascade. With respect to antithrombin III, the OC-induced decrease is about 10%, far short of the profound reduction needed to form a clot. [102] Changes in blood coagulation depend on the dose of estrogen. The decrease of the ethinylestradiol content to below 50 µg/day, common in current OC preparations, has considerably limited the changes in blood coagulation factors that were observed in OCs with a higher ethinylestradiol content. [103] Venous Thromboembolism and Leiden Factor V.

During the normal coagulation process, protein C and its cofactor S prevent hypercoagulation by inhibiting the activity of coagulation factors V and VII. The Leiden mutation, a genetic mutation of factor V consisting of an alteration of a single amino acid, makes factor V resistant to the action of protein C. The Leiden mutation of factor V occurs in 5% of the U.S. white population and is less frequent in black and Hispanic women. Its presence predisposes the carrier to VTE (Table 17-9) . In women of reproductive age, the rate of VTE increases to 5.7 VTE events per 10,000 woman-years, and in OC users the increase amounts to 28.5 events per 10,000 woman-years. The identification of the Leiden factor V mutation is the first instance in which increased VTE events in OC users could be linked to a concrete defect in a coagulation cascade. However, screening for the Leiden mutation would be impractical because examination of 1 million potential OC users would detect only 50 women at risk. In addition, 62,000 women would have false-positive results. [104] [105] Physicians generally consider a personal history of venous thrombosis an absolute contraindication to the use of OCs. Screening TABLE 17-9 -- Oral Contraception and Factor V Leiden: Risk of Idiopathic Venous Thromboembolism Population Relative Risk Incidence per 10,000 Woman-Years Controls

1

0.8

Oral contraception only

3.8

3.0

Factor V Leiden mutation

7.9

5.7

Factor V Leiden mutationoral contraceptive users

34.7

28.5

Modified from Vandenbroucke JP, Koster T, Briet E, et al. Increased risk of venous thrombosis in oral-contraceptive users who are carriers of factor V Leiden mutation. Lancet 1994; 344:14531457; Vandenbroucke JP, van der Meer FJ, Helmerhorst FM, Rosendaal FR. Factor V Leiden: should we screen oral contraceptive users and pregnant women? BMJ 1996; 313:11271130. for factor V Leiden may be justified in women with a strong family history of venous thrombosis. The presence of superficial varicose veins that are not a consequence of previous venous thrombosis is not a contraindication to the use of oral contraception. [106] In conclusion, although significant strides have been made in accumulating knowledge about VTE and OCs, the phenomenon remains as enigmatic as before. Reducing the dose of ethinylestradiol in the combination pill to less than 50 µg/day, along with other preventive measures, has substantially lowered the risk of VTE although it has not been eliminated entirely. There are no substantive data supporting increased safety for products containing 20 µg versus 30 to 35 µg despite the logical appeal. In-depth molecular biologic research is needed to understand VTE and pave the road to its rational prevention. Stroke and Oral Contraception

Stroke has been recognized as one of the serious complications of OC use, although its incidence has been rare. In 1976, Vessey and Doll [107] reported 41 to 45 strokes per 100,000 woman-years in OC users, a fourfold to fivefold increase over the rate of stroke in nonusers. Later studies had more reassuring outcomes. In 1996, a large epidemiologic study demonstrated that women using OCs with a low estrogen content (90%), approximately half of the daily turnover being recovered as urinary 17-ketosteroids and the other half as a series of polar compounds, including hydroxylated metabolites and conjugates. [113] These various excretory metabolites are thought to be largely inactive. Androgen Action

Current concepts of androgen action in target cells are summarized in Figure 18-10 . Major androgen functions include regulation of gonadotropin secretion by the hypothalamic-pituitary

717

Figure 18-9 Metabolism of plasma testosterone in extraglandular tissues. Testosterone can be metabolized to either active or excretory metabolites. Active metabolites such as dihydrotestosterone may be further metabolized to excretory metabolites. HSD, hydroxysteroid dehydrogenase. (From Griffin JE, Wilson JD. The testis. In Bondy PK, Rosenberg LE [eds]. Metabolic Control and Disease, 8th ed. Philadelphia, WB Saunders, 1980, pp 15351578.)

system, initiation and maintenance of spermatogenesis, formation of the male phenotype during sexual differentiation, promotion of sexual maturation at puberty, and control of sexual drive and potentia in men (see Chapter 24) . Testosterone is believed to enter cells by passive diffusion. Inside cells that express 5-reductase, testosterone can be converted to dihydrotestosterone. Testosterone and dihydrotestosterone bind to the same high-affinity androgen receptor, and the hormone-receptor complexes attach to hormone response elements in deoxyribonucleic acid (DNA) to initiate biologic responses. ( See Chapter 5 for a discussion of the ancillary proteins involved in the regulation of transcription by the androgen-receptor complex.) Few of the presumed large number of acceptor sites in DNA have been defined, and the factors that determine the specificity of hormone response are poorly understood. [114] However, interactions between the hormone-receptor complex and the hormone response elements either increase or decrease gene transcription. [115] The model of androgen action shown in Figure 18-10 is based on studies of androgen metabolism in animals and humans of various ages and on studies of single-gene mutations that impair androgen action. [116] [117] The testosterone-receptor complex regulates gonadotropin secretion and virilization of the wolffian ducts during male sexual differentiation and is probably responsible for sexual dimorphism of muscle development. The dihydrotestosterone-receptor complex controls external virilization during embryogenesis and the development

Figure 18-10 Schematic diagram of androgen action. Testosterone, secreted by the testis, binds to the androgen receptor in a target cell, either directly or after conversion to dihydrotestosterone. Dihydrotestosterone binds more tightly than testosterone. The major actions of androgens, shown on the right, are mediated by testosterone ( solid lines) or by dihydrotestosterone (broken lines). (From Griffin JE. Androgen resistancethe clinical and molecular spectrum. N Engl J Med 326:611618, 1992. Copyright 1992, Massachusetts Medical Society. All rights reserved.)

of most male secondary sexual characteristics during puberty, including androgen-mediated hair growth and loss. The question of which hormone is involved in spermatogenesis is unresolved. On the basis of studies of androgen metabolism in rodent testis, it is generally believed that testosterone is the active hormone for this function; however, dihydrotestosterone is formed in the spermatogenic tubule [118] and sperm production is impaired in subjects with 5-reductase 2 deficiency, [119] raising the possibility that dihydrotestosterone may play a role in human spermatogenesis. Androgen receptors are present in highest concentration in androgen target tissues such as the accessory organs of male reproduction [120] and some areas of the brain. Tissues such as skeletal muscle, [121] heart and vascular smooth muscle, [122] and placenta [123] have small amounts of receptor, and in the testis androgen

receptors are present in both Sertoli cells [124] and Leydig cells. [125] Whether the presence of androgen receptors identifies a tissue as androgen-responsive is not clear. The amount of receptor present in a tissue may be affected by androgen or estrogen, by age, and by single-gene mutations and genetic polymorphisms. A single androgen receptor binds both testosterone and dihydrotestosterone, [126] and the receptor is encoded by a gene on the long arm of the X chromosome. [127] The complementary DNA encoding the human androgen receptor [128] [129] [130] predicts a protein of 917 amino acids and a molecular mass of about 99 kd (Fig. 18-11) . In its amino acid sequence, the receptor shares a high degree of homology with the progesterone, glucocorticoid, and mineralocorticoid receptors in the hormone and DNA binding domains. The N-terminus is only weakly homologous to comparable regions of other steroid hormone receptors and contains glutamine, proline, and glycine homopolymeric sequences. The length of the glutamine repeat is polymorphic in normal populations, and more than 90% of normal women are heterozygous at this locus. [131] Very long glutamine repeat sequences are associated with a neurologic disease termed spinobulbar muscular atrophy (Kennedy's syndrome). [132] The active form of the androgen receptor is a homodimer that forms as a result of interacting sites in the N-terminal and C-terminal domains of the protein; the homodimer in turn interacts with other proteins to form an active transcription regulatory complex. If a single receptor mediates the action of both testosterone and dihydrotestosterone, why is dihydrotestosterone formation important for normal androgen action? A partial answer is that the affinity of the human androgen receptor for testosterone is less than it is for dihydrotestosterone, a difference that results in more rapid dissociation for the testosterone-receptor complex. [133] Testosterone-receptor complexes are also less stable and transform to the DNA-binding state less efficiently. [133] Dihydrotestosterone formation may serve fundamentally to amplify the androgen signal rather than to allow interaction of

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Figure 18-11 Schematic diagram of the normal androgen receptor.

the hormone-receptor complex with specific DNA target sequences. [134] Many issues in androgen action remain unresolved. For example, most acceptor sites for binding androgen-receptor complexes in nuclei, the so-called hormone response elements (HREs), are palindromes in DNA sequences located 5' to genes under control of the hormone and recognize specific sequences in the DNA binding domains of the receptors (see Chapter 4) . However, the characterized glucocorticoid and androgen regulatory elements respond in vitro to either hormone-receptor complex, and other factors determine specificity of action for these hormones in vivo, including the site of location of the HRE in relation to the coding sequence under hormonal control, [135] differences in binding affinities of the hormone-receptor complexes to the HREs, [136] and participation of additional transcription regulatory proteins including suppressor proteins in the active transcription complex. [114] It is noteworthy that in most target tissues (the human and dog prostate glands being notable exceptions) androgen action is limited; for example, under the control of androgen, the penis increases approximately 10-fold in size during male puberty, but when some maximal level is achieved growth ceases regardless of the androgen status of the individual. This cessation of growth corresponds temporally with a decrease in the level of the androgen receptor in the penis, [137] but it is not clear whether the decrease in androgen receptor level is the cause or the consequence of growth cessation.



Estrogen Physiology

As discussed earlier, estrogens may be either secreted directly by the testes or formed in peripheral tissues from 19-carbon precursors. The role of estrogen as an independent hormone in male physiology is incompletely understood but includes closure of the epiphyses, acceleration of the pubertal growth spurt, accrual and maintenance of bone density, influence on gonadotropin secretion, a role in male sexual drive or potentia or both, and control of epididymal function. [138] [139] Estrogen excess in men causes gynecomastia (see later). These various actions appear to be mediated predominantly through the estrogen receptor . In addition, estrogen interacts with androgen action by mechanisms that are not fully understood. In the prostate, estrogens may enhance androgen action by increasing the number of androgen receptors, [93] whereas under physiologic conditions estrogens do not cause growth of the male breast because androgens appear to act as weak antiestrogens and prevent the binding of estrogen to the estrogen receptor. [94]



Spermatogenesis and Fertilization Spermatogenic Cycle

Spermatogenesis involves three processes: (1) multiplication of the germ cells, (2) reduction of the number of chromosomes from the diploid to the haploid state (meiosis), and (3) formation of a superstructure that allows sperm motility as well as generation of energy to drive motility. This superstructure also protects the chromosomes against environmental damage and provides sperm with the capacity to penetrate the ovum. [140] After migration of the germ cells to the genital ridge is completed during the second month of gestation (see Fig. 18-2) , the total number of spermatogonia is approximately 3 × 105 per gonad, and by puberty this number increases to about 6 × 10 8 per testis. As a result of the hormonal events accompanying puberty, a profound cellular proliferation ensues [141] ; the net result is the production of approximately 1 billion sperm each day from the completion of puberty to extreme old age, a total of more than 1 trillion sperm during the usual male reproductive life span. Although the process of spermatogenesis is similar among species, there are major histologic differences, including differences between man and other primates. As illustrated in Figure 18-12 , each spermatogonium undergoing differentiation after puberty gives rise to 16 primary spermatocytes, each of which then enters meiosis and gives rise to four spermatozoa. Thus, 64 spermatozoa can develop from each spermatogonium. In the steady state at least 1.5 million spermatogonia begin this cycle each day, and because nearly half of potential sperm production is lost during meiosis, the actual number of spermatogonia that commence meiosis may be closer to 3 million/day. [142] The commitment of spermatogonia to differentiation does not occur randomly; indeed, because clumps or groups of adjacent cells share a similar if not identical degree of histologic development, contiguous groups of spermatogonia undertake differentiation simultaneously. During spermatogenesis, cytokinesis is frequently incomplete and cytoplasmic bridges form between differentiating spermatocytes and spermatids. This interconnection facilitates coordinated development of groups of germ cells, the so-called wave or synchrony of histologically distinct stages. Clermont [143] identified six typical cellular associations in human seminiferous tubules; thus, one or two generations of spermatids at given steps of spermatogenesis are always associated with one or two generations of spermatocytes and with specific groups of spermatogonia. The succession of these six stages in any one area of tubular epithelium constitutes the cycle of the seminiferous epithelium. [143] [144] The ultrastructural changes during spermatogenesis involve a reorganization of nucleus and cytoplasm and development of the flagellum [145] (Fig. 18-13) . The chromatin becomes progressively more dense, and the nucleus comes to occupy an eccentric position at the cranial pole of the spermatid adjacent to

Figure 18-12 Cell divisions during spermatogenesis. The overall number of cell divisions is much higher than that during oogenesis.

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Figure 18-13 Schematic diagram illustrating conversion of spermatocyte to spermatid to spermatozoon.

the acrosomal cap. The latter is probably formed from the Golgi apparatus and is believed to be essential for the penetration by the sperm of the zona pellucida of the ovum. The core of the sperm tail develops from a centriole near the Golgi apparatus and consists of nine outer fibers and two inner fibers. Mitochondria form a helix around the cilia from the neck to the annulus of the tail. The terminal region of the tail consists of the axial filament surrounded by the cell membrane; most of the cytoplasm is shed as spermatids are released into the lumen of the tubule. Spermatogenesis takes approximately 70 days from the beginning of the differentiation of the spermatocyte to the formation of motile sperm. [146] The transport of the sperm through the epididymis to the ejaculatory duct requires an additional 12 to 21 days [147] and is a journey that involves peristaltic movement, bulk fluid drag, and intrinsic sperm motility. Sperm leaving the testes are relatively immature and have a poor capacity to fertilize. Sperm maturation during passage through the epididymis involves development of the capacity for sustained motility, modification of the structural state of the nuclear chromatin and the tail organelles, and loss of remnant cytoplasm (the cytoplasmic droplet). [148] Acquisition by the sperm of the capacity to fertilize (capacitation) is poorly understood and may be completed in the female genital tract. Energy to drive motility is derived from the hydrolysis of adenosine triphosphate generated in mitochondria in the middle piece of the tail (see Fig. 18-13) . The axial structure of the tail contains a central pair of microtubules surrounded by nine doublet tubules and nine dense fibers; the doublets are attached to the central tubules by a series of radial spokes, to each other by dynein arms, and to the axonemal membrane by so-called Y links. Motility is believed to involve a sliding action of the microtubules, analogous to the interaction of actin and myosin in muscles. The dynein arms contain a powerful adenosine triphosphatase, and sliding is generated by interaction of the dynein arms and is restricted by the radial spokes. [149] Mutations that influence the doublet arms, the spokes, or the spoke heads can lead to the immotile cilia syndromes (see later). [150] Control of Spermatogenesis

Spermatogenesis does not occur in the hypophysectomized state, and restoration of spermatogenesis after hypophysectomy and its initiation at puberty require LH and FSH. FSH acts directly on the spermatogenic tubule, whereas LH enhances spermatogenesis indirectly by increasing testosterone formation in Leydig cells. [151] FSH and testosterone act in the testis by the same general mechanisms as peptide and steroid hormones in other tissues ( see Chapter 4 and Chapter 5 ). For example, FSH binds to receptors on the surface of Sertoli cells and spermatogonia and stimulates adenylate cyclase, resulting in increased intracellular cAMP levels, activation of protein kinases, and phosphorylation of a variety of proteins. [151] FSH stimulates spermatogenesis at several levels. [152] For example, it stimulates mitosis of Sertoli cells, increasing their number during puberty, and promotes maturation and development of tight junctions between Sertoli cells. FSH actions in Sertoli cells include increased production of androgen-binding protein, transferrin, inhibin, aromatase, and plasminogen activators and enhanced uptake of glucose and enhanced conversion of glucose to lactate. FSH receptor mRNA levels vary during different stages of the spermatogenic cycle, suggesting that the levels are regulated and may play a role in spermatogenesis. However, the fact that spermatogenesis and fertility can occur in the presence of loss-of-function mutations that impair FSH and its receptor suggests that the principal role of FSH in spermatogenesis is a quantitative one. [152] Androgen receptors are present in Sertoli cells, Leydig cells, and peritubular myoid cells, and androgen receptor levels in Sertoli cells vary with the stage of spermatogenesis and appear to be under the control of androgen. [153] Genetic evidence has provided insight into the role of androgen in spermatogenesis; mouse

chimeras in which sperm lack a functional androgen receptor can produce fertile sperm provided that the Sertoli cells express normal androgen receptor. [154] Thus, androgen appears to act on the Sertoli cell and not on spermatogonia themselves. Androgen receptors are present in Sertoli cells, but some androgen actions in the cells appear to be indirectly mediated. The Sertoli cell cytoplasm provides the milieu for germ cell maturation, and the tight junctions between Sertoli cells (see Fig. 18-4) ensure the proper conditions for spermatogenesis. Some molecules enter the tubules readily, whereas others are excluded. For example, testosterone and glucose enter rapidly, whereas peptide hormones are generally excluded. Some peptides secreted into the tubular lumen are retained there and probably do not function as endocrine factors outside the testis. Because of the barrier, the testes cannot rely solely on the delivery of hormones, nutrients, and growth factors from the circulation and produce several paracrine-autocrine regulatory factors. Indeed, as mentioned earlier, a multitude of endocrine, paracrine, and autocrine factors have been described in the testes, including neuropeptides, vasoactive peptides, growth factors, and immune-derived cytokines. [82] [83] [84] The large number of testicular cell types and of cell interactions, the bicompartmental organization of the testis, and the many factors being formed (and released) by various cells have led to the widespread assumption that coordination of the two functions of the testis (steroidogenesis and spermatogenesis) involves tissue-specific communication strategies utilizing the unique anatomic features of the testis. However, most of the postulated paracrine-autocrine effects are based on in vitro studies and may not provide insight into the process in intact animals. To date, no integrated view of the relevance of these factors for testicular function or for the treatment of testicular disease has been generated. [82] Understanding the role of paracrine interactions may come through improved in vitro culture systems, from stem cell biology, and from the use of transgenic animals.

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The hormonal requirements for the initiation of spermatogenesis in maturing animals differ from those for maintenance in adults or for reinitiation after hypophysectomy. After hypophysectomy in the adult male, spermatogenesis can be restored by treatment with FSH (human menopausal gonadotropin [hMG]) plus hCG. After spermatogenesis is restored, it can usually be maintained by hCG treatment alone. [151] The latter phenomenon, together with the finding that in otherwise normal subjects with suppressed FSH activity spermatogenesis can be restored by LH alone, [155] suggests that FSH is essential for initiation but not maintenance of spermatogenesis. However, FSH may be necessary for quantitatively normal sperm production in men, [156] and a hypophysectomized man with an activating mutation of the FSH receptor had sustained spermatogenesis. [157] Fertilization

Fertilization normally takes place within the fallopian tube, and spermatozoa usually require a period in the female genital tract before they can fertilize. This functional change, termed capacitation, is believed to consist of at least two components: (1) enhancement of the rate of flagellar beat with acceleration of sperm movement and (2) development of the capacity to undergo an acrosome reaction and consequently allow the plasma membrane of the sperm to fuse with the ovum.[158] The time required for optimal capacitation of normal sperm can vary from 2 hours to more than 6 hours. [159] Whether capacitation is an absolute requirement in the human or serves only to enhance fertilizing capabilities is not known. Because fertilization can take place in vitro when sperm and eggs are combined with no preincubation, the minimal time required for some spermatozoa to undergo capacitation must be short. [159] Capacitation appears to involve a change in the intracellular concentration or metabolism of calcium or cAMP. [160] The acrosome reaction may also involve calcium. [158] Neither the fallopian tube nor the egg itself appears to be essential for the acrosome reaction, which begins as a fusion between the acrosomal membrane and the overlying plasmalemma and is followed by calcium influx into the sperm. Subsequently, the acrosome fragments and disappears. The acrosome is derived from lysosomes, and its disintegration causes release of hydrolytic enzymes and proteases. The fact that the acrosome reaction is followed within a few hours by a loss of sperm motility means that variability in the timing of capacitation in a sperm population relative to the moment of insemination increases the chance of successful fertilization. Ordinarily,

Figure 18-14 Schematic diagram of the different phases of male sexual function during life as indicated by mean plasma testosterone level and sperm production at different ages. (From Griffin JE, Wilson JD. The testis. In Bondy PK, Rosenberg LE [eds]. Metabolic Control and Disease, 8th ed. Philadelphia, WB Saunders, 1980, pp 15351578.)

about a fifth of motile spermatozoa recovered from the oviduct at variable times after insemination have undergone the reaction. The net effect of the enhanced motility and the acrosome reaction is that sperm acquire the capacity to penetrate the formidable vestments of the ovum. [158] One consequence of the sequential acceleration of motility and initiation of the acrosome reaction is that sperm transport to the site of fertilization in the fallopian tube is a culling process. Only a small number of the millions of sperm that are ejaculated reach the site of fertilization. The features that distinguish spermatozoa that reach the ampulla and fertilize the egg are not known, but these sperm are presumed to exhibit the fastest motility and the most delayed initiation of the acrosome reaction. Understanding of the mechanism of sperm penetration is based largely on studies of fertilization of human eggs in vitro, a situation that may not be identical to the phenomenon in intact humans.[161] Ovulated eggs are surrounded by layers of cumulus cells embedded in a matrix of hyaluronic acid. The mechanism by which spermatozoa tunnel through the cumulus is not known. Possibly, hyaluronidase is released by the degenerating acrosome, and the mechanical agitation of the flagellum may disperse the cumulus cells. Under in vitro conditions, prior disposal of the cumulus with hyaluronidase is necessary to allow penetration of the zona pellucida and hence to permit fertilization by the sperm.



Phases of Normal Testicular Function

The phases of normal testicular function can be delineated in terms of the plasma testosterone concentration (Fig. 18-14) . In the male embryo, the production of testosterone by the testes begins to rise at the end of the second month of gestation and shortly thereafter reaches a maximal value that is maintained until late in gestation and then decreases. [162] [163] At the time of birth, the plasma testosterone level is only slightly higher in males than in females. [164] [165] Shortly afterward, the plasma testosterone level again commences to rise in the male infant and remains elevated for approximately 3 months, falling to low levels by 1 year. [164] [165] The plasma level then remains low (but higher in boys than in girls) until the onset of puberty, when it again increases in boys and reaches adult levels by about age 17.[166] Plasma levels remain more or less constant in the adult until middle age and then gradually decline during the later decades of life. place after puberty. The physiologic events during these various periods differ, as do

[167] [168]

Sperm production takes

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the consequences of testicular derangements at different stages of life. Embryonic Male Sexual Differentiation

The process of sexual differentiation is described in Chapter 22 . In brief, the embryos of both sexes develop in an identical fashion until the seventh week of gestation. Thereafter, the anatomic development and the physiologic development diverge, with formation of the male or female phenotypes. As formulated by Jost,[169] normal sexual development in the mammalian embryo depends on three sequential processes. The first involves the establishment of genetic sex, which is defined by the sex chromosome constitution established at the time of conception. The heterogametic sex (XY) in mammals is male, whereas the homogametic sex (XX) is female. In the second phase, the sex chromosomes determine whether the indifferent gonad differentiates into a testis in the male or an ovary in the female. The third step involves the translation of gonadal sex into phenotypic sex and is the direct consequence of the type of gonad formed; that is, testicular secretions determine that the urogenital tract and external genitalia will be male in character. The internal genitalia in the two sexes are derived from the wolffian and müllerian ducts that exist side by side in early embryos of both sexes. [170] The wolffian ducts serve as the excretory ducts of the mesonephric kidney and are physically attached to the indifferent gonad, whereas the müllerian duct has no continuity with the gonad. In the male, the wolffian ducts give rise to the epididymis, vasa deferentia, seminal vesicles, and ejaculatory ducts and the müllerian ducts disappear. In the female, the fallopian tubes, uterus, and upper vagina are derived from the müllerian ducts and the wolffian ducts disappear. The external genitalia and the urethra in the two sexes develop from common anlagen: the urogenital sinus and the genital tubercle, folds, and swelling. The urogenital sinus gives rise to the prostate and prostatic urethra in the male and to the lower portion of the vagina and urethra in the female. The genital tubercle becomes the glans penis in the male and the clitoris in the female. The urogenital swelling becomes the scrotum or the labia majora, and the genital folds develop into the shaft of the penis or the labia minora. In the absence of the testis, as in the normal female or in the male embryo castrated before the onset of phenotypic differentiation, the development of phenotypic sex proceeds along female lines. [169] Thus, masculinization of the fetus requires the action of testicular hormones, whereas the female phenotype develops in the absence of gonadal secretions. Under ordinary circumstances, chromosomal sex, gonadal sex, and phenotypic sex are concordant; that is, chromosomal sex determines gonadal sex and gonadal sex in turn determines phenotypic sex, without deviation from the chromosomal program. Control over the formation of the male phenotype is vested in the action of three hormones. [170] Two of the three, AMH and testosterone, are secretory products of the fetal testis. AMH, a glycoprotein hormone of the embryonic testis, acts ipsilaterally in the male embryo to suppress the müllerian ducts and consequently prevents development of the uterus and fallopian tubes. [171] Testosterone converts the wolffian ducts into the epididymides, vasa deferentia, and seminal vesicles and is also the precursor for the third fetal hormone, dihydrotestosterone. [170] The latter hormone, which is formed within the urogenital sinus and lower urogenital tract from circulating testosterone, acts in the urogenital sinus to induce formation of the male urethra and prostate and in the genital tubercle, swelling, and folds to cause the midline fusion, elongation, and enlargement that eventuate in the male external genitalia. [172] Thus, androgens function during fetal life to induce the formation of the accessory organs of male reproduction. Testosterone and dihydrotestosterone act through the same receptor mechanism during embryogenesis and in the adult [172] (see Fig. 18-10) . The formation of the male phenotype is largely completed by 12 to 15 weeks of gestation, but at the time of completion of the male urethra, the external genitalia in the two sexes do not differ in size. [170] Descent of the testes and differential growth of the external genitalia in the male take place largely during the second half of gestation. The control of testosterone formation by the embryonic testis is incompletely understood. By the 13th week of human gestation, testosterone secretion appears to be regulated by LH from the fetal pituitary gland or by placental hCG in the fetal circulation, or by both. [173] The decrease in testosterone synthesis late in gestation correlates both with a decline in the number of LH-hCG receptors in the testis [174] and with a decrease in the level of hCG and LH in the fetal circulation. [173] Castration of the male rhesus monkey during late gestation results in a further decrease in plasma testosterone and elevation of plasma gonadotropins. [175] Anencephaly and other forms of congenital hypopituitarism cause the syndrome of microphallus. [176] Taken together, these findings indicate that testosterone production during the second half of gestation is regulated by LH or hCG and that LH production itself is under negative-feed-back control by testosterone. The mechanism by which testosterone production is controlled between gestational weeks 8 and 12, when male phenotypic development takes place in the human embryo, is not clear. In the rabbit embryo, testosterone production during the analogous phase of male development appears to be independent of gonadotropin, but for technical reasons this phase of embryonic development in human gestation has not been adequately examined. The fact that most male infants with anencephaly, congenital hypopituitarism, or both have normal male urethras suggests either that androgen synthesis during early gestation is independent of gonadotropins or that chorionic gonadotropin, which apparently is not present in the rabbit, acts as a fail-safe mechanism to guarantee normal male development in the absence of LH from the fetal pituitary gland. [176] However, the fact that loss-of-function mutations of the LH receptor gene cause impairment of the virilization of the male external genitalia (but not of the wolffian ducts) indicates that LH or hCG plays a role in early androgen synthesis. [177] In addition to their role in male phenotypic development, androgens secreted during fetal or neonatal life (or both) exert at least two types of effects on the CNS in some species: regulation of the hypothalamic-pituitary system and control of diverse sexually dimorphic behavior patterns. [178] Androgens are presumed to act in brain through the same receptor as in the urogenital tract and other androgen target tissues. Social imprinting also plays a critical role in sex-specific behavior in some species. Male sexual development, apart from spermatogenesis, is remarkably complete during embryogenesis. For example, male infants have periodic erections during the later phases of gestation, which indicates that the complex neurogenic pathways that regulate this process have developed by that time. The extent to which androgen action in the human CNS influences human sexual behavior has not been established. There is no evidence for permanent imprinting by fetal androgens on the hypothalamic control of gonadotropin production in the human, and it is not established whether gonadal hormones have any direct effect on gender identity or gender behavior apart from their role in anatomic development of the sexual phenotype. Nevertheless, both androgens and cultural factors probably play important roles in the development of characteristic male behavior. [178] Therefore, in making clinical decisions about sex assignment in subjects with ambiguous genitalia, it is important to undertake a thorough diagnostic evaluation and appropriate therapeutic intervention as early as possible, preferably in the newborn nursery, to ensure that the

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psychosocial variables are consonant with biologic and anatomic development. Neonatal Life

The neonatal surge in testosterone secretion is the consequence of a rise in plasma gonadotropin levels, but neither the cause of the increase in gonadotropin levels nor the precise function of the temporary increase in testosterone secretion is understood. Serum inhibin levels increase in boys in the first year of life in parallel with testosterone levels. [179] In some species, the neonatal testosterone surge is believed to be responsible for two aspects of male development: (1) permanent virilization of the hypothalamus so that it secretes LH tonically rather than cyclically as in the female and (2) the priming of androgen target tissues for subsequent androgen-mediated growth and maturation in later life. [180] Blockade of the neonatal activation of the pituitary-testicular axis in male monkeys resulted in subnormal increases in LH and testosterone and impairment of testicular enlargement at the time of puberty. [181] However, in humans there is no evidence that neonatal deprivation or excess of androgen has any permanent effect on hypothalamic-pituitary function. [182] Whether neonatal androgen plays a specific role in the male gender identity or gender role behavior is likewise uncertain. [178] Indirect evidence suggests that neonatal androgen influences the subsequent androgen-mediated growth of the male urogenital tract. Boys who are born with microphallus caused by deficient androgen biosynthesis may have subnormal androgenmediated growth of the external genitalia if androgen replacement therapy is not started until the time of normal male puberty. However, their response may be normal if androgen is administered temporarily during infancy. [183] Such observations are consistent with the view that late fetal or neonatal androgen primes the male urogenital tract by promoting early growth and potentiating maturational effects of the hormone at puberty. Puberty

In the prepubertal years, the plasma levels of gonadotropins and gonadal steroids are low. Maturation of adrenal androgen secretion, termed adrenarche, results in enhanced secretion of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), and androstenedione in boys as early as age 6 or 7, several years before maturation of the hypothalamic-pituitary-gonadal axis. [184] The secretion of these androgens is probably under the control of corticotropin (also called adrenocorticotropic hormone [ACTH] or adrenocorticotropin) and appears to be independent of activation of the pituitary-gonadal axis. In part, the prepubertal growth spurt and the early development of axillary and public hair are mediated by these adrenal androgens, which are believed to bind to the androgen receptor only after conversion to testosterone or dihydrotestosterone in target tissues (see Fig. 18-10) . Before the onset of puberty, the low levels of plasma gonadotropins are under feedback control by the small amounts of androgen secreted by the testes, as evidenced by the fact that castration at this time results in a rise in plasma gonadotropins to levels similar to those of the postpubertal castrate. [185] Gonadotropins in children, as in adults, are secreted in a pulsatile fashion, with the pulses occurring at 2- to 3-hour intervals. [186] These facts suggest that the negative-feedback control of gonadotropin secretion is exquisitely sensitive to plasma testosterone levels before puberty and subsequently changes during puberty development. The factors that determine the onset of puberty are poorly understood and may reside in the hypothalamic-pituitary system, in the testis itself, in the adrenal gland, or at some undefined

Figure 18-15 Ontogeny of luteinizing hormone (LH) secretion. Plasma LH concentrations were sampled every 20 minutes for 24 hours in three normal males at different stages of development. Top, Pattern in an adult man with frequent secretory episodes throughout the 24-hour period and no significant sleep-related augmentation. Middle, Secretory pattern in midpuberty in which marked secretory episodes occur during sleep. Bottom, Pattern in prepuberty in which there are no significant secretory episodes at any time throughout the sampling period. (From Griffin JE, Wilson JD. The testis. In Bondy PK, Rosenberg LE [eds]. Metabolic Control and Disease, 8th ed. Philadelphia, WB Saunders, 1980, pp 15351578. Courtesy of R.M. Boyar.)

level (see Chapter 24) . The sequence of pubertal maturation has, however, been well characterized. Its onset is heralded by sleep-associated pulses of LH secretion (Fig. 18-15) and, to a lesser extent, by increases in the episodic secretion of FSH. [187] Later in puberty, the increased plasma gonadotropin levels become sustained throughout the day, as do the resulting increases in plasma testosterone and dihydrotestosterone levels. The rise in gonadotropin secretion is believed to be the consequence of both an increase in GnRH secretion and an increase in sensitivity of the pituitary to GnRH. [164] Plasma levels of bioactive LH increase even more than those of the immunoreactive hormone.[188] The pubertal changes in gonadotropin and steroid hormone levels in plasma are compatible with the concept that with maturation, the hypothalamic-pituitary system becomes less sensitive to feedback inhibition by circulating androgens, which results in a higher mean plasma androgen concentration. The maturational change in the hypothalamic-pituitary system appears to be triggered by the attainment of a critical body mass or percent body fat, [189] [190] possibly mediated by an increase in plasma leptin levels. [191] The pubertal changes in the testes are illustrated in Figure 18-16 . In prepubertal testes, the interstitial cells consist of an undifferentiated mesenchyme with immature tubules. After puberty, the cytoplasm of the functioning Leydig cells develops a characteristic foamy appearance and the various stages of spermatogenesis can be delineated within the tubule. Initiation of spermatogenesis early in puberty is associated with a rise in

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Figure 18-16 Photomicrographs of representative testicular biopsy specimens (×115). A, Immature tubule development and undifferentiated interstitial (Leydig) cells in a normal prepubertal boy. B, Full spermatogenesis and mature Leydig cells in a normal adult man. C, Klinefelter's syndrome, with marked fibrosis and hyalinization of tubules. D, Complete testicular feminization, with abundant Leydig cells and incomplete tubule maturation. E, Sertoli cellonly syndrome (germinal cell aplasia), with normal Leydig cells and no germinal cells demonstrable within tubules. F, Maturation arrest at spermatid stage in an adult man. (From Griffin JE, Wilson JD. The testis. In Bondy PK, Rosenberg LE [eds]. Metabolic Control and Disease, 8th ed. Philadelphia, WB Saunders, 1980, pp 15351578. Courtesy of F. Vellios and B. Fallis.)

serum inhibin B levels. [192] As indicated by the appearance of sperm in centrifuged urine samples from boys entering puberty, sperm production is an early pubertal event and can occur when testicular growth has just begun. [193] The anatomic and functional changes of puberty are largely the consequence of the action of testicular androgens. It is probable that all androgenic actions are mediated by dihydrotestosterone or testosterone and that other naturally occurring 19-carbon steroids act as androgens only if they are converted to testosterone or dihydrotestosterone within extraglandular tissues. The physiologic effects of androgens have been classified as either androgenic (maturation of the male urogenital tract and spermatogenesis) or anabolic (promotion of growth in muscle and other somatic tissues). These various effects are mediated by the same androgen receptor

rather than being the consequence of different mechanisms of action. Androgen is responsible for many effects at puberty. Rugal folds appear in scrotal skin. The testes, penis, and scrotum enlarge, and the penis and scrotum become pigmented. The prostate, seminal vesicles, and epididymis increase in size. Growth of the various accessory organs of reproduction accounts for about a fourth of androgen-mediated nitrogen retention in puberty. [194] One consequence of this growth and maturation process is the transformation of the cuboidal epithelia of the secretory tissues of the urogenital tract into columnar epithelia. The characteristic hair growth of male puberty involves development of the mustache and beard; regression of the scalp line; appearance of truncal, extremity,

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TABLE 18-2 -- Stages of Puberty Genital Stage

Pubic Hair Stage

Stage 1: Preadolescent. Testes, scrotum, and penis are about the same size and proportion as those in early childhood.

Stage 1: Preadolescent. Vellus over the pubes is no further developed than that over the abdominal wall, i.e., no pubic hair.

Stage 2: Scrotum and testes have enlarged, and there is a change in the texture of scrotal skin and some reddening of scrotal skin.

Stage 2: There is sparse growth of long, slightly pigmented, downy hair, straight pigmented, downy hair, straight or only slightly curled, appearing chiefly at base of penis.

Stage 3: Growth of the penis has occurred, at first mainly in length but with Stage 3: Hair is considerably darker, coarser, and more curled and spreads sparsely some increase in breadth. There has been further growth of the testes and the over junction of pubes. scrotum. Stage 4: The penis is further enlarged in length and breadth, with Stage 4: Hair is now adult in type, but the area covered by it is smaller than that in development of glans. The testes and the scrotum are further enlarged. There most adults. There is no spread to the medial surface of the thighs. is also further darkening of scrotal skin. Stage 5: Genitalia are adult in size and shape. No further enlargement takes place after stage 5 is reached.

Stage 5: Hair is adult in quantity and type, distributed as an inverse triangle. There is spread to the medial surface of the thighs but not up the linea alba or elsewhere above the base of the inverse triangle.

Data from Marshall WA, Tanner JM. Variations in the pattern of pubertal changes in boys. Arch Dis Child 1970; 45:1323. and perianal hair; and extension of the pubic hair upward into a diamond-shaped pattern. Growth of axillary and pubic hair, which was already initiated at adrenarche, is enhanced. Enlargement of the larynx and thickening of the vocal cords cause lowering of the pitch of the voice. Acceleration of linear growth from about 5 to 8 cm/year is accompanied by growth of muscle and connective tissue, which accounts for the major portion of pubertal nitrogen retention and is followed by closure of the epiphyses. In the human, the principal androgen-sensitive muscles are those of the pectoral region and the shoulder. [195] Testosterone and its metabolites also appear to be necessary for the development of normal bone density with sexual maturation. [196] The hematocrit value increases, and plasma high-density lipoprotein (HDL) levels decrease. [197] These various growth and maturation processes reach some limiting value, and the administration of even supraphysiologic amounts of exogenous androgen has little if any somatic effect after puberty is completed. The androgen-mediated pubertal growth spurt is thought to be the consequence of increased growth hormone pulse amplitude [198] and the secondary increases in the levels of IGF-I. [199] A variety of behavioral and psychological changes, including development of sexual potency and libido, take place at puberty. The extent to which the behavioral changes are the result of effects of steroids on the brain, indirect consequences of the anatomic changes at puberty, or cultural conditioning has not been defined.

[178]

The events encompassing puberty vary in regard to both the time frame during which the process is initiated and completed and the sequence in which various changes take place. Because there is also variability in the end results, namely differences in

Figure 18-17 Diagram of sequence of events at puberty. The ranges of age during which changes occur in normal boys are indicated by figures below each bar. (Data from Marshall WA, Tanner JM. Variations in the pattern of pubertal changes in boys. Arch Dis Child 1970; 45:1323.)

secondary sexual characteristics that depend on genetic, psychosocial, and nutritional factors, definition of the limits of normal puberty constitutes one of the most difficult and important problems of adolescent endocrinology. The system for staging pubertal development developed by Marshall and Tanner [200] is summarized in Table 18-2 and Figure 18-17 . Adult male levels of plasma testosterone, LH, and FSH are usually achieved by Tanner stage 4. [201] However, most studies apply to only one ethnic group or nationality and should be applied to other groups with caution because the timing of this process is influenced by ethnic background, [202] possibly the consequence of differences in body weight among different groups. In the United States, male sexual maturation occurs on a similar time scale in black and white persons and is independent of socioeconomic status.[203] A major problem remains defining abnormalities of these various functions, that is, separating pathologic delay of puberty from normal variation. For this evaluation, the family history of the pattern of development (that of siblings and parents) may help. Comparisons with age-adjusted 90% confidence limits for testicular volume [203] [204] and penis size [ 202] are also useful. In some instances, measurement of sleep-related surges in plasma LH and testosterone levels can provide evidence that puberty is commencing, [187] but observation over time may be required to determine whether delayed puberty is a normal variant (for the use of specific diagnostic procedures in this regard, see Chapter 24 ). Adulthood

On average, reproductive capacity is attained between the ages of 16 and 19 years. As indicated in Figure 18-17 , most anatomic changes are also completed by this time. However, androgen-mediated growth of body hair is usually not maximal until the middle to late 20s. The various physiologic actions of androgen during puberty and adulthood can be separated into two general types: permanent and concurrent. Permanent effects encompass anatomic actions that are irreversible and do not regress if androgen production ceases, such as the effects on the larynx. Concurrent effects are those that require a continuing male level of the hormones, such as the enhancement of erythropoietin production and hemoglobin levels. Other physiologic effects of androgen have both permanent and concurrent components; for example, beard growth slows but rarely stops in men who are castrated postpubertally. Many features of castration have been described in anecdotal

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form only, but several aspects have been studied in some detail: First, postpubertal castration results in a negative nitrogen balance. The source and exact magnitude of the nitrogen loss have not been established, but probable

sites of loss include the secretory tissues of the male urogenital tract and, to some extent, other androgen target tissues such as muscle. Androgen replacement in castrated men restores nitrogen balance and the secretory capacity of the epididymis, seminal vesicles, and prostate. Second, castration is followed by a progressive decline in male sexual drive so that only rare castrated subjects can have intercourse after a few years. In such individuals, physiologic androgen replacement results in a rapid and predictable restoration of male sexual activity. Third, two thirds of men subjected to surgical or medical castration experience hot flushes that may persist for 5 years or longer. [205] Long-term consequences of castration in men include osteoporosis, gynecomastia, profound shrinkage (apparent disappearance) of the prostate, and enlargement of the pituitary gland (assumed to be due to hyperplasia of LH-secreting cells). [206] At the completion of puberty, the plasma testosterone levels have attained the adult male level of 10 to 35 nmol/L (3 to 10 ng/mL), sperm production has reached a steady level, and plasma concentrations of gonadotropins are in the adult range. Thus, the mature feedback regulatory system shown in Figure 18-5 (Figure Not Available) is established and is sustained in normal men for approximately 40 years. Even under the best circumstances, the system can be perturbed, usually temporarily, by a variety of influences at the level of both the testis and the hypothalamic-pituitary system. One of the most important of these influences is scrotal temperature; spermatogenesis is exquisitely sensitive to alterations in temperature, and temporary increases in systemic or local temperature (as in a hot bath) can be followed by temporary decreases in sperm production. [207] Spermatogenesis can also be influenced by diet, drugs, environmental agents, and a variety of psychological stresses. Testosterone production is more stable than spermatogenesis but can also be impeded by drugs (see later). Old Age

The term male climacteric implies an analogy to the complete cessation of ovarian function in women at the time of menopause. Men do not experience a relatively rapid total cessation of Leydig cell or seminiferous tubule function with old age, nor do men with intact testes experience the hot flashes characteristic of menopause. Male sexual function does decrease with age, but this decline does not appear to coincide with hormonal changes. Nevertheless, in older men carefully screened to exclude major health problems or medication use, serum testosterone levels do decline with age, although most older men still have serum testosterone levels within the range of normal for young men. The normal circadian rhythm in serum testosterone levels is lost with age, [208] and levels of bioavailable testosterone, namely serum testosterone not bound to SHBG, and Leydig cell reserve, as assessed by response to hCG, are decreased in older men. [209] [210] In a cross-sectional analysis of the Massachusetts Male Aging Study, testosterone parameters began to change around age 40 years; free testosterone levels decreased about 1.2%/year and SHBG levels increased about 1.2%/year, so that the total testosterone levels do not reflect the true level of bioavailable testosterone in older men. [211] A longitudinal follow-up of these subjects also showed a fall in total and free testosterone levels of about 1.5%/year. [212] In another study, serum levels of bioavailable estradiol and testosterone both declined with age so that most men older than 65 had low levels of bioavailable testosterone as compared with young men. [213] When controlled for ejaculatory frequency, sperm density does not change with age, but older men have lower ejaculate volumes, decreased sperm motility, and an increased percentage of abnormal sperm. [214] Total daily sperm production, as assessed histologically, also declines with age. [211] However, most older men have values for these parameters that are within the normal range for young fertile men, and decreased sperm production in older men does not correlate with Leydig cell number.[215] Levels of serum inhibin B decrease after age 35. [216] Serum LH and FSH levels increase in elderly men in keeping with some decrease in bioavailable testosterone and sperm production. [215] [216] [217] Studies of gonadotropin secretion indicate a decreased LH pulse frequency but no change in pulse amplitude with age. [218] Levels of bioactive and immunoreactive LH are increased to a similar degree in elderly men. [210] These observations suggest that the hypothalamic-pituitary responsiveness to the decreased Leydig cell function is appropriate. In one study, the bioactive pituitary LH reserve was decreased in elderly men, as indicated by the response to GnRH and tamoxifen [219] ; in another study, the response to clomiphene citrate was normal. [210] Basal levels of bioactive FSH are similar in young and elderly men, but total levels of FSH are higher in elderly men.[220] Administration of clomiphene citrate increases bioactive and immunoreactive FSH levels to a similar degree. [211] The modest increase in serum gonadotropin levels with age is less than would be predicted for the decline in bioavailable testosterone levels, possibly a consequence of enhanced negative feedback of androgens. [221] Veldhuis and colleagues [222] have suggested that many of the endocrine changes with age are due to impaired synchrony of neuroendocrine function. The close coupling between sleep stage and nocturnal penile tumescence and episodic LH and testosterone secretion becomes blurred in the elderly. [222] Pulsatile administration of GnRH to older men returns LH pulse frequency and amplitude and plasma LH levels to normal, but plasma testosterone levels do not increase equivalently. [223] These findings suggest that defects may occur in the Leydig cell and in the CNS with aging. The role of these changes in other aspects of aging is yet to be determined. In healthy men aged 73 to 94 years, levels of total and bioavailable testosterone correlate positively with muscle strength [224] and total body bone mineral density (BMD) and negatively with fat mass. Levels of plasma estrone and estradiol correlate positively with BMD, and the relation between testosterone levels and BMD is independent of estradiol levels. [224] Estrogen appears to play a major role in preventing bone resorption, whereas both estrogen and testosterone regulate bone formation. [225] In other studies, the decline in bioavailable testosterone in elderly men correlated with depressed mood[226] and with changes in cognitive function. [227] The possible relation between testosterone levels and body composition provides some rationale for testosterone replacement in healthy older men with low testosterone levels (see later).



ASSESSMENT OF TESTICULAR FUNCTION Leydig Cell Function History and Physical Examination

The assessment of androgen status should include an inquiry about the following:

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1. 2. 3. 4.

Presence of developmental abnormalities at birth (e.g., hypospadias, microphallus, cryptorchidism) (see later). Timing and extent of sexual maturation at puberty. Rate of beard growth. Current libido, sexual function, muscle strength, and energy.

Inadequate androgen production or action during embryogenesis impairs development of the normal male phenotype, and Leydig cell failure before puberty impairs sexual maturation and causes eunuchoidism, namely an infantile amount and distribution of body hair, poor development of skeletal muscles, and failure of closure of the epiphyses so that the arm span is more than 5 cm greater than height and the lower body segment (heel to pubis) is more than 5 cm longer than the upper body segment (pubis to crown). Detection of Leydig cell failure that begins after puberty requires a high index of suspicion and appropriate laboratory assessment. One reason for difficulty in detecting this condition is that decreased sexual function in adult men is more common than Leydig cell failure. Erectile dysfunction with preservation of normal libido is usually not due to testosterone deficiency but rather to the adverse effects of systemic disease or of medications prescribed to treat such disease. Likewise, decreased libido may not be recognized by individual patients and may be revealed only by discussion with sexual partners. A second reason is that some manifestations of testosterone deficiency are nonspecific, including depression, loss of drive in the workplace, decreased stamina, increased irritability, hostility, and nervousness. [228] Another reason that androgen deficiency is missed is that some functions that require androgens for initiation continue unabated with Leydig cell failure and some functions that eventually regress do so slowly. The frequency of shaving may not decrease for many months or years because of slow decline in the rate of beard growth once established. Finally, some consequences of testosterone deficiency such as increased abdominal obesity and decreased muscle mass may not be recognized as out of the range of normal variation. [229] Assessment of Plasma Luteinizing Hormone, Androgens, and Sex HormoneBinding Globulin

Because the LH level must be interpreted in light of plasma testosterone, it is usually appropriate to measure both hormones by using a pool formed by combining equal quantities of blood obtained from three or four samples at 15- to 20-minute intervals. In this way, only a single pooled sample of plasma is submitted to the laboratory and the averaging of values is accomplished before the assay. Dual-site immunometric assays using an appropriate international standard or reference preparation have largely replaced competitive radioimmunoassays. The usual normal range of plasma LH in adult men is 1.3 to 13 IU/L. Plasma testosterone is also measured by immunoassay. Like LH, testosterone is secreted in a pulsatile fashion. [230] Because the diurnal variation of plasma testosterone is significant in many men, [79] it is desirable to measure testosterone in the morning along with plasma LH in pooled samples as just described. The normal range in adult men is 10 to 35 nmol/L (3 to 10 ng/mL). The plasma testosterone level is higher in prepubertal boys than girls, the normal range in boys being 0.2 to 0.7 nmol/L (0.05 to 0.2 ng/mL). The start of puberty is marked by a rise in plasma testosterone at night as a consequence of sleep-related nocturnal gonadotropin surges, [187] and daytime levels of plasma testosterone gradually increase with the stages of puberty. In young men, the plasma testosterone is higher in the morning than in the late afternoon. [208] In healthy middle-aged and elderly men, morning testosterone levels tend to remain stable over long periods. [231] Plasma dihydrotestosterone is also measured by immunoassay. In normal young men, the plasma concentration averages about 10% of the testosterone value. Testicular function cannot be assessed by measurement of urinary 17-ketosteroids, which are composed of metabolites of testicular and adrenal androgens.

[113]

Measurement of the SHBG level is sometimes useful for interpretation of levels of total plasma testosterone. Binding capacity of SHBG can be assessed with the use of radioactive androgen, and the protein is measured by immunoassay. In most situations, measurement of total testosterone provides an adequate assessment of Leydig cell function. However, in men with altered levels of SHBG (men older than 55 years of age and men with human immunodeficiency virus [HIV], abnormal liver function, or marked obesity), it is appropriate to assess the free or bioavailable testosterone level. Measurement of free testosterone levels by equilibrium dialysis is rarely done, and the analogue free testosterone assay is unreliable because it is influenced by the SHBG level. [232] It is more practical to measure bioavailable (non-SHBG-bound) testosterone; this assay is performed by precipitating SHBG and measuring testosterone in the supernatant. [233] Alternatively, free or bioavailable testosterone can be estimated from the levels of total testosterone and SHBG, using the affinity constants of binding of testosterone to albumin and SHBG. [234] In most circumstances, these calculated values correspond closely to directly measured levels. [234] Dynamic Tests of the Hypothalamic-PituitaryLeydig Cell Axis

To assess Leydig cell function before puberty, it is common to measure the response of plasma testosterone to gonadotropin stimulation as an index of Leydig cell reserve. [235] Normal prepubertal boys respond to 3 to 5 days of injection of hCG at 1000 to 2000 IU/day with an increase of plasma testosterone to about 7 nmol/L (2 ng/mL); the magnitude of the response increases with the initiation of puberty and peaks in early puberty. In some circumstances, the response of plasma LH to GnRH is measured to assess the functional integrity of the hypothalamic-pituitaryLeydig cell axis. Before puberty, the responses of LH and FSH are similar, and with puberty the LH response to acute administration of GnRH increases whereas the FSH response remains the same. The amount of LH released after acute administration of GnRH is believed to reflect the amount of hormone stored in the pituitary. Administration of 100 µg of GnRH to normal men causes LH levels to increase fourfold to fivefold with a peak level at 30 minutes. [236] However, the range of response is broad, and the peak LH after a single dose of GnRH usually correlates with the basal level. In men with primary testicular failure, measurement of basal LH is usually sufficient and assessment of the GnRH response adds little information. Men with pituitary or hypothalamic disease may have either a normal or an abnormal LH response to an acute dose of GnRH, and consequently a normal response is of no value in determining the presence of secondary disease or in distinguishing hypothalamic from pituitary disease. A subnormal response to an acute dose indicates that an abnormality exists but provides no evidence concerning the site of the abnormality. Measurement of long-term GnRH responsiveness is useful for evaluation of men with secondary hypogonadism and a subnormal LH response to an acute dose of GnRH. If daily

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infusions of GnRH for 1 week lead to the development of a normal LH response to an acute dose of GnRH, a hypothalamic cause of the hypogonadism is likely. More often, a hypothalamic or pituitary defect is diagnosed with imaging studies.


[237]


Seminiferous Tubule Function History and Physical Examination

Leydig cell dysfunction usually results in defective spermatogenesis, and men with the clinical features of Leydig cell dysfunction are usually infertile. In contrast, men with primary disorders of the seminiferous tubules can present with infertility as the sole clinical manifestation. Examination of the testes is an essential portion of the physical examination. The seminiferous tubules account for about 60% of testicular volume. The prepubertal testis is about 2 cm in length (and 2 mL in volume, as assessed by the Prader orchidometer) and increases in size with puberty to reach the adult size by about age 16. When damage to the seminiferous tubules occurs before puberty the testes are small and firm, whereas postpubertal damage characteristically results in small, soft testes. The normal adult testis is at least 4 cm in length with a volume of 15 to 25 mL. Considerable damage can occur before overall size shrinks below the lower limits of normal. Testis size varies among ethnic groups. Asian men have smaller testes than European men independent of differences in overall body size. [238] Testicular size can be assessed with a ruler or with a Prader orchidometer. [239] Because of the frequent occurrence of varicocele among infertile men and its possible causal role in infertility, the testes should be carefully palpated while the patient is standing. Seminal Fluid Examination

Routine evaluation of the seminal fluid assesses parameters that do not necessarily reflect the functional capacity of the sperm. Seminal fluid should be obtained by masturbation into a clean glass or plastic container. Collection in a condom or after coitus interruptus may be incomplete and is not recommended. The volume of the normal ejaculate is 2 to 6 mL. The seminal fluid coagulates immediately after ejaculation and then liquefies within 15 to 30 minutes. The specimen should be analyzed within an hour. Motility is estimated by examining a drop of undiluted seminal fluid and recording the percentage of motile forms. The quality of motility can be graded 1 to 3. Spermatozoa with grade 3 motility move rapidly across the field, grade 2 spermatozoa move aimlessly, and grade 1 spermatozoa have a beating tail but do not move. Normally, 60% or more of sperm should be motile, with an average quality of motility of grade 2.5 or more. After it liquefies, the semen sample should be mixed well and diluted 1:20 either in water or with dilute sodium bicarbonate containing 1% phenol to immobilize the spermatozoa. A drop of this specimen is placed on a standard blood-counting chamber, and the spermatozoa are counted within five blocks containing 16 squares each. This number multiplied by 10 6 represents the count per milliliter. Sperm density can also be estimated by using an electronic particle counter. The normal value is usually considered to be greater than 20 million/mL, with total sperm per ejaculate greater than 60 million. Random sperm counts are complicated by the effects of factors such as hot baths, acute febrile illness, and medications. The net result is that it is difficult to define the minimally adequate ejaculate. When 24 to 36 hours of sexual rest is specified and ejaculates are examined at 2-week intervals, average semen quality and sperm output are lower than previously considered normal for fertile men. [240] Three ejaculates are usually required to determine sperm number and cytologic features, and six estimates or more may be necessary for valid assessment if the initial ejaculates are of equivocal quality. [240] Seminal fluid cytology can provide a useful index of fertility. The seminal fluid smear is prepared similarly to a blood smear but with special stains. Normal spermatozoa have symmetrically oval heads, middle pieces that are larger at the proximal ends and inserted symmetrically into the heads, and tails 7 to 15 times longer than the heads. Some abnormal spermatozoa are present in all semen. The best correlations between histologic abnormalities and infertility occur when a single anomaly (e.g., lack of the acrosome) is found in a large percentage of sperm. More than one abnormality may be present. Although there is no clear definition of the minimal structural features compatible with fertility, 60% or more of the spermatozoa should have normal morphology. [240] Evaluation of sperm structure by electron microscopy, if available, is useful for identifying specific defects in immotile sperm (see later). [241] Testicular Biopsy

In men with azoospermia, testicular biopsy may be helpful if intracytoplasmic sperm injection (ICSI) is contemplated for treatment of infertility. [242] It is most appropriate to perform such biopsies at the time ICSI is scheduled. Fine-needle aspiration can provide biopsy material and sperm for ICSI. [243] In most men with infertility associated with oligospermia, testicular biopsy is of little value. The diagnosis of Klinefelter's syndrome related to chromosomal mosaicism limited to the testes can be established only by tissue culture and karyotypic analysis of the biopsy material. The histologic features of several testicular disorders are illustrated in Figure 18-16 . Plasma Follicle-Stimulating Hormone and Inhibin B

Levels of plasma FSH usually correlate inversely with spermatogenesis; that is, elevations of FSH occur in men with intact hypothalamic-pituitary axes when there is severe damage to the germinal epithelium. An inverse relationship also exists between levels of plasma inhibin B and FSH in men, [60] including semen donors, infertile men, and men with elevated FSH levels. FSH is measured by immunoassay using the appropriate international standard or reference preparation. As with LH assays, dual-site immunometric assays are now commonly used and the usual range in normal men is 0.9 to 15 IU/L. Oligospermia caused by primary testicular defects is usually associated with elevated FSH levels. Chromosomal Analysis

Examination of buccal mucosa cells for the presence of chromatin clumps on the nuclear membrane (the Barr body) provides evidence for the number of X chromosomes. In general, there is one Barr body for every X chromosome in excess of one. The Barr body, which represents the second X chromosome in XX individuals, is identifiable in 20% or more of the nuclei of cells in normal females and in less than 2% of cells of normal males. If buccal mucosa cells are stained with quinacrine or its mustard derivative and examined by fluorescence microscopy, the Y chromosome can be identified. This method provides a rapid and accurate means of determining the sex chromosome complement under some circumstances such as suspected male pseudohermaphroditism. Analysis of the chromosomal karyotype, the most accurate means of determining the chromosome complement, involves

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culture of peripheral blood leukocytes or of tissue fibroblasts in medium containing an agent such as phytohemagglutinin that induces the cells to divide. A spindle poison such as colchicine, which arrests mitosis at metaphase, is added; the cells are harvested and stained; and the number and histologic characteristics of the chromosomes are assessed in several cells. This technique is valuable for establishing the exact chromosome complement, the presence of mosaicism, the presence of structural chromosome alterations, and the sex chromosome composition. The study of multiple tissues may be necessary to establish chromosome mosaicism. In a given tissue, 20 cells must be examined to exclude with 95% confidence a mosaicism of 15% or greater.



Estrogenic Function History and Physical Examination

Gynecomastia (enlargement of the male breast), the most consistent feature of feminizing states in men, is the consequence of proliferation of glandular tissue. The physician should seek the presence of gynecomastia by examining the patient while he is in the sitting position; the fingers are used to grasp the glandular tissue. Palpation with the flat part of the hand while the patient is supine may result in failure to detect early or minimal breast enlargement. In obese men, it is important to try to detect the edge of the rim of glandular tissue that separates it from the adipose tissue of the chest wall. Ultrasonography or mammography may be useful in separating true gynecomastia from lipomastia. Plasma Estrogens

As discussed earlier, most estradiol and estrone in normal men are formed by extraglandular aromatization of circulating androgens. As assessed by immunoassay, plasma estradiol is usually less than 180 pmol/L (50 pg/mL) in normal men and plasma estrone is somewhat higher but usually less than 300 pmol/L (80 pg/mL). A recombinant cell bioassay has been developed to measure estradiol at low levels. [244]



ABNORMALITIES OF ANDROGEN METABOLISM AND TESTICULAR FUNCTION Abnormalities of testicular function have different consequences, depending on the phase of sexual life in which they are first manifested. Although there are problems inherent in all classifications and although some assignments are arbitrary, such a categorization of testicular diseases has a sound physiologic rationale. For example, although Klinefelter's syndrome is a disorder of chromosomal sex, it is usually diagnosed in individuals when manifestations become apparent after the time of expected puberty. Although such limitations must be recognized, disorders of the testes can be classified as abnormalities of fetal development, puberty, adult life, and senescence. Fetal Life Abnormalities of Male Sexual Differentiation

Disturbances in sexual differentiation can arise from a variety of mechanisms: 1. 2. 3. 4.

Environmental insult, as in the ingestion of a virilizing drug during pregnancy. Nonfamilial aberrations of the sex chromosomes, as in 45,X/46,XY chromosomal mosaicism. Developmental birth defects of multifactorial origin, as in most cases of hypospadias. Hereditary disorders resulting from single-gene mutations, as in the testicular feminization syndrome.

The disorders of sexual differentiation and their management are described in Chapter 22 , but because 46,XX men and men with Klinefelter's syndrome ordinarily present with problems of undervirilization or infertility, they are also discussed in this chapter. Cryptorchidism

Descent of the testes is essential to normal function because spermatogenesis requires the lower temperature that is present in the scrotum. Failure can occur at any site in the normal pathway of descent, from high in the abdomen to the scrotum itself. The implications and sequelae of cryptorchidism differ, depending on the site at which descent ceases. [245] A large portion of the literature in this field is difficult to interpret because of imprecise definitions. Cryptorchidism can be defined as a testis that is not 4 cm or more below the public tubercle in an infant of normal size and subclassified, depending on the location of the maldescended testis, as follows: 1. The intra-abdominal testis (10%) cannot be felt. It is usually located just above the internal inguinal ring. Infants with bilateral intra-abdominal testes can be distinguished from female pseudohermaphrodites by assessment of the chromosomal karyotype and from boys with bilateral anorchia by demonstrating that the plasma testosterone level increases after administration of hCG. Unilateral intra-abdominal testes must be separated from the syndrome of mixed gonadal dysgenesis, in which a testis is present on one side and an intra-abdominal streak gonad is present on the other. 2. The canalicular testis (20%) has traversed the internal inguinal ring and is present in the inguinal canal; it may move intermittently between the canal and the upper scrotum. Such testes are small or would not be able to pass the external inguinal ring. When the testis is in the canal, the aponeurosis of the external oblique muscle forms such a firm barrier that the testis can rarely be palpated. 3. The high scrotal testis (40%) is farther along the pathway of descent but does not reach the bottom of the scrotum. It is characteristically smaller than its normal partner and has a limited range of motion so that it can retract into the groin but not past the internal ring. Retraction may make accurate diagnosis and classification difficult. 4. The obstructed testis (30%) is a fourth category in which failure of descent appears to be due to a physical barrier formed by a cord of fascia between the inguinal pouch and the inlet of the scrotum. Another category is the ectopic testis. On rare occasions, the testis may deviate from its normal pathway of descent and become ectopic in location. [246] The five most frequent sites of ectopia are the perineum, the femoral canal, the superficial inguinal pouch, the suprapubic area, and the opposite scrotum. Testicular ectopia is believed to be caused by an abnormality of the gubernaculum. [247] In most situations, the higher the location of the testis or the more extreme the ectopia, the more difficult surgical repair becomes. About 3% of full-term male infants have at least one cryptorchid testis at birth. Completion of descent usually occurs during the first few weeks after birth so that the incidence of cryptorchidism at 6 to 9 months and in adult men is about

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0.7% to 0.8%. Accurate diagnosis and classification may require careful, repeated observations by a single observer to be certain that a normally (or partially) descended testis has not retracted into the groin. The concept that spontaneous descent can occur after a few months of age is a misconception that arose because of the failure to recognize that many normal testes are retractile in young boys and that elicitation of the cremasteric reflex can cause at least partial retraction of fully descended testes in three fourths of boys. The incidence of retraction declines with age, and it rarely occurs after midpuberty. It is important to appreciate that a testis in the superficial inguinal pouch may be a temporarily retracted normal testis, a temporarily retracted high scrotal testis, a transiently palpable canalicular testis, or an obstructed testis and that differentiation among these possibilities is not always simple. Pathogenesis

The cause of testicular maldescent is not well understood. The cryptorchid testis functions poorly in regard to both androgen secretion and spermatogenesis, but it is not always clear whether the testis functions poorly because of maldescent or fails to descend completely because it was abnormal to begin with. Maldescent of the testis occurs with increased frequency in many congenital defects, including virtually all disorders that impair virilization or prevent development of normal intra-abdominal pressure. Although maldescent is common in individuals with severe impairment of the androgen receptor, mutations of the androgen receptor gene are rare in boys with isolated cryptorchidism. [248] Likewise, defects of the gubernaculum are common in cryptorchid testes, [249] but mutations of the INSL 3 gene have not been detected in men with idiopathic cryptorchidism. [250] In some instances, a clear relation exists between maldescent and malfunction of the testis. For example, in the obstructed testis, in which a physical barrier prevents descent,[247] and in syndromes in which intra-abdominal pressure is inadequate because the abdominal muscles are absent or incomplete, such as the prune-belly syndrome,[14] inadequate testicular function in later life is the consequence of impeded descent. Conversely, in all series testes appear to have been abnormal from the first, and it is reasonable to assume that the defect in these instances plays a causal role in the maldescent. As many as half of boys with a unilateral nonpalpable testis and a contralateral descended testis have an absent (vanishing) testis rather than a cryptorchid testis. [251] The diagnosis of vanishing testis is suggested when compensatory testicular hypertrophy causes the contralateral descended testis to be larger than the mean for the age. [252] Cryptorchid testes can sometimes be identified by magnetic resonance imaging or ultrasonography [246] but definitive diagnosis may require laparoscopy. [253] Sequelae

About 10% of testicular tumors arise in an undescended testis, whereas cryptorchidism is present in fewer than 1% of adult men. [247] Thus, malignancy is more likely to develop in an undescended testis than in a fully descended one. The greatest risk of malignancy is associated with intra-abdominal testes. Such malignancies commonly involve the germ cells, most commonly seminomas or embryonal cell carcinomas. Surgical correction of cryptorchidism does not remove this risk because

malignancy may develop in a previously cryptorchid testis many years after orchiopexy. Moreover, the contralateral normally descended scrotal testis is the site of development of malignancy in approximately a fifth of tumors associated with unilateral cryptorchidism. The frequency of malignancy in cryptorchid testes should not be exaggerated because the chance of tumor development in any individual with cryptorchidism is low, but lifelong follow-up is required. Each cryptorchid testis should be surgically placed in a site that allows ready examination, and if this is not possible the cryptorchid testis should be removed. Periodic examination of the testes should be mandatory in the routine care of men with a history of cryptorchidism. [247] In unilateral cryptorchidism, regardless of whether surgical correction has been undertaken, overall androgen production and levels are generally normal, presumably because malfunction of one testis can be compensated by the other testis. [254] Although as many as 60% of men in whom bilateral cryptorchidism was corrected in childhood can father children, [255] cryptorchidism is associated with defective spermatogenesis. Mean sperm density is lower in adult men after surgical repair of cryptorchid testes in childhood, [254] and spermatogenesis can also be decreased in the normally descended testis in men in whom one testis is cryptorchid. Basal FSH levels and FSH responsiveness to GnRH are higher on average in such men. [254] Considered together, these types of evidence support the concept that testicular malfunction, as evidenced by impaired spermatogenesis, is a major factor in maldescent. Management

Cryptorchidism should be treated by surgical or medical means, or both, and although correction may not prevent all sequelae, it is generally believed that correction should be undertaken before age 5 years. In the case of intra-abdominal testes (unilateral or bilateral), the issue is to exclude vanishing testes as the cause and bring intra-abdominal testes into the scrotum, where they can be monitored by physical examination. Measurement of levels of serum inhibin B [179] and AMH[256] may be useful in distinguishing bilateral intra-abdominal testes from vanishing testes. The testes that cannot be brought into the scrotum should be removed. Likewise, obstructed testes must be treated surgically. The unresolved issue concerns the role of medical therapy in boys with canicular or high scrotal testes; in relatively large randomized trials, treatment of such boys with hCG, GnRH, or both agents in seriatim was said to cause descent of the testes into a normal position in about a fifth of cases. [257] However, the stimulation of apoptosis of germ cells by hCG and GnRH may have long-term deleterious consequences. [258] [259] [260] Apoptosis is increased 1 month after hCG treatment but returns to normal subsequently. [258] In one study, however, the level of apoptosis in the original testicular biopsy correlated many years later negatively with testis volume and positively with serum FSH levels, whereas sperm density was not affected. [259] Treatment of cryptorchid boys with GnRH or hCG before orchiopexy caused a similar decrease in germ cells per tubule. [260] Whether any hormonal treatment of cryptorchidism affects subsequent fertility is not known.



Neonatal Life

It is not clear whether abnormality in the neonatal surge in testosterone secretion results in pathologic consequences in humans. As mentioned earlier, however, temporary inhibition of the pituitary-testicular axis in the neonatal primate is associated with impaired testicular function at puberty. [181]



Puberty

The central issue in dealing with disorders of puberty in both sexes is separating subjects with true absence or precocity of pubertal development from those at the extreme limits of normal variation. Normal puberty in the male is variable in onset, duration, and sequence of events. The spectrum of normal

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puberty and the disorders of puberty are discussed in detail in Chapter 24 . (Feminizing states in prepubertal and pubertal boys can result from either absolute or relative increases in estrogen levels, as discussed later.) However, partial impairments of puberty may not be recognized until adulthood, as in Klinefelter's syndrome (see Chapter 22) , in some androgen resistance syndromes in men (see Chapter 22) , and in isolated gonadotropin deficiency (see Chapter 24) .



Adulthood

Adult abnormalities of testicular function can be due to hypothalamic-pituitary defects, testicular disorders, or abnormalities in sperm transport (Table 18-3) . Most such abnormalities are associated with underandrogenization and infertility, but some exhibit isolated infertility. Even partial defects in Leydig cell function can cause infertility because spermatogenesis requires androgen action. Therefore, although the evaluation of infertility differs from that of the underandrogenized man, it is essential to exclude the presence of subtle Leydig cell dysfunction in every man with infertility. Certain factors or conditions (e.g., hyperprolactinemia, radiation, cyclophosphamide administration, environmental toxins, autoimmunity, paraplegia, androgen resistance) can cause either isolated infertility or a combined defect in testicular function (see Table 18-3) . In addition to the manifestations of androgen deficiency described earlier in relation to assessment of Leydig cell function, testosterone deficiency can cause osteoporosis [261] [262] and mild elevations of serum total and LDL cholesterol and triglyceride levels. [263] Infertility with Impaired Virilization

Hypothalamic and Pituitary Disorders

Disorders of the hypothalamus and pituitary gland can impair secretion of gonadotropins and cause secondary decreases in androgen production and spermatogenesis, either as an isolated defect or as part of more complex pituitary insufficiency (see Chapter 8) . Thus, destructive lesions of the hypothalamus and pituitary gland such as infarction, pituitary macroadenomas, metastatic or suprasellar tumors, infections, granulomatous processes, or radiation injury can cause panhypopituitarism and lead to a secondary testicular defect. Likewise, isolated, functional impairment of hypothalamic GnRH secretion can occur with fasting or critical illness. [264] [265] [266] Congenital isolated gonadotropin deficiency occurs in both sporadic and familial forms. [267] [268] The incidence of the disorder is not established, but in most centers it is second only to Klinefelter's syndrome as a cause of hypogonadism in men. The disorder was originally described by Kallmann as a familial syndrome associated with anosmia and can be manifest in childhood as microphallus or cryptorchidism. Male urethral development is usually complete. Because most penile growth occurs during the latter two thirds of gestation, the presence of microphallus in this disorder has been interpreted as evidence for a role of pituitary gonadotropin in regulating testosterone production during the later portion of gestation. Growth in childhood is normal although bone age is usually retarded. Most affected individuals are identified because of failure to undergo puberty. Some individuals, particularly familial cases, have associated defects such as cleft lip or palate, or both; hearing defect; colorblindness; and eye movement abnormalities. [269] Less severely affected individuals have only partial defects in the production of FSH or LH. A variant in which Leydig cell function is impaired despite testes of normal or near-normal size was originally known as the fertile eunuch TABLE 18-3 -- Adult Abnormalities of Testicular Function Infertility with Undervirilization Infertility with Normal Virilization Hypothalamic-Pituitary Fasting, critical illness

Isolated FSH deficiency

Isolated gonadotropin deficiency

Congenital adrenal hyperplasia Androgen administration

Adrenal hypoplasia congenita GnRH receptor mutations LH/FSH mutations Cushing's syndrome Hyperprolactinemia Hemochromatosis Panhypopituitarism Testicular Developmental and structural defects LH receptor mutations

Germinal cell aplasia

Klinefelter's syndrome

AZF mutations of Y chromosome

XX male syndrome FSH receptor mutations Cryptorchidism Varicocele Immotile cilia syndrome Acquired defects Viral orchitis

Mycoplasma infections

Trauma Radiation Drugs (e.g., spironolactone, alcohol, ketoconazole, cyclophosphamide)

Radiation Drugs (e.g., cyclophosphamide, sulfasalazine)

Environmental toxins

Environmental toxins

Autoimmumity

Autoimmunity

Granulomatous disease Defects associated with systemic disease Liver disease

Febrile illness

Renal failure

Celiac disease

Sickle cell disease Immunologic disease (HIV, rheumatoid arthritis)

Neurologic disease (myotonic dystrophy, paraplegia, spinobulbar muscular atrophy)

Neurologic disease (paraplegia)

Androgen resistance Undervirilized man

Infertile man

Sperm Transport Obstruction of epididymis/vas deferens (cystic fibrosis, congenital absence, vasectomy, diethylstilbestrol exposure) AZF, azoospermia factor; FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; HIV, human immunodeficiency virus. syndrome, which is even harder to separate from delayed puberty than is the typical disorder. [270] Isolated gonadotropin deficiency of all types is less common in women than in men.[267] The underlying defect is in the pulsatile release of gonadotropins. [267] The most common pattern is total absence of detectable LH pulses associated with profound impairment of pubertal maturation. Other individuals exhibit a developmental arrest pattern with nocturnal pulses similar to those of early puberty and some testicular growth. Alternatively, the defect appears to impair LH pulse amplitude or pulse frequency. Consequently, the disorder encompasses a spectrum of defects in LH secretion.

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The underlying defect in most patients is at the hypothalamic level; short-term administration of GnRH causes an increase in plasma LH and FSH levels in about half of individuals, and GnRH treatment for 5 days or longer increases plasma gonadotropin levels to the normal range in most men with isolated gonadotropin deficiency but not in individuals with panhypopituitarism. [271] The more severe the deficiency, the longer GnRH must be administered to correct gonadotropin secretion. [272] Isolated gonadotropin deficiency can be inherited as an autosomal dominant, autosomal recessive, or X-linked trait. The X-linked form with anosmia is the best characterized. The neurons that secrete GnRH originate in the olfactory placode of the fetus and migrate into the brain with the olfactory, terminalis, and vomeronasal nerves. A defect in the KAL gene in the X-linked disorder impairs the migration of these nerves and thus causes GnRH deficiency, the olfactory disturbance, and hypoplasia of the olfactory bulbs. The genetic locus has been assigned to Xp23.3, and the gene encodes a protein, anosmin, that has homology to neural cell adhesion molecules. [273] In a study of 104 individuals with isolated gonadotropin deficiency, Oliveira and colleagues [274] identified KAL mutations in 3 of 21 familial and in 4 of 39 sporadic cases but in no instance in the absence of anosmia and concluded that the majority of mutations do not involve KAL. In the presence of olfactory disturbances, other midline defects, a positive family history, or a combination of these factors, the diagnosis of the KAL disorder is not difficult to establish either in an infant with microphallus or in an under-virilized adult. In men with anosmia or hyposomia, defects of the rhinencephalon may be demonstrated by magnetic resonance imaging. [267] In older individuals without midline abnormalities or anosmia and with uninformative family histories, the diagnosis can be made (after the presence of a pituitary tumor is excluded) by documenting a normal acute response to GnRH administration after a week of GnRH treatment. This approach is rarely followed in practice. In the middle teen years, separation of individuals with hypogonadotropic hypogonadism from those with delayed puberty may require prolonged observation (see Chapter 24) . Additional mutations that cause gonadotropin deficiency encompass a variety of different mechanisms. One involves a mutation on the X chromosome that causes the X-linked form of adrenal hypoplasia congenita in which adrenal insufficiency is associated with hypogonadotropic hypogonadism and which is due to mutations or deletions of the DAX1 gene (see earlier), which encodes a member of the nuclear hormone receptor family of transcription regulatory factors that are expressed in the hypothalamus, pituitary gland, adrenal glands, and gonads. [268] [275] The fact that the response of LH to GnRH is variable suggests underlying defects in both the hypothalamus and pituitary gland. [276] One individual with adrenal hypoplasia congenita virilized in response to hCG, but 3 years of combination therapy with hCG and hMG did not induce spermatogenesis.[277] The adrenal insufficiency is manifested during infancy, and the hypogonadotropic hypogonadism is recognized at the time of expected puberty, although rarely both may be incomplete or late in onset. [278] Mutations in the GnRH receptor can cause an autosomal recessive form of congenital isolated gonadotropin deficiency unassociated with anosmia. [279] The phenotype is variable, even within a given family, with some patients having partial responses to GnRH and partial hypogonadism. One family exhibited a decrease in the amplitude of LH pulses. [279] A functional form of hypogonadotropic hypogonadism has been described in a family with a mutation in the LH gene. [280] The proband did not undergo spontaneous puberty and had low plasma testosterone and elevated immunoreactive LH levels, but testosterone levels increased after administration of exogenous LH and hCG. A missense mutation in the LH gene impaired binding of LH to the LH receptor. Heterozygous male carriers for the mutation may have low testosterone levels and impaired fertility. Similarly, a partial deletion of the coding sequence of the FSH gene caused a truncation of the molecule and hypogonadism in one man. [281] Serum FSH was undetectable, and LH levels were elevated despite the low testosterone level, suggesting that the absence of FSH impaired Leydig cell function. Hypogonadotropic hypogonadism also occurs in the Prader-Willi syndrome (obesity, short stature, mental retardation, and hypotonia) caused by partial deletions or uniparental disomy of chromosome 15.[282] [283] Three forms of therapy have been used for hypogonadotropic hypogonadism: 1. Androgen replacement to virilize. 2. Gonadotropin therapy to induce fertility. 3. Intermittent administration of GnRH analogues (except in patients with mutations in the GnRH receptor gene). In the infant or the young child with microphallus, administration of testosterone for limited periods (3 months) may cause enlargement of the penis to the normal range without affecting linear growth or causing other significant virilization. [284] In the older child or the adult, long-acting testosterone esters are administered parenterally, as with other forms of hypogonadism (see later). As in other forms of androgen deficiency, the closer to the time of onset of normal puberty that replacement therapy is begun, the more effective the promotion of normal virilization. Administration of hCG over the long term also causes serum testosterone levels to increase to normal adult male levels. [285] In men with severe (prepubertal) hypogonadotropic hypogonadism, however, the induction of fertility usually requires the administration of FSH, in the form of human menopausal gonadotropin, in addition to hCG.[286] The response to gonadotropin therapy is not influenced by prior testosterone therapy but is a function of the initial testis size, men with testes less than 4 mL in volume responding less favorably. [287] Once a normal sperm count is achieved, it may be maintained by use of hCG or, occasionally, by testosterone esters. In rare cases of partial defects in gonadotropin secretion, spermatogenesis can be promoted by testosterone therapy alone. [267] The long-term administration of GnRH in a pulsatile manner to men with hypogonadotropic hypogonadism results in normal plasma testosterone levels, normal pulsatile secretion of LH, normal mean levels of plasma LH and FSH, and, in most, mature sperm in the ejaculate. [267] Acquired isolated gonadotropin deficiency can be caused by pathologic states that impair the hypothalamus or pituitary secondarily. For example, elevated plasma cortisol levels, as in Cushing's syndrome, can depress LH secretion independently of a space-occupying lesion of the pituitary. [266] Likewise, chronic administration of exogenous glucocorticoids can lower testosterone levels by inhibiting GnRH secretion. [266] Hyperprolactinemia associated with both pituitary microadenomas and macroadenomas also causes secondary testicular dysfunction. Macroadenomas may cause hyperprolactinemia either because of direct secretion by prolactinomas or because of interference with the delivery of inhibitory hormones from the hypothalamus to

the pituitary gland by the mass effect of a nonsecretory tumor. Hypogonadism can result from hyperprolactinemia itself, destruction of the normal pituitary gland, or a combination of these effects. Prolactin excess by itself can cause underandrogenization, infertility, and impotence, probably

732

by impairing GnRH release. Administration of low doses of bromocriptine to men with microadenomas caused an initial increase in plasma LH level and a subsequent increase in serum testosterone level. [288] The preferred treatment is bromocriptine or cabergoline (see Chapter 8) . Hemochromatosis causes iron deposition in the pituitary gland and testes, and about half of affected men have hypogonadism, usually accompanied by testicular atrophy. Abnormal testicular function in this disorder may result in part from the associated liver disease, but most testicular dysfunction is due to hypogonadotropic hypogonadism.[266] The pituitary nature of the hypogonadism was recognized because of the lack of response of LH to GnRH administration and the normal response of plasma testosterone to hCG. A primary testicular abnormality may also occur. [266] Acquired transfusional iron overload can cause similar abnormalities of the pituitary-testicular axis. [289] In both states, reduction of iron stores may result in recovery of gonadotropin secretion. Hypothalamic or pituitary injury can occur after head trauma even in the absence of fracture, and the most common manifestation is deficiency of gonadotropins and human growth hormone, although multiple deficiencies can be present. Clinical evidence of hormone deficiency may be apparent immediately after injury or not until years later. [290] In conditions in which testosterone levels are decreased despite normal LH levels, the mechanism is less clear. Men with massive obesity have decreased SHBG levels and decreased levels of total and bioavailable testosterone that return toward normal with weight loss. [291] For example, in men with a body mass index greater than 40, free testosterone levels, LH, and LH pulse amplitude are decreased, implying malfunction of the hypothalamic-pituitary system. [292] Obesity may be the cause of decreased testosterone levels in the pickwickian syndrome. [293] Men with temporal lobe seizures may also have hormonal findings consistent with hypogonadotropic hypogonadism.[294] Finally, acquired hypogonadotropic hypogonadism may be idiopathic. [295] [296] Testicular Disorders

Abnormalities of testicular function in the adult can be grouped into developmental and structural defects of the testes, acquired testicular defects, abnormalities associated with systemic or neurologic diseases, and androgen resistance. Developmental and Structural Defects

LH Receptor Mutations.

Inactivating mutations of the LH receptor gene cause variable defects in testosterone formation. [297] The more severe defects in LH receptor function result in an autosomal recessive form of male pseudohermaphroditism in which 46,XY individuals have a female phenotype, a blind-ending vagina, and inguinal testes with absence of Leydig cells (Leydig cell hypoplasia) (see also Chapter 22) . Less severe impairments of LH receptor function are associated with variable defects, including a male phenotype and microphallus. Testosterone levels are low in the presence of elevated gonadotropins. In most subjects, the phenotypic defects correlate with both the basal level of the LH receptor and the response of plasma testosterone to hCG, [298] but in one individual with a male phenotype associated with a deletion of exon 10 of the LH receptor gene, serum testosterone levels increased after treatment with hCG. [299] Klinefelter's Syndrome.

The most common developmental defect of the testis is Klinefelter's syndrome (see also Chapter 22) .[300] The disorder is characterized by small, firm testes; various degrees of impaired sexual maturation; azoospermia; gynecomastia; and elevated gonadotropin levels. The underlying TABLE 18-4 -- Characteristics of Patients with Classic Versus Mosaic Klinefelter's Syndrome * Characteristic 47,XXY (%)

47,XY/47,XXY (%)

Abnormal testicular histologic features

100

94

Decreased length of testis

99

73

Azoospermia

93

50

Decreased testosterone level

79

33

Decreased facial hair

77

64

Increased gonadotropin level

75

33

Decreased sexual function

68

56

Gynecomastia

55

33

Decreased axillary hair

49

46

Decreased length of penis

41

21

Data from Gordon DL, Krmpotic E, Thomas W, et al. Pathologic testicular findings in Klinefelter's syndrome. 47,XXY vs. 46,XY-47, XXY. Arch Intern Med 1972; 130:720729. *Table based on 519 47,XXY patients and 51 46,XY/47,XXY patients. Significantly different at P < .05 or better.

defect is the presence of an extra X chromosome, the usual chromosomal karyotype being either 47,XXY (classic form) or 46,XY/47,XXY (mosaic form). The incidence is approximately 1 in 500 males. Prepubertal boys with Klinefelter's syndrome have small testes with a decreased number of spermatogonia but are endocrinologically normal; the diagnosis at this age may be made on the basis of the cognitive features (see later). The diagnosis is usually made after the time of expected puberty because of gynecomastia or underandrogenization and later by infertility (Table 18-4) . Damage to the seminiferous tubules and azoospermia are consistent features of the 47,XXY variety. The small, firm testes are usually less than 2 cm in length and less than 4 mL in volume. Histologic changes in the testes include hyalinization of the tubules, absence of spermatogenesis, and an apparent increase in the number of Leydig cells (see Fig. 18-17 C) . Mean body height is increased because of a longer lower body segment; the presence of this feature before puberty suggests that it is not secondary to androgen deficiency but is probably related to the underlying chromosomal abnormality. Gynecomastia occurs in about 85% of affected individuals, develops during adolescence, is usually bilateral and painless, and may become disfiguring. Klinefelter's syndrome can cause learning disabilities and poor impulse control. These tendencies may explain the increased frequency of the disorder among men in mental and penal institutions. Indeed, although many men with the disorder have above average or superior intelligence, poor school performance is common, with decreased verbal scores and a higher incidence of dyslexia and attention-deficit disorder. [301] The risk of breast cancer is increased, presumably because of the presence of gynecomastia, [302] [303] and there is an increased prevalence of extragonadal germ cell tumors in the mediastinum and brain. [304] [305] Autoimmune disorders appear to be more common, perhaps related to the altered levels of gonadal hormones. [300]

Varicose veins, venous stasis ulcers, and thromboembolic disease are also more common. Levels of plasminogen activator inhibitor 1 were reported to be elevated in subjects with Klinefelter's syndrome with leg ulcers but not in those without leg ulcers. [306] Forty percent of men with Klinefelter's syndrome have taurodontism, an abnormality of the dental pulp that predisposes to early tooth decay. [300] Most have a male psychosexual orientation and function sexually as men. The syndrome may go undiagnosed in the majority of affected men, even as adults. [307]

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46,XY/47,XXY mosaicism is the cause of about one fourth of cases of Klinefelter's syndrome, as estimated by chromosomal karyotypes of peripheral blood leukocytes.[308] The true prevalence may be underestimated because chromosomal mosaicism can be present in the testes in individuals in whom the chromosomal karyotype of peripheral leukocytes is normal. As summarized in Table 18-4 , the manifestations of the mosaic form are usually less severe, and the testes may be normal in size. The endocrine abnormalities are also less severe, and gynecomastia and azoospermia are less common, with occasional men being fertile. Additional karyotypic varieties of Klinefelter's syndrome have been described (see Chapter 22) . The 47,XXY form of Klinefelter's syndrome is due to meiotic nondisjunction of the chromosomes during gametogenesis. About 40% of the responsible meiotic nondisjunction occur in the father and 60% occur in the mother. Advanced maternal age is a predisposing factor in the latter cases. [300] In contrast, mitotic nondisjunction after fertilization of the zygote causes the mosaic form and can arise in either a 46,XY zygote or a 47,XXY zygote. Characteristic endocrine changes include elevation of plasma FSH and LH levels. FSH shows the best discrimination, and little overlap occurs with normal individuals, a consequence of the consistent damage to the seminiferous tubules. In the late teens, the plasma testosterone level may be normal. By the middle 20s, the plasma testosterone level averages half the normal value, but the range is broad and overlaps the normal range. [300] Mean plasma estradiol levels are elevated, and SHBG levels are about twice normal. [309] The net result is a variable degree of feminization and insufficient androgenization. The feminization, including development of gynecomastia, is thought to depend on the ratio of circulating estrogen to androgen (see later). Before puberty, plasma gonadotropin levels and the response to GnRH are normal, but by the time of expected puberty plasma gonadotropins and the response to GnRH are elevated. [310] Older men with untreated Klinefelter's syndrome may have an enlarged or an abnormal sella turcica, presumably secondary to impairment of gonadal steroid feedback and gonadotrope hyperplasia. [311] Optimally, affected boys should be identified in childhood to allow treatment of the testosterone deficiency early before it has an effect on BMD and to prevent adverse psychological effects of incomplete sexual maturation. The commencement of testosterone replacement before age 20 results in a normal BMD, whereas institution of therapy in older individuals does not enhance BMD. [312] Androgen replacement has no effect on fertility but has the same general benefits as in other forms of male hypogonadism.[313] Plasma LH levels usually return to normal with therapy, sometimes after many months. [314] Men with rare spermatozoa in the ejaculate or with spermatids or more advanced stages of spermatogenesis on biopsy may achieve fertility with in vitro fertilization using ICSI (see later). However, as many of 15% of the sperm produced by men with the disorder contain a 24,XY chromosome composition, which may result in an increased incidence of the disorder in offspring. [315] XX Male Syndrome.

The XX male syndrome, a variant of Klinefelter's syndrome, occurs in about 1 in 20,000 male births. [308] [316] The testes are small and firm, generally less than 2 cm long; gynecomastia is usual; the penis is normal to small in size; and azoospermia and hyalinization of the seminiferous tubules are present. Affected individuals have male psychosexual identification and absence of female internal genitalia. The mean plasma testosterone level is low, and levels of plasma estradiol and gonadotropins are high. [317] The phenotype differs from that of Klinefelter's syndrome in that the average height is less than that of normal men, the incidence of cognitive impairment is not increased, and hypospadias or ambiguous genitalia may be present. Four theories were proposed to explain male development in the absence of a Y chromosome: (1) mosaicism in some tissues for a Y-chromosomecontaining cell line, (2) a gain-of-function mutation for some autosomal gene, (3) deletion or inactivation of some gene or genes that normally suppress testicular development, and (4) interchange of a portion of the Y chromosome with the X chromosome. [318] Evidence has now been obtained for the presence of mechanisms 1, 2, and 4 in men with this disorder. Y-chromosome sequences are detectable in approximately 80% of 46,XX men and are usually located in the distal region of the X chromosome. [319] [320] Thus, the etiology in most XX males is analogous to that in the sxr mouse, in which a fragment of the Y chromosome has been translocated to the X chromosome. [321] Mosaicism involving an intact Y chromosome was present in 1% of the cells in one individual. [319] The other third of 46,XX men are Y-negative and lack sequences for SRY. The Y-negative group is more likely to have ambiguity of the external genitalia, whereas the Y-positive group has the Klinefelter phenotype. [318] [319] [320] The translated region of the Y can be quite small and involve only the SRY gene itself. The SRY-negative variant is sometimes familial and may occur in families with true hermaphroditism, suggesting that these disorders are due to variable manifestations of the same genetic defect. [322] Such a defect could either be autosomal or X-linked. One 46,XX male had a duplication of the SOX9 gene (a downstream gene involved in SRY-mediated testicular differentiation), indicating an autosomal mechanism. [322] In one SRY-positive man with genital ambiguity, the presence of a duplication of the SOX9 gene, a downstream target of SRY in testicular differentiation, indicated autosomal inheritance. [323] Most SRY-positive 46,XX males do not have genital ambiguity, and in one SRY-positive man with genital ambiguity more than 90% of the of the Yp fragment containing SRY was located on the inactive X chromosome in blood lymphocytes. [324] In parallel studies of a 46,XX man with no ambiguity, the Y p fragment was located predominately in the active X chromosome. [324] These findings document the complexity and heterogeneity of the disorder. The management is similar to that for Klinefelter's syndrome. Acquired Defects

Mumps.

The most common cause of acquired testicular failure in the adult is viral orchitis. Mumps virus is most frequent, but echovirus, lymphocytic choriomeningitis virus, and group B arboviruses also cause orchitis. The disorder is due to invasion of the tissue by the virus rather than to indirect effects of infection. Orchitis is common in mumps, occurring in as many as a fourth of adult men with the disease. [325] In about two thirds of cases it is unilateral. It usually develops 4 to 8 days after the onset of parotitis but occasionally precedes it. After the acute inflammatory phase, the testis gradually decreases in size, although swelling can persist for months. The testis may return to normal size and function or undergo atrophy. The atrophy results from both the direct effects of the virus and ischemia caused by pressure and edema within the taut tunica albuginea. The histologic features of the atrophic testis include progressive tubular sclerosis and hyalinization. [325] The degree of atrophy is not necessarily proportional to the severity of the orchitis. It is usually apparent within 1 to 6 months after the orchitis subsides, but the full extent of damage may not be evident for many years. Atrophy occurs in approximately a third of men with orchitis and is bilateral in about a tenth. The hormonal changes associated with gynecomastia related to mumps orchitis include normal estrogen and decreased testosterone production. [326] The frequency with which mumps results in infertility is not

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known. Almost 50% of men with unilateral mumps orchitis have sperm densities of less than 10 million/mL in the first 3 months, but the sperm count returns to normal within 1 to 2 years in about 75%. [325] In contrast, semen parameters return to normal in less than a third of men with bilateral orchitis. The initial treatment is bed rest and scrotal support. If pain is severe, administration of prednisone can reduce swelling and pain. Glucocorticoid therapy does not appear to have a beneficial effect on the return of the sperm count to normal. [325] In one study, treatment with interferon shortened the duration of symptoms and

prevented atrophy. [327] Trauma.

Trauma is second to viral orchitis as a cause of testicular atrophy in the adult. The exposed position of the testis in the scrotum renders it uniquely susceptible to both thermal and physical damage. In a prospective study, testicular atrophy occurred in half of men after blunt trauma to the scrotum. [328] Radiation.

Both spermatogenesis and testosterone production are sensitive to radiation; impaired secretion of testosterone appears to result from decreased testicular blood flow.[329] The incidence of radiation-associated damage to Leydig cells is directly related to the dosage and inversely related to age at treatment. [330] Most prepubertal boys have normal plasma testosterone levels and normal pubertal maturation after receiving 12 Gy of radiation to the testes, but the presence of an increased LH level in some suggests that compensatory changes are involved in the achievement of normal testosterone levels. In most prepubertal boys, radiation doses greater than 20 Gy cause permanent testosterone deficiency. [330] In contrast, radiation doses above 30 Gy cause testosterone deficiency in only half of adolescent boys and young adults. (Also see "Infertility with Normal Virilization.") Drugs.

Drugs can cause underandrogenization and infertility in several ways: direct inhibition of testosterone synthesis, blockade of the peripheral actions of androgen, and enhancement of estrogen levels. In addition, agents such as propranolol and guanethidine can impair erectile function in men whose hypothalamic-pituitary-testicular axis is normal.[266] Two drugs that in high doses block testosterone synthesis are spironolactone and cyproterone, both of which interfere with the late reactions in testosterone biosynthesis. Spironolactone appears to impair CYP17 activity. [331] Plasma testosterone levels do not change appreciably, however, during usual therapeutic regimens. The antifungal agent ketoconazole blocks testosterone synthesis, also by inhibiting CYP17 activity. [332] The decrease in testosterone after a single dose of ketoconazole is transient, with the nadir occurring within 4 to 8 hours and testosterone returning to baseline within 24 hours as ketoconazole levels fall. However, with doses of ketoconazole greater than 400 mg/day, depression of plasma testosterone levels may be sustained. Impairment of libido is common in men with epilepsy, partly as a consequence of medication. [333] Enzyme-inducing antiepileptic drugs such as phenytoin and carbamazepine lower bioavailable testosterone, raise plasma SHBG and LH levels, and decrease the metabolic clearance of testosterone. [334] The effect is more pronounced with multiple-drug regimens. [333] Valproic acid does not appear to have as severe an adverse effect in this regard. [266] Independent of its effects on the liver, ethanol ingestion reduces testosterone levels acutely and chronically, [335] the result of inhibition of testosterone synthesis. [336] The inhibition of steroidogenesis appears to occur at the 3-HSD reaction as the result of a decrease in the concentration or availability of the pyridine nucleotide cofactors for the reaction, an effect probably mediated by the ethanol metabolite acetaldehyde. [337] The fact that ethanol lowers testosterone levels without causing appropriate elevations of plasma LH suggests that hypothalamic-pituitary function is also impaired. [335] Ethanol can also impair spermatogenesis. [338] Antineoplastic and chemotherapeutic agents, especially cyclophosphamide, commonly induce infertility (see later). Combination chemotherapy for acute leukemia, Hodgkin's disease, and other malignancies may also impair Leydig cell function. [339] This toxic effect on the Leydig cell seems to be produced primarily by alkylating agents. Treatment with alkylating agents during the prepubertal years does not interfere with testicular function in later life, but elevated LH levels develop in many men after treatment, implying the presence of subclinical Leydig cell dysfunction. [330] Treatment of adult men with alkylating agents does not alter LH or testosterone levels.[339] High-dose interleukin-2 therapy for metastatic cancer causes a transient reduction in serum testosterone levels. [340] Plasma testosterone levels may be low in men ingesting large amounts of marijuana, heroin, or methadone. [341] [342] Plasma LH is usually normal, suggesting combined hypothalamic-pituitary and testicular defects. Hyperprolactinemia may contribute to the lowering of testosterone levels. [266] Elevated plasma estradiol and decreased plasma testosterone levels may occur in men taking digitalis preparations, the mechanism being unclear. [266] Drugs can interfere with gonadotropin production either as the result of a direct inhibition [343] [344] (as in medroxyprogesterone acetate administration) or as a secondary consequence of enhanced prolactin secretion (as with phenothiazine therapy). [345] Medroxyprogesterone may also impair testosterone secretion at the testicular level. [346]

Several drugs inhibit androgen action by competition at the receptor level. Although spironolactone can inhibit testosterone synthesis, in the usual dosage regimens it acts primarily by antagonizing the binding of androgen to the androgen receptor, which leads to gynecomastia and impotence. [331] Cyproterone also acts as an androgen antagonist. The most commonly administered androgen antagonist is cimetidine. [347] [348] Gynecomastia can occur in men who are treated with the drug, and decreased sperm density and elevated basal testosterone levels are accompanied by impairment of the LH response to GnRH. Ranitidine appears to be a less potent antiandrogen. [348] Omeprazole can also cause gynecomastia and impotence. [349] Environmental Toxins.

Prolonged exposure to lead results in direct testicular toxicity and an impaired pituitary response of plasma LH.

[350]

Autoimmune Disorders.

Testicular failure can occur as part of a generalized autoimmune disorder in which multiple primary endocrine deficiencies coexist and circulating antibodies to the basement membrane of the testes are present (see Chapter 37) .[351] [352] Granulomatous Disease.

The testes can also be involved in granulomatous disease. Testicular atrophy occurs in 10% to 20% of men with lepromatous leprosy as the result of invasion of the tissue (and in some instances of paratesticular structures as well) by the bacilli. The result is a decreased plasma testosterone level and elevated plasma LH and FSH levels.[353] Destruction of the testis is less common with other systemic granulomatous diseases. Defects Associated with Systemic Diseases

Abnormalities of the hypothalamic-pituitary-testicular axis occur in a number of systemic diseases. Given the chronic ill health and generalized wasting that may coexist, it is often difficult to distinguish specific effects of the underlying condition

735

(e.g., renal failure) from those of malnutrition. The inflammatory cytokines interleukin-1, tumor necrosis factor , and interleukin-6 lower testosterone and have variable effects on plasma LH levels. [266] [354] [355] [356] In in vitro preparations, these agents inhibited several steroidogenic enzymes and StAR protein. [354] [355]

Renal Failure.

About 50% of men undergoing dialysis for renal failure experience decreased libido and impotence associated with impairments in both spermatogenesis and testosterone biosynthesis. The defect in spermatogenesis varies from partial to total destruction of the germ cells. [357] The decrease in plasma testosterone and increase in plasma LH and FSH levels indicate a defect at the testicular level. [266] Plasma testosterone production rates are decreased, and the response of plasma testosterone to hCG is subnormal. [357] After dialysis, plasma testosterone levels and testosterone production rates improve but usually not to the normal range. [266] Hyperprolactinemia occurs in 25% of men who undergo long-term dialysis. [357] In contrast, successful renal transplantation results in a return of testosterone and prolactin levels to normal and a slight decrease in LH and FSH levels. [266] Most men experience improved sexual function after transplantation, and half have sperm densities of more than 10 million/mL. [358] Hepatic Disease.

Cirrhosis of the liver can impair testicular function apart from the direct toxic effects of ethanol. Gynecomastia and testicular atrophy occur in half of men with cirrhosis, and 75% of men with hepatic cirrhosis are impotent. Decreased spermatogenesis and peritubular fibrosis are present in about 50% of patients. Plasma estradiol levels are usually elevated, and plasma testosterone levels are decreased. [266] The net result is a ratio in serum of unbound estradiol to unbound testosterone that is about 10 times normal. Levels of SHBG are about twice normal. The metabolic clearance and production rates of testosterone are decreased, and estradiol production is increased. Extraglandular conversion of androgens, primarily adrenal androgens, to estradiol and estrone is increased about three-fold, presumably because of decreased hepatic extraction of androgens. Basal levels of LH and FSH range from normal to moderately elevated. In men with low testosterone levels pulsatile LH secretion is impaired, implying a defect at the hypothalamic-pituitary level, whereas dynamic tests of the pituitary-testicular axis and hCG responsiveness tests point to a testicular defect. Gonadotropin levels decrease as liver function fails. [266] The reason for abnormal testicular and hypothalamic-pituitary function is uncertain. Elevated estrogen levels can cause both defects. Testosterone therapy has been tried. [266] [359] Although estradiol levels increase (in correlation with the severity of the cirrhosis) after administration of testosterone enanthate, the estrogen/androgen ratio may become normal [359] and gynecomastia may regress.[266] Men with alcoholic cirrhosis may experience spontaneous recovery of sexual function with abstinence from alcohol despite persistent liver abnormalities. Men with alcoholic cirrhosis and testicular atrophy, however, are less likely to experience improvement in sexual function with abstinence from alcohol. Sexual function and testosterone levels are also decreased in men with other forms of liver disease. [266] The hormonal abnormalities in the pituitary-testicular axis can be reversed by liver transplantation. Sickle Cell Anemia.

Sexual maturation can be impaired in boys with sickle cell anemia. [266] Furthermore, in adult men with sickle cell anemia, secondary sexual characteristics are subnormal in most and testicular atrophy occurs in about a third. [360] Testicular biopsy in two men revealed maturation arrest of spermatogenesis. The defect may be either testicular or hypothalamic. Chronic Illness.

Abnormal Leydig cell function, frequently accompanied by decreased sperm counts, occurs in a many systemic diseases including protein-calorie malnutrition,[266] advanced Hodgkin's disease and cancer before chemotherapy, [266] cystic fibrosis, [361] chronic pulmonary disease, [266] and amyloidosis. [362] Such disorders usually cause a low plasma testosterone level and either a normal or slightly increased plasma LH level, suggesting combined hypothalamic-pituitary and testicular defects. The low plasma testosterone level is not the result of plasma factors that inhibit its binding to SHBG and hence is not analogous to that in the euthyroid sick syndrome (see Chapter 10) . Indeed, because mean plasma SHBG levels are elevated, bioavailable testosterone may be lower than total testosterone. These changes in testosterone and LH may be nonspecific effects of illness because similar changes occur after surgery, myocardial infarction, and severe burns.[266] Thyrotoxicosis.

The changes in the hypothalamic-pituitary-testicular axis in thyrotoxicosis may be secondary to increased estrogen levels and include decreased sperm count and semen volume, increased plasma total testosterone level, and normal levels of unbound testosterone. [266] The testosterone response to hCG is blunted, and basal LH levels are increased. Immune Disorders.

Immune disease may cause testicular dysfunction, and primary or secondary hypogonadism is common in men with acquired immunodeficiency syndrome (AIDS), about half of whom have low testosterone levels with normal or appropriately increased plasma LH. [266] Although the disease may involve the testes directly, the hormonal changes suggest a nonspecific response to systemic illness and perhaps the toxic effects of immune cytokines. Many HIV-positive men who progress to AIDS have a transient state of increased LH and normal testosterone levels before testosterone levels become low. Serum SHBG levels are increased in HIV-positive men independent of CD4 count, [363] but bioavailable testosterone levels decrease progressively as CD4 counts decline. [364] Levels of bioavailable testosterone decline in men with AIDS before wasting occurs. [365] The current practice is to provide androgen replacement in men with AIDS when testosterone deficiency becomes manifest. Testosterone replacement in such men results in an increase in lean body mass, [366] [367] improved quality of life, [366] [368] and increased muscle strength. [369] Men with rheumatoid arthritis may have low serum testosterone, particularly during disease flares and while they are receiving glucocorticoids. testosterone levels are usually normal in men with long-standing, stable rheumatoid arthritis.

[370]

In contrast,

Neurologic Disease.

Neurologic disease can cause testicular abnormalities. Men with myotonic dystrophy usually have small testes, low plasma testosterone levels, and elevated plasma LH and FSH levels. [371] Spinobulbar muscular atrophy (Kennedy's syndrome) is a form of adult-onset degenerative motor neuropathy associated with gynecomastia, testicular atrophy, a hormonal profile suggestive of androgen resistance (see later), and an expansion of the homopolymeric glutamine repeat region in the amino-terminal end of the androgen receptor gene. [132] Although the effects are variable, spinal cord lesions that cause quadriplegia or paraplegia initially cause diminished plasma testosterone levels that usually return toward normal, but semen parameters may be permanently abnormal.[266] [372] Some paralyzed men retain the capacity to have erections and ejaculate, depending on the extent of involvement of the lumbosacral spinal cord. [373] Men with trisomy 21 (Down's syndrome) have impairment of both germinal and Leydig cell function and elevation of FSH and LH levels. [374]

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Androgen Resistance

Partial androgen resistance can cause underandrogenization and infertility in men with normal external genitalia. In such men, androgen resistance is manifested by increased testosterone production, elevated plasma LH levels in some, and abnormal androgen receptors in cultured genital skin fibroblasts. [375] The presence of elevated testosterone or LH levels, or both, is not a reliable predictor of which men have a receptor defect. Testicular biopsy reveals maturation arrests or germinal

cell aplasia similar to that shown in Figure 18-16 D and E. In some families, affected men have had gynecomastia and undervirilization but, in some, preserved fertility. [375] [376] [377] Point mutations have been identified in the androgen receptor in men with the isolated infertility and undervirilized, fertile male phenotypes (see Chapter 22) . [117] [376] [377] Infertility with Normal Virilization

Isolated infertility with normal Leydig cell function is caused by a separate group of disorders. Isolated infertility can be due to defects in the hypothalamic-pituitary system, the testis, or the sperm transport system (see Table 18-3) . Hypothalamic-Pituitary Disorders

Isolated FSH deficiency has been reported in men in whom virilization and plasma LH and plasma testosterone levels were normal but plasma FSH levels were persistently low. [378] [379] Plasma FSH levels in such men may increase [378] or remain undetectable [379] after GnRH administration. One man with no FSH response to GnRH had a point mutation in the FSH gene. [379] In some men with chronic untreated or undertreated congenital adrenal hyperplasia related to CYP21 deficiency, suppression of gonadotropin secretion by adrenal androgens causes infertility. [380] This diagnosis is suggested by the presence of small testes, normal to elevated levels of testosterone, and suppressed levels of gonadotropins and is confirmed by finding elevated plasma levels of 17-hydroxyprogesterone and androstenedione ( see Chapter 14 and Chapter 22 ). When androgens are administered in pharmacologic doses to normal men, gonadotropins are suppressed and about 50% of the men have azoospermia (see Chapter 17) . Although men who present with isolated infertility are unlikely to be receiving testosterone replacement therapy, the use of androgens by weight-lifters and body-builders is common. Self-prescribed regimens may include parenteral testosterone esters and a variety of oral and parenteral substituted androgens, often termed anabolic steroids (see later). Supraphysiologic androgen administration can cause reversible azoospermia in normal men. [266] [381] Testicular Disorders

Developmental and Structural Defects

Germinal Cell Defects.

Also known as the Sertoli cellonly syndrome, germinal cell aplasia is a poorly understood defect of the testis. The disorder encompasses histologic features that can have several causes, one of which may be a single-gene defect. Other men with typical histologic and clinical features have a history of viral orchitis, cryptorchidism, [382] alcoholism, [383] or androgen resistance. [376] Testicular biopsy reveals complete absence of germinal elements (see Fig. 18-16E) . The clinical features include azoospermia, normal virilization, absence of gynecomastia, normal to small testes, and normal chromosomal complement. Plasma testosterone and LH values are usually normal, and plasma FSH values are high. The concept of germinal cell aplasia became even more complex with the recognition that one or more Y chromosome determinants other than the SRY gene are essential for spermatogenesis. [384] Some men have a deletion of the long arm of the Y chromosome that includes an azoospermia factor (AZF) that maps to Yq11.23.[385] As many as 18% of men with azoospermia (occasionally severe oligospermia) have chromosome microdeletions in this region. [386] Testicular histology varies from germinal cell aplasia to maturation arrest, and the plasma FSH is elevated. Candidate genes for AZF have been identified by positional cloning; the first is a family of genes termed Y-located RNA recognition motif (YRRM) genes[387] that encode RNA-binding proteins. The YRRM genes are expressed in germ cells, but the fact that multiple genes exist in this family makes it hard to assess their function. The second AZF candidate, termed DAZ (deleted in azoospermia), also encodes a testis-specific RNA recognition motif. [388] In one study, microdeletions in the AZF region were present in a third of men with idiopathic azoospermia and a fourth of men with oligospermia of unknown origin. [389] These microdeletions include the DAZ and YRRM regions and additional sequences. [389] Microdeletions were present in 7% of 46,XY men with known causes of infertility. [390] A mutation in the FSH receptor gene in several Finnish families is associated with variable defects in spermatogenesis and infertility.

[391]

Histologic findings in men with azoospermia include hypospermatogenesis or spermatogenic arrest (see Fig. 18-16F) . Familial male infertility with hypospermatogenesis or maturation arrest can be inherited as X-linked [392] or autosomal recessive traits. [393] In most men, however, the family history is uninformative[393] and the cause of infertility is unknown. In both familial and sporadic cases, meiosis is defective. Cryptorchidism.

Unilateral cryptorchidism, even when corrected before puberty, is associated with abnormal semen in many individuals (see earlier). This finding suggests that the testicular abnormality is bilateral even in unilateral cryptorchidism. Varicocele.

Varicocele is believed to be the most common treatable cause of male infertility, possibly of causative importance in a third of infertile men. [394] Varicocele is caused by retrograde flow of blood into the internal spermatic vein and results in a progressive, often palpable, dilation of the peritesticular pampiniform plexus of veins. It is thought to be due to incompetence of the valve between the internal spermatic vein and the renal vein and is more common (85%) on the left. The incidence of varicocele is about 10% to 15% in the general population and 20% to 40% in men with infertility. The findings on semen analysis are usually nonspecific, but sperm density is often decreased with medium or large varicoceles. [395] Most men with varicocele are fertile and have no detectable abnormality of the hypothalamic-pituitary-testicular axis. The leading theory concerning the adverse effect is that varicocele leads to an increased scrotal (and testicular) temperature, and the elevated scrotal surface temperatures in men with both unilateral and bilateral varicoceles can improve after surgical repair. [396] The testis on the side of the varicocele may be small. [397] Men with varicocele can also have unrelated causes of infertility. [398] Of men with varicocele and sperm counts less than 5 million/mL, about a fifth have microdeletions in the Y chromosome. [398] On average, semen quality improves after surgical repair of varicoceles, but the effect on fertility is inconsistent in that impregnation rates after varicocele repair are probably less than 50%. In one large study of almost 1000 men there was an association between subsequent fertility and preoperative sperm density, and the men with preoperative sperm densities greater

737

than 10 million/mL had a 70% impregnation rate after repair. [399] Immotile Cilia Syndrome.

The immotile cilia syndrome is a hereditary disorder characterized by defective motility of the cilia in the airways and elsewhere in the body and either immotile or poorly motile sperm.[150] The disorder is usually inherited as an autosomal recessive trait. In the airways, defective cilia cause chronic sinusitis and bronchiectasis and the immotile sperm cannot fertilize. Kartagener's syndrome is a subcategory of the syndrome and is associated with situs inversus. The structural abnormalities that impair motility of cilia can be defined by electron microscopy and include missing or abnormally short dynein arms, short spokes with no central sheath, missing central microtubules, and displacement of one of the microtubule doublets. Cilia from epithelia and sperm from the same individual usually exhibit the same defects, but some mutations can apparently result in immotile sperm without impairment of cilia in the lung. [400] In evaluating sperm for structural

abnormalities, the physician should take care to examine a number of axonemes and to confirm the structural defect because axonemal structure can vary in normal respiratory cilia and sperm. The infertility should be treatable, at least theoretically, by empirical methods (see later). Acquired Defects

Mycoplasma Infection.

A role for Mycoplasma (Ureaplasma urealyticum) in infertility is inferred because infections are common in women whose infertility is associated with a "male factor," suggesting that genital tract mycoplasma infection may cause male infertility. [401] Eradication of the infection improves the pregnancy rate despite the fact that the severity of mycoplasma infection in men does not correlate with any specific alteration in sperm density or morphology. [402] Other evidence suggests that the presence of mycoplasma in the lower urogenital tract may represent silent colonization rather than infection. [403] Radiation.

Radiation can cause isolated infertility, with damage sometimes being demonstrable after only 0.15 Gy (15 rad). Doses higher than 1 Gy (100 rad) can cause extreme oligospermia or azoospermia, and higher doses decrease sperm counts and damage spermatids. A return to baseline sperm density takes 9 to 18 months after doses of 1 Gy (100 rad) or less, 30 months for doses of 2 to 3 Gy (200 to 300 rad), and 5 years or more for doses of 4 to 6 Gy (400 to 600 rad). [404] Fractionated radiation may have a more profound effect on the testes than single doses. [405] Permanent infertility can occur after radiation for malignant lymphoma of the abdomen despite shielding of the testes. [406] Administration of radioactive iodine to men for thyroid cancer can also impair spermatogenesis and elevate plasma FSH levels; recovery occurs in about 2 years. [407] Prior suppression of testicular function by administration of testosterone or GnRH, or both, does not protect the testes from radiotherapy (or cytotoxic drugs). [408] Drugs.

The principal drugs that cause isolated infertility are alkylating agents such as cyclophosphamide.[409] Spermatocytes and spermatogonia may disappear completely, causing the picture of germinal cell aplasia with only Sertoli cells lining the tubular lumen. Serum FSH levels can increase fivefold and serve as a marker for germ cell loss; levels of inhibin B decline. [410] Serum LH and testosterone levels usually remain within normal limits. Cessation of cyclophosphamide therapy is followed by return of spermatogenesis within 3 years in about half of azoospermic men. [411] Vinblastine, doxorubicin, procarbazine, and cisplatin are also toxic to the germinal epithelium. Combination regimens such as mechlorethamine, vincristine, procarbazine, and prednisone (MOPP) have an even more profound impact on spermatogenesis. [409] The combination of doxorubicin, bleomycin, vinblastine, and dacarbazine is less toxic than MOPP. [412] Combination chemotherapy that includes cyclophosphamide or procarbazine causes postpubertal azoospermia in about half of prepubertal boys, [413] whereas etoposide in combination chemotherapy causes less toxicity. [414] Chemotherapy-induced azoospermia after treatment with vinblastine, bleomycin, and cisplatin for testicular cancer is usually reversible within 2 years of stopping treatment [415] ; cisplatin is thought to be the primary mediator of the toxicity. [416] Sulfasalazine and methotrexate can also cause oligospermia and infertility. [417] [418] Environmental Toxins.

Because of the potential toxicity of physical and chemical agents, the occupational and recreational history should be carefully evaluated in all men with infertility. Known environmental toxins include chemicals such as the nematocide dibromochloropropane and related compounds, ethylene glycol, cadmium, lead, and organic chloride compounds.[419] In a large meta-analysis of studies of normal men, sperm density was said to have declined from 113 million/mL in 1940 to 66 million/mL in 1990.[420] This report has subsequently been confirmed [421] and refuted (reviewed in reference [419] ). Environmental toxins that might act as estrogens or antiandrogens have been proposed as the cause. Cigarette smoking may also contribute to decreasing sperm density. [419] Autoimmunity.

Although autoimmunity may cause combined underandrogenization and infertility, the usual manifestation is isolated infertility, and antibodies to the basement membrane of the seminiferous tubules or to the sperm themselves may be responsible. Antisperm antibodies of the immunoglobulin A (IgA) class may prevent the penetration of cervical mucus by sperm. [422] IgG and IgA antibodies may impair the acrosome reaction and the binding of sperm to the zona pellucida of the oocyte. Therapy usually involves in vitro fertilization (see later). Development of antisperm antibodies is not always a primary phenomenon because the antibodies have been identified in men with both bilateral and unilateral obstruction of the vas deferens [423] and after vasectomy. Defects Associated with Systemic Diseases

Temporary impairment of semen quality, particularly decreased sperm density, is common after acute febrile illness. This is one reason that several semen analyses must be obtained in the work-up for men with infertility to be confident that true basal parameters have been determined (see earlier). Men with celiac disease have a distinct testicular abnormalitynamely, endocrine features typical of androgen resistance with elevated plasma testosterone and LH levels.[424] [425] [426] Improvement in gluten enteropathy may reverse the androgen resistancelike state. [424] [425] [426] As discussed earlier, spinal cord injury is commonly associated with isolated infertility. [266] Androgen Resistance

Androgen resistance may cause infertility without underandrogenization (see earlier). [117] It was originally thought on the basis of functional studies that androgen receptor defects might account for 10% to 20% of idiopathic azoospermia or severe oligospermia, [427] [428] but loss-of-function mutations in the androgen receptor gene are present in only about 2% of such men. [429] [430] [431] Expansion of the CAG repeat sequence in the N-terminal region of the androgen receptor (see earlier) appears

738

to be more common in men with azoospermia or severe oligospermia.[432] [433] Impairment of Sperm Transport

Disorders of sperm transport may cause as much as 6% of male infertility. [394] Such disorders can be unilateral or bilateral, congenital or acquired. Infertility in men with unilateral obstruction may be due to antisperm antibodies. [423] Obstructive azoospermia at the level of the epididymis also occurs in association with chronic infections of the paranasal sinuses and lungs.[434] In polycystic kidney disease, dilated cysts of the seminal vesicles may obstruct semen transport. [435] Tuberculosis, leprosy, and gonorrhea can obstruct the ejaculatory system, and sperm transport can be obstructed by deep midline müllerian cysts.[436] Congenital defects of the vas deferens can cause azoospermia or oligospermia in sons of women given diethylstilbestrol during pregnancy.[437] Congenital bilateral absence of the vas deferens is common in men with cystic fibrosis, and mutations in the gene responsible, the transmembrane conductor regulator (CFTR) gene, can cause bilateral absence of the vas deferens without other manifestations of the disease. [438] Congenital unilateral absence of the vas deferens may be an incomplete form of the bilateral disorder. [438] [439] Thus, congenital absence of the vas deferens and cystic fibrosis are variable manifestations of mutations of the same gene.

Idiopathic Infertility

In large series, known causes were identified for only about 60% of cases of infertility in males, with the remainder classified as idiopathic (Table 18-5) .[394] [440] Because at best only about half of infertile men with a varicocele achieve fertility after surgical repair, it is likely that even a larger fraction of infertile men have idiopathic infertility. Some may have androgen resistance, and as many as a fifth may have disorders involving the AZF gene (see earlier). Others have oligospermia or azoospermia with normal plasma LH and testosterone but elevated FSH levels in the absence of cryptorchidism, radiation, or drug exposure. Studies of such men indicate that isolated FSH elevation may be associated with a decreased GnRH pulse frequency pulsatile GnRH therapy. [442] [443] Men with oligospermia and normal FSH levels

[441]

and that FSH levels may be corrected with

TABLE 18-5 -- Relative Frequency of Causes and Associated Conditions in Men Who Present with Infertility Cause or Condition % in Study of Greenberg et al.[394] (n = 425) % in Study of Baker et al. [440] (n = 1041) Hypogonadotropic hypogonadism

0.9

0.6

Klinefelter's syndrome

1.6

1.9

Cryptorchidism

6.1

6.4

Varicocele

37.4

40.3

Immotile sperm

0.5

0.6

Viral orchitis

1.9

1.6

Radiation-chemotherapy Obstruction of epididymis or vas deferens

0.5 6.1

4.1

Androgen resistance Coital disorders Idiopathic disorders

0.1 4.0

0.5

41.5*

43.4

*Includes miscellaneous abnormalities, 10.2%, and undiagnosed primary testicular failure, 5.9.% Includes possible obstruction, 4.5%.

may also have altered pulsatile secretion of gonadotropins and testosterone, [444] and testosterone production rates are said to be low in selected infertile men with isolated FSH elevations and normal total serum testosterone. [445] Whether these abnormalities are of causative significance is unknown. A subset of men with severe idiopathic oligospermia associated with a decreased ratio of serum testosterone to estradiol have shown an increase in sperm density after treatment with an aromatase inhibitor. [446] Testicular biopsies of men with idiopathic infertility have shown increased apoptosis associated with maturation arrest and hypospermatogenesis, [447] [448] decreased expression of the c-kit receptor,[448] and increased mutations consistent with abnormal DNA repair mechanisms. [449] Management of Infertility

The management of infertility is usually unsatisfactory because the number of potentially correctable causes is small (see Table 18-5) . When appropriate, however, associated hormonal disorders and coexisting medical conditions may be treated and offending drugs can be discontinued. Although claims of success have been made for a variety of empirical therapies for infertility with oligospermia, most such claims fail to take into account the spontaneous fertility rate in untreated oligospermic men (25% in 1 year). [240] The fact that treatment-independent pregnancy occurs in all forms of human infertility (male and female factors) makes it necessary for all therapies to be evaluated by randomized clinical trials. [450] When several forms of empirical therapyincluding testosterone rebound, nonaromatizable androgen (mesterolone), gonadotropin, antiestrogen (clomiphene), antibiotics, bromocriptine, varicocele repair, artificial insemination, and no therapywere compared in one large retrospective analysis of oligospermic men, none were effective. [451] In Vitro Fertilization

The only effective empirical therapy for male infertility is in vitro fertilization. Standard techniques of in vitro fertilization require about 500,000 motile sperm/mL of ejaculate. Although the fertilizing capacity of sperm from men with abnormal sperm parameters is diminished, conventional techniques can result in 10% or more live births per attempt in men with mild to moderate abnormalities. [452] Such rates are threefold to fivefold higher than natural impregnation rates in such men. However, standard in vitro fertilization is ineffective in men with more severe defects in spermatogenesis. In the Melbourne experience, fertilization rates were low in men with severe oligospermia, poor motility, and increased numbers of abnormal forms. [453] Intracytoplasmic Sperm Injection

Better results have been obtained with the development of intracytoplasmic sperm injection (ICSI)namely, fertility rates of 50% or more using poor quality semen, including decreased sperm number, impaired motility, increased abnormal forms, and combined defects. [454] In men with obstructive azoospermia in whom sperm must be aspirated from the epididymis, fertilization rates are nearly normal. In the past, men with nonobstructive azoospermia, typically with elevated plasma FSH levels, were not treatable. Such patients include men with maturation arrest, postcryptorchidism tubular atrophy, mumps orchitis, and Klinefelter's syndrome. However, when minute amounts of sperm could be identified by testicular biopsy, fertilization and impregnation have been achieved with rare spermatozoa or spermatids retrieved from such biopsy specimens using ICSI. [454] ICSI should not be undertaken until men with abnormal semen undergo a

739

complete work-up so that hypogonadotropic hypogonadism or some other treatable condition is not missed. Furthermore, ICSI may increase the chances of transmitting the father's disorder to offspring, as has been reported in men with Klinefelter's syndrome and men with AZF mutations. [455] [456]



Old Age

The role of the decrease in total and bioavailable testosterone and the increase in estradiol in the decline of male sexual function with aging is not clear (see the following). However, this changing hormonal milieu may be involved in the pathogenesis of breast enlargement in elderly men and in the development of prostatic hyperplasia. Prostatic Hyperplasia

Enlargement of the prostate to the extent that it obstructs urethral outflow is common in elderly men. [457] [458] The gland weighs a few grams at birth, and at puberty androgen-mediated growth causes the prostate to reach the adult size of about 20 g by age 20. This growth is accompanied by transformation of the cuboidal epithelium of the acini to a columnar, secretory epithelium and by initiation of secretion of the prostatic component of the ejaculate. The weight of the gland remains stable for about 25 years. Beginning in the fifth decade of life, a hyperplastic phase of prostatic growth ensues in most men. The second growth phase, unlike the earlier growth, which involves the gland diffusely, typically begins in the periurethral region as a localized proliferation of glandular and stromal elements. The hyperplasia may remain limited in scope, but in many men growth continues and eventually compresses the remaining normal portion of the prostate. The progressive increase in gland size can cause lower urinary tract symptoms and urinary obstruction, but the correlation between symptoms and anatomic changes can be unpredictable. On the one hand, hyperplasia primarily of the periurethral region can obstruct urine outflow in the absence of gross prostatic enlargement; on the other hand, men with gross enlargement of the gland can be asymptomatic. The second growth spurt, like the growth at puberty, requires a functioning testis. Dihydrotestosterone formed within the prostate from testosterone mediates the embryonic development, pubertal growth, and hyperplastic growth of the prostate. [459] Administration to animals of a 5-reductase inhibitor to block dihydrotestosterone formation caused involution of the prostate despite elevation of testosterone levels within the gland. [460] [461] Furthermore, although plasma testosterone may decline with age, the level of dihydrotestosterone in the hyperplastic gland either remains constant or increases. [462] Prostatic hyperplasia also occurs in the aging male dog, and most research on its pathogenesis has been done in that species. Administration to the castrated dog of androgens that cause an increase in the prostatic dihydrotestosterone level caused prostatic enlargement comparable to that seen in the naturally occurring disorder.[463] Estrogen acts synergistically with dihydrotestosterone to enhance prostatic growth in the dog [463] because estrogen increases the amount of androgen receptor in the tissue. [459] Thus, two hormones participate in the development of prostatic hyperplasia in the dog; dihydrotestosterone is responsible for prostate growth, and estradiol enhances dihydrotestosterone action. Three types of evidence suggest that dihydrotestosterone and estradiol are also involved in human prostatic hyperplasia: 1. Either surgical or pharmacologic castration causes a decrease in the size of the hyperplastic prostate gland, [464] [465] indicating that continuing androgen action is essential to maintain the hyperplastic state. 2. Inhibition of prostatic 5-reductase with agents such as finasteride causes a profound decrease in prostatic dihydrotestosterone levels [466] and a 20% to 30% decrease in prostate volume after 3 to 6 months of therapy. This effect is maintained for up to 4 years and is associated with few side effects. [467] 3. There is a temporal relation between the development of prostatic hyperplasia and the increase in plasma estradiol with age, but a causal relation between estradiol and human prostatic hyperplasia has not been established. [468] Demonstration that hormones play a role in prostatic hyperplasia does not necessarily provide insight into its pathogenesis because their action could be permissive rather than causal, and the reason that the disorder varies so markedly in its manifestations is unclear. Similarly, the therapeutic role of 5-reductase inhibitors is not established because there is a strong placebo effect on urinary symptoms, there is no clear-cut relation between symptoms and urine flow, and the natural history of the disorderparticularly how to predict which subset of men will develop significant obstructionis not understood. As a consequence, although a variety of minimally invasive or medical therapies are now available, the indications for surgical or medical management, compared with watchful waiting, are sometimes unclear. [457] [458] [469]

Prostatic Cancer

The endocrine aspects of prostatic cancer are discussed in Chapter 39 .



Disorders of All Ages Testicular Tumors

Tumors of the testes occur with an incidence of 2 to 3 per 100,000 men per year in the United States and account for about 1% of cancer deaths in men. [470] The incidence in most Western countries has risen since the 1930s, particularly in adults, [471] but mortality rates have declined. [471] [472] The frequency shows a trimodal curve, with peaks in childhood (embryonal carcinomas and teratocarcinomas), young adulthood, and old age (seminomas). The incidence in blacks is a sixth or less that in whites, but overall the tumors are the second most common malignancy (after leukemia) in men between ages 20 and 35 years. The tumors are commonly bilateral (either simultaneous or sequential, e.g., a seminoma developing in one testis many years after the removal of the other). [473] Occurrence is familial in 1% to 2% of cases. [474] The presence of an isochromosome of the short arm of chromosome 12 is characteristic of germ cell tumors of all subtypes.[475] Several factors predispose to testicular malignancy. Men with cryptorchidism have a fivefold increased risk of development of such tumors, men with intra-abdominal testes being more at risk than those with high inguinal testes, [476] so that in one series 10 of 131 men with testicular cancer had antecedent maldescent. [477] Three fourths of tumors associated with maldescent are seminomas, the remainder being other germ cell tumors. Early orchiopexy facilitates detection, but whether it reduces the incidence of tumor development is not clear. [478] Testicular malignancy may be more frequent in individuals with abnormal sexual development (i.e., 45,X/46,XY mixed gonadal dysgenesis or 46,XY testicular feminization) than with other forms of testicular maldescent.[479] Occupational exposure to extremely high or low temperature can increase the risk. [480] Estrogen administration to pregnant women may be a predisposing factor in male offspring, [477] and Down's syndrome,[481] Klinefelter's syndrome, [482] and HIV infection [483] are associated with an increased incidence.

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TABLE 18-6 -- Classification of Testicular Tumors I. Germ cell tumors (95%) A. Single-celltype tumors (60%) 1. Seminomas 2. Yolk sac tumors (embryonal cell tumors) 3. Teratomas 4. Choriocarcinoma B. Combination tumors (40%) II. Tumors of gonadal stroma (12%) A. Leydig cell B. Sertoli cell C. Primitive gonadal structures III. Gonadoblastomas A. Germ cell + stroma cell Data from Mostofi FK. Pathology of germ cell tumors of testis: a progress report. Cancer 1980; 45:17351754.

Most testicular tumors in men with congenital adrenal hyperplasia related to steroid 21-hydroxylase (CYP21) deficiency consist of adrenal cell rests, are dependent on corticotropin for growth and secretion, and occur in men who are inadequately treated and hence have elevated plasma corticotropin levels. [484] However, the tumors can be difficult to separate histologically from interstitial cell tumors. Diagnosis

Most testicular cancers produce local symptoms, but delay in making a diagnosis is common because of oversights by both physicians and patients. Testicular cancers usually occur before age 45, and men should be educated about the need to seek prompt medical advice for any change in a previously normal testis, including enlargement, pain or a feeling of heaviness, swelling, or other unusual findings. [485] Pain occurs in half of affected men, and to reduce delay physicians should consider any testicular mass to be a tumor until proved otherwise and to obtain surgical consultation if symptoms and signs persist. [486] Classification

The most widely used classification is that of Mostofi [487] (Table 18-6) and is based on the cell type from which the tumor originates. Germ Cell Tumors

Germ cell tumors are the most common types. Seminomas are characterized by large cells with clear cytoplasm in a delicate fibrovascular stroma infiltrated with lymphocytes; the granulomatous reaction around the tumor can be so intense as to suggest a graft-versus-host reaction. [488] These tumors account for at least half of all testicular neoplasms and can be subdivided into spermatocytic seminomas, which occur in older men and are associated with a 90% to 95% 5-year survival, and anaplastic seminomas, which have a poor prognosis. Embryonal carcinomas are the most common testicular tumors in boys, resemble embryonal carcinomas of the ovary, and are associated with 5-year survivals of about 70% in infants and 25% in adults. Choriocarcinomas contain syncytiotrophoblastic cells and usually occur in the second and third decades of life; the prognosis is poor. Teratomas contain at least two germ cell layers and may be either benign or malignant; they are second in frequency to embryonal carcinomas in childhood and are unusual in adults. Tumors that contain combinations of germ cell types account for 40% of germ cell tumors; the biology of such tumors is determined by the least differentiated (most malignant) element. Of mixed tumors that contain cells of germinal and stromal origin, perhaps the most distinctive is the gonadoblastoma, which consists of germ

cells, sex cords, and, usually, Leydig cells. [489] Gonadoblastomas usually occur in dysgenetic testes containing a Y chromosome and synthesize androgen. Germ cell tumors of all types can also originate in extragonadal sites, including the mediastinum [490] and the brain. [491] These extragonadal tumors are presumed to arise from aberrant migration of germ cells early in embryogenesis; from some common precursor stem cell line that normally gives rise to germ cells, thymus, and pineal gland; or from migration of transformed gonadal germ cells. [492] Testicular germ cell tumors usually occur as a nodule or painless swelling of the testis but may be identified as the result of metastases or because of the peripheral manifestations of hCG secretion by the tumor. After diagnosis, the tumors are staged either by surgical exploration or by computed tomographic scanning or magnetic resonance imaging. Stage I is limited to the testes, stage II involves metastases to infradiaphragmatic lymph nodes but not beyond, stage III involves supradiaphragmatic lymph nodes, and stage IV involves extralymphatic metastases. Germinomas can secrete several distinct tumor cell markers into plasma, including hCG and its subunit, -fetoprotein, lactate dehydrogenase, carcinoembryonic antigen, and placental alkaline phosphatase. [493] Virtually all germ cell tumors synthesize hCG and its subunits, but the hormone is secreted in large amounts only by some nonseminoma germ cell tumors (choriocarcinomas, teratocarcinomas, and yolk sac tumors). [494] -Fetoprotein is a marker of tumors containing yolk sac elements, and teratomas can secrete carcinoembryonic antigen. [495] An elevated level of one of these tumor markers in the plasma of a patient whose tumor has been classified as a pure seminoma usually indicates that the tumor is actually a combination tumor. These markers are particularly useful for following the response to therapy. [493] Secreted hCG may be endocrinologically active and cause enhanced formation of testosterone [496] and, more important, of estradiol [497] by the testes. The net result can be a feminizing syndrome and inhibition of the secretion of LH and FSH by the pituitary (see later). [498] The treatment of germ cell tumors constitutes a major triumph of cancer therapy. Appropriate therapeutic strategies include debulking of the tumor mass, resection of involved lymph nodes, administration of chemotherapy (usually combinations of cisplatin, vinblastine, etoposide, and bleomycin), radiation, and monitoring of tumor cell markers.[499] The cure rates for patients with seminomas are approximately 90% for stage I disease, and individuals with stage III nonseminoma tumors, which were previously uniformly lethal, now have good survival rates. [500] Because young men with germ cell tumors may have infertility related to castration, radiation, chemotherapy, or a combination, cryopreservation of semen before treatment has been advocated as a means of preserving fertility, [501] but many men have adequate sperm production after chemotherapy. [502] Treatment is associated with a small risk of recurrence and the development of secondary solid tumors and leukemia [503] and with the late sequelae of chemotherapy such as nephrotoxicity and neurotoxicity. [504] Stromal Cell Tumors

Stromal tumors (Leydig cell tumors, Sertoli cell tumors) account for 1% to 2% of testicular tumors, and both cell types may coexist within the same tumor. Rarely, adrenal rest tumors occur in the testes. [505] As would be expected, Leydig cell tumors

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commonly secrete testosterone and thus may cause virilization in prepubertal boys (precocious pseudopuberty); many of the tumors secrete estradiol as well and cause mixed signs of feminization and virilization during the prepubertal years and feminizing signs in adult men. The hormones from such tumors can suppress levels of endogenous gonadotropins and testosterone and can cause azoospermia and decreased size of the contralateral testis. [506] Because the tumors may be so small as to be recognized only by ultrasonography, documenting that the testis is the site of increased estrogen production may require selective catheterization of the testicular veins. Sertoli cell tumors show a bimodal age distribution, most patients being younger than 1 year or between ages 20 and 45 years. The tumors are frequently bilateral and familial (usually as a component of the Peutz-Jeghers syndrome). [507] [508] Gynecomastia occurs in about 25% of patients, and estrogen secretion can impair spermatogenesis and cause shrinkage of the contralateral testis. Leydig cell hyperplasia can occur in the area around the tumor, implying either that the tumor is of mixed cell origin or that Sertoli cells secrete some factor that stimulates Leydig cell development. [509] Complete cure and regression of feminizing signs usually follow surgical resection. Approximately 10% of stromal tumors are malignant. [510] Rete Testis Tumors

Adenocarcinoma of the rete testis is rare but tends to be highly malignant. [511] Summary

Testicular tumors can enhance production of estradiol and testosterone by more than one mechanism. When tumors produce steroid hormones autonomously, plasma gonadotropin levels and androgen secretion by uninvolved portions of the testes are depressed, and azoospermia is common. When hCG is secreted by the tumor, production of estradiol and testosterone is increased in unaffected areas of the testes, and azoospermia is uncommon. Furthermore, occasional choriocarcinomas that cannot synthesize steroids de novo nevertheless convert circulating androgens to estrogens. When hormones are formed directly or indirectly by the tumors, the response varies depending on the pattern of hormones produced and the age of the subject.



ABNORMALITIES IN ESTROGEN METABOLISM Gynecomastia

Administration of large amounts of estrogen to men, as for carcinoma of the prostate or in preparation for sex change surgery, causes a variety of side effects, including fluid retention and congestive heart failure, hypertension, electrocardiographic changes, myocardial infarction, and thromboembolic disease. [512] At lower levels, as can occur with estrogen-secreting testicular tumors, estrogen excess suppresses gonadotropin secretion, secondarily impairs testosterone production, and inhibits spermatogenesis. However, the most common manifestation of estrogen excess in men is gynecomastia (breast enlargement). Sexual dimorphism in breast development at the time of puberty is due to the ovarian secretion of estrogen, and estrogen excess at any stage of life can cause breast enlargement in men. In the absence of a progestagen, the breast acini and lobules do not undergo complete female development, [513] probably explaining why galactorrhea is unusual in men. Clinical Features

At the clinical level, gynecomastia is complicated by problems of definition. The common view has been that any palpable breast tissue in men is abnormal except for three situations: transient gynecomastia of the newborn, pubertal gynecomastia, and gynecomastia that occasionally occurs in elderly men. [514] However, this view was challenged by Nuttall [515] and Niewoehner and Nuttall, [516] who reported that 36% of normal adult men and two thirds of hospitalized men have palpable breast tissue. The prevalence may have increased because of some unrecognized cause. A confounding problem is that it can be difficult to distinguish enlargement of breast tissue from lipomastia, in which enlargement is caused by adipose tissue. [517] True gynecomastia can be separated from lipomastia by mammography[518] or sonography. [519] Autopsy data are not of much help in establishing the frequency of gynecomastia because they do not provide information about what fraction of gynecomastiaactive or inactiveis theoretically palpable. [520] For the purposes of this discussion, breast enlargement in men (other than in the three so-called physiologic states) may be indicative of an underlying endocrinopathy and deserves at least a limited evaluation. Histopathology and Etiology

Gynecomastia is frequently asymmetrical, and unilateral gynecomastia can be temporary in that one breast may enlarge months or years before the other. The process begins with proliferation of the stroma and the duct system, which elongates, buds, and duplicates. With time, progressive fibrosis and hyalinization are accompanied by regression of epithelial cells so that the ducts decrease in number. [521] On correction of the cause, resolution involves reduction in size and cell content of the epithelia followed by gradual disappearance of the ducts, leaving hyaline bands that may persist or eventually disappear. Gynecomastia is generally viewed as the consequence of absolute or relative estrogen excess, [522] and it can be classified as either physiologic or pathologic (Table 18-7) . Physiologic Gynecomastia

During three phases of male life, breast enlargement can be a normal finding. Gynecomastia in the Newborn

Enlargement of the breast in the newborn is probably due to maternal or placental estrogens, or both. The swelling may or may not be associated with milk production and usually disappears in a few weeks but can persist longer. [523] Adolescent Gynecomastia

Transient enlargement of the breast occurs in about 40% of adolescent boys. [524] The median age at onset is 14 years, the breasts may be asymmetrical and tender, and by age 20 only a small number of men have palpable vestiges of gynecomastia. The most severe disorder, termed pubertal macromastia (breast tissue > 4 cm), may persist into adulthood and is more commonly associated with an underlying endocrinopathy. [525] The cause of pubertal breast enlargement is uncertain. Plasma estradiol levels in boys normally reach the adult range before plasma testosterone, plasma estradiol levels are higher in boys with gynecomastia. [527] As a result, the

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TABLE 18-7 -- Classification of Endocrine Gynecomastia Physiologic Gynecomastia Gynecomastia in the newborn Adolescent gynecomastia Gynecomastia of aging Pathologic Gynecomastia Relative estrogen excess Congenital defects Congenital anorchia Klinefelter's syndrome Androgen resistance (testicular feminization and Reifenstein's syndrome) Defects in testosterone synthesis Secondary testicular failure (viral orchitis, trauma, castration, neurologic and granulomatous diseases, renal failure) Increased estrogen production Increased testicular estrogen secretion Testicular tumors Bronchogenic carcinoma and other tumors producing hCG

[526]

and average

True hermaphroditism Increased substrate for extraglandular aromatase Adrenal disease Liver disease Starvation Thyrotoxicosis Increase in extraglandular aromatase Drugs Estrogens or drugs that act like estrogens (diethylstilbestrol, estrogen-containing cosmetics, birth control pills, digitalis, estrogen-contaminated foods, phytoestrogens) Drugs that enhance endogenous estrogen formation (gonadotropins, clomiphene) Drugs that inhibit testosterone synthesis and/or action (ketoconazole, metronidazole, cimetidine, etomidate, alkylating agents, cisplatin, flutamide, spironolactone) Drugs that act by unknown mechanisms (busulfan, isoniazid, methyldopa, calcium channelblocking agents, captopril, tricyclic antidepressants, penicillamine, diazepam, marijuana, heroin) Idiopathic Gynecomastia hCG, human chorionic gonadotropin. plasma ratios of estradiol to testosterone and of estrone to adrenal androgens tend to be high in boys with pubertal gynecomastia. within the breast may also play a role in the gynecomastia of puberty. [529]

[ 528]

Local formation of estrogen

Gynecomastia of Aging

Gynecomastia can occur in otherwise healthy elderly men, but because gynecomastia can also be due to underlying pathology, the diagnosis of involutional gynecomastia is one of exclusion. Approximately 40% of elderly men at autopsy have true gynecomastia, [514] and the prevalence is approximately 70% in hospitalized men aged 50 to 69 years.[516] Because many older men take medications and have concurrent disorders, gynecomastia of aging, if it exists, may be caused by coexisting medical problems rather than by age itself. Changes in estrogen and androgen metabolism in men older than 70 years include decreases in mean levels of total and bioavailable plasma testosterone, elevation of plasma SHBG, increase in the rate of peripheral aromatization, decrease in the ratio of androgen to estrogen, increase in levels of plasma LH and FSH, and blunting or loss of the circadian rhythm of plasma testosterone levels (see earlier). Such changes may cause a sufficient alteration of the ratio of testosterone to estradiol to induce breast enlargement in the absence of disease. Pathologic Gynecomastia

Pathologic gynecomastia can be due to a relative (as in testosterone deficiency) or absolute increase in estrogen formation (as in Leydig cell tumors), to drugs, or to unknown causes. Relative Estrogen Excess

Failure of testosterone synthesis or action causes elevated plasma gonadotropin levels, and relative estrogen excess ensues because of the extraglandular aromatization of adrenal androgens and on occasion a secondary increase in testicular estrogen secretion (see Fig. 18-18A) . Congenital Defects

Congenital Anorchia.

Congenital anorchia is a rare, often familial disorder in which the testes are missing in phenotypically normal 46,XY males (see Chapter 22) . Affected individuals are thought to have bilateral cryptorchidism at birth, but no testes are located on surgical exploration of the abdomen. Because testicular hormones are necessary for male phenotypic development and because the penis is normal in this disorder, it is believed that testes are present and function normally until late in embryonic life and then regress for unknown reasons. Approximately half of anorchid men develop gynecomastia. In some anorchid men, Leydig cells secrete small amounts of testosterone into the circulation even if testes cannot be found at surgery. [530] Other men with congenital anorchia have profound testosterone deficiency and a small amount of estradiol formed by the indirect pathway androstenedione estrone estradiol in extraglandular tissues.[531] These findings imply that the critical factor for feminization is not the absolute level of estrogen but rather some ratio of testosterone to estradiol. Androgen appears to block estrogen action by competing with estradiol for binding to the estrogen receptor. [532] Klinefelter's Syndrome.

Approximately half of nonmosaic and a third of mosaic men with Klinefelter's syndrome have gynecomastia after the expected time of puberty. [300] Plasma FSH and LH levels are high, and the average plasma testosterone level is half normal, although some have normal testosterone levels. Variations in plasma levels of testosterone and estradiol are associated with variable degrees of androgenization and feminization in the disorder. [309] The causes of elevated plasma estradiol are complex. [533] [534] Early in adolescence, plasma testosterone is usually in the normal male range as the result of elevated plasma LH, which also causes enhanced estradiol secretion by the testes. Testicular function becomes progressively impaired with time, so that after age 15 years, serum testosterone and estrogen levels begin to decline [310] and the end stage resembles anorchia (see earlier). Diminished estrogen clearance may further increase estrogen/androgen ratios. Androgen Resistance (Testicular Feminization and Reifenstein's Syndrome).

Hereditary defects in the X-linked gene that encodes the androgen receptor cause a spectrum of syndromes of incomplete virilization in 46,XY men who have testes and male testosterone levels but who are resistant to their own and to exogenous androgens (see Chapter 22) . In the most severe form, affected individuals are phenotypic women with testicular feminization. If the impairment of receptor function is less complete, the phenotype is that of men with Reifenstein's syndrome (hypospadias and gynecomastia) or men with undervirilization or infertility, or both. [117] Women with complete testicular feminization and men with Reifenstein's syndrome have normal or elevated production

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Figure 18-18 Four different patterns of abnormal androgen-estrogen dynamics can result in the development of gynecomastia. The altered component in each pattern is highlighted in black, and specific examples of each type of abnormality are listed at the bottom of each panel. Details of normal androgen-estrogen dynamics are shown in Figure 18-8 (Figure Not Available) . HCG, human chorionic gonadotropin; HSD, hydroxysteroid dehydrogenase.

rates for testosterone and estradiol, presumably because of increased secretion by the testes in response to elevated plasma gonadotropin levels (Fig. 18-18B) .[117] FSH and LH levels are elevated because of resistance at the hypothalamic-pituitary level to negative-feedback control by testosterone. However, there is no direct relation between the rates of estrogen secretion in these disorders and the degree of feminization that results, probably because the degree of feminization is influenced by other factors such as the severity of the androgen resistance and the variable elevation of plasma androgen levels. Defects in Testosterone Synthesis.

Five inherited defects impair testosterone synthesis and prevent normal virilization of the male embryo (see Chapter 22) . Each of the defects involves a critical biochemical step in the conversion of cholesterol to testosterone. The completeness of the defects and the severity of clinical manifestations vary, but gynecomastia is common in two of the disorders, 3-HSDII deficiency and 17-HSDIII deficiency. Feminization in these disorders can arise from more than one mechanism. For example, normal or low levels of plasma estrogen can cause feminization in the presence of diminished androgen production, [535] analogous to the situation in congenital anorchia. Alternatively, estrogen production may be increased because of increased availability for extragonadal aromatization of steroids such as androstenedione that accumulate proximal to the enzymatic block. [536] Partial deficiency of 17-HSDIII and late-onset 3-HSDII deficiency are rare causes of gynecomastia in otherwise phenotypically normal men. Testicular Failure

Viral orchitis is the most common cause of testicular failure after puberty, and mumps is the most frequent etiology (see earlier). In men with gynecomastia and bilateral testicular atrophy related to orchitis, testosterone production is severely impaired, whereas production of estradiol and estrone is normal, arising almost entirely from extraglandular sources [326] (Fig. 18-18A) . The second most common cause of acquired testicular atrophy in the adult is trauma, and gynecomastia can result. [537] Trauma to the testes can be associated with elevated levels of plasma estradiol many years later. [537] Neurologic disease, including myotonic dystrophy and spinal cord injury, can also cause testicular atrophy. [538] Testicular atrophy, decreased plasma testosterone levels, elevated gonadotropin levels, and gynecomastia are also common in leprosy.[539] Gynecomastia is present in approximately half of men undergoing hemodialysis for renal failure.[357] [540] Plasma LH and FSH levels are elevated, the plasma testosterone level is low,

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and plasma prolactin levels are elevated. Estradiol levels may also be high (see earlier). Increased Estrogen Production

Estrogen production in men can increase because of (1) increased testicular secretion, (2) increased availability of substrate for extraglandular formation, or (3) increased activity of extraglandular aromatase itself. Increased Testicular Estrogen Secretion(see Fig. 18-18B)

Testicular Tumors.

Testicular tumors can feminize in three ways. First, germinal cell tumors ( embryonal carcinomas, choriocarcinomas, teratomas, and rarely seminomas) can produce hCG or fragments of hCG, which can act in uninvolved areas of the testes to stimulate the synthesis of estradiol and testosterone, [494] which in turn suppress plasma LH and FSH. Second, stromal cell tumors (Leydig and Sertoli cell tumors) can secrete testosterone and estradiol autonomously. About 20% of men with Leydig cell tumors have gynecomastia, [541] and gynecomastia may be even more common with Sertoli cell tumors.[498] [509] [542] Feminization can occur before such tumors are detectable by physical examination, but even small tumors can usually be identified by ultrasonography. [543] [544] Similarly, in choriocarcinomas [545] and in hepatocellular carcinomas,[546] aromatase in the tumor tissue can convert circulating adrenal and testicular androgens to estrogens. Third, some Sertoli cell tumors stimulate adjacent Leydig cells to secrete androgens that serve as substrate for aromatase in the tumor cells. discussion of diagnosis and management of testicular tumors.)

[ 547]

(See earlier for

Bronchogenic Carcinoma.

Lung cancer can cause an increase in hCG levels in plasma, and gynecomastia in this condition correlates with the amount of estradiol secreted by the testes. Indeed, hCG secretion by any tumor, such as by transitional cell tumors of the urinary tract, can cause feminization. [549]

[548]

True Hermaphroditism.

In true hermaphroditism (see Chapter 22) , both the ovarian and the testicular components of the gonads are endocrinologically active and cause a mixed pattern of feminization and virilization at puberty. [550] Gynecomastia is due to gonadal estrogen secretion (see Fig. 18-18B) , presumably by the ovarian elements of the ovotestes.[550] Increased Substrate for Peripheral Aromatase(see Fig. 18-18C)

Adrenal Disease.

In feminizing adrenal carcinoma, estrogen production is usually due to massive increases in the levels of the adrenal androgens androstenedione and DHEA, which serve as substrates for extraglandular aromatization. In rare instances, adrenal tumors secrete estrogen. [551] [552] Feminization in boys with congenital adrenal hyperplasia (as in CYP21 or CYP11A2 deficiency) is usually the consequence of increased production of androstenedione by the adrenal glands and hence of increased substrate for peripheral aromatase. [553] [554] In some instances, decreased testosterone levels may play a role in the gynecomastia. [555] Increased androstenedione is also the usual cause of feminization in men with 17-HSDIII deficiency.[536] Androgen Administration.

Administration of testosterone to children commonly causes gynecomastia, correlating with an increase in estrogens, whereas replacement with conventional doses in adult men increases plasma estradiol but rarely causes gynecomastia. [556] In contrast, testosterone administration to men with impaired liver function can cause

profound increases in plasma estrogen levels. In addition, administration of supra-physiologic amounts of aromatizable androgens can increase estradiol levels as much as sevenfold in normal men,[557] and gynecomastia is common in users of anabolic steroids. [558] [559] In probing patients' histories for possible causes of gynecomastia, it should be remembered that some androgens are not aromatizable or are weak substrates for the enzyme (see the following). Liver Disease.

Liver disease is a common cause of feminization. Gynecomastia is thought to be largely a result of over-production of estrogen. However, the liver is not the direct source of the estrogens, which are mainly due to decreased hepatic catabolism of androstenedione and the consequent increased availability of androstenedione for extrasplanchnic aromatization. [560] In carcinoma of the liver, feminization can be the consequence of increased aromatase activity in the tumor itself. [561] Starvation.

Gynecomastia was common in American prisoners of war during World War II. [562] About a third of the cases occurred during refeeding after release, other instances were associated with temporary improvements in the food supply during imprisonment, and most regressed within a few months. The pathophysiology of starvation gynecomastia may be similar to that with liver disease (see earlier). Thyrotoxicosis.

Thyrotoxicosis can cause gynecomastia. [563] Elevation of plasma estradiol levels is probably due to increased androstenedione production rates and increased formation of estrogen in extraglandular sites. [564] [565] Increase in Extraglandular Aromatase(see Fig. 18-18D)

Increased activity of aromatase enzymes in peripheral tissues [566] [567] [568] can increase estrogen production as much as 50-fold, [566] and in at least one family the trait appeared to be inherited in an autosomal dominant pattern through three generations, being manifested in females by precocious puberty and macromastia and in males by gynecomastia.[568] A characteristic feature is that the onset of gynecomastia correlates with the onset of adrenarche and occurs before the time of normal puberty. A similar trait is present in the Sebright bantam chicken, in which an autosomal dominant gene increases extraglandular aromatization more than 100-fold. [569] Drugs

Drugs can cause gynecomastia by direct action as estrogens, by enhancement of testicular production of estrogens, by inhibition of testosterone synthesis or action, or by unknown mechanisms. Estrogens and Estrogen Mimetics

Estrogens given to men in any form can cause gynecomastia, as in the treatment of prostatic cancer with diethylstilbestrol [570] and of transsexual men with estrogens.[571] Young men and boys are particularly sensitive to estrogens, and gynecomastia can develop as a result of industrial exposure or dermal ointments containing estrogens. [572] Identifying the source may require a high index of suspicion, as in the case of a barber who massaged the scalps of customers with ointment containing estrogen, [573] factory workers who manufacture oral contraceptives, [574] children of workers in a diethylstilbestrol manufacturing plant who absorbed the drug from the clothing of their fathers, [575]

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and offspring of women who use topical estrogen preparations. [576] Sufficient estrogen to induce gynecomastia can be absorbed by men during sexual intercourse with partners who use vaginal creams containing estrogen. [577] In the United States, no federal regulations cover estrogens in cosmetics, and estradiol levels may be as high as 18 ng/g in creams and 50 mg/dL in lotions. [578] Epidemics of gynecomastia among children have resulted from the ingestion of milk or meat from estrogen-treated cows, [579] raising the possibility that long-term exposure to small amounts of estrogens may be a cause of idiopathic gynecomastia. Sources may include meat and dairy products from animals treated with estrogens other than diethylstilbestrol, [580] endogenous estrogens in animal tissues, [581] or plant or fungal estrogens in foods. [582] About 10% of men given digitalis for a year had gynecomastia [583] ; however, abnormal liver function is common in such men, and gynecomastia is said to correlate better with congestive heart failure than with administration of digitalis. [584] Nevertheless, digitalis preparations associated with gynecomastia also have estrogenic effects on the vaginal epithelium in postmenopausal women. [585] Digitalis binds to the estrogen receptor and may act as a direct estrogen agonist. [586] Drugs That Enhance Endogenous Estrogen Formation

Administration of hCG can cause gynecomastia as a consequence of increased estradiol secretion by the testes. [587] Clomiphene citrate (both an estrogen agonist and antagonist) has been used to treat gynecomastia in boys, but paradoxically it can cause gynecomastia on withdrawal, presumably by increasing LH secretion and consequently increasing estradiol secretion by the testes. [588] Drugs That Inhibit Testosterone Synthesis or Action

The antifungal drug ketoconazole and other imidazoles block steroid hormone synthesis. [589] The inhibition of steroid synthesis by ketoconazole is transient, and plasma testosterone values return to normal after blood levels of the drug fall. Gynecomastia occurs only if the drug causes prolonged lowering of plasma androgen levels.[590] Gynecomastia is presumably due to altered ratios of estradiol to testosterone. [591] Antineoplastic agents can cause long-term impairment of testosterone synthesis, presumably through toxic effects on Leydig cells; such damage may occur when the therapy is for systemic neoplasms (e.g., alkylating agents for Hodgkin's disease) or for testicular cancers. [592] The cause of gynecomastia has not been elucidated, but it may be due to elevated plasma gonadotropin levels secondary to testicular damage and enhancement of testicular estrogen synthesis. [592] Gynecomastia is common in men treated with spironolactone. [593] At low doses, the drug prevents the binding of androgen to its receptor, and at high dose it inhibits testosterone synthesis.[331] [594] Antiandrogens, including cyproterone, flutamide, zanoterone, and bicalutamide, inhibit testosterone binding to the receptor and can cause gynecomastia. [595] [596] [597] [598] Gynecomastia is a common side effect of treatment with cimetidine, [599] which also blocks the binding of androgen to the androgen receptor and may inhibit the catabolism of estradiol. [600] Gynecomastia is less common in subjects receiving ranitidine. Suggestive evidence for induction of gynecomastia by an environmental antiandrogen has come from studies of an epidemic of temporary gynecomastia that affected Haitian refugees in five detention centers in the United States in 1981.[601] The delousing agent used in these centers binds to the androgen receptor and acts as an antiandrogen in rats. [602] All antiandrogens are believed to impair the feedback control of gonadotropin production and cause elevation of plasma LH, which in turn increases estradiol secretion from the testes. Drugs That Act by Unknown Mechanisms

A variety of drugs cause gynecomastia by unknown mechanisms. For example, gynecomastia occurred in boys and in men given human growth hormone. [603] [604] Many drugs are associated with gynecomastia with a frequency that is probably not coincidental; these include busulfan, calcium channelblocking agents, angiotensin-converting enzyme inhibitors, diazepam, isoniazid, methyldopa, omeprazole, penicillamine, tricyclic antidepressants, and a variety of antiviral agents, particularly protease inhibitors used for the treatment of HIV. [605] [606] [607] [608] [609] [610] [611] [612] Some of these agents may act by altering liver function. Both marijuana and

heroin are suspected causes of gynecomastia, but a direct causal relation has not been established.

[613] [614]

Idiopathic Gynecomastia

In all published series, 50% or more of subjects evaluated for gynecomastia did not have an endocrine or drug cause identifiable at autopsy [615] or by laboratory evaluation. [616] If one adds the instances in which the designated cause is tenuous, the idiopathic category may account for 75% of cases. It is not known whether men with idiopathic gynecomastia are in fact normal (as proposed by Nuttall [515] ), whether a feminizing factor had been transiently present but had disappeared at the time of evaluation, whether the gynecomastia is due to long-term exposure to small amounts of one or more environmental estrogens or antiandrogens, or whether the gynecomastia is the consequence of subtle, unrecognized endocrine disease. The extent to which minor endocrine disorders are not recognized with current methodologies is uncertain. The fact that gynecomastia can develop as the result of subtle environmental exposure to estrogens or antiandrogens (as described earlier) raises the possibility that a large fraction of idiopathic gynecomastia may be the consequence of unrecognized exposure to endocrine disruptors. [602] The critical clinical point is that, whatever the cause, the diagnosis of idiopathic gynecomastia carries no known import related to health. Lack of Role of Prolactin in Gynecomastia

Plasma prolactin levels are usually normal in men with gynecomastia of diverse causes, and men who have prolonged elevation in plasma prolactin secondary to use of psychotropic drugs do not commonly have gynecomastia. [617] [618] Consequently, prolactin is not believed to play a direct role in the disorder. This conclusion is in keeping with the fact that prolactin is not a growth hormone for the breast. Furthermore, when gynecomastia develops in men with prolactin-secreting tumors of the pituitary gland and high plasma prolactin levels or in men taking psychotropic agents, the gynecomastia is probably the consequence of secondary testicular failure as a result either of the effects of the tumor mass or of inhibition of LH secretion by prolactin. Diagnosis

The dilemma is to distinguish men with significant endocrine disease from those with idiopathic gynecomastia. In general, only men with symptomatic gynecomastia are evaluated; however, if there is a question about whether the gynecomastia is real, the issue is best solved by mammography or ultrasonography. [518] [519]

746

Most of the known causes of gynecomastia can be identified by a work-up that includes the following: 1. A careful drug history that encompasses potential environmental and indirect exposures to endocrine substances. 2. A detailed physical examination including the testes (the finding of small testes bilaterally suggests testicular insufficiency, and asymmetrical testes raise the possibility of testicular tumors). 3. Evaluation of liver function. 4. A limited endocrine work-up, including (a) measurement of plasma DHEAS or urinary 17-ketosteroids (usually elevated in adrenal feminizing states), (b) measurement of plasma estradiol (helpful if elevated but usually normal), (c) assessment of plasma hCG (sometimes elevated with testicular tumors), and (d) measurement of plasma LH and testosterone. * If these parameters are normal, as is frequently the case, the usual recourse is to observe the patient without treatment. If the symptoms persist or worsen and if the enlargement is progressive, a more extensive evaluation may have to be undertaken. Treatment

The difficulty in treating gynecomastia is inherent in its natural history. If the feminizing process persists for a long period, the initial glandular hyperplasia is replaced by progressive fibrosis and hyalinization that do not regress after the source of excess estrogen is corrected. [522] Consequently, surgery remains the mainstay of therapy and is frequently indicated for psychological and cosmetic reasons. Such surgery is usually accomplished through a circumareolar approach. [619] Medical management is most successful when it is addressed to gynecomastia of recent onset or to prevention of its development. Testosterone administration has inconsistent effects in Klinefelter's syndrome but can cause dramatic improvement in other forms of testicular failure (e.g., anorchia, viral orchitis). Several drugs have been tried for gynecomastia, including the antiestrogens tamoxifen [620] and clomiphene, [621] the aromatase inhibitor testolactone, [622] and danazol, [623] a weak androgen that inhibits gonadotropin secretion. In one study, tamoxifen was about twice as effective in treating idiopathic gynecomastia as danazol [624] ; in another study, tamoxifen was uniformly effective in treating the gynecomastia induced by antiandrogen treatment in men with prostatic carcinoma. [625] Treatment with dihydrotestosterone (which cannot be aromatized to estrogen) was also reported to provide symptomatic improvement. [626] Perhaps the most effective therapy for gynecomastia is to prevent its development by radiating the breasts before the institution of estrogen therapy in men with prostatic carcinoma[627] or of antiandrogen therapy in male sex offenders. [628] This therapy is about 90% effective, and the complication rate is low.



Impairment of Estrogen Formation or Action

The study of men with single-gene mutations that impair estrogen formation or action has provided insight into the role of estrogen in male physiology. forms of estrogen deficiency are rare, but the fact that the phenotypes in the two disorders are similar establishes the importance of this role.

[138] [629]

These

Aromatase Deficiency

Aromatase deficiency is the consequence of autosomal recessive loss-of-function mutations in the CYP19 gene. [56] [630] In the two reported men with this disorder, childhood development was considered normal but skeletal growth continued into the 20s despite pubertal maturation and resulted in tall stature. This growth pattern was associated with failure of epiphyseal closure, marked delay in bone age, and osteopenia. One man had undetectable estrogen in plasma and elevated levels of testosterone, dihydrotestosterone, and gonadotropins; testicular volume was normal, and semen analysis was declined.[56] The other affected man was evaluated because of tall stature, infertility, and skeletal pain associated with severe osteopenia; the testicular volume was 8 mL bilaterally, the sperm density was very low with many immotile sperm, and testicular biopsy revealed a maturation arrest at the spermatocyte stage.[630] The etiology of the infertility in the latter patient was not clear because a brother was infertile in the presence of a normal CYP19 gene, suggesting that the infertility in this family may be due to some other disorder. The man responded dramatically to estradiol therapy with an increase in bone density to the normal range, resolution of bone pain, and lowering of elevated levels of total and LDL cholesterol and triglycerides. [630] These men had different homozygous missense mutations in the CYP19 gene that caused single amino acid substitutions and resulted in the formation of mutant enzymes with 0.2% to 0.4% of the activity of normal enzyme.[56] [630] Estrogen Receptor Deficiency

An autosomal recessive, loss-of-function mutation in the estrogen receptor gene has been described in a man with tall stature, unfused epiphyses, osteopenia, and acanthosis nigricans. [55] Virilization was normal, and he had a normal level of plasma testosterone. Plasma levels of estradiol, estrone, FSH, and LH were elevated, and semen analysis revealed a normal sperm density but decreased sperm motility. Serum lipoprotein levels were normal, but the presence of hyperinsulinemia and impaired glucose tolerance indicated insulin resistance. He did not respond to treatment with high-dose, transdermal estradiol sufficient to raise the plasma free estradiol 10-fold above normal, as indicated by no change in plasma gonadotropins or in bone density after 6 months. He was homozygous for a missense mutation in exon 2 of the estrogen receptor gene that resulted in a premature termination codon and hence precluded the formation of functional receptor. In summary, the evidence from these rare disorders of estrogen formation and action indicates that estrogen plays a major role in controlling skeletal maturation and both the accrual and maintenance of bone mass in men. In both conditions, there was no pubertal spurt in growth; growth was instead steady and continued in association with failure of epiphyseal closure. Despite testosterone levels that were normal or increased, gonadotropin levels were elevated. These findings are in accord with studies of aromatase inhibitors, which indicate that estrogens are important for feedback control of gonadotropin secretion at the level of the pituitary and the hypothalamus.[631] Abnormalities of carbohydrate and lipid metabolism in these patients appear to be inconsistent. The fact that gender identity and gender role behavior are male in both conditions indicates that estrogen does not play a critical role on these parameters. [629] *High LH and normal or low testosterone levels suggest testicular insufficiency; low LH and low testosterone levels suggest hypopituitarism, estrogen secretion from a tumor, or an exogenous source of estrogen; and high LH and high testosterone levels suggest androgen resistance).

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HORMONAL THERAPY Androgen Therapy

When administered by mouth, testosterone is absorbed into the portal blood and degraded by the liver so that only a small fraction reaches the systemic circulation. Parenterally injected testosterone is also rapidly absorbed and degraded. As a consequence, effective androgen therapy requires either administration of testosterone in a slowly absorbed form (transdermal or micronized oral preparations) or administration of chemically modified analogues. Such chemical alterations either retard absorption or catabolism to maintain effective blood levels or enhance the androgenic potency of each molecule so that physiologic effects can be achieved at a lower plasma level of drug. Three general types of modification of testosterone are clinically useful: 1. Esterification of the 17-hydroxyl group (type A). 2. Alkylation at the 17 position (type B). 3. Modification of the A, B, or C rings, particularly substitutions at the 1, 2, 9, and 11 carbons (type C) (Fig. 18-19) . Most agents actually contain combinations of ring structure alterations and either 17 alkylation or esterification of the 17-hydroxyl group. Esterification of testosterone with various carboxylic acids decreases the polarity of the steroid, makes it more soluble in the fat vehicles that are used for injection, and hence slows release of the injected steroid into the circulation. [632] The esters of 19-nortestosterone have particularly slow release and turnover rates. [381] The longer the carbon chain in the ester, the more fat soluble the steroid becomes and hence the more prolonged the action. For example, testosterone propionate must be injected daily, whereas testosterone cypionate and testosterone enanthate can be administered every 2 or 3 weeks. [633] Even more slowly hydrolyzed esters are under investigation,

Figure 18-19 Types of androgen preparations available for clinical use. Type A derivatives are esterified in the 17 position. Type B steroids have alkyl substitutions in the 17 position. Type C derivatives involve a variety of alterations of ring structure that enhance activity, impede catabolism, or influence both functions. Most androgen preparations involve combinations of type AC or type BC changes.

such as testosterone buciclate, which is administered every 12 weeks, [634] and testosterone undecanoate, which is administered every 6 weeks. [635] Testosterone cypionate or enanthate was for many years the treatment of choice for male hypogonadism. Although testosterone esters can be detected in plasma, they must be hydrolyzed before the hormone acts so that the effectiveness of therapy can be monitored by assaying the plasma level of testosterone after administration. Most esters must be injected, but twomethenolone acetate and testosterone undecanoatecan be administered by mouth. Testosterone undecanoate is absorbed through the lymphatic system into the systemic circulation, and physiologic blood levels of testosterone can be achieved at doses of approximately 120 mg/day. [636] Because of rapid turnover, testosterone undecanoate must be administered two to three times a day.[637] The reason for the oral effectiveness of methenolone acetate (and of mesterolone) is not entirely clear. [638] The use of transdermal testosterone formulations makes it possible to sustain serum testosterone levels in the normal male range while avoiding the necessity for parenteral administration. These formulations include a scrotal patch, two nonscrotal patches, and a gel. The scrotal patch Testoderm is designed to deliver either 4 or 6 mg of testosterone over 24 hours and takes advantage of the fact that absorption across the scrotal skin is efficient in the absence of permeation enhancers. [639] After application in the morning, serum levels peak in 2 to 3 hours and are maintained throughout the day. The nonscrotal patches, Androderm and Testoderm TTS, differ in recommended application sites and times to peak serum levels, but both provide physiologic testosterone levels throughout the day. [640] [641] [642] Androderm, available in 2.5- and 5-mg doses, is applied at bedtime; peak levels are achieved in 8 hours, and the application site must be rotated to avoid skin irritation. Testoderm TTS delivers 5 mg of testosterone from a larger surface area, is applied in the morning, results in a maximal serum level in 2 to 3 hours, and appears to cause minimal skin irritation. [641] In a randomized comparative study, Androderm therapy did not cause the temporary supraphysiologic levels of estradiol and of total and bioavailable testosterone that occur after injections of testosterone enanthate. [642] The transdermal preparation resulted in more normal testosterone levels, less frequent suppression of plasma LH, and less frequent elevation of plasma hemoglobin levels. [642] A transdermal 1% testosterone gel has been developed for the application of 50 to 100 mg of testosterone to the shoulders or abdomen each morning, the usual dose being 50 mg.[643] [644] The absorption of testosterone appears to be largely independent of the surface area to which it is applied, and steady-state serum levels are achieved within a few days. The hands must be washed carefully after each application to avoid inadvertent transmission of the hormone, but the application site should not be washed for 6 hours to maintain absorption efficiency. Application site skin-to-skin transfer may occur with close physical contact. In comparison with the 5-mg nonscrotal patch, 50 mg of testosterone gel causes somewhat higher serum testosterone levels. [644] Administration of a 5-mg preparation of testosterone cyclodextrin sublingually three times a day also results in normal plasma testosterone levels in hypogonadal men.[645] 17-Alkylated androgens, such as methyltestosterone and methandrostenolone, are effective by mouth because alkylated steroids are absorbed into the portal circulation, are slowly catabolized by the liver, and reach the systemic circulation in effective amounts. For this reason, 17-methyl or 17-ethyl substitution is present in most orally active androgens. Because 17-alkylated androgens are believed to act within the cell as such (i.e., the alkyl groups are not removed), because assays are not routinely available for monitoring blood levels, and because

748

they can cause abnormal liver function, these steroids have a limited role in medicine.

[646]

Other alterations of the ring structure either alter the metabolism or enhance the potency of a given molecule. For example, the potency of fluoxymesterone, 19-nortestosterone, and 1-methylsubstituted steroids may be enhanced because they are poor precursors for estrogen formation in extraglandular tissues. [647] Similarly, 19-nortestosterone is a more potent androgen than testosterone because its more planar ring structure, like that of dihydrotestosterone, fits more tightly into the binding site of the androgen receptor. [648] 7-Methyl-19-nortestosterone cannot be 5-reduced and may be useful when androgen replacement is needed with minimal effects on the prostate.[649] [650] As with 17-alkylated steroids, androgens with ring alterations are not converted to testosterone in vivo, and specific assays for each must be used to monitor blood levels. One orally effective androgen, mesterolone, is neither esterified nor alkylated in the 17 position and cannot be aromatized to estrogens in peripheral tissues. Consequently, effective androgen replacement can be achieved by oral administration without causing abnormalities of liver function; unfortunately, the steroid is ineffective in regulating gonadotropin secretion and consequently is a poor agent for routine androgen replacement therapy. [638]

The subcutaneous implantation of testosterone-filled silicone elastomer (Silastic) capsules results in slow release of hormone into plasma for long periods, [651] but this mode is impractical because of the large size of such capsules. When large amounts of testosterone are given by mouth in microparticulate form (200 to 400 mg/day), physiologic blood levels can be achieved, but the preparation has to be taken several times a day [652] and these doses induce hepatic drug-metabolizing enzymes, the long-term effects of which are uncertain. [653] Androgens for Normal Men

When the plasma testosterone level is raised above the normal range, both the basal levels of LH and FSH and the peak levels after GnRH administration are diminished. As a consequence, the testicular volume is decreased about 20%, sperm production is uniformly decreased by 90% or more, and the volume of the ejaculate remains unchanged. [654] [655] Acne is common, and the serum estradiol level increases twofold. [654] Administration of usual replacement doses of testosterone enanthate (100 mg/week) to normal men caused significant decreases in truncal and total body fat and increases in BMD in the spine, [656] [657] effects that are probably the consequence of the temporary increases in testosterone levels above the normal range after the injections. Administration of six times this dose (600 mg/week) of testosterone enanthate to normal men caused an increase in fat-free body mass, triceps and quadriceps muscle size, and muscle strength. [658] [659] In a similar study, there was no change in levels of prostate-specific antigen (PSA). [660] In a dose-response study in healthy young men, testosterone enanthate caused increases in fat-free body mass, muscle strength, and hemoglobin levels and decreases in fat mass and HDL cholesterol levels, beginning with a dose that was just above replacement levels (125 mg/week). [661] Sexual function, visual-spatial cognition, mood, and PSA levels did not change at any dose. [661] Androgens for Hypogonadal Men

The aim of androgen therapy in hypogonadal men is to restore or normalize male secondary sexual characteristics (beard, body hair, external genitalia) and male sexual behavior and to promote normal male somatic development (hemoglobin, voice, muscle mass, nitrogen balance, and epiphyseal closure). Because a reliable assay for plasma testosterone is widely available for monitoring therapy, the treatment of androgen deficiency is straightforward and almost universally successful. The parenteral administration of a long-acting testosterone ester, such as 100 to 300 mg of testosterone enanthate at 1- to 3-week intervals, results in a sustained increase in plasma testosterone concentration to the normal male range or slightly above. [314] [633] [642] The usual replacement regimen is 200 mg every 2 weeks. [633] Similar effects are obtained with the transcutaneous administration of testosterone. [640] [642] [644] Such regimens usually reduce the plasma LH level (if elevated) and maintain serum testosterone within the normal range. [633] Serum testosterone should be measured after 4 to 6 weeks of therapy to assess adequacy of dosage; the trough level is measured in men receiving intramuscular testosterone, and midmorning levels are assessed in men receiving transdermal formulations. If the hypogonadism is primary and of long duration (as in Klinefelter's syndrome), suppression of the plasma LH value to the normal range may not occur for many weeks, if at all. [314] In postpubertal testicular failure, even of many years' duration, resumption of normal sexual activity is usual after adequate replacement, primarily because of increased libido [662] and increased frequency of erections. [663] Androgen therapy does not restore spermatogenesis in hypogonadal states, but the volume of the ejaculate, derived largely from the prostate and seminal vesicles, and other secondary sexual characteristics return to normal. Treatment of hypogonadal men with testosterone results in growth of the prostate to the same degree as that of age-matched controls. [664] Testosterone replacement in such men can cause dramatic changes in body composition, strength, and BMD, although maximal effects may not be seen for as long as 2 years. [665] [666] [667] [668] [669] [670] Improvement in BMD involves both trabecular and cortical bone and is independent of the age at which replacement is started. [670] In men of all ages in whom hypogonadism develops before expected puberty (such as in hypogonadotropic hypogonadism), it is appropriate to bring plasma testosterone into the adult range slowly. When therapy is begun at the time of expected puberty, the normal events of male puberty proceed in the usual fashion. If therapy is delayed until after the time of usual puberty, the degree of virilization is variable, but many such men undergo a late but relatively complete anatomic and functional male maturation (Fig. 18-20) . Intermittent androgen therapy is sometimes given to prepubertal hypogonadal boys with microphallus to stimulate penile growth[183] [284] (also see Chapter 22) , and such therapy does not appear to have an adverse effect on final penile size. [671] If patients are monitored closely and androgen is given for only short periods, such therapy also probably has no effect on somatic growth. In boys of pubertal age with either isolated hypogonadotropic hypogonadism or primary testicular deficiency, the initial administration of small doses of testosterone esters followed by a gradual increase to doses of 100 to 150 mg/m 2 of body surface area per month results in a normal pubertal growth spurt. [672] Penile growth, deepening of the voice, and appearance of other secondary sexual characteristics usually commence during the first year of treatment. Puberty in normal boys extends over several years, and treatment that is designed to replicate normal development cannot shorten the process greatly (see Chapter 24) . The usual practice in hypogonadal boys is to institute androgen therapy between the ages 12 and 14 years, depending on their subjective need for sexual development. Testosterone exerts its full action only in the presence of a balanced hormonal environment and particularly in the presence of adequate levels of growth hormone. Consequently, hypogonadal boys with coexisting growth hormone deficiency have a diminished

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Figure 18-20 Effect on penile size of 200 mg of testosterone cypionate intramuscularly every 2 weeks for 11 months in a previously untreated 22-year-old man with microphallus caused by hypogonadotropic hypogonadism. (From Griffin JE, Wilson JD. Disorders of sexual differentiation. In Walsh PC, Retik AB, Stamey TA, et al [eds]. Campbell's Urology, 6th ed. Philadelphia, WB Saunders, 1992, pp 15091542.)

response to androgens in regard to both growth and the development of secondary sexual characteristics unless growth hormone is given simultaneously. noted earlier, the promotion of growth by testosterone is the consequence of enhanced secretion of growth hormone and IGF-I. [199] [674]

[672] [673]

As

The presence of prostate or breast cancer is a contraindication to androgen therapy, and men older than 50 should be screened for preexisting prostate cancer by digital rectal examination and measurement of the serum PSA level. Indeed, in men with severe hypogonadism, biopsy can reveal occult prostate cancers in which PSA levels are not elevated because of androgen deficiency. [675] Consequently, older men should have a repeated digital rectal examination and PSA measurement within 2 to 3 months of initiation of testosterone therapy and at 6- to 12-month intervals thereafter. Androgen replacement does not cause an increase in prostate size or PSA level above that of age-matched men, [664] and benign prostatic hyperplasia is not a contraindication to androgen replacement. However, switching androgen replacement from testosterone esters to transdermal testosterone causes lowering of PSA levels,[676] suggesting that more physiologic levels of testosterone provide less stimulation to the prostate. The presence of polycythemia or obstructive sleep apnea may be a relative contraindication to testosterone therapy (see "Toxic Side Effects" following). Androgens for Healthy Older Men with Decreased Bioavailable Testosterone Levels

Because many of the changes in body composition, libido, and erectile function with aging also occur with male hypogonadism, androgen administration has been evaluated in healthy older men. The criterion for inclusion in such studies is a low level of total testosterone or of bioavailable (nonSHBG-bound) testosterone. In one study of older men with a total serum testosterone less than 14 nmol/L (10 4 ) at adolescence to initiate breast development. [668] Conceptions have been documented despite extensive karyotypic studies revealing only a 45,X cell line in multiple tissues.[669] [670] [671] In a large study of Turner's syndrome patients older than 12 years, including, in addition to 45,X patients, those with X chromosome mosaicism and structural abnormalities, spontaneous pubertal development occurred in 16%, including menarche at a mean age of 13 years. [672] Regular menses occurred 9 years post menarche in just over one third of this group. The presence of a second X chromosome was much more evident in those with spontaneous puberty. The figure for spontaneous pregnancy appears to be 3% to 5%. [672] [673] In addition to variability in the rate of follicular atresia, another possible explanation for the presence of

oogonia in 45,X individuals is that a certain number of 45,X germ cells may undergo mitotic nondisjunction with the formation of 46,XX oogonia. [674] This process normally occurs in the female creeping vole and serves as a sex-determining mechanism in this species. Alternatively, some fertile 45,X patients may be unrecognized sex chromosome mosaics. Women with a 45,X cell line have increased fetal wastage and an increased number of chromosomally abnormal liveborn infants, including those with gonadal dysgenesis or Down's syndrome. [671] [675] [676] [677] Adrenarche in patients with gonadal dysgenesis is associated with a normal rise in adrenal androgen production in childhood but sparse pubic and axillary hair. Before the age of 10 years, the plasma concentration of adrenal androgens is normal, [678] but levels of dehydroepiandrosterone (DHEA), testosterone, and androstenedione are lower than normal after age 15, reflecting absence of the gonadal contribution. [679] Adrenarche occurs independently of gonadarche. [678] Rarely, enlargement of the clitoris may be present at birth or develop at puberty. Secretion of androgens by "Leydig cells" in the gonadal streak is a possible cause, as is the presence of a cryptic Y cell line. Males with a 45,X karyotype have a Y to X chromosome or a Y-autosome translocation involving variable segments of the euchromatic (sex-determining) region of the Y chromosome.[680] [681] [682] [683] [684] [685] Translocations have been reported involving the short arm of chromosomes 5, 14, 15, and 18 and the X chromosome. [680] [681] [682] [683] [684] [ 685] Most patients have had either minor or major

892

anomalies not usually associated with the syndrome of gonadal dysgenesis, such as the cri-du-chat syndrome. These additional anomalies are no doubt related to the autosome involved in the translocation and to the degree of deletion involved. Incidence in Abortuses, Newborns, and Twins.

The incidence of gonadal dysgenesis is approximately 1 per 2000 live female births, [686] [687] and approximately 50% of the patients have a 45,X karyotype. There is, in addition, a considerable loss of 45,X embryos and fetuses. [688] About 7% of spontaneous abortuses have a 45,X constitution. [688] It is estimated that the frequency of 45,X zygotes is 2%, probably the most common chromosome anomaly in humans but fewer than 1% of 45,X conceptuses survive to term. [689] Hook and Warburton [690] have analyzed chromosome karyotypes in embryonic and fetal deaths and demonstrated a significant disparity between the 45,X karyotype and those with mosaicism and/or an isochromosome for the long arm of the X chromosome (Xqi). They postulated a "fetoprotective" effect of more than one dose of some locus or loci on the long arm of the X chromosome. [690] Associated Disorders.[613 ] [ 691]

Turner's syndrome carries a threefold increase in mortality and a reduction in life expectancy of 6 to 13 years; it is less in 45,X patients than in non-45,X Turner syndrome.[692] The incidence of autoimmune disorders is increased; the most prevalent is autoimmune thyroiditis and Graves' disease, which occurs in 15% to 30%. [613] The prevalence of thyroid antibodies and hypothyroidism (or hyperthyroidism) increases during childhood and adolescence [693] [694] and in adulthood may approach 50% of patients (a 10-fold relative risk). [691] Early diagnosis may be facilitated by monitoring levels of thyroid antibody and basal thyroid-stimulating hormone with the use of sensitive assays. Basal and thyrotropin-releasing hormoneinduced concentrations of prolactin may be elevated in euthyroid patients with gonadal dysgenesis. Prevalences of rheumatoid and psoriatic arthritis and inflammatory bowel disease are increased in patients with a 45,X karyotype. [613] [695] Carbohydrate intolerance with mild insulin resistance is common, especially after age 16 years and may become worse with obesity or during treatment with growth hormone or oxandrolone.[696] [697] The risk of type 2 diabetes mellitus is increased fourfold and that of type 1, onefold. [691] Mean cholesterol levels may be elevated after 11 years of age, independent of age and body mass index. [698] The prevalence of chronic liver disease is increased; Gravhold and co-workers [691] report a relative risk of 5.7 for cirrhosis; 44% of women had an elevated concentration of serum liver enzymes. [613] The risk of developing Crohn's disease and ulcerative colitis is increased and the inflammatory bowel disease is often severe. [613] As discussed earlier, congenital renal anomalies are common (about ninefold greater than the general population) and the risk of the obstructive uropathy and pyelonephritis is greatly increased. In addition, vascular malformations involving the kidney are more prevalent. During childhood, problematic otitis media is common and may result in conductive hearing loss. [699] Abnormalities in the growth of the temporal bone, condylar cartilage, and spheno-occipital synchondrosis result in an abnormality in the positioning of the external auditory meatus and the relation of the middle ear to the eustachian tube in patients with the syndrome of gonadal dysgenesis. [699] These changes, along with abnormalities in the shape of the palate, are thought to be responsible for the increased incidence of otitis media. Sensorineural deafness is present in about two thirds of adult patients. [700] The frequent episodes of otitis media and the sensorineural hearing loss are independent variables in gonadal dysgenesis; the sensorineural hearing loss may be related to loss of genes on the X chromosome responsible for the gonadal dysgenesis phenotype. [701] In a Danish study, Turner's syndrome women had a fivefold increased risk of developing cancer of the colon and rectum. anorexia nervosa is increased; its onset usually coincides with the initiation of estrogen treatment.

[ 691]

As noted previously, the prevalence of

Origin of 45,X Constitution and Phenotype.

A 45,X chromosome constitution ( see Fig. 22-1A and Fig. 22-4 ) may be a consequence of nondisjunction [42] or chromosome loss during gametogenesis in either parent that results in a sperm or ovum lacking a sex chromosome. Although errors of mitosis in a normal zygote often lead to mosaicism, a purely 45,X constitution may arise at the first cleavage division from anaphase lag with loss of a sex chromosome or, less likely, from mitotic nondisjunction with failure of the complementary 47,XXX or 47,XYY cell line to survive (see Fig. 22-4) . Loss of one X or Y chromosome between fertilization and the first cleavage division may be a frequent but not the only cause of a 45,X embryo. [42] Several lines of evidence support the hypothesis of a mitotic error (as well as meiotic errors) in this syndrome: (1) the lack of association with advanced maternal age, in contrast to XXY Klinefelter's syndrome (indeed the incidence of 45,X conceptuses is increased in teenage pregnancies) [702] [703] ; (2) the prevalence of sex chromosome mosaicism; (3) the increased frequency of twinning in sibships with a 45,X individual [704] ; and (4) the occurrence of a 46,XY monozygotic cotwin of a 45,X individual. [705] Family studies of X-linked traits (e.g., color blindness, Xg blood group) indicate that loss of the paternally derived X chromosome is more common than would be expected with random loss of either the maternally or the paternally derived X chromosome; in informative pedigrees, 77% of 45,X individuals have loss of the paternal sex chromosome (45,XM ), and 23% have loss of the maternal X chromosome (45,XP ). Similar findings have been obtained with the use of molecular cytogenetic analysis. [706] [707] The deviation in parental origin of the retained X chromosome [713] raised the possibility that retention of the maternal X chromosome might play a role in survival in addition to affecting the phenotype through "imprinting." [708] However, the percentage of aborted fetuses with a 45,X M karyotype is the same as in liveborn infants. [709] [710] Therefore, imprinting does not appear to affect fetal survival in 45,X individuals. Although some studies showed no effect of imprinting on phenotype, [711] 45,XM patients appear to have an increased prevalence of cardiovascular anomalies and webbed neck; furthermore, the height in 45,X M correlates more strongly with maternal height than with midparental height. [712] A putative imprinted gene on the maternal X chromosome, [623] which affects social cognition, is discussed earlier in the section on clinical aspects. The very high embryonic and fetal mortality of 45,X conceptuses, as opposed to those with 45,X/46,XX or 45,X/46,XY mosaicism or a structurally abnormal X

chromosome, raised the possibility that all liveborn individuals with the gonadal dysgenesis syndrome are mosaics. [714] This hypothesis has not been supported by cytogenetic data [715] or by molecular analysis with X chromosome probes. [716] [717] However, the possibility of prenatal mosaicism and nonrandom loss of structurally abnormal sex chromosomes postnatally has not been ruled out. Molecular analysis with the use of Y-specific probes has been inconclusive. [47] From 0% to 33% of patients with gonadal dysgenesis whose karyotype is other than 45,X/46,XY have a low percentage of Y chromosome material. [47] [718] Chu and colleagues [47] studied 87 patients with the use of multiple Y chromosome probes and found 3.4% to be positive for low-percentage

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mosaicism for all or part of the Y chromosome. The similar results obtained by Binder and colleagues [719] indicate a low level of Y chromosome mosaicism in 45,X and chromatin-positive individuals with gonadal dysgenesis. The significance of low-percentage mosaicism for Y chromosome material and its relation to gonadal differentiation and the risk of malignancy are still to be determined. Cryptic Y chromosome identification by sensitive PCR techniques may, however, not show concordance between clinical signs of hyperandrogenism and molecular studies on gonad material. In such Y-negative gonads there is hilus cell hyperplasia. The routine screening of all Turner's syndrome patients for the presence of SRY is not recommended. The classic gonadal dysgenesis phenotype is usually associated with absence of all or a proximal portion of the short arm of the X or Y chromosome. The haploinsufficiency of genes in these loci that are not inactivated is postulated to cause the phenotype. Page and co-workers suggested that the genes that encode ribosome protein S4X (RPS4X), and its homologue on the Y chromosome (RPS4Y), are candidate genes. [124] However, the location of this gene on the long arm of the X chromosome and the fact that it is expressed in patients with 46,XXp and 46X,Xqi karyotypes make it an unlikely candidate. [720] Zinn and associates using both cytogenetic and molecular analyses found evidence of a critical region, Xp11.2p22.1, that included loci for short stature (in addition to the more distal SHOX gene), gonadal failure, high arched palate, and thyroid autoimmunity. [721] Ogata and Matsuo[54] postulated that the gonadal dysgenesis phenotype is multifactorial in origin. According to their construct, the phenotype is related to (1) quantitative loss or alteration of euchromatic or noninactivated genes (haploinsufficiency), leading to global nonspecific developmental defects; (2) haploinsufficiency of pseudoautosomal and/or Y-specific growth genes and lymphogenic genes, resulting in short stature and the "deformative" stigmata; and (3) oocyte loss and gonadal dysgenesis from impaired or failed chromosome pairing during meiotic prophase. [54] [722] The underlying cause of this sex chromosome abnormality is not known. An increased frequency of thyroid autoimmunity in patients with the syndrome of gonadal dysgenesis and in their parents suggests that the genetic predisposition to develop autoantibodies in one or both parents is associated with an increased prevalence of the 45,X constitution and other chromosome abnormalities in the offspring. Infants with the syndrome of gonadal dysgenesis have been born after artificial insemination and in vitro fertilization. [723] Familial occurrence of 45,X gonadal dysgenesis is rare. Diagnosis and Treatment.[ 613]

Phenotypic females with the following features should have a karyotype analysis: (1) short stature (>2.5 SD below the mean height value for age), (2) somatic stigmata associated with gonadal dysgenesis, and (3) delayed adolescence with increased plasma or urinary gonadotropin levels. Although determination of the X chromatin pattern (Barr body) is a rapid method of screening, karyotype analysis is the definitive procedure. The concentration of plasma FSH and inhibins is useful in assessing the functional status of the gonads. Even though many severely affected 45,X individuals with prominent dysmorphic features (lymphedema, loose folds of skin over the back of the neck) or coarctation of the aorta are recognized at birth or early infancy, the diagnosis in the less severely affected may be delayed until childhood when short stature is evident or until the age of puberty when secondary sex characteristics fail to appear. It is important to obtain a karyotype on all girls with unexplained short stature especially those with even subtle dysmorphic features of the syndrome of gonadal dysgenesis. Savendahl and Davenport found that (excluding

Baseline Karyotype

TABLE 22-10 -- Suggested Follow-up of Adults with Syndrome of Gonadal Dysgenesis (Turner's Syndrome) Annual Physical examination (e.g., BMI, blood pressure, CVS)

35 Yearly Echocardiography Bone densitometry

Renal and pelvic ultrasound

Thyroid function

Echocardiography

Fasting lipids

Thyroid autoantibodies

Fasting blood glucose

Gonadotropins

Liver function

Audiogram

Renal function BMI, Body mass index; CVS, cardiovascular system. From Elsheikh M, Dunger DB, Conway GS, Wass JA. Turner's syndrome in adulthood. Endocr Rev 2002; 23:120140. those in whom the diagnosis was made in infancy) overall delay in diagnosis was 7.7 years. [724] Early diagnosis is important for more optimal long-term management, treatment, and counseling, including, if appropriate and with informed parental consent, the use of recombinant hGH treatment before the child falls below -2.0 SD in height, to achieve better growth in childhood and potentially normal adult stature. The following studies should be done in women when the diagnosis of the syndrome of gonadal dysgenesis is made: an intravenous pyelogram or ultrasonographic examination to exclude a renal anomaly; an echocardiogram or MRI study to assess cardiovascular function; periodic hearing examination and evaluation of thyroid function and thyroid antibodies; regular measurements of plasma glucose levels after adolescence; and monitoring for scoliosis and bone density in late adolescence and adulthood for evidence of osteopenia. [725] Guidelines for the diagnosis and management for Turner's syndrome in childhood and adulthood have been updated following an international consensus workshop [726] and reviewed by Elsheikh and co-workers. [613] There is particular emphasis on long-term monitoring for cardiovascular disease (including hypertension, aortic dilatation and the risk of aortic dissection), [613] [727] regular screening for thyroid dysfunction, recognition of hearing impairment which worsens in adult life and early planning for any request for assisted conception. Table 22-10 presents the suggested follow-up of adults with Turner's syndrome. [613] Therapy is directed toward augmenting stature, correcting somatic anomalies, inducing secondary sexual characteristics and menses, and counseling. As noted, the short stature in gonadal dysgenesis is not related to a deficiency of growth hormone, insulin-like growth factors, thyroid hormone, or adrenal or gonadal steroids. However, administration of pharmacologic doses of biosynthetic hGH increases growth rate and augments final height by a mean of 5 to 10 cm. [659] [728] [729] [730] [731] The heterogeneity in response appears to be related to the chronologic age at the start of therapy, the duration of therapy, the dose and frequency of growth hormone administration, the use of oxandrolone and/or estrogen, the growth standards used, the height of the parents, and the growth hormone peak elicited by pharmacologic stimuli. [659] [728] [729] [730] [731] [732] [733] [734] Rosenfeld and co-workers have the longest and most extensive study. [728] [735] Starting in 1983, 70 patients between the ages of 4.7 and 12.4 years with normal growth hormone responses to provocative stimuli were studied. [728] They were randomly assigned to (1) a control group for 1 year; (2) the anabolic steroid oxandrolone at a dose of 0.125 mg/kg by mouth daily; (3) growth hormone 0.125 mg/kg subcutaneously three times per week; or (4) a combination of oxandrolone and growth hormone. After 12 to

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24 months, all groups except the growth hormone alone group were placed on combination therapy; however, the oxandrolone dose was lowered to 0.0625 mg/kg because of signs of virilization. After 3 years, most patients received daily growth hormone rather than thrice weekly; however, the dose of 0.375 mg/kg per week was unchanged. The mean height in patients who completed therapy was 151.7 cm, for a net mean gain of 9 cm over projected height and historical controls. Long-term studies now indicate that when growth hormone treatment alone is started early enough, most girls will benefit and some will achieve normal final height. [735] [736] [737] [738]

Attaining a final height of 150 cm is now a realistic target. [738] It is important to individualize the dose of growth hormone depending on the patient's growth response to the usual dose (0.375 mg/kg per week in six or seven divided doses). Growth hormone treatment should be considered when the patient's height falls below -2.0 SD on the growth curve for normal girls. Growth hormone therapy is approved by the U.S. Food and Drug Administration for the treatment of patients with Turner's syndrome, and it was approved earlier in Japan and many Western European countries. It is prudent to discuss growth hormone therapy with the parents and patients, including its efficacy and side effects, in all patients with gonadal dysgenesis whose height is more than 2.0 SD below the mean value for age, especially those whose growth rate is less than 5 cm/yr. Studies are in progress to evaluate the effects of initiating growth hormone therapy at 5 to 6 years of age before the growth deficit is severe. Growth hormone therapy is usually continued until the growth rate falls to less than 2 cm/yr or the bone age exceeds 15 years. Supraphysiologic doses of growth hormone induced insulin resistance but not hyperglycemia; the increased insulin values returned to the normal range when growth hormone treatment was discontinued. [739] Estrogen therapy has commonly been deferred until age 15 or later on the assumption that treatment at an earlier age leads to rapid skeletal maturation and diminished height. This premise was based largely on the fact that pharmacologic doses of estrogens can accelerate bone maturation and lead to premature epiphyseal fusion without a proportionate increase in height. Studies in patients with aromatase deficiency and in a patient with a mutation in the estrogen receptor indicate that estrogen rather than androgen is the principal gonadal hormone involved in bone maturation and fusion and bone mineral accretion. [358] [740] [741] We examined the effect of early low dose, conjugated estrogen therapy on linear growth, bone age, and the development of secondary sexual characteristics in a group of patients with gonadal dysgenesis. [654] Low-dose conjugated estrogens (9 µg/kg body weight per day) or ethinyl estradiol (141 ng/kg body weight per day) was given to 21 patients with the syndrome of gonadal dysgenesis who had a mean age of 13 years and mean bone age of 10.7 years. Growth rate was transiently accelerated but declined to below the pretherapy rate after 12 months of therapy. The final height of the patients treated with low-dose estrogen was not different from that of control nontreated patients or that of a group of six girls with Turner's syndrome in whom normal ovarian function was present and spontaneous puberty ensued. Hence, no increase or decrease in mean final height was noted in our study. However, girls who received estrogen with a bone age of less than 11 years were shorter than the girls who began low-dose estrogen therapy after a bone age of 11 years. [654] Similar results have been obtained subsequently in other studies. [743] [744] Initially it is important to use the lowest dose of an estrogen preparation (including an estradiol patch) [745] that gradually will induce pubertal development. Serious psychological effects are frequently associated with a prolonged delay in the treatment of sexual infantilism. [746] The institution of low-dose, conjugated estrogen, synthetic estrogen therapy or transdermal estradiol patch [745] alone at approximately 13 years of age (bone age >11 years) elicits a brief growth spurt without inordinate advancement of skeletal maturation or reduction in final height and induces the development of secondary sexual characteristics at an age comparable to that of normal peers, thereby obviating the undesirable psychological consequences and deficient bone mineralization of a prolonged delay in sexual maturation. Studies in which growth hormone treatment was combined with early estrogen therapy (i.e., in patients younger than 12 years of age) indicate a shorter final height than in patients who received growth hormone treatment and "late" estrogen substitution therapy. [747] The number of years of growth hormone therapy before estrogen therapy is a critical factor in predicting height gained, and hence the time of initiation of estrogen therapy in the growth hormonetreated patient has an important influence on final height. [747] Earlier introduction of growth hormone therapy or the use of higher doses of growth hormone or both to induce normal or near-normal height for age [738] [748] permits the initiation of estrogen therapy by about 13 years of age, an important psychologic consideration. Therefore, in the treatment of girls with Turner's syndrome, the goal of increased adult stature must be balanced against the desire for sexual maturation in each individual patient. Estrogen replacement therapy may improve certain neuropsychologic deficits (nonverbal processing speed, motor function, and memory). neurocognitive deficits in adult women with Turner's syndrome were not altered significantly by estrogen replacement. [751]

[749] [750]

However, the

A number of instances of endometrial carcinoma have been reported in patients with gonadal dysgenesis. [752] The evidence suggests that estrogens, especially when unopposed by progesterone, can produce a progression of histologic changes from endometrial hyperplasia to invasive carcinoma (see also Chapter 16) . To clarify the relation between estrogen therapy and endometrial pathology in gonadal dysgenesis, Rosenwaks and colleagues [753A] studied 41 patients receiving estrogen replacement therapy. Increased risk of abnormal endometrial histology correlated with (1) a lifetime dosage of conjugated estrogens of more than 2500 mg, (2) more than 7 years of estrogen therapy, and (3) a daily dose of conjugated estrogens greater than 1.25 mg. Progestagens can modify the effect of estrogens on endometrial histology. It is therefore prudent to treat patients with gonadal dysgenesis with low-dose cyclic estrogen replacement therapy, with progestagen added at the end of each cycle. Further studies are necessary to assess the optimal dose of estrogen that reduces the risk of endometrial carcinoma while concurrently preventing osteoporosis. Rarely, patients with a 45,X karyotype and no cytogenetic evidence of Y chromosome material develop gonadoblastomas. [754] However, a study employing multiple Y-specific DNA probes indicates that 3.4% of apparent 45,X patients have Y chromosomal material present. [47] These 45,X patients may be at risk for gonadoblastoma formation. Most patients with a 45,X karyotype have little or no risk of neoplastic transformation of the streak gonads. Replacement Therapy.

We routinely initiate therapy (depending on the height) at about 13 years of age with 0.3 mg of conjugated estrogen or 5 µg of ethinyl estradiol by mouth or very low dose transdermal 17-estradiol. [745] The oral dose is gradually increased over the next 2 to 3 years to 0.6 to 1.25 mg of conjugated estrogens or 10 µg of ethinyl estradiol daily for the first 21 days of the month. The patient is maintained on the minimal dose of estrogen needed to maintain secondary sexual characteristics, permit withdrawal bleeding, and prevent osteopenia. Medroxyprogesterone acetate, 5 to 10 mg/day, is given from the 10th through the 21st day of the month to ensure more physiologic menses and to reduce the risks of endometrial and breast cancer. There is only limited clinical

895

experience on the use of transdermal estradiol patch in adolescent girls, but with this approach (while more expensive) the natural estradiol reaches the systemic circulation directly without first undergoing metabolism by the intestine and liver. [745] The common late adolescent and adult oral dose of estrogen replacement therapy often fails to increase to normal the size of the uterus (especially an adult fundal-cervical ratio as assessed by pelvic ultrasonography). [746] The attainment of a mature heart-shaped uterine configuration is important only if the patient elects to become pregnant by oocyte donation and in vitro fertilization. [747] An important part of the management is the education of the patient and family. [748] A frank discussion with the parents of the pathophysiology of the condition is appropriate when the diagnosis is made and reinforced at later sessions. Thereafter, the child should be given as much information about her condition as she can comprehend to allay any false fears or anxieties. An honest assessment of reproductive function based on clinical findings as well as hormone levels should be given to the patient when appropriate. Advances in in vitro fertilization and embryo transplantation make pregnancy possible for these patients, but the miscarriage rate is high[749] and the risk of aortic rupture is increased during pregnancy. The importance of medical and psychosocial management throughout life in patients with the syndrome of gonadal dysgenesis must be emphasized. Social and psychosocial support from the parents and the physician usually results in a well-adjusted woman. Partial Sex Chromosome Monosomy and Clinical Variants of the Syndrome of Gonadal Dysgenesis

Partial sex chromosome monosomy may or may not modify the expression of the classic 45,X phenotype.[46] [749A] Forty to 50 percent of patients with the typical syndrome of gonadal dysgenesis are X chromatin positive. This group usually has a structurally abnormal X chromosome or sex chromosome mosaicism involving a 45,X cell line. Chromatin-positive and chromatin-negative variants of gonadal dysgenesis are discussed here in relation to the more usual types of sex chromosome aberrations with which they may be associated. The diagram in Figure 22-43 shows the variable effect of partial sex chromosome monosomy (haploinsufficiency) on the typical features of the syndrome. In patients with sex chromosome mosaicism, the ratio in each gonad of 45,X primordial germ cells and blastemal components to those with a normal 46,XX or 46,XY constitution is probably the major determinant of whether the ultimate gonadal structure is a streak, a dysgenetic or hypoplastic ovary or testis, or a relatively normal gonad.[46] [88] [154] [749] The weight of evidence supports the idea that, after migration into the primitive gonad, primordial germ cells that bear a 45,X constitution degenerate more rapidly, quite likely by apoptosis, than do 46,XX cells, resulting in a streak, hypoplastic, or normal ovary. [79] Similarly, if the gonadal blastemal components, in particular the Sertoli cells, do not contain an appropriate number of 46,XY cells, testicular development does not take place [750] (Fig. 22-44) . The quantitative relation between 45,X cells and those with a 46,XX or 46,XY pattern in peripheral tissues may also be responsible for the variable effect of mosaicism on stature and associated somatic stigmata. [46]

In patients with a single cell line (euploid) containing a structurally abnormal sex chromosome, the somatic and gonadal consequences appear to be related to the nature and degree of the short or long arm deficiency of the second X or Y chromosome. Table 22-11 summarizes the correlation between structural abnormalities of the X and Y chromosomes and the clinical manifestations. The use of deletion mapping of the human sex chromosomes to clarify the relation of phenotype to karyotype has limitations. Structural abnormalities are often associated with mosaicism because of loss of the structurally abnormal sex chromosome from the stem cell line. Furthermore, structural rearrangements of chromosomes are complex. However, the advent of chromosome banding and molecular genetic techniques has facilitated the analysis of structurally abnormal sex chromosomes. At present, the data suggest that (1) ovarian determinants are located on both the long and short arms of the X chromosome and that patients with short arm deletions proximal to band Xp21 or long arm deletions proximal to band Xq25 usually have streak gonads and sexual infantilism [79] [95] [154] [749] [750] ; and (2) the short arm of the X chromosome (and to a lesser extent the long arm) contains loci that, if deleted, result in short stature and the somatic stigmata of the syndrome of gonadal dysgenesis (see section on biologic functions of the X chromosome and Y chromosome). [79] [95] [154] [749] [750] X Chromatin-Positive Variants of Gonadal Dysgenesis

45,X/46,XX, 45,X/47,XXX, and 45,X/46,XX/47,XXX Mosaicism.

45,X/46,XX mosaicism is the most common finding in patients with chromatin-positive gonadal dysgenesis and is second in frequency only to 45,X; the mosaic karyotype arises through loss of one X chromosome in a 46,XX conceptus. [46] [754A] Patients with this form of mosaicism usually exhibit fewer of the associated somatic anomalies, are not invariably short, may menstruate, and may even be fertile. One gonad may be of the streak type, and the contralateral gonad may be either a hypoplastic or a normal ovary; alternatively, both ovaries may be either normal or hypoplastic. During a family survey for a leukocyte anomaly, a normal grandmother with 45,X/46,XX/47,XXX mosaicism was discovered fortuitously. Some appreciation of the variable clinical features may be gleaned from nine patients with these forms of mosaicism studied by Morishima and Grumbach.[46] All had normal female external genitalia. Of seven who attained pubertal age, four developed some female secondary sexual characteristics, and two menstruated regularly. One of the two has had three pregnancies. In some, no important somatic abnormalities were detected, and two were of normal stature. One of the 45,X/46,XX patients had a webbed neck, coarctation of the aorta, and other gonadal dysgenesis stigmata but was of normal stature and menstruated regularly. A 12-year-old 45,X/46,XX/47,XXX patient had primary hypothyroidism and autoimmune thyroiditis. 46,XXqi and 45,X/46,XXqi.

Patients with the Xqi structural abnormality (isochromosome for the long arm of the X) have an X chromosome that consists primarily of two long arms (Xq) and lacks a short arm (Xp); it arises mainly as a consequence of a break in sequence in the proximal short arm and not by centromere misdivision [48] (Fig. 22-45) and occurs in about 15% of Turner's syndrome individuals (about 1 in 13,000 female live births). The Xqi chromosomes may be either monocentric or dicentric X. [754A] [754B] In a review of 89 cases, 29 were monocentric. Of these, only 5 of 17 were associated with mosaicism for a 45,X cell line. [753] In contrast, 49 of 60 patients with a dicentric isochromosome had a 45,X cell line. Dicentric X isochromosomes are more unstable than monocentric forms and probably result more frequently in sex chromosome mosaicism through loss of the heteromorphic dicentric X chromosome. In 14 patients studied with molecular biologic techniques, Xp markers were found in three dicentric Xqi chromosomes and in three monocentric Xqi chromosomes. [754] In five instances the Xqi was paternally derived. Isochromosome for the long arm of the X is the most common form of structural rearrangement of the X chromosome. Patients with a long arm X isochromosome are invariably

896

Figure 22-43 The range of phenotypic and gonadal expression in variants of the syndrome of gonadal dysgenesis and its relation to sex chromosome constitution. Typical phenotypic and gonadal findings in monosomic 45,X gonadal dysgenesis may be modified by the presence of a mosaic chromosomal constitution or by the presence of a structurally abnormal second sex chromosome. For example, 45,X/46,XX and 45,X/47,XXX mosaicism may be associated with normal stature, minimal somatic features of gonadal dysgenesis, and varying degrees of ovarian differentiation, or with a clinical picture indistinguishable from that of classic 45,X gonadal dysgenesis. Phenotype and gonadal differentiation apparently depend on the proportion of 45,X to 46,XX or 47,XXX cells in somatic and germ cells during differentiation. Similarly, the presence of a structurally abnormal X chromosome frequently modifies some features of the classic syndrome. When 45,X/46,XY mosaicism or a structurally abnormal Y chromosome is present, varying degrees of testicular differentiation may be found. The spectrum of clinical findings may extend from phenotypic male through ambiguous genitalia to phenotypic female, depending on the degree of fetal testicular insufficiency. In addition, beneficial effects of a normal XY cell line or presence of some part of a Y chromosome may lead to normal stature and a modification of the somatic defects associated with 45,X monosomy. (From Jones HW Jr, Grumbach MM. Developmental disorders [females]. In Cooke RE [ed]. Biologic Basis of Pediatric Practice. New York, McGraw-Hill, 1968, pp 10871093. Reproduced with permission of McGraw-Hill, Inc.)

short and have streak gonads, [46] [79] [88] although some menstruate spontaneously. [668] [755] In general, the somatic stigmata of gonadal dysgenesis are less severe than in 45,X patients. Coarctation of the aorta and severe lymphedema of the hands and feet are less common in 46,XXqi patients. Webbing of the neck, if present, is usually slight. The findings indicate that absence of the short arm on the second X, even in the presence of an X chromosome composed of two long arms, leads to short stature, failure of ovarian development, and some somatic stigmata of gonadal dysgenesis. The prevalence of autoimmune thyroiditis, [756] decreased glucose tolerance, and inflammatory bowel disease may be higher in patients with structural abnormalities of the X chromosome, especially 46,XXqi, than in 45,X individuals. Structurally abnormal X chromosomes are usually late replicating (except in balanced X-autosome translocations), and they give rise to the X chromatin body. Therefore, X chromatin bodies are larger than normal in patients with a 46,XXqi constitution but their increased size may be less evident in buccal smears than in other tissues. Karyotype analysis reveals a metacentric X chromosome with two arms of equal length whose banding pattern is similar to that of the long arm of the normal X chromosome. 46,XXpi.

There is controversy about the existence of an isochromosome for the short arm of the X chromosome. Of the 11 reported cases, 3 have been revised to long arm deletions, 4 were reported as presumptive, and 2 have been questioned on cytogenetic grounds. [757] The controversy revolves around the difficulty in distinguishing Xpi from deletions of the long arm

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Figure 22-44 The loss of germ cells during migration to or after seeding of the indifferent gonad in a 45,X individual gives rise to a gonadal streak, because germ cells are necessary for ovarian development of the indifferent gonad; evidence suggests that loss occurs after implantation of germ cells. In the presence of 45,X/46,XX mosaicism, gonadal differentiation may vary from that of an ovary to that of a gonadal streak. Similarly, in 45,X/46,XY mosaics, depending on the sex chromosome constitution of the germ cells and gonadal blastema, gonadal differentiation may vary from that of a testis to that of a gonadal streak. In 47,XXY individuals, germ cells become implanted in the primitive testis, but a marked loss of spermatogonia seems to occur in the perinatal period and infancy. (From Jones HW Jr, Grumbach MM. Developmental disorders [females]. In Cooke RE [ed]. Biologic Basis of Pediatric Practice. New York, McGraw-Hill, 1968, pp 10871093. Reproduced with permission of McGraw Hill, Inc.)

of the X chromosome, because the banding pattern of Xp is quite similar to that of Xq from the centromere to Xq24. There have been no reports of either high-resolution chromosome banding or molecular genetic analysis in a patient with 46,XXpi. 46,XXr or 45,X/46,XXr.

A ring X chromosome usually occurs as part of 45,X/46,XXr mosaicism or a more complex karyotype (see Fig. 22-45) .[758] Short stature is present in most patients, and most have minor stigmata of gonadal dysgenesis; none has a webbed neck or coarctation of the aorta. Approximately one third have spontaneous menses and develop secondary sexual characteristics. A mother and daughter with 45,X/46,XXr have been described. [759] Although most patients with 45,X/46,XXr have the gonadal dysgenesis phenotype, patients with severe mental retardation, syndactyly, and abnormal facies have been reported. [760] XIST, a gene that is transcribed only by the inactive X chromosome, is usually not expressed in these severely affected patients. [141] [758] [761] [762] All genes analyzed on the proximal short and long arms of the ring X were expressed, suggesting that the abnormal phenotype in these patients is caused by disomy for these genes on the X chromosome resulting from lack of dosage compensation. [141] A ring X chromosome is associated with significant learning difficulties and some behavior problems. [763] The degree of mental impairment is usually related to the size of the active ring. The variable phenotype associated with active or inactive ring X chromosomes confounds predicting prognosis in an affected fetus detected by prenatal diagnosis. [758] [760] The proportion of X chromatin-positive cells is decreased in patients with a ring X chromosome, and the X chromatin bodies tend to be small. The ring X chromosome, with rare exceptions, exhibits late DNA replication. [46] The ring X chromosome arises by loss of both ends (telomeres) of the chromosome and union of the proximal breaks; as a consequence, a variable amount of chromatin material is lost from each arm (see Fig. 22-5) . Ring chromosomes are unstable, and the size of the ring varies in different cells of the same subject. In relation to gonadal dysgenesis, studies of patients with a ring X chromosome have established that loss of both telomeres of an X chromosome need not lead to the development of streak gonads. It is sometimes difficult to be sure of the cytogenetic origin of the ring chromosome, a distinction that is critical in view of the increased risk of gonadal tumors associated with dysgenetic gonads and Y cell lines. Molecular cytogenetic analysis with specific X and Y chromosome probes have made identification easier.

[764] [765]

46,XXp and 45,X/46,XXp.

Deletions of the short arm of the X chromosome (Xp) are rare and are frequently associated with 45,X mosaicism. Phenotypic-karyotypic analysis of 40 nonmosaic patients indicated considerable variation in somatic stigmata and gonadal function. [94] [721] [766] [767] [768] [769] [770] Patients with a terminal deletion of the short arm of the X (distal to Xp22) can have normal ovarian function and no somatic stigmata of gonadal dysgenesis with the possible exception of a modest degree of short stature. [141] [771] Patients with deletions proximal to Xp22 usually have short stature, variable stigmata of gonadal dysgenesis, and gonadal dysfunction (Fig. 22-46) . A lymphedema critical region is proposed at Xp11.4. [94] The abnormal X chromosome is usually the late DNA-replicating X and is responsible for the small X chromatin body in interphase nuclei in these patients. Of interest is the report of a familial group of seven patients with the syndrome of gonadal dysgenesis secondary to a deletion of the short arm of an

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TABLE 22-11 -- Relation of Structural Abnormalities of X and Y to Clinical Manifestations of Gonadal Dysgenesis Type of Sex Chromosome Karyotype Phenotype Sexual Short Somatic Anomalies of Syndrome of Gonadal Abnormality Infantilism Stature Dysgenesis Loss of an X or Y

45,X

Female

+

+

+

Isochromosome for long arm of an X

46,X(Xq)

Female

+ (occ. ±)

+

+

Deletion of short arm of an X*

46X,del(X)(p21)

Female

+, ±, or -

+ (-)

+ (-)

Deletion of long arm of an X*

46X,del(X)(q21)

Female

+

- (+)

-

Deletion of both arms of an X (ring X)

46,Xr(X)(q22;q25) Female

- or +

+

+

Loss of short arm of Y

46X,del(Y)(p11)

+

+

+

Ambiguous

*In Xp and Xq, extent and site of deleted segment are variable, and the number after the p or q indicates the deletion site in the long (q) or short (p) arm.

X chromosome in which the disorder was transmitted by carriers of a balanced translocation between the X and chromosome 1. [772] Familial Xp deletions have been detected in seven additional families with a variable phenotype and short stature as the only major phenotypic abnormality. [773] 46,XXq and 45,X/46,XXq.

Patients have been reported with a deletion of the long arm of the X chromosome (Xqi). In general, patients with only a 46,XXq cell line are normal in stature or have moderately short stature and exhibit few manifestations of gonadal dysgenesis but have primary amenorrhea, sexual infantilism, and streak gonads. We have studied two patients with 46,XXq karyotypes, and the findings in one are summarized in Figure 22-47 . Exceptions to the rule that 46,XXq patients lack stigmata of gonadal dysgenesis and are of normal height were reported before chromosome banding techniques became available. Such cases may represent either hidden mosaicism or complex structural rearrangements of the X chromosome, including inversions and interstitial rather than terminal deletions. Studies with FISH and Southern blotting suggest that gonadal dysgenesis stigmata are absent when the break point is distal to Xq24 and that deletions distal to Xq25-q26 do not reduce stature. [774] [775] Most Xq patients sooner or later have ovarian failure or premature menopause irrespective of the size of the deletion. [79] [776] Isodicentric X.

Isodicentric X chromosomes are large X chromosomes with two C bands. These chromosomes replicate

Figure 22-45 Structural anomalies of the X chromosome. The normal X at the left is G banded. A dark band on the short arm and two major dark bands on the long arm are visible. The first Xq and the ring X chromosome (Xr) are not banded. They show late replication with tritiated thymidine. Note symmetry of the arms of the second Xq. Even with G banding, it is difficult to distinguish this chromosome from a possible short arm isochromosome. The long arm isochromosome (Xqi) appears to be dicentric. The two chromosomes to the far right are isodicentric X chromosomes. Both have two C bands but only one functional centromere. There is a mirror-like band pattern on both sides of a point between the two C bands. The first isodicentric X chromosome presumably represents a break in the long arm of X at q22 with fusion of chromatids and duplication of the entire chromatid. The second isodicentric X chromosome appears to represent a terminal break in the short arm so that reduplication of the chromatids has produced what appears to be almost two X chromosomes.

late, form a large bipartite sex chromatin body, and apparently have one functionally suppressed centromere (see Fig. 22-45) . The banding pattern of isodicentric X chromosomes reveals a mirror image about a point between the two centromeres (C bands). These chromosomes are usually associated with mosaicism for a 45,X cell line and presumably arise by chromatid break and fusion of sister chromatids. This event would produce an acentric fragment that would be lost during cell division and thereby result in a 45,X cell line. Phenotypic-karyotypic correlations in these patients are similar to those in Xp and Xq patients. [79] [776]

X-Autosome Translocations.

X-autosome translocations have been reviewed. [79] [777] [778] In general, women with a break in the X chromosome between Xp13 and Xp26 manifest infertility, confirming the belief that this region contains genes critical to gonadal differentiation and function. [79] Male carriers of a balanced X-autosome translocation with an X chromosome break in the "critical region," Xp13 to Xp26, are usually infertile. X Chromatin-Negative Variants of Gonadal Dysgenesis

The pattern of sex chromosome mosaicism and structural abnormalities of the Y chromosome is similar to that for the X chromosome. Usually, as a consequence of its effect on gonadal differentiation, a Y-bearing cell line modifies the typical female phenotype of the syndrome by causing a variable degree of masculine differentiation of the genital tract.

899

Figure 22-46 Variable gonadal function and phenotypic stigmata in three patients with a deletion of the short arm of the X chromosome (Xp) of different degrees. A, A 13-year-old phenotypic female of short stature (-3.5 SD) with low-set ears, high-arched palate, low hairline, broad chest with wide-spaced areolae, cubitus valgus, puffy hands and feet, and short fourth metacarpals. There was no evidence of secondary sexual characteristics. The plasma FSH level was elevated at 26 µg/L (LER-869); the plasma estradiol level was less than 22 pmol/L (6 pg/mL). The buccal smear contained a normal proportion of X chromatin bodies in interphase nuclei, which were conspicuously small. Karyotype analysis and autoradiography revealed a 46,XXp karyotype. The abnormal X chromosome appeared to lack the entire short arm. B, A phenotypic female, aged 17 years, 4 months, with the stigmata of the syndrome of gonadal dysgenesis. Her height was 151 cm (-3 SD), and she had multiple nevi, cubitus valgus, and a short fourth metacarpal on the right hand. At age 13 the patient noted spontaneous onset of breast development, which did not progress. Plasma gonadotropin levels were elevated: LH 7.3 µg/L (LER-960) and FSH 53 µg/L (LER-869). The concentration of plasma estradiol was 70 pmol/L (19 pg/mL). On buccal smear, the cells had a normal proportion of X chromatin bodies, which appeared small. Karyotype analysis and autoradiography indicated an Xp chromosome that had been deleted close to the centromere, but a small segment of the short arm was visible distal to the centromere. C, A 20-year-old phenotypic female with a chief complaint of dysfunctional uterine bleeding. She had short stature, slight puffiness of hands and feet, and short fourth metacarpals. Female secondary sexual characteristics appeared at age 11, and menarche at age 13 was followed by regular menses, which later became irregular. The buccal smear contained nuclei with a normal proportion of small sex chromatin bodies. Bilateral ovaries were identified grossly and histologically during an appendectomy. Karyotype was 46,XXp. The extent of deletion of the short arms of the abnormal X chromosome in this patient is less than that seen in patients in A and B. A segment of the short arm is readily discernible above the centromere. It appears that, in these three patients with XXp karyotypes, somatic and gonadal manifestations of the syndrome of gonadal dysgenesis correlated with the magnitude of deletion of the short arm of the X chromosome. 45,X/46,XY, 45,X/47,XYY, 45,X/46,XY/47,XYY, and Related Abnormalities (Table 22-12) .

A highly diverse phenotype is encountered in these forms of mosaicism, [46] [779] [780] ranging from phenotypic females to individuals with ambiguous external genitalia to phenotypic males (Fig. 22-48) . As in 45,X/46,XX mosaicism, short stature and the associated somatic abnormalities, although frequently present, are inconsistent features and may vary independently of each other and of gonadal differentiation. In the review by Zah and co-workers [779] of 60 patients with 45,X/46,XY mosaicism, two thirds were reared as females. Of nine patients with 45,X/46,XY or 45,X/46,XY/47,XYY

900

Figure 22-47 A, A 22-year-old tall female with a chief complaint of primary amenorrhea had a deletion of the long arm of one X chromosome, Xq. At age 12 she developed sparse pubic hair. Breast development did not occur, and she remained sexually infantile. Height was 178 cm (+2.6 SD), and weight was 70 kg (+1.2 SD). No somatic stigmata of the syndrome of gonadal dysgenesis were noted. Plasma gonadotropin levels were elevated: LH was 5.6 µg/L (LER-960), and follicle-stimulating hormone level was 36.5 µg/L (LER-869). The buccal smear showed a normal proportion of X chromatin bodies that were slightly small. B, A Giemsa-stained Xq, which exhibited (C) the late-labeling pattern characteristic of an X chromosome.

mosaicism studied by Morishima and Grumbach, [46] one was a phenotypic female, one was a phenotypic male, and seven had ambiguous external genitalia (Table 22-13) . The gonads varied from bilateral streaks in the phenotypic female to bilateral dysgenetic testes. In others the development was asymmetric: one patient had a streak in one mesosalpinx and a rudimentary testis on the contralateral side (so-called mixed gonadal dysgenesis); another had a normal testis in the scrotum and a herniated streak, fallopian tube, and vestigial uterus (hernia uteri inguinale) in the contralateral inguinal region. In several cases, the streak gonad contained a few primordial follicles. However, the presence of sparse primordial follicles in a gonad is insufficient for a diagnosis of true hermaphroditism. [780] [781] TABLE 22-12 -- Clinical Features of 45,X/46,XY Mosaicism Karyotype: 45,X/46,XY Genitalia:

Female ambiguous male with normal gonadal function

Wolffian duct derivatives:

Duct differentiation, contingent on functional integrity of homolateral fetal gonad (i.e., streak) uterus, fallopian tubes; dysgenetic testis variable structures; testis wolffian duct derivatives

Müllerian duct derivatives: Gonad:

Streak gonads dysgenetic testes normal testes; Streak gonad + dysgenetic testis"mixed gonadal dysgenesis"; risk gonadal neoplasm (gonadoblastoma)

Habitus:

Short, stigmata of gonadal dysgenesis; genitalia: streak gonads female with sexual infantilism at puberty; dysgenetic testes ambiguous genitalia; if gonadoblastoma present gynecomastia secondary to estradiol production; testes normal male differentiation

Hormone profile:

Increased plasma FSH and LH; decreased testosterone concentrations

FSH, follicle-stimulating hormone; LH, luteinizing hormone.

During screening of a family for bone marrow transplantation donors, we discovered an adult with 45,X/46,XY mosaicism. Except for short stature, no stigmata of the syndrome of gonadal dysgenesis were present, and he had normal adult male genitalia (Fig. 22-49) . Plasma gonadotropin and testosterone levels, both basally and in response to LHRH, were within the normal range for adult men. On pelvic ultrasonography, müllerian duct derivatives were absent, and the testes appeared normal and homogeneous. The sperm count was 17 million/mL, and the patient demonstrated fertility, as evidenced by the fathering of a "normal" 46,XY fetus. The finding of a short, otherwise normal fertile male with 45,X/46,XY mosaicism extends the phenotypic spectrum of this disorder. Ascertainment bias may be responsible for the lack of well-differentiated males in reports of this disorder. [782] A review of the chromosome analyses of 58 patients in the literature who were ascertained because of ambiguous genitalia shows a preponderance of 45,X cells, [782A] suggesting that only individuals whose abnormality

occurred in the first few cell divisions are represented in this group of patients. However, 90% of the fetuses diagnosed by amniocentesis and confirmed postnatally as 45,X/46,XY mosaics have normal male genitalia. [782] [783] [784] Follow-up of these patients is limited, and hypothalamic-pituitary-gonadal function has not been characterized. However, their lack of presentation at a later date with either gonadal tumors or gonadal dysfunction suggests that most (at least 75%), like our patient, have normal hypothalamic-pituitary-gonadal function and are at low risk for testicular tumors. The restricted local or paracrine action of the testes on the differentiation of genital ducts is well demonstrated in patients with asymmetric gonadal development. In such patients, development of male ducts and involution of the müllerian structures are also asymmetric and parallel the degree of testicular development on each side. As discussed previously, local action of the testis on müllerian duct regression is mediated through AMH, whereas unilateral differentiation of male ducts is mediated by high local levels of testosterone in the wolffian ducts and their derivatives. The presence of Sertoli cells in the ipsilateral gonad correlates with the absence of müllerian structures on the same side in patients with 45,X/46,XY mosaicism. [785] This observation is consistent with the local secretion of AMH by embryonic and fetal Sertoli cells. Male differentiation of the external genitalia is, however, brought about by the systemic effects of testosterone secreted by a fetal testis and

901

Figure 22-48 Three patients with 45,X/46,XY sex chromosome mosaicism who illustrate the highly variable phenotype in this variant of the syndrome of gonadal dysgenesis. (Numbers of the patients refer to designation in Table 22-13 .) A, Patient 1, a phenotypic female, was age 15 years, 4 months. She had short stature (-3.1 SD), an increased number of pigmented nevi, puffiness over the dorsa of fingers, and broad and short hands, and she was sexually infantile (breast development seen in photograph followed estrogen therapy) except for sparse pubic and axillary hair. The urinary gonadotropins were markedly elevated. B, Patient 3, aged 3 years, 1 month, had ambiguous external genitalia, perineal hypospadias, and undescended gonads. He was of average height and had a broad chest and a duplication of the left kidney. C, Patient 9, aged 8 years, 1 month, was a phenotypic male with a penile urethra and unilateral undescended gonad, average height, cubitus valgus, short fourth metacarpals, and puffiness of dorsa of fingers. By age 15, male secondary sexual characteristics were well advanced and a left scrotal testis, which was normal in histologic appearance, measured 4.0 × 2.4 cm.

converted locally to 5-dihydrotestosterone. Hence, the external phenotype may range from a simulant female to a completely male configuration. Although the secretion of androgenic hormones at adolescence is usually predictable from the degree of masculinization of the external genitalia in utero, virilization may occur at puberty in patients with a female phenotype. Breast development at or after the age of puberty occurs in about one fourth of cases and is usually associated with a gonadal neoplasm. We studied two adolescent 45,X/46,XY subjects who had breast development and had pubertal levels of plasma estradiol; at laparotomy a gonadoblastoma that secreted estradiol was found (Fig. 22-50) . The propensity of patients with 45,X/46,XY mosaicism to develop gonadal tumors is high, and prophylactic removal of the streak gonads or dysgenetic undescended testes is indicated. Gonadoblastoma, a complex tumor composed of large germ cells, Sertoli cells, and stromal derivatives, is the neoplasm most often found, and it can give rise to a malignant germinoma. Therefore, after the removal of the gonads, serial sections should be examined for evidence of a tumor. The risk of tumor is 15% to 20%[786] [787] [788] and is age related. In a population study of 114 phenotypic females with Turner's syndrome, PCR with primers spanning the Y chromosome detected Y chromosomal material in 12.2%; the occurrence of gonadoblastoma was estimated at 7% to 10% of those with Y chromosome DNA. [788] None of these individuals had signs of virilization. [788] Four 45,X/46,XY patients with incomplete virilization have been described in whom carcinoma in situ (CIS) was present in biopsies of the gonads. [786] [787] CIS is thought to be a premalignant lesion leading to germ cell tumors. [787] [788] Because 45,X/46,XY mosaics not only may harbor gonadoblastomas, roughly 30% of which are associated with germ cell tumors, but also have an increased prevalence of CIS, gonadal biopsy is indicated in all individuals with a male phenotype and 45,X/46,XY mosaicism. If the testis is histologically normal and is in the scrotum or can be placed in the scrotum, it can be retained. However, careful, close follow-up is mandatory. Müller, Skakkeback, and associates recommend ultrasonography of the gonads and biopsy of the retained testis at the start of puberty. If biopsy and ultrasonography show lack of evidence of CIS, they suggest an annual ultrasonographic examination and a second biopsy at 20 years of age. [787] The absence of CIS at age 20 suggests that the risk of a gonadal germ cell tumor is minimal. Page and his group proposed the location of a gonadoblastoma susceptibility locus on the Y chromosome in deletion interval 3 on the short arm and a proximal segment of the long arm of the Y chromosome. [72] Subsequently, the TSPY (testis-specific

902

TABLE 22-13 -- Genital Structures in Nine Patients with 45,X/46,XY Mosaicism Genital Ducts Case 1

2

3

4

External Genitalia Female

Ambiguous

Ambiguous

Ambiguous

Urogenital Sinus

Phallus Enlargement

-

-

+

+

+

+

+

+

Gonads

Female

Rt. streak?

Rt. fallopian tube?

Lt. streak?

Lt. fallopian tube?

Rt. testis

Rt. fallopian tube

Lt. streak

Lt. fallopian tube

Rt. not found

Rt. fallopian tube

Lt. streak

Lt. fallopian tube

Rt. dysgenetic testis

Rt. fallopian tube

Male Uterus

Rt. Lt.

Uterus

Rt. vas deferens Lt.

Uterus

Rt. Lt.

Vestigial uterus

Lt. fallopian tube


Lt. dysgenetic testis 5

Ambiguous

+

+


Rt. fallopian tube

Uterus

Lt. fallopian tube

Rt. vas deferens Lt. vas deferens


Ambiguous

+

+


Rt. fallopian tube

Uterus

Rt.

Lt. fallopian tube

Lt.


Rt.

Rt. vas deferens

Lt. dysgenetic testis

Lt.

Lt. vas deferens


Rt. fallopian tube


8

Ambiguous

Ambiguous

+

+

+

+

Uterus

Lt. fallopian tube


Lt. streak 9

Male

-

Normal penis

Rt. streak

Rt. fallopian tube

Lt. testis

Lt.

Vestigial uterus

Rt. Lt. vas deferens

From Morishima A, Grumbach MM. The interrelationship of sex chromosome constitution and phenotype in the syndrome of gonadal dysgenesis and its variants. Ann NY Acad Sci 1968; 155:695715.

Figure 22-49 The external genitalia of a normally differentiated male with 45,X/46,XY mosaicism. Karyotype analyses revealed 16% and 68% mosaicism for a 45,X cell line in blood and skin, respectively. Gonadotropin levels, both basal and LHRH stimulated, and plasma testosterone levels were normal. Fertility was documented in vitro and by the conception of a normal male fetus.

903

Figure 22-50 45,X/46,XY mosaicism with a feminizing gonadoblastoma. A, A 20-year-old female with many stigmata of the syndrome of gonadal dysgenesis, including short stature, multiple nevi, cubitus valgus, and hyperconvex, small nails. The buccal smear was X chromatin negative; on fluorescence microscopy, 30% of interphase nuclei had a single Y body. Karyotype was 45,X/46,XY. The patient had spontaneous development of public and axillary hair at age 12. At age 18, breast development was noted. Her height was 139 cm (-5.1 SD) and weight 39 kg (-2.5 SD). Bone age was 17 years; an intravenous pyelogram was normal. The concentration of plasma gonadotropins at 20 years of age was elevated; plasma luteinizing hormone was 8 µg/L (LER-960) and follicle-stimulating hormone was 50 µg/L (LER-869). The concentration of plasma estradiol was 95 pmol/L (26 pg/mL), and that of estrone was 117 pmol/L (32 pg/mL); the plasma testosterone level was less than 0.7 nmol/L (0.2 ng/mL). On exploratory laparotomy, normal-appearing fallopian tubes and a uterus were found. The right gonad was a typical "streak," with whorls of fibrous connective tissue. B, The left gonad was replaced by a 1.3 × 1 × 1 cm tumor mass, which, on histologic section, revealed well-defined nests and islands of Sertoli-Leydig-like cells and germ cells, as well as calcification consistent with diagnosis of gonadoblastoma. C, Higher magnification illustrates aggregates of germ cells and small epithelial cells resembling immature Sertoli cells, as well as cells indistinguishable from Leydig cells. After gonadectomy the concentration of plasma estradiol was prepubertal ( 0.9 cm increase in phallic length Raise 46,XY male pseudohermaphrodites as males except those with: Complete androgen insensitivity syndrome. The dilemma presented by partial androgen insensitivity syndrome Completely female genitalia (?) Compelling reasons for sex assignment as female, including parents' informed decision



967

Reassignment of Sex After the Newborn Period

Children may be assigned an inappropriate sex because of errors in diagnosis or ignorance of the principles that should properly determine this choice. In such cases, the knotty decision to change the sex of rearing or to leave matters undisturbed depends largely on the age of the child and the degree to which the gender identity has been established. Money has stated that a change in the sex of rearing is feasible until the age of 18 months and is sometimes successful until 30 months, [462] [491] but thereafter, in our culture, serious and sometimes complex psychiatric and social consequences may be encountered. This concept has been challenged. Nevertheless, change of gender assignment in children should be undertaken after 18 months only after a review of alternatives and with the provision of close supervision and long-term counseling of the patient, parents, and siblings. Before, at, or during adolescence, the patient may reach the decision that he or she has been reared in the wrong sex and may request assistance in changing his or her sex of assignment. If there are sufficient grounds for this belief, the request should be considered seriously and honored. Some patients may have serious psychological disturbances, and both psychiatric and legal counsel should be sought. [1197]



Reconstructive Surgery

Because the presence of ambiguous external genitalia is likely to reinforce doubt about the sexual identity of the infant or child, it is desirable to initiate reconstructive surgery as early as is medically and surgically feasible. The functional result, rather than the cosmetic, is paramount. It is highly desirable that surgery on the external genitalia be initiated before 6 months of age when practicable. [507] [508] [923] [1012] [1553] [1554] The management of clitoromegaly in female pseudohermaphrodites and in male pseudohermaphrodites reared as females has been controversial. Two different operative approaches have been used: clitoral recession, first reported by Lattimer in 1961 to replace the then widely used clitorectomy, [1013] [1555] and clitoroplasty. [396] [507] [508] [ 1015] Clitoridectomy [857] [1556] has long been abandoned as a mutilating procedure. Documentation of the role of the clitoris as an erotic organ in women [ 1557] makes it clear that clitoridectomy must be avoided. Clitoral recession has significant drawbacks mainly because of painful clitoral erections. [1013] Clitoroplasty, as recommended by Donahoe,[507] Rink,[508] Hutson,[1015] and Baskin[396] requires excision of the shaft and corpora with retention of the glans. This procedure and modifications of it [1012] are used most widely at present. Long-term data are still necessary to evaluate the efficacy of this procedure with respect to appearance and sexual function. The extent of the initial repair of the urogenital sinus and vagina depends in large part on the skill and experience of the surgeon. These are not procedures that should be undertaken by surgeons or urologists who have not had training and experience with these techniques and are not part of a team of professionals that address clinical issues. Even when the initial repair has been done in the past by an experienced surgeon, it has not been uncommon for patients who have had vaginoplasties performed at age 18 months or earlier to require secondary operations because of stenosis of the introitus. [1554] We believe that reconstruction of a vagina in male pseudohermaphrodites reared as females and in female pseudohermaphrodites can be deferred until adolescence or until requested by the patient. A small vaginal pouch often can be enlarged by daily manipulations with a suitable mold. [1559] Even if the vagina remains too shallow for satisfactory coitus, manual dilatation makes it easier to carry out subsequent surgical correction.

TABLE 22-46 -- Removal of the Gonads Retain histologically normal and functional scrotal testes in 45,X/46,XY male pseudohermaphrodites. Follow closely. Consider biopsy postpubertally to detect carcinoma in situ and perform periodic ultrasonography of testes. Complete androgen insensitivity: testes may be retained until after puberty. Partial androgen insensitivity or biosynthetic defects: if female sex of rearing is selected by parents, we recommend gonadal removal before puberty to prevent virilization.

A male with hypospadias no longer requires multiple procedures to create a phallic urethra. In recent years, new surgical techniques have reduced the number of operations and the length of hospitalization and have increased the success rate and parental expectations for "normality" in both the short and the long term. [506] Circumcision should be avoided to preserve as much tissue as possible. Laparoscopy can be undertaken (if necessary) simultaneously with the initial operation. It is often desirable to insert prosthetic testes to give the scrotum dependency and to improve cosmetic appearance. These may be changed to adult-sized prostheses in adolescence.



Removal of the Gonads

A high incidence of gonadal tumors in patients with certain forms of gonadal dysgenesis and dysgenetic male pseudohermaphroditism especially makes it mandatory that an evaluation of this risk be given priority in deciding whether and when the gonads should be removed. Although the incidence of gonadoblastomas and germinomas (seminomas or dysgerminomas) increases near the normal time of adolescence, tumors are sometimes discovered during the first decade. Because temporizing serves no useful purpose and may expose the child to hormone secretions inappropriate to the chosen sex for rearing, it is advisable to proceed with gonadectomy concurrently with the initial repair of the external genitalia in patients who are at high risk, especially in the instance of intra-abdominal rudimentary testes. We are evaluating the use of MRI of the pelvis every 1 to 2 years to screen for gonadal neoplasms in children at risk. Although prevalence of gonadal tumors has been reported to be as high as 9% in patients with the androgen resistance syndrome based on studies over three decades ago, some patients who developed tumors may have had atypical forms of gonadal dysgenesis; a modern survey is not available. The prevalence of gonadal malignancy before age 25 years in patients with androgen resistance appears to be relatively low. If the patient has a hernia and surgical repair is indicated, we recommend gonadectomy at that time to avoid a second operation. Otherwise, in the patient with complete androgen resistance, the undescended testes may be left in situ until after puberty. Thereafter, a frank and open discussion with the patient of the pathophysiology of androgen resistance and an assessment of the risk of gonadal malignancy needs to be undertaken to obtain informed consent for gonadectomy (Table 22-46) . There is a risk of some degree of virilization at the time of puberty in patients with the partial form of androgen resistance and in those with other forms of male pseudohermaphroditism with retained testes in whom a female sex for rearing has been assigned. In these cases, gonadectomy before puberty has been advanced and should be considered and discussed with the patient and parents. In some male pseudohermaphrodites who are raised as males, at least partial development of male secondary sexual characteristics will occur at the expected time of puberty. Provided the testes are not dysgenetic

968

and are sufficiently descended to permit palpation, it is reasonable to leave the testes in situ. Such patients should be carefully examined at regular intervals for the presence of a tumor. MRI, ultrasonography, and examination of testicular biopsy specimens are useful in the early diagnosis of CIS and testicular neoplasm. [1560] Hormone substitution therapy in hypogonadal patients should be prescribed in such a way that secondary sexual characteristics emerge appropriately in both timing and sequence. The goal of therapy should be to approximate normal adolescent development as closely as possible. In females, including patients with the syndrome of gonadal dysgenesis, estrogenic hormone substitution therapy is initiated with low oral doses of estrogen (0.3 mg conjugated estrogens or 5 µg ethinyl estradiol daily) or a transdermal estradiol patch. [745] Breast enlargement and growth of the uterus frequently occur within 3 months. Usually, cyclic therapy with estrogen and an oral progestagen is begun after 6 to 12 months of estrogen therapy or sooner if breakthrough bleeding occurs (see section on treatment of gonadal dysgenesis). Development of male secondary sexual characteristics is usually better with repository injections of testosterone or a transdermal testosterone patch than with oral preparations. Few data are available on the use of dermal testosterone therapy in childhood or adolescence. Many oral synthetic androgens have the added disadvantage of predisposing to biliary stasis, jaundice, and hepatic tumors. Rapid virilization is usually inadvisable, and it is preferable to promote virilization gradually over many months in a manner similar to that in normal boys. The effect of gonadal steroids on skeletal maturation is dose related, whereas the effect on linear growth is less so. The relation between attained stature and skeletal maturation at the inception of therapy and the dose of androgen prescribed determine the ultimate effect of this therapy on adult height. An initial intramuscular dose of 50 mg of testosterone enanthate or other long-acting testosterone ester may be given monthly, beginning at age 12 to 13 years. Thereafter, the dose should be increased gradually over 3 to 4 years to the adult replacement dose of 200 mg every 2 weeks, usually reaching the adult level after a bone age of 17 years has been attained (see Table 22-45) . In selected patients we have used the transdermal testosterone patch; initially the 2.5-mg patch is applied overnight for 8 hours. Subsequently the length of application is gradually increased to 24 hours.



Psychological Management

The newborn with ambiguous genitalia presents a clinical, social, and psychological challenge. [1561] Initially, in infants with ambiguous genitalia it is best for the physician to admit uncertainty regarding the "true sex" of the child and to urge the parents not to immediately assign a name and send out birth announcements. The filing of the birth certificate with the name should be delayed until a definite gender assignment and name has been given to the child. Clinical, cytogenetic, hormonal, and radiologic evaluation should be undertaken expeditiously. Thereafter, clearly presented, comprehensive, and informative discussions with the parents should ensue with all members of the team (i.e., endocrinologist, infant's physician, geneticist, surgeon, mental health specialist, social worker) present, if possible. This discussion should take into account the anxieties, religious views, social mores, cultural background, and level of understanding of the parents to make the best gender assignment for the infant and to obtain informed consent for the decision from the parents. A simple explanation of the normal process of sexual differentiation with appropriate illustrative material is useful because it lays the groundwork for the concept that all fetuses are bipotential initially and that sex differentiation is a complex process that may not TABLE 22-47 -- Management of Ambiguous Genitalia Hormone therapy at puberty if necessary. Progressive, step-by-step, age-appropriate discussion of diagnosis, pathophysiology, gender, and potential for fertility with the patient from childhood through adolescence, as well as with the parents. Secrecy is unwarranted and counterproductive. Involve the patient in decisions about surgery and sex hormone replacement therapy. Provide continuing psychosocial and endocrinologic support to the patient and the family. Long-term follow-up data on the outcome of modern management needs to be a high priority. be completed in utero. An analogy to other so-called birth defects (e.g., cleft lip, congenital heart disease) is accurate, easily understood, and less psychologically threatening. It should be stated clearly that the anatomic abnormalities can be surgically repaired by an experienced surgeon, that hormone replacement can be given if necessary, and that psychological support is available (Table 22-47) . Continuing follow-up by the team members should address any questions that arise during infancy and childhood. In this age of "freedom of information," it is prudent to discuss in an age-appropriate manner and in progressive stages all aspects of the diagnosis, pathophysiology, management, and treatment of the ambiguous genitalia with the patient as soon as his or her level of increased understanding allows for this. The implications of these observations for the management of intersex are debated in a series of published papers in the Journal of Clinical Ethics.[1552]



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1536. Hecker

BR, McGuire LS. Psychosocial function in women treated for vaginal agenesis. Am J Obstet Gynecol 1977; 129:543547.

1537. Meyer-Bahlburg 1538. Reiner

HF. Gender assignment and reassignment in 46,XY pseudohermaphroditism and related conditions. J Clin Endocrinol Metab 1999; 84:34553458.

W. To be male or femalethat is the question. Arch Pediatr Adolesc Med 1997; 151:224225.

1539. Bradley

SJ, Oliver GD, Chernick AB, Zucker KJ. Experiment of nurture: ablatio penis at 2 months, sex reassignment at 7 months, and a psychosexual follow-up in young adulthood. Pediatrics 1998; 102:e9 (E91E95). 1540. Kuhnle

U, Krahl W. The impact of culture on sex assignment and gender development in intersex patients. Perspect Biol Med 2002; 45:85103.

1541. Warne

GL, Zajac JD. Disorders of sexual differentiation. Endocrinol Metab Clin North Am 1998; 27:945967.

1542. Zucker

KJ. Intersexuality and gender identity differentiation. J Pediatr Adolesc Gynecol 2002; 15:313.

1543. Dittmann 1544. Lee

RW. Ambiguous genitalia, gender-identity problems, and sex reassignment. J Sex Marital Ther 1998; 24:255271.

PA. Should we change our approach to ambiguous genitalia? Endocrinologist 2001; 11:118123.

1545. Daaboul 1546. Dreger 1547. Taha

J, Frader J. Ethics and the management of the patient with intersex: a middle way. J Pediatr Endocrinol Metab 2001; 14:15751583.

AD. Hermaphrodites and the Medical Invention of Sex. Cambridge: Harvard University, 1998.

SA. Male pseudohermaphroditism: factors determining the gender of rearing in Saudi Arabia. Urology 1994; 43:370374.

1548. Forest

MG. Pattern of the response of testosterone and its precursors to human chorionic gonadotropin stimulation in relation to age in infants and children. J Clin Endocrinol Metab 1979;

49:132137. 1549. Aaronson

IA. Micropenis: medical and surgical implications. J Urol 1994; 152:414.

1550. McMahon

DR, Kramer SA, Husmann DA. Micropenis: does early treatment with testosterone do more harm than good? J Urol 1995; 154:825829.

1551. Sutherland 1552. Howe 1553. Lim

RS, Kogan BA, Baskin LS, et al. The effect of prepubertal androgen exposure on adult penile length. J Urol 1996; 156:783787.

EG, Dreger AD, Groveman SA, et al. Special Issue: Intersexuality. J Clin Ethics 1998; 9:337430.

YJ, Batch JA, Warne GL. Adrenal 21-hydroxylase deficiency in childhood: 25 years' experience. J Paediatr Child Health 1995; 31:222227.

1554. Krege

S, Walz KH, Hauffa BP, Korner I, Rubben H. Long-term follow-up of female patients with congenital adrenal hyperplasia from 21-hydroxylase deficiency, with special emphasis on the results of vaginoplasty. BJU Int 2000; 86:253259.

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1555. Lattimer 1556. Gross

JK. Relocation and recession of the enlarged clitoris with preservation of the glans: an alternative to amputation. J Urol 1961; 86:113116.

RE, Randolph J, Crigler JF Jr. Clitorectomy for sexual abnormalities: indications and technique. Surgery 1966; 59:300308.

1557. Masters

HW, Johnson VE. Human Sexual Response. Boston: Little, Brown, 1966.

1558. Creighton 1559. Wabrek

SM, Minto CL, Steele SJ. Objective cosmetic and anatomical outcomes at adolescence of feminizing surgery for ambiguous genitalia done in childhood. Lancet 2001; 358:124125.

AJ, Millard R, Wilson WB Jr, et al. Creation of a neovagina by the Frank nonoperative method. Obstet Gynecol 1971; 37:408413.

1560. Thomsen

C, Jensen KE, Giwercman A, et al. Magnetic resonance: in vivo tissue characterization of the testes in patients with carcinoma in situ of the testis and healthy subjects. Int J Androl 1987; 10:191198. 1561. Reiner

WG. Assignment of sex in neonates with ambiguous genitalia. Curr Opin Pediatr 1999; 11:363365.



1003

Chapter 23 - Normal and Aberrant Growth Edward O. Reiter Ron G. Rosenfeld

NORMAL GROWTH ASSESSMENT OF GROWTH Childhood is a time of growth, a process that is complex and involves the interaction of multiple, diverse factorsthe "cumulative sum of millions of unsynchronized cell replications." [1] [2] [3] Growth is common to all multicellular organisms and occurs by cell replication and enlargement along with the nonhomogeneous processes of cell and organ differentiation. The overall morphologic development, the rates of cellular division in different organ systems at different times, and the ultimate outcome are determined by the genetic composition of the individual interacting with external factors, including nutrition and psychosocial and economic factors. The very nature of linear height growth, whether occurring as a continuous process or with periodic bursts of growth and arrest, [3] [4] [5] has been hard to characterize definitively. During 1 year of growth monitoring, there may be marked seasonal variations of height and weight gain, with several monthly bursts of weight and then height growth. [6] Some normal children may have a broad growth channel, with many showing diverse but characteristic tracks. [7] [8] Nonetheless, even though the process of growth is multifactorial and complex, children usually grow in a remarkably predictable manner. Deviation from such a normal pattern of growth can be the first manifestation of a wide variety of disease processes, including both endocrine and nonendocrine disorders and involving virtually any organ system of the body. Frequent and accurate assessment of growth is, therefore, of primary importance in the care of children. Phases of Normal Growth

Growth occurs at differing rates during intrauterine life, early and middle childhood, and adolescence, prior to its cessation after fusion of long bone and vertebral epiphyseal growth plates. Prenatal growth averages 1.2 to 1.5 cm/week but varies dramatically (Fig. 23-1) ; midgestational length growth velocity of 2.5 cm/week falls to almost 0.5 cm/week immediately prior to birth. Growth velocity ( Fig. 23-2 and Fig. 23-3 ) averages about 15 cm/year during the first 2 years of life and slows to about 6 cm/year during middle childhood. Pubertal growth begins earlier in girls than in boys but is 3 to 5 cm greater in magnitude in boys than in girls. The peak height velocity during the pubertal growth spurt is comparable to the rate of growth during the second year of life. The time of onset of the pubertal growth spurt varies in normal children, reflecting the concept of a tempo of growth or rate of maturation, as emphasized by Tanner and associates. [9] [10] [11] [12] [13] In most normal children, the final height is not influenced by the chronologic time of the onset of the pubertal growth spurt, although the sex-related differences in adult height of approximately 13 cm are due to an earlier cessation of growth in females. [12] Growth ceases when the skeleton achieves adult maturity. Karlberg and associates have resolved the normal linear growth curve into three additive, partially superimposable phases. [14] [15] The components of this model include (1) an infancy phase, starting in midgestation and then rapidly decelerating to about 3 to 4 years of age; (2) a childhood phase, slowly decelerating during early adolescence; and (3) a sigmoid-shaped puberty phase that involves the adolescent growth spurt. Hormonal concomitants of these phases have been suggested, but as seen in this chapter, the interplay of the growth hormone (GH)/insulin-like growth factor (IGF) axis, gonadal steroids, and thyroxine (T 4 ) is complex, and an attempt to

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Figure 23-1 Rate of linear growth and weight gain in utero and during the first 40 weeks after birth. Length velocity is expressed in centimeters per week. The solid line depicts actual linear growth rate; the dashed line connecting the prenatal and postnatal length velocity lines depicts the theoretical curve for no uterine restriction late in gestation. The lighter dashed line depicts weight velocity. (Data from Tanner JM. Fetus into Man. Cambridge, Mass, Harvard University Press, 1978.)

define individual predominance of one hormone at any time of life is likely an oversimplification.



Measurement

Assessment of growth requires accurate and reproducible determinations of height. [16] [17] [18] Supine length is routinely measured in children younger than 2 years of age, and erect height is assessed in older children. The inherent inaccuracies involved in measuring length in infants are often obscured by the rapid skeletal growth during this period. For measurement of supine length (Fig. 23-4) , it is best to use a firm box with an inflexible board against which the head lies with a movable footboard on which the feet are placed perpendicular to the plane of the supine length of the infant. Optimally, the child should be relaxed, the legs should be fully extended, and the head should be positioned in the Frankfurt plane, with the line connecting the outer canthus of the eyes and the external auditory meatus perpendicular to the long axis of the trunk. When children are old enough (and physically capable) to stand erect, it is best to employ a wall-mounted "Harpenden" stadiometer, similar to that designed by Tanner and Whitehouse for the British Harpenden Growth Study. Free-standing stadiometers are also available but require frequent recalibration. The traditional measuring device of a flexible arm mounted to a weight balance is notoriously unreliable and does not provide reliably accurate serial measurements. As with length measurements in infants, positioning of the child in the stadiometer is critical (Fig. 23-5) . The child should be fully erect, with the head in the Frankfurt plane; the back of the head, thoracic spine, buttocks, and heels should touch the vertical axis of the stadiometer, and the heels should be together. Every effort should be made to correct discrepancies related to lordosis or scoliosis. Ideally, serial measurements should be made at the same time of day because standing height may undergo diurnal variation. [19] A trained individual, rather than an inexperienced member of the staff, should determine height. We recommend that lengths and heights be measured in triplicate, that variation is

Figure 23-2 Height velocity chart for boys constructed from longitudinal observations of British children. The 97th, 50th, and 3rd percentile curves define the general pattern of growth during puberty. Shaded areas define velocities of children who have peak velocities at ages up to 2 standard deviations (SDs) before or after the average age depicted by the percentile lines. Arrows and diamonds mark the 97th, 50th, and 3rd percentiles of peak velocity when the peak occurs at these early or late limits. (Modified from charts prepared by J. M. Tanner and R. H. Whitehouse from data published in Tanner JM, et al. Arch Dis Child 1966; 41:613635 [10] ; Iranmanesh A, et al. J Clin Endocrinol Metab 1991; 73:10811088[ 172] ; and Tanner JM, et al. Arch Dis Child 1976; 51:170179[1586] ; reproduced with permission of J. M. Tanner and Castlemead Publications, Ward's Publishing Services, Herts, UK.)

no more than 0.3 cm, and that the mean height is recorded. To determine height velocity, when several measurements are being made within a short period, the same individual should perform the determinations to eliminate interobserver variability. Even when every effort is made to obtain accurate height measurements, a minimum interval of 6 months is necessary for meaningful height velocity computation. Nine to 12 months' data are preferable so that errors of measurement are minimized and the seasonal variation in height velocity [20] is assimilated into the data.



Growth Charts

Evaluation of a child's height must be done in the context of normal standards. Such standards can be either cross-sectional or longitudinal. Most pediatric endocrine clinics in the United States continue to use the cross-sectional data, provided by the National Center for Health Statistics (NCHS), originally introduced in 1977. [21] [22] Epidemiologic limitations exist in these growth charts. The original infant charts, for example, were derived from a private study of a group of subjects who were primarily white, formula-fed, middle-class infants from southwestern Ohio. Data employed for older children came from national health examination surveys conducted from 1963 to 1974.

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Figure 23-3 Height velocity chart for girls (see legend for Figure 23-2) . (Modified and reproduced with permission of J. M. Tanner and Castlemead Publications, Ward's Publishing Services, Herts, UK.)

The NCHS, now part of the Centers for Disease Control and Prevention (CDC), has recently provided a set of 16 new growth charts (8 each for boys and girls), representing revisions of 14 existing charts, and has introduced new charts ( Fig. 23-6 Fig. 23-7 Fig. 23-8 Fig. 23-9 Fig. 23-10 Fig. 23-11 Fig. 23-12 Fig. 23-13 ) for body mass index[23] :

The charts show little change in average height over the last 25 years despite the perception that today's children are taller than those from three decades ago. These charts compare individual children with the 5th, 10th, 25th, 50th, 75th, 90th, and 95th percentiles of normal children in the United States. There are, however, two major limitations of these charts when applied to the individual child. First, they do not satisfactorily define children below the 5th or above the 95th percentiles, the very children in whom it is most critical to define the degree to which they deviate from the normal growth centiles. The NCHS data are useful in

Figure 23-4 Technique for measuring recumbent length. A device suitable for measurement of length of infants is available from Raven Equipment Ltd., UK.

(Courtesy of Noel

Cameron.)

Figure 23-5 Technique for measuring erect height using the Harpenden stadiometer with direct digital display of height. Devices of this type are available from Seritex, Inc., Carlstadt, N.M., and from Holtain Ltd., Wales, UK.

computing standard deviation scores (SDSs), which are more helpful, because a short child can be described as, for example, -4.2 or -2.5 SDS from normal. A height SDS for age is calculated as follows:

Because these are defined by cross-sectional data, however, 1006

Figure 23-6 Length-for-age and weight-for-age percentiles for boys (birth to 36 months). Developed by the National Center for Health Statistics in collaboration with the National Center for Chronic Disease Prevention and Health Promotion (2000). http://www.cdc.gov/growthcharts

childhood SDSs are not directly comparable with SDS during adolescence, when variation in growth rate and maturational tempo can be large. Second, cross-sectional data are of greater value during infancy and childhood than in adolescence because differences in the timing of pubertal onset can considerably influence normal growth rates. To address this issue, Tanner and colleagues [9] developed longitudinal growth charts, combining longitudinal data to construct the curve shapes with centile widths obtained from a large cross-sectional survey, thus accounting for variability in the timing of puberty. Such charts are of particular value in assessing growth during adolescence and puberty and for plotting sequential growth data on any given child. The data from cross-sectional and longitudinal growth studies have been employed to develop height velocity standards, enhancing the value of linear growth velocity measurements in an individual ( see Fig. 23-2 and Fig. 23-3 ). [9] [24] Carefully documented height velocity data are invaluable in assessing the child with abnormalities of growth. Although there is considerable variability in the normal height velocity in children of different ages, between age 2 years and the onset of puberty, children

normally grow with remarkable fidelity relative to the normal growth curves. The physician should note any "crossing" of height percentiles during this age period, and abnormal height velocities always warrant further evaluation.

Figure 23-7 Head circumferencefor-age and weight-for-length percentiles for boys (birth to 36 months). Developed by the National Center for Health Statistics in collaboration with the National Center for Chronic Disease Prevention and Health Promotion (2000). http://www.cdc.gov/growthcharts

Syndrome-specific growth curves have been developed for a number of clinical conditions associated with growth failure (e.g., Turner's syndrome, [25] achondroplasia, [26] Down's syndrome[27] ). Such growth profiles are invaluable for tracking the growth of children with these clinical conditions. Deviation of growth from the appropriate disease-related growth curve suggests the possibility of a second underlying cause.



Body Proportions

Many abnormal growth states, including both short stature and excessive stature, are characterized by disproportionate growth. The following determinations should be made as part of the evaluation of short stature: 1. 2. 3. 4.

Occipitofrontal head circumference. Lower body segment: distance from top of pubic symphysis to the floor. Upper body segment: sitting height (height of stool should be subtracted from standing height). Arm span.

Published standards exist for these body proportion measurements, which must be evaluated relative to the patient's age. [28] The upper segment/lower segment ratio, for example, ranges from 1.7 in the neonate to slightly below 1.0 in the adult. 1007

Figure 23-8 Length-for-age and weight-for-age percentiles for girls (birth to 36 months). Developed by the National Center for Health Statistics in collaboration with the National Center for Chronic Disease Prevention and Health Promotion (2000). http://www.cdc.gov/growthcharts



Skeletal Maturation

The growth potential in the tubular bones can be assessed by evaluation of the progression of ossification within the epiphyses. The ossification centers of the skeleton appear and progress in a predictable sequence in normal children, and skeletal maturation can be compared with normal age-related standards. This forms the basis of bone age or skeletal age, the only readily available quantitative determination of net somatic maturation and thus a mirror of the tempo of growth and maturation. It is not clear which factors determine this normal maturational pattern, but it is certain that genetic factors and multiple hormones, including T 4 , GH, and gonadal steroids, are involved. [29] [30] [31] Recent studies in patients with mutations of the gene for the estrogen receptor [32] or for the aromatase enzyme [33] [34] [35] have shown that estrogen is primarily responsible for epiphyseal fusion, [36] although it seems unlikely that estrogen is solely responsible for all aspects of skeletal maturation. After the neonatal period, a radiograph of the left hand and wrist is commonly used for comparison with the published standards of Greulich and Pyle. [37] An alternative method for assessing bone age from radiographs of the left hand involves a scoring system for developmentally identified stages of each of 20 individual bones,[38] a technique that has been adapted for computed assessment. [39] [40] The left hand is used because radiographs of the entire skeleton would be tedious and expensive and would involve additional radiation exposure. However, the

Figure 23-9 Head circumferencefor-age and weight-for-length percentiles for girls (birth to 36 months). Developed by the National Center for Health Statistics in collaboration with the National Center for Chronic Disease Prevention and Health Promotion (2000). http://www.cdc.gov/growthcharts

hand does not contribute to height, and accurate evaluation of growth potential sometimes calls for radiographs of the legs and spine. A number of important caveats concerning bone age must be considered. Experience in determination of bone age is essential to minimize intraobserver variance, and clinical studies involving bone age generally benefit from having a single reader perform all interpretations. The normal rate of skeletal maturation differs between boys and girls and among different ethnic groups. The standards of Greulich and Pyle are separable by sex but were developed in American white children between 1931 and 1942. Finally, both the Greulich and Pyle and the Tanner and Whitehouse standards involved normal children [41] and may not be applicable to children with skeletal dysplasias, endocrine abnormalities, or other forms of growth retardation.



Prediction of Adult Height

The extent of skeletal maturation observed in an individual can be employed to predict the ultimate height potential. Such predictions are based on the observation that the more delayed the bone age (relative to chronologic age), the longer the time before epiphyseal fusion prevents further growth. The most commonly used method for height prediction, based on Greulich and Pyle's Radiographic Atlas of Skeletal Development,

[37]

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Figure 23-10 Stature-for-age and weight-for-age percentiles for boys (2 to 20 years). Developed by the National Center for Health Statistics in collaboration with the National Center for Chronic Disease Prevention and Health Promotion (2000). http://www.cdc.gov/growthcharts

was developed by Bayley and Pinneau [42] and relies on bone age, height, and a semiquantitative allowance for chronologic age (Table 23-1) . The system of Tanner and colleagues[38] [43] employs height; bone age; chronologic age; height and bone age increments during puberty in the previous year; and menarchal status. Roche and associates [44] employ the combination of height, bone age, chronologic age, midparental height, and weight. Further, attempts have been made to calculate final height predictions without requiring the use of skeletal age [45] [46] by using multiple regression analyses with available data such as height, weight, birth measurements, and midparental stature. All of these systems are, by nature, empirical and are not absolute predictors. The more advanced the bone age, the greater the accuracy of the adult height prediction because a more advanced bone age places a patient closer to final height. All methods of predicting adult height are based on data from normal children, and none has been documented to be accurate in children with growth abnormalities. For this kind of precision, it would be necessary to develop disease-specific (e.g., achondroplasia, Turner's syndrome) atlases of skeletal maturation.



Parental Target Height

Because genetic factors are important determinants of growth and height potential, it is useful to assess a patient's stature relative to that of siblings and parents. Tanner and associates developed a growth chart modifying the heights of children, aged 2 to 9 years, by the midparental height. [47] Further, the child's predicted adult height (see earlier) may be related to a parental target height, or the mean parental height with the addition or subtraction of 6.5 cm for boys and girls, respectively. The two standard deviation (2 SD) range for this calculated parental target height is about ± 10 cm, so that calculated target heights, like predicted adult heights, are approximations. Recent statistical reassessment shows a tendency for regression to the mean of the children's height as related to the midparental target height. [48] Failure to realize this may lead to inappropriately using short parental height as an explanation for marked short stature in a child. Nevertheless, when a child's growth pattern clearly deviates from that of parents or siblings, the possibility of underlying pathology should be considered. Although it is certainly important to measure the heights of parents and siblings, rather than accept their statural claims, one must recall as well that it is not always possible to know the heights of the true biologic parents.



ENDOCRINE REGULATION OF GROWTH The Pituitary Gland

The concept of the pituitary as a "master gland," controlling the endocrine activities of the body, has been replaced by recognition of the importance of the brain and, particularly, the hypothalamus in regulating hormonal production and secretion. Nevertheless, the pituitary gland is central to understanding the regulation of growth. Embryologically, the pituitary gland is formed from two distinct sources [49] : Rathke's pouch, a diverticulum of the primitive oral cavity (stomodeal ectoderm), gives rise to the adenohypophysis. The neurohypophysis (posterior pituitary) originates in the neural ectoderm of the floor of the forebrain, which also develops into the third ventricle. The adenohypophysis normally constitutes 80% of the weight of the pituitary and consists of anterior, intermediate, and infundibular lobes. In humans, the anterior lobe is the largest component and houses the most hormone-producing cells. Rathke's pouch, the origin of the adenohypophysis, can be identified in the 3-mm embryo during the 3rd week of pregnancy. GH-producing cells can be found in the adenohypophysis by 9 weeks of gestation. [50] Vascular connections between the anterior lobe of the pituitary and the hypothalamus develop about this time, [51] [52] although hormone production can occur in the pituitary in the absence of connections with the hypothalamus. Somatotrophs can frequently be demonstrated in the pituitary in anencephalic newborns. [53] Nevertheless, the initiation of development of the anterior pituitary is probably dependent on responsiveness of the oral ectoderm to inducing factors from the ventral diencephalon (Fig. 23-14) .[54] [55] [56] [57] [58] [59] [60] A complex orchestration of temporally sequenced and geographically restricted expression of multiple extracellular signaling peptides and intracellular transcription factors regulates this developmental process. [60] [61] [62] The developing pituitary gland and hypothalamus are in close anatomic juxtaposition, and their embryonic development is likely to be codependent. Some of the diencephalic factors that have been identified to be critical in formation and patterning of Rathke's pouch, which, in the mouse, is initiated on embryonic day 8 (e8) are bone morphogenetic proteins 4 and 2 (BMP-4/2), Wnt5a, and fibroblast growth factor 8 (FGF-8). [54] [60] [63] The dorsal neuroepithelial

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Figure 23-11 Stature-for-age and weight-for-age percentiles for girls (2 to 20 years). Developed by the National Center for Health Statistics in collaboration with the National Center for Chronic Disease Prevention and Health Promotion (2000). http://www.cdc.gov/growthcharts

signal, BMP-4, is needed for "organ commitment" of the pituitary, whereas a BMP-2 (ventral) and FGF-8 (dorsal) gradient determines pituitary cell phenotypes (i.e., gonadotrophs and the Pit-1dependent lines, somatotrophs, lactotrophs, and thyrotrophs [ventral] and melanotrophs and corticotrophs [dorsal]). [54] [55] [57] [60] It seems that reciprocal interaction of at least two transcription factors, Pit-1 and GATA-2, are important in implementing the cell-determination signals of BMP-2 and FGF-8.

[54]

[61]

Explant studies in the mouse have demonstrated that if Rathke's pouch is removed from the oral ectoderm on e10.5 and incubated in appropriate culture medium, differentiation of each of the pituitary cell types continues, indicating that by that point, organogenesis of the anterior pituitary is no longer dependent on hypothalamic signals,[60] although such signals may remain critically involved in pituitary hormone production. A number of pituitary-specific transcription factors are involved in the determination of pituitary cell lineages and cell-specific expression of anterior pituitary hormones[57] [58] [60] [64] [65] [66] To date, defects in several homeodomain transcription factors shown to be involved in human anterior pituitary development and differentiation have now been associated with various combinations of pituitary hormone deficiencies ( see Fig. 23-14 and Table 23-5 ). Because additional gene defects have been implicated in abnormal murine pituitary development, it seems likely that the number of human genetic defects will expand. In the adult, the mean pituitary size is 13 × 9 × 6 mm. [67] The mean weight is 600 mg (range, 400 to 900 mg), is slightly greater in women than in men, and increases during pregnancy. [68] In the newborn, pituitary weight averages about 100 mg. Normally, the pituitary resides in the sella turcica, immediately above and partially surrounded by the sphenoid bone. The volume of the sella turcica is a good index of pituitary size and may be reduced in the child with pituitary hypoplasia [69] or increased in some with PROP1 defects (see Table 23-5) . The anatomic proximity between the optic chiasm and the pituitary is important because hypoplasia of the optic chiasm may occur together with hypothalamic/pituitary dysfunction in the syndrome of septo-optic dysplasia. [70] Children with congenital blindness or nystagmus should be monitored carefully for hypopituitarism, and suprasellar growth of a pituitary tumor may initially manifest with visual complaints or evidence of decreases in peripheral vision. The existence of a portal circulatory system within the pituitary gland is critical for normal pituitary function. The blood supply of the pituitary is shown in Figure 23-15 . [51] [52] Hypothalamic peptides, produced in neurons that terminate in the infundibulum, enter the primary plexus of the hypophyseal portal circulation and are transported via the hypophyseal portal veins to the capillaries of the anterior pituitary. This portal system thus provides a means of communication between the neurons of the hypothalamus and the anterior pituitary.



Growth Hormone Chemistry

Human GH is produced as a single chain, 191amino acid, 22-kd protein (Fig. 23-16) . [71] [72] It is not glycosylated, but it does contain two intramolecular disulfide bonds. GH shares sequence homology with prolactin, chorionic somatomammo-tropin (CS) (placental lactogen), and a 22-kd GH variant (GH-V) secreted only by the placenta [73] that differs from pituitary GH by 13 amino acids. The genes for these proteins have probably evolved from a common ancestral gene, even though the genes are located on different chromosomes (chromosome 6 for prolactin, chromosome 17 for GH). [74] The genes for GH, prolactin, and placental lactogen share a common structural organization, with four introns separating five exons. In fact, the GH subfamily contains five members, whose genes are located on a 78-kb section of chromosome 17; the 5' to 3' order of the genes are GH, a CS pseudogene, CS-A, GH-V, and CS-B. [75] Normally, about 75% of GH produced by the pituitary is of the mature, 22-kd form. Alternative splicing of the second codon results in deletion of amino acids 32 to 46, yielding a 20-kd form, which normally accounts for 5% to 10% of pituitary GH. [74] [76] The remainder of pituitary GH includes desamidated and N-acetylated forms and various GH oligomers. Secretion

The pulsatile pattern characteristic of GH secretion largely reflects the interplay of two hypothalamic regulatory peptides, growth hormonereleasing hormone (GHRH)[77] [78] and somatostatin (somatotropin releaseinhibiting factor [SRIF]), [79] with presumed modulation by putative other GH-releasing factors. [80] GHRH activity is species-specific, presumably reflecting the specificity of binding to a G proteinrelated receptor on the pituitary somatotrophs. Regulation of GH production by GHRH is mediated largely at the level of transcription and is enhanced by increases in intracellular cyclic adenine monophosphate (cAMP) levels. The GHRH receptor is a member of the G proteincoupled receptor family B-III, also called the secretin family, and has partial sequence identity with receptors for vasoactive intestinal

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Figure 23-12 Body mass indexfor-age percentiles for boys (2 to 20 years). Developed by the National Center for Health Statistics in collaboration with the National Center for Chronic Disease Prevention and Health Promotion (2000). http://www.cdc.gov/growthcharts

polypeptide, secretin, calcitonin, and parathyroid hormone. [81] [82] In the dwarf transgenic mouse model with diminished GHRH production, pituitary somatotroph proliferation is markedly decreased. [83] Mutations of the GHRH gene itself have not yet been reported, but anatomic and functional abnormalities of the connection between the hypothalamus and the anterior pituitary, which prevent interaction of GHRH with its receptor on the somatotroph, are the most important causes of clinical growth hormone deficiency (GHD). The Gsh-1 homeobox gene, which is expressed in the developing central nervous system (CNS) [84] but not in the pituitary, plays an important role, nonetheless, in mouse pituitary development. Mice with mutations in this gene do not produce GHRH and, presumably, do not produce gonadotropin-releasing hormone (GnRH) because anterior pituitary hypoplasia and deficiencies of GH, prolactin, and luteinizing hormone (LH) are present. [85] The effects of this gene on hypothalamic releasing factors are analogous to those of the Pit-1 or PROP1 genes (see later) at the pituitary level. Instead of regulating GH synthesis, somatostatin appears to affect the timing and amplitude of pulsatile GH secretion. The pulsatile secretion of GH in vivo is believed to result from a simultaneous reduction in hypothalamic somatostatin release and increase in GHRH release. [86] Conversely, a trough of GH secretion occurs when somatostatin is released in the face of diminished GHRH activity. The regulation of the reciprocal secretion of GHRH and somatostatin is imperfectly understood. Multiple neurotransmitters

Figure 23-13 Body mass index-for-age percentiles for girls (2 to 20 years). Developed by the National Center for Health Statistics in collaboration with the National Center for Chronic Disease Prevention and Health Promotion (2000). http://www.cdc.gov/growthcharts

and neuropeptides are involved in the regulation of the release of these hypothalamic factors. [87] These factors influence GH secretion with stress, sleep, hemorrhage, fasting, hypoglycemia, and exercise and form the basis for a number of GH-stimulatory tests employed in the evaluation of GH secretory capacity/reserve GH secretion is also influenced by a variety of nonpeptide hormones, including androgens, [88] [89] estrogens, [90] T 4 ,[91] and glucocorticoids. [92] [93] The mechanisms by which these hormones regulate GH secretion may involve actions at both the hypothalamic and pituitary levels. Practically speaking, hypothyroidism and glucocorticoid excess each may blunt spontaneous and provocative GH secretion. Gonadal steroids appear to be responsible for the rise in GH secretion characteristic of puberty. [94]

Synthetic hexapeptides capable of stimulating GH secretion are termed GH secretagogues (GHSs).[95] [96] [97] [98] [99] [100] [101] [102] These peptides stimulate GH release and enhance the GH response to GHRH, although they work at receptors distinct from those for GHRH, at hypothalamic and pituitary sites. [100] [101] [102] [103] Finding 40% to 60% homology to the G proteincoupled GHS receptor in the pufferfish indicates that structure and function of this receptor have been highly conserved for about 400 million years, certainly suggesting a fundamental role for the natural ligand of this receptor. [102] Kojima and co-workers[104] identified a putative endogenous ligand, a 28amino acid with the serine 3 residue N-octanoylated, referred to as ghrelin. It is found primarily in the stomach (and throughout the gastrointestinal tract [105] ) but also in

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TABLE 23-1 -- Prediction of Adult Stature: Fraction of Adult Height Attained at Each Bone Age Girls Bone Age (years/months)

Boys

Retarded Average* Advanced Retarded Average* Advanced

60

0.733

0.720

0.680

63

0.742

0.729

0.690

66

0.751

0.738

0.700

69

0.763

0.751

0.709

70

0.770

0.757

0.172

0.718

0.695

0.670

73

0.779

0.765

0.722

0.728

0.702

0.676

76

0.788

0.772

0.732

0.738

0.709

0.683

79

0.797

0.782

0.742

0.747

0.716

0.689

80

0.804

0.790

0.750

0.756

0.723

0.696

83

0.813

0.801

0.760

0.765

0.731

0.703

86

0.823

0.810

0.771

0.773

0.739

0.709

89

0.836

0.821

0.784

0.779

0.746

0.715

90

0.841

0.827

0.790

0.786

0.752

0.720

93

0.851

0.836

0.800

0.794

0.761

0.728

96

0.858

0.844

0.809

0.800

0.769

0.734

99

0.866

0.853

0.819

0.807

0.777

0.741

100

0.874

0.862

0.828

0.812

0.784

0.747

103

0.884

0.874

0.841

0.816

0.791

0.753

106

0.896

0.884

0.856

0.819

0.795

0.758

109

0.907

0.896

0.870

0.821

0.800

0.763

110

0.918

0.906

0.883

0.823

0.804

0.767

113

0.922

0.910

0.887

0.827

0.812

0.776

116

0.926

0.914

0.891

0.832

0.818

0.786

119

0.929

0.918

0.897

0.839

0.827

0.800

120

0.932

0.922

0.901

0.845

0.834

0.809

123

0.942

0.932

0.913

0.852

0.843

0.818

126

0.949

0.941

0.924

0.860

0.853

0.828

129

0.957

0.950

0.935

0.869

0.863

0.839

130

0.964

0.958

0.945

0.880

0.876

0.850

133

0.971

0.967

0.955

0.890

0.863

136

0.977

0.974

0.963

0.902

0.875

139

0.981

0.978

0.968

0.914

0.890

140

0.983

0.980

0.972

0.927

0.905

143

0.986

0.983

0.977

0.938

0.918

146

0.989

0.986

0.980

0.948

0.930

149

0.992

0.988

0.983

0.958

0.943

150

0.994

0.990

0.986

0.968

0.958

153

0.995

0.991

0.988

0.973

0.967

156

0.996

0.993

0.990

0.976

0.971

159

0.997

0.994

0.992

0.980

0.976

160

0.998

0.996

0.993

0.982

0.980

163

0.999

0.996

0.994

0.985

0.983

166

0.999

0.997

0.995

0.987

0.985

169

0.9995

0.998

0.997

0.989

0.988

170

1.00

0.999

0.998

0.991

0.990

173 176

0.993 0.9995

179 180

0.9995

0.994 0.995

1.00

0.996

183

0.998

186

1.00

From Post EM, Richman RA. A condensed table for predicting adult stature. J Pediatr 1981; 98:440442; based on data of Bayley N, Pinneau SR. J Pediatr 1952; 40:423441. These tables have been organized in an easy to use slide-rule format (Adult Height Predictor, copyright 1987 Ron G. Rosenfeld). *Average: Bone age within 1 year of chronologic age.

the hypothalamus, heart, lung, and adipose tissue. Administration of ghrelin stimulates food intake and obesity [106] and raises plasma GH concentrations [104] [107] [108] [109] and, to a lesser extent, adrenocorticotropic hormone (ACTH). [109] These data suggest that ghrelin is an important stimulus for nutrient allocation for growth and metabolism and a central component of the GH regulatory system. Pituitary adenylate cyclase-activating peptide (PACAP), a hypothalamic peptide possibly involved in the regulation of GH secretion, is a member of the PACAP/glucagon superfamily. [110] [111] The developmental abnormality holoprosencephaly, which can be associated with GHD, may be caused by gene defects affecting PACAP and PACAP receptor expression. [111] [112]

The synthesis and secretion of GH are also regulated by the IGF peptides. [113] [114] [115] Receptors specific for IGF-I and IGF-II have been identified in varied pituitary cell systems. [116] [117] Inhibition of GH secretion by IGF-I and IGF-II in rat anterior pituitary cells has been demonstrated in a perfusion system, [118] and spontaneous GH secretion is diminished in humans treated with synthetic IGF-I. [119] [120]

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Figure 23-14 Development of pituitary cell lineages. A, Schematic representation of pituitary cell precursors showing the expression of prevalent transcription factors at each stage of development. Terminally differentiated cells are shown as larger and shaded circles together with the hormones produced (lineage-specific transcription factors are highlighted in bold in these cells). The interaction with transcription factors and signaling molecules in the hypothalamus is also noted. Transcription factors are represented in lower case (except for SF1 and GATA2), whereas signaling molecules appear in upper case. B, Schema showing the timing of appearance and disappearance of pituitary transcription factors during mouse embryogenesis. BMP4, bone morphogenic protein 4; e, embryonic day; ER, estrogen receptor; FGF8, fibroblast growth factor 8; FSH, follicle-stimulating hormone; GHRH, growth hormone-releasing hormone; GnRH, gonadotropin-releasing hormone; GSU, -glycoprotein subunit; LH, luteinizing hormone; POMC, pro-opiomelanocortin; PRL, prolactin; SF1, steroidogenic factor 1; TRH, thyrotropin-releasing hormone; TSH, thyrotropin (thyroid-stimulating hormone). (From Lopez-Bermejo A, Buckway CK, Rosenfeld RG. Genetic defects of the growth hormoneinsulin-like growth factor axis. Trends Endocrinol 2000; 11:43.) Studies of GH Secretion in Humans

The episodic release of GH from pituitary somatotrophs results in intermittent increases in serum levels of GH that are separated by periods of low or undetectable levels, during which time GH secretion is minimal. [121] [122] [123] The pulsatile nature of GH secretion has been demonstrated by frequent serum sampling, coupled with the use of immunofluorometric or chemoluminescent assays of GH. [123] [124] [125] [126] Under normal circumstances, serum GH levels are less than 0.04 µg/L between secretory bursts. Consequently, it is impractical to assess GH secretion by random serum sampling. Extensive sampling studies in people at different ages, in healthy people, and in many patients with abnormal conditions have defined GH pulses, basal secretion, and diurnal variability. [94] [121] [122] [127] [128] [129] [130] [131] [132] [133] [134] Computer programs have been developed to indicate whether changes in GH levels in various life periods and under diverse clinical circumstances occur because of a change in a secretory mass or pulse frequency, an altered clearance, or a combination of these processes. [121] [123] [132] [133] [135] Deconvolution techniques allow accurate estimates of the quantity of GH secreted per burst, GH clearance kinetics, pulse amplitudes and frequencies, and an overall calculation of endogenous GH production. Approximate entropy, a model-free measure, is applied to quantify the degree of orderliness of GH release patterns. [136] [137] The impact of the specific nature of pulsatile GH secretion on its biologic actions is under study. [121] [123] [138] For example, it appears that better statural growth is associated with large swings of GH output but of relatively uniform magnitude in an irregular sequence (high approximate entropy). [139] [140] By 9 to 12 weeks of gestation, GH-secreting cells have been identified, and by 7 to 9 weeks, immunoreactive pituitary GH is present. [50] [141] [142] By 5 weeks of gestation, fetal pituicytes secrete GH in vitro, [143] before the hypothalamic-portal vascular system is differentiated. [144] [145] [146] By at least 6 weeks of gestation, Pit-1 messenger RNA (mRNA) and Pit-1 protein are expressed; its abundant presence early in gestation suggests an important role in cytodifferentiation and cell proliferation. [147] By the end of the first trimester, GH can be identified in

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Figure 23-15 Main components of the hypothalamic-pituitary portal system. (From Guyton AC, Hall JE. Human Physiology and Mechanisms of Disease, 6th ed. Philadelphia, WB Saunders, 1997, p 600, with permission.)

fetal serum, with peak levels of around 150 µg/L in midgestation. [141] [142] [148] Throughout the latter part of pregnancy, serum levels fall and are lower in term than in premature infants, perhaps reflecting feedback by the higher serum levels of IGF peptides characteristic of the later stages of gestation. [149] [150] Through childhood and early puberty, mean levels of GH decrease from values of 25 to 35 µg/L in the neonatal period to approximately 5 to 7 µg/L. [94] [127] [142] During adolescence, 24-hour GH secretion peaks, [94] undoubtedly contributing to the high serum levels of IGF-I characteristic of puberty. During mid to late puberty, the increase in GH production is due both to enhanced pulse amplitude and increased mass of GH per secretory burst rather than to a change in pulse frequency

Figure 23-16 Covalent structure of human growth hormone. (From Chawla RK, Parks JS, Rudman D. Structural variants of human growth hormone: biochemical, genetic, and clinical aspects. Annu Rev Med 1983; 34:519547.)

( Fig. 23-17 and Fig. 23-18 ).[94] [121] [127] [136] Greater irregularity in GH secretion corresponds to greater linear growth. [151] In the face of stable levels of the GH-binding protein (GHBP), [152] [153] the enhanced pubertal GH production is associated with higher levels of free GH (Fig. 23-19) , facilitating the production of IGF-I. By late adolescence, [132] GH and IGF production begins to decline and continues to fall throughout adult life. Normal young adult men generally experience 6 to 10 GH secretory bursts per 24 hours, a value similar to that in younger children and in adolescents. [94] [123] [127] In contrast, 24-hour GH production rates for normal men range from 0.25 to 0.52 mg/m 2 surface area, [93] [154] about 20% to 30% of pubertal levels; this is largely due to decreased GH pulse amplitude with age. [94] [127] Indeed, puberty may be considered, with some justification, a period of "acromegaly," whereas aging, with its decrease in GH secretion, has been termed the somatopause. [90] [155] [156] Physiologic states that affect GH secretion, in addition to maturation and aging, include sleep, [157] [158] nutritional status, [159] fasting, exercise, [160] stress, [160] and gonadal steroids. [88] [89] Maximal GH secretion occurs during the night, especially at the onset of the first slow-wave sleep (stages III and IV). Rapid-eyemovement sleep, however, is associated with low GH secretion. [157] [158] [161] A circadian rhythm of somatostatin secretion, on which is superimposed episodic bursts of GHRH release, may help explain the nocturnal augmentation of GH production. [162] When testosterone was administered to boys with delayed puberty, spontaneous GH release was enhanced, but such a change was not duplicated by administration of nonaromatizable androgens, emphasizing the possible unique importance of estrogen on GH secretion. [13] [132] [163] [164] [165] [166] [167] [168] The effects of testosterone on serum IGF-I levels may, in part, be independent of GH because individuals with mutations of the GH receptor (GHR) still experience a rise in serum IGF-I during puberty. [169] With a combination of deconvolution analysis, approximate

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Figure 23-17 Relation between 24-hour (24h) mean growth hormone (GH) levels and age in boys and men. Bars represent values for the 24-hour mean (± SE) levels of GH (left axis) from 60 24-hour GH profiles of healthy boys and men subdivided according to chronologic age. An idealized growth velocity curve reproduced from the 50th percentile values for whole-year height velocity of North American boys (9) is superimposed. (From Martha PM Jr, Rogol AD, Veldhuis JD, et al. Alterations in the pulsatile properties of circulating growth hormone concentrations during puberty in boys. J Clin Endocrinol Metab 1989; 69:563570.)

entropy, and cosine regression analysis, Veldhuis and associates [136] [167] carefully evaluated intensive GH sampling data, derived from measurements in sensitive GH assays, in prepubertal and pubertal boys and girls. In addition to the amplified secretory burst mass originating from jointly increased GH pulse amplitude and duration, they found that sex steroids selectively affected facets of GH neurosecretory control; estrogen increases basal GH secretion rate and irregularity of GH release patterns, whereas testosterone stimulates greater GH secretory burst mass and IGF-I concentrations. Obesity is characterized by markedly decreased GH production, reflected by nearly a marked decrease in number of GH secretory bursts and of half-life duration. [159] [170] Obesity in childhood and adolescence, similarly, is characterized by decreased GH production but normal IGF and increased GHBP levels and often increased linear growth. [170] The hyperinsulinism associated with obesity causes lowered IGF-binding protein (IGFBP)-1 and, perhaps, higher free IGF-I levels. [171] Endogenous GH secretion and levels achieved during provocative tests in these obese subjects [165] approximate the diagnostic range of GHD. Fasting increases both the number and amplitude of GH secretory bursts, presumably reflecting decreased somatostatin secretion and enhanced GHRH release while lowering GHBP concentrations. Rapid changes in levels of IGFBPs in response to altered nutrition and changes of insulin levels may modify the effect of IGF-I on its negative feedback and effector sites.[123] [170] Body mass also influences GH production in normal prepubertal and pubertal children and adults. [127] [134] [172] Growth Hormone Receptor/Growth HormoneBinding Protein

Leung and colleagues [173] cloned both the rabbit and human complementary DNAs (cDNAs) for the GHR. Each contains an open reading frame of 638 amino acids and encodes a mature receptor of 620 amino acids and a predicted molecular

Figure 23-18 A, The mean (± SE) 24-hour (24h) levels of growth hormone (GH) for groups of normal boys at varied stages of pubertal maturation. B, The mean (± SE) area under the GH concentration versus time curve for individual GH pulses, as identified by the Cluster pulse detection algorithm. C, The number of GH pulses (± SE), as detected by the Cluster algorithm, in the 24-hour GH concentration profiles for boys in each of the pubertal study groups. Note: The mean 24-hour GH concentration changes are largely mediated by changes in the amount of GH secreted per pulse rather than the frequency of pulses. In each panel, bars bearing the same letter are statistically indistinguishable. (From Martha PM Jr, Rogol AD, Veldhuis JD, et al. Alterations in the pulsatile properties of circulating growth hormone concentrations during puberty in boys. J Clin Endocrinol Metab 1989; 69:563570.)

Figure 23-19 Levels of growth hormone (GH) and growth hormonebinding protein (GHBP) measured in normal pubertal boys throughout adolescence. GHBP levels do not significantly change during puberty, but there is a significant increment of GH production and, therefore, of GH levels during this same time. These data suggest that there may be greater amounts of "free GH" during this period, leading to greater production of insulin-like growth factor I. (Data from Martha PM Jr, et al. J Clin Endocrinol Metab 1989; 69:563570; and Martha PM Jr, et al. J Clin Endocrinol Metab 1991; 73:175181.)

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Figure 23-20 (Figure Not Available) A model depicting intracellular signaling intermediates induced by binding of growth hormone (GH) with the GH receptor (GHR). ERKS, extracellular signal-regulated kinase; Grb, growth factor receptorbinding protein; JAK, Janus kinase; IRS, insulin receptor substrate; MAP, mitogen-activated protein kinase; MEK, MAPK-ERK kinase; PKC, protein kinase C; SHP-2, protein tyrosine phosphatase; SOCS, suppressors of cytokine signaling; STAT, signal transducer and activator of transcription. (From Le Roith D, Bondy C, Yakar S, et al. The somatomedin hypothesis. Endocr Rev 2001; 22:5374. © The Endocrine Society.)

weight of 70 kd before glycosylation. There are three domains: (1) an extracellular, hormone-binding domain, (2) a single membrane-spanning domain, and (3) a cytoplasmic domain. In humans, the most important circulating GH-binding protein appears to be derived from proteolytic cleavage of the extracellular domain of the receptor. [174] In the mouse[175] [176] and rat,[177] however, there are multiple transcripts for the GHR; the larger, 3.4 to 4.8 kb, transcript codes for the intact receptor and the 1.2 to 1.9 kb transcript codes for the soluble GHBP. The coding and 3' untranslated regions of the human GHR are encoded by nine exons, numbered 2 to 10. [178] [179] The gene for the human GHR is located on chromosome 5p13.1-p12, where it spans more than 87 kb.[180] The GHR shows sequence homology with the prolactin receptor and with receptors for interleukin (IL)-2, IL-3, IL-4, IL-6, and IL-7, as well as receptors for erythropoietin, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon. [178] The GHR is a member of the class 1 hematopoietic cytokine family. [181] Examination of the crystal structure of the GH/GHR complex revealed that the complex consists of one molecule of GH bound to two GHR molecules, indicating a GH-induced receptor dimerization, which is necessary for GH action. [182] After binding to its receptor, GH stimulates phosphorylation of a protein with an apparent molecular weight ratio of 120 kd. [183] Although it was originally suspected that the GHR might be capable of autophosphorylation, it is now apparent that the major tyrosine-phosphorylated protein is associated with the receptor rather than being the receptor itself. JAK2 has been recently identified as the critical GHRassociated tyrosine kinase. [184] The presumed sequence of steps in GH action is shown in Figure 23-20 (Figure Not Available) : 1. 2. 3. 4. 5. 6.

Binding of GH to the membrane-associated GHR. Sequential dimerization of the GHR through binding to each of two specific sites on GH. Interaction of the GHR with JAK2. Tyrosine phosphorylation of both JAK2 and the GHR. Changes in cytoplasmic and nuclear protein phosphorylation and dephosphorylation. Stimulation of target gene transcription.

GH-dependent and JAK2-dependent phosphorylation and activation have been demonstrated for many cytoplasmic signaling molecules that, after forming homodimers or heterodimers, translocate into the nucleus, bind DNA, and activate transcription. [185] [186] [187] How all of these seemingly redundant pathways interact to mediate the various anabolic and metabolic actions of GH remains to be elucidated. The major GHBP in human plasma binds GH with high specificity and affinity but with relatively low capacity, because about 45% of circulating GH is bound. [153] [188] [189] [190] [191] The GHBP is, in essence, the extracellular domain of the GHR and has an apparent molecular weight ratio of approximately 55 kd. An additional GHBP, not

related to the GHR, binds approximately 5% to 10% of circulating GH with lower affinity. [153] [191] GHBP prolongs the half-life of GH, presumably by impairing its glomerular filtration, and modulates its binding to the GHR. In general, GHBP levels reflect GHR levels and activity;

1016

that is, low levels are associated with states of growth hormone insensitivity (GHI).[153] [191] In the rapidly growing child, however, levels of GHBP are quite low. [192] [193] Initial assays for GHBP involved incubation of serum with [ 125 I]-GH and separation of bound from free radioligand. [153] Carlsson and co-workers[194] have developed a ligand-mediated immunofunctional assay for measurement of GHBP. Levels of GHBP are low in early life, rise through childhood, and plateau during the pubertal years and adulthood. [152] [191] [192] [193] Once puberty is reached, levels are usually constant for a given individual. [152] Impaired nutrition, diabetes mellitus, hypothyroidism, chronic liver disease, and a spectrum of inherited abnormalities of the GHR are associated with low levels of GHBP, whereas obesity, refeeding, early pregnancy, and estrogen treatment can cause elevated levels of GHBP. [153] [191] A direct correlation exists between GHBP levels and body mass index. [195] Serum GHBP levels correlate inversely with 24-hour GH production [152] ; this reciprocal relationship between GH production and GHBP in normal subjectsand in subjects with idiopathic short stature (ISS) [196] [197] may result from adjustments of GH secretion to accommodate GHR levels that may be genetically determined or modulated by environmental factors such as nutritional status. [195] [198] Assays of serum levels of GHBP are useful in identifying subjects with GH insensitivity due to genetic abnormalities of the GHR. [199] [200] Patients with GHI due to nonreceptor abnormalities, defects of the intracellular domain of the GHR, or inability of the receptor to dimerize may, however, have normal serum levels of GHBP. [169] [201] [202] [203] Inhibition of GH signaling by several members of the GH-inducible suppressors of cytokine signaling (SOCS) family has been reported. [204] The importance of SOCS proteins in controlling growth is demonstrated by the finding of gigantism in SOCS-2 knockout mice. [205] Endotoxin and proinflammatory cytokines, such as IL-1 and tumor necrosis factor (TNF-), which can also induce SOCS proteins, [206] produce GHI. SOCS-3 induced by IL-1 and TNF- or by endotoxin in vivo may play a role in the GHI induced by sepsis. [207] Critically ill patients with septic shock treated with GH had increased mortality, [208] possibly related to induction of GHI in specific tissues as a consequence of endotoxinemia and cytokinemia. Growth Hormone Actions

According to the somatomedin hypothesis, the anabolic actions of GH are mediated through the IGF peptides. [209] [210] Although this theory is largely true, GH is also capable of inducing effects that are independent of IGF activity. Indeed, the actions of GH and IGF are, on occasion, contradictory, as evident in the diabetogenic actions of GH[211] [212] and the glucose-lowering activity of IGFs. Green and colleagues [213] have attempted to resolve some of these differences in a dual-effector model, in which GH stimulates precursor cells, such as prechondrocytes, to differentiate. When differentiated cells or neighboring cells then secrete IGFs, these peptides act as mitogens and stimulate clonal expansion. This hypothesis is based on the ability of IGF peptides to work not only as hormones that are transported through the blood but also as paracrine or autocrine growth factors. GH has a variety of metabolic actions, some of which appear to be independent of IGF production, such as enhancement of lipolysis, [214] stimulation of amino acid transport in diaphragm [215] and heart, [216] and enhancement of hepatic protein synthesis. Thus, there are multiple sites of GH action, and it is often not entirely clear which of these actions are mediated through the IGF system and which might represent IGF-independent effects of GH. [217] These sites of action include the following: 1. Epiphysis: stimulation of epiphyseal growth. 2. Bone: stimulation of osteoclast differentiation and activity, stimulation of osteoblast activity, and increase of bone mass by endochondral bone formation. 3. Adipose tissue: acute insulin-like effects, followed by increased lipolysis, inhibition of lipoprotein lipase, stimulation of hormone-sensitive lipase, decreased glucose transport, and decreased lipogenesis. 4. Muscle: increased amino acid transport, increased nitrogen retention, increased lean tissue, and increased energy expenditure. The concept of IGF-independent actions of GH is supported by in vivo studies, in which IGF-I cannot duplicate all of the effects of GH, such as nitrogen retention and insulin resistance. [218] The administration of GH for 1 to 3 weeks to calorically restricted normal or obese men results in significant nitrogen retention, although this effect does not persist with prolonged therapy. [219] The effects of GH in normal human aging [220] and in catabolic states [221] are subjects of active investigation.



Insulin-Like Growth Factors Historical Background

The IGFs (or somatomedins) are a family of peptides that are, in part, GH-dependent and that mediate many of the anabolic and mitogenic actions of GH. Originally identified in 1957 by their ability to stimulate [ 35 S] sulfate incorporation into rat cartilage and termed sulfation factor,[209] concurrent investigations indicated that only a component of the insulin-like activity of normal serum could be blocked by the addition of anti-insulin antibodies. The remaining activity, termed nonsuppressible insulin-like activity (NSILA), was subsequently demonstrated to contain two soluble, low-molecular-weight (7-kd) forms, named NSILA-I and NSILA-II. [222] [223] A third line of investigation arose from studies by Dulak and Temin [224] on the mitogenic nature of bovine serum; the mitogenic factor was termed multiplication-stimulating activity (MSA) and shares metabolic and mitogenic activities with both sulfation factor and NSILA. In 1972, the restrictive labels of sulfation factor and NSILA were replaced by the term somatomedin.[225] The following criteria for a somatomedin were established: 1. 2. 3. 4.

The concentration in serum must be GH-dependent. The factor must possess insulin-like activity in extraskeletal tissues. The factor must promote the incorporation of sulfate into cartilage. The factor must stimulate DNA synthesis and cell multiplication.

Purification yielded two somatomedin peptides: a basic peptide (somatomedin-C) and a neutral peptide (somatomedin-A). [226] [227] In 1978, Rinderknecht and Humbel[228] [229] isolated two active somatomedins from human plasma, and after demonstrating a striking structural resemblance to proinsulin, renamed them insulin-like growth factors (IGFs). IGF Structure and Molecular Biology

IGF-I, a basic peptide of 70 amino acids, correlates with somatomedin-C, and IGF-II is a slightly acidic peptide of 67 amino acids. The two peptides share 45 of 73 possible amino acid positions and have approximately 50% amino acid homology to insulin. [210] [228] [229] Like insulin, both IGFs have A and B chains connected by disulfide bonds. The connecting C-peptide region is 12 amino acids long for IGF-I and 8 amino acids

1017

for IGF-II, bearing no homology with the C-peptide region of proinsulin. IGF-I and IGF-II also differ from proinsulin in possessing carboxy-terminal extensions, or D-peptides, of 8 and 6 amino acids, respectively. This structural similarity explains the ability of both IGFs to bind to the insulin receptor and of insulin to bind to the type I IGF receptor (see later). On the other hand, structural differences probably also explain the failure of insulin to bind with high affinity to the IGFBPs (see later). IGF Variants

There are several variants of the two IGF peptides. Rinderknecht and Humbel [229] reported that up to one fourth of the IGF-II isolated from human plasma lacked the N-terminal alanine. Jansen and colleagues [230] demonstrated that an IGF-II cDNA isolated from a human liver library predicted an IGF-II variant in which Ser 29 was replaced by Arg-Leu-Pro-Gly, and Zumstein and associates [231] identified this variant peptide subsequently in human plasma. Zumstein and associates isolated a 10-kd IGF-II variant from human plasma that contains a 21-residue carboxy extension, representing a portion of the E domain of pro-IGF-II (see later). In one peptide fragment isolated, Ser 33 was replaced by Cys-Gly-Asp. A 25-kd IGF-II variant was isolated by Gowan and colleagues, presumably representing a carboxyl-terminal extension.[232] The significance of "big" IGF-II forms is still uncertain. In general, these variants appear capable of binding to IGF and insulin receptors and to IGFBPs and can participate in formation of the 150-kd IGF/IGFBP-3/acid-labile subunit (ALS) ternary complex. Big IGF-II can be produced by mesenchymal tumors and can cause nonislet-cell tumor hypoglycemia (NICTH). Daughaday and co-workers[233] described a patient with a leiomyosarcoma and recurrent hypoglycemia, in whom 70% of serum IGF-II was in higher-molecular-weight forms. Removal of the tumor eliminated big IGF-II from the serum and corrected the hypoglycemia. The presence of big IGF-II in NICTH has been confirmed in multiple laboratories, but it is unclear why hypoglycemia occurs in the face of normal total serum IGF-II levels. Zapf[234] has proposed that NICTH occurs when secretion of big IGF-II results in suppression of GH, insulin, and 7-kd IGF-II, leading to decreased production of IGF-I, IGFBP-3, and the ALS and increased production of IGFBP-2. This leads to a shift in the distribution of IGF-II from the 150-kd ternary complex to the 40- to 50-kd molecular-weight complex, composed of IGFBP-3, IGFBP-2, and a number of other low-molecular-weight IGFBPs. It is presumed that this results in increased bioavailability of IGF-II to target tissues, enhanced glucose consumption, and decreased hepatic glucose production. Big forms of IGF-I have not been as thoroughly documented as with IGF-II. Powell and associates, [235] however, have reported that IGF-I forms with an apparent molecular weight ratio as high as 19 kd may be found in uremic serum. Large molecular forms of IGF-I have also been identified in conditioned media of human fibroblast cell lines. Two IGF-I precursor molecules have been identified. [210] The first 134 amino acids of each are identical, comprising the signal peptide (48 amino acids), the mature IGF-I molecule (70 amino acids), and the first 16 amino acids of the E domain of the precursor. IGF-IA has additional 19 amino acids, and IGF-IB has additional 61 amino acids (total 195 residues). Alternative splicing of the IGF-I gene presumably generates the two mRNAs. The primary IGF-II translation product in human, rat, and mouse contains 180 amino acids, including a 24-residue signal peptide, the 67amino acid mature IGF-II sequence, and a carboxyl-terminal E peptide of 89 amino acids. The IGF-I Gene

The IGF genes (Fig. 23-21) are expressed differently in the embryo, fetus, child, and adult. [210] [236] [237] [238] Single large genes encode both IGF-I and IGF-II. The human IGF-I gene is located on the long arm of chromosome 12, [239] [240] and it contains at least six exons. Exons 1 and 2 encode alternative signal peptides, probably each containing several transcription start sites. Exons 3 and 4 encode the remaining signal peptide, the remainder of the mature IGF-I molecule, and part of the trailer peptide (E peptide). Exons 5 and 6 encode, alternatively, used segments of the trailer peptide (resulting in the IGF-IA and IGF-IB forms) and 3' untranslated sequences with multiple different polyadenylation sites. The wide diversity of IGF-I mRNAs thus reflects the following: 1. Multiple leader exons and transcription start sites. 2. Alternative splicing of exons 5 or 6. 3. Multiple polyadenylation sites in exon 6. The IGF-II Gene

The human IGF-II gene (Fig. 23-22) is located on the short arm of chromosome 11 [239] [240] [241] adjacent to the insulin gene and spans 35 kb of genomic DNA, containing 9 exons. Exons 1 to 6 encode 5' untranslated RNA; exon 7 encodes the signal peptide and most of the mature protein; and exon 8 encodes the

carboxy-terminal portion of the protein and part of the trailer peptide, whose coding is completed in exon 9. Thus, multiple mRNA species exist for both IGF-I and IGF-II, allowing for tissue-specific expression of specific transcripts and for developmental and hormonal regulation. The mechanisms involved in the regulation of IGF gene expression include the existence of multiple promoters, heterogeneous transcription initiation within each of the promoters, alternative splicing of various exons, differential RNA polyadenylation, and variable mRNA stability. Translation of IGF-I genes may also be under complex control. Regulation of IGF Gene Expression

GH appears to be the primary regulator of IGF-I gene transcription, which begins as early as 30 minutes after intraperitoneal injection of GH into hypophysectomized rats.[242] Transcriptional activation by GH affects both IGF-I promoters equivalently, resulting in a 20-fold rise in IGF-I mRNA. This coordinated, rapid induction of all IGF-I mRNA species coincides with induction of Spi 2.1 gene by GH, although the relationship between these two processes is still not clear. [242] Furthermore, there may be tissue-to-tissue variability in GH-induced expression of IGF-I mRNA. [243] Other factors that influence IGF-I gene expression include estrogen, which stimulates IGF-I mRNA expression in the uterus but inhibits GH-stimulated IGF-I transcription in the liver. [244] The pubertal rise in serum IGF-I levels reflects the effect of gonadal steroids on IGF-I transcription, some of which results from the pubertal rise in GH secretion and some of which is due to a direct effect of gonadal steroids on IGF synthesis or secretion, because a pubertal rise in serum IGF levels is also observed in patients with GHI. The factors involved in the regulation of IGF-II gene expression are less clear. [245] In humans and rats, IGF-II gene expression is high in fetal life and has been detected as early as the blastocyst stage in mice. [246] Serum levels of IGF-II are high in midgestation in pregnant rabbits. [247] Fetal tissues generally

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Figure 23-21 Structure of the IGF-I gene. A, The organization of the genes encoding human, rat and chicken insulin-like growth factor I (IGF-I) is depicted. Exons are represented by boxes (coding regions are in black, noncoding regions are in white), polyadenylation sites by arrows, and promoter regions by a bracket and the letter P. The full extent of the second and last human exons and the second rat exon has not been determined, as indicated by the dotted lines. B, Structure and expression of the human IGF-I gene. The structure of the different human IGF-I messenger RNAs (mRNAs) is displayed below the map of the gene. Sites of pre-mRNA processing are indicated by the thin lines. Sites of differential polyadenylation are marked at the 3' end of the gene by vertical arrows and in the mRNAs by horizontal boxes of varying length. (A and B, From Rotwein P. Structure, evolution, expression, and regulation of insulin-like growth factors I and II. Growth Factors 1991; 5:318.)

have high IGF-II mRNA levels that decline postnatally, although brain IGF-II mRNA remains high in the adult rat. [248] IGF-II mRNA is expressed constituitively in a number of mesenchymal and embryonic tumors, including Wilms' tumor, [249] [250] rhabdomyosarcoma, neuroblastoma, pheochromocytoma, hepatoblastoma, leiomyoma and leiomyosarcoma, liposarcoma, and colon carcinoma. [251] [252] [253] [254] [255] Production of big IGF-II by these tumors may cause NICTH (see earlier). [233] A tumor suppressor gene associated with Wilms' tumor (WT1) has been mapped to 11p13, close to the IGF-II locus (11p15.5), consistent with the possibility of a direct effect of WT1 on IGF-II gene transcription and suggesting an autocrine role for IGF-II in some tumors. [256] [257] This may be relevant in the embryonal tumors of Beckwith-Wiedemann syndrome (BWS), in which there may be loss of heterozygosity in the 11p15 maternally derived chromosome and paternal isodisomy, consistent with parental imprinting and a twofold increase in gene dosage of the active IGF-II allele. [258] IGF Imprinting

Gene regulation for the IGF system may also be subject to genomic imprinting, a process that influences the expression of specific genes. Namely, certain autosomal genes are expressed only from one of the two theoretically available alleles, in a manner that is specific for the parent of origin. The result is a heritable difference in gene expression, depending on whether a specific allele is inherited from the mother or the father. Allele-specific imprinting is exemplified by abnormalities of chromosome 15q11-13, where deletions of the paternal chromosome result in Prader-Willi syndrome, and deletions of the maternal locus are associated with Angelman syndrome, two phenotypically distinct conditions. The molecular mechanisms responsible for genomic imprinting involve variable DNA methylation. The first evidence for imprinting in the IGF axis emerged from studies of targeted gene disruption of Igf2 in the mouse[259] that caused fetal growth retardation only when the disrupted allele was inherited from the father (i.e., maternally imprinted). [260] The human IGF-II gene is similarly imprinted. [258] [261] [262] In tissues where only maternal chromosomes are present, such as ovarian teratomas, no IGF-II expression is observed, whereas gene expression is observed in tissues where only paternal chromosomes are present (complete hydatidiform mole). [263] Loss of imprinting (or relaxation of imprinting) of the IGF-II gene has been observed in rhabdomyosarcomas, lung cancers, Wilms' tumors, and choriocarcinoma. [250] [251] [254] In such situations, IGF-II may act as an autocrine or paracrine growth factor for neoplastic tissue. Furthermore, in Wilms' tumor, loss of imprinting of the IGF-II gene is associated with reduced expression of the putative tumor suppressor gene H19.[257] [264] The H19 gene appears to be imprinted in a reciprocal manner to igf2/IGF2, and the two genes may be coordinately regulated, because the genes are located near each other on the same chromosome. The genes for the type II IGF receptor, which is the same as the cation-independent mannose-6-phosphate (M6P) receptor, are also imprinted, although in a different manner.[265] Thus,

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Figure 23-22 Structure of the IGF-II gene. A, The organization of human, rat, and mouse IGF-II genes is shown. Exons are represented by boxes (coding regions are in black, noncoding in white), polyadenylation sites by thick vertical arrows, and promoter regions by a bracket and the letter P. The multiple transcription initiation sites for mouse and rat exon 1 are marked by the thin vertical arrows. The locations of mouse pseudoexons 1 and 2 are indicated. B, Structure and expression of the human IGF-II gene. The structure of different human insulin-like growth factor II (IGF-II) messenger RNAs (mRNAs) is displayed below the map of the gene. The patterns of mRNA processing are indicated by the thin lines. Sites of differential polyadenylation are marked at the 3' end of the gene by vertical arrows and in the mRNAs by horizontal boxes of varying length. (A and B, From Rotwein P. Structure, evolution, expression, and regulation of insulin-like growth factors I and II. Growth Factors 1991; 5:318.)

the mouse Igf2 receptor gene and the human IGF type 2 receptor gene are both expressed by the maternal allele (i.e., paternally imprinted). If IGF-II functions as a fetal growth factor, there is potential for both maternal and paternal regulation of fetal size. Targeted Disruption of IGF Genes

The role of the IGF axis in fetal growth has been firmly established by a series of studies involving IGF and IGF receptor null mutations. [266] Unlike GH and GHR knockouts,[267] [268] which are near normal size at birth, mice with knockouts of the gene for either IGF-I or IGF-II have birth weights approximately 60% of normal. [259] [260] [277] Mouse mutants lacking both IGF-I and the GHR are only 17% of normal. [269] These observations indicate that both IGF-I and IGF-II are important embryonic

and fetal growth factors but that GH itself does have some independent role as well. Although fetal size was proportionately reduced in both situations and although morphogenesis was grossly normal, a higher neonatal death rate was observed following disruption of the gene for IGF-I. Growth delay began on day e11 for IGF-II knockouts and on day e13.5 for IGF-I knockouts. Those mice with IGF-I gene disruptions who survived the immediate neonatal period continued to have growth failure postnatally, with weights 30% of normal by 2 months of age. Indeed, postnatal growth was poorer than that observed in mice with GHR, GHRH receptor mutations, or Pit-1 mutations, indicating that both GH-dependent and GH-independent factors are necessary for normal growth. A similar prenatal and postnatal growth phenotype has been observed in the one reported case of an IGF-I gene deletion. [270] When the genes for both IGF-I and IGF-II were disrupted, weight at birth was only 30% of normal, and all animals died shortly after birth, apparently from respiratory insufficiency secondary to muscular hypoplasia. Specific ablation of hepatic IGF-I production through the Cre/loxP recombination system confirmed that the liver is the principal source of circulating IGF-I but demonstrated that an 80% lowering of serum IGF-I levels had no apparent effect on postnatal growth. [271] [272] [273] [274] Presumably, either local (paracrine) chondrocyte production of IGF-I or other tissues (possibly adipose) maintain adequate endocrine sources of IGF-I to account for growth preservation. Supportive data for the predominant role in growth of locally produced IGF-I are the modest decrement of postnatal growth seen in ALS null mice. models are

[275]

These murine

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complex as IGFBP-3 levels are reduced despite increased GH and, in contrast with the human, rise after treatment with exogenous IGF-I. Further, the free IGF-I levels are normal in these animals but do not prevent an increment of GH production. [273] [276] In another study, knockout of the gene for the type 1 IGF receptor resulted in birth weights 45% of normal and 100% neonatal lethality. [277] Concurrent knockout of genes for IGF-I and the type 1 IGF receptor resulted in no further reduction in birth size (45% of normal), consistent with the concept that all IGF-I actions in fetal life are mediated through this receptor. On the other hand, simultaneous knockout of the genes for IGF-II and the type 1 IGF receptor resulted in further reduction of birth size to 30% of normal (as with simultaneous knockouts of IGF-I and IGF-II), suggesting that some of the fetal anabolic actions of IGF-II are mediated by a secondary mechanism (perhaps placental growth). This pathway does not appear to involve the type 2 IGF receptor, because knockout of this paternally imprinted gene results in an increased birth weight but death in late gestation or at birth. [278] Because this receptor normally degrades IGF-II, increased growth reflects excess IGF-II acting through the IGF-I receptor; there is, however, variable accumulation of IGF-II in such mouse tissues. [279] Knockout of the type 2 IGF receptor plus IGF-II causes a birth weight 60% of normal (as is the case with knockout of IGF-II alone) but allows fetal survival. [280] Several conclusions can be drawn from these studies: 1. 2. 3. 4. 5. 6. 7.

IGF-I is important for both fetal and postnatal growth. IGF-I is more important than GH for postnatal growth. IGF-II is a major fetal growth factor. The type 1 IGF receptor mediates anabolic actions of both IGF-I and IGF-II. The type 2 IGF receptor is bifunctional, serving to both target lysosomal enzymes and to enhance IGF-II turnover. IGF-I production is involved in normal fertility. Placental growth is impaired only with IGF-II knockouts.

Whether these studies in mice are fully applicable to humans is yet unknown. IGF Peptide Assays

Bioassay methods for IGF activity included stimulation of [ 35 S] sulfate incorporation, using various modifications of the original method described by Salmon and Daughaday.[209] [281] [282] A wide variety of other bioassays used stimulation of the synthesis of DNA, [283] RNA,[284] or protein[285] or of glucose uptake.[285] Such assays are cumbersome, subject to interference by IGFBPs, and incapable of distinguishing between IGF-I and IGF-II. When somatomedin-C (and later, IGF-I and IGF-II) were purified, it became possible to develop radioreceptor assays (RRAs) [286] [287] and competitive proteinbinding assays [288] [289] ; development of specific antibodies permitted the development of accurate and specific measurement of IGF-I and IGF-II. [290] [291] [292] [293] [294] The issue of IGFBPs must be addressed in any IGF assay. [295] For example, the discrepant results found in uremic sera assayed for IGF by bioassay, RRA, and immunoassay are due to the interference of IGFBPs [296] ; such interference is a particular problem in conditions with a relatively high IGFBP/IGF peptide ratio and at the extremes of the assay (i.e., GHD or acromegaly). The most effective way to deal with IGFBPs is to separate them from IGF peptides by chromatography under acidic conditions [297] ; however, this is a labor-intensive procedure and has been occasionally replaced by an acid ethanol extraction procedure. [298] Although this latter method may be reasonably effective for most serum samples, it is problematic in conditions of high IGFBP/IGF peptide ratios, such as conditioned media from cell lines and sera from newborns and from subjects with GHD or uremia. Alternative methods include the use of antibodies generated against synthetic peptides, such as the C-peptide region of IGF-I or IGF-II, which does not bind to IGFBPs. In general, such antibodies have high specificity but relatively low affinity. An alternative approach, developed by Blum and colleagues, [299] involves use of an antibody with high specificity for IGF-II, which permits the addition of excess unlabeled IGF-I, to saturate endogenous IGFBPs. Bang and co-workers [300] have bypassed the interference of IGFBPs by employing a truncated IGF-I radioligand, which has decreased affinity for IGFBPs. Currently, the most practical and effective way to perform accurate IGF assays with minimal interference by IGFBPs is to use the sandwich assay method.[301] These assays, which can be performed in either enzyme-linked immunosorbent assay (ELISA) or immunoradiometric assay, do not employ a radiolabeled IGF molecule, which can bind to IGFBPs, as in conventional radioimmunoassays, and lead to erroneous readings if IGFBP levels are elevated. [302] The absolute IGF values obtained in many of the assays may be falsely high because of low purity and inconsistent amino acid analyses of local standards, but this can be avoided by the use of the World Health Organization International Reference Reagent 87/518 calibration standard. [303] Serum Levels of IGF Peptides

In human fetal serum, IGF-I levels are relatively low and are positively correlated with gestational age. [304] [305] Some,[304] [305] [306] but not all,[307] groups have reported a correlation between fetal cord serum IGF-I levels with birth weight. IGF-I levels in human newborn serum are generally 30% to 50% of adult levels. Serum levels rise during childhood and attain adult levels at the onset of sexual maturation (Fig. 23-23A and B) . [308] During puberty, IGF-I levels rise to two to three times the adult range.[309] Thus, levels during adolescence correlate better with Tanner stage (or bone age) than with chronologic age. Girls with gonadal dysgenesis show no adolescent increase in serum IGF-I, clearly establishing the association of the pubertal rise in IGF-I with the production of gonadal steroids. [310] [311] [312] The pubertal rise in gonadal steroids may stimulate IGF-I production indirectly, by first leading to a rise in GH secretion, but patients with GHI due to GHR mutations show a pubertal rise in serum IGF-I despite a decline in GH levels, thereby suggesting a direct effect of gonadal steroids on IGF-I. [120] After adolescence, or at least after 20 to 30 years of age, serum IGF-I levels demonstrate a gradual and progressive age-associated decline, [220] [313] a decline that is possibly responsible for the negative nitrogen balance, decrease in muscle mass, and osteoporosis of aging. [220] This provocative hypothesis is unproven but has generated interest in the potential use of GH and IGF-I therapy in normal aging. Human newborn levels of IGF-II are generally 50% of adult levels. By 1 year of age, however, adult levels are attained, with little, if any subsequent decline, even up to the seventh or eighth decade. This pattern of IGF-II levels in humans is different than that in the rat or mouse, in which serum IGF-II levels are also high in the fetus but rapidly decline postnatally to undetectable levels in the adult. [314] [315] Measurement of IGF Levels in Growth Disorders

The GH dependency of the IGFs was established in the initial report from Salmon and Daughaday [209] and further clarified with the development of sensitive and

specific immunoassays that distinguish between IGF-I and IGF-II. [293] IGF-I levels are more GH-dependent than are IGF-II levels and are more

1021

Figure 23-23 Normal serum levels (micrograms per liter) of insulin-like growth factor I (IGF-I) for males (A) and females (B). Lines represent the mean ± 3 SD. (Data courtesy of Diagnostic Systems Laboratories, Inc., Webster, Texas.)

likely to reflect subtle differences in GH secretory patterns. However, serum IGF-I levels, as stated earlier, are influenced by age, degree of sexual maturation, and nutritional status. As a result, construction of age-defined normative values may be misleading. IGF-I levels in normal children younger than 5 years of age are low, and there is overlap between the normal range and values in GH-deficient children. Assessment of serum IGF-II levels is less age-dependent, especially after 1 year of age, but IGF-II is less GH-dependent than is IGF-I. Moore and associates [316] performed GH stimulation tests in 78 children with heights below the 5th percentile and serum IGF-I levels lower than 0.5 U/mL. Although 19 of these children were subsequently discovered to have GH deficiency on the basis of standard provocative tests, there was an overlap of serum IGF-I levels between GH-deficient children and children with other forms of short stature and normal provocative GH levels. It was only in children with bone ages greater than 12 years that serum IGF-I levels permitted discrimination between GHD and normal short children. Similarly, Reiter and Lovinger [317] found that 4 of 16 children with low provocative GH levels had normal serum IGF-I levels, whereas 7 of 25 children with normal provocative GH levels had low serum IGF-I levels. Rosenfeld and colleagues [294] evaluated the efficacy of IGF-I and IGF-II measurements in 68 GH-deficient patients, 197 children with normal stature, and 44 normal children with short stature ( Fig. 23-24 and Fig. 23-25 ). Eighteen percent of the GH-deficient children had serum IGF-I levels within the normal range for age, and 32% of normal short children had low IGF-I levels. Low IGF-II levels were found in 52% of GH-deficient children and in 35% of normal short children. However, the use of combined IGF-I/IGF-II assays provided better discrimination. Only 4% of GH-deficient children had normal plasma levels of both IGF-I and IGF-II. Furthermore, only

1022

0.5% of normal children and 11% of normal short children had low serum levels of both IGF-I and IGF-II. The observation that many "normal short" children have low serum levels of IGF-I, IGF-II, or both calls into question the criteria by which the diagnosis of GHD is made. Given that provocative GH testing is both arbitrary and nonphysiologic and the inherent variability in GH assays, it is not surprising that the correlation between IGF-I levels and provocative GH levels is imperfect. These points are further supported by recent observations with immunoassays for IGFBP-3 (see later). IGF Receptors

A study was published in which the binding of IGFs to the insulin receptor provided an explanation for their insulin-like activity. [318] Shortly thereafter, Megyesi and co-workers[319] identified distinct receptors for insulin and IGF in rat hepatic membranes. At least two classes of IGF receptors exist; insulin, at high levels, competes for occupancy of one form of IGF receptor but has essentially no affinity for the second form of receptor. Structural characterization of these receptors documented the differences in the two forms of receptor (Fig. 23-26) .[320] [321] [322] [323] [324] [325] The type 1 IGF receptor is closely related to the insulin receptor; both are heterotetramers comprised of two membrane-spanning subunits and two intracellular subunits. The subunits contain the binding sites for IGF-I and are linked by disulfide bonds. The subunits contain a transmembrane domain and an adenosine triphosphate (ATP)-binding site and a tyrosine kinase domain that constitute the presumed signal transduction mechanism for the receptor. One mole of the full heterotetrameric receptor appears to bind one mole of ligand. Although the type 1 IGF receptor has been commonly termed the IGF-I receptor, the receptor binds both IGF-I and IGF-II with high affinity, and both IGF peptides appear capable of activating tyrosine kinase by binding to this receptor. In studies involving transfection and overexpression of the type 1 IGF receptor cDNA, the Kd for IGF-I is typically in the range of 0.2 to 1 nM; affinity for IGF-II is usually slightly less but varies from study to study. The affinity of the type 1 receptor for insulin is generally 100-fold less, thereby explaining the relatively weak mitogenic effect of insulin. Ullrich and associates [326] deduced the structure of the human type 1 IGF receptor from cDNA; the mature peptide constitutes 1337 amino acids with a predicted molecular mass of 151,869 (Fig. 23-27) . The translated - heterodimer is subsequently cleaved at an Arg-Lys-Arg-Arg sequence at positions 707 to 710, and the released and subunits are linked by disulfide bonds to form the mature () 2 -receptor in which two alpha chains are joined by secondary disulfide bonds. The subunits are extracellular and contain a cysteine-rich domain, which is critical for IGF binding. As is the case with the insulin receptor, the subunit has a short extracellular domain, a hydrophobic transmembrane domain, and the intracellular tyrosine kinase domain and ATP-binding site. Like the insulin receptor, the type 1 IGF receptor undergoes ligand-induced autophosphorylation, principally on tyrosines 1131, 1135, and 1136. [327] [328] [329] [330] Both receptors are believed to have evolved from a common ancestor protein but are encoded by genes on separate chromosomes (chromosome 15 for the type 1 IGF receptor [331] ) and chromosome 19 for the insulin receptor). The type 1 IGF receptor gene spans greater than 100 kb of genomic DNA, with 21 exons; the genomic organization resembles that of the insulin receptor gene. [326] [332] Exons 1 to 3 code for the 5' untranslated region, the signal peptide, the N-terminal region, and the cysteine-rich domain of the subunit involved in ligand binding. The remainder of the subunit is

Figure 23-24 Serum insulin-like growth factor I (IGF-I) levels in normal subjects ( a and d), normal short stature subjects (b and e), and subjects with growth disorders (c and f). (From Rosenfeld RG, Wilson DM, Lee PDK, Hintz RL. Insulin-like growth factors I and II in the evaluation of growth retardation. J Pediatr 1986; 109:428433.)

encoded by exons 4 to 10. The peptide cleavage site involved in generation of the and subunits is encoded by exon 11, and the tyrosine kinase domain of the subunit is encoded by exons 16 to 20. It is in the latter region that the type 1 IGF receptor and insulin receptor share the greatest sequence homology, ranging from 80% to 95%; interspecies homology in this region of the receptors is also high. Exon 21 encodes 3' untranslated sequences. Type 1 IGF receptor mRNA has been identified in virtually every tissue except liver. [333] [334] By Northern blot hybridization, human mRNA reveals two bands of 11 and 7 kb; rat tissues contain only the 11-kb band. [335] Type 1 IGF receptor mRNA is most abundant in embryonic tissues and appears to decrease

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Figure 23-25 Serum insulin-like growth factor II (IGF-II) levels in normal subjects ( A), normal short stature subjects (B), and subjects with growth disorders (C). (From Rosenfeld RG, Wilson DM, Lee PDK, Hintz RL. Insulin-like growth factors I and II in the evaluation of growth retardation. J Pediatr 1986; 109:428433.)

with age. The type 1 IGF receptor is present at the embryonic eight-cell stage (the type 2 IGF receptor is first demonstrable at the 2-cell stage) and becomes widely expressed postimplantation, consistent with the observation that this receptor is essential for normal fetal growth. As with other growth factor receptor tyrosine kinases, binding of ligand (IGF-I or IGF-II) induces receptor autophosphorylation of critical tyrosine residues in the type 1 receptor. [327] [328] [329] [330] Mutations of the ATP-binding site or of critical tyrosine residues in the subunit result in loss of IGF-stimulated thymidine incorporation and glucose uptake. Autophosphorylation appears to occur by transphosphorylation of sites on the opposite subunit. phosphorylating other tyrosine-containing

[336] [337]

The activated type 1 IGF receptor is capable of

Figure 23-26 Structure of the insulin-like growth factor (IGF) receptors. The insulin and IGF-I receptors are both heterotetrameric complexes composed of extracellular subunits that bind the ligands and subunits that anchor the receptor in the membrane and that contain tyrosine kinase activity in their cytoplasmic domains. The tyrosine kinase domain of the insulin receptorrelated receptor (IRR) is homologous to the tyrosine kinase domains of the insulin and IGF-I receptors. The C-terminal domain is deleted in the IRR. Hybrids consist of a hemireceptor from both insulin and IGF-I receptors. The IGF-II/mannose-6-phosphate (M6P) receptor is not structurally related to the IGF-I and insulin receptors or the IRR, having a short cytoplasmic tail and no tyrosine kinase activity. (From LeRoith D, Werner H, Geitner-Johnson D, Roberts CT Jr. Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocr Rev 1995; 16:143163. © The Endocrine Society.)

substrates, such as insulin receptor substrate 1 (IRS-1), a 185-kd protein that is the predominant substrate of the insulin receptor kinase and IRS-2 (Fig. 23-28) (Figure Not Available) . [338] IRS-1 contains specific phosphotyrosine motifs that can associate with proteins containing SH2 (src homology 2) domains, such as PI3-kinase (phosphatidylinositol-3 kinase), [339] Grb2 (growth factor receptor-bound protein 2), [340] Syp (a phosphotyrosine phosphatase), [339] and Nck (an oncogenic protein). [341] The substrates, which are phosphorylated by the IGF receptor, include the members of the IRS family, particularly IRS-1 and IRS-2, as both of the knockout mice models for these genes result in poor growth (as well as insulin resistance). [342] Other IRS molecules may have a negative feedback role in regulating IGF action.[343] 1024

Figure 23-27 Structure of the human insulin-like growth factor I (IGF-I) receptor precursor. Molecular cloning of human IGF-I receptor complementary DNAs (cDNAs) isolated from a placental library revealed the presence of an open reading frame of 4101 nucleotides. The 1367amino acid polypeptide contains, at its N-terminus, a 30-amino acid hydrophobic signal peptide, which is responsible for the transfer of the nascent protein chain in to the endoplasmic reticulum. After digestion by endopeptidases at a proteolytic cleavage site (Arg-Lys-Arg-Arg) located at residues 707 to 710, and subunits are released and linked by disulfide bonds to give the configuration of the mature heterotetrameric receptor. Shown in this diagrammatic representation are, in addition, the cysteine-rich domain of the subunit and the transmembrane and tyrosine kinase domains of the subunit. (From LeRoith D, Werner H, Geitner-Johnson D, Roberts CT Jr. Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocr Rev 1995; 16:143163. © The Endocrine Society.)

Activation of the type 1 IGF receptor also leads to tyrosine phosphorylation of Shc (src homology domaincontaining protein), [344] which then associates with Grb2 and activates Ras, leading to a cascade of protein kinases, including Raf, MAP kinase kinases, MAP kinases, and S6 kinase. [345] [346] [347] Thus, phosphorylation of IRS-1 by either the type 1 IGF or insulin receptor activates multiple signaling cascades that ultimately influence nuclear transcription and gene expression. It is, presumably, at this level that the IGF peptides exert their mitogenic and anabolic actions. Given that insulin and IGF peptides activate similar, if not identical, signaling pathways through their own specific receptors, it is unclear how the cell distinguishes between these overlapping ligands. Whether this merely reflects the relative levels of receptors or whether divergent downstream pathways exist for insulin and IGF action remain questions for future investigation. [348] Although targeted disruption of the gene for the type 1 IGF receptor causes fetal growth retardation, a clear role for this receptor in the cell cycle has not been established. Fibroblast cell lines derived from mouse embryos homozygous for the knockout gene still undergo cell cycledependent division, although at a slower rate.[349] [350] [351] On the other hand, the transformed phenotype of some cells may be critically dependent on expression of the type 1 IGF receptor. The SV40TAg is capable of inducing a transformed phenotype in a cell only in the presence of intact type 1 IGF receptors. [351] NIH 3T3 cells and Rat-1 fibroblasts that are made to overexpress the type 1 IGF receptor develop IGF-Idependent neoplastic transformation, with colony formation in soft agar and tumor formation in nude rats. [352] Prager and colleagues[353] have shown that truncation of the type 1 IGF receptor at the amino terminus increases transforming potential, suggesting that the subunit normally restricts this function and that the binding of IGF to the receptor releases constraints on mitogenic stimulation. Variants of both the and subunits are present in placenta, [354] muscle[354] [355] and brain. [356] These variants may explain seemingly anomalous competitive binding studies. [357] [358] [359] The molecular mechanisms for the formation of such receptor variants have not been identified; nor is it clear if they differentially bind IGF-I, IGF-II or insulin. The formation of IGF-insulin receptor hybrids that contain an -IGF hemireceptor disulfide-linked to an -insulin hemireceptor (see Fig. 23-26) [360] [361] [362] appears to be ligand-dependent, [363] and studies with monoclonal antibodies specific for the insulin or type 1 IGF receptor suggest that such receptors develop in cells with abundant native receptors, such as muscle and placenta. [364] [365] Such hybrids have near-normal affinity for IGF-I but decreased affinity for insulin. The physiologic significance of such hybrid receptors is unknown. The type 2 IGF receptor bears no structural homology with either the insulin or type 1 IGF receptors. It has an apparent molecular mass of 220,000 under nonreducing conditions and 250,000 after reduction, indicating that it is a monomeric protein. The cloned human type 2 receptor cDNA predicts a molecular mass of 270,294 and a lengthy extracellular domain containing 15 repeat sequences of 147 residues each, a 23-residue transmembrane domain, and a small cytoplasmic domain consisting of only 164 residues. [366] The receptor does not contain an intrinsic tyrosine kinase domain or any other recognized signal transduction mechanism. The type 2 IGF receptor is identical to the cation-independent M6P receptor, a protein involved in the intracellular lysosomal targeting of acid hydrolases and other mannosylated proteins. [367] [368] Most of these receptors are located on intracellular membranes, in equilibrium with receptors on the plasma membrane. [369] Why this receptor binds both IGF-II and M6P-containing lysosomal enzymes is unknown. Unlike the type 1 IGF receptor, which binds both IGF peptides with high affinity and insulin with 100-fold lower affinity, the type 2 receptor binds only IGF-II with high affinity, the K d ranging from 0.017 to 0.7 nM; IGF-I binds with lower affinity, and insulin does not bind at all. [369] One mole of IGF-II binds per mole of receptor. IGF-II and M6P bind to different portions of the receptor, but the two ligands do show some reciprocal inhibitory effects on receptor binding, suggesting that IGF-II may affect the sorting of lysosomal enzymes. Alternatively, this receptor may be important to the degradation of IGF-II. Knockout of the gene for the type 2 IGF receptor in mice causes macrosomia and fetal death. The mitogenic and metabolic actions of both IGF-I and IGF-II appear to be mediated through the type 1 IGF receptor because monoclonal antibodies directed against the IGF-I binding site on the type 1 IGF receptor inhibit the ability of both IGF-I and IGF-II to stimulate thymidine incorporation and cell replication. [370] [371] Similarly, polyclonal antibodies that block IGF-II binding to the type 2 IGF/M6P receptor do not block IGF-II actions. [372] [373] [374] In addition, IGF-II analogues with decreased affinity for the type 1 receptor but preserved affinity for the type II receptor are less potent than IGF-II in stimulating DNA synthesis, [228] and the M6P receptor in hepatic tissues from chicken [375] or frog[376] does not bind IGF-II. Presumably, the mitogenic actions of IGF-II in these species are mediated solely through the type 1 IGF receptor. Nevertheless, some IGF-II actions may be mediated via the type 2 IGF receptor. Rogers and Hammerman [377] suggested that the type 2 receptor is involved in

production of inositol triphosphate and diacylglycerol in proximal tubules and canine kidney membranes. Tally and co-workers [357] reported that IGF-II stimulates the growth of a K562 human erythroleukemia cell line, an action not duplicated by either IGF-I or insulin. Minniti and colleagues [358] reported that IGF-II appears capable of acting as an autocrine growth factor and cell motility factor for human rhabdomyosarcoma cells, actions apparently mediated through the type 2 receptor, and IGF-II may activate a calcium-permeable cation channel via the type 2 IGF receptor, perhaps through coupling to a pertussis toxinsensitive, guanine nucleotidebinding protein (G i protein). [359] [363] [378] [379] [380] [381] In cells transfected with the human type 2 IGF receptor cDNA, IGF-II decreased cAMP accumulation promoted by cholera

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Figure 23-28 (Figure Not Available) Schematic representation of intracellular signaling pathways of the insulin-like growth factor I receptor (IGF-IR). On binding IGF-I, the IGF receptor undergoes autophosphorylation at multiple tyrosine residues. The intrinsic kinase activity of the receptor also phosphorylates insulin receptor substrate 1 (IRS-1) at multiple tyrosine residues. Various SH domaincontaining proteins, including PI 3-kinase, Syp, Fyn, and Nck, associate with specific phosphotyrosine-containing motifs within IRS-1. These docking proteins recruit diverse other intracellular substrates, which then activate a cascade of protein kinases, including Raf-1 and one or more related kinases. These protein kinases, in turn, activate various other elements, including nuclear transcription factors. Alterations in expression of various IGF-I-responsive genes results in longer-term effects of IGF-I, including growth and differentiation. This model of signal transduction cascades also shows a potential mechanism for the inhibition of apoptosis. (From Le Roith D, Bondy C, Yakar S, et al. The somatomedin hypothesis. Endocr Rev 2001; 22:5374. © The Endocrine Society.)

toxin or forskolin. Mutations or truncation of the small cytoplasmic domain of the receptor prevented these IGF-II actions. The type 2 IGF receptor also binds other molecules such as M6P-containing enzymes (e.g., cathepsin and urokinase), which may be important in the removal of these enzymes from the cellular environment, thus modulating tissue remodeling. [382] In addition, the type 2 IGF receptor binds retinoic acid and may mediate some of the growth inhibitory effects of retinoids. [383] As discussed earlier, knockout of the type 2 IGF receptor results in excessive growth. This receptor, therefore, may act as a growth inhibitory component of the IGF system responding to and mediating multiple antimitogenic systems. [384] IGF-Binding Proteins (Fig. 23-29) (Figure Not Available)

In contrast with insulin, the IGFs circulate in plasma complexed to a family of binding proteins that extend the serum half-life of the IGF peptides, transport the IGFs to target cells, and modulate the interaction of the IGFs with surface membrane receptors. [385] [386] [387] [388] The identification and characterization of IGFBPs in body fluids[389] and in conditioned media from cultured cells have been facilitated by the development of a number of biochemical and assay techniques, including gel chromatography, RRAs, affinity cross-linking, Western ligand blotting, [390] immunoblotting, and specific radioimmunoassays. However, study of the molecular biology of the IGFBPs has provided the most information concerning their structural inter-relationship. IGF-Binding Protein Structure(Fig. 23-30)

To date, the cDNAs for six distinct human and rat IGFBPs have been cloned and sequenced. [386] [387] [391] Their structural characteristics are summarized in Figure 23-30 . The amino acid sequences of the six cloned mammalian IGFBPs are highly conserved. Within a species, the IGFBPs share an overall amino acid sequence homology in the order of 50%, and between species there is more than 80% sequence homology for individual IGFBPs. Perhaps the most impressive similarity in structure is the conservation of the number and placement of the cysteine residues. The total number of cysteines varies from 16 to 20 (18 are conserved in human IGFBPs 1 to 5; IGFBP-6 conserves 16 of the 18, and IGFBP-4 has 2 additional cysteines in the middle region of the protein), and each of the IGFBPs has cysteine-rich regions at the amino-terminus and carboxyl-terminus. Conservation of the spatial order of the cysteines presumably indicates that the secondary structure of the IGFBPs, which is dependent on disulfide bonding, must also be conserved. Disulfide bonding is essential for formation of the IGF

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Figure 23-29 (Figure Not Available) Schematic representation of the insulin-like growth factor (IGF) system, including IGF ligands (IGF-I and IGF-II), binding proteins (both high- and low-affinity binders), IGF-binding protein (IGFBP) proteases, type I and type II IGF receptors, and potential IGFBP(s) and IGFBP-related protein (IGFBP-rP) receptors. M6P, mannose-6-phosphate. (From Hwa V, Oh Y, Rosenfeld RG. The insulin-like growth factorbinding protein [IGFBP] superfamily. Endocr Rev 1999; 20:761787. © The Endocrine Society.)

binding site of each IGFBP; reduction of the disulfide proteins results in loss of IGF binding. On the other hand, the middle region of the IGFBPs is not well conserved, containing N-glycosylation sites for IGFBP-3 and IGFBP-4 and two additional cysteine residues in IGFBP-4. Some of the more specialized

Figure 23-30 Amino acid sequences of human insulin-like growth factorbinding proteins (IGFBPs) 1 to 6, deduced from nucleotide sequences. Sequences in the amino-terminal and carboxyl-terminal residues are aligned to show maximal homologies. Dashes indicate gaps. Residues that are identical in five or six of the six IGFBPs are shaded. (From Rechler MM. Insulin-like growth factor binding proteins. Vitam Horm 1993; 47:114.)

properties of the IGFBPs, such as cell association, IGF enhancement, and IGF-independent actions (see later) may be dependent on specific sequences in these midregions. An RGD (arginine-glycine-aspartic acid) sequence near the carboxyl-terminus of IGFBP-1 and IGFBP-2 [392] is the minimum

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sequence required for the binding of many extracellular matrix proteins to membrane receptors of the integrin protein family, and IGFBPs may associate with the cell surface through such amino acid sequences. [393] However, IGFBP-3, which lacks an RGD sequence, also binds to cell membranes, [394] [395] possibly to specific receptors[396] (see later). Under most conditions, the IGFBPs appear to inhibit IGF action, presumably by competing with IGF receptors for binding IGF peptides. [397] For example, IGF analogues with decreased affinity for IGFBPs have increased biologic potency. [398] [399] [400] In studies involving transfection of the human IGFBP-3 gene into fibroblasts, increased expression of IGFBP-3 inhibited cell growth, even in the absence of added IGF, suggesting a direct inhibitory role of the binding protein. [401] Under some conditions, however, the IGFBPs appear to enhance IGF action, perhaps by facilitating the delivery of IGF to target receptors. [402] The discovery of several groups of cysteine-rich proteins that contain domains similar to the amino-terminus of the IGFBPs has led to the proposal of an IGFBP superfamily,[403] which includes the family of six high-affinity IGFBPs, as well as a number of IGFBP-related proteins (IGFBP-rPs). Three of the IGFBP-rPs (Mac25/IGFBP-rP1; connective tissue growth factor, CTGF/IGFBP-rP2; NovH/IGFBP-rP3) have been shown to bind IGFs, although with considerably lower affinity than the IGFBPs. Like the IGFBPs, the IGFBP-rPs are modular proteins and the highly preserved amino-terminal domain appears to represent the consequence of exon shuffling of an ancestral gene. The role, if any, of the IGFBP-rPs in normal IGF physiology is unclear, but it seems likely that they influence cell growth by IGF-independent and IGF-dependent mechanisms. Analysis of IGFBPs is further complicated by the presence of IGFBP proteases, which degrade IGFBP. [404] [405] Initially reported in the serum of pregnant women, [404] [405] proteases for IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5 are present in serum, seminal plasma, [406] cerebrospinal fluid, [407] and urine.[408] It is likely that multiple IGFBP proteases exist, including calcium-dependent serine proteases, kallikreins, cathepsins, [409] and matrix metalloproteases. [410] Proteolysis of IGFBPs complicates their assay and must be taken into consideration when measuring the various IGFBPs in biologic fluids. [411] The physiologic significance of limited proteolysis of IGFBPs remains to be determined, although protease activity usually decreases the affinity of the IGFBP for IGF peptides (Fig. 23-31) and may enhance the mitogenic and anabolic effects of IGF peptides in this way. In prostate epithelial cells [412] [413] [414] prostate-specific antigen acts as a potent IGFBP-3 protease ( Fig. 23-32 and Fig.

23-33 ), and in rat granulosa cells, [415] follicle-stimulating hormone (FSH) induces an IGFBP-5 protease. IGF-Binding Proteins as Carrier Proteins

Given the high affinity of the IGFBPs for IGF-I and IGF-II (K d 10-10 to 10-11 M), virtually all IGF-I and IGF-II in serum is complexed to IGFBPs. [416] In normal adult serum, 75% to 80% of the IGF peptides are carried in a ternary complex consisting of one molecule of IGF plus one molecule of IGFBP-3 plus one molecule of an 88-kd protein termed the acid-labile subunit (ALS).[417] [418] Binding of ALS to form the full ternary complex occurs after the binding of IGF by IGFBP-3 or IGFBP-5, although ALS may bind to IGFBP-3 even in the absence of IGF. [419] The 150-kd ternary complex is too large to leave the vascular compartment and extends the half-life of IGF peptides from approximately 10 minutes for IGF alone to 1 to 2 hours for IGF in the IGFIGFBP-3 binary complex to 12 to 15 hours for IGF in the ternary complex. [420] The fact that both IGFBP-3 and ALS are GH-dependent

Figure 23-31 Schematic representation of the effect of insulin-like growth factorbinding protein (IGFBP) proteases on IGF action. In this model, proteolysis of IGFBPs results in a reduction in their affinity for IGF ligands, resulting in enhanced binding of IGF peptides by IGF receptor. (From Cohen P, Rosenfeld RG. The IGF axis. In Rosenbloom AL [ed]. Human Growth Hormone: Basic and Scientific Aspects. Boca Raton, Fla, CRC Press, 1991, pp 4358.)

provides an additional mechanism for GH regulation of the IGF axis. Although IGF-I administration to hypophysectomized rats increases serum levels of IGFBP-3, [421] [422] [423] no sustained increase in serum levels of IGFBP-3 occurs in humans following administration of IGF-I. [120] [424] ALS levels may decline even after IGF-I administration, presumably reflecting IGF feedback inhibition of pituitary GH secretion. [424] [425] Thus, in serum of patients with GHD or GHI, little IGF is present in the 150-kd ternary complex, most being found in the lowermolecular-weight IGFIGFBP-3 complex or bound by other IGFBPs. Although GH administration to GH-deficient patients shifts IGF from the 40- to 50-kd low-molecular-weight peak to the 150-kd high-molecular-weight peak, [426] [427] a similar phenomenon is not observed following IGF-I treatment. [426] IGF peptides in the 40- to 50-kd molecular-weight peak may not be restricted to the vascular compartment. IGFBP-1, IGFBP-2, and IGFBP-4, at least, can probably cross endothelial barriers. [428] In the fetus and neonate, whose IGFBP-3 levels are relatively low, and in GHD and GHI, binding of IGFs by IGFBP-1, IGFBP-2, IGFBP-4, and IGFBP-5 may predominate over binding to IGFBP-3. [314] [426] [429] Similarly, in tumorinduced hypoglycemia associated with increased serum levels of IGF-II, ternary complex formation may be decreased, and most IGF peptides are found in the low-molecular-weight peak. IGF-Binding Proteins as Modulators of IGF Action

In general, the binding affinity of IGFBPs for IGF peptides is higher than that of IGF receptors, implying that IGFBPs can modulate IGF binding to its receptors, thereby modulating IGF biologic actions (Fig. 23-34) .[416] Coincubation of cells with IGF-I and a molar excess of IGFBP-3 results in an inhibition of IGF-Istimulated thymidine incorporation in human fibroblasts, lipogenesis in rat epididymal adipocytes, [430] and glucose consumption in mouse fibroblasts. [431] Termination of inhibition apparently requires dissociation of IGFs from the IGF-IGFBP complex by mass action, proteolysis, or other mechanisms. IGFBP proteases have been identified in a wide

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Figure 23-32 The effect of insulin-like growth factorbinding protein 3 (IGFBP-3) proteolysis by prostate-specific antigen (PSA) on IGFBP-3 affinity for IGF-I (A) and IGF-II (B). (From Cohen P, Peehl DM, Graves HC, Rosenfeld RG. Biological effects of prostate-specific antigen as an insulin-like growth factorbinding protein-3 protease. J Endocrinol 1994; 142:407415.)

variety of body fluids and cell culture media [409] [410] [412] [413] [415] and are postulated to play a role in altering IGF availability by lowering the affinities of IGFBPs for their ligand, thereby increasing the availability of IGFs to cell membrane receptors (see earlier). [414] [432] Under certain conditions, the IGFBPs potentiate IGF action. In human and bovine fibroblasts, DNA synthesis and -aminoisobutyric acid transport are potentiated when cells are preincubated with IGFBP-3, whereas IGFBP-3 is inhibitory if added at the same time as IGF-I. [433] [434] These observations have suggested that cell association of IGFBP-3 during preincubation is essential for its IGF-potentiating effect, perhaps allowing IGFBP-3 to serve as a reservoir for IGFs and bringing the ligand into closer proximity to the type 1 IGF receptors. This cell surface association of IGFBP-3 may involve interaction with heparin and heparin sulfate proteoglycans on the cell membrane or specific IGFBP-3 receptors. [396] IGF-Independent Actions of IGF-Binding Proteins

The IGFBPs are bioactive molecules that, in addition to binding IGF, have a variety of IGF-independent functions. These include growth inhibition in some cell types,[435] growth stimulation in other tissues, [436] direct induction of apoptosis, [437] and modulation of the effects of other non-IGF growth factors. These effects of IGFBPs are mediated by binding to their own receptors. The IGFBP signaling pathways are currently being unraveled and involve interaction of IGFBPs with nuclear retinoid receptors as well as with other molecules on the cell surface and in the cytoplasm. [438] IGFBP-3, itself, appears to have intrinsic inhibitory effects on cells, independent of its interaction with IGF. Villaudy and co-workers [439] found that the stimulation of DNA synthesis by basic FGF is inhibited by simultaneous treatment with IGFBP-3, even in the presence of levels of insulin, suggesting that sequestration of IGF peptides from type 1 IGF receptors is not the only means whereby IGFBP-3 inhibits cell growth. IGFBP-3 is also more effective than immunoneutralization of IGF-I in inhibiting serum-stimulated DNA synthesis, and IGFBP-3 inhibits FSH-stimulated DNA synthesis in cultured ovarian granulosa cells, with or without added IGF. [440] Under the same conditions, IGFBP-2 is less inhibitory, despite its higher affinity for IGF peptides. Expression of a transfected human IGFBP-3 cDNA in mouse fibroblasts inhibits both IGF-stimulated and insulin-stimulated cell proliferation (Fig. 23-35) .[401] Similar studies in fibroblasts derived from mouse embryos homozygous for a targeted disruption of the type 1 IGF receptor again demonstrated inhibition with overexpression of IGFBP-3. [441] These studies strongly support an IGF-independent action for IGFBP-3 (Fig. 23-36) . IGFBP-3 binds with high affinity to the surface of various cell types, including human breast cancer cells and rat chondrocytes, and inhibits monolayer growth of these cells in an IGF-independent manner (Fig. 23-37) .[396] [442] [443] Furthermore, transcriptional regulation of IGFBP-3 expression may be the mechanism for the inhibition of breast cancer cell growth by both transforming growth factor 2 (TGF-2) and retinoic acid (Fig. 23-38) .[444] [445] [446] [447] Reduction of IGFBP-3 production through the use of IGFBP-3 antisense oligodeoxynucleotides decreases the inhibitory effects of both TGF-2 and retinoic acid, suggesting that IGFBP-3 production may be a common pathway for multiple hormones and growth factors involved in the modulation of cell growth. [444] For example, estrogen inhibits expression and secretion of IGFBP-3, whereas antiestrogens stimulate production of IGFBP-3 in estrogen receptorpositive human breast cancer cells. [448] Similarly, the mitogenic action of epidermal growth factor (EGF) in human cervical epithelial cells is associated with inhibition of IGFBP-3 expression, and the inhibitory effect of retinoic acid is accompanied by increased IGFBP-3 expression. [449] Regulation of IGFBP-3 gene expression plays a role in signaling by p53, a potent tumor suppressor protein. [450] The presence of cell membrane proteins or receptors that specifically bind IGFBP-3 provides a potential mechanism for IGF-independent growth inhibitory actions of IGFBP-3 (Fig. 23-39) . [396] IGFBP-3 may inhibit cell growth both by sequestering IGF ligands (IGF-dependent action of IGFBP-3) and also by binding to the cell surface (IGF-independent action of IGFBP-3). IGFBP-3 proteases may not only degrade intact

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Figure 23-33 The effect of insulin-like growth factorbinding protein 3 (IGFBP-3) proteolysis by prostate-specific antigen (PSA) on the ability of IGFBP-3 to inhibit IGF-I (A) and IGF-II (B) action. (From Cohen P, Peehl DM, Graves HC, Rosenfeld RG. Biological effects of prostate-specific antigen as an insulin-like growth factorbinding protein-3 protease. J Endocrinol 1994; 142:407415.)

IGFBP-3 to forms with lower affinities for IGFs but also generate IGFBP-3 fragments with enhanced affinity for cell surface IGFBP-3interacting proteins or receptors. A proteolytic fragment of IGFBP-3 that fails to bind IGFs still retains its ability to inhibit cell proliferation. [451] Characteristics of IGF-Binding Proteins 1 to 6

IGF-Binding Protein 1

IGFBP-1 was the first of the IGFBPs to be purified and to have its cDNA cloned. [452] The protein was actually identified and purified from several different tissues, including amniotic fluid [453] and Hep G2 conditioned media, [454] placental membranes (placental protein 12), [455] and endometrium (pregnancy-associated 1 -globulin). [456] Its gene is 5.2 kb long, located on the short arm of chromosome 7, and composed of four exons. [457] The mature protein is 30 kd and is nonglycosylated. mRNA for IGFBP-1 is strongly expressed in decidua (although not in placental trophoblasts), liver, and kidney. IGFBP-1 may be involved in reproductive functions, including endometrial cycling, [458] oocyte maturation,[459] and fetal growth.[429] [460] It is the major IGFBP in fetal serum in early gestation, reaching levels as high as 3000 µg/L by the second trimester. Levels of IGFBP-1 in newborn serum are inversely correlated with birth weight, consistent with an inhibitory role on fetal IGF action. IGFBP-1 also appears to have an important metabolic role, because its gene expression is enhanced in catabolic states, [314] [461] [462] and serum levels undergo diurnal variation.[463] Insulin suppresses and glucocorticoids enhance IGFBP-1 mRNA levels. [461] [464] The acute modulation of serum IGFBP-1 levels may regulate the free fraction of circulating IGF peptides. [461] [463] For example, administration of IGFBP-1 transiently reduces the glucose-lowering capability of IGF-I in rats. [465] Although most in vitro studies are consistent with an inhibitory effect of IGFBP-1 on IGF actions, presumably reflecting interference with IGF ligand-receptor interactions,[388] IGFBP-1 potentiates IGF effects in certain cell systems, [466] possibly as the result of the binding of IGFBP-1 to cell membranes through its Arg-Gly-Asp (RGD) sequence; RGD is an integrin receptor recognition sequence that presumably allows IGFBP-1 to associate with the 5 1 integrin (fibronectin) receptor. [393] The ability of IGFBP-1 to inhibit or potentiate IGF action may depend on post-translational modifications of IGFBP-1, such as phosphorylation, which appears to enhance IGFBP-1 affinity for IGF-I and thereby inhibit IGF action. [467] IGF-Binding Protein 2

The IGFBP-2 gene is located on the long arm of chromosome 2. [468] [469] A single 1.6-kb mRNA yields a mature protein of approximately 34 kd. Like IGFBP-1, IGFBP-2 is highly expressed in fetal tissues, particularly in the CNS. [470] IGFBP-2 is also similar to IGFBP-1 in its lack of N-glycosylation and in the presence of an RGD sequence, perhaps allowing cell association and potentiation of IGF action. [471] Nevertheless, knockout of the IGFBP-2 gene[472] or overexpression of IGFBP-1 in transgenic mice[473] appears to have little effect on phenotype, possibly reflecting "redundancy" in the IGF BP system, in which one IGFBP can compensate for loss of another. The existence of a low-molecular-weight IGFBP in cerebrospinal fluid was inferred from studies demonstrating a 34-kd IGFBP that did not react with antibodies to IGFBP-1 (or IGFBP-3).[474] This IGFBP appeared to be consistent with a previous observation of CSF IGFBPs with preferential affinity for IGF-II. [475] IGFBP-2 is expressed in secretory endometrium and endometrial tumors [458] and is the major IGFBP in seminal fluid and in the conditioned media of prostatic epithelial cells. [476] Interestingly, IGFBP-2 gene expression is markedly reduced in prostatic stromal cells from patients with benign prostatic hyperplasia, suggesting that IGFBP-2 may inhibit stromal growth. [477] Serum levels of IGFBP-2 are frequently elevated in patients with prostatic carcinoma. [478] IGF-Binding Protein 3

The IGFBP-3 gene is located on chromosome 7 in proximity to the gene for IGFBP-1. [479] It contains four exons homologous to those of IGFBP-1 and IGFBP-2 and a fifth exon, consisting of 3' untranslated sequences. In all human tissues studied to date, a single 2.6-kb mRNA has been observed, whereas an additional 1.7-kb mRNA species suggests alternative splicing in baboons. [480] mRNA levels are high in liver, but IGFBP-3 appears to be synthesized in hepatic endothelia (portal venous and sinusoidal) and Kupffer cells, whereas ALS is synthesized in hepatocytes. [481] [482] IGFBP-3 is GH-dependent, due to either a direct GH effect or regulation by IGF. IGF-I administration to hypophysectomized rats increases serum levels of IGFBP-3.[421] [422] [423] On the other hand, IGF-I treatment of patients with GHI does not alter serum IGFBP-3 levels, [120] [393] [409] whereas GH treatment of GH-deficient patients does increase serum levels. Whether these observations mean that GH has a direct effect on IGFBP-3 or reflect GH regulation of ALS and ternary complex formation is unclear.

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Figure 23-34 Affinity cross-linking of [ 125 I]IGF-I (A) and [125 I]IGF-II (B) to membranes from Hs578T breast cancer cells. In the absence of unlabeled insulin-like growth factor (IGF) peptide (lane 1), IGF was predominantly bound to 40- to 45-kd IGFBP-3; no type I or type II IGF receptors were observed. Iodinated IGF was readily displaceable by unlabeled IGF-I or IGF-II (lanes 2 to 5) but not by unlabeled IGF-I/insulin hybrid molecule (lanes 6 and 7) or by an IGF analogue with decreased affinity for IGF-binding proteins (QAYL, lanes 9 and 10 in A). However, addition of [Leu27] IGF-II, which has decreased affinity for the type I IGF receptor (lanes 11 and 12 in A and lanes 7 and 8 in B), resulted in "unmasking" of the 130-kd subunit of the type I IGF receptor (A) and the 250-kd type II IGF receptor. (From Oh Y, Muller HL, Lamson G, Rosenfeld RG. Insulin-like growth factor [IGF]-independent action of IGFbinding protein 3 in hs578T human breast cancer cells: cell surface binding and growth inhibition. J Biol Chem 1993; 268:1496414971.)

The mature IGFBP-3 protein has a molecular weight of approximately 29 kd; however, because it is N-glycosylated, it normally migrates as a doublet-triplet of 40 to 46 kd. Glycosylation does not appear to alter its affinity for IGF-I or -II. [483] IGFBP-3 also undergoes serine phosphorylation of IGFBP-3, although its physiologic significance is uncertain. [484] Perhaps the most significant post-translational modification of IGFBP-3 is proteolysis (see earlier). Discrepancies between immunoblot analyses and radioimmunoassays for IGFBP-3 reflect the altered affinity of IGFBP-3 fragments for IGF ligands, although some proteolytic fragments of IGFBP-3 are capable of ternary complex formation.[411] In pregnancy serum the predominant form of IGFBP-3 is a glycosylated 29-kd fragment. A similarsized IGFBP-3 fragment is present in serum from patients who are postsurgical or catabolic [485] and from patients with noninsulin-dependent diabetes mellitus (non-IDDM). [486] IGFBP-3 is the predominant IGFBP in adult serum, where it carries approximately 75% of the total IGF, primarily as part of the 150-kd ternary complex. Serum levels

are reduced in patients with GHD or GHI, conditions in which assays for serum IGFBP-3 have important diagnostic value (see later). IGFBP-3 associates with cell membranes. Affinity cross-linking studies employing [ 125 I]IGF-I and a human breast cancer cell line have demonstrated no binding to the type 1 IGF receptor but rather to membrane-associated 45-kd IGFBP-3 (see Fig. 23-34) . When IGF analogues with selective affinity for IGFBPs were added, a typical 135-kd subunit of the type 1 IGF receptor was uncovered, demonstrating that membrane-associated IGFBP-3, with its high affinity for IGF peptides, normally "masks" the IGF receptors. Oh and associates [444] demonstrated that the binding of IGFBP-3 to cell membrane proteins was specific, cation-dependent, and of high affinity. Whether these proteins constitute genuine IGFBP-3 receptors remains to be demonstrated, although they may mediate IGF-independent actions of IGFBP-3. Alternatively, IGFBP-3 may associate with heparin-containing proteoglycans both in the extracellular matrix and in the cell membrane, because both IGFBP-3 and IGFBP-5 contain heparin-binding consensus sequences in their COOH termini. [487] However, treatment of cell monolayers with heparinase or chondroitinase has only minor effect on IGFBP-3 binding. Like other IGFBPs, IGFBP-3 inhibits IGF action, especially when the binding protein is present in excess. Presumably, inhibition of IGF action by IGFBP-3 reflects a sequestering of IGF peptides away from the type 1 receptor. Proteolysis of IGFBP-3, resulting in a decrease in affinity for IGF ligands, decreases the inhibitory effects of the binding protein. IGF-Binding Protein 4

The IGFBP-4 gene, located on chromosome 17, contains four exons. [488] A single 2.6-kb mRNA has been identified with high expression in liver. The protein is the smallest of the IGFBPs with 237 amino acids in humans, including 20 cysteines and one N-linked glycosylation site. In immunoblots of most biologic fluids, IGFBP-4 is a 24/28-kd doublet; deglycosylation eliminates the 28-kd band. [489] IGFBP-4 appears to interact with connective tissues, [490] but there is no evidence of membrane association, consistent with a primary role for IGFBP-4 as a soluble, extracellular IGFBP. IGFBP-4 was initially isolated on the basis of its ability to inhibit IGF-stimulated cell proliferation in bone, [491] and there is no evidence for any IGF-potentiating effects. The inhibitory effects of IGFBP-4 are reduced by proteolysis of the protein, much as has been observed with IGFBP-3 degradation. IGFBP-4 proteases are produced by a wide variety of cells, including neuroblastoma, [492] smooth muscle,[493] fibroblasts, [494] osteoblasts, [495] and prostatic epithelium. [496] Activation of IGFBP-4 proteolysis occurs in the presence of IGF-I or IGF-II, presumably reflecting a conformational change in IGFBP-4 resulting from IGF occupancy. [497] [498] The clinical use of IGFBP-4 measurements is minor.[499] IGF-Binding Protein 5

Complementary DNAs for IGFBP-5 have been isolated and sequenced from rat ovary and human placenta and from a human osteosarcoma. [391] [500] The gene is located on chromosome 5 and contains four exons. A single 6.0-kb mRNA is expressed in a wide variety of tissues, particularly in kidney. Mature IGFBP-5 is produced as a 252-amino acid protein with no N-linked glycosylation sites but with one O-linked glycosylation site. [501]

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Figure 23-35 Effect of transfection of Balb/c fibroblasts with a human insulin-like growth factorbinding protein 3 (IGFBP-3) complementary DNA (cDNA) (Tx-BP-3) or with the control plasmid (Tx-P) on cell growth. Transfection with the IGFBP-3 cDNA resulted in a decreased cell proliferation (A) and increased cell doubling time (B). The latter effect could not be overcome with insulin, supporting the concept that the inhibitory effects of IGFBP-3 are IGF independent. (From Cohen P, Lamson G, Okajima T, Rosenfeld RG. Transfection of the human insulin-like growth factorbinding protein 3 gene into Balb/c fibroblasts inhibits cellular growth. Mol Endocrinol 1993; 7:380386. © The Endocrine Society.)

The addition of excess IGFBP-5 to human osteosarcoma cells inhibits IGF-Istimulated DNA and glycogen synthesis. [502] However, when IGFBP-5 adheres to fibroblast extracellular matrix, it potentiates the growth-stimulatory effects of IGF on DNA synthesis. [503] The affinity of IGFBP-5 for IGF-I is reduced approximately sevenfold when the binding protein is associated with extracellular matrix, providing a potential mechanism for release of IGFs to cell surface receptors. Association of IGFBP-5 with extracellular matrix also appears to protect it from proteolysis. [504] Addition of IGFBP-5 to conditioned medium from fibroblasts results in proteolysis to a 21-kd fragment that does not potentiate IGF action, whereas the deposition of IGFBP-5 in extracellular matrix of fibroblasts makes it relatively resistant to degradation. Andress and Birnbaum[505] purified a 23-kd IGFBP-5 fragment from U-2 osteosarcoma cells that has reduced affinity for IGFs but enhances IGF-Istimulated mitogenesis. The 23-kd IGFBP-5 fragment stimulates mitogenesis in an IGF-independent manner, presumably by binding to a specific "receptor" on the cell membrane. Unlike proteolysis of IGFBP-4, which is enhanced by addition of IGFs, degradation of IGFBP-5 is inhibited by the binding of IGF peptides. [46] [415] [501] Proteolysis of IGFBP-5 results in the formation of 16- to 23-kd fragments demonstrated on immunoblots. Degradation of IGFBP-5 may have particular importance in the regulation of granulosa cell activity. In healthy ovarian follicles, neither IGFBP-4 nor IGFBP-5 is expressed, whereas both binding proteins are expressed in atretic follicles, thereby providing a mechanism for intrafollicular regulation

Figure 23-36 Theoretical mechanisms of cellular insulin-like growth factorbinding protein (IGFBP) actions.

of IGF action. [506] [507] Furthermore, FSH enhances IGF action in the ovary and stimulates IGFBP-5 proteolysis.

[415]

IGFBP-5 and IGFBP-4 also appear to be major IGFBPs in bone, where, in addition to inhibiting IGF actions, IGFBP-5 may promote IGF-receptor interactions. Thus, depending on the conditions, IGFBP-5 can either inhibit or potentiate IGF actions. IGF-Binding Protein 6

The human IGFBP-6 gene is located on chromosome 12 and contains four exons. IGFBP-6 transcripts include a major 1.3-kb mRNA and a minor 2.2-kb transcript. [508] The mature peptide contains 216 amino acids and has a molecular mass of approximately 23 kd, although it may migrate at a higher molecular weight on SDS gels, presumably reflecting O-glycosylation. [509] Although IGFBP-6 binds both IGF-I and IGF-II, it has a significantly greater affinity for IGF-II. [510] IGFBP-6 is found in relatively high levels in cerebrospinal fluid, as is also the case for IGFBP-2, which also binds IGF-II with selectively high affinity. IGFBP-6 may also have a role in regulating ovarian activity, perhaps by functioning as an antigonadotropin. [511] Radioimmunoassays for the IGF-Binding Proteins

Specific radioimmunoassays have been developed for IGFBP-1, [512] [513] [514] IGFBP-2, [478] IGFBP-3,[411] [515] [516] IGFBP-4, [517] IGFBP-5, and IGFBP-6. Measurement of IGFBP-3 appears to have the greatest clinical value because it is GH-dependent (Fig. 23-40) . Blum and colleagues [516] have suggested that immunoassay of serum levels of IGFBP-3 may be superior to IGF-I assays in the diagnosis of GHD, because normal levels of IGF-I are so low in young children and many "normal" short children have low levels of IGF-I. Because IGFBP-3 determinations reflect the levels of both IGF-I and IGF-II, their age dependency is not nearly as striking as that of IGF-I; even in young children normal levels are higher than 500 µg/L. The use of IGFBP assays in the evaluation of IGF deficiency and GHD is discussed later. Measurement of IGFBP levels in biologic fluids may be useful for evaluation of malignancies or other pathologic states where the IGFBP levels may be altered.



Gonadal Steroids

Although androgens and estrogens do not contribute substantially to normal growth before puberty, the adolescent rise

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Figure 23-37 Inhibition of Hs578T breast cancer cell growth by insulin-like growth factorbinding protein 3 (IGFBP-3) is IGF independent. Recombinant IGFBP-3 from Escherichia coli results in decreased cell number and cannot be overcome by the addition of an IGF analogue with normal affinity for IGF receptors but decreased affinity for IGFPB-3 (QAYL-Leu-IGF-II). On the other hand, IGF-II, which itself does not stimulate cell proliferation in Hs578T cells, partially releases cells from the growth-inhibitory effects of IGFBP-3, presumably by causing dissociation of IGFBP-3 from the cell membrane. (From Oh Y, Muller HL, Lamson G, Rosenfeld RG. Insulin-like growth factor [IGF]-independent action of IGF-binding protein 3 in Hs578T human breast cancer cells. J Biol Chem 1993; 268:1496414971.)

Figure 23-38 Transforming growth factor 2 (TGF-2) inhibits Hs578T cell growth by transcriptional regulation of insulin-like growth factorbinding protein 3 (IGFBP-3). Reduction in IGFBP-3 messenger RNA (mRNA) and protein levels through the use of an IGFBP-3 antisense oligodeoxynucleotide resulted in significant reduction in the growth inhibitory actions of TGF-2. (From Oh Y, Muller HL, Ng L, Rosenfeld RG. TGF-2induced cell growth inhibition in human breast cancer cells is mediated through IGFBP-3 action. J Biol Chem 1995; 270:1358913592.)

in serum gonadal steroid levels is an important part of the pubertal growth spurt. States of androgen or estrogen excess prior to epiphyseal fusion cause rapid linear growth and skeletal maturation. Thus, just as growth deceleration requires evaluation, growth acceleration can be as abnormal and may be a sign of precocious puberty or virilizing congenital adrenal hyperplasia. A GH-replete state is obligatory for a normal growth response to gonadal steroids, and children with GHD do not have a normal growth response to either endogenous or exogenous androgens. Gonadal steroids work, in part, by enhancing GH secretion and also stimulate IGF-I production directly, as evidenced by the rise in serum IGF-I levels and pubertal growth spurt in children with mutations of the GHR. [169] Both androgens and estrogens increase skeletal maturation. It is likely that androgens primarily act in this regard after conversion to estrogens by aromatase in extraglandular tissues but may also have independent action. [518] Indeed, mutation of the estrogen receptor in a man was associated with tall stature and open epiphyses, [32] and similar findings occur in patients with mutations of the gene encoding the aromatase enzyme. [33] [34] In addition, women with an estrogen receptor variant have increased height, [519] whereas estrogen receptor polymorphisms, but not serum estradiol levels, are related to bone density and height in males. [520]

Figure 23-39 Schematic diagram of insulin-like growth factor (IGF)-independent and IGF-dependent actions of IGF-binding protein 3 (IGFBP-3), the latter being mediated through a putative membrane-associated IGFBP-3 receptor.

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Figure 23-40 Normal serum levels of insulin-like growth factorbinding protein 3 (IGFBP-3) (micrograms per milliliter) by age for males (A) and females (B). The lines represent the mean ± 3 SD. (Data courtesy of Diagnostic Systems Laboratories, Inc., Webster, Texas.)

Skeletal development, in terms of bone mass accretion, is an important pubertal phenomenon and is largely mediated by estrogen action. [521] [522] [523] [524] A longitudinal analysis of pubertal calcium accretion found that approximately 26% of adult calcium is laid down during the two adolescent years of maximal growth. [525] More than 90% of skeletal mass is present by 18 years of age, [526] [527] and estrogens appear to regulate the timing of the growth spurt, stabilization of bone modeling, and endosteal mineral apposition. [521] [528] Rubin [30] described a schema by which early and mid puberty are a time of linear growth, increased bone mineral content, and mineral density (a reflection of bone growth, perhaps largely mediated by the GH-estrogen synergism). It appears that pubertal males have stronger bones than females, perhaps due to greater bone deposition on the periosteal surface in contrast with increased bone on the endocortical surface in females. [529] Late puberty is characterized by bone maturation (epiphyseal closure) and increased volumetric density (true bone mineral density that is not size based), which are apparently mediated more clearly by estrogen. Late menarche and delayed puberty appear to be risk factors for later osteopenia, [530] but some data exist to suggest that low bone mass may already be present prepubertally in these individuals. [531] Independent and synergistic effects of gonadal steroids, GH, and IGF-I contribute to the attainment of peak bone mass in adults. [30] [31] [521] [528] [532] [533]



Thyroid Hormone

Thyroid hormone is a major contributor to postnatal growth, although, like GH, it is of relatively little importance to growth of the fetus. Hypothyroidism postnatally can cause profound growth failure and virtual arrest of skeletal maturation. In addition to a direct effect on epiphyseal cartilage, thyroid hormones appear to have a permissive effect on GH

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TABLE 23-2 -- Classification of Growth Retardation I. Primary Growth Abnormalities A. Osteochondrodysplasias B. Chromosomal abnormalities C. Intrauterine growth retardation II. Secondary Growth Disorders A. Malnutrition B. Chronic disease C. Endocrine disorders 1. Hypothyroidism 2. Cushing's syndrome 3. Pseudohypoparathyroidism 4. Rickets a. Vitamin Dresistant rickets 5. IGF deficiency a. GHD due to hypothalamic dysfunction b. GHD due to pituitary GH deficiency c. GH resistance (1) Primary GH insensitivity (2) Secondary GH insensitivity d. Primary defects of IGF synthesis e. Primary defects of IGF transport and clearance f. IGF insensitivity (1) Defects of the type 1 IGF receptor (2) Postreceptor defects III. Idiopathic Short Stature A. Genetic short stature B. Constitutional delay of growth and maturation C. Heterozygous defects of the GH receptor GH, growth hormone; GHD, growth hormone deficiency; IGF, insulin-like growth factor. secretion. Patients with hypothyroidism have decreased spontaneous GH secretion and blunted responses to GH provocative tests. Treatment with thyroid hormone results in rapid "catch-up" (accelerated) growth, which is typically accompanied by marked skeletal maturation, potentially causing overly rapid epiphyseal fusion and compromise of adult height.



GROWTH RETARDATION A classification of growth retardation is shown in Table 23-2 . Growth disorders are subdivided into these categories: 1. Primary growth abnormalities, in which the defect(s) appears to be intrinsic to the growth plate. 2. Secondary growth disorders, or growth failure resulting from chronic disease or endocrine disorders (the newly introduced category of "IGF deficiency" can result from GHRH deficiency, GHD, or GH or IGF insensitivity). 3. ISS, including variants of normal (constitutional delay of growth and maturation [CDGM] and genetic short stature), heterozygous mutations of the GH receptor gene (GHR gene, a variant of GHI) and as yet unclarified mutations throughout the growth-related genome.



PRIMARY GROWTH ABNORMALITIES Osteochondrodysplasias

The osteochondrodysplasias encompass a heterogeneous group of disorders characterized by intrinsic abnormalities of cartilage or bone, or both. conditions share the following features:

[ 534] [535]

These

1. Genetic transmission. 2. Abnormalities in the size and shape of bones of the limbs, spine, and/or skull. 3. Radiologic abnormalities of the bones (generally). More than 100 osteochondrodysplastic conditions have been identified to date on the basis of physical features and radiologic characteristics, and biochemical, molecular, and genetic studies of these conditions will undoubtedly lead to the recognition of additional types. An international classification for the osteochondrodysplasias, developed in 1970 [536] and revised in 1978 [537] and 1992,[534] is summarized in Table 23-3 . Of note, the category of dysostosis has been dropped from the classification, which focuses on developmental disorders of bone and cartilage. Diagnosis of osteochondrodysplasias can be difficult. Although the underlying molecular and biochemical defects have been identified in many of these conditions, clinical and radiologic evaluation remain central to the diagnosis. Frequently, the clinical features are characteristic, and the diagnosis can be made at birth or even prenatally by ultrasonography. The family history is critical, although many cases are due to fresh mutations, as is generally the case in the classical autosomal dominant achondroplasia and hypochondroplasia. Measurement of body proportions should include arm span, sitting height, upper and lower body segments, and head circumference. Clinical and radiologic evaluation should be used to determine whether involvement is of the long bones, skull, and vertebrae and whether abnormalities are primarily at the epiphyses, metaphyses, or diaphyses. Two of the more common osteochondrodysplasias are achondroplasia and hypochondroplasia. [538]

TABLE 23-3 -- Classification of Osteochondrodysplasias I. Defects of the Tubular (and Flat) Bones and/or Axial Skeleton A. Achondroplasia group B. Achondrogenesis C. Spondylodysplastic group (perinatally lethal) D. Metatropic dysplasia group E. Short rib dysplasia group (with/without polydactyly) F. Atelosteogenesis/diastrophic dysplasia group G. Kniest-Stickler dysplasia group H. Spondyloepiphyseal dysplasia congenita group I. Other spondylo epi-(meta)-physeal dysplasias J. Dysostosis multiplex group K. Spondylometaphyseal dysplasias L. Epiphyseal dysplasias M. Chondrodysplasia punctata (stippled epiphyses) group N. Metaphyseal dysplasias O. Brachyrachia (short spine dysplasia) P. Mesomelic dysplasias Q. Acro/acro-mesomelic dysplasias R. Dysplasias with significant (but not exclusive) membranous bone involvement S. Bent bone dysplasia group T. Multiple dislocations with dysplasias U. Osteodysplastic primordial dwarfism group V. Dysplasias with increased bone density W. Dysplasias with defective mineralization X. Dysplasias with increased bone density II. Disorganized Development of Cartilaginous and Fibrous Components of the Skeleton III. Idiopathic Osteolyses

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Achondroplasia

Achondroplasia is the most common of the osteochondrodysplasias, with a frequency of about 1:26,000. Although transmitted as an autosomal dominant disorder, 80% to 90% of cases appear to be due to new mutations. Achondroplasia is due to a mutation in a transmembranous domain of the gene for FGF receptor 3 (FGF-R3) [539] [540] located on the short arm of chromosome 4(4p16.3).[539] [541] [542] [543] Most cases identified to date are caused by activating mutations at a "hot spot" at nucleotide 1138 (codon380, gly380arg) of the FGF-R3 gene,[538] [539] [540] [544] [545] and because these mutations create new recognition sites for restriction enzymes, they can be easily diagnosed. The mutation rate reported at this site indicates that it may be the most mutable gene in the human genome.[544] [545] The homogeneity of mutation in achondroplasia probably explains the minimal heterogeneity in its phenotype. Infants homozygous for this condition have severe disease, typically dying in infancy from respiratory insufficiency due to the small thorax. Diminished growth velocity is present from infancy, although short stature may not be evident until after 2 years of age. Mean adult heights in males and females are 130 and 120 cm, respectively. [546] Growth curves for achondroplasia have been developed and are of value in following patients. [26] With increasing age, the diagnosis of achondroplasia becomes easier because these patients have characteristic abnormalities of the skeleton, including

megalocephaly, low nasal bridge, lumbar lordosis, short trident hand, and rhizomelia (shortness of the proximal legs and arms) with skin redundancy. Radiologic findings include small, cuboid-shaped vertebral bodies with short pedicles and progressive narrowing of the lumbar interpedicular distance. The iliac wings are small, with narrow sciatic notches. The small foramen magnum may lead to hydrocephalus, and spinal cord and/or root compression may result from kyphosis, stenosis of the spinal canal, or disc lesions. [547] [548] GH secretion in these children is comparable to that in normal children. [549] In a mouse with an equivalent FGF-R3 mutation showing many features of human achondroplasia, there is ligand-independent dimerization and phosphorylation of FGF-R3 with activation of STAT proteins and up-regulation of cell cycle inhibition. [550] Additionally, such mutant mice also exhibit down-regulation of expression of the Indian hedgehog and PTHrP receptor genes, which are also involved in bone formation. [551] As a result of the overexpression of this receptor activity, there is abnormal chondrogenesis and osteogenesis during endochondral ossification. Hypochondroplasia

Hypochondroplasia is an autosomal dominant disorder, previously described as a "mild form" of achondroplasia, that frequently results from a mutation (Asn540Lys) in the FGF-R3 gene.[538] [552] [553] [554] The two disorders do not occur in the same family. [555] About 70% of affected individuals are heterozygous for a mutation in the FGF-R3 gene, but locus heterogeneity exists as other unidentified mutations cause a similar pheotype. [538] [556] Mullis and co-workers, using restriction enzyme analysis, suggested that the IGF-I gene may be a candidate gene for hypochondroplasia, [557] but other molecular abnormalities are likely to be found. The facial features of achondroplasia are absent, and both the short stature and rhizomelia are less pronounced. Adult heights typically are in the 120- to 150-cm range. In contrast with achondroplasia, poor growth may not be evident until after 2 years of age, but stature then deviates progressively from normal. Occasionally, the disproportionate short stature is not apparent until adulthood. Outward bowing of the legs may be accompanied by genu varum. Lumbar interpedicular distances diminish between L1 and L5, and, as with achondroplasia, there may be flaring of the pelvis and narrow sciatic notches. The diagnosis is exceedingly difficult to make in young children. Mild variants of the syndrome may not be clinically distinguishable from normal, and radiologic studies should be performed if a question arises.



Chromosomal Abnormalities

Abnormalities of autosomes or sex chromosomes may cause growth retardation, frequently associated with somatic abnormalities and mental retardation, as in deletion of chromosome 5 or trisomy 18 or 13. Such abnormalities, however, may be subtle, and the diagnosis of Turner's syndrome must be considered in any girl with unexplained short stature. In many cases, the precise cause of growth failure is not clear because the genetic defects do not affect known components of the GH-IGF system. The chromosomal lesion may directly influence normal tissue growth and development or, indirectly, modulate local responsivity to IGF. Down's Syndrome

Trisomy 21, or Down's syndrome, is probably the most common chromosomal disorder associated with growth retardation, affecting approximately 1 in 600 live births. On average, newborns with Down's syndrome have birth weights 500 g below normal and are 2 to 3 cm shorter. Growth failure continues postnatally and is typically associated with delayed skeletal maturation and a delayed and incomplete pubertal growth spurt. Adult heights range from 135 to 170 cm in men and 127 to 158 cm in women.[27] The cause of growth failure in Down's syndrome and in other autosomal defects is unknown. Attempts to find underlying hormonal explanations for growth retardation have been unsuccessful, even though hypothyroidism due to Hashimoto's thyroiditis is more common than normal in Down's syndrome and should be sought. Marginal levels of GH secretion and low serum levels of IGF-I have been reported in Down's syndrome, [558] [559] [560] [561] and exogenous GH may be efficacious in the short term. [562] [563] [564] [565] [566] It is more likely, however, that the growth failure reflects a generalized biochemical abnormality of the epiphyseal growth plate. Gonadal Dysgenesis

In girls with gonadal dysgenesis (Turner's syndrome), short stature is the single most common feature, occurring more frequently than delayed puberty, cubitus valgus, or webbing of the neck. [567] [568] [569] In large series of such individuals, short stature occurs in 95% to 100% of girls with a 45,X karyotype (see Chapter 22) .[570] [571] [572] Several distinct phases of growth have been identified in girls with Turner's syndrome [573] [574] [575] : 1. Mild intrauterine growth retardation (IUGR), with mean birth weights and lengths of 2800 g and 48.3 cm, respectively. 2. Slow growth during infancy falling to -3 SDS by 3 years of age. 3. Delayed onset of the "childhood phase" of growth [14] [15] [575] and progressive decline in height velocity from 3 years of age until approximately 14 years of age, resulting in further deviation from normal height percentiles. 4. A prolonged adolescent growth phase, characterized by a partial return toward normal height, followed by delayed epiphyseal fusion. Mean adult heights in the United States and Europe range from 142.0 to 146.8 cm (lower in Asia). There are important genetic and ethnic influences on growth in these girls. Parental height correlates well with final patient height, [576] [577] and a cross-cultural study in 15 countries demonstrated a strong correlation

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(r = 0.91) between final height in Turner's syndrome and in the normal population with an approximate 20-cm deficit. [570] The cause of growth failure in Turner's syndrome remains unclear. Girls have a skeletal dysplasia and are haploinsufficient for the SHOX gene (short stature homeobox-containing gene) located in the pseudoautosomal region of the short arm of the X chromosome. [578] Mutations and deletions of this gene are associated with poor height growth and several syndromes of skeletal dysplasia including Madelung's deformity, which is also seen in Turner's syndrome. [578] [579] [580] The incidence of IUGR is much greater in girls lacking two copies of the SHOX gene (46% versus 7%). [581] Most patients have normal GH and IGF levels during childhood; reports of low GH or IGF levels in adolescents with Turner's syndrome are likely due to low serum levels of gonadal steroids. [582] Growth impairment is evident prior to the period when activity of the GH-IGF axis is decreased. Nevertheless, GH therapy is capable of both accelerating short-term growth and increasing adult height. [572] [583] [584] This diagnosis must be considered in all girls with unexplained growth failure and especially in girls who are short for family but are growing between the 5th and 10th percentiles in the first decade of life. Nonetheless, a recent study found that the mean age at diagnosis lagged 5.3 years behind the age at which patients with Turner's syndrome fell below the 5th percentile, [585] where its frequency is approximately 1/100. Such data affirm the need for vigorous assessment of all girls who are either absolutely short or relatively small for family heights. 18q Deletions

Deletion of the long arm of chromosome 18 has an estimated prevalence of 1 in 40,000 live births. In a review of 50 cases, 64% of children (mean age 5.8 ± 4.5 years) had heights greater than 2 SD below the mean, with only 6% greater than 0 SDS. [586] Fifteen percent had serum IGF-I concentrations, and 9% had IGFBP-3 concentrations below -2 SD. Seventy-two percent of children had reduced GH responses to provocative testing, although such testing was not always rigorous. Clinical trials of GH therapy in such cases are currently in progress.



Intrauterine Growth Retardation

Infants with IUGR comprise a heterogeneous group with birth weight and/or length below the 3rd or 10th percentile for gestational age, depending on the study. [587] [588] [589] [590] [591] They may also be referred to as small-for-gestational-age (SGA) infants, in contrast with those who are appropriate for gestational age (AGA). The importance of this distinction, in addition to a number of issues influencing neonatal morbidity, is in the prediction of later growth: Most AGA low-birth-weight infants experience catch-up growth during the first 2 years of life, in contrast with the slower, attenuated growth of SGA infants who may have persistent height deficits throughout childhood and adolescence. [589] [590] [591] [592] First-trimester growth failure has been closely associated with low birth weight and low-birth-weight percentile. [593] The earlier in gestation that fetal growth is impaired, the less likely that complete recapture of lost growth will occur. IUGR can arise from abnormalities in the fetus, the placenta, or the mother (Table 23-4) . Factors affecting fetal growth include nutrition provided by the maternal-placental system, alterations of fetal IGF production, and as yet unclarified genes. Although it is understandable why uterine constraint or twin pregnancies might result in limited fetal growth, the reason for abnormal fetal growth in most cases of IUGR is unclear.

TABLE 23-4 -- Causes of Intrauterine Growth Retardation I. Intrinsic Fetal Abnormalities A. Chromosomal disorders B. Syndromes associated with primary growth failure 1. Russell-Silver syndrome 2. Seckel's syndrome 3. Noonan's syndrome 4. Progeria 5. Cockayne's syndrome 6. Bloom's syndrome 7. Prader-Willi syndrome 8. Rubenstein-Taybi syndrome C. Congenital infections D. Congenital anomalies II. Placental Abnormalities A. Abnormal implantation of the placenta B. Placental vascular insufficiency; infarction C. Vascular malformations III. Maternal Disorders A. Malnutrition B. Constraints on uterine growth C. Vascular disorders 1. Hypertension 2. Toxemia 3. Severe diabetes mellitus D. Uterine malformations E. Drug ingestion 1. Tobacco 2. Alcohol 3. Narcotics

The implications of IUGR may extend beyond fetal life. Although most SGA infants exhibit catch-up growth by 2 years of age, a large subgroup remains small. In a retrospective study of 47 individuals who had IUGR, 23 men had an adult height of 162 cm and 24 women had an adult height of 148 cm. [594] Larger studies [589] [590] [591] demonstrated that SGA children had a fivefold to sevenfold greater chance of short stature than AGA children did. Ten percent to 15% of SGA infants will have short stature, and this group makes up as much as 20% of all short children. In a study of a more severely affected neonatal intensive care unit SGA population, 27% had not yet achieved catch-up by 6 years of age. [595] Final adult height is -0.8 to -0.9 SDS, which is a mean deficit of 3.6 to 4 cm when adjusted for family stature. [590] The endocrinologic mechanisms of the poor growth are varied but may include abnormalities of GH production and secretory patterns [589] and insensitivity to GH and IGF-I action. [596] The childhood and adolescent endocrine disorders associated with the SGA children include premature adrenarche, insulin resistance, functional ovarian hyperandrogenism, and an attenuated pubertal growth spurt. [589] [597] Furthermore, SGA infants have an increased risk of hypertension, maturity-onset diabetes mellitus, and cardiovascular disease later in life. [598] [599] [600] Not all of these problems appear to occur in those IUGR babies who do not have catch-up growth, although insulin resistance has been described. [601] Whether IUGR is causally related to these disorders or is a symptom of an underlying inborn metabolic disorder is not yet known. Intrinsic Fetal Factors

In contrast with the role of the endocrine system in postnatal growth, intrauterine growth is less dependent on fetal pituitary hormones. infants are of normal length and weight at birth. Pituitary GH is synthesized and secreted by the latter half of the first trimester, with

[602] [603]

Athyreotic and agonadal

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midgestational levels peaking at 150 µg/L and then falling to around 30 µg/L at term. [141] Because the anencephalic fetus is normal in size, the pituitary was thought to be unnecessary for fetal growth. [604] [605] However, documentation of birth size of rats and humans with congenital GHD [606] [607] [608] [609] and of human newborns with mutations of the GH or GHR genes[169] indicate that GH from the fetal pituitary makes a small contribution to birth size. Infants with neonatal GHD are around -0.5 to

-1.5 SD below the mean in length and are heavy for this length. [607] [608] [609] These observations should not be interpreted to mean that the IGF axis is unimportant in fetal growth. Gene knockout studies show that causing elimination of paracrine/autocrine production of IGF-I, and IGF-II, as well as of the type 1 IGF receptor, impairs fetal growth, although placenta size is normal. [259] [260] [277] [610] Circulating IGF-I levels in fetal and cord blood correlate with fetal size and are reduced in IUGR, especially in situations associated with decreased growth velocity.

[150]

[306] [611] [612] [613] [614]

Similar data suggestive of marked GHI are found in the first week of life following severe fetal malnutrition. [615] The molar ratio of IGF-II to the IGF-II receptor was also found to be related to birth and placenta weight. [616] The initial case of a deletion of the IGF-I gene had profound intrauterine growth failure. [270] [617] The implication of that report is that local tissue production of IGF-I is critical for intrauterine growth and that its regulation is largely GH independent. Human umbilical cord lymphocytes have increased numbers of IGF receptors, [618] and mRNAs for both IGF-I and -II are abundant in fetal tissues. [619] [620] Exogenous IGF-I administration increases neonatal growth rate and protein and fat accretion in pigs with IUGR. [621] Hepatic levels of GHR mRNA and of GHR are low, [141] [622] [623] perhaps explaining the modest impact of GH on IGF production and linear growth. In neonates with IUGR, GH levels are elevated, [624] and exogenous GH treatment has little or no effect on growth, body composition, or energy expenditure, [625] [626] further supporting a state of relative insensitivity to GH at this developmental stage. With defects of the GHR, neonatal IGF levels are low, [141] suggesting a role for GH in regulating IGF production. Similarly, IGFBPs are identifiable in serum and other biologic fluids in the fetus and newborn.[429] However, serum levels of IGFBP-3 and ALS, the major serum carriers of IGF peptides in the adult, are low in the fetus and newborn. Thus, the components of the IGF system are apparently regulated directly by glucose levels or indirectly by fetal insulin secretion, with less impact of GH levels. [611] [627] [628] The role of insulin production in fetal growth is demonstrated by somatic overgrowth of the hyperinsulinemic infants of mothers with diabetes and of infants with the syndrome of persistent neonatal hyperinsulinemic hypoglycemia (nesidioblastosis). [587] [629] [630] In contrast, infants with pancreatic agenesis or with abnormalities of the insulin receptor in the "leprechaun" syndrome are SGA. [587] Further, the inverse relationship of insulin and IGFBP-1 levels and the finding that fetal IGFBP-1 levels are elevated in IUGR [628] support an important role for insulin in fetal growth regulation. In addition to these well-characterized endocrine profiles, cord blood cortisol levels are inversely related to IGF-I and directly to IGFBP-3 concentrations. [614] In infants with IUGR, a close correlation ( r = -0.54) was observed between cord blood cortisol levels and length growth during the first 3 months of life. This is a period during which substantial catch-up growth occurs in some, but clearly not all, IUGR infants. [589] Infants with IUGR exhibiting poor postnatal growth, particularly when the abnormalities are intrinsic to the fetus, have frequently been categorized as having "primordial growth failure." Several syndromes are briefly noted in the following sections. Russell-Silver Syndrome

Russell-Silver syndrome (RSS) is a condition that was independently described by Russell [631] and by Silver and associates. [632] Although this syndrome is probably due to a heterogeneous group of disorders, the common findings include IUGR, postnatal growth failure, congenital hemihypertrophy, and small, triangular facies. [633] [634] [635] [ 636] Nonspecific findings include clinodactyly, precocious puberty, delayed closure of the fontanels, and delayed bone age. [633] [634] [635] Adults are short, with final heights about -4 SD below the mean. [634] [636] Endogenous GH secretion in prepubertal children with RSS is similar to that in other short IUGR children and less than in AGA short children. [589] [637] Because no genetic or biochemical basis for this disorder has been identified, RSS is often used incorrectly as a designation for IUGR of unknown etiology. Maternal uniparental disomy of chromosome 7 exists in 7% to 10% of cases. [63] [638] [639] [640] Candidate genes in chromosome region 7p11-13, such as those for IGFBP-1, IGFBP-3, and EGF-R, all show biallelic expression, but a growth suppression gene, GRB10, which binds to the insulin and IGF-I receptors and replicates asynchronously, remains a candidate gene for overexpression. [640] [641] Paternally expressed imprinted genes at 7q32, PEG/MEST, and g2-COP are other candidates. [639] [642] [ 643]

Seckel's Syndrome

Originally described by Mann and Russell in 1959, [644] the condition most commonly termed Seckel's syndrome is also known as Seckel's bird-headed dwarfism.[645] The syndrome is an autosomal recessive disorder characterized by IUGR and severe postnatal growth failure, combined with microcephaly, prominent nose, and micrognathia. Final height is typically 90 to 110 cm, with moderate to severe mental retardation. The nature of the underlying defect is unknown, although the gene defect may be at 3q22.1-q24. [646] Noonan's Syndrome

Although Noonan's syndrome shares certain phenotypic features with Turner's syndrome, the two disorders are clearly distinct. [647] [648] In Noonan's syndrome, the sex chromosomes are normal and transmission is apparently autosomal dominant; neither the gene locus nor product has been identified, although linkage with chromosome 12 has been demonstrated. Both males and females may be affected, which may explain the misleading terms Turner-like syndrome and male Turner's syndrome. Affected individuals typically have webbing of the neck, a low posterior hairline, ptosis, cubitus valgus, and malformed ears. Cardiac abnormalities are primarily right-sided (pulmonary valve) rather than the left-sided lesions (aorta, aortic valve) characteristic of Turner's syndrome. Although birth weight is generally within the normal range, mean growth in length and weight is below the 3rd percentile through much of childhood with a falling height velocity not dissimilar to that seen in Turner's syndrome except for the late and attenuated pubertal increments. [649] [650] GH secretory abnormalities do not account for the short stature, although endogenous GH production may be reduced somewhat. [651] [652] Microphallus and cryptorchidism are common, and puberty may be delayed or incomplete. Mental retardation of variable degrees is present in approximately 25% to 50% of patients.

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Progeria

The senile appearance characteristic of progeria (Hutchinson-Gilford syndrome) is generally apparent by 2 years of age. [653] There is a progressive loss of subcutaneous fat, accompanied by alopecia, hypoplasia of the nails, joint limitation, early onset of atherosclerosis, typically followed by angina, myocardial infarction, hypertension, and congestive heart failure. Skeletal hypoplasia results in severe growth retardation, which usually becomes evident by 6 to 18 months of age. Cockayne's Syndrome

Cockayne's syndrome, like progeria, is characterized by a premature senile appearance. [654] Retinal degeneration, photosensitivity of the skin, and impaired hearing may also be present. Growth failure typically appears at 2 to 4 years of age. Transmission is as an autosomal recessive disorder. Prader-Willi Syndrome

Growth failure in Prader-Willi syndrome [655] [667] may be evident at birth and is more impressive postnatally. It is considered at length in the discussion of IGF deficiency syndrome caused by hypothalamic dysfunction. Other syndromes associated with moderate to profound growth failure include Bloom's syndrome, de Lange's syndrome, leprechaunism (mutations of the insulin receptor gene), Ellis-van Creveld syndrome, Aarskog's syndrome, Rubenstein-Taybi syndrome, mulibrey nanism (Perheentupa's syndrome), Dubowitz's syndrome, and Johanson-Blizzard syndrome. [656] It is interesting that the gene for the ghrelin receptor is close to the mapped location of de Lange's syndrome. [657]



Maternal and Placental Factors

Maternal factors and placental insufficiency can impair fetal growth. Although such affected infants have better growth potential compared with infants with "primordial growth failure," postnatal growth is not always normal. Maternal nutrition is an important contributor to fetal growth and to the child's growth during the first year of life.[658] Fetal growth retardation may also result from alcohol consumption during pregnancy [659] [660] [661] and from use of cocaine,[663] marijuana,[663] and tobacco.[664] The mechanisms of such drug-induced fetal growth retardation are unclear but probably include uterine vasoconstriction and vascular insufficiency, placental abruption, and premature rupture of membranes. Although maternal tobacco use is, statistically, a major contributor to reduced fetal size, it is unlikely, by itself, to result in severe IUGR. The maternal hormonal milieu is affected by placental steroids and peptides, especially placental GH and human placental lactogen (hPL), also called human chorionic somatomammotropin (hCS), which influence the production of maternal IGF-I. [611] Maternal IGF affects placental function and may facilitate transport of nutrients to the fetus and maternal IGF-I levels correlate with fetal growth. [665] [666] Hasegawa and colleagues [662] have found increased levels of free (non-IGFBP bound) IGF-I levels during normal human pregnancy, possibly due to accelerated proteolysis of IGFBP-3. The placenta has multiple functions, including the transport of nutrients, oxygen, and waste and production of hormones, and it consumes oxygen and glucose brought to it by the uterine circulation. Placental GH affects maternal IGF production that in turn affects placental function. The finding of normal levels of placental GH and IGF-I in a woman with Pit-1 deficiency supports the importance of placental GH action. [668] Additionally, ghrelin message and peptide are present in human placentae during the first trimester. [657] hPL is a major regulator of glucose, amino acid, and lipid metabolism in the mother, aiding in the mobilization of nutrients for transport into the fetus. Damage to the placenta by vascular disease, infection, or intrinsic abnormalities of the syncytiotrophoblasts can impair these important functions. Sometimes, but not always, examination of the placenta can reveal diagnostic information as to the pathogenesis of IUGR. An X-linked homeobox gene, Esx1, detected only in extraembryonic tissues and human testis, is a chromosomally imprinted regulator of placental morphogenesis. [669] [670] [671] Heterozygous and homozygous mutant mice were born 20% smaller than normal and had large edematous placentae. [670] Vasculature was abnormal at the maternal-fetal interface, presumably causing the growth retardation.



SECONDARY GROWTH DISORDERS Malnutrition

Given the worldwide presence of undernutrition, it is not surprising that inadequate caloric and protein intake is the most common cause of growth failure. [672] Marasmus refers to cases with an overall deficiency of calories, including protein malnutrition. Subcutaneous fat is minimal, and protein wasting is marked. Kwashiorkor refers to inadequate protein intake, although it may also be characterized by some caloric undernutrition. In both conditions, multiple deficiencies of vitamins and minerals are apparent. [673] Frequently, the two conditions overlap. Decreased weight growth generally precedes the failure of linear growth by a very short time in the neonatal period and by several years at older ages. Stunting of growth in early life has life-long consequences, resulting in diminished height growth.[674] Both acute and chronic malnutrition affects the GH-IGF system. [675] [676] The impaired growth is usually associated with elevated basal and stimulated serum GH levels,[677] but in generalized malnutrition (marasmus) GH levels may be normal or low. [678] In both conditions, serum IGF-I levels are reduced. [679] [680] Malnutrition may consequently be considered a form of GHI, with serum IGF-I levels reduced despite normal or elevated GH levels. GHBP levels, as a reflection of GHR content, are decreased. [675] [676] GHI may be an adaptive response, whereby protein is spared by the lipolytic and anti-insulin actions of GH. [681] [682] Reduced serum IGF-I levels would serve to shift calories from anabolic to survival requirements. These adaptive mechanisms are accompanied by changes in serum IGFBPs to further limit IGF action during periods of malnutrition. [676] [683] Inadequate calorie and protein intake complicates many chronic diseases that are characterized by growth failure. Anorexia is a common feature of renal failure and inflammatory bowel disease and occurs with cyanotic heart disease, congestive heart failure, CNS disease, and other illnesses. Some of these conditions, furthermore, may be characterized by deficiencies of specific dietary components, such as zinc, iron, and vitamins, necessary for normal growth and development. Undernutrition may also be voluntary, as with dieting and food fads (Fig. 23-41) .[684] Caloric restriction is especially common in girls during adolescence, when it may be associated with anxiety concerning obesity, and in gymnasts and ballet dancers. Anorexia nervosa and bulimia are extremes of "voluntary" caloric deprivation and are commonly associated with impaired growth, prior to epiphyseal fusion, that may result in diminished final adult height. [685] [686] [687] [688] [689] Adolescent bone mineral accretion is impaired and significant osteopenia may persist

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Figure 23-41 Curves of weight and height of a child with growth failure resulting from prolonged self-imposed caloric restriction because of a fear of becoming obese. The crossing of percentiles on the weight curve preceded that for the height curve; when the caloric intake was normalized (arrow), the gain in weight occurred before the improvement in linear growth. At the end of the prolonged period of caloric restriction, weight age (10.2 years) was less than height age (12 years). (From Pugliese MT, Lifshitz F, Grad G, et al. Fear of obesity: A cause of short stature and delayed puberty. N Engl J Med 1983; 309:513518.)

into adulthood. [31] [690] Later in adolescence, malnutrition may cause delayed puberty and menarche and a variety of metabolic alterations. In anorexia nervosa, hormonal profiles are similar to those in protein-energy malnutrition, [685] [686] [687] [688] [689] [691] [692] with high basal levels of GH but low levels of IGF-I, IGFBP-3, and GHBP. GHBP and IGFBP-3 levels correlate with body mass index, as in normal children. [152] [685] [693] The hormones of the GH-IGF axis return to normal levels with refeeding. [676] [685] [693]



Chronic Diseases[694] Malabsorption and Gastrointestinal Diseases

Intestinal disorders that impair absorption of calories or protein cause growth failure, for many of the reasons cited earlier. [681] [695] [696] [697] Growth retardation may predate other manifestations of malabsorption and chronic inflammatory bowel disease. Accordingly, celiac disease (gluten-induced enteropathy) and regional enteritis (Crohn's disease) should be considered in the differential diagnosis of unexplained growth failure. Serum levels of IGF-I may be reduced, [681] [698] [699] [700] reflecting the malnutrition, and it is crucial to discriminate between these conditions and GHD or related disorders causing IGF-I deficiency. Documentation of malabsorption requires demonstration of fecal wasting of calories, especially fecal fat, along with other measures of gut dysfunction such as the D-xylose or breath hydrogen studies. In celiac disease (Fig. 23-42) , impaired linear growth may be the first manifestation of disease, [681] [701] [702] [703] [704] [705] [706] although the degree of growth impairment may be similar in patients with or without gastrointestinal symptoms. [681] In European studies, celiac disease is the cause of unexplained growth impairment in 5% to 20% of unselected patients. [703] [704] [705] [706] The onset and progression of puberty may be delayed, and menarche may be late. [695] [701] Accordingly, a screening test for celiac disease is needed to obviate the standard diagnostic tests involving multiple intestinal biopsies [707] in assessments of asymptomatic patients. Both immunoglobulin G (IgG) and IgA antigliadin and IgA antiendomysial antibodies have relatively high sensitivity and specificity [701] [708] but have been largely superseded by IgA tissue transglutaminase antibodies. [709] IgA deficiency is the most common immunodeficiency in assessments of antibody data. Nonetheless, the diagnosis of celiac disease ultimately requires demonstration of the characteristic mucosal flattening in small bowel biopsy. Changes in the serologic profiles mirror the clinical status obviating the need for subsequent biopsies. Gluten withdrawal is a highly effective treatment for celiac disease and results in rapid catch-up growth and decreased clinical symptoms during the first 6 to 12 months of treatment. [681] [701] Low IGF-I and IGFBP-3 levels return to normal during this period. [700] [703] [710] Most children who receive appropriate dietary management ultimately achieve a normal final height. [711] [712] Growth failure in Crohn's disease is probably due to a combination of malnutrition from malabsorption and anorexia, chronic inflammation, [696] [697] inadequacy of trace minerals in the diet, and use of glucocorticoids. [713] IGF-I levels are low, especially with impaired growth. [681] [697] One third to two thirds or more of children with Crohn's disease have impaired growth at diagnosis, [681] [697] [714] [715] and occasional patients have significant growth failure as the first evidence of Crohn's disease. [681] [713] Osteopenia is common.[31] [716] An elevated erythrocyte sedimentation rate, anemia, and low serum albumin are useful clues, but diagnosis ultimately requires colonoscopy and biopsy along with gastrointestinal imaging studies. Long-term treatment includes enteral and parenteral nutrition, anti-inflammatory agents, alternate-day steroid therapy, and judicious operative intervention. Newer therapeutic

[ 681] [713]

Figure 23-42 Catch-up growth in a girl with gluten-induced enteropathy (celiac disease). After 8 years of growth impairment, the patient was placed on a gluten-free diet and demonstrated substantial catch-up growth. Note the return to the previous growth percentiles. (Courtesy of J. M. Tanner.)

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alternatives may include GH. [717] Permanent impairment of linear growth and deficits of final height may occur in 30% of patients. [713] [718] Chronic Liver Disease

Impaired linear growth and short stature with chronic liver disease in childhood [719] [720] [721] [722] [723] [724] are caused by decreased food intake, fat and fat-soluble vitamin malabsorption, trace element deficiencies, and abnormalities of the GH-IGF system. [723] [724] [725] Decreased levels of IGF-I, IGF-II, and IGFBP-3 and increased GH secretion define the acquired GHI syndrome. [726] [727] [728] [729] [730] [731] A close correlation between GH-dependent peptides and liver function indicates the dominant regulatory role of damaged hepatocytes in end-stage liver failure. [726] Low levels of full-length and truncated GHRs in the cirrhotic liver and consequent diminished release of GHBP substantiate the GHI. [728] [729] [732] Despite provision of adequate calories, insensitivity to the action of GH persists. [724] [727] Liver transplantation prolongs life expectancy, [733] [734] [735] but linear growth is variably improved in the early post-transplantation years. [722] [730] [736] [737] [738] Exogenous glucocorticoid administration presumably plays a major role in the continued growth retardation [722] [738] ; GH and IGF-I production are normal, but the amount of "free IGF" may be decreased because IGFBP-3 levels are relatively high. [730] [737] Post-transplantation growth is inversely correlated with age and directly correlated with degree of growth impairment at transplantation. [721] [722] Exogenous GH treatment, for a period of 18 months, enhances growth rates and increases median height SDS by 0.7 unit.[739] [740] Cardiovascular Disease

Congenital heart disease with cyanosis or chronic congestive failure can cause growth failure. [741] [742] [743] As many as 27% of children with varied cardiac lesions were below the 3rd percentile for height and weight in one survey, [744] and 70% were lower than the 50th percentile in another. [745] Because cardiac defects are usually congenital, many infants have dysmorphic features and IUGR. [746] Inadequate caloric intake is the most common cause of growth impairment in children with congenital heart disease [743] [745] [747] frequently associated with anorexia and vomiting. Chronic congestive heart failure is associated with malabsorption that includes protein-losing enteropathy, intestinal lymphangiectasia, and steatorrhea. [743] [748] [749] Greater cardiac and respiratory work and the relatively higher ratio of metabolically active, energy-utilizing brain and heart to the growth-retarded body mass (cardiac cachexia [750] ) causes an increased basal metabolic rate in these children. [751] [752] [753] Food intake that appears adequate for the child's weight is thus inadequate for normal growth. The degree of cyanosis or hypoxia does correlate with the degree of growth impairment. [743] [745] [754] Decreased levels of IGF-I and IGFBP-3,[755] [756] and normal levels of GH and hepatic GHRs in chronically hypoxemic newborn sheep, [756] suggest GHI distal to the GHR. Linear growth and pubertal maturation depend on left ventricular function in children and adolescents with complicated rheumatic heart disease. [757] Corrective surgery may restore normal growth, frequently after a phase of catch-up growth with normalization of energy expenditure. [752] [758] Surgery must, on occasion, be delayed until the infant reaches an appropriate size, resulting in the conundrum that surgery corrects growth failure but cannot be performed because the infant is too small. In these situations, meticulous attention to caloric support and alleviation of hypoxia and heart failure is necessary to promote growth prior to surgery. Fortunately, this problem has diminished because of operative successes in the neonatal period. The nutritional management of these infants includes calorie-dense feedings, because of the need to restrict fluids; calcium supplementation, because of the use of

diuretics that may cause calcium loss in the urine; and iron, to maintain an enhanced rate of erythropoiesis.

[ 751]

Renal Disease

All conditions that impair renal function can impair growth. [759] [760] [761] [762] [763] Uremia and renal tubular acidosis can cause growth failure before other clinical manifestations become evident. The growth impairment results from multiple mechanisms, including inadequate formation of 1,25-dihydroxycholecalciferol (1,25 [OH] 2 D) with resultant osteopenia, decreased caloric intake, loss of electrolytes necessary for normal growth, metabolic acidosis, protein wasting, insulin resistance, chronic anemia, and compromised cardiac function as well as from impaired GH and IGF production and action. In nephropathic cystinosis, acquired hypothyroidism contributes to the inadequate growth. [764] Sixty percent to 75% of patients with chronic renal failure treated prior to the GH therapeutic era had a final adult height more than -2 SD below the mean.[765] Children and adolescents have normal or elevated circulating levels of GH, depending on the degree of renal failure. [760] [761] [766] [767] [768] [769] [770] In children with end-stage renal disease (ESRD) on dialysis or preterminal chronic renal failure (CRF), the half-life of GH is prolonged twofold. [726] The number of secretory bursts was increased in ESRD by twofold to threefold over chronic renal failure and controls and mean levels of GH were 2.5-fold higher in ESRD than in chronic renal failure patients or controls. Overall, children with ESRD produced substantially more GH than either chronic renal failure patients or controls. Early reports of decreased serum IGF levels in uremia were an artifact due to inadequate separation of IGF from IGFBPs prior to assay. [296] Decreased hepatic IGF production [771] is possibly due to low hepatic GHR gene expression.[772] Additionally, the uremic state may cause a postreceptor defect in GH signal transduction by diminishing phosphorylation and nuclear translocation of GH-activated STAT proteins. [773] Serum IGF-I and -II levels are, however, usually normal, [760] [766] [767] [774] but increases in serum IGFBPs, especially IGFBP-1, [760] [761] [766] [774] [775] may inhibit IGF action. [776] [777] [778] [779] In patients with nephrotic syndrome, serum levels of IGF-I and IGFBP-3 are low because of urinary loss of IGF-IGFBP complexes. [762] Chronic glucocorticoid therapy for a variety of renal disorders can exacerbate growth retardation by diminishing GH release and blunting IGF-I action at growth plates. [778] [780] [781] [782] Chronic renal disease, especially ESRD, with increased GH levels and production, low levels of IGF-I, and poor growth is thus a state of relative resistance to GH and, in some instances, to IGF-I. [765] [776] [777] [778] [779] [783] Even after successful renal transplantation, growth may not be normal. [784] [785] [786] [787] [788] In the large cohort of patients in the North American Pediatric Renal Transplant Cooperative Study, [786] [788] [789] mean height increased after transplantation by only 0.11 SD in the first 4.5 years. The youngest age group (200 years) contact among the families from the Indian subcontinent. The most probable explanation for all four families is that of a "founder effect," or one-time mutations in each group with propagation within geographically isolated gene pools. [1134] In an analysis of 30 families with isolated GHD type IB, Salvatori and colleagues found new missense mutations in transmembrane and intracellular domains of GHRH receptor in three families (10%) with two affected members in each. [1136] Transfection experiments indicated normal cellular expression of these mutant receptors.

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Mutation of the gene for GHRH receptor in its ligand binding domain has also been identified in the little mouse (lit/lit), [1137] leading to dwarfism and decreased numbers of somatotrophs.[1114] [1138] [1139] In this model, the fetal somatotroph mass is normal and hypoplasia, but not absence, of the somatotrophs is evident only after birth. [1114] [1116] [1138] [1139] Such data suggest that GHRH is not an essential factor for fetal differentiation of the somatotrophs and that GHRH-independent cells persist or that mutation does not cause total loss of GHRH function. Genetic Abnormalities of Growth Hormone Production and Secretion Resulting in Isolated Growth Hormone Deficiency.

Four forms of isolated GHD due to errors of the GH gene have been reported (see Table 23-5) .[970] [1096] The gene encoding GH (GH1) is located on chromosome 17q23 in a cluster that includes two genes for hPL: (1) a pseudogene for hPL and (2) the GH2 gene that encodes placental GH. [969] [1095] GH1 and GH2 differ in mRNA splicing pattern: GH1 generates 20-and 22-kd proteins (of approximately equal bioactivity), whereas GH2 yields a protein differing from GH1 in 13amino acid residues. Isolated GHD type IA (GHD-IA) results primarily from large deletions, with rare microdeletions and single base-pair substitutions of the GH1 gene that prevent synthesis or secretion of the hormone. [1140] GHD-IA is inherited as an autosomal recessive trait and affected individuals have profound congenital GHD. Because GH is not produced even in fetal life, patients are immunologically intolerant of GH and, typically, develop anti-GH antibodies when treated with either pituitary-derived or recombinant DNAderived GH. When antibodies prevent patients from responding to GH, GHD-IA can be viewed as a form of GHI, and such patients are candidates for IGF-I therapy. The less severe form of autosomal recessive GHD (isolated GHD type IB) also may result from mutations or rearrangements of the GH1 gene. These mutations cause production of an aberrant GH molecule that retains some function or at least generates immune tolerance. Patients usually respond to exogenous GH therapy without antibody production. The very low frequency (1.7%) of GH1 gene mutations in familial type 1B isolated GHD suggests the importance of studying the GH1 gene promoter region.[1096] [1141] In a group of 65 children with isolated GHD-IB, the GHRH receptor gene was normal in domains coding for the extracellular region, [1142] but more recently, mutations in the transmembranous and intracellular gene domains were found in 10% of families with isolated GHD-IB. [1136] Isolated GHD-II is inherited as an autosomal dominant trait. Such patients may have splice site, intronic, and missense mutations of the GH1 gene. The most common cause appears to be those mutations that inactivate the 5' splice donor site of intron 3, resulting in skipping of exon 3. [64] [1096] It is likely that they function in a dominant-negative manner with the GH mutant suppressing intracellular accumulation and secretion of wild-type GH. [1143] [1144] [1145] [1146] In patients with missense mutations in exon 4 or 5, clinical presentation is quite variable with some evidence for reversibility of the impairment of intracellular GH storage and secretion by GH treatment.[1147] Type III GHD, transmitted as an X-linked trait with associated hypogammaglobulinemia, [1148] has not yet been related to a mutation of the GH1 gene. Bioinactive Growth Hormone.

Serum GH exists in multiple molecular forms, the consequences of alternative post-transcriptional or post-translational processing of the mRNA or protein, respectively. Some of these forms are presumed to have defects in the amino acid sequences required for binding of GH to its receptor and different molecular forms of GH may have varying potencies for stimulating skeletal growth, although this remains to be rigorously proven. Short stature with normal GH immunoreactivity but reduced biopotency has been suggested, [1149] [1150] but the molecular abnormalities have been characterized only in a few such situations. [1151] [1152] In one child with extremely short stature (-6.1 SDS), a mutant GH caused by a single missense heterozygous mutation (cys to arg, codon 77 of GH1 gene) bound with greater affinity than normal to GHBP and the GHR and inhibited the action of normal GH. The child grew more (6 versus 3.9 cm/year) during a period of therapy with exogenous GH in moderate dosage. Strangely, the father had the same genetic abnormality but did not express the mutant hormone. In the second patient [1152] with marked short stature (-3.6 SDS), a heterozygous A-to-G substitution on exon 4 of GH leads to a glycine to arginine substitution. This mutation is located in site 2 of GH molecular binding with its receptor and leads to failure of appropriate sequential receptor dimerization and subsequent diminished tyrosine phosphorylation and the GH-mediated intracellular cascade of events. Bioactivity determined in a mouse B cell lymphoma line was about 33% of immunoreactivity. [1153] Exogenous GH substantially increased growth velocity (4.5 to 11.0 cm/year). There remain other patients, however, in whom diminished bioactivity by sensitive in vitro assays is not reflected by comparable immunoreactivity, but who do not have GH1 mutations, suggesting the importance of abnormal post-translational modifications of GH or other peripheral mechanisms. [398] [1154] Trauma.

See earlier topics. Inflammation.

See earlier topics. Tumors Involving the Pituitary Gland.

Many tumors that impair hypothalamic function also impair pituitary secretion of GH. In addition, craniopharyngiomas are a major cause of pituitary insufficiency. [1155] [1156] [1157] These tumors arise from remnants of Rathke's pouch, the diverticulum of the roof of the embryonic oral cavity that normally gives rise to the anterior pituitary. Genetic defects in this condition, although certainly reasonable to suspect, have not yet been identified. This tumor is a congenital malformation present at birth and gradually grows over the ensuing years. The tumor arises from rests of squamous cells at the junction of the adenohypophysis and neurohypophysis, and it forms a cyst as it enlarges, which contains degenerated cells and may calcify but does not undergo malignant degeneration. The cyst fluid ranges from a "machinery oil" to a shimmering cholesterol-laden liquid, and the calcifications may be microscopic or gross. [1158] About 75% of craniopharyngiomas arise in the suprasellar region, the remainder resembling pituitary adenomas. [1158] [1159] [1160] [1161] [1162] [1163] Craniopharyngiomas can cause manifestations at any age from infancy to adulthood but usually in middle childhood. The most common presentation is due to increased intracranial pressure, including headaches, vomiting, and oculomotor abnormalities. Visual field defects result from compression of the optic chiasm and papilledema or optic atrophy may be present. Visual and olfactory hallucinations have been reported, as have seizures and dementia. Most children with craniopharyngiomas show evidence of growth failure at the time of presentation. GH and the gonadotropins are the most commonly affected pituitary hormones in children and adults, but deficiency of thyrotropin and/or ACTH may also occur; diabetes insipidus is present in 25% to 50%. [1158] [1159] [1160] [1164] Fifty percent to 80% of patients have abnormalities of at least one anterior pituitary hormone at diagnosis. [1159] [1164]

Although lateral skull films may demonstrate enlargement or distortion of the sella turcica, frequently accompanied by suprasellar calcification(s), some children have normal plain films. MRI is the most sensitive diagnostic technique, allowing identification of cystic and solid components and delineation of

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TABLE 23-6 -- Proposed Classification of Growth Hormone Insensitivity Syndromes * 1. Primary GH insensitivity (hereditary defects) a. GH receptor defect (may be positive or negative for GH-binding protein) (1) Extracellular mutation (2) Cytoplasmic mutation (3) Intracellular mutation b. GH signal transduction defect (distal to cytoplasmic domain of GH receptor) c. Insulin-like growth factor I (IGF-I) synthetic defect ( IGF-I gene deletion) d. IGF-I transport defect e. IGF-I receptor defect f. Bioinactive GH molecule 2. Secondary GH insensitivity (acquired defects) a. Circulating antibodies to GH that inhibit GH action b. Antibodies to the GH receptor c. GH insensitivity caused by conditions such as malnutrition, liver disease, catabolic states d. Other conditions that cause GH insensitivity To supplement this revised classification, the following definitions are proposed: GH insensitivity: Clinical and biochemical features of IGF-I deficiency and resistance to exogenous GH, associated with GH secretion that would not be considered abnormally low GH insensitivity syndrome: GH insensitivity associated with the recognizable dysmorphic features described by Laron et al

[ 1172]

Partial GH insensitivity: GH insensitivity in the absence of dysmorphic features described by Laron et al IGF-I, insulin-like growth factor I; GH, growth hormone. *Data from Savage MO, Rosenfeld RG. Acta Paediatr Suppl 1999; 428:147. Data from Laron Z, et al. J Pediatr 1993; 122:241. [1172]

[1264]

anatomic relationships necessary for a rational operative approach. Operative intervention either via craniotomy or transsphenoidal resection may result in partial or almost complete removal of the lesion. Postoperative radiation, especially when tumor resection is incomplete, is commonly used. In some patients, especially those who become obese, a syndrome of normal linear growth without GH may occur. The circulating growth-promoting substances in this condition include insulin and other poorly characterized mitogens. [1165] The long-term childhood and adolescent consequences of craniopharyngioma are substantial, with many quality of life issues exacerbating the hypopituitarism. Pituitary adenomas (see Chapter 8) are infrequent during childhood and adolescence, accounting for fewer than 5% of operated patients at large centers. [1162] [1163] [1166] Nearly two thirds of tumors immunochemically stain for prolactin, and a small number stain for GH. GH-secreting pituitary adenomas are exceedingly unusual in youth. There is a variable experience as to the invasive nature of pituitary adenomas, although the prevailing opinion is that they are less aggressive in children than in adults. [1162] [1166] In 56 patients at the Mayo Clinic with non-ACTH secreting adenomas removed transsphenoidally, macroadenomas were about one third more frequent than microadenomas, with girls outnumbering boys 3.3 to 1. [1162] The incidence of hypopituitarism in patients with macroadenoma was about 50%, compared with none in patients with microadenomas; long-term cure rates were 55% to 65% for both tumor sizes. The localized or generalized proliferation of mononuclear macrophages (histiocytes) characterizes Langerhans cell histiocytosis, a diverse disorder occurring at all ages, with peak incidence at ages 1 to 4 years. [1167] Endocrinologists are more familiar with the term histiocytosis X, which includes three related disorders: (1) solitary bony disease (eosinophilic granuloma), (2) Hand-Schüller-Christian disease (chronic disease with diabetes insipidus, exophthalmos, and multiple calvarial lesions), and (3) disseminated histiocytosis X (Letterer-Siwe, with widespread visceral involvement). These syndromes are characterized by an infiltration and accumulation of Langerhans cells in the involved areas, such as skull, hypothalamic-pituitary stalk, CNS, and viscera. Although these disorders, especially Hand-Schüller-Christian disease, are classically associated with diabetes insipidus, approximately 50% to 75% of patients in selected series have growth failure and GHD at the time of presentation. [1168] [1169] [1170] In contrast, only 1% of unselected children with Langerhans cell histiocytosis living in Canada during a 15-year period [1171] had GHD. Isolated GHD or deficiencies of other anterior pituitary hormones may occur. Peripheral: Inherited and Acquired Syndromes of Insensitivity to Growth Hormone Action (Table 23-6)

Growth Hormone Insensitivity.

The term GHI describes patients with the phenotype of GHD but with normal or elevated serum GH levels and diminished production of IGF-I (Table 23-7) (Fig. 23-49) . [169] [1172] These individuals clearly have IGF-I deficiency. Primary GHI denotes (1) abnormalities of the GHR, including the extracellular GH binding domain, the extracellular GHR dimerization domain, or the intracellular domain; (2) postreceptor abnormalities of GH signal transduction; (3) primary defects of IGF-I biosynthesis; and (4) genetic insensitivity to IGF-I action. Secondary GHI is an acquired and relatively common condition that can be due to (1) malnutrition; (2) hepatic, renal, and other chronic diseases; (3) circulating antibodies to GH; and (4) antibodies to the GHR. Illness-related GHI is discussed in the specific text sections. Abnormalities of the Growth Hormone Receptor.

The initial report of primary GHI by Laron and colleagues described TABLE 23-7 -- Clinical Features of Growth Hormone Insensitivity Growth and Development Birth weight: near normal

Birth length: may be slightly decreased Postnatal growth: severe growth failure Bone age: delayed, but may be advanced relative to height age Genitalia: micropenis in childhood; normal for body size in adults Puberty: delayed 37 years Sexual function and fertility: normal Craniofacies Hair: sparse before age 7 years Forehead: prominent; frontal bossing Skull: normal head circumference; craniofacial disproportion due to small facies Facies: small Nasal bridge: hypoplastic Orbits: shallow Dentition: delayed eruption Scleras: blue Voice: high-pitched Musculoskeletal/Metabolic/Miscellaneous Hypoglycemia: in infants and children; fasting symptoms in some adults Walking and motor milestones: delayed Hips: dysplasia; avascular necrosis of femoral head Elbow: limited extensibility Skin: thin, prematurely aged Osteopenia

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Figure 23-49 The normal GH-IGF axis (A) and the GH-IGF axis showing four potential biochemical defects capable of causing GH insensitivity (B): (1) abnormalities of the GH receptor, binding protein, or both; (2) abnormal signal transduction, resulting from a defect in the intracellular domain of the GH receptor or postreceptor; (3) defect of IGF synthesis (3); and (4) defect of IGF secretion. GH, growth hormone; GH-BP, GH-binding protein; GHRH, GH-releasing hormone; IGF, insulin-like growth factor; IGFBP, IGF-binding protein; SMS, somatostatin. (From Rosenfeld RG, Rosenbloom AL, Guevara-Aguirre J. Growth hormone [GH] resistance due to primary GH receptor deficiency. Endocr Rev 1994; 15:369390. © The Endocrine Society.)

"three siblings with hypoglycemia and other clinical and laboratory signs of GHD, but with abnormally high levels of immunoreactive serum GH." [1173] To date, approximately 250 cases have been identified worldwide, [64] [169] [1145] most from the Mediterranean region or from Ecuador (probably from Spanish Conversos, or Jews who converted to Christianity during the Inquisition). [1174] These individuals do not respond to exogenous GH, in terms of growth, metabolic changes, or increases in serum levels of IGF-I and IGFBP-3. [120] Cellular unresponsiveness to GH was demonstrated in vitro by the failure of GH to stimulate erythroid progenitor cells from the peripheral blood of patients, [1175] and direct evidence of receptor dysfunction was provided by the demonstration that microsomes obtained by liver biopsy do not bind radiolabeled GH. [1176] GHBP activity is usually (in 75% to 80% of cases) undetectable in the sera of patients with this disorder. [199] [200] Studies of the GHR gene in Israeli patients indicate that some, but not most, contained gene deletions, [1177] and a wide variety of homozygous point mutations in this gene (missense, nonsense, and abnormal splicing) have been identified subsequently. [169] [1145] [1178] [1179] Most of the mutations are in the extracellular (GH binding) domain of the GHR; at least one mutation of the extracellular domain does not affect GH binding but prevents dimerization of the receptor. [202] In these situations, the genotype does not uniformly characterize the phenotype. [1145] Mutations of the intracellular and transmembrane domains are much less frequent. One patient had two separate amino acid substitutions in the intracellular domain, but because both mutations are on the same allele, which comes from the unaffected mother, the diagnosis of GHI in this patient was in doubt. [1180] Chujo and associates, [1181] additionally, found that a specific heterozygous missense exon 10 (which codes for most of the GHR intracellular domain) mutation, [1180] was present in 14 of 96 volunteers; there was no significant effect on stature, suggesting that this mutation represents a normal polymorphism. Another subject with compound heterozygous mutations in exon 10 was extremely short (-4 SDS) but grew in response to a very high dose of GH. [1182] At this time, data are inadequate to determine whether these intracellular domain substitutions represent genuine mutations or innocent polymorphisms. Another profoundly short patient, fully resistant to GH but highly responsive to IGF treatment, had coexistent heterozygous mutations affecting exons 6 and 9. similarly short girl (-8 SDS) had compound heterozygosity in exons 8 and 10. [1184]

[1183]

A

Woods and colleagues [203] described two cousins with severe GHR and homozygous mutations at the 5' splice donor site of intron 8, resulting in a mutant GHR without functional transmembrane or intracellular domains. A similar defect was found in a Druse girl with a mutation of the 3' acceptor site of intron 7. [1185] Serum levels of GHBP were elevated because the mutant receptor protein apparently becomes detached from the cell receptor surface. Two defects directly affecting the intracellular domain have been reported to result in dominantly inherited GHI. In one, a white girl and her mother (de novo mutations), both with short stature and biochemical evidence of GHI, were found to

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Figure 23-50 Serum growth hormone (GH) and GH-binding protein levels in sera of patients with GH receptor deficiency from Ecuador. (From Rosenfeld RG, Rosenbloom AL, Guevara-Aguirre J. Growth hormone [GH] resistance due to primary GH receptor deficiency. Endocr Rev 1994; 15:369390. © The Endocrine Society.)

be heterozygous for a single G-to-C transversion in the 3' splice acceptor site of intron 8, resulting in a truncated GHR 1277 lacking most of the intracellular domain.[1186] A second report described high serum GHBP concentrations in two Japanese siblings and their mother who were characterized by partial GHI. [1187] The

patients and their mother had a heterozygous point mutation that disrupted the 5' splice donor site of intron 9, causing skipping of exon 9 and the appearance of a premature stop codon in exon 10 and resulting in the same GH 1277 receptor molecule as described by Ayling and co-workers. [1186] Under in vitro conditions, the Japanese mutation has been shown to result in a GHR molecule that behaves in a dominant negative manner, inhibiting GH-induced tyrosine phosphorylation of STAT5. [1188] Heterozygosity for defects of the GHR may cause relative GHI, [962] [963] [1189] [1190] with modest growth occurring only in response to high doses of GH. Such observations raise the important question of whether heterozygosity for GHI can result in a clinically important phenotype and whether some children labeled as "idiopathic short stature" (ISS) may harbor such mutations. Given the requirement for dimerization of the GHR, there is the potential for an abnormal protein to have varying degrees of dominant negative effect. Ross and associates [1191] described a truncated (1-279) GHR splice variant whose differential production could act to regulate GHBP production and, more important, to modulate GHR signaling in a negative fashion. In summary, the clinical features of GHI due to GHR deficiency are identical to those of other forms of severe IGF deficiency, such as congenital GHD. As with GHD, however, there is a wide range of clinical phenotypes. Basal serum GH levels are typically elevated in children but may be normal in adults (Fig. 23-50) . Most patients have decreased serum GHBP levels, but a normal or even elevated serum GHBP concentration does not exclude the diagnosis of GHRD because mutations of the GHR dimerization domain and in the intracellular domain have been described. Patients with measurable GHBP tend to be taller. [1145] Serum IGF-I, IGF-II, and IGFBP-3 levels are profoundly reduced (Fig. 23-51) , but partial clinical and biochemical phenotypes have been described, typically but not always related to milder mutations of the GHR gene, resulting in only a modest reduction in binding activity and receptor action. [64] [1145] [1185] Postreceptor Abnormalities of Growth Hormone Signal Transduction.

Children with profound short stature, failure to respond to GH in vivo or in vitro, but with normal GH binding and GHBP levels, may have abnormalities of activation of postreceptor signal transduction. [1192] [1193] [1194] Studies in two families described failure to activate the STAT pathway. In one, there was a presumed defect at the level of JAK2 (height -6.8 SDS), but there was a more distal defect in the other, affecting MAPK activation (height -4 and -3.4 SDS). [1194] Primary Defects of IGF-I Biosynthesis.

Woods and colleagues [270] described a 15-year-old boy with a partial deletion of the IGF-I gene yielding a truncated IGF-I molecule. Severe prenatal and postnatal (-6.7 SDS) growth retardation and insensitivity to exogenous GH were consistent with the expectations of the phenotype of IGF-I deficiency. Sensorineural deafness, mental retardation, and microcephaly suggest a role for prenatal IGF-I in CNS development. Hyperinsulinism and insulin resistance were presumably due to overproduction of GH. IGF-I levels were exceedingly low, but IGFBP-3 and GHBP levels were normal. The patient was homozygous for deletions of exons 4 and 5 of the IGF-I gene, with both parents being heterozygous carriers and perhaps mildly affected themselves. Although unresponsive to GH therapy, the patient was able to achieve accelerated growth velocity, improved body composition, increased bone mineralization, and decreased insulin resistance on treatment with IGF-I. [1195] [1196] Genetic Insensitivity to IGF Action.

Abnormalities of the distal arm of the GH-IGF axis are quite uncommon. Genetic defects of the GH molecule with lowered secretion and impaired bioactivity of the GHR and of GH signal transduction have been described above. Finally, conditions of insensitivity to IGF-I action exist and include abnormalities of (1) IGF transport and clearance that would alter presentation of IGF to its receptor, (2) the IGF receptor itself, and (3) postreceptor signaling activation. Such patients would be expected to be exceedingly small, both during prenatal and postnatal life, have elevated GH and normal to high IGF-I levels, and poor growth responses to GH and (presumably) IGF-I administration. Primary Defects of IGF Transport and Clearance.

In fibroblasts from a single short child of 127 studied, Tollefsen and

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Figure 23-51 Serum levels of insulin-like growth factor I (IGF-I), IGF-II, IGF-binding protein 2 (IGFBP-2) and IGFBP-3 in patients with GH receptor deficiency from Ecuador. (From Rosenfeld RG, Rosenbloom AL, Guevara-Aguirre J. Growth hormone [GH] resistance due to GH receptor deficiency. Endocr Rev 1994; 15:369390. © The Endocrine Society.)

co-workers[1197] found a marked resistance to IGF-I stimulated -aminoisobutyric acid uptake and thymidine incorporation. And IGF-variant with 600-fold lower binding affinity for IGFBPs stimulated the fibroblasts of this child and normal children, thus eliminating a primary IGF-I receptor defect. This patient's fibroblasts secreted more IGFBPs than normal and had a 10-fold increase in a cell surface protein similar in size though not immunoreactivity to IGFBP-1. Barreca and associates [1198] studied a short boy (-6 SDS) with increased GH, normal IGF-I, and 20- to 30-fold elevated IGFBP-1 levels. Growth failure seemed due to inhibition of IGF-I action by IGFBP-1. Short-term treatment with GH led to suppression of IGFBP-1, increased ternary-complexed IGF-I, and a marked increase of growth rate. Primary Defects of IGF-I Receptor Production or Responsivity.

In mouse knockout models, homozygous mutations of the IGF-I receptor result in profound growth failure and neonatal mortality. Heterozygous mutations are phenotypically similar to wild-type mice. In the African Efe pygmies, a series of studies [1199] demonstrated extreme insensitivity to the in vitro growth-enhancing effects of IGF-I. Reduced IGF-I receptor transcripts and sites with resultant diminished tyrosine phosphorylation and postreceptor signaling, although no definable receptor mutation, are suggested explanations. [1200] In leprechaunism, a syndrome of growth failure and insulin receptor dysfunction, IGF-I insensitivity is variable. [1199] [1201] The profound abnormality of the insulin receptor suggests that heterodimeric insulin-receptor and IGF-I receptor combinations could possibly lead to failed activation of the IGF-I signaling cascade. As the IGF-I receptor gene resides at 15q26.3, deletions of the distal long arm of chromosome 15 or ring chromosome 15 may lead to hemizygosity for the IGF-I receptor. [1199] [1202] Although such patients may have IUGR and striking postnatal growth failure, lack of a biologic response to IGF-I has not been conclusively demonstrated. [1202] Whether growth failure in such patients is due to altered levels of IGF-I receptor or represents the net effect of the loss of other genes located on 15q remains to be determined. Diagnosis of IGF Deficiency Syndrome: Growth Hormone Deficiency

Because the proper means of diagnosing GHD is controversial, [1203] the concept of the IGF deficiency syndrome becomes even more relevant. With availability of highly specific assays for the IGF peptides and binding proteins and with increasing understanding of the GH-IGF axis, evaluation of patients with growth failure should include a combination of careful auxologic assessment and appropriate measures of the GH-IGF system. Documenting a deficiency of IGF levels and concomitant alterations in serum concentrations of IGFBPs suggests an abnormality of GH secretion or activity and makes necessary a thorough evaluation of hypothalamic-pituitary-IGF function. The foundation for the diagnosis of IGF deficiency is the careful documentation of serial heights and determination of height velocity. In the absence of other evidence of pituitary GH secretory dysfunction, it is usually unnecessary to perform tests of GH secretion. Thus, even in children below the 5th percentile in height (which, obviously, applies to 5% of the normal population), documentation of a normal height velocity (above the 25th percentile for several years) makes the diagnosis of IGF

deficiency and GHD highly unlikely. Assessment of pituitary GH production is difficult because GH secretion is pulsatile, with the most consistent surges occurring at times of slow-wave electroencephalographic rhythms during phases 3 and 4 of sleep. The regulation of GH secretion

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Stimulus

Dosage

TABLE 23-8 -- Tests to Provoke Growth Hormone Secretion * Times Samples Are Taken (minutes)

Comments

Exercise

Step climbing; exercise cycle for 10 min.

0, 10, 20

Observe child closely when on the steps

Levodopa

30 kg: 500 mg Clonidine

0.15 mg/m2

0, 30, 60, 90

Arginine HCl (IV)

0.5 g/kg (max 30 g)

0, 15, 30, 45, 60

10% arginine HCl in 0.9% NaCl over 30 min Insulin (IV)

0.050.1 unit/kg

0, 15, 30, 60, 75, 90, 120

Hypoglycemia, requires close supervision

Glucagon (IM)

0.03 mg/kg (max 1 mg)

0, 30, 60, 90, 120, 150, 180

Nausea, occasional emesis

GHRH (IV)

1 µg/kg

0, 15, 30, 45, 60, 90, 120

Flushing, metallic taste

GHD, growth hormone deficiency; GHRH, growth hormonereleasing hormone; IM, intramuscular; IV, intravenous. *Tests should be performed after an overnight fast. Many investigators suggest that prepubertal children should be "primed" with gonadal steroids, e.g., 5 mg Premarin orally the night before and the morning of the test or with 50100 µg/day ethinyl estradiol for 3 consecutive days before testing or 100 ng depot testosterone 3 days before testing. This, of course, alters the patient's steady state and performs the provocative test in a steroid-rich environment. Patients must be euthyroid at the time of testing. Insulin-induced hypoglycemia is a potential risk of this procedure, which is designed to lower the blood glucose by at least 50%. Documentation of appropriate lowering of blood glucose is recommended. If GHD is suspected, the lower dosage of insulin is usually administered, especially in infants. D 10 W and glucagon should be available.

involves at least two hypothalamic factorsGHRH and somatostatinand multiple other peptides and neurotransmitters. Spontaneous GH secretion varies with gender, age, pubertal stage, and nutritional status, all of which must be factored into the evaluation of GH production. Between normal pulses of GH secretion, serum GH levels are low (often 0.5 over 1 year in children >2 years of age A height velocity < -2 SD over 1 year A height velocity >1.5 SD below the mean sustained over 2 years Signs indicative of an intracranial lesion Signs of multiple pituitary hormone deficiency Neonatal symptoms and signs of growth hormone deficiency *See Growth Hormone Research Society. Consensus guidelines for the diagnosis and treatment of growth hormone (GH) deficiency in childhood and adolescence: summary statement of the GH Research Society. J Clin Endocrinol Metab 2000; 85:39903993.

for GHD permits concomitant assessment of ACTH/cortisol secretion during insulin-induced hypoglycemia. The diagnosis of GHD in a newborn is especially challenging. The presence of micropenis in a male newborn should always lead to an evaluation of the GH-IGF axis. A GH level must be measured in the presence of neonatal hypoglycemia occurring in the absence of a metabolic disorder, such as hyperammonemia or carnitine deficiency syndromes. A level below 20 mg/L in a polyclonal radioimmunoassay suggests GHD. The use of standard GH stimulation tests, except for the glucagon test, is not recommended in neonates. Normative data are not available for stimulated serum GH levels, but a cut-off of 25 ng/mL is probably appropriate and stimulated values lower than 20 ng/mL certainly should raise suspicion. MRI is essential when the diagnosis is suspected, and useful clinical information defining developmental abnormalities of the hypothalamic-pituitary area may be available sooner than GH assay data. An IGFBP-3 level is of value for the diagnosis of neonatal GHD, but IGF-I levels are rarely helpful. [1261] In fact, serum IGFBP-3 should be performed as the test of choice in suspected neonatal GHD. In summary, a child should be considered a candidate for GH therapy if he or she meets one of these auxologic criteria, supported by biochemical evidence of GHD based on sex steroidprimed provocative tests or evidence of IGF deficiency based on measurement of IGF-I and IGFBP-3 concentrations. Such patients should also

undergo MRI studies of the hypothalamus-pituitary and assessment of other pituitary hormone deficiencies. It is understood that this approach will result in GH treatment of some children with idiopathic, isolated GHD or IGF deficiency and that such cases require careful monitoring of both pituitary status and responsiveness to GH treatment. The latter can be assessed relative to recently developed predictive models [1262] [1263] and the diagnosis of GHD reconsidered in the child with idiopathic, isolated GHD, normal MRI findings, and a subnormal clinical response to GH. Diagnosis of IGF Deficiency Syndrome: Growth Hormone Insensitivity

The combination of decreased serum levels of IGF-I, IGF-II, and IGFBP-3 plus increased serum levels of GH suggests a diagnosis of GHI. [169] The possibility of GHR deficiency is supported by a family history consistent with autosomal recessive transmission. Savage and associates [1257] [1264] devised a scoring system for evaluating short children for the diagnosis of GHR deficiency, based on five parameters: 1. 2. 3. 4. 5.

Basal serum GH higher than 10 µ/L (5 µg/L). Serum IGF-I below 50 µg/L. Height SDS below -3. Serum GHBP less than 10%, based on binding of ( 125 I)GH. A rise in serum IGF-I levels after GH administration of less than twofold the intra-assay variation (10%).

Blum and colleagues [1265] proposed that these criteria could be strengthened by: 1. 2. 3. 4.

Evaluating GH secretory profiles, rather than isolated basal levels. Employing an age-dependent range and the 0.1 percentile as the cut-off level for evaluation of serum IGF-I concentrations. Using highly sensitive IGF-I immunoassays and defining a failed GH response as the inability to increase serum IGF-I levels by at least 15 µg/L. Measuring both basal and GH-stimulated IGFBP-3 levels.

These criteria fit well with the population of patients with GHR deficiency in Ecuador, but that is a homogeneous population with severe GHI. [169] [1256] The applicability of these criteria elsewhere remains to be evaluated. An important biochemical marker is the response of IGF-I (and, possibly, IGFBP-3) to GH stimulation. Normal ranges and age-defined responses of serum IGF-I levels have not been established. [1266] [1267] Decreased serum levels of GHBP suggest the diagnosis of GHR deficiency, but some individuals with GHR deficiency have normal serum concentrations of GHBP. [169] [202] [ 203] Such cases represent mutations in the dimerization site or in the intracellular domain of the receptor or abnormalities of postreceptor signal transduction mechanisms. On the other hand, polymorphisms of the GHR gene, without associated reductions in levels of IGF-I or IGFBP-3, should not be considered examples of GHI. At this point, definitive diagnosis of GHI requires (1) the classic phenotype, (2) decreased serum levels of IGF-I and IGFBP-3, and (3) identification of an abnormality of the GHR gene. Idiopathic Short Stature

Many children and early adolescents are short (150 cm in females) (Table 23-10) , although the correlation between predicted and final height is imperfect and must be viewed with caution.[1277] [1278] [1279] When CDGM occurs in the context of familial short stature (see later), however, children may experience both a delayed adolescent growth spurt and a short final height. As stated earlier, some have attributed the diminished growth in the peripubertal period in CDGM to a transient GHD or to a "lazy" pituitary, a concept that is probably due TABLE 23-10 -- Criteria for Presumptive Diagnosis of Constitutional Delay of Growth and Maturation 1. No history of systemic illness 2. Normal nutrition 3. Normal physical examination, including body proportions 4. Normal thyroid and GH levels 5. Normal CBC, sedimentation rate, electrolytes, BUN 6. Height at or below the 3rd percentile, but with annual growth rate above the 5th percentile for age 7. Delayed puberty a. Males: failure to achieve Tanner G2 stage by age 13.8 years or P2 by 15.6 years b. Females: failure to achieve Tanner B2 stage by age 13.3 years 8. Delayed bone age

9. Normal predicted adult height a. Males: >163 cm (64 inches) b. Females: >150 cm (59 inches) BUN, blood urea nitrogen; CBC, complete blood count; GH, growth hormone. to the inadequacies of GH testing, especially to the failure to pretreat patients with a brief course of gonadal steroids. [132] [1230] Low serum levels of IGF-I and IGFBP-3 or a poor GH response to provocative testing (after priming with gonadal steroids) should mandate an investigation for underlying pathology, such as intracranial tumors. Genetic (Familial) Short Stature

The control of growth in childhood and the final height attained are polygenic in nature. For this reason, familial height affects an individual's growth, and evaluation of a specific growth pattern must be placed in the context of familial growth and stature. Formulas have been developed for determination of parental target height, and growth curves that relate a child's height to parental height are available. [47] As a general rule, a child who is growing at a rate that is inconsistent with that of siblings or parents warrants further evaluation. Furthermore, many organic diseases characterized by growth retardation are genetically transmitted. This list includes multiple causes, such as GHI due to mutations of the GHR gene, GH gene deletions, mutations of the PROP1 or POUF1 gene, pseudohypoparathyroidism, diabetes mellitus, and some forms of hypothyroidism. Inherited nonendocrine diseases characterized by short stature include osteochondrodysplasias (see earlier), dysmorphic syndromes associated with IUGR (see earlier), inborn errors of metabolism, renal disease, and thalassemia (see later). Identifying short stature as inherited thus does not, by itself, relieve the physician of responsibility for determining the underlying cause of growth failure. Nonetheless, a constellation of clinical findings describes a normal variant referred to as genetic short stature (GSS) (or familial short stature) that differs from the syndrome of CDGM discussed earlier. In GSS, childhood growth is at or below the 5th percentile but the velocity is generally normal. The onset and progression of puberty are normal or even slightly early and more rapid than normal, so that skeletal age is concordant with chronologic age. Parental height is short (both parents are often below the 10th percentile), and pubertal maturation is normal. Final heights in patients with GSS are short and in the target zone for the family. [1276] The GHIGF system is normal, but exogenous GH therapy during middle childhood years may increase linear growth velocity substantially without disproportionate augmentation of skeletal maturation. Whether long-term GH treatment enhances final height outcome, however, is not clear. Heterozygous Mutations of the Growth Hormone Receptor

The level of the GHR may be genetically determined, although modulated by such factors as nutritional status; GH production appears to be inversely related to GHR/GHBP levels. [195] [198] Accordingly, GHBP levels have been assessed in subjects with ISS. [196] [197] [1283] Serum levels of GHBP in 90% of children with ISS are lower than the normal mean, 20% being below the normal range, especially a subgroup with low IGF-I and higher mean 12-hour levels of GH. [196] [197] Such data raise the possibility that an abnormality of GHR content or structure might impair GH action. The inverse relationship of GHBP levels to GH production is consistent with this hypothesis. [152] In a small group of patients with growth failure, low levels of IGF-I, and poor response to exogenous GH, heterozygous GHR mutations were present in 28%. [962] In contrast with the rarity of homozygous GHR mutations in GHR, heterozygosity is more common and may be a frequent cause of short stature. [963] [1189] In heterozygotes, protein from the mutant allele may disrupt the normal

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dimerization that occurs when GH interacts with its receptor, leading to diminished GH action and growth impairment. The IGF-I/IGFBP-3 generation test following 4 days of GH administration may reveal individual patients with findings of low basal and provoked peptides and modestly elevated GH levels that might represent partial GHI. [1284] [1285] Biochemical confirmation of insensitivity is mandatory in such cases.



TREATMENT OF GROWTH RETARDATION When growth failure is the result of a chronic underlying disease, such as renal failure, CF, or malabsorption, therapy must be directed at treatment of the underlying condition. Although growth acceleration may occur in such children with GH or IGF-I therapy, complete catch-up requires correction of the primary medical problem. If treatment of the underlying condition involves glucocorticoids, growth failure may be profound and is unlikely to be correctable until steroids are reduced or discontinued. Correction of growth failure associated with chronic hypothyroidism requires appropriate thyroid replacement. As discussed earlier, thyroid therapy causes dramatic catch-up growth but also markedly accelerates skeletal maturation, potentially limiting adult height. More gradual thyroid replacement or the use of gonadotropin inhibitors to delay puberty, or both, may be necessary to obtain maximal final height. Treatment of Constitutional Delay

CDGM is a normal variant, with (by definition) potential for a normal (although delayed) pubertal maturation and a normal (albeit diminished for target zone) adult height. Most subjects can be managed by careful evaluation to rule out other causes of abnormal growth and delayed puberty combined with appropriate explanation and counseling. The skeletal age and Bayley-Pinneau table are often helpful in explaining the potential for normal growth to the patient and parents. A family history of constitutional delay is also a source of reassurance. On occasion, however, the stigmata of short stature and delayed maturation may be psychologically disabling for the preadolescent or teenager. Some adolescents with delayed puberty have poor self-images and limited social involvement. [1286] In such patients and in some in whom pubertal delay is predicted on the basis of the overall clinical picture, there is a role for the judicious use of short-term gonadal steroids. Two aspects of this syndrome are addressed by androgen treatment: short stature, especially in boys between ages 10 and 14, and delayed puberty after age 14 years. In the younger group, in whom CDGM is apparent, the orally administered, synthetic androgen, oxandrolone, has been used extensively. [1287] In several controlled studies, [1288] [1289] [1290] [1291] [1292] [1293] oxandrolone therapy for 3 months to 4 years increased linear growth velocity of 3 to 5 cm/year without adverse affects or decreasing either actual [1293] [1294] [1295] or predicted[1290] [1294] [1296] final height. The growth-promoting effects of oxandrolone appear related to its androgenic and anabolic effects rather than to augmentation of the GH-IGF axis. [1297] [1298] Currently recommended treatment is 0.1 mg/kg orally per day. In older boys, in whom delayed pubertal maturation is unbearable and anxiety-provoking, testosterone enanthate has been administered intramuscularly with success. [1287] [1288] [1299] Criteria for therapy of such adolescents should include: 1. 2. 3. 4.

A minimal age of 14 years. Height below the 3rd percentile. Prepubertal or early Tanner G2 stage with a early morning serum testosterone lower than 3.5 nmol/L ( the 90th percentile for gestational age). Even in the absence of clinical symptoms or a family history, the birth of an excessively large infant should lead to evaluation for maternal (or gestational) diabetes mellitus.



Sotos' Syndrome and Beckwith-Wiedemann Syndrome

Two relatively rare syndromes can also cause LGA infants. Children with cerebral gigantism (Sotos' syndrome)[1549] [1550] [1551] are typically above the 90th percentile for both length and weight at birth. Additional clinical features include a prominent forehead; dolichocephaly; macrocephaly; high-arched palate; hypertelorism with an unusual slant to the eyes; prominent ears, jaw and chin; large hands and feet with thickened subcutaneous tissue; mental retardation; and motor incoordination. Although such children continue to grow rapidly during the early years of childhood, puberty is usually early and causes premature epiphyseal fusion. Most patients have a final height within the normal population range. [1551] GH secretion and serum IGF levels are normal, and no cause of the overgrowth in cerebral gigantism has been identified. Beckwith-Wiedemann syndrome (BWS) is the most common (1/13,700) of a group of disorders. It is associated with excessive somatic and specific organ growth, collectively referred to as overgrowth syndromes, apparently caused by excess availability of the growth factor IGF-II encoded by the gene Igf2.[701] BWS is characterized by fetal macrosomia with omphalocele, [1552] with its clinical features due to selective organomegaly, including macroglossia, renal medullary hyperplasia, and neonatal hypoglycemia due to islet cell hyperplasia. [1553] As with cerebral gigantism, excessive fetal, neonatal, and childhood growth ultimately

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leads to early epiphyseal fusion, without an increase in adult height.

[1554]

Although an association between BWS and disordered regulation of IGF-II gene transcription exists, to date no consistent postnatal abnormality of the GH-IGF axis has been identified. [1555] [1556] The paternally derived gene for IGF-II is overexpressed, and the maternally transmitted gene is not active. [1557] Four children with somatic overgrowth but not the diagnostic features of BWS had IGF-II gene overexpression.[1558] Various lines of investigation have localized "imprinted" genes involved in BWS and associated childhood tumors to chromosome 11p15.5. These include, in addition to Igf2, the gene H19, which is involved in Igf2 suppression as well as the gene WT-1 (the Wilms' tumor gene). [257] Mutations in GPC3, a glypican gene, which codes for an IGF-II neutralizing membrane receptor, cause the related Simpson-Golabi-Behmel overgrowth syndrome.[1559] [1560]



POSTNATAL STATURAL OVERGROWTH As stated earlier, both cerebral gigantism and BWS are associated with rapid perinatal growth, but rapid growth usually ends by early to middle childhood. Nevertheless, these conditions should be considered when tall stature in childhood is accompanied by the characteristic phenotypic features or with a history of unexplained fetal overgrowth. As in the case of the child with growth failure, crossing height percentiles between infancy and the onset of puberty is an indication for further evaluation. Although such growth patterns are frequently not of concern to parents, that overly rapid statural growth can indicate serious underlying pathology. Furthermore, as with short stature, children with tall stature must be evaluated in the context of familial growth and pubertal patterns. Familial (Constitutional) Tall Stature

GH secretion and levels of IGF-I and IGFBP-3 in familial tall stature (FTS) are often in the upper normal range. [1255] Tauber and co-workers[1561] divided 65 children with FTS into a subset with high GH secretion rates (5.4 ± 2.3 mg/L/mm) and frequent secretory bursts (5.1 ± 1.6/day) and another subset with lower GH secretion (2.1 ± 0.5 mg/L/mm) and fewer episodic spikes (3.3 ± 1.3/day). IGF-I levels were higher in the group producing more GH and were normal in the low GH group. The investigators postulated that both enhanced secretion of GH and greater efficiency of GH-mediated IGF-I production might be potential causes of FTS. Like children with short stature, children with tall stature must be evaluated relative to familial growth patterns and parental target height. [1562] When a family history of tall stature is available, support and reassurance may be all that are required. A careful assessment of pubertal status and bone age facilitates prediction of adult height and usually obviates the need for hormonal therapy. Standard height prediction using Bayley-Pinneau tables, especially for children younger than 12 years of age, tend to overestimate final height with large confidence limits, [1563] [1564] [1565] particularly in boys. We discourage therapy for boys with predicted adult heights less than 198 cm (6 feet, 6 inches) and girls with predicted adult heights less than 183 cm (6 feet). Indeed, societal changes in attitudes toward tall individuals appear to discourage treatment except in extreme circumstances. The number of patients treated in the United States has fallen markedly since 1970. Therapy, when necessary, is aimed at the acceleration of puberty to cause premature epiphyseal fusion. [1566] [1567] Accordingly, the optimal time for treatment is before the onset of puberty. The earlier the intervention, the more likely that adult height can be decreased, although patients are not usually referred until late childhood or early puberty. Although some success with lower dosages had been reported, administration of ethinyl estradiol at a dose of 0.15 to 0.3 mg/day is a reasonable starting level in girls and can be increased, if necessary and well-tolerated, to 0.5 mg/day. Conjugated estrogens, 7.5 to 10 mg/day, have also been successful. If breakthrough bleeding occurs, cyclic progestagens may be added to the estrogen therapy. Treatment should be continued until epiphyses fuse, because post-treatment growth may be substantial if treatment is stopped early. [1565] The mechanism of estrogen action is probably complex, because estrogen can affect both GH secretion and serum IGF levels and, more importantly, acts directly on the epiphysis. Estrogen mediates epiphyseal fusion in both girls and boys. [32] [33] [34] In prepubertal girls, estrogen therapy reduces adult height by as much as 5 to 6 cm, relative to predictions. When therapy is initiated after the onset of puberty, the decrement in adult height is not likely to be as large. The use of high-dose estrogen in otherwise normal children must be weighed against the known (and unknown) toxicity of such therapy, [1568] including nausea, weight gain, edema, and hypertension. During the initial phases of therapy, growth is paradoxically accelerated as the child rapidly progresses through puberty. Other potential problems, such as thromboembolism, cystic hyperplasia of the breast, endometrial hyperplasia, and cancer, have not been definitively related to estrogen therapy in children but should be discussed with the patient and family. Therapy in boys with tall stature is even more problematic. For the reasons discussed earlier, estrogen is likely to be most efficacious in accelerating epiphyseal fusion but is obviously undesirable in males. Androgens also accelerate skeletal maturation, presumably via aromatization to estrogen but at the price of rapid virilization.



Obesity

Obesity is frequently associated with rapid skeletal growth and early onset of puberty. [1569] Patients with obesity tend to have diminished overall GH production but normal high GHBP and IGF-I levels maintaining adequate or enhanced linear growth velocity. Early activation of adrenal androgenesis and premature pubarche are common. Bone age is usually modestly accelerated so that both puberty and epiphyseal fusion occur early and adult height is normal. This association between obesity and growth is so characteristic that the child with obesity and short stature should always be evaluated for underlying pathology, such as hypothyroidism, GHD, Cushing's syndrome, or Prader-Willi syndrome.



Excess Growth Hormone Secretion

Pituitary gigantism is a rare condition analogous to acromegaly in adults (see Chapter 8) . [1570] [1571] [1572] Typically, GH-secreting tumors of the pituitary are eosinophilic or chromophobe adenomas. Their etiology is uncertain, although many result from somatic mutations that generate constitutively activated G proteins with reduced guanosine triphosphatase activity. [1573] The resulting increase in intracellular cAMP in the pituitary leads to increased GH secretion. McCune-Albright syndrome, which is also caused by mutations resulting in constitutive activation of G proteins, may also be characterized by somatotropic tumors and excess GH secretion. [1574] [1575] GH-secreting

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tumors have also been reported in multiple endocrine neoplasia and in association with neurofibromatosis and tuberous sclerosis (see Chapter 8) .[1576] GH excess that occurs prior to epiphyseal fusion results in rapid growth and attainment of adult heights above the expected genetic potential. When GH hypersecretion is accompanied by gonadotropin deficiency, accelerated linear growth may persist for decades, as in the case of the Alton giant, who reached a height of 280 cm by the time of his death in his 20s. [1577] Manifestations typical of acromegaly may also appear, such as soft tissue swelling, enlargement of the nose, ears and jaw with coarsening of the facial features, pronounced increases in hand and foot size, diaphoresis, galactorrhea, and menstrual irregularity. Serum IGF-I levels are elevated, although high IGF-I levels may also be a normal manifestation of puberty. Basal serum GH levels may be normal or increased, but serum GH is not suppressed by administration of glucose (1.75 g/kg of body weight, up to a maximum of 100 g). Although abnormalities of the sella turcica are often evident on lateral skull films, the demonstration of increased GH-IGF secretion should lead to radiologic evaluation of the hypothalamus and pituitary by MRI or computed tomography. Definitive therapy requires surgical ablation of the tumor. Fortunately, this can usually be accomplished by a transsphenoidal pituitary surgery, although macroadenomas may require a more aggressive surgical approach. As described in Chapter 8 , the use of somatostatin analogues, dopamine agonists, and novel GHR antagonists is an important component of treatment programs for GH excess.



Precocious Puberty

Precocious puberty, whether mediated centrally (increased gonadotropin secretion, GnRH-dependent) or peripherally (increased androgen and/or estrogen secretion GnRH-dependent), results in accelerated linear growth in childhood, mimicking the pubertal growth spurt. Because skeletal maturation is also accelerated, adult height is frequently compromised. The diagnostic evaluation and management of precocious puberty are discussed in Chapter 24 .



Miscellaneous Causes of Tall Stature

Marfan's syndrome, an autosomal dominant disorder of collagen metabolism, is characterized by hyperextensible joints, dislocation of the lens, kyphoscoliosis, and dissecting aortic aneurysm and often leads to long, thin bones that result in arachnodactyly and moderately tall stature. Homocystinuria, an autosomal recessive disorder, phenotypically resembles Marfan's syndrome, although patients are usually mentally retarded. The rate of linear growth may increase modestly in hyperthyroidism. Tall stature has been found in patients with familial ACTH resistance due to a defective ACTH receptor. [1578] Dosage effects of the SHOX gene may result in tall stature. [1579] In females with three copies of the SHOX gene and gonadal dysgenesis, adult stature was +2 to +2.9 SDS.[1580] [1581] In women with 47,XXX karyotype, mean final heights are around 5 to 10 cm taller and in men with 47,XXY karyotype (Klinefelter's syndrome), about 3.5 cm taller than population means. [10] [1580] [1582] Males with an XYY karyotype may also have moderate tall stature. In addition to the SHOX effects, however, the variable degree of estrogen production in some of these syndromes must influence skeletal maturation and final height. [36] It is worth commenting that although delayed puberty may be associated with short stature in childhood, failure to enter puberty and complete sexual maturation may result in sustained growth during adult life with ultimate tall stature and a characteristic eunuchoid habitus. The description of tall stature with open epiphyses resulting from mutation of the estrogen receptor or from aromatase deficiency underscores the fundamental role of estrogen in promoting epiphyseal fusion and termination of normal skeletal growth. [32] [33] [34]



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HP, Schmid C, Zapf J, Froesch ER. Effects of recombinant insulin-like growth factor-I on insulin secretion and renal function in normal human subjects. Proc Natl Acad Sci USA 1989; 86:28682872. 1540. Laron

Z, Klinger B, Erster B, Anin S. Effects of acute administration of insulin-like growth factor-I in patients with Laron-type dwarfism. Lancet 1988; 2:11701172.

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35:145150. 1542. Laron

Z, Anin S, Klipper-Auerbach Y, Klinger B. Effects of insulin-like growth factor-I on linear growth, head circumference, and body fat in patients with Laron-type dwarfism. Lancet 1992; 339:12581261. 1543. Walker

J, Van Wyk JJ, Underwood LE. Stimulation of statural growth by recombinant insulin-like growth factor-I in a child with growth hormone insensitivity syndrome (Laron type). J Pediatr 1992; 121:641646. 1544. Wilton

P. Treatment with recombinant insulin-like growth factor-I of children with growth hormone receptor deficiency (Laron syndrome). Acta Paediatr Suppl 1992; 282:137141.

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C, Preece MA, Rose SJ, et al. Growth response to rhIGF-I 80 µg/kg twice daily in children with growth hormone insensitivity syndrome: relationship to severity of clinical phenotype. Clin Endocrinol (Oxf) 1999; 51:787792. 1546. Ranke

MB, Savage MO, Chatelain PG, et al. Long-term growth of patients with growth hormone insensitivity syndrome with IGF-I: results of the European Multicentre Study. The Working Group on Growth Hormone Insensitivity Syndromes. Horm Res 1999; 51:128134. 1547. Backeljauw

PF, Underwood LE, and The GHIS Collaborative Group. Therapy for 6.57.5 years with recombinant insulin-like growth factor I in children with growth hormone insensitivity syndrome: a clinical research center study. J Clin Endocrinol Metab 2001; 86:15041510. 1548. Guevara-Aguirre

J, Vasconez O, Martinez V, et al. A randomized, double-blind, placebo-controlled trial on safety and efficacy of recombinant human insulin-like growth factor-I in children with growth hormone receptor deficiency. J Clin Endocrinol Metab 1995; 80:13931398. 1549. Sotos 1550. Wit

JF, Cutler EA, Dodge P. Cerebral gigantism. Am J Dis Child 1977; 131:625627.

JM, Beemer FA, Barth PG, et al. Cerebral gigantism (Sotos syndrome): compiled data of 22 cases. Eur J Pediatr 1985; 144:131140.

1551. Agwu

JC, Shaw NJ, Kirk J, et al. Growth in Sotos syndrome. Arch Dis Child 1999; 80:339342.

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C, Gonzalez-Crussi F, Fowler JW. Complete and incomplete forms of Beckwith-Wiedemann syndrome: their oncogenic potential. J Pediatr 1980; 96:4750.

1553. Elliott

M, Bayly R, Cole T, et al. Clinical features and natural history of Beckwith-Wiedemann syndrome: presentation of 74 new cases. Clin Genet 1994; 46:168174.

1554. Weng

EY, Moeschler JB, Graham JM Jr. Longitudinal observations on 15 children with Wiedemann-Beckwith syndrome. Am J Med Genet 1995; 56:366373.

1555. Drummond

IA, Madden SL, Rohwer-Nutter GI, et al. Repression of the insulin-like growth factor II gene by the Wilms' tumor suppressor WT1. Science 1992; 257:674678.

1556. Schofield

PN, Nystrom A, Smith J, et al. Expression of a high-molecular-weight form of insulin-like growth factor II in a Beckwith-Wiedemann syndrome associated adrenocortical adenoma. Cancer Lett 1995; 94:7177. 1557. Kubota

T, Saitoh S, Matsumoto T, et al. Excess functional copy of allele at chromosomal region 11p15 may cause Wiedemann-Beckwith syndrome. Am J Med Genet 1994; 49:378383.

1558. Morison

IM, Becroft DM, Taniguchi T, et al. Somatic overgrowth associated with overexpression of insulin-like growth factor II. Nature Med 1996; 2:311316.

1559. Cano-Gauci

DF, Song HH, Yang H, et al. Glypican-3deficient mice exhibit developmental overgrowth and some of the abnormalities typical of Simpson-Golabi-Behmel syndrome. J Cell Biol 1999; 146:255264.

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1560. Veugelers

M, Cat BD, Muyldermans SY, et al. Mutational analysis of the GPC3/GPC4 glypican gene cluster on Xq26 in patients with Simpson-Golabi-Behmel syndrome: identification of loss-of-function mutations in the GPC3 gene. Hum Mol Genet 2000; 22:13211328. 1561. Tauber

M, Pienkowski C, Rochiccioli P. Growth hormone secretion in children and adolescents with familial tall stature. Eur J Pediatr 1994; 153:311316.

1562. Dickerman 1563. Josse

Z, Loewinger J, Laron Z. The pattern of growth in children with constitutional tall stature from birth to age 9 years: a longitudinal study. Acta Paediatr Scand 1984; 73:530536.

EE, Temperli R, Mullis PE. Adult height in constitutionally tall stature: accuracy of five different height prediction methods. Arch Dis Child 1992; 67:13571362.

1564. Ignatius

A, Lenko HL, Perheentupa J. Oestrogen treatment of tall girls: effect decreases with age. Acta Paediatr Scand 1991; 80:712717.

1565. De

Waal WJ, Greyn-Fokker MH, Stijnen TH, et al. Accuracy of final height prediction and effect of growth-reductive therapy in 362 constitutionally tall children. J Clin Endocrinol Metab 1996; 81:12061216. 1566. Bierich 1567. Sorgo

JR. Estrogen treatment of girls with constitutional tall stature. Pediatrics 1978; 62(suppl):11961201.

W, Scholler K, Heinze F, et al. Critical analysis of height reduction in oestrogen-treated tall girls. Eur J Pediatr 1984; 142:260265.

1568. Trygstad 1569. Forbes

O. Oestrogen treatment of adolescent tall girls: short-term side effects. Acta Endocrinol 1986; 113(suppl 279):170173.

GB. Nutrition and growth. J Pediatr 1977; 91:40.

1570. Spence

HJ, Trias EP, Raiti S. Acromegaly in a 9 1/2-year-old boy. Am J Dis Child 1972; 123:504506.

1571. AvRuskin

TW, Sau K, Tang S, Juan C. Childhood acromegaly: successful therapy with conventional radiation and effects of chlorpromazine on growth hormone and prolactin secretion. J Clin Endocrinol Metab 1973; 37:380. 1572. DeMajo

SF, Onativia A. Acromegaly and gigantism in a boy: comparison with three overgrown non-acromegalic children. Pediatrics 1960; 57:382

1573. Lefkowitz 1574. Lightner 1575. Geffner

RJ. G proteins in medicine. N Engl J Med 1995; 332:186187.

ES, Winter JSD. Treatment of juvenile acromegaly with bromocriptine. J Pediatr 1981; 98:494496.

ME, Nagel RA, Dietrich RB, Kaplan SA. Treatment of acromegaly with a somatostatin analog in a patient with McCune-Albright syndrome. 1987; J Pediatr 3:740743.

1576. Hoffman

WH, Perrin JS, Halac E, et al. Acromegalic gigantism and tuberous sclerosis. J Pediatr 1978; 93:478

1577. Daughaday

WH. Extreme gigantism: analysis of growth velocity and occurrence of severe peripheral neuropathy and neuropathic arthropathy (Charcot joints). N Engl J Med 1977; 297:12671269.

1578. Elis

LLK, Huebner A, Metherell LA, et al. Tall stature in familial glucocorticoid deficiency. Clin Endocrinol (Oxf) 2000; 53:423430.

1579. Ogata

T, Kosho T, Wakui K, et al. Short stature homeoboxcontaining gene duplication on the der(X) chromosome in a female with 45X/46,Xder(X), gonadal dysgenesis, and tall stature. J Clin Endocrinol Metab 2000; 85:29272930. 1580. Ogata

T, Matsuo N. Sex chromosome aberrations and stature: deduction of the principal factors involved in the determination of adult height. Hum Genet 1993; 91:551562.

1581. Nakamura 1582. Tanner 1583. Post

Y, Suehiro Y, Sugino N, et al. A case of 46,X, der(X)(pter q21::p21pter) with gonadal dysgenesis, tall stature, and endometriosis. Fertil Steril 2001; 75:12241225.

JM, Whitehouse RH, Takaishi M. Standards from birth to maturity for height, weight, height velocity, and weight velocity: British children, 1965. Arch Dis Child 1966; 41:454471.

EM, Richman RA. A condensed table for predicting adult stature. J Pediatr 1981; 98:440442.

1584. Takeuchi

T, Suzuki H, Sakurai S, et al. Molecular mechanism of growth hormone (GH) deficiency in the sponmtaneous dwarf rat: detection of abnormal splicing of GH messenger ribonucleic acid by the polymerase chain reaction. Endocrinology 1990; 126:3138. 1585. Dana

K, Baptista J, Blethen SL. Updated National Collaborative Growth Study (NCGS) data. Personal communication, 2001.

1586. Tanner

JM, Whitehouse RH. Clinical longitudinal standards for height, weight, height velocity, weight velocity, and the stages of puberty. Arch Dis Child 1976; 51:170179.

1587. Rotwein 1588. Cohen

P. Structure, evolution, expression, and regulation of insulin-like growth factors I and II. Growth Fact 1991; 5:318.

P, Rosenfeld RG. The IGF axis. In Rosenbloom AL (ed). Human Growth Hormone: Basic and Scientific Aspects. Boca Raton, Fla, CRC Press, 1995, pp 279285.



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Chapter 24 - Puberty: Ontogeny, Neuroendocrinology, Physiology, and Disorders Melvin M. Grumbach Dennis M. Styne

Puberty should not be considered as a de novo event but rather as a phase in the continuum of the development of gonadal function and the ontogeny of the hypothalamic-pituitary-gonadal system in the fetus, through puberty, to the attainment of full sexual maturation and fertility. By puberty, secondary sexual characteristics appear and the adolescent growth spurt occurs, which result in the striking sexual dimorphism of mature individuals, fertility is achieved, and profound psychological effects ensue. [1] These changes are a consequence of stimulation of the gonads by pituitary gonadotropins and increase in gonadal steroid output. Adolescence, a term usually considered to relate to the psychosocial aspects of the teenage years, is accompanied by the onset of adult patterns of sociosexual and economic behavior.[2] The human being is the most reproductively successful of mammals, and many anthropologists have attributed this success to the prolonged pattern of human growth and development[3] [4] and the delay in attaining full sexual maturity. [2] [5] The evolution of the human scheme of growth involves the development of two stages: a childhood stage and an adolescent stage that includes an adolescent or pubertal growth spurt (Fig. 24-1) . Not even our closest biologic relative, the chimpanzee, which matures twice as rapidly as the human, unequivocally exhibits these two stages including the unique human adolescent growth spurt. (The estimated date for divergence of the chimpanzee and human lineages is 4 million to 5 million years ago.) Evolution theorists proposed that a critical part of human success and of many biosocial characteristics emanates from the learning and practice of adult behaviors related to sex and childrearing, particularly provisioning children (not just infants) with food, [2] which is unique to humans. These include learning skills related to production of food, cooperative hunting, division of labor according to sex, sharing food, tool making, and adjusting to the social organization and cultural environment. Bogin, on the other hand, noting that tool making preceded the evolutionary development of adolescence, suggested that, in addition, the evolution and value of human childhood and adolescence and this unique pattern of growth and development have had a significant role in the comparatively striking reproductive advantage and success of the human being. [2] [5] [6] [7] Mayr[8] has called this process of selection "selection for reproductive success." Historical evidence suggests that puberty occurs at an earlier age today than in the past, usually reflected by age of menarche, which is removed by several years from the first sign of secondary development in girls. [9] [10] [11] [12] [13] The average age of menarche in industrialized European countries has decreased 2 to 3 months per decade over the past 150 years, and in the United States the decrease has been approximately 2 to 3 months per decade in the last century [10] [11] [12] [14] (Fig. 24-2) . However, this secular trend has slowed or ceased in "developed" countries such as the United States, Australia, and Western Europe (e.g., Britain and Holland) since approximately 1940, presumably because of improved socioeconomic status and health and the benefits of urbanization. [10] [11] [12] [13] [15] [16] [17] The social class difference in

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Figure 24-1 A summary and proposed scheme of the evolution of the human pattern of postnatal growth and development during the first 20 years of life. A. afar, Australopithecus afarensis, a "bipedal chimpanzee"; A. africa, Australopithecus africanus; H. habilis, Homo habilis (the toolmaker); H. erec 1, early Homo erectus; H. erec 2, late Homo erectus; H. sapiens, Homo sapiens. The early hominid australopithecine specimens from South Africa date to about 3.0 to 1.5 million years ago. H. afarensis, while a hominid (the family of all human species), retained many anatomic features of nonhominid species, for example, an adult brain size of about 400 mL compared with H. habilis (650 to 800 mL), early H. erectus (850 to 900 mL), late H. erectus (up to 1100 mL), and modern H. sapiens (about 1400 mL). Infancy is defined as the period when the mother's breast milk is the sole or most important source of nutrition and in preindustrialized societies ends at about 36 months. Childhood is the period after weaning, when the child is dependent on others for food and protection; this period ends when the growth of the brain in weight is almost complete, at about age 7 years. The juvenile stage is defined as prepubertal individuals who are no longer dependent on their parents for survival. The adolescent stage that begins with the onset of puberty, ends when adult height is attained. [2] [5] The pattern in A. afarensis is no different from that in the chimpanzee (Pan troglodytes). Note the first appearance of the childhood stage, H. habilis (arising about 2 million years ago) and the first appearance of the adolescent stage in H. erectus 2 (about 500,000 years ago); H. sapiens arose about 120,000 to 150,000 years ago. (Modified from Bogin B. Growth and development: recent evolutionary and biocultural research. In Boaz NT, Wolfe LD [eds]. Biological Anthropology: The State of the Science. Bend, Ore, International Institute for Human Evolutionary Research, 1995, pp 4970.)

menarcheal age has narrowed or disappeared in most countries, [18] [19] [20] whereas in Denmark, Spain, and Brazil there remains evidence for a continued decline, at least in certain local regions. [21] [22] [23] In the nomadic Lapp culture, in which the standard of living changed little between 1870 and 1930, no trend toward earlier menarche was found. [24] According to a 1973 survey by the U.S. National Center for Health Statistics, the age of menarche in the United States is 12.8 years, [25] [26] and data published in 1997 [27] indicate that this age remains true for white but not for black girls, in whom the mean age of menarche is 6 months earlier. [28] At present there remains a difference in the age of attainment of stages of puberty in different countries even if stability was reached; for example, Japanese boys undergo changes in testicular size about 1 year earlier than Swiss boys reach the same stages. [29] Remarkably, there is a reverse secular trend in Northern Italy (in women born between 1950 and 1959) and other areas of Europe leading to a later age of menarche; this has been attributed putatively to a resurgence of physical and psychological stress. [30] [31] The method of ascertainment of the age of menarche is of importance. Contemporaneous recordings are performed with the probit method of asking, "yes" or "no," are you menstruating? These may be incorrect because of social pressures of the culture and socioeconomic group considered. [32] Recalled ages

Figure 24-2 Changes in age at menarche, 1840 to 1978, illustrating the advance in the age at menarche in Western Europe and the United States since 1840 and the slowing of this trend since about 1965. (Modified from Tanner M, Eveleth PB. Variability between populations in growth and development at puberty. In Berenberg SR [ed]. Puberty, Biologic and Psychosocial Components. Leiden, HE Stenfert Kroese, 1975, pp 256273. Reprinted by permission of Kluwer Academic Publishers.)

of menarche are used in other studies and considered to be accurate within 1 year (in 90% of cases) during the teenage years

[33] [ 34]

and in older women, too.[35]

Earlier menarche within the normal range may have health consequences. International studies show that earlier age at menarche is associated with a greater risk of development of breast cancer [36] [37] [38] ; indeed, the risk for women with menarche at age younger than 12 years is increased by about 50% compared with those with menarche at 16 years. [39] [40] [41] Further, there is indirect evidence relating earlier menarche to increasing likelihood of hepatocellular carcinoma. [42]

If the age of puberty was later in past centuries than at present, the age of attaining adult height was also later. Surveys of army recruits, schoolchildren, and workers in Europe and America, as well as records of slaves in the United States, show that large portions of the population in the past two centuries continued to grow a considerable amount into their early 20s, whereas modern adolescents cease to grow and reach stable heights by about 17 years of age. [10] [11] The adult heights attained during the 18th and 19th centuries were often at modern 25th percentiles or less. The secular trend toward increased height is more marked than the secular trend toward earlier puberty. Dietary modification may affect the age of menarche and other aspects of puberty. The Harvard Longitudinal Studies of Childhood Health and Development related dietary intake to menarche and found that girls had earlier menarche if they were taller and consumed more animal protein and less vegetable protein as early as 3 to 5 years of age; further, girls had earlier peak growth if they had a history of higher dietary fat intakes at 1 to 2 years of age and higher animal protein intakes at 6 to 8 years. Girls had higher peak velocity if, controlling for body size, they consumed more calories and animal protein 2 years before peak growth. [43] Moderate obesity (up to 30% above normal weight for age) is associated with earlier menarche [44] [45] and conversely adult women with obesity had a history of a tendency toward an earlier age of menarche [46] ; which is the cause and which is the effect is not clear. Pathologic obesity is associated with delayed

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menarche. [47] Black American girls are advanced in secondary sexual development compared with white American girls of the same age during the first three stages of puberty; this may be related, in part, to the higher prevalence of obesity in black girls, although, as stated later, there appears to be a genetic influence in ethnic differences as well. [44] [45] [48] The interaction of socioeconomic conditions, nutrition, energy expenditure, states of health, and puberty is of particular importance in areas of the world where nutrition is suboptimal. [49] [50] [51] South America and Africa have a pattern in which rural children fare better and have earlier puberty and taller stature than urban children, the opposite of the pattern found in previous eras, demonstrating a disturbing trend of adverse nutritional conditions in crowded urban centers. [52] [53] The onset of secondary sexual development in girls of the Kikuyu in Kenya is 13.0 years with menarche at 15.9 years, [54] in contrast to the age of onset of puberty in black American girls of 8.9 years with menarche at 12.2 years [55] ; the Kikuyu start later and have a shorter time of transition to menarche than the American girls. Kikuyu boys enter puberty before or at the same age as Kikuyu girls. [54] Further, boys of the Hadza of Tanzania enter puberty 2 years earlier than girls. [56] In contrast, in the United States and wherever else puberty has been studied in the Western world, girls enter puberty before boys by an average of at least 6 months and often more. Other studies demonstrate the effect of chronic disease on the age of menarche. Delay can occur in any serious chronic condition that is not adequately treated; for example, celiac disease can delay menarche as well as decrease growth in childhood, as does infection with Helicobacter pylori. [57] [58] The earlier suggestion that blindness may advance the age of menarche[59] is not supported by more recent studies. Puberty starts at a later age and the period of pubertal development lasts longer at high altitudes than at low altitudes even when nutritional status is similar. [60] Strenuous physical activity in girls, especially, but not necessarily, when associated with low body weight, can delay or arrest puberty. [61] On the contrary, inactive, bedridden children with mental retardation reach menarche at an earlier age and at a lower proportion of body fat value than do similarly retarded children who are more active.[62] There is evidence that even the living environment might influence menarche. A convergence of the onset of menses in women or girls living together was noted. [63] Several publications followed that studied the timing of the spontaneous onset of menses in women living together or when axillary odor scent (presumably containing pheromones) was given to women; some studies noted synchrony and others did not. Subsequently, the methodology of the studies was criticized and the positive findings deemed incorrect. [64] Nonetheless, one group of investigators found and studied menstrual synchrony in a variety of conditions in college women and older subjects and found that closeness of sleeping conditions was not a prerequisite to synchronizing menstrual cycles but that being close friends was of significance in the phenomenon.[65] [66] Genetic factors play an important role in the onset of puberty, as illustrated by the similar age of menarche in members of an ethnic population and in mother-daughter and sibling pairs. [67] Further support for the influence of genetics on the age of menarche is found in the concordance of the ages of pubertal developmental stages and menarche, which are closer between monozygotic than dizygotic twins. [68] [69] [70] [71] [72] Secondary sexual development occurs earlier in black girls than in white girls in the United States, and although we have alluded to the influence of body mass index (BMI) upon menarche, genetics appears of importance although there is no apparent effect of social or economic factors on this relationship (see later). Thus, when socioeconomic and environmental factors lead to good nutrition, general health, and infant care, the age of onset of puberty in normal children is determined largely by genetic factors. [67] The influence of genetics on mother-daughter comparisons of age of menarche may be subject to complicating factors. In one study of white girls, there was a trend for maternal age at menarche to predict adolescent's age at menarche, but breast development, weight, family relations (including the absence of a father), and depressive affect were predictive of age at menarche in this group with family relations more strongly predicting the age at menarche than the influence of breast development or weight. This study also raised the question of whether psychological stress can decrease the age of menarche or whether stress is likely to occur because of the earlier menarche. [73] [74] [75] On the other hand, another study of age of menarche confirmed the influence of the age of menarche in mothers on the age of menarche in daughters but found no influence of stress related to family problems upon early menarche. [76] Thus, environmental influences should be considered in the study of the age of menarche, although only some of these influences appear to have greater significance than genetic patterns.



PHYSICAL CHANGES OF PUBERTY Secondary Sexual Characteristics

Comparative description of the physical changes between individuals and populations requires an objective and reproducible method of describing the maturation of secondary sexual characteristics. Tanner [12] developed standards of the most useful signs of sexual maturation that have been widely used throughout the world ( Fig. 24-3 Fig. 24-5 ). Self-assessment scales of adolescent sexual maturation are available and are used in some studies to avoid the embarrassment of a secondary sexual examination in normal children and adolescents. [77] [78] However, the answers to self-assessments may be influenced by the subjects' views of what is considered normal or by wishes to conform with normal development [79] [80] [81] and may be less accurate in some ethnic groups than others. [82] Female

Two distinct phenomena occur in the female. The development of the breast and its modified apocrine glands [83] [84] is primarily under the control of estrogens secreted by the ovaries (see Fig. 24-3) ; the growth of pubic and axillary hair (see Fig. 24-4) is mainly under the influence of androgens secreted by the adrenal cortex and the ovary. The glandular and connective tissue of the mammary gland begins to develop at the onset of pubertal maturation. Thus, lobules composed of small ductules and cellular connective tissue develop to a more pronounced degree in the female at puberty. Proliferation of fatty and connective tissue accounts for 80% of the volume of the adult, nonlactating female breast. [83] The classification of the stages of breast development [85] depends on specific characteristics common to the female breast but does not include size or inherent shape of the breasts, which are determined by genetic and nutritional factors (see Fig. 24-3) . Four stages were described by Stratz, [85] a fifth was added by Reynolds and Wines, [86] and modifications were made to the schema by Tanner,[12] who produced the most widely utilized staging. The initial breast development may be unilateral

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Figure 24-3 Stages of breast development according to Marshall and Tanner [153] and Reynolds and Wines. [86] Stage 1: preadolescent; elevation of papilla only. Stage 2: breast bud stage; elevation of breast and papilla as a small mound, enlargement of areolar diameter. Stage 3: further enlargement of breast and areola with no separation of their contours. Stage 4: projection of areola and papilla to form a secondary mound above the level of the breast. Stage 5: mature stage; projection of papilla only, resulting from recession of the areola to the general contour of the breast. (Photographs from Van Wieringen JD, Wafelbakker F, Verbrugge HP, et al. Growth Diagrams 1965 Netherlands: Second National Survey on 024 Year Olds. Netherlands Institute for Preventative Medicine TNO. Groningen, Wolters-Noordhoff, 1971. © Wolters-Noordhoff, Groningen.)

for several months and may be cause for unfounded concern by girls or parents. Indeed, surgical biopsies have been performed inappropriately in girls in whom it was not appreciated that asymmetrical development is normal. There are unusual cases of agenesis of the breast in which no glandular or fat enlargement occurs regardless of the level of estrogen stimulation. On the other hand, virginal breast hypertrophy, extreme and rapid increase in breast size at the onset of puberty, is rare. It has been attributed, at least in part, to increased sensitivity to estrogen action [87] or to increased local estrogen synthesis and growth factors. Changes in the diameter of the papilla of the nipple are sequential and linked to stages of pubertal development. Nipple papilla diameter does not increase much during pubic hair stage 1 to 3 or breast stage 1 to 3 (diameter is 3 to 4 mm) but

Figure 24-4 Stages of female pubic hair development, according to Marshall and Tanner, [2128] Reynolds and Wines, [86] and Dupertuis and colleagues.[2129] Stage 1: preadolescent; the vellus over the pubes is not further developed than that over the anterior abdominal wall; that is, there is no pubic hair. Stage 2: sparse growth of long, slightly pigmented, downy hair, straight or only slightly curled, appearing chiefly along the labia. This stage is difficult to see on photographs. Stage 3: hair is considerably darker, coarser, and curlier. The hair spreads sparsely over the junction of the pubic region. Stage 4: hair is now adult in type, but the area covered by it is still considerably smaller than in most adults. There is no spread to the medial surface of the thighs. Stage 5: hair is adult in quantity and type, distributed as an inverse triangle of the classical feminine pattern. The spread is to the medial surface of the thighs but not up the linea alba or elsewhere above the base of the inverse triangle. (Photographs from Van Wieringen JD, Wafelbakker F, Verbrugge HP, et al. Growth Diagrams 1965 Netherlands: Second National Survey on 024 Year Olds. Netherlands Institute for Preventative Medicine TNO. Groningen, Wolters-Noordhoff, 1971. © Wolters-Noordhoff, Groningen.)

does increase after breast stage 3, providing an objective method of differentiating stage 4 from stage 5 (final diameter is approximately 9 mm)

[ 88]

(Table 24-1) .

The stage of breast development is usually comparable to the stage of pubic hair development in normal girls, but as different endocrine organs control these two processes, these features mature at different ages, and discordance can occur in disease states, the stages should be classified separately ( Table 24-2 and Table 24-3 and Fig. 24-6 and Fig. 24-7 ). Areolar diameter also increases in boys at puberty, and most boys have palpable glandular enlargement of the breast, transient gynecomastia (see later in this chapter). Although rarely evident clinically in individual girls because of its subtle nature, increase in height velocity (rather than

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Figure 24-5 Stages of male genital development and pubic hair development, according to Marshall and Tanner, [2128] Reynolds and Wines, [ 86] and Dupertuis and colleagues.[ 2129] Genital development: Stage 1: preadolescent. Testes, scrotum, and penis are about the same size and proportion as in early childhood. Stage 2: the scrotum and testes have enlarged; the scrotal skin shows a change in texture and also some reddening. Stage 3: growth of the penis has occurred, at first mainly in length but with some increase in breadth; there is further growth of the testes and

scrotum. Stage 4: the penis is further enlarged in length and breadth with development of the glans. The testes and scrotum are further enlarged. The scrotal skin has further darkened. Stage 5: genitalia are adult in size and shape. No further enlargement takes place after stage 5 is reached. Public hair development: Stage 1: preadolescent; the vellus over the pubic region is not further developed than that over the abdominal wall; that is, there is no pubic hair. Stage 2: sparse growth of long, slightly pigmented, downy hair, straight or slightly curled, appearing chiefly at the base of the penis. Stage 3: hair is considerably darker, coarser, and curlier and spreads sparsely over the junction of the pubes. Stage 4: hair is now adult in type, but the area it covers is still considerably smaller than in most adults. There is no spread to the medial surface of the thighs. Stage 5: hair is adult in quantity and type, distributed as an inverse triangle. The spread is to the medial surface of the thighs but not up the linea alba or elsewhere above the base of the inverse triangle. Most men will have further spread of the pubic hair. (Photographs from Van Wieringen JD, Wafelbakker F, Verbrugge HP, et al. Growth Diagrams 1965 Netherlands: Second National Survey on 024 Year Olds. Netherlands Institute for Preventative Medicine TNO. Groningen, Wolters-Noordhoff, 1971. © Wolters-Noordhoff, Groningen.)

breast development) is actually the first sign of puberty in girls; however, breast budding is what is first noted by most lay or medical observers. There are changes in the appearance of the vaginal opening at puberty. [89] Dulling and thickening of the vaginal mucosa from the prepubertal reddish glistening appearance are due TABLE 24-1 -- Nipple Diameter Compared with Breast and Pubic Hair Stages: Comparison of Longitudinal and Cross-Sectional Data Nipple Size (mm) * Stage

Cross-Sectional Data

Longitudinal Data

Breast 1

2.89 (0.81)

3.0 (0.77)

2

3.28 (0.89)

3.37 (0.96)

3

4.07 (1.32)

4.72 (1.40)

4

7.74 (1.64)

7.25 (1.46)

5

9.94 (1.38)

9.41 (1.45)

1

2.95 (1.02)

3.14 (1.31)

2

3.32 (0.91)

3.69 (1.34)

3

4.11 (1.54)

4.44 (1.17)

4

7.15 (1.81)

6.54 (1.47)

5

9.66 (1.59)

8.98 (1.56)

Public hair

Reprinted by permission of Elsevier Science Publishing Co., Inc. from Papilla (nipple) development during female puberty, by Rohn RD. Journal of Adolescent Health Care, Vol. 2, pp. 217220. Copyright 1982 by The Society for Adolescent Medicine. *Results are means ± standard deviation (SD; in parentheses). Significantly different from previous stage, P 5 SD), correlate with the magnitude of the hypothalamic damage visualized by cranial MRI [1043] and quite likely are manifestations of injury to the hypothalamic ventromedial nuclei (associated with increased parasympathetic activity and hyperinsulinemia) or the paraventricular nuclei, or both. [1044] Ten reported cases of aberrant sleep patterns followed treatment for craniopharyngioma with awakenings at night and in some cases daytime somnolence. [1045] A Rathke's cleft cyst can produce symptoms and signs indistinguishable from those of a craniopharyngioma. The usual treatment is surgical drainage and excision of the cyst wall. [1046] Other Extrasellar Tumors

Sexual infantilism may be caused by other extrasellar tumors that arise in or encroach on the hypothalamus. Germinomas (previously termed pinealomas, ectopic pinealomas, atypical teratomas, or dysgerminomas)[1047] or other germ cell tumors of the CNS are the extrasellar tumors that most commonly cause sexual infantilism, although, when all primary CNS tumors are considered, germinomas are rare. The diagnosis is usually made during the second decade of life. Polydipsia and polyuria are among the most common symptoms,[1048] followed by visual difficulties and abnormalities of growth and puberty. [1049] The most common endocrine abnormalities are deficiencies of vasopressin and GH, but other anterior pituitary hormone deficiencies (including gonadotropin deficiency) and elevated serum prolactin levels are frequent. The concentrations of hCG, in spinal fluid especially and in serum, and of -fetoprotein are useful tumor markers in children and adolescents with a CNS germ cell tumor. Rather than delaying puberty, germ cell tumors in boys may cause isosexual precocity by secretion of hCG (see section on sexual precocity). A single case of an hCG-secreting suprasellar teratoma that produced mild sexual precocity in a 6-year-old girl who had nondetectable serum concentrations of LH and FSH was reported. This pubertal development was thought to be possibly related to aromatase activity of the teratoma; with therapy and regression of the tumor, the breast budding disappeared. [1050] A germ cell tumor may arise in the suprasellar hypothalamic region, in the pineal region, or in another area of the CNS. Subependymal spread along the lining of the third ventricle is common, and seeding may lead to involvement of the lower spinal cord and corda equina. MRI scans with contrast enhancement are useful in the diagnosis of tumors more than 0.5 cm in diameter and detection of isolated enlargement of the pituitary stalk, an early finding on MRI scans. [1048] Periodic MRI monitoring for further development of a tumor is indicated whenever thickening of the pituitary stalk is encountered. Hypothalamic-pituitary abnormalities on MRI are related to functional defects such as diabetes insipidus. [1048] [1049] [1051] [1052] Unlike the size of the pituitary gland, which increases 100% between years 1 and 15, the size of the pineal gland does not change after age 1 in normal individuals, and thus any enlargement after that time is suspicious of a mass lesion. [1053] Pure germ cell tumors (germinomas) are radiosensitive, and radiation is the preferred treatment; the clinical features and the response to radiation therapy are so characteristic that surgery is rarely indicated except for biopsy to establish a tissue diagnosis. [1048] [1054] When a mixed germ cell tumor is found, both radiation therapy and chemotherapy are recommended. Hypothalamic and optic gliomas or astrocytomas, occurring either as part of neurofibromatosis (von Recklinghausen's disease) or independently, can also cause sexual infantilism.[1055] [1056] [1057]

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Pituitary Tumors

Pituitary adenomas are rare in childhood and adolescence as only 2% to 6% of all pituitary adenomas occur in this age group. In one study, 50% of pituitary adenomas occurring before adulthood were prolactinomas, 20% were GH-secreting adenomas, and 30% were chromophobe adenomas. [1058] Hyperprolactinemia related to microprolactinomas or macroprolactinomas of the pituitary is uncommon in childhood and adolescence and is a rare cause of delayed puberty in both boys and girls.[1059] [1060] [1061] [1062] Among our patients, only 2 of 29 had delayed onset of puberty, [1062] although primary amenorrhea was the presenting symptom in 13 of 20 pubertal females. Galactorrhea may be absent by history but is often demonstrable by manual manipulation of the nipples (because serum prolactin may rise after manipulation of the nipples, samples should be obtained before examination or many hours later). Transsphenoidal resection of microprolactinomas in children and adolescents was an effective treatment with an 89% cure rate. [1062] The dopamine agonist bromergocryptine is used by some as a method of decreasing serum prolactin concentrations and decreasing the size of the tumors [1063] ; we use this approach in children and adolescents in whom resection of the adenoma is incomplete and to reduce the size of large macroprolactinomas before attempted surgical removal. Pubertal progression in affected boys and girls as well as normal menstrual function in girls usually follows the reduction in serum prolactin levels. In a series of prolactinomas in children and adolescents, there was a preponderance of microadenomas in girls and of macroadenomas in boys, and these larger tumors led to local symptoms related to their size Other Central Nervous System Disorders Leading to Delayed Puberty

Langerhans' Cell Histiocytosis (Hand-Schüller-Christian Disease, or Histiocytosis X)

This disorder, now thought to be a clonal proliferative disorder of Langerhans' histiocytes or their precursors, [1064] [1065] is characterized by the infiltration of lipid-laden histiocytic cells or foam cells in the skin, viscera, and bone. [1066] [1067] [1068] Diabetes insipidus, usually resulting from infiltration of the hypothalamus or the pituitary stalk or both, is the most common endocrine manifestation. [1069] However, GH deficiency and delayed puberty may occur. [1070] [1071] There may be visceral involvement including the lung, liver, and spleen. Other findings include cyst-like areas in flat bones of the skull, the ribs, the pelvis, and the scapula; in the long bones of the arms and legs; and in the dorsolumbar spine. Lesions of the mandible lead to the radiographic impression of "floating teeth" within rarefied bone and the clinical finding of absent or loose teeth. Infiltration of the orbit may lead to exophthalmos, and mastoid or temporal bone involvement may lead to chronic otitis media. [1072] Treatment with glucocorticoids, antineoplastic agents, and radiation is promising in terms of survival, but more than 50% of patients have late sequelae or progression. [1068] [1072] [1073] [1074] The natural waxing and waning course of this disease makes evaluation of therapy difficult. [ 1075] [ 1076] Postinfectious Inflammatory Lesions of the Central Nervous System, Vascular Abnormalities, and Head Trauma

These are unusual causes of hypogonadotropic hypogonadism. Rarely, tuberculous or sarcoid granulomas of the CNS are associated with delayed puberty. Hydrocephalus may cause delayed puberty that can be reversed with decompression, [1078] [1079] as can pressure from a subarachnoid cyst as noted earlier.

[ 1077]

Radiation of the Head

Radiation of the head for treatment of CNS tumors, leukemia, or neoplasms of the head and the face may result in gradual onset of hypothalamic-pituitary failure. [1080] Although GH deficiency is the most common hormone disorder resulting from radiation, gonadotropin deficiency also occurs. [898] [1081] Decreased growth caused by GH deficiency with early onset of puberty can lead to a decrease in the final height of children with acute lymphocytic leukemia treated with CNS radiation. [1082] The advance in the age of onset of puberty is positively correlated with the age of diagnosis of the condition for which the radiation was given and positively correlated with BMI at diagnosis.[1083] Newer radiation treatment regimens using 18 Gy instead of 24 Gy may have less influence on advancing the age of menarche and may lead to less long-term morbidity. [1084] [1085] One study found that girls receiving 25 Gy of CNS irradiation after 7 years of age were more likely to have delayed puberty whereas in those treated earlier the onset of puberty was not affected, although they ultimately had diminished height [1086] ; the later part of this study contrasts with the more frequently reported advance of puberty with radiation therapy as noted earlier. Developmental Defects

Midline malformations of the head and the CNS are associated with a variety of endocrine deficiencies. Septo-optic or optic dysplasia is caused by abnormal development of the prosencephalon. The optic nerve is usually affected, leading to small, dysplastic, pale optic discs and pendular (evenly moving side to side) nystagmus; severely affected patients may be blind. The midline hypothalamic defect may lead to GH deficiency and diabetes insipidus and may be associated with deficient ACTH, TSH, and gonadotropin secretion; short stature and delayed puberty result, although true precocious puberty is an alternative outcome (see later). [1087] The septum pellucidum is often absent in association with optic hypoplasia or dysplasia, which is readily demonstrable by imaging techniques. [118] [1088] The pituitary gland may be hypoplastic, presumably because of the lack of hypothalamic stimulatory factors, and in some patients the neurohypophysis may have an ectopic location. [1089] In our series, the syndrome is associated with decreased maternal age. A mutation in the HESX1 gene is a rare cause of septo-optic dysplasia. [1090] [1091] Other developmental defects of the anterior pituitary gland associated with hypogonadotropic hypogonadism and other pituitary hormone deficiencies are caused by autosomal recessive mutations in homeobox genes encoding transcription factors involved in the early aspects of pituitary development. These include, in addition to HESX1, mutations in LHX3[1092] and PROP1.[1093] [1094] PROP1 mutations, which cause GH and TSH deficiency, can be associated in affected males and females with delayed puberty or late onset of secondary hypogonadism in adulthood. [1094] In one study of 73 patients with "idiopathic" multiple pituitary hormone deficiencies, 35 had a mutation in PROP1.[1095] Less commonly ACTH deficiency is a feature of PROP1 mutations.[1096] Other congenital midline defects ranging from complete dysraphism and holoprosencephaly to cleft palate or lip are also associated with hypothalamic-pituitary dysfunction. [1078] Homozygous mutations in the LHX3 gene, which encodes a member of the LIM class of homeodomain proteins, are associated with multiple pituitary hormone deficiencies including LH and FSH and severe

restriction of head rotation. [1092] Twenty cases of duplication of the hypophysis were reported with delayed puberty present in at least one. [1097] Individuals with myelomeningocele (myelodysplasia) have an increased frequency of endocrine abnormalities, including hypothalamic hypothyroidism, hyperprolactinemia, and elevated gonadotropin concentrations, and some patients demonstrate true precocious puberty. [1098] [1099]

1177

Isolated Gonadotropin Deficiency

Isolated hypogonadal hypogonadism is characterized by selective deficiency of gonadotropins owing to a defect at the level of the hypothalamus involving the LHRH pulse generator or the gonadotrophs, or both, without an anatomic lesion ( Table 24-23 and Table 24-24 ). [950] [954] [1029] [1059] [1100] [1101] [1102] [1103] As a consequence, signs of puberty fail to occur by age 14 years in boys and age 13 years in girls or the pubertal maturation is incomplete or transient. In boys, micropenis or undescended testes or both signs are evidence of a fetal testosterone deficiency. The heterogeneous disorders that lead to isolated hypogonadotropic hypogonadism are typically associated with a prepubertal concentration of gonadal sex steroid values (testosterone in boys; estradiol in girls) and low or normal gonadotropin levels. In the severe form, the concentration of gonadal sex steroids and gonadotropins is low, pulsatile secretion of LH is absent or virtually so, and the LH response to the administration of LHRH is deficient. The testes, if palpable, are small and the concentration of serum inhibin B and estimate of seminiferous tubule function are low.[1104] [1105] Isolated gonadotropin deficiency may occur in families (about 20% to 30% of patients) or sporadically. The pattern of inheritance in affected families is that of an autosomal dominant, autosomal recessive, or X-linked recessive trait. [1106] [1107] [1108] In contrast to patients with CNS tumors, who usually have associated GH deficiency and growth failure, and to patients with constitutional delay in growth and adolescence, who are short for chronologic age, patients with isolated gonadotropin deficiency are usually of appropriate height for their age (Fig. 24-48) . Because their concentrations of gonadal steroids are too low for the epiphyses to fuse at the normal age, these patients develop increased arm span for height and decreased upper/lower ratios (eunuchoid body proportions) and, if untreated, usually become tall adults. [1109] An autosomal recessive form has been described in the mouse ( hyg/hyg) in which there is a deletion of a part of the LHRH gene. The mutant RNA is incapable of generating functional LHRH. [1110] Kallmann's Syndrome

This genetically heterogeneous syndrome (Table 24-25) is the most common form of isolated hypogonadotropic hypogonadism with delayed puberty in which anosmia or hyposmia resulting from agenesis or hypoplasia of the olfactory lobes or sulci, or both, is associated with LHRH deficiency. [1111] [1112] The prevalence in boys is about four times that in girls (Fig. 24-49) . Although the extent of the defect in olfaction usually seems to correlate with the degree of LHRH deficiency, even in patients with complete anosmia the LHRH deficiency may be partial (the fertile eunuch syndrome). [1105] [1113] Rarely, affected men who had a severe delay in puberty may recover spontaneously, experience an increase in testicular size, and enter full puberty. [1114] [1115] The magnitude of the LHRH deficiency correlates with the size of the testes[1104] (Fig. 24-50) . Affected individuals often do not notice impaired olfaction; testing with graded dilutions of pure scents is useful to discriminate the magnitude of the deficit in olfaction. [1116] Undescended testes and gynecomastia are common in this and all types of hypogonadotropic hypogonadism in boys. [1104] About one half of males with Kallmann's syndrome are born with a micropenis. [1117] Associated defects inconsistently present are cleft lip, cleft palate, imperfect facial fusion, seizure disorders, short metacarpals, pes cavus, neurosensory hearing loss, cerebellar ataxia and nystagmus, ocular motor abnormalities, [1118] and, limited to the X-linked form, unilateral or rarely bilateral renal aplasia or dysplasia, [1119] and mirror movements of the upper extremities (synkinesia)[1111] [1120] (see Table 24-24) . All of these structures and organs are sites of expression of the KAL gene in the human fetus[1121] (see Table 24-24) . Coronal and axial cranial MRI scans of the olfactory bulbs and sulci are useful as an ancillary approach to diagnosis, [1122] [1123] especially in affected infants and children of prepubertal age. [1123] In a review of MRI findings in 64 individuals with Kallmann's syndrome, 56% had bilateral agenesis of the olfactory bulbs (in 2% the agenesis was unilateral) and 56% had absent or abnormal olfactory sulci bilaterally (in 17% the abnormality was unilateral). [1124] Altogether, in Kallmann's syndrome less than 10% have normal cranial MRI findings. Serum LH and FSH are indistinguishable from those in prepubertal children except for lack of or diminished nocturnal pulses of gonadotropin in patients with Kallmann's syndrome. [1125] This syndrome is genetically heterogeneous and can be transmitted as an X-linked, autosomal dominant, or autosomal recessive trait. Only 14% of familial cases of Kallmann's syndrome and 11% of sporadic cases have mutations in the KAL gene on the X chromosome, but these cases are more likely to have complete absence of gonadotropin secretion pulses and have absence of migration of GnRH neurons to the hypothalamus. [1126] The autosomal pattern is more frequent in families in which there are both anosmic and hyposmic individuals as well as patients with normosmic findings. Reports of affected males who were infertile suggested an X-linked mode of inheritance, [1111] and X linkage has been substantiated by gene mapping techniques in some patients. The Xp22.3 locus is the site of the KAL1 gene, an X-linked gene that escapes X inactivation and maps 1.5 megabases proximal to the steroid sulfatase gene at the same locus. The KAL1 gene encodes a 680-amino-acid glycoprotein, named anosmin-1, with characteristics of an extracellular neural adhesion molecule that could putatively function as a pathfinder in the guidance of LHRH neurons to the medial basal hypothalamus (see earlier discussion). The developmental distribution of anosmin-1 by immunostaining and by in situ hybridization with KAL1 mRNA in the human embryo and fetus provides evidence of its widespread distribution including the olfactory placode and forebrain by week 5 to 6. Anosmin-1 is an extracellular matrix component, with the implication that its action is mainly local. The human embryonic and fetal tissues in which it has been detected include the mesonephros and metanephros, precartilaginous skeleton, inner ear, and cerebellum. [1127] Only the molecular genetics of the X-linked form is well established. [1128] A variety of deletions and mutations of the KAL gene have been described, including large and small (exon) deletions, [924] [1129] [1130] point mutations, and a variety of nonsense mutations leading to frameshift and premature stop codons. [924] [1124] A small proportion of familial cases in which X-linked inheritance is well documented apparently do not have a mutation in the coding region of the KAL gene; the defect in some of these patients may be located in the promoter region of the KAL gene.[1131] Contiguous gene TABLE 24-23 -- Isolated Gonadotropin Deficiency Males more commonly affected Familial or sporadic Height normal for age; tall adult height if untreated Eunuchoid skeletal proportions Delayed bone age Small, often cryptorchid testes: Diameter 2.5 cm in diameter Serum testosterone concentration > 50 ng/dL Pubertal LH response to LHRH bolus Pubertal pattern of LH pulsatility LH, luteinizing hormone; LHRH, LH-releasing hormone. only when the fat is retracted can the full extent of phallic development be assessed. (This feature is among the most common causes of inappropriate referral for hypogonadism.) The extent of pubic and axillary hair is noted, as is the degree of acne. The possibility of cryptorchidism or retractile testes should be differentiated if no testes are palpated in the scrotum. Neurologic examination, including examination of the optic discs and visual fields by frontal confrontation perimetry and evaluation of olfaction, may reveal findings suggesting the presence of a CNS neoplasm or a developmental defect (Kallmann's syndrome). The stigmata of gonadal dysgenesis (Turner's syndrome) or the small testes and gynecomastia of Klinefelter's syndrome may suggest one of these diagnoses. Complete physical examination including the lungs, heart, kidney, and the gastrointestinal tract is also important in the search for a chronic disorder that may delay puberty. Laboratory studies (Table 24-28) include determination of plasma LH and FSH concentrations, measurement of the rise in LH level after LHRH administration, determination of testosterone concentrations in boys and estradiol levels in girls, and measurements of T 4 and prolactin concentrations in boys and girls if the clinical features warrant. It is important to use one of the few national endocrine laboratories for the determinations of the hormones of puberty because most local laboratories are interested only in differentiating normal, higher, adult values from inappropriately low levels and not the low concentrations characteristic of the early stage of pubertal development. For example, levels of estradiol below 15 pg/mL are not measured routinely or with confidence in many clinical laboratories despite the availability of methods and commercial kits to measure accurately values as low as 1.5 pg/mL.

1199

TABLE 24-28 -- Endocrine and Imaging Studies in Delayed Adolescence Initial assessment Plasma testosterone or estradiol Plasma FSH and LH Plasma thyroxine (and prolactin) Bone age and lateral skull roentgenograph Test of olfaction Follow-up studies Karyotype (short, phenotypic females) MRI with contrast enhancement Pelvic ultrasonography (females) LHRH test hCG test (males) Pattern of pulsatile LH secretion Visual acuity and visual fields

FSH, follicle-stimulating hormone; hCG, human chorionic gonadotropin; LH, luteinizing hormone; MRI, magnetic resonance imaging.

Radiographic examination includes bone age determination and, if the diagnosis is at all consistent with a CNS lesion, an MRI of the brain with specific attention to the pituitary and hypothalamic area using contrast; only advanced pituitary tumors or significantly calcified craniopharyngiomas appear on lateral skull films, and, although a positive result is useful, a negative radiograph cannot rule out a CNS defect. [1565] In contrast to MRI scans, CT scanning can detect calcification. Ultrasound evaluation of the uterus and ovaries is not usually indicated initially in work-up of delayed puberty but provides useful information about the state of development of these structures.[127] Again, it is important that the ultrasonographer has experience with children and young adolescents. Regrettably, individuals with normal internal genital organs have been told that they lack a uterus or ovaries, or both, by ultrasonographers inexperienced with this age group. One study demonstrated streak gonads in 50% of a group of 70 patients with Turner's syndrome. [1566] Assessment of chromosomal karyotype should be considered in all short girls, even in the absence of somatic signs of Turner's syndrome and especially if puberty is delayed, and in boys with suspected Klinefelter stigmata or behavior. A presumptive diagnosis of constitutional delay in growth and adolescence is made if the history and growth chart reveal a history of short stature but consistent growth rate for skeletal age (and no signs or symptoms of hypothalamic lesions), if the family history includes parents or siblings with delayed puberty, if the physical examination (including assessment of the olfactory threshold) is normal, if optic discs and visual fields are normal, and if the bone age is significantly delayed. In classical cases, an MRI scan of the hypothalamic-pituitary region may not be necessary. The rate of growth in these patients is usually appropriate for bone age; a decrease in growth velocity occurs in some normal children just before the appearance of secondary sexual characteristics and may awaken concerns if such a pattern occurs in these subjects. Further, in these individuals the onset at puberty correlates better with bone age than with chronologic age. Elevated concentrations of gonadotropins and gonadal steroids to early pubertal levels precede secondary sexual development by several months; thus, measurements of serum LH, FSH, estradiol, or testosterone levels may help in predicting future development. The third-generation LH assays are reported to be sufficiently sensitive to allow the determination of the onset of endocrine puberty with a single blood sample in most boys, but an LHRH test is still often performed. An increase in the concentration of LH of more than 7.5 IU/L (2 ng/mL LER-960) determined by conventional polyclonal radioimmunoassay after intravenous administration of 100 µg of LHRH usually precedes the first physical sign of sexual maturation by less than 1 year. Clomiphene citrate, an antiestrogen with weak estrogenic effects, decreases secretion of gonadotropins in prepubertal patients but increases gonadotropin secretion in pubertal patients and in adults. However, we have not found administration of clomiphene citrate to be useful in the diagnosis of constitutional delay of growth and adolescence. Various tests have been proposed for differentiating hypogonadotropic hypogonadism from constitutional delay in puberty. Trials assessing the prolactin response to TRH,[1567] [1568] chlorpromazine,[1569] metoclopramide,[1570] or domperidone [1571] for differential diagnosis either failed or gave inconsistent results. [1570] [1571] The combination of the prolactin response to metoclopramide and the gonadotropin response to LHRH has been suggested, [1572] as has the use of priming doses of LHRH with evaluation of the gonadotropin response to a subsequent dose of LHRH [1573] [1574] [1575] or to a superactive LHRH agonist. [1576] [1577] The FSH response is higher in patients with hypothalamic-pituitary deficiencies who undergo pubertal development. [1578] A sensitive immunofluorometric assay for LH may help to distinguish between constitutional delay of growth and adolescence and hypogonadotropic hypogonadism better than the polyclonal LH radioimmunoassay. [1579] Urinary gonadotropin excretion is lower in hypogonadotropic patients than in delayed puberty, but this method of differential diagnosis may require years of observation before the difference is apparent. [1255] Although some methods are promising, their efficacy remains to be confirmed. There is a tendency for hypogonadotropic patients to undergo adrenarche at a normal age and to have a higher DHEAS concentration than those with constitutional delay in growth, and this pattern is helpful in the differential diagnosis. [969] [1580] [1581] Measurement of 8 AM serum testosterone is proposed to be an accurate indication of impending pubertal development; a value greater than 0.7 nmol/L (20 ng/dL) predicts enlargement of testes to greater than 4 mL by 12 months in 77% of cases and by 15 months in 100% of cases, whereas of those with a value less than 0.7 nmol/L only 12% entered puberty in 12 months and only 25% entered puberty in 15 months. This technique may help predict spontaneous pubertal development but still requires considerable watching and waiting. [1582] At present, there does not appear to be a practical and reliable endocrine test for indisputably differentiating between constitutional delay in growth and adolescence and hypogonadotropic hypogonadism. Watchful waiting remains the procedure of choice. A typical patient with isolated gonadotropin deficiency is of average height for age and has eunuchoid proportions; low plasma concentrations of gonadal steroids, LH, and FSH; and no increase or a blunted response of LH after LHRH administration. The amplitude and usually the frequency of LH pulses are decreased when serial blood samples are studied over a 24-hour period. In some but not all forms of Kallmann's syndrome, the sense of smell is absent or impaired. However, differentiation of isolated gonadotropin deficiency in the absence of hyposmia or anosmia from constitutional delay in puberty may be difficult at initial study. Gonadotropin-deficient patients may be as short as those with constitutional delay in growth and adolescence, and concentrations of LH and FSH in hypogonadotropic hypogonadism may be indistinguishable from those of normal prepubertal children or children with constitutional delay. Sometimes years of observation are necessary to detect the appearance of spontaneous and progressive signs of secondary sexual development or to document rising concentrations of gonadotropins or gonadal steroids before the diagnosis is clear. In general, but not in all

1200

cases, absence of the first signs of sexual maturation or failure of a rise in gonadotropins or gonadal steroid levels by age 18 in the presence of a normal concentration of serum DHEAS for chronologic age supports the diagnosis of isolated gonadotropin deficiency. Patients with deficiency of gonadotropins combined with deficiency of other pituitary hormones require careful evaluation for a CNS neoplasm. Visual field or optic disc abnormalities support the diagnosis of CNS tumor; even if these tests are normal, cranial MRI should be performed to evaluate the pituitary gland and stalk and the hypothalamic region. CT scans but especially MRI scans of the head are valuable in detecting mass lesions and developmental abnormalities of the hypothalamic-pituitary region. [1565] [1583] Treatment of Delayed Puberty and Sexual Infantilism

Treatment of delayed puberty (Table 24-29) depends on the diagnosis and the nature of the disorder. Patients with constitutional delay in growth and adolescence ultimately have spontaneous onset and progression through puberty. Often, reassurance and continued observation to ensure that the expected sexual maturation occurs are sufficient. However, the stigma of appearing less mature than one's peers can cause psychological stress; such individuals may be unable to participate in the dating activities their friends are starting, smaller size may lead them to avoid participation in athletics, immature appearance may lead to ridicule especially in the locker room, and school work may suffer because of their poor self-image. [1584] [1585] Some children feel such intense peer pressure and low self-esteem that only the appearance of signs of puberty reassures them and enables them to participate in sports and social activities with their peers. Poor self-image in late-maturing boys may carry into adulthood even after normal puberty ensues. [1584] [1585] The growth retardation is often responsible for most of the stress rather than the delay in pubertal development itself. [1586] For psychological reasons, in boys of age 14 or older who show no signs of puberty, a 4- to 6-month course of testosterone enanthate, cypionate, or cyclopropionate (50 to 100 mg intramuscularly every 4 weeks) may be helpful. [1587] [1588] [1589] The low dose of testosterone enanthate is generally considered to be safe but can raise LDL and lower HDL cholesterol values as an expected effect. Oral treatment with 2.5 mg of fluoxymesterone (Halotestin) for 6 to 60 months allows increased pubertal development without adverse effects on final height, although the necessity to take a daily dose may decrease compliance. [437] Low-dose oxandrolone (2.5 mg/day orally) [1590] is sometimes used as an oral alternative to intramuscular testosterone enanthate; this agent increases growth through androgenic effects reflected by suppression of LH and FSH but does not stimulate GH secretion as it is not aromatized to estrogen. [1591] The temporary increase in growth velocity found with oxandrolone does not affect final height. [1592] [1593] [1594] Short-term treatment with fluoxy-mesterone (2.5 mg/day orally) was also reported to be a safe treatment that does not compromise adult height. [437] Testosterone undecanoate at 40 mg/day is likewise an effective but expensive treatment for those choosing oral therapy. [1595] [1596] [1597] Transdermal testosterone may be applied as a daily patch or a gel, although experience with these forms of androgen is more limited than with the other forms. Preliminary experience suggests that overnight (approximately 8 to 9

hours) or every other night use of 2.5 mg is effective. Testosterone gel is being investigated as a daily topical preparation to advance pubertal development. For girls of age 13 or older, a 3- to 4-month course of ethinylestradiol (5 µg/day orally) or conjugated estrogens (0.3 mg/day orally) may be used to initiate maturation of the secondary sexual characteristics without unduly advancing bone age or limiting final height. [1598] [1599] A fourth-generation aromatase inhibitor, letrozole, administered along with testosterone in a randomized controlled trial in boys with constitutional delay in puberty and growth decreased the advancement in bone age, an effect that will presumably lead to a greater adult height [1600] by strikingly decreasing the synthesis of estradiol from testosterone. Estradiol [172] is the sex steroid that has the major effect on skeletal maturation. Letrozole does not block the virilizing effects of testosterone. This promising treatment is experimental; it may improve the decreased adult height in some boys with constitutional delay compared with their predicted genetic potential, [1011] [1012] [1014] [ 1015] [1601] a decrease that testosterone cannot overcome. If, during the 3 to 6 months after discontinuing gonadal steroid therapy, spontaneous puberty does not ensue or the concentrations of plasma gonadotropins and plasma testosterone in boys or plasma estradiol in girls do not increase toward pubertal values, the treatment may be repeated. Usually, only one or two courses of therapy are necessary. When treatment is discontinued after bone age has advanced, for example, to 12 to 13 years in girls or 13 or 14 years in boys, patients with constitutional delay usually continue pubertal development on their own, whereas those with gonadotropin deficiency do not progress and may, in fact, regress. Alleviating the underlying problem treats functional hypogonadotropic hypogonadism associated with chronic disease. Delayed puberty in this situation is usually a result of inadequate nutrition and low weight; when weight returns to normal values, puberty usually occurs spontaneously. Treatment with T 4 allows normal pubertal development in hypothyroid patients with delayed puberty. Congenital or acquired gonadotropin deficiency as a result of a lesion or surgery requires replacement therapy with gonadal steroids at an age approximating the normal age of onset of puberty ( Table 24-30 and Table 24-31 ). An exception may occur when GH deficiency coexists with gonadotropin deficiency; if bone age advancement and epiphyseal fusion are brought about by testosterone or estradiol replacement before therapy with GH causes adequate linear growth, adult height is compromised. However, if puberty is not initiated early enough, the patient may well suffer psychological damage. It is generally advisable to initiate puberty in such patients with low-dose TABLE 24-29 -- Management and Treatment of Delayed Puberty Objectives Determine site and etiology of abnormality Induce and maintain secondary sexual characteristics Induce pubertal growth spurt Prevent the potential short-term and long-term psychological, personality, and social handicaps of delayed puberty Ensure normal libido and potency Attain fertility Therapy Concerned but not anxious or socially handicapped adolescent: Reassurance and follow-up (tincture of time) Repeat evaluation (including serum testosterone or estradiol) in 6 mo Psychosocial handicaps, anxiety, highly concerned: Therapy for 4 mo with Boys: testosterone enanthate 100 mg intramuscularly every 4 wk at 1414.5 yr of age, or overnight transdermal testosterone patch Girls: ethinyl estradiol 510 µg daily by mouth or conjugated estrogens 0.3 mg daily by mouth or overnight ethinyl estradiol patch at 13 yr of age No therapy for 46 mo; reevaluate status including serum testosterone or estradiol; if indicated repeat treatment regimen

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TABLE 24-30 -- Hormonal Substitution Therapy in Boys with Hypogonadism Goal: to approximate normal adolescent development when diagnosis is established Initial therapy: at 13 yr of age, testosterone enanthate (or other long-acting testosterone ester) 50 mg intramuscularly every month for about 9 mo (612 mo) Over the next 3 to 4 yr: gradually increase dose to adult replacement dose of 200 mg every 23 wk Begin replacement therapy in boys with suspected hypogonadotropic hypogonadism by bone age 14 yr To induce fertility at appropriate time: pulsatile LHRH or FSH and hCG therapy gonadal steroids by age 14 in boys and age 13 in girls regardless of the definitive diagnosis of gonadotropin deficiency; thus, these children with GH deficiency would be treated similarly to those with isolated delayed puberty. Patients with isolated GH deficiency may have a delayed onset of puberty; with GH administration, puberty usually occurs at an appropriate age but may progress faster than in normal individuals. [1602] [1603] A study of over 200 children with GH deficiency treated with hGH showed a correlation between the age of onset of induced puberty and final height in patients who were also gonadotropin-deficient, whereas those who underwent spontaneous puberty, which occurred earlier than the age of hormone-induced puberty in the gonadotropin-deficient children, had a lower final height; this supports the advisability of waiting to initiate puberty in GH- and gonadotropin-deficient subjects. [1604] Height at the onset of puberty is also correlated with final height in GH-deficient children. [1605] Clinical trials are in progress to determine the effects of artificially delaying puberty with an LHRH analogue to attempt to achieve a greater final height in patients with isolated GH deficiency treated with hGH[1606] (see earlier). Micropenis resulting from fetal androgen deficiency caused by a primary testicular defect or gonadotropin deficiency [1607] can be successfully treated with small doses of testosterone enanthate (25 to 50 mg/month intramuscularly) administered for short periods during infancy [1146] [1607] (also see Chapter 22) . Patients with isolated congenital GH deficiency occasionally have micropenis that may be successfully treated with GH replacement alone. [1608] As discussed earlier, episodic administration of LHRH can elicit pulsatile LH and FSH release and gonadal stimulation in prepubertal children or hypogonadotropic patients. [946] [949] [950] Portable pumps have been used to administer LHRH in episodic fashion over prolonged periods. Pulsatile LHRH therapy can induce puberty and promote the development of secondary sexual characteristics and spermatogenesis in men [1609] [1610] [1611] [1612] [1613] and ovulation in women [1614] [1615] ; pregnancy has been achieved with this regimen in women with hypogonadotropic hypogonadism. A lower frequency of LHRH administration favors FSH secretion and a higher frequency favors LH secretion and, ultimately, has been associated with a PCOS-like picture. [1616] A comparison of two different frequencies of LHRH administration did not reveal a difference between an LH pulse given subcutaneously every 3 hours or every 45 minutes in the rapidity of onset of pubertal development or serum LH, FSH, or sex steroid concentrations; this indicates that the hypothalamic-pituitary-gonadal axis is sufficiently robust to accommodate various frequencies of LHRH secretion. [1617] The use of pulsatile LHRH administration is not practical for the routine induction of puberty in adolescent boys and girls with gonadotropin deficiency. Both hCG and human menopausal gonadotropin can be used as effective substitutes for recombinant human pituitary LH and FSH to produce full gonadal TABLE 24-31 -- Hormonal Substitution Therapy in Girls with Hypogonadism

When diagnosis of hypogonadism is firmly established (e.g., girls with 45,X gonadal dysgenesis), begin hormonal substitution therapy at 1213 yr of age Goal: to approximate normal adolescent development Initial therapy: ethinyl estradiol 5 µg by mouth or conjugated estrogen 0.3 mg (or less) by mouth daily for 46 mo After 6 mo of therapy (or sooner if "breakthrough" bleeding occurs) begin cyclic therapy: Estrogen: first 21 days of month Progestagen: (e.g., medroxyprogesterone acetate 5 mg by mouth) 12th to 21st day of month Gradually increase dose of estrogen over next 23 yr to conjugated estrogen 0.61.25 mg or ethinyl estradiol 1020 µg daily for first 21 days of month In hypogonadotropic hypogonadism: to induce ovulation at appropriate time: pulsatile LHRH or FSH and hCG therapy maturation, especially in those with pituitary pathology. But, again, this regimen is cumbersome and expensive. therapy is the treatment of choice for hypothalamic or pituitary gonadotropin deficiency until fertility is achieved.

[1618]

Thus, long-term gonadal steroid replacement

[1619]

Hypergonadotropic hypogonadism is treated by replacement of testosterone in boys and estradiol in girls. For treatment of gonadal dysgenesis, estrogen therapy should be initiated when the patient is age 13 (bone age > 11 years) to allow secondary sexual development at an appropriate chronologic age. The Klinefelter syndrome is compatible with varying degrees of masculinization at puberty, but some patients require testosterone replacement. The concentrations of plasma testosterone and LH should be monitored every 6 months during puberty and yearly thereafter. If the LH level rises more than 2.5 SD above the mean value or the testosterone level decreases below the normal range for age, testosterone replacement therapy is indicated. Patients receiving gonadal steroid replacement follow the same treatment regimen whether the diagnosis is hypogonadotropic hypogonadism or hypergonadotropic hypogonadism ( see Table 24-30 and Table 24-31 ). Various testosterone preparations with several routes of administration are available. [1620] Alkylated testosterone preparations are to be avoided because of the risk of peliosis hepatis (hemorrhagic liver cysts), which is not related to dose or duration of treatment; although regression is possible with discontinuation of testosterone treatment, progression to liver failure can occur. [1621] [1622] Males may receive testosterone enanthate, propionate, or cypionate, 50 to 100 mg every 4 weeks intramuscularly at the start; later the dosage is gradually increased to 200 to 300 mg every 2 to 3 weeks. Low-dose replacement therapy is appropriate until well into the pubertal growth spurt. [168] Testosterone may be administered by cutaneous patch on scrotal skin or nonsexual skin to cause secondary sexual development in hypogonadal adolescents; patches may be given at night to recreate the diurnal variation of testosterone seen in early puberty. Physiologic values of serum testosterone are possible along with secondary sexual development.[1623] [1624] A teenage boy may be less likely to apply a patch daily, and biweekly or monthly injections may allow better compliance; nonetheless, we and others find that 2.5- and 5-mg dermal testosterone patches may be useful in motivated teenagers. New testosterone gel preparations, usually rubbed onto the forearms, are approved for adults and may be used in a similar manner. Testosterone ointment may be used as therapy for microphallus to enlarge the size of the phallus intentionally, [1625] but an infant coming in contact with the skin of an individual

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with testosterone gel (before it is absorbed into the intended subject's skin) runs the risk of unplanned testosterone effects. Initially, girls 12 to 13 years of age are given ethinylestradiol, 5 µg/day orally, or conjugated estrogens, 0.3 mg/day by mouth, on the first 21 days of the month. The dose is gradually increased over the next 2 to 3 years to 10 µg of ethinylestradiol or 0.6 to 1.25 mg of conjugated estrogen for the first 21 days of the month. The maintenance dose should be the minimal amount to maintain secondary sexual characteristics, sustain withdrawal bleeding, and prevent osteoporosis. After breakthrough bleeding occurs, or no later than 6 months after the start of cyclic therapy, a progestagen (e.g., medroxyprogesterone acetate, 5 mg/day) is added on days 12 through 21 of the month. Undesirable effects are uncommon but may include weight gain, headache, nausea, peripheral edema, and mild hypertension. Application of portions of transdermal 17-estradiol patches at night was shown to mimic levels of estrogen produced in early puberty and to bring about breast development slowly[1626] ; other therapeutic schedules are possible. [1627] [1628] [1629] [1630] As with testosterone, there must be care that the preparation is not placed in contact with young children or untoward estrogen effects may occur. There is concern about the increased risk of endometrial and breast carcinoma in patients receiving chronic estrogen replacement therapy including patients with Turner's syndrome. This is not an issue in adolescents or young adults but is a consideration in older women. The use of progestational agents to antagonize the effect of estrogens reduces the risk of endometrial cancer, but knowledge of the optimal dose of estrogen and progesterone to enhance development without unduly increasing the risk of cancer must come from future studies. Estrogen replacement is important for its antiosteoporotic action on bone. Surprisingly, we lack controlled studies on optional sex steroid replacement regimens in adolescent women. Patients with hypopituitarism may complain of sparse pubic hair growth or, in girls, total absence of public hair. Pubic hair thickens further in affected males with hCG treatment that adds the testicular contribution of testosterone to the exogenous testosterone therapy. GH therapy in males with GH and gonadotropin deficiency enhances the steroidogenic response of the testes to hCG administration. [1631] Further, adolescent or young adult women have been given low doses (25 mg) of long-acting intramuscular testosterone every 4 weeks to stimulate the growth of pubic hair without virilization. [1632] The result of treatment with testosterone in boys with radiation-induced primary testicular failure is normal final height, although in a group of patients with concomitant spinal radiation, the upper/lower segment ratio was much reduced, indicating impaired spinal growth. [1633] The results of clinical trials of biosynthetic hGH therapy in Turner's syndrome indicate that an increase in growth rate with a substantial increase in final height into the lower range of the normal growth curves is possible, especially with a dose higher than used in GH deficiency (see more detailed earlier discussion in the section on Turner's syndrome). [1462] [1463] [1464] [1465] [1634] [1635] There is some degree of improvement of the abnormal body proportions of Turner's syndrome with hGH treatment, but the disproportionate growth of the foot may dissuade some girls from continuing treatment to maximal benefit for height. [1469] The addition of estrogen therapy at low doses has been reported either to exert no effect on adult height or to reduce the adult height obtained with GH therapy administered alone.[1634] [1635] [1636] [1637] [1638] [1639] [1640] [1641] [1642] Indeed, the length of time of exposure to GH before estrogen treatment is said to be the major determinant of whether GH and estrogen treatment increased final height. [1643] However, it is postulated that if GH is started early enough (e.g., 2 to 8 years of age), estrogen therapy may be added at an age (13 years) appropriate for the institution of puberty (see discussion in Turner's syndrome section). [1644] Counseling and a peer support group are exceedingly important components of the long-term management. [1603] The bone density is decreased in Turner's syndrome, at least in part, because of hypogonadism at puberty, and this tendency becomes more severe with age in patients who discontinue or do not receive estrogen replacement therapy. [1645] [1646] Transdermal estrogen was shown to increase bone density in subjects with Turner's syndrome who have finished statural growth. [1647]



DISORDERS OF PUBERTY (Continued) Sexual Precocity

Sexual precocity (Table 24-32) is defined as the appearance of any sign of secondary sexual maturation at an age more than 2.0 SD below the mean; in the past, the ages of 8 years in girls and 9 years in boys were considered the lower limits of the normal onset of puberty. [154] [1648] Present data detailed previously indicate that the limits in normal boys remain at 9 years but that the lower limit for white girls is 7 years and for black girls is 6 years, assuming that there is no sign or symptom of CNS disorders or other serious or chronic disease that might cause sexual precocious puberty. These guidelines are similar to those proposed by the Drug and Therapeutics and Executive Committees of the Lawson Wilkins Pediatric Endocrine Society. [100] The new data noted in the first section of this chapter show that breast development and pubic hair development may occur in girls as young as 6 years in substantial numbers, especially in black girls, leading to a need for careful evaluation and conservatism, even in these young years, in evaluating and treating girls with only minimal, relatively non-progressive signs of sexual precocity. If the sexual precocity results from premature reactivation of the hypothalamic LHRH pulse generatorpituitary gonadotropingonadal axis, the condition is called complete isosexual precocity or true or central precocious puberty and is LHRH-dependent. Pulsatile LH release has a pubertal pattern in this form, and the rise in the concentration of LH after LHRH administration is indistinguishable from the normal pubertal pattern of serum LH. If extrapituitary secretion of gonadotropins or secretion of gonadal steroids independent of pulsatile LHRH stimulation leads to virilization in boys or feminization in girls, the condition is termed incomplete isosexual precocity, pseudoprecocious puberty, or LHRH-independent sexual precocity. The production of excessive estrogens in males leads to inappropriate feminization, and the production of increased androgen levels in females leads to inappropriate virilization; these conditions are termed contrasexual precocity (also termed heterosexual precocity). Hence, the disorders that cause sexual precocity can be separated into those in which the increased secretion of gonadal steroids depends on LHRH stimulation of pituitary gonadotropins and those in which it is unrelated to activation of the hypothalamic LHRH pulse generator. In all forms of sexual precocity, the increased gonadal steroid secretion increases height velocity, somatic development, and the rate of skeletal maturation and, because of premature epiphyseal fusion, can lead to the paradox of tall stature in childhood but short adult height. Data on the final height in true precocious puberty are scarce (see Table 24-32) , but several studies of untreated females with idiopathic central precocious puberty demonstrated a mean final height of 151 to 155 cm.[871] [1649] [1650] [1651] [1652] [1653] [1654] [1655] [1656] There are few reports of final height in boys with untreated precocious puberty [1649] (Table 24-33) . In the boys followed to adult stature by Thamdrup,[1650] the mean height was 155.4 cm ± 8.3 (SD) and all were well below midparental height and far below the fathers' height. Blood pressure matches that of height- and weight-related

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normal subjects rather than age-matched normal persons; thus, elevated blood pressure for age in patients with sexual precocity may not indicate hypertension (blood pressure in normal children is best related to height rather than to age, and this is an extension of the concept). Serum alkaline phosphatase and IGF-I concentrations reflect the degree of sexual development rather than chronologic age. [391] True or Central Precocious Puberty: Complete Isosexual Precocity (LHRH-Dependent Sexual Precocity)

In our series of over 200 patients with true precocious puberty, [871] girls had true precocious puberty five times more commonly than boys and the idiopathic form was eight times more common in girls than in boys (Table 24-34) . Neurologic abnormalities occurred at least as often as idiopathic true precocious puberty in boys, whereas in girls neurologic lesions were a fifth as common as idiopathic disorders. Thus, it is essential to search for a neurologic etiology for true precocious puberty, especially in boys. In some cases, sexual precocity may be the only manifestation of a CNS tumor [871] [875] [1657] [1658] (Table 24-35) .

TABLE 24-32 -- Classification of Sexual Precocity True Precocious Puberty or Complete Isosexual Precocity (LHRH-Dependent Sexual Precocity or Premature Activation of the Hypothalamic LHRH Pulse Generator) Idiopathic true precocious puberty CNS tumors Optic glioma associated with neurofibromatosis type 1 Hypothalamic astrocytoma Other CNS disorders Developmental abnormalities including hypothalamic hamartoma of the tuber cinereum Encephalitis Static encephalopathy Brain abscess Sarcoid or tubercular granuloma Head trauma Hydrocephalus Arachnoid cyst Myelomeningocele Vascular lesion Cranial irradiation True precocious puberty after late treatment of congenital virilizing adrenal hyperplasia or other previous chronic exposure to sex steroids Incomplete Isosexual Precocity (Hypothalamic LHRH-Independent) Males Gonadotropin-secreting tumors hCG-secreting CNS tumors (e.g., chorioepitheliomas, germinoma, teratoma) hCG-secreting tumors located outside the CNS (hepatoma, teratoma, choriocarcinoma) Increased androgen secretion by adrenal or testis Congenital adrenal hyperplasia (CYP21 and CYP11B1 deficiencies) Virilizing adrenal neoplasm

Leydig cell adenoma Familial testotoxicosis (sex-limited autosomal dominant pituitary gonadotropin-independent precocious Leydig cell and germ cell maturation) Cortisol resistance syndrome Females Ovarian cyst Estrogen-secreting ovarian or adrenal neoplasm Peutz-Jeghers syndrome In Both Sexes McCune-Albright syndrome Hypothyroidism Iatrogenic or exogenous sexual precocity (including inadvertent exposure to estrogens in food, drugs, or cosmetics) Variations of Pubertal Development Premature thelarche Premature isolated menarche Premature adrenarche Adolescent gynecomastia in boys Macro-orchidism Contrasexual Precocity Feminization in Males Adrenal neoplasm Chorioepithelioma CYP11B1 deficiency Late-onset adrenal hyperplasia Testicular neoplasm (Peutz-Jeghers syndrome) Increased extraglandular conversion of circulating adrenal androgens to estrogen Iatrogenic (exposure to estrogens) Virilization in Females Congenital adrenal hyperplasia CYP21 deficiency CYP11B1 deficiency 3-HSD deficiency Virilizing adrenal neoplasm (Cushing's syndrome) Virilizing ovarian neoplasm (e.g., arrhenoblastoma) Iatrogenic (exposure to androgens) Cortisol resistance syndrome Aromatase deficiency LHRH, luteinizing hormone-releasing factor (GnRH); CNS, central nervous system; CYP21, 21-hydroxylase; CYP11B1, 11-hydroxylase; 3-HSD, 3-hydroxysteroid dehydrogenase 4,5-isomerase. Modified from Grumbach MM. True or central precocious puberty. In Kreiger DT, Bordin CW, (eds). Current Therapy in Endocrinology and Metabolism, 19851986. Toronto, BC Decker, 1985, pp 48.

Long-Term Follow-up of True Precocious Puberty

Pregnancy has occurred in patients with true or central precocious puberty as early as 5 years of age. [1659] Of course, such pregnancies are in fact the result of childhood sexual abuse of a child with true precocious puberty, a fact rarely reported by the sensational press. Fertility in later life is less well documented, but in our experience as well as that of others, normal pregnancies have occurred in women who had idiopathic true precocious puberty, [1659] [1660] a CNS abnormality triggering true precocious puberty, [871] [1654] or premature menarche. In the isosexual precocity of the McCune-Albright syndrome, there are also reports of adult fertility. [675] [1661] [1662]

Idiopathic True or Central Precocious Puberty

By common definition, 2.5% of normal children develop signs of puberty before the lower limits of normal. The Hermann-Giddens study found that 27% of black and 7% of white girls have some manifestation of secondary sex development by 7 years of age. Thus, as stated previously, a useful definition of sexual precocity is onset before 6 years in black girls, before 7 years in white girls, and before 9 years in boys, assuming there

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TABLE 24-33 -- Historical Controls of Untreated Children with True Precocious Puberty No. of Patients (Women/Men) Reference

Final Ht (cm) *

Women

Men

26/8

151.3 ±8.8

155.4 ± 8.3

Hayles[1654]

40/11

152.7 ± 8.0

156.0 ± 7.3

Werder et al [1655]

4/0

150.9 ± 5.0

Lee[1653]

15/0

155.3 ± 9.6

UCSF

8/4

153.8 ± 6.8

Thamdrup[1650] Sigurjonsdottir and

159.6 ± 8.7

Total

93/23

152.7 ± 8.6

155.6 ± 7.7

From Paul D, Conte FA, Grumbach MM, Kaplan SL. Long-term effect of gonadotropin-releasing hormone agonist therapy on final and near-final height in 26 children with true precocious puberty treated at a median age of less than 5 years. J Clin Endocrinol Metab 1995; 80:546551. *Mean ± 1 SD.

is no sign or symptom of CNS or other serious disease. Although this definition is a useful guideline, a significant proportion of girls 6 to 8 years of age with idiopathic precocious puberty represent one end of the bell-shaped curve for normal puberty onset, as described at the beginning of the chapter, and are examples of early normal puberty, just as those with constitutional delay in growth and adolescence are healthy but late maturers who fall in the older age segment of the normal distribution. The nature of the striking sex difference in the prevalence of idiopathic true precocious puberty (females >> males) in contrast to constitutional delay in growth and puberty (males >> females) is poorly understood. There may be a history of early maturation in the family; rarely, true precocious puberty is transmitted as an autosomal recessive trait in boys and girls. [871] [1663] A larger group of children, however, develop true precocious puberty with no familial tendency toward early maturation and no signs of organic disease; these children have idiopathic true precocious puberty. This condition, which may be manifest in infancy, is about nine times more common in girls than in boys (see Table 24-35) and is commonly associated with electroencephalographic abnormalities. [1664] The age at onset in girls in about 50% of cases is 6 to 7 years, in about 25% is 2 to 6 years, and in 18% is in infancy [871] (Fig. 24-57) . Organic forms of true precocious puberty, especially if associated with hypothalamic hamartoma, have an earlier mean age of onset than the idiopathic form. [871] [889] In boys (Fig. 24-58) the testes usually enlarge under gonadotropin stimulation before any other signs of puberty are TABLE 24-34 -- Distribution by Sex of Children with Idiopathic and Neurogenic Precocious Puberty Idiopathic Series

Neurogenic

Male Female Male Female

Thamdrup (1961)[1650]

4

34

7

11

Wilkins (1965) [675]

13

67

10

5

Sigurjonsdottir and Hayles (1968) [1654]

8

54

16

16

University of California, San Francisco (1981) *

13

121

26

45

*Unpublished.

TABLE 24-35 -- Etiology of True Precocious Puberty * Etiology Idiopathic

Number and Sex 121F, 13M

Other causes CNS-hypothalamic tumors including hamartomas

11F, 15M

Arachnoid cyst

2F, 1M

Hydrocephalus

6F, 1M

Head trauma (child abuse)

1F

Perinatal asphyxia, cerebral palsy

3F, 1M

Encephalitis or meningitis

3F, 1M

Sex chromosome abnormalities (47,XXY; 48,XXXY)

2M

Nonspecific seizure disorder or mental retardation

26F, 16M

Degenerative CNS disease

3M

Congenital virilizing adrenal hyperplasia with secondary true precocious puberty

3M

From Kaplan SL, Grumbach MM. Pathogenesis of sexual precocity. In Grumbach MM, Sizonenko PC, Aubert ML, (eds). Control of the Onset of Puberty. Baltimore, Williams & Wilkins, 1990, pp 620660. © 1990, the Williams & Wilkins Co., Baltimore. *Data from University of California, San Francisco, Pediatric Endocrine Clinic.

seen; in girls (see Fig. 24-57) an increase in the rate of growth, the appearance of breast development, enlargement of the labia minora, and maturational changes in the vaginal mucosa are the usual presenting signs, with variable manifestations of public hair depending on the age at onset. Progression of secondary sexual maturation is often more rapid than the normal pattern of pubertal maturation. A waxing and waning course of development may be encountered. [1665] The rapid growth is associated with the rise in estrogen synthesis and secretion and the increased GH secretion and elevation of serum IGF-I levels because of stimulation by estradiol. [172] [173] [ 192] [ 209] The ratio of bone age to chronologic age and the rise of IGF-I above normal values for age are predictive of outcome; more mildly affected children progress less rapidly and tend to maintain their target height. [1666] Patients with slowly progressive

Figure 24-57 Age at onset of idiopathic true precocious puberty in 106 children. Open bars, female; hatched bars, male. At all ages, the frequency is greater in females than in males. The peak prevalence in girls is between ages 6 and 8 years. (From Kaplan SL, Grumbach MM. The neuroendocrinology of human puberty: an ontogenetic perspective. In Grumbach MM, Sizonenko PC, Aubert ML [eds]. Control of the Onset of Puberty. Baltimore, Williams & Wilkins, 1990, pp 168. © 1990, the Williams & Wilkins Co., Baltimore.)

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Figure 24-58 Left, A boy 2 years, 5 months of age with idiopathic precocious puberty. He had pubic hair and phallic and testicular enlargement by 10 months of age. At 1 year of age, his height was 86 cm (+4 SD); the phallus measured 10 × 3.5 cm, and the testes measured 2.5 × 1.5 cm. Plasma luteinizing hormone (LH) was 1.9 ng/mL (LER-960); follicle-stimulating hormone (FSH) 1.2 ng/mL (LER-869); and testosterone 416 ng/dL. After 100 µg of LH-releasing hormone (LHRH), the plasma LH increased to 8.4 ng/mL and FSH to 1.8 ng/mL, a pubertal response.

When photographed, the patient had been treated with medroxyprogesterone acetate for 1.5 years. His height was 95.2 cm (+ 1 SD), the phallus was 6 × 3 cm, and the testes were 2.4 × 1.3 cm. Basal concentrations of LH (LER-960) were 0.9 ng/mL; FSH (LER-869) 0.8 ng/mL; and testosterone 7 ng/dL. After 100 µg of LHRH, LH concentrations rose to 2.3 ng/mL, whereas FSH concentrations did not change when he was on treatment with medroxyprogesterone acetate. For conversion to SI units, see the legends of Figure 24-17 and Figure 24-18 . (Left, From Styne DM, Grumbach MM. Puberty in the male and female: its physiology and disorders. In Yen SCC, Jaffe RB, [eds]. Reproductive Endocrinology, 2nd ed. Philadelphia, WB Saunders, 1986, pp 313384.) Right, A 3 3/12-year-old girl with idiopathic true precocious puberty who had recurrent vaginal bleeding since 9 months of age. Height age, 4 5/12 years; bone age, 8 10/12 years.

or unsustained puberty have little or no loss of predicted final height [1665] and are characterized by the presence of normal or only slightly elevated estrogen and IGF-I concentrations. [1666] If height prediction is normal at the time of diagnosis rather than reduced, the patient does not require therapy. [1667] [1668] [1669] Spermatogenesis in males and ovulation in females often occur, and fertility is possible. The uterus and ovaries increase in size in true precocious puberty. The ovaries may also develop a multicystic appearance that may remain even after successful treatment with an LHRH agonist. [1670] True precocious puberty in females does not lead to premature menopause. However, in girls there is an increased risk for the development of carcinoma of the breast [36] [37] [38] [39] [41] in adulthood. Psychosexual development [1671] [1672] is advanced only modestly in patients with sexual precocity (about 1½ years in girls with idiopathic true precocious puberty). [1673] The pituitary gland undergoes hypertrophy in early infancy, puberty, and pregnancy and is increased in size on MRI in patients with central precocious puberty. [175] [1674] [1675] T1 images indicate a convex upper border of the pituitary gland in patients in normal or central precocious puberty, indicating the similarity in the physiologic changes of both conditions. Two sisters have been reported with the rare finding of pituitary gland hyperplasia (height greater than 1 cm) in central precocious puberty. [1676] Although empty sella may be associated with central precocious puberty, the empty sella syndrome is less frequently observed in patients with central precocious puberty than in patients with pituitary hypofunction. Although empty sella was found in 10% of children imaged for suspected hypothalamic-pituitary disorders including hypogonadotropic hypogonadism, the incidence in the general population is not known. [1677] [1678] [1679] The gonadotropin and gonadal steroid concentrations in plasma, the LH response to LHRH administration, and the amplitude and frequency of LH pulses are in the normal pubertal range [492] [940] [945] ( Fig. 24-59 and Fig. 24-60 ; see Fig. 24-41 ). The new third-generation gonadotropin assays may allow the diagnosis of true precocious puberty by determination in a single serum sample for LH in the basal state or 40 minutes after a single subcutaneous dose of LHRH. [1680] [1681] [1682] The notable improvement in the discriminatory power and specificity of

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Figure 24-59 Left, Mean basal plasma luteinizing hormone (LH) level (LER-960) and mean peak and increment after intravenous LH-releasing hormone (LHRH) (100 µg) in normal prepubertal and pubertal females and in females with idiopathic true precocious puberty. The mean peak and increments of plasma LH are higher in true precocious puberty than in normal puberty. Right, Basal follicle-stimulating hormone (FSH) level (LER-1364) and mean peak and increment after intravenous LHRH (100 µg) in normal prepubertal and pubertal females with true precocious puberty. The concentration of FSH and the response to LHRH were greater in females with true precocious puberty and normal puberty than in prepubertal females. (From Kaplan SL, Grumbach MM. Pathogenesis of sexual precocity. In Grumbach MM, Sizonenko PC, Aubert ML [eds]. Control of the Onset of Puberty. Baltimore, Williams & Wilkins, 1990, pp 620660. © 1990, the Williams & Wilkins Co., Baltimore.)

Figure 24-60 Left, Serial determinations of plasma estradiol in three girls with idiopathic true precocious puberty. Note the striking fluctuations in values. Right, Serial determinations of plasma testosterone in three boys with true precocious puberty (B.L. and J.C. have a hypothalamic hamartoma; M.D. has the idiopathic form). For conversion to SI units, see the legends of Figure 24-17 and Figure 24-18 . (From Kaplan SL, Grumbach MM. Pathogenesis of sexual precocity. In Grumbach MM, Sizonenko PC, Aubert ML [eds]. Control of the Onset of Puberty. Baltimore, Williams & Wilkins, 1990, pp 620660. © 1990, the Williams & Wilkins Co., Baltimore.)

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the new commercial LH and FSH assays in the diagnosis of true precocious puberty is well illustrated by the observations of Brito and co-workers. [1683] The mean basal concentration of LH in 100 prepubertal children (60 males, 40 females) was 0.6 mIU/mL, the minimal detectable concentration. In Tanner stage 2 (breast development in girls, testes size in boys), the LH levels ranged from less than 0.6 to 2.7 mIU/mL in boys and less than 0.6 to 1.2 mIU/mL in girls. In 58 children with true precocious puberty, the mean basal LH concentration was 1.6 mIU/mL in boys, with a sensitivity of 71% and a specificity of 100%. In girls the sensitivity was 62% and the specificity 100%. In 10 children with LHRH-independent sexual precocity, the LH level was undetectable (2.5 nmol/L (>75 ng/dL) in boys younger than 8 yr of age determined by sensitive, specific immunoassay A plasma estradiol, recurrently 36 pmol/L (10 pg/mL) determined by a sensitive, specific assay capable of quantifying low concentrations of estradiol Onset of menarche (and recurrent menses) in girls younger than 9 yr of age Psychosocial factors and parental anxiety, including evidence that the child's psychosocial well-being is adversely affected In children with neurogenic or organic true precocious puberty, especially those with associated GH deficiency, the course is almost invariably progressive and LHRH treatment should not be delayed

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TABLE 24-43 -- Potential Use of Aromatase Inhibitors or Estrogen Receptor Antagonists to Restrain Skeletal Maturation in Disorders of Growth and Sexual Maturation Growth disorders or variants of normal growth (to restrain epiphyseal maturation) Isolated growth hormone deficiency Genetic short stature/constitutional delay in growth Sexual precocity Congenital virilizing adrenal hyperplasia in male and female To reduce dose of glucocorticoid To inhibit conversion of C19 steroids to estrogens (or estrogen action) With/without use of C17/20 lyase inhibitor or anti-androgen Testotoxicosis To inhibit conversion of C19 steroids to estrogens McCune-Albright syndrome To inhibit conversion of C19 steroids to estrogens (or estrogen action) Adolescent gynecomastia To inhibit estrogen synthesis (or estrogen action) From Grumbach MM. Estrogen, bone, growth, and sex: a sea change in conventional wisdom. J Pediatr Endocrinol Metab 2000; 13(suppl 6):14391455. support the choice of LHRH agonists over surgical intervention. The LHRH agonists are useful in conjunction with GH in the management of organic or neurogenic true precocious puberty, especially when associated with GH deficiency (usually as a result of radiation of the brain). Such a regimen allows a longer period of GH treatment before epiphyseal fusion. [1606] As the course of neurogenic true precocious puberty is almost invariably progressive, after appropriate evaluation of the tempo of puberty, it is advisable to initiate LHRH agonist treatment. A few, usually short-term, studies utilized GH and LHRH agonist in short normal children or those with intrauterine growth retardation in an attempt to increase final height. Some, but not all, studies suggest that an increase in predicted final height is possible. This regimen is experimental; its cost-effectiveness needs to be considered (see the earlier section considering the hormonal control of the pubertal growth spurt). [1023] [1024] [1572] [1823] [1824] [1825] [1826] [1827] On the other hand, the combination of LHRH agonist and GH is useful for increasing final height in GH-deficient patients of pubertal age. [1828] An aromatase inhibitor, such as letrozole, decreases or eliminates the effect of estrogen on bone age advancement. This effect may be useful to improve height prognosis in sexual precocity in boys. [172] [173] Controlled studies are necessary to establish safety and efficacy (Table 24-43) . Psychosocial Aspects

Psychological management is a critical aspect of the care of children with true precocious puberty. [875] [1585] [1722] [1817] With the advanced physical maturation for chronologic age, they tend to seek friends closer to their size, strength, and physical development. Difficulties may arise because they lack the social skills of older children. Sex education of the child and the family is essential and must be given in a skillful, sensitive, and explicit manner; the risks of sexual abuse in both sexes and of pregnancy in girls need to be discussed. The parents need to be informed about the management of menses. The onset of sexual activity may be earlier than average but generally remains within the normal range. [1671] It is imperative to provide support in handling the increased height, advanced sexual maturation, and effects of gonadal steroids on behavior, activity, and emotional

stability. The unrealistic demands and expectations that arise from the discrepancy between the physique and the chronologic, mental, and psychosexual age require wise counseling, as do the reaction to ridicule by peers and the concern about being different from age mates. Some of these problems have been mitigated by school acceleration, advancing the child one or two grades, if this is consistent with the mental and emotional development. These comments are applicable to children with all forms of sexual precocity. The effectiveness of LHRH agonists has reduced but not eliminated many of these issues in true precocious puberty. [1722] Incomplete Form of Isosexual Precocity: Luteinizing HormoneReleasing HormoneIndependent Sexual Precocity

In this group of disorders, the secretion of testosterone in boys and of estrogen in girls is independent of the hypothalamic LHRH pulse generator (see Table 24-32) . Affected individuals do not exhibit a pubertal-type LH response to administration of LHRH or a pubertal pattern of pulsatile LH secretion, nor do they respond to chronic administration of an LHRH agonist with suppression of gonadal steroid output. Incomplete isosexual precocity or precocious pseudopuberty is a consequence of gonadal or adrenal steroid secretion independent of LHRH, of iatrogenic exposure to gonadal steroids, or, in boys, of rare hCG- or LH-secreting tumors. Boys

Chorionic Gonadotropin-Secreting Tumors

Several types of germ cell tumors can secrete a glycoprotein hormone that has the bioactivity of LH or hCG and can cross-react in some polyclonal LH radioimmunoassay systems. Studies using highly specific antisera to the subunit of hCG, however, confirm that the gonadotropin is hCG. Boys with these hCG-secreting neoplasms may have slightly enlarged testes (although not usually to a size consonant with the size of the phallus and other male secondary sex characteristics) and may be difficult to differentiate from boys with true precocious puberty on the basis of physical examination alone. [871] [1829] [1830] However, plasma hCG levels are elevated without an increase in the concentration of FSH or LH. [1829] Hepatomas and hepatoblastomas are among the most serious of these tumors and cause firm, irregular nodular or smooth hepatic enlargement (Fig. 24-69) . The hCG has been localized to the multinucleated tumor giant cells; in one case, -fetoprotein was found in the embryonal-type tumor cells spread throughout the hepatoblastoma. [1831] The average survival is only 10.7 months after diagnosis; the mean age at onset is 2 years, 8 months. [1830] [1831] [1832] [1833] Some teratomas, chorioepitheliomas, or mixed germ cell tumors in the hypothalamic region (or in the mediastinum, the lungs, the gonads, or the retroperitoneum) and certain hypothalamic pineal tumors (usually a germ cell tumor or mixed germ cell tumor, [1829] [1834] [1835] less commonly a chorioepithelioma or its variants) cause sexual precocity in boys by secreting hCG rather than by activating the hypothalamic LHRH pulse generator and the pituitary gonadotropingonadal axis. [1829] The prevalence of hCG-secreting embryonal neoplasms, especially of the mediastinum, is increased in boys with 47,XXY or mosaic Klinefelter's syndrome. About 20% of mediastinal germ cell tumors occur in boys with Klinefelter's syndrome, a prevalence 30 to 50 times that in unaffected boys. [1359] [1836] [1837] Plasma -fetoprotein is a useful additional marker for yolk sac (endodermal sinus) or mixed germ cell tumors [1838] ; the cells in the tumor that secrete -fetoprotein appear to differ from those that secrete hCG. Intracranial germ cell tumors are 2.6 times more common in males than females [1839] ; in females, germ cell tumors do not cause gonadotropin-induced isosexual precocity because of the paucity of

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Figure 24-69 A 1 5/12-year-old boy with a human chorionic gonadotropin (hCG)secreting hepatoblastoma. Note the outline of the large liver (left) and the penile enlargement (right). The testes were 2 × 1 cm, and public hair was stage 2. The plasma hCG level was 50 mIU/mL; plasma testosterone 168 ng/dL; and plasma -fetoprotein 160,000 ng/mL. Metastatic lesions in both lungs were seen on the radiograph of the chest. To convert testosterone values to SI units, see the legend of Figure 24-17 . To convert hCG values to international units per liter, multiply by 1.0. To convert -fetoprotein values to micrograms per liter, multiply by 1.0. (From Kaplan SL, Grumbach MM. Pathogenesis of sexual precocity. In Grumbach MM, Sizonenko PC, Aubert ML [eds]. Control of the Onset of Puberty. Baltimore, Williams & Wilkins, 1990, pp 620660. © 1990, the Williams & Wilkins Co., Baltimore.)

effects of hCG in prepubertal females. However, they can cause true precocious puberty through disinhibition of the hypothalamic LHRH pulse generator by local effects; rarely, the germ cells contain sufficient aromatase activity to convert circulating C 19 precursors (of adrenal origin after adrenarche) to estradiol, which in some instances is sufficient to induce breast development. [1050] [1840] [1841] [1842] "True" pure CNS germ cell tumors (germinomas) secrete insufficient hCG to be readily detectable in the circulation, but in some patients hCG can be detected in the cerebrospinal fluid. [1048] In mixed germ cell tumors, on the other hand, hCG is commonly present in the blood as well as in cerebrospinal fluid. In children, germ cell tumors in the suprasellar-hypothalamic region do not exhibit a sex predominance and are generally associated with pituitary hormone deficiencies including diabetes insipidus. [1048] Germ cell tumors that secrete hCG are rarely located in the thalamus and basal ganglia. Intracranial germ cell tumors account for 3% to 11% of malignant CNS tumors in children and adolescents, with a predominance in the Far East. [1843] [1844] Germ cell tumors of the hypothalamus or pineal region constitute less than 1% of primary CNS tumors in Western countries but account for 4.5% of such tumors in Japan. [1845] Mixed germ cell tumors and especially pure germinomas are radiosensitive, and regression of sexual precocity may occur if the bone age is less than 11 years, only to progress later into normal puberty. [1829] Long-term survival was reported to be 88% after appropriate therapy. [1846] Calcification of the pineal is found in 8% to 11% of 8- to 11-year-old children and by itself is not indicative of a tumor. Gonadotropin-secreting pituitary adenomas are exceedingly rare in children. An LH- and prolactin-secreting pituitary adenoma caused sexual precocity in two boys. [1845] [1847] The concentration of serum LH was strikingly elevated (900 IU/L) and did not rise further after the administration of LHRH. The elevated serum testosterone (7 nmol/L, 200 ng/dL) and prolactin (215 µg/L) levels and the high concentration of LH fell to prepubertal values after removal of a "chromophobe" adenoma with suprasellar extension. Precocious Androgen Secretion Caused by Congenital Adrenal Hyperplasia, Virilizing Adrenal Tumor, or Leydig Cell Tumor

Virilizing congenital adrenal hyperplasia caused by a defect in 21-hydroxylation (CYP21, cytochrome P450 c21 deficiency) leads to elevated androgen concentrations and masculinization and is a common cause of LHRH-independent sexual precocity in boys [1330] (see Chapter 22) .[1848] Approximately 75% of patients with P450 c21 deficiency have salt loss resulting from impaired aldosterone secretion and have low serum sodium and high serum potassium concentrations. Increased plasma concentrations of 17-hydroxyprogesterone, increased levels of urinary 17-ketosteroids and pregnanetriol, and advanced bone age and rapid growth are characteristic. Treatment with glucocorticoids suppresses the abnormal androgen secretion and arrests virilization; treatment with mineralocorticoids, when necessary, corrects the electrolyte imbalance. A rarer form of virilizing adrenal hyperplasia is usually accompanied by hypertension and is caused by 11-hydroxylase deficiency; the progressive virilization ceases and the blood pressure falls to normal with glucocorticoid therapy. All forms of congenital adrenal hyperplasia are inherited as autosomal recessive traits. [1330] Virilizing congenital adrenal hyperplasia, if untreated, can cause anovulatory amenorrhea in females and oligospermia in males; with treatment, the infertility is usually corrected (see Chapter 22) . Treatment of virilizing congenital adrenal hyperplasia may unmask LHRH-dependent sexual precocity (secondary true precocious puberty) as a consequence of the advanced somatic and presumably hypothalamic

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maturation because of exposure to androgen before glucocorticoid therapy is initiated. Virilizing adrenal carcinomas or adenomas secrete large amounts of DHEA and DHEAS and on occasion testosterone. Glucocorticoids do not suppress the increased secretion of adrenal androgens or the urinary excretion of 17-ketosteroids to the normal range for age in carcinoma, but they readily decrease plasma 17-hydroxyprogesterone or 11-deoxycortisol levels and 17-ketosteroid excretion in congenital adrenal hyperplasia. Cushing's syndrome resulting from adrenal

carcinoma may cause isosexual precocity and growth failure in boys. Rarely, an adrenal adenoma may produce both testosterone and aldosterone, leading to sexual precocity and hypertension with hypokalemia. [1849] Adrenal rests, or heterotopic adrenal tissue in the testes, may enlarge with endogenous ACTH stimulation in boys with untreated or inadequately treated virilizing congenital adrenal hyperplasia and may mimic bilateral or unilateral interstitial cell tumors. Rarely, the adrenal rests may lead to massive enlargement of the testes (see Chapter 22) . MRI sonography including Doppler flow studies of the testes is useful in defining the extent and nature of the testicular masses. In boys in whom the testicular tumors are unresponsive to glucocorticoid therapy or improved compliance, surgical management including enucleation of the tumor has been useful in preventing further damage to the testes and improving the potential for fertility. [1850] LH receptors have been detected on adrenal or cortical cells. [1851] [1852] [1853] [1854] Although some of the testicular masses may become autonomous, it seems possible that LH stimulation is a factor in some patients. Infrequently, a Leydig cell tumor in boys is the cause of sexual precocity; unilateral enlargement (often nodular) of the testis usually occurs in this neoplasm (although 5% to 10% are bilateral), in contrast to the usually normal size of both testes for chronologic age in boys with congenital adrenal hyperplasia or a virilizing adrenal tumor.[675] [1855] Of interest, an LH receptoractivating mutation was detected in three boys with a sporadic Leydig cell adenoma (see later). [1856] Women with a previous history of congenital adrenal hyperplasia or a virilizing tumor may exhibit ovarian hyperandrogenism associated with persistent elevation of LH in spite of successful treatment of their initial virilizing condition in childhood: this is not usually the case in women who have late-onset congenital adrenal hyperplasia.[1857] Familial or Sporadic Testotoxicosis (Familial Male-Limited Gonadotropin-Independent Sexual Precocity with Premature Leydig Cell and Germ Cell Maturation)

A unique form of sexual precocity in males is pituitary gonadotropinindependent familial premature Leydig cell and germ cell maturation, or testotoxicosis. [1845] [1858] [1859] [1860] [1861] [1862] [ 1863] [1864] Although it has been recognized as an LHRH-independent form of male isosexual precocity only since 1981, this disorder was described more than 50 years ago. Indeed, Andrew Shenker brought to our attention a report by Stone in 1852. [1865] Affected boys have secondary sexual development with penile enlargement, which may be present at birth, [1859] and bilateral enlargement of testes to the early or midpubertal range, although the testes are often smaller than expected in relation to penile growth and pubertal maturation (Fig. 24-70) . The testes show premature Leydig and Sertoli cell maturation and spermatogenesis; in some instances, Leydig cell hyperplasia is present. [1858] [1859] [1861] The rate of linear growth is rapid, skeletal maturation is advanced, and muscular development is prominent. Serum hormone determinations reveal prepubertal basal and LHRH-stimulated gonadotropin concentrations and lack of a pubertal pattern of LH pulsatility, whether measured by immunologic or bioassay techniques [1859] (Table 24-44) . In affected boys, plasma testosterone values are in the normal pubertal or adult range with normal clearance of testosterone. The onset of adrenarche and its biochemical marker, serum DHEAS, correlates with bone age rather than chronologic age. Treatment with an LHRH agonist does not suppress the testicular function or maturation. [1859] [1863] When most untreated affected individuals reach late childhood or early adolescence, fertility is achieved and an adult pattern of LH secretion and response to LHRH is demonstrable [1860] ; secondary LHRH-dependent true precocious puberty is superimposed on the substrate of testotoxicosis. [1860] [1861] [1866] In some adults, impaired spermatogenic function is associated with elevated concentrations of plasma FSH.[1860] This disorder, although it occurs sporadically, quite likely as a consequence of a germ line mutation or even a postzygotic one, is inherited as a sex-limited autosomal dominant trait [1860] and probably accounts for the earlier descriptions of "true" precocious puberty in families in which only males were affected. A kindred with nine generations of affected males has been reported [1860] ; obligatory female carriers of the trait were unaffected as constitutional activation of the LH receptor on the ovary causes no ill effects. [1860] [1866] In 1993, Shenker [1867] and Kremer [1868] and their colleagues independently described heterozygous activating mutations of the heterotrimeric Gs proteincoupled LH/CG receptor that in concert transduce the LH/CG signal to the main effector, adenyl cyclase (Fig. 24-71) . The receptors for pituitary and placental gonadotropins and TSH belong to a subfamily of the seven-transmembrane-spanning, G proteincoupled receptors. The LH receptor, first cloned from the rat [1869] and pig [1870] and later the human,[1871] [1872] is a glycoprotein of 80 to 90 kd. It is encoded by a gene localized to chromosome 2p21 (the same as the FSH receptor) that spans at least 70 kb and contains 11 exons separated by 10 introns. The large glycosylated aminoterminal extracellular hormone-binding domain of the 701-amino-acid LH/hCG receptor [1872] is encoded by exons 1 to 10. A single exon, the large exon 11, encodes the entire G-linked transmembrane domain with its 7-helical segments connected by alternating extracellular and intracellular loops, the intracellular domain, and the 3' untranslated regionalmost two thirds of the receptor [1384] [1873] [1874] [1875] (see Fig. 24-71) . Thirteen constitutively activating heterozygous missense mutations (in over 60 reported patients) all residing within exon 11 have been reported (see Fig. 24-71) ; six involve the transmembrane helix VI, two involve the flanking third cytoplasmic loop, there is one each in helix V and helix II, and less commonly there are mutations in the first transmembrane helix.[1867] [1868] [1876] [1877] [1878] [1879] [1880] Thus, nine mutations are between amino acid residues 542 and 581, suggesting a hot spot. There appears to be a limited repertoire of mutations in American boys, consistent with a founder effect; European pedigrees are more diverse. [1881] [1882] A model of the transmembrane domain of the receptor provides novel suggestions concerning the structural and functional effects of these activating mutations. [1883] Transfected cultured cells with these mutations exhibited increased basal cAMP production in the absence of agonist, observations consistent with a constitutive activating mutation. [1881] The conformational changes in the LH receptor that lead to its constitutive activation have yet to be established, but various possibilities have been considered. [1884] LHRH-dependent puberty usually ensues in adolescence. Infertility related to testicular damage can occur in adult men. [1562] [1860] Inactivating mutations of the LH/CG receptor and their clinical consequences are discussed in Chapter 22 . In one Polish family, the disorder, a mutation of M298T in the second transmembrane domain of the LH receptor, led to sexual precocity in one boy but not in the mother, who carried

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Figure 24-70 Familial testotoxicosis. Left, A 5½-year-old boy and his 28-year-old father with the disorder. The boy exhibited signs of sexual precocity by 3 years of age. Height was 130.6 cm (+4.8 SD); bone age 12½ years. The plasma testosterone level was 267 ng/dL; dihydrotestosterone 46 ng/dL; dehydroepiandrosterone sulfate (DHEAS) 23 µg/dL. The plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were low, and neither rose after treatment. Pulsatile LH secretion was not demonstrable. Treatment with deslorelin, an LHRH agonist, was without effect. The father had begun sexual maturation by 3 years of age and had reached a final height of 162.6 cm in his early teens. The plasma testosterone level was 294 ng/dL; LH 0.5 ng/mL (LER-960); and FSH 0.5 ng/mL (LER-869). The father had an adult-type LH and FSH response to LHRH; the LH level increased to 7.5 ng/mL, and the FSH level to 2 ng/mL. At least 28 male family members over nine generations are affected. To convert dihydrotestosterone values to nanomoles per liter, multiply by 0.03467. For other conversions to SI units, see the legends of Figure 24-17 and Figure 24-18 . Center, External genitalia of the 5½-year-old boy. The penis measured 12 × 2.8 cm; the right testis was 4 × 2 cm, and the left testis 3.5 × 2.5 cm. Right, Testis of the boy showed Leydig cell maturation without Reinke crystalloids and spermatogenesis (Mallory trichome).

the same mutation, or in her father or his son, the maternal uncle, suggesting the involvement of epigenetic factors. [1885] Three boys with sexual precocity related to a sporadic Leydig cell adenoma had an Asp 578 His mutation in the tumor [710] [1856] (Fig. 24-72) (Figure Not Available) . The remarkable association of testotoxicosis and pseudohypoparathyroidism type Ia due to a mutation in the subunit of Gs is considered in the following. Boys with LHRH-independent, pituitary gonadotropinindependent maturation of the testes do not respond to chronic administration of an LHRH TABLE 24-44 -- Testotoxicosis: Clinical and Laboratory Characteristics Sex-limited autosomal dominant inheritance; activating mutation in the gene encoding the LH receptor Early onset of sexual precocity in boys with bilateral testicular enlargement Prepubertal immunologic and biologic LH response to LHRH, prepubertal LH pulse secretory pattern Concentration of plasma testosterone in pubertal range Premature Leydig cell and seminiferous tubule maturation No CNS, adrenal, or testicular abnormalities demonstrable by radiologic or hormonal studies Lack of suppression of plasma testosterone or physical signs of puberty by LHRH agonist

CNS, central nervous system; LH, luteinizing hormone; LHRH, LH-releasing hormone. agonist with suppression of testosterone secretion, in contrast to the characteristic response in patients with true precocious puberty. [1859] However, testosterone secretion, height velocity and rate of bone maturation, and aggressive and hyperactive behavior have been decreased by treatment with oral medroxyprogesterone acetate.[875] [1859] Two other therapies have been used (Table 24-45) . Ketoconazole, an orally active substituted imidazole derivative, suppresses gonadal and adrenal biosynthesis at several steps. [1886] At the dosage used in testotoxicosis (200 mg every 8 to 12 hours orally), [1862] [1887] ketoconazole mainly inhibits the enzyme cytochrome P450 c17 , which regulates both 17-hydroxylation and the scission (17,20-lyase) of -hydroxypregnenolone to dehydroepiandrosterone (see adrenarche in this chapter). However, even at the recommended dose, the agent produces a mild transient decrease in cortisol secretion and interferes with binding of testosterone to TeBG. Secondary true precocious puberty often occurs when the bone age advances to or has already reached the pubertal range (usually > 11.5 years), at which time addition of an LHRH agonist is appropriate. [1862] Ketoconazole can cause hepatic injury, which is usually mild and reversible, but hepatotoxicity is rarely severe. [1886] Further, reversible renal injury, rash, and interstitial pneumonia have been reported in a patient who tolerated lower doses, suggesting a dose-response effect. [1888] Another therapeutic approach has been the use of the antiandrogen (and antimineralocorticoid) spironolactone combined with testolactone, an inhibitor of cytochrome P450 aromatase

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Figure 24-71 A, The serpentine seven transmembrane G s protein coupled hLH/hCG receptor with its large extracellular domain and the intracellular domain. The seven helical transmembrane domains are indicated by Roman numerals. B, The two-dimensional seven-transmembrane topology of the hLH/hCG receptor with positions of constitutively activating mutations causing testotoxicosis (male-limited autosomal dominant sexual precocity). The mutations are indicated by solid circles and the residue number. Note the cluster of mutations in the VI transmembrane helix and third cytoplasmic loop. The aspartine 578glycine mutation is the most common. (Redrawn from Yano K, Kohn LD, Saji M, et al. A case of male limited precocious puberty caused by a point mutation in the second transmembrane domain of the luteinizing hormone choriogonadotropin receptor gene. Biochem Biophys Res Commun 1996; 220:10361042.)

(CYP19), the key enzyme in the conversion of androgens to estrogens. [1889] Because these boys often experience secondary central precocious puberty after control with spironolactone and testolactone, the addition of an LHRH agonist is a useful step to suppress pituitary gonadotropin secretion. [1890] More potent and specific nonsteroidal antiandrogens such as flutamide and nilutamide [1891] and aromatase inhibitors, such as letrozole [1892] [1893] to inhibit the rate of skeletal maturation and linear growth by suppressing estradiol synthesis, are now available and have potentially greater therapeutic efficacy. [172] [173] Table 24-45 lists the various agents used in the treatment of testotoxicosis; which of these agents or combination of agents will be effective for long-term treatment and safe remains to be determined. Girls

Incomplete isosexual precocity in girls (see Table 24-32) is caused by conditions in which estrogen is secreted autonomously by an ovarian cyst or tumor, by an adrenal neoplasm, or because of inadvertent exposure to estrogen. In a pure hCG-secreting tumor in girls, signs of isosexual precocity are absent. Girls harboring a teratoma or teratocarcinoma (or a CNS germ cell tumor) that secretes hCG have had sexual precocity caused by concurrent estrogen secretion by the tumor; these girls may also have galactorrhea, especially if chorionic somatomammotropin (human chorionic somatomammotropin, human placental lactogen) is also secreted. Autonomous Ovarian Follicular Cysts

The most common estrogen-secreting ovarian mass and ovarian cause of sexual precocity is the follicular cyst. [1894] Antral follicles up to about 8 mm in diameter are common in the ovaries of normal prepubertal girls [122] [1895] [1896] [1897] and may be seen in third-trimester fetuses and newborn infants. [1898] [1899] [1900] [1901] [1902] They may appear and regress spontaneously. Large follicular cysts may be discovered because of the presence of an abdominal mass or abdominal pain, especially after torsion or as an unexpected finding on pelvic sonography performed for other reasons. Occasionally, the antral follicles secrete estrogen and may enlarge to form large masses, or the follicular cysts may recur and cause recurrent signs of sexual precocity and acyclic vaginal bleeding. Enlarged antral follicles or cysts occur in premature thelarche, true precocious puberty, and transient or incomplete sexual precocity. [871] [1689] [1903] [1904] [1905] With some ovarian follicular cysts, the transient or recurrent sexual precocity is LHRH-independent (Fig. 24-73) . The concentration of estradiol fluctuates, usually correlating with changes in the size of the follicular cyst when monitored by pelvic sonography, [1906] and may increase to levels found in a granulosa cell tumor. [492] [871] [1905] The concentration of LH is suppressed, a pubertal pattern of pulsatile LH secretion is absent, and the LH rise induced by LHRH is prepubertal. [871] [1689] [1904] [1905] It is curious that a constitutive activating mutation of the FSH receptor is undescribed in a female, especially because a heterozygous mutation, Asp 567 Gly, has been detected in the third intracellular loop of the FSH receptor in a hypophysectomized man who, despite the gonadotropin deficiency, was fertile and had normal-sized testes. [1907] This is a site of activating mutations in the LH receptor. Accordingly, the possibility that some girls with recurrent ovarian cysts harbor an activating mutation of the FSH receptor seems worthy of study. The McCune-Albright syndrome needs to be considered in any girl with recurrent ovarian cysts, even with apparent initial absence of other features of this disorder, because of somatic activating mutations in the gene encoding the subunit of the heterotrimeric Gs protein (see below).

1222

Figure 24-72 (Figure Not Available) Mutations in the luteinizing hormone (LH) receptor protein. Schematic structure of the LH receptor protein and localization of the inactivating ( open squares) and activating (filled circles) mutations currently known in the human LH receptor. The short lines across the amino acid chain separate the 11 exons. (From Themmen APN, Huhtaniemi IT. Mutations of gonadotropins and gonadotropin receptors. Endocr Rev 2000;21(5):551583.)

An unusual syndrome of estradiol-secreting ovarian cysts in preterm infants born before 30 weeks of gestation is associated with edema of the labia majora and, in some instances, of the lower abdominal wall. [1900] In four preterm neonates the syndrome appeared weeks after birth and 1 to 4 weeks before the putative date of a full-term gestation. The follicular cysts, which may be unilateral or bilateral, were detected by abdominal

Disorder

TABLE 24-45 -- Pharmacologic Therapy for Sexual Precocity Treatment

Action and Rationale

LHRH dependent True or central precocious puberty

LHRH agonists

Desensitization of gonadotropes; blocks action of endogenous LHRH

Medroxyprogesterone acetate

Inhibition of ovarian steroidogenesis; regression of cyst (inhibition of FSH release)

LHRH independent Incomplete sexual precocity Girls Autonomous ovarian cysts

McCune-Albright syndrome

Medroxyprogesterone acetate *

Inhibition of ovarian steroidogenesis; regression of cyst (inhibition of FSH release)

Third-generation aromatase inhibitor, e.g., letrozole

Inhibition of P-450 aromatase; blocks estrogen synthesis

Ketoconazole*

Inhibition of P-450-c17 (CYP17) (mainly 17,20-lyase activity)

Spironolactone * or flutamide and letrozole

Antiandrogen

Boys Familial testotoxicosis

Inhibition of aromatase; blocks estrogen synthesis Medroxyprogesterone acetate *

Inhibition of testicular steroidogenesis

LHRH, luteinizing hormonereleasing hormone. Modified from Grumbach MM, Kaplan SL. Recent advances in the diagnosis and management of sexual precocity. Acta Paediatr Jpn 1988; 30(suppl):155175. *If true precocious puberty develops, an LHRH agonist can be added.

and pelvic sonography. The LH and FSH response to LHRH suggested that the cysts were LHRH-dependent. Treatment with medroxyprogesterone acetate was associated with regression of the cysts. LHRH agonists are useful in the treatment of ovarian follicular cysts associated with true precocious puberty (LHRH-dependent) but not so-called autonomous cysts. However, girls with autonomously functioning 1223

Figure 24-73 A 4 10/12-year-old girl with recurrent "autonomous" follicular cysts of the ovary. MPA, medroxyprogesterone acetate (oral). For conversion to SI units, see the legend of Figure 24-17 . (From Kaplan SL, Grumbach MM. Pathogenesis of sexual precocity. In Grumbach MM, Sizonenko PC, Aubert ML [eds]. Control of the Onset of Puberty. Baltimore, Williams & Wilkins, 1990, pp 620660. © 1990, the Williams & Wilkins Co., Baltimore.)

ovarian follicular cysts, whether recurrent or an isolated episode, often respond to treatment with oral medroxyprogester-one acetate but not to LHRH agonists. Medroxyprogesterone acetate also seems to prevent recurrence and accelerate involution of the follicular cysts [871] [1905] and reduce the risk of torsion. The use of one of the new, potent aromatase inhibitors such as letrozole to reduce estradiol secretion is another potential approach to treatment. [1908] Surgical intervention is rarely indicated; a large or persistent cyst can be reduced by puncture at laparoscopy. The size of the cyst can be monitored readily by pelvic sonography. Plasma estradiol concentrations in girls with recurrent cysts (>7 cm) may increase to high levels indistinguishable from those in granulosa cell tumors of the ovary, [871] [1905] but they do not have increased plasma granulosa cell tumor markers such as AMH and inhibin. Alternatively, the levels of estrogen in blood and urine may be in the early pubertal range. A characteristic feature in girls with recurrent cysts is waxing and waning of estrogen levels that correlate with changes in the appearance of the ovary on pelvic sonography. Pelvic sonography is useful for visualization of ovarian cysts and estimation of functional activity. [1906] [1909] In occasional patients, exploratory laparotomy or laparoscopy may be necessary to differentiate these cysts from ovarian neoplasms or to rupture the cyst; the latter, if indicated, can also be performed by percutaneous aspiration guided by sonography. The luteinization of follicular cysts may be related to subtle elevations and increased pulses of plasma FSH. A cyst that secretes estrogen autonomously differs from the follicular cysts that may occur in girls as a result of true precocious puberty. In the latter case, removal or reduction of the cyst does not correct the sexual precocity. [1905] [1910] Furthermore, the autonomously secreting cysts are not associated with augmented pulsatile LH secretion or with a pubertal LH response to LHRH administration. Ovarian cysts and sexual precocity have been associated with the fragile X syndrome in girls. [1911] Granulosa Cell Tumor of the Ovary

This tumor is rare in childhood, and theca cell tumors are even less common. [1910] [1912] Juvenile granulosa cell tumors have distinctive features that differentiate them from the tumors in adults. Characteristic histologic features include nodular architecture, follicle formation, abundant interstitial and intrafollicular acid mucopolysacchariderich fluid, irregular microcysts, individual cell necrosis, and high mitotic activity (mean activity, 11 mitotic figures per 10 high-power fields). Size can vary from 2.5 to 25 cm with a mean diameter of 12 cm. The interstitial mucinous fluid consists predominantly of hyaluronic acid. [1913] The prognosis is good as only about 3% of patients die of the disease. Approximately 80% of granulosa cell tumors can be palpated on bimanual examination. Less than 5% are bilateral or clinically malignant. The concentration of plasma estradiol may increase to high levels [492] ; FSH and LH concentrations are usually suppressed. The tumor secretes AMH and inhibin, which are sensitive tumor markers. [1914] [1915] [1916] [1917] [1918] Sonograms of the ovary facilitate diagnosis. After surgical removal, measurements of plasma estradiol and AMH levels are a useful screen for metastases; if the patient is younger than 9 years an elevated estradiol and at any age an abnormal rise in concentration of plasma AMH or inhibin suggests recurrence or metastasis. Occasionally, gonadoblastomas in streak gonads, rare lipoid tumors, cystadenomas, and ovarian carcinomas secrete estrogens, androgens, or both hormones. Even with successful resection of a gonadal sex steroidsecreting neoplasm, the child is at risk for secondary central precocious puberty developing in the future. Gonadal tumors composed of a mixture of germ cells and sex cord stromal cells that are distinct from gonadoblastoma are usually benign when discovered in female infants or children with 46,XX karyotypes, [1919] [1920] although neoplastic transformation is a risk. [1921] [1922] Two cases of metastasizing malignant mixed germ cellsex cordstromal tumors have been described in prepubertal girls with isosexual precocity. [1922] Some of these neoplasms secrete -fetoprotein and other tumor markers. Ovarian tumors are rare in the prepubertal period, accounting for about 1% of all tumors in girls younger than 17 years, and most are benign. [1923] [1924] [1925] The majority of ovarian tumors arise from germ cells or sex cord stromal cells in childhood and less than 20% are of epithelial origin, whereas in adults the majority of tumors are of epithelial origin. [1926] [1927] Early diagnosis of most childhood tumors of the

1224

ovary, unlike ovarian cancer in women, allows successful cure. [1928] Peutz-Jeghers Syndrome

This autosomal dominant syndrome of mucocutaneous pigmentation of the lips, buccal mucosa, fingers, and toes; gastrointestinal hamartomatous polyposis; and a predisposition to malignancy is associated with a rare, distinctive sex cord tumor with annular tubules in both boys and girls. [1929] [1930] [1931] Estrogen secretion by the tumor may lead to feminization and incomplete sexual precocity in boys as well as girls. Less frequently, an epithelial tumor of the ovary, dysgerminoma, or a feminizing Sertoli-Leydig cell tumor has been found in patients with Peutz-Jeghers syndrome. [1932] [1933] Children with this disorder should be examined at regular intervals for the presence of gonadal tumors by pelvic sonography. The syndrome is due to mutations in the gene on 9p13.3 encoding a serine/threonine protein kinase STK11 leading to haploinsufficiency of this novel tumor-suppressing gene. [1934] [1935] [1936] [1937] Sex cordstromal tumors derive from the coelomic epithelium or mesenchymal cells of the embryonic gonads and are composed of granulose, theca, Leydig, and Sertoli cells. Estrogen secretion from these tumors can cause pseudoprecocious puberty, and androgen secretion can cause virilization. Both inhibin A and B activin are produced as well as antimüllerian factor; all serve as useful tumor markers. [1916] [1938] [1939] Sex cordstromal tumors not associated with Peutz-Jeghers syndrome are malignant in 25% of patients; these tumors may grow quite large, whereas those associated with Peutz-Jeghers syndrome are often small and multiple, and contain

calcifications.

[1940]

Adrenal Adenomas

Adrenocortical tumors are rare in childhood (reported to be 0.6% of all childhood tumors and 0.3% of all malignant childhood tumors), but most produce steroid hormones in childhood whereas those in adults usually do not. The median age of diagnosis is 4 years, with various studies stating mean ages of 2, 4.3, and 5 years. Forty-one percent appear before 2 years and 71% before 5 years of age. Most cause virilization or Cushing's syndrome, but adrenal tumors may produce estrogen as well as androgens and cause sexual precocity in a girl or gynecomastia in a boy. One adrenal adenoma found in a 7-year-old girl expressed the gene for aromatase, demonstrating that the tumor could directly produce estrogen. [1941] There was substantial production of estrogen leading to a serum estradiol concentration of 145 pg/mL, a value in the range that is found in adrenal carcinomas as well as adenomas. Incomplete Sexual Precocity: Boys and Girls

McCune-Albright Syndrome

This sporadic syndrome,[1942] [1943] which occurs about twice as often in girls as in boys, is due to somatic activating mutations in the gene ( GNAS1) encoding the subunit of the trimeric guanosine triphosphate (GTP)-binding protein (G s ) that stimulates adenyl cyclase. It is characterized by the triad of irregularly edged hyperpigmented macules (café-au-lait spots); a slowly progressive bone disorder, polyostotic fibrous dysplasia, that can involve any bone and is frequently associated with facial asymmetry and hyperostosis of the base of the skull; and, more commonly in girls, LHRH-independent sexual precocity [1662] [1944] [1945] ( Fig. 24-74 and Table 24-46 ). Autonomous hyperfunction most commonly involves the ovary, but other endocrine involvement includes thyroid (nodular hyperplasia with thyrotoxicosis or, remarkably, with euthyroid status), [1365]

Figure 24-74 A 7 4/12-year-old girl with luteinizing hormonereleasing hormone (LHRH)independent sexual precocity associated with McCune-Albright syndrome. She had breast development since infancy, and it increased noticeably at about 3 years of age; 6 months later episodes of recurrent vaginal bleeding began. Growth of pubic hair was noted at about 4 to 5 years of age. At age 5 1/12 years the bone age was 6 11/12 years; height was +1 SD above the mean value for age. By 6½ years of age, when she was seen at the University of California, San Francisco, the bone age had advanced to 9 years, and height was at +1 SD. Breasts were at Tanner stage 4; pubic hair at stage 3. Extensive irregular café-au-lait macules cover the right side of the face, left lower abdomen and thigh, and both buttocks. A bone survey showed widespread involvement of the long bones with typical polyostotic fibrous dysplasia, and the floor of the anterior fossa of the skull was sclerotic and the diploetic space widened. She has had two pathologic fractures through bone cysts in the right upper femur. Note the osseous deformities. Plasma estradiol concentrations were consistently in the pubertal range; LH response to LHRH was prepubertal. Results of thyroid function studies were normal, including the thyrotropin response to thyrotropin-releasing hormone administration and antithyroid antibodies were not detected. Treatment with oral medroxyprogesterone acetate suppressed menses and arrested pubertal development but did not slow skeletal maturation. Her final height is 142 cm (-2.5 SD). Menstrual cycles are regular.

adrenal (multiple hyperplastic nodules with Cushing's syndrome), [1945] pituitary (adenoma or mammosomatotroph hyperplasia with gigantism and acromegaly and hyperprolactinemia), [1946] and parathyroids (adenoma or hyperplasia with hyperparathyroidism). [1662] In addition, hypophosphatemic vitamin Dresistant rickets or osteomalacia can occur in this syndrome because of either overproduction of a phosphaturic factor, phosphatonin, [1947] secreted by the bone lesions or an 1225

TABLE 24-46 -- Clinical Manifestations of McCune-Albright Syndrome in 158 Reported Patients Age at Diagnosis Manifestation

*

Patients (%) (n = 158)

Male (n = 53)

Female (n = 105)

(yr)

(range)

Fibrous dysplasia

97

51

103

7.7

(052)

Polyostotic more common than monostotic

Café-au-lait lesion

85

49

86

7.7

(052)

Variable size and number of lesions, irregular border ("coast of Maine")

Sexual precocity

52

8

74

4.9

(0.39)

Common initial manifestation

Acromegaly/gigantism

27

20

22

14.8

(0.242)

17/26 with adenoma on MRI/CT

Hyperprolactinemia

15

9

14

16.0

(0.242)

23/42 of acromegalic with PRL

Hyperthyroidism

19

7

23

14.4

(0.537)

Euthyroid goiter is common

Hypercortisolism

5

4

5

4.4

(0.217)

All primary adrenal

Myxomas

5

3

5

34

(1750)

Extremity myxomas

Osteosarcoma

2

1

2

36

(3437)

At site of fibrous dysplasia, not related to prior radiation therapy

Rickets/osteomalacia

3

1

3

27.3

(852)

Cardiac abnormalities

11

8

9

Hepatic abnormalities

10

6

10

1.9

Comments

Responsive to phosphorus plus calcitriol

(0.166)

Arrhythmias and CHF reported

(0.34)

Neonatal icterus is most common

MRI, magnetic resonance imaging; CT, computed tomography; PRL, prolactin; CHF, congestive heart failure. Modified from Ringel MD, Schwindinger WF, Levine MA. Clinical implication of genetic defects in G proteins: the molecular basis of McCune-Albright syndrome and Albright hereditary osteodystrophy. Medicine (Baltimore) 1996; 75:171184. *Evaluations include clinical and biochemical data; other rarely described manifestations include metabolic acidosis, nephrocalcinosis, developmental delay, thymic and splenic hyperplasia, and colonic polyps.

intrinsic renal abnormality leading to excess generation of nephrogenous cAMP in the proximal tubule and, as a result, decreased reabsorption of phosphate. [1948] At least two of the features must be present to consider the diagnosis. Hepatocellular dysfunction may be due to expression of the mutant-activating gene in liver cells. [1949] This is a sporadic condition that can be concordant or discordant in monozygotic twins. [1950] The skin manifestations may not be conspicuous in infancy, although the majority of patients have pigmented skin lesions in infancy that usually increase in size along with body growth.[1951] The irregular-bordered café-au-lait macules usually do not cross the midline, are often located on the same side as the main bone lesions, and have a segmented distribution. [1942] The skeletal lesions in the cortex are dysplastic and are filled with spindle cells with poorly organized collagen support; they take the form of scattered cystic areas of rarefaction on radiography and often result in pathologic fractures and progressive deformities [1952] (Fig. 24-75) (Fig. 24-75) . Technetium bone scintigraphy has been the most sensitive approach to the detection of bone lesions before they are visible radiographically. If the skull is involved, there may be entrapment and compression of optic or auditory nerve foramina, which can lead to blindness, deafness, facial asymmetry, and ptosis. Fifty percent of affected children in one series

manifested bone abnormalities by 8 years of age.

[ 1951]

Increased serum GH levels have an adverse effect on the skull deformities.

The sexual precocity, the onset of which is often in the first 2 years of life and is frequently heralded by menstrual bleeding, is due to an autonomously functioning luteinized follicular cyst of the ovary (see Table 24-47) . [871] [1662] The ovaries contain multiple follicular cysts but not corpora lutea and commonly exhibit asymmetrical enlargement as a result of a large solitary cyst that characteristically enlarges and spontaneously regresses only to recur (Fig. 24-76) .[871] [1662] [1863] [1945] [1953] [1954] Serum estradiol is elevated (at times to extraordinarily high levels); in contrast, the LH response to LHRH is prepubertal and the pubertal pattern of nighttime LH pulses is absent at the onset and during the initial years. [871] [1955] [1956] Later in the course of the sexual precocity, when the bone age approaches 12 years, the LHRH pulse generator becomes operative and ovulatory cycles ensue. Thus, an affected girl may progress from LHRH-independent puberty to LHRH-dependent puberty [871] [1955] [1957] (see Table 24-45) . LHRH agonists are not effective for treatment in the LHRH-independent stage. Testolactone (40 mg/kg per day orally), [1958] a relatively weak aromatase inhibitor, has been of equivocal usefulness [1959] and some patients become resistant to the drug. [1960] The new, highly potent, specific, third-generation aromatase inhibitors, for example, letrozole, should be more effective. [172] [173] Antiestrogens offer another method of control of the estrogenic effects of the disorder. A single case report of treatment with tamoxifen showed decreases in bone age advancement, growth rate, menses, and pubertal development. [1961] Multicenter trials of antiestrogen and antiaromatase therapy in this disorder are in progress. Sexual precocity is rare in boys with McCune-Albright syndrome. [1662] [1944] [1962] [1963] Affected boys may have asymmetrical enlargement of the testes in addition to signs of sexual precocity. The histologic changes are reminiscent of those in testotoxicosis; the seminiferous tubules are enlarged and exhibit spermatogenesis, and Leydig cells may be hyperplastic. [1962] The LH response to LHRH was prepubertal in two cases. The hormonal data (although scant) and the testicular findings appear similar to those in boys with familial testotoxicosis. [1662] A 3.8-year-old boy with McCune-Albright syndrome (Arg 201 His mutation detected in bone and testis tissue) had the unusual feature of macro-orchidism (right testis 9 mL, left testis 7 mL) and absence of sexual precocity. He had several café-au-lait lesions on the back and a radiograph of the skeleton showed polyostotic fibrous dysplasia. Gonadotropins, the LHRH stimulation test, and sex steroid levels were prepubertal but serum inhibin B and AMH concentrations were strikingly elevated. The testes on histology showed that most seminiferous tubules were "slightly" increased in diameter and filled with Sertoli cells but lacked a lumen. The tubules stained intensively for inhibin B B subunit; mature Leydig cells were absent. [1964] The pathogenesis of the sporadic McCune-Albright syndrome was uncertain since its first description. It may occur concordantly or discordantly in monozygotic twins; familial cases have not been described. In 1986, Happle [1965] posited that the disorder is caused by an autosomal dominant lethal gene that results in loss of the zygote in utero and that cells bearing

1226

Figure 24-75a Bone lesions in McCune-Albright syndrome. A, The skull with severe thickening primarily at the base due to fibrous dysplasia. The auditory and optic nerves could be caught in narrowed foramina but that is not the case in these patients. B and C, distortions of the long bones, which can develop into a "shepherd's crock" appearance; note the multiple bone cysts.

this mutation survive only in embryos mosaic for the lethal gene. The early somatic mutation would lead to a mosaic pattern of the distribution of cells containing the mutation. The severity of the disorder would depend on the proportion of mutant cells in various embryonic tissues. The description of somatic mutations in human endocrine tumors that convert the peptide chain of the Gs protein into a putative oncogene (referred to as a gsp mutation)[1966] raised the possibility of a similar defect in the McCune-Albright syndrome that both affects a differentiated function such as a signaling pathway and mediates the regulation of proliferation. These hypotheses have now been established. Mutations in the gene encoding 1227

Figure 24-75b D, Bone scan showing the areas of remodeling that "light up" depending upon the area affected in individual patients. There are examples of patients primarily affected in the craniofacial area, in the appendicular area, and in both areas as well as the axial skeleton. (Courtesy of Michael T. Collins, M.D., National Institutes of Health, Bethesda, Maryland and Sandra Gorges, M.D., University of California, Davis.)

the subunit of the stimulatory G protein for adenyl cyclase were identified in the tissues of children with the McCune-Albright syndrome. The heterotrimeric guanine nucleotidebinding proteins (G proteins) are a subfamily within the large superfamily of GTP-binding proteins and serve to transduce signals from a large number of cell-surface receptors with a common structural motif of seven membrane-spanning domains to their intracellular effector molecules, including enzymes and ion channels; in essence, they couple serpentine cell-surface receptors to effectors (Fig. 24-77) . For Gs, the stimulatory G protein, the effector is adenyl cyclase, which is controlled by Gs and an inhibitory (Gi) G protein. [1967] [1968] [1969] The heterotrimer is composed of (1) an subunit (39 to 45 kd) that binds GTP and has intrinsic GTPase activity, which converts GTP to guantosine diphosphate (GDP), and (2) a subunit (35 to 36 kd) and a smaller subunit (7 to 8 kd) that are tightly but noncovalently associated with each other. A distinct gene encodes each of the subunits. The G proteins function as conformational switches. The GDP-ligand subunit is bound to the () subunits and is in an inactivated state. When its ligand or agonists activate the cell-surface receptor, the GDP is catalytically released from the subunit and enables GTP to bind. This leads to dissociation of the GTP-activated subunit, its dissociation from the bound () subunits, and activation of the effector, adenyl cyclase. When GTP is hydrolyzed by the intrinsic GTPase activity of G s , the and () subunits reassociate and the subunit is now in the off or inactive conformation. The three-dimensional structure of the heterotrimeric G proteins has been determined.[1970] [1971] [1972] [1973] [1974] Activating heterozygous mutations in the G s subunit that occurred as an early postzygotic event have now been described in the McCune-Albright syndrome. The somatic constitutive activating mutation, which leads to excess cAMP production and in some tissues cAMP-induced hyperplasia, [1973] has a mosaic pattern and the proportion of the hyperactive mutant to normal cells varies in different tissues, contributing, at least in part, to the varied clinical findings, its severity, its sporadic nature, and the discordant occurrence in monozygotic twins. A germ line mutation is presumed to be lethal to the embryo. Two gain-of-function somatic missense mutations have been described in this disorder, both of which involve the arginine 201 residue of the subunit, [1967] the site of covalent modification by cholera toxin: arginine 201 with either a cysteine or a histidine substitution (see Fig. 24-77) .[1944] [1975] [1976] [1977] [1978] The arginine

1228

Chronologic Age Bone Age (yr) (yr)

TABLE 24-47 -- A Patient with McCune-Albright Syndrome and Recurrent Ovarian Cysts Height Physical Signs* Basal and Post-LHRH Plasma Estradiol, (cm) pmol/L (pg/mL)

Radiograph, Long Bones

1 4/12

1 3/12

81.1

Café au lait pigmentation, B2, PH1

LH 0.61.3 (LER-960)

40 (11)

Normal

5566 (1518)

Normal

5195 (1426)

Normal

7.37.3 (2020)

Polyostotic fibrous dysplasia of femurs

FSH 1.93.2 (LER-869) Vaginal bleeding (× 2 mo) 1 8/12 2 6/12

(DHEAS 1.8 mL volume and length > 36 mm) is rare. Measurement of the ellipsoid volume of the uterus (V = longitudinal diameter × anteroposterior diameter × transverse diameter × 0.523) is the most sensitive and specific discriminator between premature thelarche and early true precocious puberty [128] and provides better early discrimination than the LH response to LHRH. Growth in stature is normal. [675] [2039] [2043] [2044] [2045] Premature thelarche is a benign, self-limited disorder compatible with normal pubertal development at an appropriate age; only reassurance and follow-up are usually necessary. The appearance of premature thelarche can, however, be the harbinger of further sexual maturation in a minority of cases as discussed earlier. [675] [2040] [2043] [2046] [2047] Because the development may be unilateral, it is important to consider the condition in girls with unilateral breast development so that needless worry about a breast neoplasm is not stimulated in the parents and no unnecessary surgical procedure is carried out. Indeed, the removal of tissue in premature thelarche may leave the child with no possibility of future breast development. [1564] In selected instances, sonography of the breast is useful in distinguishing unilateral premature

thelarche from less benign conditions. [2048] The most common cause of breast mass in the pubertal girl is fibroadenoma; although metastatic disease may locate in the pubertal breast, breast carcinoma is exceedingly rare. [2048] Plasma estradiol levels are slightly high for age in premature thelarche when determined by a highly sensitive estrogen assay. [2049] However, there is usually no increase in plasma levels of TeBG or in thyroxine-binding globulin, indicators of estrogen action on circulating plasma proteins, [2050] although a modest increase of TeBG for age has been reported. [1596] The urocytogram often reveals an estrogen effect on squamous epithelial cells in the urine. [492] [2051] The concentration of serum FSH may be in the pubertal range, nocturnal FSH pulsatility has been detected, and the rise in FSH elicited by the administration of LHRH may be augmented for chronologic age, with an FSH/LH ratio higher in precocious thelarche than in normal individuals or girls with true precocious puberty. [945] [1690] [2039] [2052] However, these results overlap those in normal prepubertal girls. Sonograms of the ovary often show one or several cysts larger than 0.5 cm that disappear and reappear, usually correlating with changes in the size of the breasts,[1909] [2052] but the volume of the ovary and uterus is prepubertal. [128] [2053] As noted, there is evidence for intermittent secretion of small amounts of estrogen from the ovary. Thus, as postulated for some recurrent ovarian cysts, premature thelarche appears to result from the ovarian response to transient increases in FSH levels and possible variations in ovarian sensitivity to FSH. [945] [2051] The LH response to LHRH is prepubertal in all cases. [945] [2054] Plasma inhibin and activin concentrations have not been reported; the possible role of a paracrine-acting pituitary factor in stimulating FSH independently of LHRH is not known. "Exaggerated" thelarche is described as premature thelarche with the added findings of advanced bone age and increased growth rate, estrogen effects in addition to thelarche. The endocrine measurements in the basal state are in the normal prepubertal range, whereas after LHRH agonist stimulation, the FSH but not LH rose higher than in control subjects or those with true precocious puberty. [2055] Premature Isolated Menarche

Rarely, girls begin periodic vaginal bleeding at age 1 to 9 without any other signs of secondary sexual development. [2056] [2057] The bleeding can recur for 1 to 6 years and then cease. At the normal age of puberty (3 to 11 years later), secondary sexual development and menses ensue and follow a normal pattern, as does stature. Fertility was later demonstrated after a normal onset of puberty in women with this variant of pubertal development. The etiology is uncertain, but it may be a counterpart of premature thelarche. Isolated menarche may appear before other manifestations of sexual precocity in the McCune-Albright syndrome and in the premature sexual maturation that can occur in juvenile hypothyroidism. Before the diagnosis of premature menarche is accepted, all other causes of vaginal bleeding and precocious estrogen secretion and of exposure to exogenous estrogens should be excluded, including neoplasms, granulomas, infection of the vagina or cervix, or a foreign body. [2058] In a series of 50 girls who had vaginal bleeding before age 10, a local lesion was found in about 50%; half of the latter had a malignant neoplasm (usually a rhabdomyosarcoma) and the other half had no discernible cause. [2059] In another report, a foreign body was responsible for 25% of vaginal bleeding in prepubertal girls. [2060] A careful examination for trauma, such as that caused by sexual

1238

abuse, is indicated. Urethral prolapse may be misdiagnosed as vaginal bleeding. Premature Adrenarche (Pubarche)

Premature adrenarche[2061] [2062] [2063] is the precocious appearance of public hair or axillary hair, or both, and less commonly an apocrine odor, comedones, and acne without other signs of puberty or virilization; it is characterized by premature and mild adrenal hyperandrogenism. [671] In the past, this designation was assigned to the appearance of these clinical features before age 8 in girls or age 9 in boys. Although in boys the age of 9 still seems appropriate, the age of 8 can no longer be used for American girls. In a well carried out cross-sectional study involving 17,077 girls with physical examination by practitioners, striking ethnic differences were detected in public hair (and breast) development between black and white girls. At 6 years of age, 9.5% (range 5.7% to 16.4%) and at 8 years of age, 34.3% of black girls had Tanner stage 2 or greater pubic hair whereas 1.4% (range 0.9% to 2.2%) and 7.7% of white girls at these ages, respectively, had public hair [27] (mean ages are shown in Table 24-3 ). Accordingly, we recommend that the diagnosis of premature pubarche be limited to black girls younger than 5 years of age and white American girls younger than 7 years, which should affect the age at which laboratory studies are initiated unless there are other signs of virilization such as clitoromegaly or rapid growth. Premature adrenarche is about 10 times more common in girls than boys. The prevalence is increased in children with CNS abnormalities without a clear sex difference; the electroencephalogram may be abnormal [699] [1664] in the absence of other neurologic findings. Familial transmission is uncommon. [2064] Premature adrenarche is commonly slowly progressive and does not have an untoward effect on either the onset or normal progression of gonadarche or final adult height. [224] Nonetheless, there is a relationship between reduced fetal growth leading to intrauterine growth retardation, the increased prevalence of premature adrenarche, and hyperinsulinism and ovarian hyperandrogenism in life [2065] (see later). Plasma concentrations of DHEA, DHEAS, androstenedione, testosterone, 17-hydroxyprogesterone, and 17-hydroxypregnenolone are comparable to values normally found in public hair stage 2. [971] [2061] [2066] [2067] [2068] ACTH stimulation increases serum DHEA and DHEAS concentrations and the excretion of urinary 17-ketosteroids, but the concentrations of plasma 17-hydroxyprogesterone and 17-hydroxypregnenolone do not increase to the levels found in individuals with virilizing forms of congenital adrenal hyperplasia. [2067] [2069] [2070] As in congenital adrenal hyperplasia, dexamethasone suppresses adrenal androgen and androgen precursor secretion. [2067] [2068] Serum gonadotropin levels in the basal state and after LHRH are in the prepubertal range in premature adrenarche. [945] [2071] Premature adrenarche occurs independently of gonadarche and is due to some factor other than increased secretion of LHRH or ACTH [969] (see the discussion of adrenarche). Bone age and height are slightly advanced for chronologic age but normal adult height is commonly achieved, [2072] [2073] with the rare exception of some individuals with unusually high values of adrenal androgens, hirsutism, acne, and a bone age more than 2.5 SD above the mean value for chronologic age. In a follow-up study of 20 girls, the functional adrenal hyperandrogenism in premature adrenarche was limited to childhood. [2073] In our view, premature adrenarche is a developmentally regulated, normal variation in the differentiation, growth, and function of the zona reticularis of the adrenal cortex, marked biochemically by the precocious increase in the concentration of plasma DHEAS to 40 µg/dL. [224] The latter is quite likely related to the independent increase of 17,20-lyase activity in the developing zona reticularis mediated by the increased phosphorylation of serine and threonine residues on the P450 c17 enzyme, and the increased abundance of cytochrome b5 and of electron-donating redox partners such as cytochrome P450 oxidoreductase and cytochrome b5 , essential for the 17,20-lyase activity of this functional microsomal enzyme (see Fig. 24-43) .[2069] Nonetheless, the factor stimulating the development and function of the zona reticularis, independent of ACTH, remains elusive (see adrenarche). In the past, failure to recognize the earlier onset of adrenarche, particularly the striking ethnic differences in black, Hispanic, and Latin populations, has contributed to the overdiagnosis of premature adrenarche [2072] [2074] [2075] and, in some instances, needless laboratory studies. The concept of exaggerated adrenarche [2076] was first advanced[2077] in relation to a postulated childhood antecedent of PCOS, the hallmarks of which are hyperandrogenism, hirsutism, anovulation, amenorrhea or oligomenorrhea, and insulin resistance and compensatory hyperinsulinemia with its attendant risk of major metabolic sequelae including type 2 diabetes mellitus, dyslipidemia, an increased propensity for coronary heart disease, and in about 50% of affected women obesity. [2074] [2078] [2079] [2080] [2081] [2082] It has been extended to include rare instances of premature adrenarche associated with excessive responses of 17-hydroxypregnenolone, DHEAS, and androstenedione to ACTH found in women with functional adrenal hyperandrogenism. Although premature adrenarche is usually considered a benign condition with no substantial long-term risk, accumulating observations indicate that girls with

premature adrenarche are at increased risk of developing functional ovarian hyperandrogenism and the polycystic ovarian syndrome, hyperinsulinism, acanthosis nigricans, and dyslipidemia in adolescence and adult life, especially if fetal growth was reduced and the birth weight was low. [671] Premature adrenarche is a risk factor for the later development of PCOS and functional ovarian hyperandrogenism in adolescent and adult women; the magnitude of this risk is unknown, but it appears to be rare with the exception of girls with a history of decreased fetal growth. [671] [2065] [2073] [2076] [2083] [2084] Plasma plasminogen activator inhibitor-1, a marker of risk for cardiovascular disease including women with PCOS, was increased in girls with premature adrenarche, especially those with low birth weights, and may be useful in the identification of those with a greater risk of developing PCOS. [2085] Girls with reduced fetal growth are at risk for a reduced number of ovarian primordial follicles at birth, small ovaries and uterus at puberty, and an increased serum FSH level and decreased estradiol concentrations, suggesting relative ovarian resistance to FSH. Girls from certain ethnic groups, especially black and Hispanic girls, have a higher risk of the association of premature adrenarche with syndrome X (obesity, hyperinsulinism, dyslipidemia, and later coronary heart disease) and the development of PCOS in late adolescence and early adulthood, [671] [2086] [2087] [2088] especially if decreased insulin sensitivity and acanthosis nigricans accompany the premature adrenarche. [2087] Of interest, the adrenal steroid pattern in the black and Hispanic patients in the latter study [2088] did not differ from that in children with uncomplicated premature adrenarche. As discussed previously, hyperinsulinism is associated with many metabolic and endocrine conditions and functional ovarian hyperandrogenism that in some cases is heralded by premature adrenarche. [2089] When the role of insulin resistance and hyperinsulinism was recognized in the pathogenesis of PCOS, therapeutic approaches to reduce insulin resistance were introduced, especially the use of insulin sensitizers. Among the latter, the most widely used drug is metformin because of its low prevalence of adverse effects and therapeutic efficacy. In early studies, this at present experimental agent in the treatment of PCOS decreased insulin resistance, ovarian hyperandrogenism, and hirsutism in both obese and nonobese patients. [553] [2090] [2091] [2092] The

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safety and efficacy of metformin in 82 children and adolescents with type 2 diabetes mellitus (10 to 16 years of age) were supported by a short-term randomized control trial, [2093] providing useful preliminary data to support its use in adolescents with insulin resistance and functional ovarian hyperandrogenism. Troglitazone is another potent insulin sensitizer that improved hirsutism and ovulation in PCOS in a well-controlled study. [2094] In an in vitro study, troglitazone but not metformin directly inhibited the steroidogenic enzymes P450 c17 and 3-HSD.[2095] These are promising approaches to a still poorly understood syndrome that often becomes manifest during adolescence. Many aspects need to be addressed in the management of this heterogeneous disorder and include, apart from pharmacologic agents, concern about nutrition and physical activity. Premature pubarche can be associated with nonclassical congenital adrenal hyperplasia caused by homozygous or compound heterozygous missense mutations in the CYP21 gene encoding cytochrome P450c21 [2096] and can readily be detected by a plasma 17-hydroxyprogesterone response to ACTH that is at least 6 SD above the mean value. The prevalence of nonclassical 21-hydroxylase deficiency in children with premature adrenarche is low [2073] [2076] [2097] except in some ethnic groups [2098] [2099] [2100] (e.g., Hispanics, Italians, and Ashkenazi Jews; see Chapter 22 ), in whom the prevalence may be as high as 20% to 30%. [2099] [2100] 21-Hydroxylase deficiency can be excluded by determining the plasma 17-hydroxyprogesterone response to ACTH. Premature adrenarche is also associated with the rare nonclassical 11-hydroxylase deficiency. There has been controversy about the prevalence and significance of 3-HSD2 deficiency and the pervasive belief that a mutation in the gene encoding this enzyme was a common cause of premature adrenarche and nonclassical 3-HSD deficiency. [2099] The possibility of a mutation in the open receding frame of 3-HSD of the type 2 or type 1 gene has been excluded as all but an uncommon cause of this condition. [2101] [2102] [2103] Mutations in the 3-HSD type 2 gene have been associated with a 17-hydroxypregnenolone response to ACTH that exceeds or equals the mean normal value by 6 SD. Of 26 families studied, only one family with a mutation, alanine 82 threonine, had affected females who exhibited premature pubarche; in this family the affected male was a male pseudohermaphrodite. [2104] Thus, a mutation in the 3-HSD type 2 or type 1 gene is an uncommon cause of premature pubarche, exaggerated adrenarche, and hirsutism in adolescent girls and women. The cause of the "mild deficiency" in 3-HSD activity is unknown, but it may be multifactorial and lead to a wide range in the secretory capacity of the zona reticularis. A family constellation was described with a dominant pattern of inheritance [2064] of elevated adrenal androgens and androgen precursors that occurred as premature pubarche; later affected individuals developed hirsutism and anovulation. [2143] Thus there is controversy as to the limits of differential diagnosis between 3-HSD deficiency and premature pubarche. Recent analyses of boys and girls with either congenital adrenal and/or gonadal 3-HSD deficiency and apparent premature pubarche and girls with pubertal hirsutism led to recommendations to improve the accuracy of the diagnosis of inherited 3-HSD deficiency. According to this study, ACTH-stimulated 17-hydroxypregnenolone (5-17P) levels in children with premature pubarche at or greater than 294 nmol/L equivalent to or greater than 54 SD above Tanner II pubic hair stage matched control mean level [17 ± 5 ( SD) nmol/L] or ACTH-stimulated ratio of 5-17P to F (cortisol) in children with premature pubarche at or greater than 363 equivalent to or greater than 38 SD above the control mean ratio [20 ± 9 ( SD)], support the diagnosis of 3-HSD deficiency. However, ACTH-stimulated 5-17P levels in children with premature pubarche up to 72 nmol/L equivalent to up to 11 SD above the control mean level, and ACTH-stimulated 5-17P to F ratio in children with premature pubarche up to 67, equivalent to up to 5 SD above the control mean ratio, are not consistent with 3-HSD deficiency congenital adrenal hyperplasia. These criteria are stricter than those utilized in the past. [2143] DHEA is a stimulus to sebaceous gland activity [2105] and prepubertal acne or comedones may appear in association with elevated serum DHEAS concentrations in some children without the appearance of pubic hair, suggesting a variant of premature adrenarche may be manifest in this manner. [2106] [2107] [2108] Androgen effects such as clitoral or penile enlargement, rapid growth, hirsutism, and deepening of the voice, for example, exclude premature adrenarche and indicate a more severe form of hyperandrogenism. Adolescent Gynecomastia

Normal pubertal boys, usually in the early stages of puberty, may have either unilateral breast enlargement (approximately 25% of boys) [2109] or bilateral breast enlargement (approximately 50% to 65% of boys)[2110] of varying degrees, commonly between chronologic ages 14 to 14½ years or pubic hair stages 3 and 4. In these boys the plasma concentrations of testosterone and estrogen are normal for the stage of puberty. Pubertal gynecomastia is usually associated with an elevated ratio of the concentration of serum estradiol to testosterone. [2111] [2112] [2113] [2114] In a prospective study, adolescent boys with gynecomastia had a lower mean free testosterone concentration, lower weight, higher plasma TeBG levels, and a tendency toward earlier onset of puberty and more rapid progression through puberty. [2109] In one study a significant decrease in the concentration ratio of plasma androstenedione to estrone and estradiol and a similarly low ratio of DHEAS to estrone and estradiol were described in boys with pubertal gynecomastia who had normal ratios of plasma testosterone to estrone and estradiol. It was postulated that either decreased adrenal production of androgens or more likely increased peripheral conversion of adrenal androgens to estrogens was a factor in the development of pubertal gynecomastia. [2115] An elevated ratio of testosterone to dihydrotestosterone, presumably related to a decrease in 5-reductase activity, was suggested in the etiology of gynecomastia as well. [2116] Immunoreactive estrogen, androgen, and progesterone receptors localized to the nucleus of ductal cells was detected in all of 30 patients with gynecomastia, but aromatase immunoreactivity, limited to stromal cells, was detected in only 37% of cases. [2117] Pubertal gynecomastia usually resolves spontaneously within 1 to 2 years of onset, and reassurance and continued observation are often adequate treatment. Nevertheless, some boys have conspicuous gynecomastia and sufficient psychological distress to warrant a reduction mammoplasty. [2118] Liposuction is an alternative approach, but its efficacy in adolescent gynecomastia remains to be established. Rarely, untreated gynecomastia persists into adulthood, as illustrated by a patient who had persistent unilateral gynecomastia that began during puberty and contralateral Poland's syndrome of hypoplasia of the chest, breast tissue, and nipple. [2119] Gynecomastia is a component of Klinefelter's syndrome, anorchia, primary and secondary hypogonadism, biosynthetic defects in testosterone synthesis, increased aromatase activity in adipose and other tissues (aromatase excess syndrome), Sertoli cell tumors, adventitious exposure to estrogens in meat or cosmetics, and variants of the androgen resistance syndromes, including Rosewater's syndrome (familial hypogonadism and gynecomastia) and Reifenstein's syndrome (hypospadias, hypogonadism, and gynecomastia). These disorders usually have characteristic findings that allow ready differentiation from the normal gynecomastia of puberty [1330] (see Chapter 22) . Gynecomastia has been described in association with the administration

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of drugs such as cimetidine, spironolactone, digitalis, and phenothiazines as well as with GH therapy

[ 2120]

and the use of marijuana.

Macro-Orchidism

Fragile X syndrome is the most common inherited cause of mental retardation. [2121] This condition is due to multiple repeats of a CCC expansion that leads to hypermethylation of the FMR1 gene (Xq,27fra) that prevents transcription and translation of the FMRP protein. Affected individuals have mental retardation in association with multiple physical anomalies including large testes. [2122] Eighty percent to 95% of adolescents or adults with fragile X syndrome have testicular volume greater than 30 mL with an average of 45 mL, although the 95th percentile for the syndrome is 70 mL. [2123] [2124] In most cases the enlargement begins at 8 to 9 years of age, prior to the appearance of pubic hair, although some prepubertal children already have a testicular volume greater than 4 mL. [2125] The enlarged testes are due to increased interstitial volume and excessive connective tissue, including increased peritubular collagen fibers, [2126] rather than to increase in the seminiferous tubules. Testicular biopsy has demonstrated normal Leydig and Sertoli cells, normal to slightly decreased spermatogenic cells, and an increase in testicular interstitial fluid. Although there may be subtle elevation of serum gonadotropins that might be part of the etiology of macro-orchidism, affected males are fertile, although most are not sexually active. Other associated anomalies include increased birth weight, high forehead, large ears, prognathism, pale irises, and an increased head circumference. Macro-orchidism without androgenization is a rare manifestation of the McCune-Albright syndrome. [1964] Macro-orchidism is an occasional finding in prepubertal boys with long-standing primary hypothyroidism. This form of testicular enlargement appears to result from increased FSH secretion independent of a pubertal increase in LH secretion or a pubertal LH response to LHRH (see above). Testicular adrenal rests in congenital adrenal hyperplasia (see Chapter 22) and a lymphoma can cause bilateral macro-orchidism. It was a feature of severe aromatase deficiency in a young male adult [182] and in men with an FSH-secreting pituitary macroadenoma. Bilateral megalotestis (testicular volume 26 mL) in adults can occur as a normal variant. [2127] One may speculate that some instances of bilateral macro-orchidism are due to a heterozygous constitutive activating mutation of the FSH receptor.



Disorders of Sexual Differentiation with Both Virilization and Feminization at Puberty

Virilization as well as feminization at puberty may occur in a phenotypic female who has a 46,XY karyotype in certain types of male pseudohermaphrodism (see Chapter 22) . 17-HSD type 3 deficiency (a testosterone biosynthetic defect) and incomplete forms of androgen resistance (resulting from defects in the androgen receptor) may occur in this manner; however, ambiguous genitalia are usually noted early in life in these conditions. True hermaphrodites with ovarian and testicular tissue may undergo both virilization and feminization at puberty. [1330]



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L, Potau N, de Zegher F. Ovarian hyporesponsiveness to follicle stimulating hormone in adolescent girls born small for gestational age. J Clin Endocrinol Metab 2000; 85:26242626.

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S, Lewy V, Danadian K, et al. Metformin therapy in adolescents with PCOS: lowering of adrenal cytochrome P450c17á activity and attenuation of hyperandrogenemia with reduction of an insulinemia (abstract). Pediatr Res 2001; 49:109A. 2093. Jones

KL, Arslanian S, Peterokova VA, et al. Effect of metformin in pediatric patients with type 2 diabetes: a randomized controlled trial. Diabetes Care 2002; 25:8994.

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W, Auchus RJ, Miller WL. Thiazolidinediones but not metformin directly inhibit the steroidogenic enzymes P450c17 and 3beta-hydroxysteroid dehydrogenase. J Biol Chem 2001; 276:16767-16771. 2096. Wilson

R, Mercado A, Cheng K, et al. Steroid 21-hydroxylase deficiency: genotype may not predict phenotype. J Clin Endocrinol Metab 1995; 80:23222329.

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PW, Dupont B, Rubenstein P, et al. High frequency of nonclassical steroid 21-hydroxylase deficiency. Am J Hum Genet 1985; 35:650667.

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JW, Pang S, Nelson C, et al. Genetic defects of steroidogenesis in premature pubarche. J Clin Endocrinol Metab 1987; 64:609617.

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M, Rheaume E, Mani P, et al. No evidence of mutations in the genes for type I and type II 3- hydroxysteroid dehydrogenase (3-HSD) in nonclassical 3-HSD deficiency. J Clin Endocrinol Metab 1994; 79:18111817. 2102. Chang

YT, Zhang L, Alkaddour HS, et al. Absence of molecular defect in type II 3-hydroxysteroid dehydrogenase (3-HSD) gene in premature pubarche children and hirsute female patients with moderately decreased adrenal 3-HSD activity. Pediatr Res 1995; 37:820824. 2103. Morel

Y, Mebarki F, Rheaume E, et al. Structure-function relationships of 3-hydroxysteroid dehydrogenase: contribution made by the molecular genetics of 3- hydroxysteroid dehydrogenase deficiency. Steroids 1997; 62:176184. 2104. Mendonca

BB, Russell AJ, Vasconcelos-Leite M, et al. Mutation in 3 beta-hydroxysteroid dehydrogenase type II associated with pseudohermaphroditism in males and premature pubarche or cryptic expression in females. J Mol Endocrinol 1994; 12:119122. 2105. Deplewski 2106. Lucky

D, Rosenfield RL. Role of hormones in pilosebaceous unit development. Endocr Rev 2000; 21:363392.

AW, Biro FM, Huster GA, et al. Acne vulgaris in premenarchal girls: an early sign of puberty associated with rising levels of dehydroepiandrosterone. Arch Dermatol 1994; 130:308314.

2107. Yamamoto 2108. Stewart

A, Ito M. Sebaceous gland activity and urinary androgen levels in children. J Dermatol Sci 1992; 4:98104.

ME, Downing DT, Cook JS, et al. Sebaceous gland activity and serum dehydroepiandrosterone sulfate levels in boys and girls. Arch Dermatol 1992; 128:13451348.

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FM, Lucky AW, Huster GA, Morrison JA. Hormonal studies and physical maturation in adolescent gynecomastia. J Pediatr 1990; 116:450455.

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M, Bustos J, Dale JH, et al. Gynecomastia in adolescent boys. JAMA 1961; 178:449454.

DM, Anderson DC. Twenty-four hour profiles of circulating androgens and oestrogens in male puberty with and without gynaecomastia. Clin Endocrinol (Oxf) 1979; 11:505521.

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SE. Gynecomastia. N Engl J Med 1980; 404:795799.

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SH, Parlow AF, Lippe BM, et al. Pubertal gynecomastia and transient elevation of serum estradiol level. Am J Dis Child 1975; 129:927931.

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PK, MacDonald PC. The role of extraglandular estrogen in human endocrinology. In Greep RO, Astwood EB (eds). Handbook of Physiology. Sect 7: Endocrinology. Vol II. Part 1. Female Reproductive System. Washington, DC, American Physiological Society, 1973, pp 615629. 2115. Moore

DC, Schlaepfer LV, Punier L, et al. Hormonal changes during puberty. V. Transient pubertal gynecomastia: abnormal androgen-estrogen ratios. J Clin Endocrinol Metab 1997; 58:492499.

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S, Mondragon L, Barron C, et al. Role of testosterone and dihydrotestosterone in spontaneous gynecomastia of adolescents. Arch Androl 1992; 28:171176.

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H, Kimura M, Shizawa S, et al. Aromatase and steroid receptors in gynecomastia and male breast carcinoma: an immunohistochemical study. J Clin Endocrinol Metab 1996; 81:30633067. 2118. McGrath

MH, Mukerji S. Plastic surgery and the teenage patient. J Pediatr Adolesc Gynecol 2000; 13:105118.

2119. Mohoney 2120. Glass

J, Hynes B. Concurrent Poland's syndrome and gynecomastia: a case report. Can J Surg 1990; 33:5860.

AR. Gynecomastia. Endocrinol Metab Clin North Am 1994; 23:825837.

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RJ. Neurodevelopmental Disorders. New York, Oxford University Press, 1999.

2122. Merenstein 2123. Butler

SA, Sobesky WE, Taylor AK, et al. Molecular-clinical correlations in males with an expanded FMR1 mutation. Am J Med Genet 1996; 64:388394.

MG, Brunschwig A, Miller LK, Hagerman RJ. Standards for selected anthropometric measurements in males with the fragile X syndrome. Pediatrics 1992; 89:10591062.

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RJ. Physical and behavioral phenotype. In Hagerman RJ, Cronister A (eds). Fragile X Syndrome: Diagnosis, Treatment and Research. Baltimore, Johns Hopkins University Press,

1996, pp 387. 2125. Lachiewicz 2126. Chudley

AE, Hagerman RJ. Fragile X syndrome. J Pediatr 1987; 110:821830.

2127. Meschede 2128. Marshall

AM, Dawson DV. Do young boys with fragile X syndrome have macroorchidism? Pediatrics 1994; 93:992995.

D, Behre HM, Nieschlag E. Endocrine and spermatologic characteristics of 135 patients with bilateral megalotestis. Andrologia 1995; 207212.

WA, Tanner JM. Variations in the pattern of pubertal changes in boys. Arch Dis Child 1970; 45:1323.

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CW, Atkinson WB, Elftman H. Sex differences in pubic hair distribution. Hum Biol 2002; 16:137142.

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2131. Roy

MP, Sempe M, Orssaud E, Pedron G. [Clinical course of puberty in girls (somatic longitudinal study of 80 adolescents)] Evolution clinique de la puberte de la fille (etude longitudinale somatique de 80 adolescentes. Arch Fr Pediatr 1972; 29:155168. 2132. Van

Wieringen JD, Wafelbakker F, Verbrugge HP. Growth Diagrams 1965 Netherlands: Second National Survey on 024 Year Olds. Netherlands Institute for Preventative Medicine TNO. Groningen, Wolters-Noordhoof Publishing, 1971. 2133. Neyzi

O, Alp H, Yalcindag A, Yakacikli S, Orr DP. Sexual Maturation in Turkish boys. Ann Hum Biol 1975; 2(3):251259.

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SF, Martorell R, Mendoza F. Sexual maturation of Mexican-American adolescents. Am J Hum Biol 1989; 1:8795.

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J, Engstrom I, Lichtenstein H, Svennberg RI. VI. Somatic pubertal development. Acta Paediatr Scand [Suppl] 1976; 121135.

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PE, Thorsen T, Stoba C, et al. Studies in normal male puberty. Acta Paediatr Scand [Suppl] 1974; 136.

2137. Bachrach 2138. Oerter

LK, Hastie T, Wang M, et al. Bone mineral acquisition in healthy Asian, Hispanic, black and Caucasian youth: A longitudinal study. J Clin Endocrinol Metab 1999; 84:47024712.

KE, Manasco P, Barnes KM, Jones J, Hill S, Cutler GBJ. Adult height in precocious puberty after long-term treatment with deslorelin. J Clin Endocrinol Metab 1991; 73:12351240.

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PA, Crowley WFJ. Gonadotrophin-releasing hormone analogues as therapeutic probes in human growth and development: Evidence from children with central precocious puberty. Acta Paediatr Scand [Suppl] 1991; 372:3338. 2140. Rappaport

R, Fontoura M, Brauner R. Treatment of central precocious puberty with an LHRH agonist (Buserelin): Effect on growth and bone maturation after three years of treatment. Horm Res 1987; 28:149154. 2141. Suwa

S, Hibi I, Kato K, Nakazima H. LH-RH agonistic analog (buserelin) treatment of precocious puberty: Collaborative study in Japan. Acta Paediatr Jpn 1988; 30(Supp):176184.

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AS, Holland FJ, Costigan DC, Jenner MR, Wielgosz G, Fazekas AT. Intranasal and subcutaneous treatment of central precocious puberty in both sexes with a long-acting analog of luteinizing hormone-releasing hormone. J Clin Endocrinol Metab 1984; 58:966972. 2142A. Oostdijk

W, Hummelink R, Odink RJ, Partsch CJ, Drop SL, Lorenzen F, et al. Treatment of children with central precocious puberty by a slow-release gonadotropin-releasing hormone agonist. Eur J Pediatr 1990; 149:308313. 2143. Donaldson

MD, Stanhope R, Lee TJ, Price DA, Brook CG, Savage DC. Gonadotrophin responses to GnRH in precocious puberty treated with GnRH analogue. Clin Endocrinol (Oxf) 1984;

21:499503. 2144. Bourguignon

JP, Van Vliet G, Vandeweghe M, et al. Treatment of central precocious puberty with an intranasal analogue of GnRH (buserelin). Eur J Pediatr 1987; 146:555560.

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JL, Zumsteg U, Blumberg A, Hadziselimovic F, Girard J, Zurbrugg RP. Long-term treatment of central precocious puberty with an intranasal LHRH analogue: Control of pituitary function by urinary gonadotropins. Eur J Pediatr 1988; 147:263269. 2146. Kauli

R, Pertzelan A, Ben-Zeev Z, Lewin RP, Kaufman H, Schally AM, et al. Treatment of precocious puberty with LHRH analogue in combination with cyproterone acetate-further experience. Clin Endocrinol (Oxf) 1984; 20:377387. 2147. Stanhope

R, Pringle PJ, Brook CG. Growth, growth hormone and sex steroid secretion in girls with central precocious puberty treated with a gonadotrophin releasing hormone (GnRH) analogue.

Acta Paediatr Scand 1988; 77:525530.



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Chapter 25 - Endocrinology and Aging Steven W. J. Lamberts

The average length of human life is currently 75 to 78 years and may increase to 85 years during the coming ten years, [1] but it is not clear whether these additional years will be satisfactory. Most data indicate a modest gain in the number of healthy years lived but a far greater increase in years of compromised physical, mental, and social function. [2] The number of days with restricted activity and admissions to hospitals and nursing homes increases sharply after 70 years of age. [3] The U.S. National Health Interview Survey indicated that more than 25 million aging people suffer from physical impairment and the number of persons requiring assistance with the activities of daily living increases from 14% at ages 65 to 75 years to 45% in people older than age 85 years. [4] [5]



AGING AND PHYSICAL FRAILTY Throughout adult life, all physiologic functions start to decline gradually. [6] There is a diminished capacity for cellular protein synthesis, a decline in immune function, an increase in fat mass, a loss of muscle mass and strength, and a decrease in bone mineral density. [6] Most older adults die of atherosclerosis, cancer, or dementia, but in an increasing number of the "healthy" oldest old, loss of muscle strength is the limiting factor that determines their chances of an independent life until death. Age-related disability is characterized by generalized weakness, impaired mobility and balance, and poor endurance. In the oldest old, this state is termed physical frailty, defined as "a state of reduced physiological reserves associated with increased susceptibility to disability." [7] Clinical correlates of physical frailty include falls, fractures, impairment in activities of daily living, and loss of independence. Falls contribute to 40% of admissions to nursing homes. [8] Loss of muscle strength is an important factor in the development of frailty. Muscle weakness can be caused by aging of muscle fibers and their innervation, osteoarthritis, and chronic debilitating diseases. [9] A sedentary lifestyle, decreased physical activity, and disuse, however, are also important determinants of the decline in muscle strength. In a study of 100 frail nursing home residents (average age, 87 years), lower extremity muscle mass and strength were closely related. [10] Supervised resistance exercise training (45 minutes three times a week for 10 weeks) doubled muscle strength and significantly increased gait velocity and stair-climbing power. This finding demonstrates that frailty in the elderly population is not an irreversible effect of aging and disease but can be influenced and perhaps even prevented. [10] Further, in nondisabled elderly persons living in the community, objective measures of lower extremity function are highly predictive of subsequent disability. [11] Prevention of frailty can be achieved only by working (training). However, exercise is difficult to implement in the daily routine of the aging population, and the number of dropouts from exercise programs is very high. Part of the aging process involving body composition (i.e., loss of muscle [strength] and bone, increase in fat mass) might also be related to changes in the endocrine system.[6] Current knowledge has shed light on the effects of long-term hormonal replacement therapy on body composition as well as on atherosclerosis, cancer formation, and cognitive function.



THE ENDOCRINOLOGY OF AGING The two most important clinical changes in endocrine activity during aging involve the pancreas and the thyroid gland. Approximately 40% of individuals aged 65 to 74 years and 50% of those older than 80 years have impaired glucose tolerance or diabetes mellitus, and in nearly 50% of elderly adults with diabetes the disease is undiagnosed. [12] These adults are at risk for development of secondary, mainly macrovascular, complications at an accelerated rate. Pancreatic, insulin receptor, and postreceptor changes associated with aging are critical components of the endocrinology of aging. Apart from decreased (relative) insulin secretion by the beta cells, peripheral insulin resistance related to poor diet, physical inactivity, increased

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Figure 25-1 During aging, declines in the activities of a number of hormonal systems occur. PRL, prolactin; T 4 , thyroxine; TSH, thyrotropin. Left, A decrease in growth hormone (GH) release by the pituitary gland causes a decrease in the production of insulin-like growth factor I (IGF-I) by the liver and other organs (somatopause). Middle, A decrease in release of gonadotropin luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and decreased secretion at the gonadal level (from the ovaries, decreased estradiol [E 2 ] from the testicle, decreased testosterone [T]) cause menopause and andropause, respectively. (Immediately after the initiation of menopause, serum LH and FSH levels increase sharply.) Right, The adrenocortical cells responsible for the production of dehydroepiandrosterone (DHEA) decrease in activity (adrenopause) without clinically evident changes in corticotropin (adrenocorticotropic hormone, ACTH) and cortisol secretion. A central pacemaker in the hypothalamus or higher brain areas (or both) is hypothesized, which together with changes in the peripheral organs (the ovaries, testicles, and adrenal cortex) regulates the aging process of these endocrine axes.

abdominal fat mass, and decreased lean body mass contributes to the deterioration of glucose metabolism. [12] Dietary management, exercise, oral hypoglycemic agents, and insulin are the four components of treatment for these patients, whose medical care is costly and intensive (see Chapter 27) . Age-related thyroid dysfunction is also common. [13] Lowered plasma thyroxine (T4 ) and increased thyrotropin concentrations occur in 5% to 10% of elderly women. [13] These abnormalities are mainly caused by autoimmunity and are thus an expression of age-associated disease rather than a consequence of the aging process. Normal aging is accompanied by a slight decrease in pituitary thyrotropin release but especially by decreased peripheral degradation of T 4 , which results in a gradual age-dependent decline in serum triiodothyronine (T 3 ) concentrations without changes in T 4 levels. [13] This slight decrease in plasma T 3 concentrations occurs largely within the broad normal range of the healthy elderly population and has not been convincingly related to functional changes during the aging process. At present, the question of whether healthy aging subjects might benefit from T 3 replacement therapy remains unresolved. Changes in insulin sensitivity and thyroid function that occur in the aging population are frequently of clinical importance and recognized and treated as diseases. Three other hormonal systems exhibit lowered circulating hormone concentrations during normal aging, and these changes have thus far been considered mainly physiologic ( Fig. 25-1 and Fig. 25-2 ). Hormone replacement strategies have been developed, but many aspects remain controversial, and replenishing hormone blood levels to those found in 30- to 50-year-old patients has not yet uniformly proved beneficial and safe. The most dramatic and rapidly occurring change in women around age 50 years is menopause.[14] Cycling estradiol production during the reproductive years is replaced by very low, constant estradiol levels. For many years, the prevailing view was that menopause resulted from exhaustion of ovarian follicles. An alternative perspective is that age-related changes in the central nervous system and the hypothalamic-pituitary unit initiate the menopausal transition. The evidence that both the ovary and the brain are key pacemakers in menopause is compelling. [14] Changes in the activity of the hypothalamic-pituitary-gonadal axis in men are slower and more subtle. During aging, a gradual decline in serum total and free testosterone levels occurs. [15] Andropause is characterized by a decrease in testicular Leydig cell numbers and their secretory capacity as well as by

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Figure 25-2 Changes in the hormone levels of normal women ( left) and men (right) during the aging process. A and B, Estrogen secretion throughout an individual normal woman's life (expressed as urinary estrogen excretion) (A) and mean free testosterone (T) index (the ratio of serum total T to sex hormonebinding globulin levels) during the life span of healthy men (B). (From Guyton AC. In Guyton AC [ed]. Textbook of Medical Physiology, 8th ed. Philadelphia, WB Saunders, 1991, pp 899914. [22] ) C and D, Serum dehydroepiandrosterone sulfate (DHEAS) concentrations in 114 healthy women (C) and 163 healthy men (D). (Adapted from Ravaglia G, et al. J Clin Endocrinol Metab 1996; 81:11731178. [ 19] ) E and F, The course of serum insulin-like growth factor I (IGF-I) concentrations in 131 healthy women ( E) and 223 healthy men (F) during aging. Note the difference in the distribution of ages in the different panels. (Adapted from Corpas E, et al. Endocr Rev 1993; 14:2039. [21] )

an age-related decrease in episodic and stimulated gonadotropin secretion.

[16] [ 17]

The second hormonal system demonstrating age-related changes is adrenopause, a term that describes the gradual decline in circulating levels of dehydroepiandrosterone (DHEA) and its sulfate (DHEAS). [18] [19] Adrenal secretion of DHEA gradually decreases over time, while corticotropin secretion, which is physiologically linked to plasma cortisol levels, remains largely unchanged. The decline in DHEA and DHEAS levels in both sexes, therefore, contrasts with the maintenance of plasma cortisol levels and seems to be caused by a selective decrease in the number of functional zona reticularis cells in the adrenal cortex instead of being regulated by a central (hypothalamic) pacemaker of aging. [20] The third endocrine system that gradually declines in activity during aging is the growth hormone (GH)insulin-like

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TABLE 25-1 -- Lifetime Disease Probability for a 50-Year-Old White Woman with or without Treatment with Long-Term Hormone Replacement Lifetime Probability * (%)

Disease

No Treatment

E2 + P

Relative Risk

Coronary heart disease

46.1

30.4

0.66

Stroke

19.8

19.3

0.96

Fractures

3040

1528

0.500.70

Dementia

16.3

11.5

0.71

Breast cancer

10.2

13.518.4

1.351.80

Endometrial cancer

2.6

2.6

82.8

83.8

Life expectancy (years)

1.00

A number of limitations and assumptions must be considered when interpreting this table: The duration of the use and dose regimens of E 2 + P varied considerably between studies included in the meta-analysis (duration, 210 years). It was assumed that the addition of progestagen to the estrogen regimen would increase the risk for breast cancer from 1.35 to 1.80. [31] E2 + P, estrogen plus progestagen. From a meta-analysis by Grady D, Rubin SM, Petitti DB, et al. Hormone therapy to prevent disease and prolong life in postmenopausal women. Ann Intern Med 1992; 117:10161037. *The estimated lifetime probabilities of developing the conditions mentioned have been derived from mortality and incidence data from the 1987 Vital Statistics of the United States. The relative risks are the best estimates of the relative risk for developing each condition in long-term hormone users compared with nonusers. These estimates were derived from a model of the risks and benefits of hormone therapy developed by Grady et al. [26] Data from Barrett-Connor. BMJ 1998; 317:457461. [27]

growth factor I (IGF-I) axis (see Fig. 25-2) .[6] [21] Mean pulse amplitude and duration and fraction of GH secreted, but not pulse frequency, gradually decrease during aging. In parallel, a progressive drop in circulating IGF-I levels occurs in both sexes. [21] [23] There is no evidence for a peripheral factor in this process of somatopause, and its triggering pacemaker seems mainly localized in the hypothalamus because pituitary somatotropes, even of the oldest old, can be restored to their youthful secretory capacity by treatment with GH-releasing peptides (see later). It is unclear whether changes in gonadal function (menopause, andropause) are interrelated with the processes of adrenopause and somatopause, which occur in both men and women. Also, functional correlates (muscle size and function, fat and bone mass, progression of atherosclerosis, and changes in cognitive function) have not been related to these changes in endocrine activity. However, a number of effects of normal aging closely resemble features of (isolated) hormonal deficiency (hypogonadism, GH deficiency), which in subjects in middle adulthood are successfully reversed by replacement of the appropriate hormone. [24] [25] Although aging does not simply result from a variety of hormone deficiency states, medical intervention in the processes of menopause, andropause, adrenopause, or somatopause might prevent or delay some aspects of the aging process.



MENOPAUSE Menopause is the permanent cessation of menstruation resulting from the loss of ovarian follicular function and is diagnosed retrospectively after 12 months of amenorrhea. In most women, vasomotor reactions, depressed mood, and urogenital complaints accompany this period of estrogen decline. In the subsequent years, the loss of estrogens is followed by a high incidence of cardiovascular disease, loss of bone mass, and cognitive impairment. The average age of menopause (51.4 years) has not changed over time and seems to be largely determined by genetic factors. The use of hormone replacement therapy (HRT), consisting of estrogen or estrogen plus progestagen, can alleviate the symptoms of menopause ( perimenopausal use), but long-term use of HRT (5 to 10 years or more) may also be advantageous in preventing cardiovascular disease, bone loss, and cognitive impairment. [26] [27] [28] [29]

Perimenopausal Use of Hormone Replacement Therapy

Typical symptoms that result from the sudden decrease in estrogen production around menopause are menstrual cycle disorders, vasomotor changes (hot flushes, night sweats), and urogenital complications (atrophic vaginal irritation and dryness, dyspareunia, atrophic urethral epithelium leading to micturition disorders). Additional symptoms are irritability, mood swings, joint pain, and sleep disturbances. Frequency, severity, onset, and duration of symptoms vary widely between individuals and between ethnic groups. About 75% of women in Western societies experience so few troublesome symptoms during the menopausal transition that HRT is not needed or requested. [27] HRT rapidly alleviates the symptoms of menopause. Hot flushes and vasomotor instability as well as symptoms of urogenital atrophy rapidly disappear upon the start of HRT.



Long-Term Hormone Replacement Therapy

Because life expectancy is increasing, the time a woman spends after menopause constitutes more than one third of her life. Long-term use of HRT (5 to 10 years) seems to have advantages with regard to the prevention of the three chronic disorders most common in the elderly: (1) cardiovascular diseases, (2) osteoporosis, and (3) dementia. There are, however, also important adverse effects of long-term estrogen-progestagen replacement therapy after menopause. The most compelling problem is the increased incidence of breast cancer. [26] [28] [30] [31] Lifetime probabilities of disease occurrence for a 50-year-old white woman entering menopause without or with subsequent HRT are presented in Table 25-1 .[26] Coronary Heart Disease and Stroke

Nearly every observational study has demonstrated a decreased risk of heart disease in women who ever used estrogen. Meta-analyses of 25 published studies of women who used estrogen and 7 studies that separately assessed estrogen plus progestagen treatment found summary relative risks of 0.70 and 0.66, respectively, for coronary heart disease among women.[27] The apparent benefit is largely limited to current or recent estrogen use. HRT does not play a role in secondary prevention; progression of coronary atherosclerosis in women with established disease was not influenced. [32] HRT was not consistently associated with a reduced risk of stroke. [26] The mechanism of cardioprotection remains uncertain but probably involves multiple actions. Estrogen is an antioxidant and calcium blocker and induces beneficial effects on concentrations of serum low-density lipoprotein (LDL) cholesterol (lowering) and high-density lipoprotein (HDL) cholesterol (increasing). Added progestagen attenuates these estradiol-mediated effects experimentally, but epidemiologically there is no convincing evidence for a decreased effect of combined therapy

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in comparison with estrogen alone in the primary prevention of coronary disease.

[27]

Bone Loss

Osteoporosis is characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to increased bone fragility and, therefore, to fracture susceptibility. The lifetime risk of fractures in 50-year-old white women is 30% to 40% (see Table 25-1) . The efficacy of HRT on the main sites of osteoporotic fractures has been documented in case-control and cohort studies but only in a few prospective controlled trials.[26] [33] Current use of HRT, especially long-term use, is associated with a reduction in the risk of hip fractures by about 30% and of spine fractures by about 50%. Most osteoporotic fractures occur after age 65. Long-term use is necessary to decrease the incidence of fractures substantially. After HRT is stopped, bone loss resumes within a year and bone turnover increases to the levels observed in untreated women within 3 to 6 months, which probably accounts for the lack of fracture protection in past users. [34] Estrogen reduces bone turnover and increases bone density in postmenopausal women in part because it improves calcium homeostasis. The addition of calcium potentiates the effect of estrogen on bone mass. The further addition of androgens (low-dose testosterone) or an antiresorptive drug (bisphosphonates) may further increase bone formation in women most at risk for fractures. Dementia

A number of experimental studies indicate that estrogens may directly influence the brain by a number of mechanisms, including activation of the cholinergic system, inhibition of oxidative stress and neuronal apoptosis, and an increase in synaptic plasticity. Estrogen may also, through its effects on the cardiovascular system, reduce the risk of vascular dementia. [35] Several studies have suggested that HRT improves cognition, prevents development of dementia, and decreases the severity of dementia, but other studies have not shown this benefit of estrogen use. [36] [37] It is now well recognized that cognition improves in perimenopausal women using HRT. However, most studies suggest that this improvement occurs because menopausal symptoms are alleviated and that there is no clear beneficial effect of HRT on cognition in asymptomatic women. [37] Ten observational studies have measured the effect of postmenopausal estrogen use on the risk of development of dementia. A meta-analysis of these studies suggested a 29% decreased risk of dementia among estrogen users. [37] However, results of eight small uncontrolled trials of estrogen use in women with dementia or Alzheimer's disease did not demonstrate a clear benefit for cognition. [38] Given the limited data, no definite conclusions can be reached about the effect of HRT in reducing cognitive decline and dementia in older women. Although the data available are promising, HRT is not currently recommended for the prevention or treatment of dementia. [39] Other Benefits

HRT is associated with slightly longer overall survival. Apart from the clear, rapidly occurring effect on menopausal symptoms, no improvement in quality of life was observed in asymptomatic older women receiving HRT. Breast and Endometrial Cancer

Late menopause has long been known to be associated with an increased risk, and early menopause with a reduced risk, of breast cancer. This observation is consistent with the idea that prolonged exposure to endogenous estrogen is an adverse risk factor. For every 1-year increase in age at menopause, there is about a 3% increase in the risk of breast cancer. [30] Most studies have found no increased risk of breast cancer in women who had ever used estrogen, usually for less than 2 years in the perimenopausal period. The relative risk increase for breast cancer in HRT users seems largely confined to current or recent use. A meta-analysis of more than 50 studies clearly demonstrated that the risk of breast cancer increases with long-term estrogen use. [40] Among women who used estrogen for 5 years or longer (median use, 11 years), the summary relative risk for breast cancer was 1.35. Among 1000 women who used HRT continuously for 10 years starting at age 50 years, it was estimated that there would be an additional six breast cancers, raising the incidence from a background of 45 cases to 51 cases. However, these data mainly refer to the use of estrogens only. In the Breast Cancer Detection Demonstration Project, in which 46,000 women participated, the estrogen-progestagen regimens were associated with greater increases in breast cancer risk compared with estrogen alone. [31] The excess risk increased by 8% for each year of combined hormone use and by 1% for each year of estrogen-only use. Thus, risk of breast cancer would be predicted to increase by approximately 80% after 10 years of estrogen-progestagen use. [41]

An association between endometrial cancer and estrogen use was observed many years ago. Ten years of unopposed estrogen use increases the risk for endometrial cancer 10-fold. [26] For this reason, the HRT regimens were supplemented with progestagens, which almost completely prevented this excess risk for endometrial cancer. Other Risks

HRT doubles a woman's risk of needing gallbladder surgery. It also doubles the risk of deep vein thrombosis and pulmonary embolism; however, the absolute risk is low, about 3 cases per 10,000 treated women per year.



Hormone Replacement Regimens

As described by Barrett-Connor, [27] presently advised doses of estrogen were designed to prevent bone loss, and progestagen regimens were proposed to prevent endometrial cancer. The advice, however, has not been based on studies of a wide range of doses. Several estrogen and progestagen preparations are available for HRT (Table 25-2) . Components of available preparations vary in their effects on different target tissues. Commercial preparations differ in their clinical effects by design, and individual women differ in their responses. HRT can be administered orally, transdermally, topically, intranasally, or as subcutaneous implants. Estrogen has distinct route-dependent effects on somatotropic action. Oral estrogens probably lower serum IGF-I concentrations through impairment of hepatic IGF-I production. This effect does not occur after transdermal estrogen administration. [43] Increasing evidence suggests that transdermally administered estrogen thus has more beneficial effects on protein metabolism and body composition. Prevention of endometrial hyperplasia and cancer induction by estrogen depends on both dose and duration of progestagen use. Uterine protection requires 12 days of cyclic progestagens or combined continuous regimens. The former causes scheduled

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TABLE 25-2 -- Hormone Replacement Regimens Bone-Conserving Estrogen Doses

*

Conjugated equine estrogens

0.625 mg/day

Estrogen sulfate

1.5 mg/day

Estradiol 17 Oral

12 mg/day

Transdermal

0.05 mg/day

Implant

50 mg 6 monthly

Oral Progesterone Doses for Endometrial Protection Norgestrel

0.15 mg

Noresthisterone

1 mg

Medroxyprogesterone acetate

10 mg

Dydrogesterone

10 mg

Micronized progesterone

200 mg

Data from Barrett-Connor E. BMJ 1998; 317:457461 243245.[42]

[27]

; Clinical Synthesis Panel on HRT. Lancet 1999; 354:152155 [28] ; and Stevenson JC. Menopause 1996;

*These are average doses for a postmenopausal woman in her sixth decade. Younger women may require higher doses; older women may require less. These minimum doses are protective when given for 12 days per calendar month. Equally protective as 2.5 mg daily continually throughout calendar month.

bleeding and the latter causes unpredictable spotting or bleeding, which usually resolves within 9 months.

[28] [42]

Initially after the start of HRT, side effects, including mastalgia, bloating, bleeding, premenstrual tension, and depression, can occur. To prevent these side effects but also to increase compliance, it is generally recommended that the patient start with half the estrogen dose. Data indicate a close relationship between endogenous circulating estrogen levels and bone loss, bone mineral density, and fractures. [44] Women with detectable serum estradiol concentrations (8 to 92 pmol/L; 5 to 25 pg/mL) had higher bone mineral density, significantly less bone loss, and a lower risk for subsequent hip fractures than women with undetectable estradiol levels. These findings suggest that much lower estrogen doses might be sufficient to maintain bone than those indicated in Table 25-2 .



Indications for Hormone Replacement Therapy

Perimenopausal or menopausal HRT is strongly indicated for women having premature menopause, women with clinically important symptoms of the menopausal transition, and women entering menopause with osteoporosis. The concept that long-term HRT after menopause is an effective risk reduction strategy for coronary heart disease, fractures, and cognitive decline has to be balanced against the increased risk of breast cancer. The decision to start long-term HRT should be based on an individual's risk factors, attitude toward hormonal treatment, and knowledge of its risks and benefits. Individualization of the treatment decision is mandatory. Both knowledge and education influence the decision to start HRT; in a Swedish study, only 24% of women 54 years of age but 72% of female general practitioners and 88% of female gynecologists were receiving estrogen-progestagen replacement therapy. [45]



Selective Estrogen Receptor Modulators

A new development in the search for optimal hormone replacement therapy during menopause came from observations that tamoxifen has variable antiestrogenic and estrogenic actions in different tissues. [46] [47] Tamoxifen suppresses the growth of estrogen receptorpositive breast cancer cells. Long-term treatment of menopausal patients with breast cancer with tamoxifen also lowered the incidence of new (contralateral) breast cancer by 40%. In addition, the number of cardiovascular incidents decreased by 70%, and the age-related decrease in bone mineral density was partially prevented. [48] [49] [50] These initially puzzling observations were explained by the fact that tamoxifen and other compounds such as raloxifene have selective estrogen receptormodulating effects, exerting antiestrogenic actions on normal and cancerous breast tissue but agonistic actions on bone, lipids, and the blood vessel walls. [51] [52] [53] These effects of tamoxifen and raloxifene may be explained by differential stabilization of the conformation of the estrogen receptor but might also be related to the activation of different estrogen receptor forms, in which the form is the classical estrogen receptor, whereas a form mediates the vascular and bone effects of estrogens. [54] [55] Raloxifene is the second selective estrogen receptor modulator (SERM) available for clinical use in menopausal women. It demonstrates estrogen agonist activity on bone and lipid metabolism and has estrogen antagonist activity in uterine and breast tissue. The 60-mg dose is currently approved for the prevention and treatment of postmenopausal osteoporosis. The efficacy and safety of raloxifene for the prevention of osteoporosis in postmenopausal women were proved in a study that demonstrated a 2.5% increase in bone mineral density in the lumbar spine and hip in a group of postmenopausal nonosteoporotic women treated with raloxifene for 2 years. [56] A significant reduction of vertebral fracture risk by raloxifene was subsequently demonstrated. [57] A total of 7705 postmenopausal women with existing osteoporosis were studied. After 36 months, bone mineral density at the hip and spine increased in the women treated with 60 mg of raloxifene by 2.1% and 2.6%, respectively, compared with those receiving placebo. At 36 months, 7.4% of women had at least one new vertebral fracture, including 10.1% of women receiving placebo and 6.6% of those receiving raloxifene at 60 mg/day. Compared with the placebo group, those receiving 60 mg of raloxifene had a relative risk for fracture of 0.7 ( P < .001). Forty-six subjects needed raloxifene at 60 mg for 3 years to prevent one vertebral fracture in menopausal women without an existing fracture; for those with an existing fracture, 16 subjects required treatment. Raloxifene has effects on lipids similar to those of estrogen, except for a relatively small effect on high-density lipoprotein cholesterol and no significant effect on triglycerides. [56] [58] Data on cardiovascular event rates and on cognitive function are not yet available. Raloxifene, in contrast to tamoxifen and estrogen, does not stimulate endometrial thickness or vaginal bleeding. increased incidence of leg cramps and hot flashes. [60]

[ 56] [59]

With regard to side effects, raloxifene causes an

A most promising effect of raloxifene is its chemoprotective action against breast cancer. Cummings and colleagues [61] reported the effects in 7705 postmenopausal women (mean age, 66.5 years) with osteoporosis who were treated for a median of 40 months with placebo or raloxifene at 60 or 120 mg/day. Thirteen cases of breast cancer were confirmed among the women assigned to raloxifene compared with 27 among the women assigned to placebo (relative risk 0.24; P < .001). To prevent one case of breast cancer, 126 women would need to be treated. Raloxifene decreased the risk of estrogen receptorpositive breast cancer by 90% but did not affect the risk of estrogen receptornegative invasive breast cancer. This important study demonstrated that among postmenopausal women with osteoporosis, the risk of invasive breast cancer was decreased by 76% during 3 years of treatment with raloxifene (Fig. 25-3) .

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Figure 25-3 Effect of raloxifene administration (60 to 120 mg/day) on the cumulative incidence of breast cancer in 7705 postmenopausal women (mean age, 66.5 years) with osteoporosis. Statistical significance of the difference between the groups was P < .001. (From Cummings SR, Eckert S, Krueger KA, et al. The effect of raloxifene on risk of breast cancer in postmenopausal women: results from the MORE randomized trial. Multiple Outcomes of Raloxifene Evaluation. JAMA 1999; 281:21892197.)



Hormone Replacement Therapy, Selective Estrogen Receptor Modulators, or No Treatment?

The issue of hormonal intervention for postmenopausal women is controversial, and many aspects remain unresolved. The idea that HRT is a global risk reduction strategy is being reevaluated. [27] [29] Although the general clinical benefits of HRT in the short term during and after the menopausal transition are clear, the balance of the beneficial effects of long-term HRT after menopause versus negative effects, especially on breast cancer incidence, remains worrisome. Currently, a vast armamentarium of pharmacologic treatments to reduce cardiovascular and bone risks is available; these include cholesterol-lowering statins, -blockers, SERMs, and bisphosphonates. An optimal choice of these different lifestyle drugs for menopausal women requires individualization of the treatment decision. Coronary artery disease, for example, is a complex disorder, resulting from an interaction of genetic predisposition and environmental factors. Risk factor modification (diet, smoking, physical activity) should be advised. Primary prevention of coronary artery disease with HRT seems effective; more effective for existing atherosclerosis are lipid-lowering drugs, aspirin, nitrates, and -blockers. [62] For women with existing osteoporosis, HRT is very effective. However, SERMs and bisphosphonates come close in their fracture-reducing effects. Recognition of an increased risk for breast cancer in menopausal women is an important consideration in the choice of SERMs. If the impressive preventive effect of raloxifene on breast cancer is confirmed to last much longer than 3 years, chemoprevention of breast cancer will probably become a major consideration in the pharmacologic choice for risk reduction in the long-term preventive treatment of postmenopausal women.



ANDROPAUSE Role of Testosterone during Aging

Age-associated hypogonadism does not develop as clearly in men at andropause as in women at menopause. The key difference is the gradual, often subtle change in androgen levels in men versus the precipitate fall of estrogen production in women. It is generally agreed that as men age, there is a decline in serum total testosterone concentration that begins after the age of 40 years. In cross-sectional studies, the annual decline in total and free testosterone is 0.4% and 1.2%, respectively. The higher decline in free testosterone levels is related to the increase in sex hormonebinding globulin (SHBG) levels with age. [15] [63] It remains unclear whether the well-known biologic changes occurring during aging in men (e.g., reduced sexual activity, muscle mass and strength, and skeletal mineralization) are causally related to these changes in testosterone bioactivity (andropause). In a group of more than 400 independently living elderly men (mean age, 78 years; range, 73 to 94 years), Beld and colleagues [64] observed a positive association between total and free serum testosterone concentrations and muscle strength as well as an inverse relationship with fat mass. Low bioavailable testosterone was associated with a depressed mood [65] in a population-based study of 856 men aged 50 to 89 years.



Testosterone Replacement Therapy

Many persuasive reports in the literature demonstrate that testosterone replacement in men of all ages (young, adult, and old) with clear clinical and biochemical hypogonadism instantly reverses vasomotor activity (flushes and sweats); improves libido, sexual activity, and mood; increases muscle mass, strength, and bone mineralization; prevents fractures; decreases fat mass; and decreases fatigue and poor concentration. [24] [63] [66] Also, the treatment of normal adult men with supraphysiologic doses of testosterone, especially when combined with resistance exercise training, increased fat-free mass and muscle size and strength. [67] Most studies reporting the results of androgen therapy in older men were small, short-term, noncontrolled, and without uniform end points. The results of a large randomized study in healthy elderly men have now been published and seem representative for effects expected of androgen therapy. [68] [69] Ninety-six men (mean age 73 years) wore a testosterone patch on their scrotum (6 mg of testosterone per 24 hours) or a placebo patch for 36 months. Mean serum testosterone concentrations in the men treated with testosterone increased from 12.7 ± 2.9 nmol/L (367 ± 7.9 ng/dL) before treatment to 21.7 ± 8.6 nmol/L (625 ± 249 ng/dL; P < .001) at 6 months of treatment and remained at that level for the duration of the study. The decrease in fat mass (3.0 ± 0.5 kg) in the testosterone-treated men during the 36 months of treatment was significantly different from the decrease (0.7 ± 0.5 kg) in the placebo-treated men ( P < .001) (Fig. 25-4) . The increase in lean mass (1.9 ± 0.3 kg) in the testosterone-treated men was significantly different from that in the placebo-treated men (0.2 ± 0.2 kg; P < .001). Changes in knee extension and flexion strength, hand grip, walking speed, and other parameters of muscle strength and function were not significantly different in the two groups. Bone mineral density in the lumbar spine increased in both the testosterone-treated (4.2% ± 0.8%) and placebo-treated (2.5% ± 0.6%) groups, but mean changes did not differ between groups (see Fig. 25-4) . However, the lower the pretreatment serum testosterone concentration, the greater the effects of testosterone treatment on lumbar spine bone density after 36 months ( P = .02). A minimal effect (0.9 ± 1.0%) of testosterone treatment on bone mineral density was observed in men with a pretreatment serum testosterone concentration of 13.9 nmol/L (400 ng/dL), but an increase of 5.9% ± 2.2% was found in men with a pretreatment testosterone concentration of 6.9 nmol/L (200 ng/dL). The subjective perception of physical function decreased significantly during the 36 months of treatment in the placebo-treated

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Figure 25-4 AC, Mean (± standard error) change from baseline in fat mass, lean mass, and bone mineral density of the lumbar spine (L2 to L4) as determined by dual-energy x-ray absorptiometry in 108 men older than 65 years who were treated with either testosterone or placebo (54 men each). The decrease in fat mass ( P < .005) and the increase in lean mass ( P < .01) in the testosterone-treated subjects were significantly different from those in placebo-treated subjects at 36 months. Bone mineral density increased significantly in both groups. (A and B, from Snyder PJ, et al. J Clin Endocrinol Metab 1999; 84:19661972[ 68] ; C, from Snyder PJ, et al. J Clin Endocrinol Metab 1999; 84:26472653. [69] )

(P < .001) but not in the testosterone-treated group. Interestingly, the effect of testosterone treatment on the perception of physical functioning varied inversely with the pretreatment serum testosterone concentration (P < .01). There was no significant difference between the two treatment groups with regard to the subjective perception of energy or sexual functions. With regard to the potential adverse effects of testosterone treatment in healthy elderly men, again the study by Snyder and colleagues [68] seems representative. The mean serum prostate-specific antigen (PSA) concentration did not change during the 36 months of treatment in the placebo-treated group but increased by a relatively small but statistically significant ( P < .001) amount by 6 months of treatment in the testosterone-treated group and remained relatively stable for the remainder of the study. The urine flow rate, volume of urine in the bladder after voiding, and number of clinically significant prostate events during the 3 years of the study were similar in the two groups. Hemoglobin and hematocrit did not change in the placebo-treated group during treatment, but both increased significantly ( P < .001) in the testosterone-treated group within 6 months and remained relatively stable for the remainder of the study. Three men treated with testosterone developed persistent erythrocytosis (hemoglobin > 17.5 g/dL; hematocrit > 52%) during treatment. Other androgen replacement studies in older men have demonstrated that lipid profiles are not adversely affected by this therapy, but the incidence of cardiovascular events in healthy elderly men who receive androgens for extended periods has not been studied. [63] [70] [71] Numerous studies of large populations of healthy men have shown a marked rise in the incidence of impotence to over 50% in men 60 to 70 years old. [72] Although this increased rate occurs in the same age group who show a clear decline in serum (free) testosterone levels, no causal relationships have been demonstrated. In most instances, testosterone replacement therapy in elderly men is not effective for the treatment of loss of libido or impotence in individuals with serum testosterone concentrations within the normal range in age-matched subjects. Other factors, such as atherosclerosis, alcohol consumption, smoking, and the quality of personal relationships, seem to be more important. [73] [74] Only in the case of clear hypogonadism is the decrease in libido and testosterone restored by potency therapy. [24] [66] This result suggests that there is a threshold level of testosterone in the low normal range below which libido and sexual function are impaired and above which there is no further enhancement of response. [75] Summarizing the available literature, the indiscriminate (preventive) treatment of healthy elderly men with testosterone at a dose that increases serum testosterone concentrations to those observed in 20- to 30-year-olds has limited anabolic effects on body composition (a slight decrease in fat mass and a slight increase in muscle mass). No beneficial effects on muscle strength or physical performance are observed. Detailed analyses of a number of studies of elderly men selected on the basis of low pretreatment serum testosterone concentrations indicated a beneficial effect of testosterone replacement therapy on muscle strength, bone mineral density, mood, and (subjective) aspects of the quality of life. [70] [73] [76] This introduces the question of how to select elderly men who might benefit from testosterone treatment. There is great interindividual variation in serum testosterone concentrations among healthy men. In adulthood, the biochemical definition of male hypogonadism is generally accepted if serum total testosterone is below 10.4 to 12.1 nmol/L (300 to 500 ng/dL), depending on the population studied [63] (Fig. 25-5) . Of healthy men between the ages of 60 to 80 years, 20% demonstrated a serum testosterone concentration below 10.4 nmol/L (300 ng/dL). In the same study, non-SHBG testosterone levels were below the lower limit for young men (50% in 24 hours) of infused magnesium, a maneuver that may be employed to assess magnesium status.[755] [756] Etiology

Intestinal Causes of Hypomagnesemia

Selective dietary magnesium deficiency does not occur, and it is remarkably difficult, in fact, to induce magnesium depletion experimentally by feeding magnesium-deficient diets, probably because renal magnesium conservation is so efficient. Large amounts of magnesium may be lost in chronic diarrheal states (this fluid may contain more than 10 mEq of magnesium per liter), or via intestinal fistulae or prolonged gastrointestinal drainage. [757] More commonly, magnesium becomes trapped within fatty acid "soaps" in disorders associated with chronic malabsorption. [758] [759] [760] [761] In a rare but informative genetic syndrome TABLE 26-11 -- Causes of Hypomagnesemia Impaired Intestinal Magnesium Absorption Primary infantile hypomagnesemia Malabsorption syndromes Increased Intestinal Magnesium Losses Protracted vomiting or diarrhea Intestinal drainage Intestinal fistulas Impaired Renal Tubular Magnesium Reabsorption Congenital magnesium-wasting syndromes Bartter's syndrome Gitelman's syndrome Magnesuria with nephrocalcinosis Acquired renal disease Tubulointerstitial disease Postobstruction, acute tubular necrosis (diuretic phase) Renal transplantation Drugs and toxins Ethanol Diuretics (loop, thiazide, osmotic) Cisplatin Pentamidine Cyclosporine Aminoglycosides Foscarnet Amphotericin B Endocrine and metabolic abnormalities Extracellular fluid volume expansion Hyperaldosteronism (primary, secondary) Inappropriate antidiuretic hormone secretion Diabetes mellitus Hypercalcemia Phosphate depletion Metabolic acidosis Hyperthyroidism Rapid Shifts of Magnesium out of Extracellular Fluid Intracellular redistribution Recovery from diabetic ketoacidosis Refeeding syndrome Correction of respiratory acidosis Catecholamines Accelerated net bone formation Following parathyroidectomy Osteoblastic metastases Treatment of vitamin D deficiency Calcitonin therapy Other losses

Pancreatitis Blood transfusions Extensive burns Excessive sweating Pregnancy (third trimester) and lactation

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termed primary hypomagnesemia, a defect in the saturable component of intestinal magnesium absorption causes hypomagnesemia that can be partially overcome by administering large amounts of oral magnesium. [762] [763] Renal Causes of Hypomagnesemia

Renal magnesium wasting may result from a primary tubular transport defect, as occurs in Bartter's syndrome and a number of other rare inherited magnesium-wasting renal tubular disorders [764] [765] [766] [767] [768] (see Table 26-11) . Most often, however, it is attributable to an acquired abnormality in tubular magnesium reabsorption. In normal persons, magnesium reabsorption is virtually complete within several days of instituting experimental dietary magnesium deficiency, even before serum magnesium has declined substantially. [769] Thus, the finding of more than 1 mEq/day of urinary magnesium in a frankly hypomagnesemic patient indicates a defect in renal tubular magnesium reabsorption. Acquired primary renal tubular magnesium wasting occurs in various tubulointerstitial disorders, recovery from acute tubular necrosis or obstruction, renal transplantation, various endocrinopathies, alcoholism, and exposure to certain drugs (see Table 26-11) . Hypomagnesemia or magnesium depletion due to subnormal renal reabsorption may complicate a variety of endocrinopathies, including hyperaldosteronism, hyperthyroidism, and disorders associated with hypercalcemia, hypercalciuria, or phosphate depletion. [756] In primary hyperparathyroidism, PTH stimulates increased tubular magnesium reabsorption, but this increase is opposed by a direct tubular effect of hypercalcemia. As a result, the serum magnesium level in primary hyperparathyroidism generally is normal or only slightly reduced. [770] In hypoparathyroidism, serum and urinary magnesium levels are low. The magnesium depletion in hypoparathyroidism is consistent with loss of both the magnesium-retaining renal action of PTH and the stimulatory effect of 1,25(OH) 2 D3 on intestinal magnesium absorption. [771] Diabetes is among the most common medical disorders associated with hypomagnesemia. [772] [773] The severity of the hypomagnesemia in diabetics correlates with indices of glycosuria and poor glycemic control, [774] which suggests that urinary losses of magnesium on the basis of glycosuria may partly explain the magnesium depletion. Rapid correction of hyperglycemia with insulin therapy causes magnesium to enter cells and may further lower the extracellular magnesium concentration during treatment. Alcoholism is another very common clinical setting in which hypomagnesemia occurs. [775] Magnesium depletion in alcoholism may result in part from nutritional deficiency of magnesium, overall caloric starvation and ketosis, and gastrointestinal losses due to vomiting or diarrhea, [730] [774] [776] but an acute magnesuric effect of alcohol ingestion probably plays the major role. [775] [777] [778] [779] This effect of alcohol is most evident when blood alcohol levels are rising and may be related to transient suppression of PTH secretion. [777] [779] Other factors that may contribute to hypomagnesemia in alcoholism include pancreatitis, malabsorption, secondary hyperaldosteronism, respiratory alkalosis, and elevation in plasma catecholamines, which increase intracellular sequestration of magnesium. [756] Numerous drugs have been identified as causes of defective renal tubular magnesium reabsorption and hypomagnesemia. [756] These agents include diuretics of all classes (especially loop diuretics), cisplatin, pentamidine, cyclosporine, aminoglycosides, foscarnet, and amphotericin. Most often, drug-induced hypomagnesemia is mild and reversible, particularly that associated with diuretic therapy. In over half of patients undergoing cisplatin therapy, hypomagnesemia is noted within days or weeks and roughly half of those who develop the abnormality exhibit persistent hypomagnesemia many months or even years later. The median duration of hypomagnesemia in cisplatin-treated patients is about 2 months, but recovery has been observed up to 2 years after treatment. [780] Cisplatin may induce a more generalized nephropathy and azotemic renal failure, but the magnesium wasting appears to be an isolated functional abnormality. There is some evidence that the renal magnesium-wasting syndrome can be prevented by intravenous magnesium administration (24 to 40 mEq) before or during cisplatin infusion. [781] Such findings suggest that cisplatin may selectively impair magnesium reabsorption by binding competitively to sites or cells involved in binding and transport of magnesium. A syndrome very similar to that seen with cisplatin therapy is observed frequently in transplant recipients who receive cyclosporin A. [782] [783] [784] The frequency of this complication has approached 100% in some series. [783] It is possible that concomitant use of other agents, especially aminoglycosides or amphotericin B, has colored the presentation in these patients. Other Causes of Hypomagnesemia

Magnesium, like phosphate, is a major intracellular ion, and significant shifts of magnesium from the extracellular compartment may therefore occur during recovery from chronic respiratory acidosis or acute ketoacidosis, with refeeding, during administration of hyperalimentation solutions, and in response to elevation of circulating catecholamines. [756] Other rapid losses of extracellular magnesium may occur during periods of greatly accelerated net bone formation (as after parathyroidectomy, during recovery from vitamin D deficiency, or with osteoblastic metastases) [775] or with large losses due to pancreatitis, [785] cardiopulmonary bypass surgery, [786] massive transfusion, [787] extensive burns, [788] excessive sweating,[789] or pregnancy or lactation. [790] Consequences of Hypomagnesemia

Most of the signs and symptoms of hypomagnesemia reflect alterations in neuromuscular function: tetany, hyperreflexia, Chvostek's and Trousseau's signs, tremors, fasciculations, seizures, ataxia, nystagmus, vertigo, choreoathetosis, muscle weakness, apathy, depression, irritability delirium, and psychosis. [730] [756] [769] Patients usually are not symptomatic unless serum magnesium concentration falls below 1 mEq/L, although occurrence of symptoms, as with levels of intracellular magnesium, may not correlate well with serum magnesium concentration. Atrial or ventricular arrhythmias may occur, as may various electrocardiographic abnormalitiesprolonged PR or QT intervals, T wave flattening or inversion, or ST segment straightening. [756] [791] Hypomagnesemia also increases myocardial sensitivity to digitalis toxicity. [792] [793]

Hypomagnesemia evokes important alterations in mineral ion and potassium homeostasis that frequently aggravate the clinical syndrome. Magnesium-deprived humans or animals develop hypocalcemia, hypocalciuria, hypokalemia (owing to impaired tubular reabsorption of potassium), and positive calcium and sodium balance. [769] [794] Sustained correction of hypocalcemia or hypokalemia cannot be achieved by administration of calcium or potassium alone, respectively, whereas both abnormalities respond to administration of magnesium. [761] [795] The etiology of hypocalcemia in the setting of hypomagnesemia may be multifactorial. Inappropriately normal or low serum PTH, despite hypocalcemia, is common and indicates a defect in PTH secretion. [575] [796] [797] Other evidence indicates that hypomagnesemia also may impair PTH action on target cells in bone and kidney, although some investigators have observed normal responsiveness, and the issue remains controversial. [574] [575] [576] [795] [796] [797] [798] Vitamin D resistance also is a feature of hypomagnesemic

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states.[760] [799] This abnormality appears to be due mainly to impaired renal 1-hydroxylation of 25(OH)D, although tissue resistance to 1,25(OH) 2 D3 also may play a role. [771] [800] The serum 1,25(OH)2 D3 concentration usually is low during hypomagnesemia, which may result from magnesium depletion per se, parathyroid insufficiency, or coexistent vitamin D deficiency. [801] [802] [803] Deficiency of 1,25(OH) 2 D3 is probably not the main cause of hypocalcemia in these patients, however,

because hypocalcemia can be rapidly corrected (within hours to days) by magnesium therapy alone, well in advance of any increase in the serum 1,25(OH) 2 D3 concentration. [801] [802] Therapy of Hypomagnesemia

Mild, asymptomatic hypomagnesemia may be treated with oral magnesium saltsMgCl 2 , MgO, or Mg(OH)2 usually given in divided doses totaling 40 to 60 mEq (480 to 720 mg) per day (see Table 26-6) . Diarrhea sometimes occurs with larger doses but generally is not a problem. The gluconate form (supplying 58 mg of magnesium per gram) is said to cause less diarrhea. [756] Patients with malabsorption or ongoing urinary magnesium losses may require chronic oral therapy to avoid recurrent magnesium depletion. Although intestinal magnesium absorption is severely impaired in renal failure, [804] oral magnesium must be administered with great caution in this setting, especially in patients receiving concomitant therapy with 1,25(OH) 2 D3 . Symptomatic or severe hypomagnesemia (magnesium concentration of 40 inches in men and >35 inches in women is a marker for the metabolic syndrome. [295A]



The Role of Increased Hepatic Glucose Production in the Hyperglycemia of Type 2 Diabetes and Impaired Glucose Tolerance

The disposal of glucose after meals depends on the ability of insulin to increase peripheral glucose uptake and simultaneously decrease endogenous glucose production. Although studies have suggested that the kidney can contribute up to 25% of endogenous glucose production, [296] [297] the defect in type 2 diabetes is primarily in defective regulation of glucose production from the liver (hepatic glucose output [HGO]). Two routes of glucose production by the liver are glycogenolysis of stored glycogen and gluconeogenesis from two- and three-carbon substrates derived primarily from skeletal muscle. [298] [299] Under different conditions and at different times postprandially, the contribution of each of these to maintenance of glucose levels may vary. Using 13 C nuclear magnetic resonance spectroscopy combined with measurement of wholebody glucose production in normal human subjects at different intervals after fasting, it was found that gluconeogenesis accounted for 50% to 96% of glucose production with the proportion increasing with increasing duration of fasting. [300] [301] The production of glucose by the liver is regulated primarily by the relative actions of glucagon and insulin to activate or suppress glucose production, respectively, although the nervous system [302] and glucose autoregulation of hepatic glucose production probably play less important roles. [303] The ability of insulin to reduce HGO is an important mechanism for maintaining normal glucose tolerance. [304] [305] Under normal circumstances, insulin suppresses up to 85% of glucose production in normal individuals by directly inhibiting glycogenolysis, especially

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Figure 29-10 Insulin suppresses hepatic glucose production by direct and indirect mechanisms. In insulin resistance, insulin's ability to suppress lipolysis in adipose tissue and glucagon secretion by alpha cells in the islet results in increased gluconeogenesis. In addition, insulin inhibition of glycogenolysis is impaired. Thus, both hepatic and peripheral insulin resistance results in abnormal glucose production by the liver.

at lower insulin concentrations. [306] Under circumstances in which glycogenolysis is enhanced by glucagon, the effects of insulin to suppress hepatic glucose production may be even greater. [307] Glucagon increases glycogenolysis by activation of the classical protein kinase cascade involving cyclic AMP (cAMP)dependent protein kinase and phosphorylase and also increases gluconeogenesis in part by increasing the transcription of phosphoenolpyruvate carboxykinase through the cAMP response element binding protein. [305] [308] [309] Insulin produces decreases in endogenous glucose production by both direct and indirect mechanisms [310] (Fig. 29-10) . In its direct action, portal insulin suppresses glucose production by inhibiting glycogenolysis through an increase in phosphodiesterase activity [311] [312] or changes in the assembly of protein phosphatase complexes.[313] [314] Insulin can also directly suppress gluconeogenesis by inhibiting the activation of phosphoenolpyruvate carboxykinase transcription through the insulin-dependent phosphorylation of the forkhead transcription factor, sequestering it in the cytoplasm. [315] [316] [317] The indirect or peripheral effect of insulin in controlling glucose production by the liver is twofold. First, insulin has a profound effect on decreasing glucagon secretion by the alpha cell of the pancreas through systemic and paracrine effects. [318] [319] The decrease in glucagon secretion decreases the activation of

Figure 29-11 Relationship between fasting hepatic glucose output and fasting plasma glucose levels. Open squares represent nondiabetic control subjects; closed squares represent diabetic subjects. (From Maggs DG, Buchanan TA, Burant CF, et al. Metabolic effects of troglitazone monotherapy in type 2 diabetes mellitus: a randomized, double-blind, placebo-controlled trial. Ann Intern Med 1998; 128:176185.)

glycogenolysis and gluconeogenesis. The second important peripheral action of insulin is decreasing FFA levels by suppressing lipolysis. FFAs increase hepatic glucose production by stimulating gluconeogenesis. [320] When the reduction in plasma FFAs during a hyperinsulinemic clamp was prevented by infusion of triglyceride emulsions with heparin (which results in increased FFA levels by activation of lipoprotein lipase), insulin-mediated suppression of HGO was reduced. [298] [321] The suppression of glucagon secretion and decrease in FFA delivery to the liver are additive in reducing liver glucose production. [322] Hepatic insulin resistance plays an important role in the hyperglycemia of type 2 diabetes, [323] [324] [325] [326] and the impairment in suppression of HGO appears to be quantitatively similar to or even larger than the defect in the stimulation of peripheral glucose disposal. [324] [327] There is a direct relationship between increases in HGO and fasting hyperglycemia [328] (Fig. 29-11) . Insulin-mediated suppression of HGO is impaired at both low and high plasma insulin levels in type 2 diabetic patients, [327] [329] [330] [ 331] and hepatic glucose production is elevated early in the course of the disease [323] but may be normal in lean, relatively insulin-sensitive type 2 diabetics. [332] Treatment of patients with metformin, which suppress hepatic glucose production, results in improvements in glucose tolerance. [333] Alterations in both the direct and indirect effects of insulin in type 2 diabetics appear to play a role in the elevation in hepatic glucose production. Defects in the direct effect of insulin to suppress hepatic glucose production that have been demonstrated in humans [334] appear to be due to a large rightward shift in the steep dose-response curve for insulin's inhibition of glycogenolysis. [335] However, peripheral insulin resistance may play the bigger role in elevated hepatic glucose production in type 2 diabetes. The resistance of adipose tissue, especially visceral fat, to suppression of lipolysis by insulin is responsible for part of the inability of insulin to suppress hepatic glucose production by the indirect route, resulting in enhanced gluconeogenesis. [336] [337] In addition, the suppression of glucagon levels in humans with insulin resistance may be impaired, again leading to an increase in endogenous glucose production. [338]



INSULIN SECRETION AND TYPE 2 DIABETES Normal insulin secretory function is essential for the maintenance of normal glucose tolerance, and abnormal insulin secretion is invariably present in patients with type 2 diabetes. In

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Figure 29-12 Mean 24-hour profiles of plasma concentrations of glucose, C peptide, and insulin in normal and obese subjects. (From Polonsky KS, Given BD, van Cauter E. Twenty-four-hour profiles and pulsatile patterns of insulin secretion in normal and obese subjects. J Clin Invest 1988; 81:442448.)

this section, the physiology of normal insulin and the alterations that are present in persons with type 2 diabetes are reviewed. Quantitation of Beta Cell Function

The measurement of peripheral insulin concentrations by radioimmunoassay is still the most widely used method for quantifying beta cell functions in vivo. [339] Although this approach provides valuable information, it is limited by the fact that 50% to 60% of the insulin produced by the pancreas is extracted by the liver without ever reaching the systemic circulation. [340] [341] The standard radioimmunoassay for the measurement of insulin concentrations is also unable to distinguish between endogenous and exogenous insulin, making it ineffective as a measure of endogenous beta cell reserve in the insulin-treated diabetic patient. Anti-insulin antibodies that may be present in patients treated with insulin interfere with the insulin radioimmunoassay, making insulin measurements in insulin-treated patients inaccurate. Conventional insulin radioimmunoassays are also unable to distinguish between levels of circulating proinsulin and true levels of circulating insulin. Insulin is derived from a single-chain precursor, proinsulin. [342] Within the Golgi apparatus of the pancreatic beta cell, proinsulin is cleaved by convertases to form insulin, C peptide, and two pairs of basic amino acids. Insulin is subsequently released into the circulation at concentrations equimolar with those of C peptide. [343] [344] In addition, small amounts of intact proinsulin and proinsulin conversion intermediates are released. Proinsulin and its related conversion intermediates can be detected in the circulation, where they constitute 20% of the total circulating insulin-like immunoreactivity. [345] In vivo, proinsulin has a biologic potency that is only about 10% of that of insulin [346] [347] and the potency of split proinsulin intermediates is between those of proinsulin and insulin. [348] [349] C peptide has no known conclusive effects on carbohydrate metabolism, [350] [351] although certain physiologic effects of C peptide have been proposed. [352] Unlike insulin, C peptide is not extracted by the liver[341] [353] [354] and is excreted almost exclusively by the kidneys. Its plasma half-life of approximately 30 minutes [355] contrasts sharply with that of insulin, which is approximately 4 minutes. Because C peptide is secreted in equimolar concentrations with insulin and is not extracted by the liver, many investigators have used levels of C peptide as a marker of beta cell function. The use of plasma C-peptide levels as an index of beta cell function is dependent on the critical assumption that the mean clearance rates of C peptide are constant over the range of C-peptide levels observed under normal physiologic conditions. This assumption has been shown to be valid for both dogs and humans,[341] [356] and this approach can be used to derive rates of insulin secretion from plasma concentrations of C peptide under steady-state conditions. [356] However, because of the long plasma half-life of C peptide, under nonsteady-state conditions (e.g., after a glucose infusion) peripheral plasma levels of C peptide do not change in proportion to the changing insulin secretory rate. [356] [357] Thus, under these conditions, insulin secretion rates are best calculated with use of the two-compartment model initially proposed by Eaton and co-workers. [358] Modifications to the C-peptide model of insulin secretion have been introduced. This approach combines the minimal model of insulin action with the two-compartment model of C-peptide kinetics and allows insulin secretion and insulin sensitivity to be derived after either intravenous or oral administration of glucose. [359] [360] [361] [362]



Signaling Pathways in the Beta Cell and Insulin Secretion

Figure 29-12 depicts the signaling pathways in the pancreatic beta cell that are involved in the stimulus-secretion coupling of insulin release. These pathways provide the mechanism whereby insulin secretion rates respond to changes in blood glucose concentrations. Glucose enters the pancreatic beta cell by a process of facilitated diffusion mediated by the glucose transporter GLUT2. Although levels of GLUT2 on the beta cell membrane are reduced in diabetic states for various reasons, it is not currently believed that this is a rate-limiting step in the regulation of insulin secretion. The first rate-limiting step in this process is the phosphorylation

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of glucose to glucose-6-phosphate. This reaction is mediated by the enzyme glucokinase. [363] [364] There is considerable evidence that glucokinase, by determining the rate of glycolysis, functions as the glucose sensor of the beta cell and that this is the primary mechanism whereby the rate of insulin secretion adapts to changes in blood glucose. According to this view, as blood glucose levels increase more glucose enters the beta cell, the rate of glycolysis increases, and the rate of insulin secretion increases. A fall in blood glucose levels results in a fall in the rate of glycolysis and a reduction in the rate of insulin secretion. Glucose metabolism produces an increase in cytosolic ATP, the key signal that initiates insulin secretion by causing blockade of the ATP-dependent K + channel (K ATP ) on the beta cell membrane. Blockade of this channel induces membrane depolarization, which leads to an increase in cytosolic Ca 2+ and insulin secretion. The biochemical events that link the increase in glycolysis to an increase in ATP are complex. Dukes and co-workers [365] proposed the glycolytic production of NADH during the oxidation of glyceraldehyde-3-phosphate as the key process because NADH is subsequently processed into ATP by mitochondria through the operation of specific shuttle systems. The rate of pyruvate generation has also been proposed as an explanation for the link between glucose metabolism and the increase in insulin secretion. [366] According to this view, pyruvate generated by the glycolytic pathway enters the mitochondria and is metabolized further in the TCA cycle. Electron transfer from the TCA cycle to the respiratory chain by NADH and reduced flavin adenine dinucleotide (FADH 2 ) promotes the generation of ATP, which is exported into the cytosol. The increase in ATP closes ATP-sensitive K + channels, which depolarizes the beta cell membrane and opens the voltage-dependent Ca 2+ channels, leading to an increase in intracellular Ca 2+ . The increase in cytosolic Ca 2+ is the main trigger for exocytosis, the process by which insulin-containing secretory granules fuse with the plasma membrane, leading to the release of insulin into the circulation. The increase in ATP not only closes K ATP channels but also serves as a major permissive factor for movement of insulin granules and for priming of exocytosis. Cyclic AMP also plays an important role in beta cell signal transduction pathways. This second messenger is generated at the plasma membrane from ATP and potentiates glucose-stimulated insulin secretion, particularly in response to glucagon, glucagon-like peptide 1 (GLP-1), and gastric inhibitory polypeptide. The cAMP-dependent pathways appear to be particularly important in the exocytotic machinery. KATP channels play an essential role in beta cell stimulus-secretion coupling. The reader is directed to an excellent review for more complete information. [367] KATP channels comprise sulfonylurea receptors (SURs) and potassium inward rectifiers, KIR6.1 and KIR6.2, that assemble to form a large octameric channel with a (SUR/KIR6.x) stoichiometry. In the pancreatic beta cell the SUR1/KIR6.2 pairs constitute the K ATP channel. KATP channels control the flux of potassium ions driven by an electrochemical potential. Opening these channels can set the resting membrane potential of beta cells below the threshold for activation of voltage-gated Ca 2+ channels when plasma glucose levels are low, thus reducing insulin secretion. Changes in the cytosolic concentrations of ATP and adenosine diphosphate (ADP) as summarized earlier lead to closure of the channels and depolarization of the beta cell membrane. Mutations in both components of the beta cell K ATP , SUR1, and KIR6.2 have been shown to lead to hypersecretion of insulin resulting clinically in either a recessive form of familial hyperinsulinemia or persistent hyperinsulinemic hypoglycemia of infancy.



Physiologic Factors Regulating Insulin Secretion Carbohydrate Nutrients

The most important physiologic substance involved in the regulation of insulin release is glucose. [368] [369] [370] The effect of glucose on the beta cell is dose-related. Dose-dependent increases in concentrations of insulin and C peptide and in rates of insulin secretion have been observed after oral and intravenous glucose loads, with 1.4 units of insulin, on average, being secreted in response to an oral glucose load as small as 12 g. [371] [372] [373] [374] The insulin secretory response is greater after oral than after intravenous glucose administration. [374] [375] [376] [377] Known as the incretin effect, [373] [378] this enhanced response to oral glucose has been interpreted as an indication that absorption of glucose by way of the gastrointestinal tract stimulates the release of hormones and other mechanisms that ultimately enhance the sensitivity of the beta cell to glucose (see following discussion of hormonal factors). In a study involving nine normal volunteers in whom glucose was infused at a rate designed to achieve levels previously attained after an oral glucose load, the amount of insulin secreted in response to the intravenous load was 26% less than that secreted in response to the oral load. [377] Insulin secretion does not respond as a linear function of glucose concentration. The relationship of glucose concentration to the rate of insulin release follows a sigmoidal curve, with a threshold corresponding to the glucose levels normally seen under fasting conditions and with the steep portion of the dose-response curve corresponding to the range of glucose levels normally achieved postprandially. [379] [380] [381] The sigmoidal nature of the dose-response curve has been attributed to a gaussian distribution of thresholds for stimulation among the individual beta cells. [381] [382] [383] When glucose is infused intravenously at a constant rate, an initial biphasic secretory response is observed that consists of a rapid, early insulin peak followed by a second, more slowly rising peak. [368] [384] [385] The significance of the first-phase insulin release is unclear, but it may reflect the existence of a compartment of readily releasable insulin within the beta cell or a transient rise and fall of a metabolic signal for insulin secretion. [386] Despite early suggestions to the contrary, [387] [388] a subsequent study demonstrated that the first-phase response to intravenous glucose is highly reproducible within subjects. [389] After the acute response, a second phase of insulin release occurs that is directly related to the level of glucose elevation. In vitro studies of isolated islet cells and the perfused pancreas have identified a third phase of insulin secretion commencing 1.5 to 3.0 hours after exposure to glucose and characterized by a spontaneous decline in secretion to 15% to 25% of the amount released during peak secretiona level subsequently maintained for more than 48 hours. [390] [391] [392] [393] In addition to its acute secretagogue effects on insulin secretion, glucose has intermediate and longer term effects that are physiologically and clinically relevant. In the intermediate term, exposure of the pancreatic beta cell to a high concentration of glucose primes its response to a subsequent glucose stimulus leading to a shift to the left in the dose-response curve relating glucose and insulin secretion. [394] [395] However, when pancreatic islets are exposed to high concentrations for prolonged periods, a reduction of insulin secretion is seen. Although all the precise mechanisms responsible for these adverse effects that have been termed glucotoxicity are not known, there is evidence that long-term exposure to high glucose reduces expression of a number of genes that are critical to normal beta cell function, including the insulin gene. [396] [397] Noncarbohydrate Nutrients

Amino acids have been shown to stimulate insulin release in the absence of glucose, the most potent secretagogues being

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the essential amino acids leucine, arginine, and lysine. [398] [399] The effects of arginine and lysine on the beta cell appear to be more potent than that of leucine. The effects of amino acids on insulin secretion are potentiated by glucose. [399] [400] [401] In contrast to amino acids, various lipids and their metabolites appear to have only minor effects on insulin release in vivo. Although carbohydrate-rich fat meals stimulate insulin secretion, carbohydrate-free fat meals have minimal effects on beta cell function. [402] Ketone bodies and short- and long-chain fatty acids have been shown to stimulate insulin secretion acutely both in islet cells and in humans. [403] [404] [405] [406] [407] The effects of elevated FFAs in the insulin secretory responses to glucose are related to the duration of the exposure. Zhou and Grill [408] first suggested that long-term exposure of pancreatic islets to FFAs inhibited glucose-induced insulin secretion and biosynthesis. This observation has been confirmed in rats. [409] In humans, it was demonstrated that the insulin resistance induced by an acute (90-minute) elevation in FFAs was compensated for by an appropriate increase in insulin secretion. [410] After chronic elevation of FFAs (48 hours), the beta cell compensatory response for insulin resistance was not adequate. Additional studies have demonstrated that the adverse effects of prolonged FFAs on glucose-induced insulin secretion are not seen in individuals with type 2 diabetes. From these results, it appears that elevated FFAs may contribute to the failure of beta cell compensation for insulin resistance. Hormonal Factors

The release of insulin from the beta cell after a meal is facilitated by a number of gastrointestinal peptide hormones, including glucose-dependent insulinotropic peptide (GIP), cholecystokinin, and GLP-1. [378] [411] [412] [413] [414] [415] [416] [417] [418] These hormones are released from small intestinal endocrine cells postprandially and travel in the blood stream to reach the beta cells, where they act through second messengers to increase the sensitivity of these islet cells to glucose. In general, these hormones are not of themselves secretagogues, and their effects are evident only in the presence of hyperglycemia. [411] [412] [413] The release of these peptides may explain why the modest postprandial glucose levels achieved in normal subjects in vivo have such a dramatic effect on insulin production, whereas similar glucose concentrations in vitro elicit a much smaller response. [418] Similarly, this incretin effect could account for the greater beta cell response observed after oral as opposed to intravenous glucose administration. Whether impaired postprandial secretion of incretin hormones plays a role in the inadequate insulin secretory response to oral glucose and to meals in IGT or diabetes is controversial, [419] [420] [421] [422] [423] [424] [425] [426] but pharmacologic doses of these peptides may have future therapeutic benefit. Subcutaneous administration of GLP-1, the most potent of the incretin peptides, lowers glucose in type 2 diabetic patients by stimulating endogenous insulin secretion and perhaps by inhibiting glucagon secretion and gastric empyting. [427] [428] Because of its short half-life, however, its longer acting analogue, exendin-4, has greater therapeutic promise. [429] Treatment with supraphysiologic doses of GIP during hyperglycemia has been shown to augment insulin secretion in normal [430] [431] but not in diabetic humans. [422] [431] Although cholecystokinin has the ability to augment insulin secretion in humans, whether it is an incretin at physiologic levels is not firmly established. [432] [433] [434] [435] Its effects are also seen largely at pharmacologic doses. [436] The postprandial insulin secretory response may also be influenced by other intestinal peptide hormones, including vasoactive intestinal polypeptide, [439] [440] [ 441] and gastrin,[438] [442] but the precise role of these hormones remains to be elucidated.

[437]

secretin,[438]

The hormones produced by pancreatic alpha and beta cells also modulate insulin release. Whereas glucagon has a stimulatory effect on the beta cell, [443] somatostatin suppresses insulin release. [444] It is currently unclear whether these hormones reach the beta cell by traveling through the islet cell interstitium (thus exerting a paracrine effect) or through islet cell capillaries. Indeed, the importance of these two hormones in regulating basal and postprandial insulin levels under normal physiologic circumstances is in doubt. Paradoxically, the low insulin levels observed during prolonged periods of starvation have been attributed to the elevated glucagon concentrations seen in this setting. [402] [445] [446] [447] [448] Other hormones that exert a stimulatory effect on insulin secretion include growth hormone, [449] glucocorticoids, [450] prolactin, [451] [452] [453] placental lactogen, [454] and the sex steroids.[455] Whereas all of the preceding hormones may stimulate insulin secretion indirectly by inducing a state of insulin resistance, some may also act directly on the beta cell, possibly to augment its sensitivity to glucose. Thus, hyperinsulinemia is associated with conditions in which these hormones are present in excess, such as acromegaly, Cushing's syndrome, and the second half of pregnancy. Furthermore, treatments with placental lactogen, [456] hydrocortisone, [457] and growth hormone[457]

are all effective in reversing the reduction in insulin response to glucose that is observed in vitro after hypophysectomy. Although hyperinsulinemia after an oral glucose load has been observed in patients with hyperthyroidism, [459] [460] the increased concentration of immunoreactive insulin in this setting may reflect elevations in serum proinsulin rather than a true increase in serum insulin. [461] [458]

Neural Factors

The islets are innervated by both the cholinergic and adrenergic limbs of the autonomic nervous system. Although both sympathetic stimulation and parasympathetic stimulation enhance secretion of glucagon, [462] [463] the secretion of insulin is stimulated by vagal nerve fibers and inhibited by sympathetic nerve fibers. [462] [463] [464] [465] [466] [467] Adrenergic inhibition of the beta cell appears to be mediated by the -adrenoceptor because its effect is attenuated by the -antagonist phentolamine [ 463] and reproduced by the 2 -agonist clonidine. [468] There is also considerable evidence that many indirect effects of sympathetic nerve stimulation play a role in regulation of beta cell function through stimulation or inhibition of somatostatin, 2 -adrenoceptors, and neuropeptides galanin and neuropeptide Y. [469] Parasympathetic stimulation of islets results in stimulation of insulin, glucagon, and pancreatic polypeptide directly and through the neuropeptides vasoactive intestinal polypeptide, gastrin-releasing polypeptide, and pituitary adenylate cyclaseactivating polypeptide. [469] In addition, sensory innervation of islets may play a role in tonic inhibition of insulin secretion through the neuropeptides calcitonin generelated peptide [470] [471] [472] and, less clearly, substance P. [473] [474] The importance of the autonomic nervous system in regulating insulin secretion in vivo is unclear. The neural effects on beta cell function cannot be entirely dissociated from the hormonal effects because some of the neurotransmitters of the autonomic nervous system are, in fact, hormones. Furthermore, the secretion of insulinotropic hormones such as GIP and GLP-1 postprandially has been shown to be under vagal [475] [476] and adrenergic [477] [478] control.



Temporal Pattern of Insulin Secretion

It has been estimated that, in any 24-hour period, 50% of the total insulin secreted by the pancreas is secreted under basal conditions and the remainder is secreted in response to

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meals.[479] [480] The estimated basal insulin secretion rates range from 18 to 32 units per 24 hours (0.7 to 1.3 mg). [356] [358] [371] [479] After meal ingestion, the insulin secretory response is rapid and insulin secretion increases approximately fivefold over baseline to reach a peak within 60 minutes ( Fig. 29-13 ; see Fig. 29-12 ). In these studies subjects consumed 20% of calories with breakfast and 40% with lunch and dinner, respectively. However, the amount of insulin secreted after each meal did not differ significantly. The rapidity of the insulin secretory response to breakfast is underscored by the fact that 71.6% ± 1.6% of the insulin secreted in the 4 hours after the meal was produced in the first 2 hours and the remainder in the next 2 hours. Insulin secretion did not decrease as rapidly after lunch and dinner and 62.8% ± 1.6% and 59.6% ± 1.4% of the total meal response were secreted in the first 2 hours after these meals. The normal insulin secretory profile is characterized by a series of insulin secretory pulses. After breakfast, 1.8 ± 0.2 secretory pulses were identified in normal volunteers and the peaks of these pulses occurred 42.8 ± 3.4 minutes after the meal. Multiple insulin secretory pulses were also identified after lunch and dinner. After these meals, up to four pulses of insulin secretion were identified in both groups of subjects. Thus, in the 5-hour time interval between lunch and dinner, an average of 2.5 ± 0.3 secretory pulses were identified, and 2.6 ± 0.2 were identified in the same period after dinner. Pulses of insulin secretion that did not appear to be meal-related were also identified. Between 11:00 PM and 6:00 AM and in the 3 hours before breakfast, on average 3.9 ± 0.3 secretory pulses were present in normal subjects. Thus, over the 24-hour period of observation, a total of 11.1 ± 0.5 pulses were identified in normal subjects. Close to 90% (87% ± 3%) of postmeal pulses in insulin secretion but only 47% ± 8% of nonmeal-related pulses were concomitant with a pulse in glucose. Oscillatory Insulin Secretion

In vivo studies of beta cell secretory function have demonstrated that insulin is released in a pulsatile manner. This behavior is characterized by rapid oscillations occurring every 8 to 15 minutes that are superimposed on slower (ultradian) oscillations occurring at a periodicity of 80 to 150 minutes. [481] The rapid oscillations persist in vitro and are therefore likely to be the result of metabolic pathways in the pancreatic beta cell that involved negative feedback loops with time lags. The rapid oscillations of insulin are of small amplitude in the systemic circulation, averaging between 0.4 and 3.2 µU/mL in several published human studies. [482] [483] [484] Because these values are close to the limits of sensitivity of most standard insulin radioimmunoassays, the characterization of these oscillations is subject to considerable pitfalls, [485] not the least of which is the need to differentiate between true oscillations of small amplitude and random assay noise. The latter problem has been overcome by the development of extremely sensitive enzyme-linked immunosorbent assays that allow the detection of extremely small changes in peripheral insulin concentrations. The application of these assays in studies involving frequent sampling from the peripheral circulation has led to a series of studies of the role of these oscillations in the overall regulation of insulin secretion. [486] [487] [488] [489] [490] These studies have suggested that increases in overall insulin secretion seen in response to a variety of secretagogues in various physiologic and pathophysiologic states are due to an increase in the amplitude of the bursts of insulin secretion. The studies have proposed that 75% of insulin secretion is accounted for by secretory bursts and the responses to GLP-1, sulfonylureas, and oral glucose are all mediated by an increase in the amplitude of insulin secretory pulses. Furthermore, consistent

Figure 29-13 Mean 24-hour profiles of insulin secretion rates in normal and obese subjects (top). The hatched areas represent ± 1 standard error of the mean. The curves in the lower panel were derived by dividing the insulin secretion rate measured in each subject by the basal secretion rate derived in the same subject. Mean data for the normal (dashed line) and obese (solid line) subjects are shown. (From Polonsky KS, Given BD, van Cauter E. Twenty-four-hour profiles and pulsatile patterns of insulin secretion in normal and obese subjects. J Clin Invest 1988; 81:442448.)

with observations made by O'Rahilly and colleagues [491] a number of years ago, relatives of patients with type 2 diabetes demonstrate a disorderly profile of the insulin secretory oscillations. A number of mathematical programs have been developed that allow these insulin secretory oscillations to be evaluated and studied. [492] The latest addition to the list is the development of ApEn and cross ApEn, which are statistics that measure temporal regularity of the oscillations in the insulin secretory profile. [493] The low amplitude of the rapid oscillations in the systemic circulation contrasts sharply with observations in the portal vein, where pulse amplitudes of 20 to 40 µU/mL have been recorded in dogs. [494] Although the physiologic importance of these low-amplitude rapid pulses in the periphery is unclear, they are likely to be of physiologic importance in the portal

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Figure 29-14 Patterns of insulin secretion in normal and obese subjects. Four representative 24-hour profiles from two normal-weight subjects (left) and two obese subjects (right). Meals were consumed at 0900, 1300, and 1800 hours. Statistically significant pulses of secretion are shown by the arrows. (From Polonsky KS, Given BD, van Cauter E. Twenty-four-hour profiles and pulsatile patterns of insulin secretion in normal and obese subjects. J Clin Invest 1988; 81:442448.)

vein. It is possible that the liver responds more readily to insulin delivered in a pulsatile fashion than to insulin delivered at a constant rate.

[ 495] [496] [497]

In contrast to the rapid oscillations, the slower (ultradian) oscillations are of much larger amplitude in the peripheral circulation. They are present under basal conditions but are amplified postprandially (Fig. 29-14) and have been observed in subjects receiving intravenous glucose, suggesting that they are not generated by intermittent absorption of nutrients from the gut. Furthermore, they do not appear to be related to fluctuations in glucagon or cortisol levels [388] and are not regulated by neural factors because these oscillations are also present in recipients of successful pancreas transplants. [498] [499] Many of these ultradian insulin and C-peptide pulses are synchronous with pulses of similar oscillatory period in glucose, raising the possibility that these oscillations are a product of the insulin-glucose feedback mechanism. Ultradian oscillations are self-sustained during constant glucose infusion at various rates, they are increased in amplitude after stimulation of insulin

secretion without change in frequency, and there is a slight temporal advance of the glucose versus the insulin oscillation. These findings suggest that the ultradian oscillations may be entirely accounted for by the major dynamic characteristics of the insulin-glucose feedback system, with no need to postulate the existence of an intrapancreatic pacemaker. [500] In support of this hypothesis, Sturis and colleagues [501] demonstrated that when glucose is administered in an oscillatory pattern, ultradian oscillations in plasma glucose and insulin secretion are generated that are 100% concordant with the oscillatory period of the exogenous glucose infusion. This close relationship between the ultradian oscillations in insulin secretion and similar oscillations in plasma glucose was further exemplified in a series of dose-response studies in which the largest amplitude oscillations in insulin secretion were observed in the subjects exhibiting the largest amplitude glucose oscillations, which in turn were directly related to the infusion dose of glucose. It has been shown that in normal humans, insulin is more effective in reducing plasma glucose levels when administered intravenously as a 120-minute oscillation than when delivered at a constant rate. These results indicate that the ultradian oscillations have functional significance. Circadian variations in the secretion of insulin have also been reported. When insulin secretory responses were measured during a 24-hour period during which subjects received three standard meals, the maximal postprandial responses were observed after breakfast. [480] [502] [503] These findings are mirrored by the results of studies in which subjects were tested for oral glucose tolerance at different times of the day and were found to exhibit maximal insulin secretory responses in the morning and lower responses in the afternoon and evening. [504] [505] [506] These diurnal differences are also noted in tests for intravenous glucose tolerance. Furthermore, although ultradian glucose and insulin oscillations are closely correlated during a constant 24-hour glucose infusion, the nocturnal rise in mean glucose levels

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Figure 29-15 Plasma insulin concentrations (A) and insulin secretion rates (B) in response to molar increments in the plasma glucose concentration during the graded glucose infusion in the insulin-resistant (dotted line) and insulin-sensitive (solid line) groups. (From Jones CNO, Pei D, Staris P, et al. Alterations in the glucose-stimulated insulin secretory dose-response curve and in insulin clearance in nondiabetic insulin-resistant individuals. J Clin Endocrinol Metab 1997; 82:18341838.)

is not accompanied by a similar increase in the insulin secretory rate. [507] It has been postulated that these diurnal differences may reflect diminished responsiveness of the beta cell to glucose in the afternoon and evening. [506]



Insulin Secretion in Obesity and Insulin Resistance

Obesity and other insulin-resistant states are associated with a substantially greater risk for the development of type 2 diabetes. The ability of the pancreatic beta cell to compensate for insulin resistance determines whether blood glucose levels remain normal in insulin-resistant subjects or whether the subjects develop glucose intolerance or diabetes. The nature of the beta cell compensation for insulin resistance involves hypersecretion of insulin even in the presence of normal glucose concentrations. This can occur only if beta cell sensitivity to glucose is increased. The increase in beta cell sensitivity to glucose seen in obesity appears to be mediated by two factors. First, increased beta cell mass is observed in obesity and other insulin-resistant states. [508] Second, insulin resistance appears to be associated with increased expression of hexokinase in the beta cell relative to the expression of glucokinase. [509] Because hexokinase has a significantly lower Michaelis constant (Km) for glucose than glucokinase, the functional effect of increased hexokinase expression is to shift the glucoseinsulin secretion dose-response curve to the left, leading to increased insulin secretion across a wide range of glucose concentrations. Assessment of the adequacy of the beta cell compensation for insulin resistance is important because this is the major determinant of the development of diabetes. In insulin-resistant states it is important to evaluate beta cell function in relation to the degree of insulin resistance. Kahn and co-workers [510] studied the relationship between insulin sensitivity and beta cell function in 93 relatively young, apparently healthy human subjects of varying degrees of obesity. The sensitivity index SI was calculated using the minimal model of Bergman as a measure of insulin sensitivity and was then compared with various measures of insulin secretion. [361] [511] The relationship between the SI and the beta cell measures was curvilinear and reciprocal for fasting insulin ( P < .0001), first-phase insulin response (AIR (acute insulin response) glucose; P < .0001), glucose potentiation slope ( n = 56; P < .005), and beta cell secretory capacity (AIRmax; n = 43; P < .0001). The curvilinear relationship between SI and the beta cell measures could not be distinguished from a hyperbola, that is, SI × beta cell function = constant. The nature of this relationship is consistent with a regulated feedback loop control system such that for any difference in SI, a proportionate reciprocal difference occurs in insulin levels and responses in subjects with similar carbohydrate tolerance. Thus, in human subjects with normal glucose tolerance and varying degrees of obesity, beta cell function varies quantitatively with differences in insulin sensitivity. The increase in insulin secretion that is observed with a fall in SI should be viewed as the beta cell compensation that allows normal glucose tolerance to be maintained in the presence of insulin resistance. The insulin resistance of obesity is characterized by hyperinsulinemia. Hyperinsulinemia in this setting reflects a combination of increased insulin production and decreased insulin clearance, but most evidence suggests that increased insulin secretion is the predominant factor. [512] [513] Both basal and 24-hour insulin secretory rates are three to four times higher in obese subjects and are strongly correlated with body mass index. Insulin secretory responses to intravenous glucose have been studied in otherwise healthy insulin-resistant subjects in comparison with insulin-sensitive subjects by means of a graded glucose infusion. Figure 29-15 depicts insulin concentrations and insulin secretion rates at each level of plasma glucose achieved, thereby constructing a glucose-insulin or glucoseinsulin secretion rate dose-response relationship. Both insulin concentrations and insulin secretion rates are increased in insulin-resistant subjects, resulting from a combination of increased insulin secretion and decreased insulin clearance. For each level of glucose, insulin secretion rates are higher in the insulin-resistant subjects, reflecting an adaptive response of the beta cell to peripheral insulin resistance. Similar compensatory hyperinsulinemia has been demonstrated using other clinical techniques such as the frequently sampled intravenous glucose tolerance test in obesity and other insulin-resistant states such as late pregnancy. [510] [514] The temporal pattern of insulin secretion is unaltered in obese subjects compared with normal subjects. Basal insulin secretion in obese subjects accounts for 50% of the total daily production of insulin, and secretory pulses of insulin occur every 1.5 to 2 hours. [503] [512] However, the amplitude of these pulses postprandially is greater in obese subjects. Nevertheless, when these postprandial secretory responses are expressed as a percentage of the basal secretory rate, the postprandial responses in obese and normal subjects are identical.



Insulin Secretion in Subjects with Impaired Glucose Tolerance

It has been suggested that insulin secretion may be normal in subjects with IGT. However, substantial defects in insulin

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Figure 29-16 Dose-response relationship between glucose and insulin secretory rate (ISR) after an overnight fast in control subjects (CON), normoglycemic subjects with a family history of noninsulin dependent diabetes mellitus (FDR), subjects with a nondiagnostic OGTT (NDX), subjects with impaired glucose tolerance (IGT), and subjects with noninsulin-dependent diabetes mellitus (NIDDM). BME, body mass index. (From Byrne MM, Sturis J, Sobel RJ, Polonsky KS. Elevated plasma glucose 2 h postchallenge predicts defects in beta-cell function. Am J Physiol 1996; 270:E572E579. The American Physiological Society, copyright 1996.)

secretion have been demonstrated in people with normal fasting glucose and glycosylated hemoglobin concentrations with glucose values greater than 140 mg/dL or 7.8 mmol/L 2 hours after ingestion of 75 g of glucose orally. Thus, defects in insulin secretion can be detected before the onset of overt hyperglycemia. Detailed study of insulin secretion in patients with IGT has demonstrated that consistent quantitative and qualitative defects are seen in this group. During oral glucose tolerance testing, there is a delay in the peak insulin response. [515] [516] [517] The glucoseinsulin secretion dose-response relationship is flattened and shifted to the right (Fig. 29-16) , and first-phase insulin responses to an intravenous glucose bolus are consistently decreased in relation to ambient insulin sensitivity. [518] [519] Further, abnormalities in first-phase insulin secretion were observed in first-degree relatives of patients with type 2 diabetes who exhibited only mild intolerance to glucose, [520] and an attenuated insulin response to oral glucose was observed in normoglycemic co-twins of patients with type 2 diabetes. [521] This pattern of insulin secretion during the so-called prediabetic phase was also seen in subjects with IGT who later developed type 2 diabetes [401] [522] [523] and in normoglycemic obese subjects with a recent history of gestational diabetes, [524] another group at high risk for type 2 diabetes. [525] Beta cell abnormalities may therefore precede the development of overt type 2 diabetes by many years. The temporal pattern of insulin secretory responses is altered in IGT and is similar to but not as pronounced as that seen in diabetic subjects (see later). There is a loss of coordinated insulin secretory responses during oscillatory glucose infusion, indicating that the ability of the beta cell to sense and respond appropriately to parallel changes in the plasma glucose level is impaired (Fig. 29-17) . Abnormalities in rapid oscillations of insulin secretion have also been observed in first-degree relatives of patients with type 2 diabetes who have only mild glucose intolerance, [491] further suggesting that abnormalities in the temporal pattern of beta cell function may be an early manifestation of beta cell dysfunction preceding the development of type 2 diabetes. Because an elevation in serum proinsulin is seen in subjects with diabetes, the contribution of proinsulin to the hyperinsulinemia of IGT has been questioned. The hyperinsulinemia of IGT has not been accounted

Figure 29-17 Oscillatory glucose infusions were administered with a periodicity of 144 minutes in representative subjects with type 2 diabetes, impaired glucose tolerance (IGT), and normal glucose tolerance. In the control subject, the insulin secretion rate (ISR) adjusts and responds to the 144-minute oscillations in glucose, resulting in sharp spectral peak at 144 minutes. In the subjects with IGT and type 2 diabetes, the ISR does not respond to the oscillatory glucose stimulus and although oscillations in insulin secretion are evident, they are irregular, resulting in markedly reduced spectral peaks at 144 minutes and small-amplitude high-frequency spectral peaks. (Adapted from O'Meara NM, Sturis J, Van Cauter E, Polonsky KS. Lack of control by glucose of ultradian insulin secretory oscillations in impaired glucose tolerance and in noninsulin-dependent diabetes mellitus. J Clin Invest 1993; 92:262271.)

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for by an increase in proinsulin, although elevations in fasting and stimulated proinsulin or proinsulin/insulin ratios have been found by many, although not all, investigators. [526] [527] [528] [529] [530] [531] Correlation of elevated proinsulin levels in IGT as a predictor of future conversion to diabetes has also been observed. [532] [533] [534]



Insulin Secretion in Type 2 Diabetes Mellitus

Because of the presence of concomitant insulin resistance, patients with type 2 diabetes are often hyperinsulinemic, but the degree of hyperinsulinemia is inappropriately low for the prevailing glucose concentrations. Nevertheless, many of these patients have sufficient beta cell reserve to maintain a eugly-cemic state by dietary restriction with or without an oral agent. The beta cell defect in patients with type 2 diabetes mellitus is characterized by an absent first-phase insulin and C-peptide response to an intravenous glucose load and a reduced second-phase response. [535] Although hyperglycemia may play a role in mediating these changes, the abnormal first-phase response to intravenous glucose persists in patients whose diabetic control has been greatly improved, [536] [537] consistent with the idea that patients with type 2 diabetes have an intrinsic defect in the beta cell. Furthermore, abnormalities in first-phase insulin secretion have also been observed in first-degree relatives of patients with type 2 diabetes who have only mild glucose intolerance, and an attenuated insulin response to oral glucose has been observed in normoglycemic co-twins of patients with type 2 diabetesa group at high risk for type 2 diabetes and who can legitimately be classified as prediabetic. [538] This pattern of insulin secretion during the so-called prediabetic phase is also seen in subjects with IGT who later develop type 2 diabetes and in normoglycemic obese subjects with a recent history of gestational diabetes, who are also at high risk for type 2 diabetes. Beta cell abnormalities may therefore precede the development of overt type 2 diabetes by many years. Type 2 diabetes also affects proinsulin levels in serum. Increased levels of proinsulin are consistently seen in association with increases in the proinsulin/insulin molar ratio. [535] The amount of proinsulin produced in this setting appears to be related to the degree of glycemic control rather than to the duration of the diabetic state, and in one series proinsulin levels contributed almost 50% of the total insulin immunore-activity in type 2 diabetes patients who had marked hyperglycemia. In addition to intact proinsulin, the beta cell secretes one or more of the four major proinsulin conversion products (split 32,33-, split 65,66-, des-31,32-, and des-64,65-proinsulin) into the circulation. These conversion products are produced within the secretory granules of the islet as a result of the activity of specific conversion enzymes at the two cleavage sites in proinsulin linking the C peptide to the A and B chains. The composition of the elevated proinsulin-like immunodeficiency (PLI) in patients with type 2 diabetes compared with control subjects has not been fully characterized. Hales and colleagues [539] have developed immunoradiometric assays for this purpose. Using these assays, split 32,33-proinsulin was reported to be the predominant proinsulin conversion product in the circulation, although des-31,32-proinsulin levels may also be elevated. Insulin, proinsulin, and conversion product concentrations were also measured with these assays 30 minutes after oral glucose in patients with type 2 diabetes. Insulin was reduced in all patients, with no overlap between patients and controls, and concentrations of proinsulin and conversion products were elevated in the diabetic patients. These data highlight the importance of the potentially confounding effects of proinsulin and proinsulin conversion products in the interpretation

Figure 29-18 Mean (± standard error of the mean [SEM]) rates of insulin secretion in type 2 diabetic patients compared with control subjects. The shaded area corresponds to 1 SEM above and below the mean in control subjects. The curves in the lower panel were derived by dividing, for each subject, the insulin secretion rate at each sampling time by the average fasting secretion rate measured between 6 AM and 9 AM in the same subject.

of circulating immunoreactive insulin in patients with type 2 diabetes and emphasize the need to measure the concentrations of the individual peptides. Abnormalities in the temporal pattern of insulin secretion have also been demonstrated in patients with type 2 diabetes. In contrast to normal subjects, in whom equal amounts of insulin are secreted basally and postprandially in a given 24-hour period, patients with type 2 diabetes secrete a greater proportion of their daily insulin under basal conditions [540] (Fig. 29-18) . This reduction in the proportion of insulin secreted postprandially appears to be related in part to a reduction in the amplitude of the secretory pulses of insulin occurring after meals rather than to a reduction in the number of pulses. In contrast to normal subjects, patients with type 2 diabetes have ultradian oscillations in insulin secretion that are less tightly coupled with oscillations in plasma glucose (Fig. 29-19) . Similar findings were observed in patients with IGT studied under the same experimental conditions and in a further group of type 2 diabetic patients studied under fasting conditions. The rapid insulin pulses are also abnormal in type 2 diabetes because the persistent regular rapid oscillations present in normal subjects are not observed. Instead, the cycles are of shorter duration and are irregular in nature. Similar findings were observed in a group of first-degree relatives of patients with type 2 diabetes who had only mild glucose intolerance, suggesting that abnormalities in oscillatory activity may be an early manifestation of beta cell dysfunction. [518] The effects of therapy on beta cell function in patients with type 2 diabetes have also been investigated. Although interpretation

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Figure 29-19 Temporal variations in postbreakfast, postlunch, and postdinner rates of insulin secretion in control and diabetic subjects. In each subject, the secretion rates during the 30 minutes before the meal and the 4 hours after breakfast or the 5 hours after lunch or dinner were expressed as a percentage of the mean rate of insulin secretion during that interval. The curves were obtained by concatenating the resulting postmeal profiles in eight representative subjects. The times at which the meals were served to successive subjects in the series are indicated by arrows. (From Polonsky KS, Given BD, Hirsch LJ, et al. Abnormal patterns of insulin secretion in noninsulin-dependent diabetes mellitus. N Engl J Med 1988; 318:12311239.)

of the results in many instances is limited by the fact that beta cell function was not always studied at comparable levels of glucose before and during therapy, the majority of the studies indicated that improvements in diabetic control are associated with an enhancement of beta cell secretory activity. This increased endogenous production of insulin appears to be independent of the mode of treatment and is in particular associated with increases in the amount of insulin secreted postprandially. [537] [541] The enhanced beta cell secretory activity after meals reflects an increase in the amplitude of existing secretory pulses rather than an increased number of pulses. Despite improvements in glycemic control, beta cell function is not normalized after therapy, suggesting that the intrinsic defect in the beta cell persists. Treatment with the sulfonylurea glyburide increases the amount of insulin secreted in response to meals but does not correct the underlying abnormalities in the pattern of insulin secretion. In particular, the abnormalities in the pulsatile pattern of ultradian insulin secretory oscillations persist on treatment with glyburide despite the increase in the amount of insulin secreted. We have also investigated the effects on insulin secretion of improving insulin resistance in subjects with IGT by using the insulin-sensitizing agent troglitazone, a thiazolidinedione. Troglitazone therapy improved insulin sensitivity, and this was associated with enhanced ability of the pancreatic beta cell to respond to a glucose stimulus as judged by improvements in the dose-response relationships between glucose and insulin secretion as well as enhanced ability of the pancreatic beta cell to detect and respond to small oscillations in the plasma glucose concentration. [542]



RODENT MODELS OF TYPE 2 DIABETES A number of spontaneous and genetically selected animal models of type 2 diabetes have been identified. Most of the models combine the two main features of type 2 diabetes, obesity-associated insulin resistance and beta cell dysfunction with or without diminished beta cell mass. As with diabetes in humans, the different rodent models of type 2 diabetes have similarities but a number of overt and subtle differences make them useful surrogates for intensive study of the syndromes associated with type 2 diabetes. An interesting observation is the striking sexual dimorphism in most rodent models of type 2 diabetes, with the male most often being affected exclusively, earlier, or more severely in most instances. In this regard, it is not like the human situation. The advent of transgenic and knockout technology in mice has produced a wide range of models of insulin resistance and beta cell dysfunction that results in hyperglycemia. It is beyond the scope of this chapter to review each of these, and the reader is referred to the primary literature for review of these animals. We limit our discussion to the well-documented spontaneous or derived models of the disease in rodents. Mouse Models of Type 2 Diabetes Leptin (Lepob) and the Leptin Receptor (db)

The ob mutation, now designated Lepob, was first described in 1950, [543] but the gene mutation responsible for the syndrome was not described until the ob mutation was found to be in the gene for leptin. [544] Mice homozygous for the ob mutation do not produce the satiety factor leptin and become markedly hyperphagic, obese, insulin-resistant, and hyperinsulinemic. They have a multitude of other hypothalamic dysfunctions that render them hypometabolic, contribute to the obesity, and also result in infertility. [545] [546] Leptin treatment of these mice results in decreased food intake and reverses many of their other metabolic defects. [547] [548] [549] [550] [551] The ob mice develop obesity at weaning that becomes progressive because of hyperphagia. Insulin resistance is seen in muscle, adipose tissue, and liver, with a variety of signaling defects that are also reversible with insulin administration. [552] The ob mouse becomes hyperglycemic and has a profound hyperinsulinemia associated with beta cell hyperplasia with up to a 10-fold increase in islet mass. [553] [554] Parabiotic experiments between the ob and db mice suggested that the db mutation would be in the receptor for ob. This was confirmed with the identification of multiple mutations in the leptin receptor in db. [555] [556] Like ob mice, db mice are hyperphagic and begin to surpass their littermates in

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weight at weaning. They are progressively hyperinsulinemic, become hyperglycemic at 6 to 8 weeks, and because of a decline in beta cell function [557] [558] [559] [560] become markedly hyperglycemic at 4 to 6 months. The reason for the more severe diabetes in the db mouse is not clear, but it may be due to background strain differences as similar defects in insulin signaling are seen in this animal model as well. [561] [562] [563] Treatment of both ob and db mice with insulin-sensitizing agents such as thiazolidinediones reversed the insulin resistance and ameliorated or prevented the onset of diabetes. [564] [565] Agouti Mouse

Dominant "yellow" mutations in the agouti gene produce obesity and hyperglycemia. Depending on the background strain, the agouti mutation has a variable phenotype. In susceptible strains, the onset of hyperinsulinemia begins at 6 weeks of age and insulin levels continue to increase with age with beta cell hyperplasia and hypertrophy. [566] [567] The agouti mutation results in systemic production of a protein normally expressed in the skin, most frequently because of a retrotransposon insertion into the promoter region of the gene. [568] Interestingly, a number of genes, including the fatty acid synthase gene, have both insulin and agouti response elements, which result in a marked increase in expression leading to increased hepatic fatty acid synthesis and enhanced fat deposition in adipocytes. [556] [569] [570] The hyperglycemia is postprandial, and the fasting glucose levels are usually normal. The exact function of the agouti gene is unknown, but the animals are hyperphagic and show enhanced growth. KK Mouse

These mice were originally bred for enhanced size but are not as obese as most other obese mice (usually less than 60 g). Breeding the KK into various background strains has produced variable insulin resistance, hyperinsulinemia, and hyperglycemia. The most studied stain is the KKA y produced in Japan. [571] This mouse has markedly increased insulin levels (>1000 µU/mL) when fed a high-fat diet. [572] [573] As the male mouse ages, glucose levels fall toward the normal range. The mutation responsible for the KK phenotype is unknown. NZO Mouse

New Zealand obese (NZO) mice were derived by inbreeding of abdominally obese outbred mice. [554] [574] [575] [576] NZO neonates have high birth weights, and mice of both sexes are large and at weaning exhibit an elevated carcass fat content. [574] Approximately 40% to 50% of group-caged NZO males, but not females, develop type II diabetes between 12 and 20 weeks of age when maintained with a chow diet containing 4.5% fat. [577] Obesity in NZO mice is characterized by widespread accumulation of subcutaneous as well as visceral fat. The obesity in these mice is accompanied by glucose intolerance in males associated with increased hepatic and peripheral insulin resistance. In contrast to those in ob and db mice, genes encoding certain gluconeogenic and glycolytic enzymes in the liver retain normal responsiveness to insulin, although there is evidence for an inappropriately active fructose-1,6-biphosphatase. [578] [579] [580] Defective beta cell insulin secretion from NZO islets in vitro and in vivo has been described. [574] There appears be a defect in the glycolytic pathway in beta cells leading to defective glucose-stimulated insulin release.[581] The genetics of NZO mice show a polygenic disorder, and none of the allelic variants have been discovered. Complicating the analysis of the model is the susceptibility of the mice to autoimmune disorders including a lupus-like syndrome [582] [583] and the insulin receptor. [584] There is also a maternal influence in the peripartum period in the development of the disorder, which may reflect substances in the maternal milk. [585] Gold Thioglucose-Induced Diabetes

Gold thioglucose induces specific lesions in the ventromedial hypothalamus and induces an initial chronic hyperinsulinemia that leads to hypoglycemia, hyperphagia, obesity, and the development of insulin resistance and hyperglycemia. [586] This model has been used as an example of pancreatic dysfunction preceding the induction of insulin resistance as opposed to pancreatic compensation for insulin resistance. Diabetes Induced by Fat Ablation

Three models of insulin-resistant diabetes have been created in which adipose tissue has been genetically eliminated by overproduction of foreign genes using the fat-specific promoter aP2. Expression of an attenuated diphtheria toxin in adipose tissue resulted in an age-dependent loss of fat, progressive insulin resistance, hyperinsulinemia, and significant diabetes. [587] [588] Adipose-specific expression of a constitutively active form of the sterol regulatory element-binding protein SREBP-1c also resulted in fat ablation. [589] Lipoatrophy has also been induced by fat-specific overexpression of a dominant-negative form of the transcription factor A-ZIP/F. [590] [591] The A-ZIP/F protein heterodimerizes with and inactivates basic zipper (B-ZIP) transcription factors, including activating protein-1 (AP-1) and CCAAT/enhancer binding protein (C/EBP) isoforms, probably disrupting normal fat development. The lack of fat in the various models leads to hepatomegaly, insulin resistance with hyperinsulinemia, hypoleptinemia and significant glucose intolerance, and diabetes. These mice represent a model of the human condition lipodystrophic diabetes and demonstrate the importance of fat in normal glucose homeostasis. It has been suggested that the lack of fat depots results in elevated

fatty acid delivery to liver and muscle and the development of insulin resistance. The diabetes in these animals can be variously treated by thiazolidinediones, [587] leptin administration, [592] and fat transplantation. [591] Interestingly, human lipodystrophy also responds to thiazolidinedione treatment, [593] suggesting that some of the effects of these compounds are not wholly dependent upon adipose tissue. C57BL/6J Mice Fed a High-Fat Diet

Male C57BL/6J (also know as B6) mice fed a high-fat, high-carbohydrate diet (58% fat by kilocalories) or a "Western" diet developed hyperglycemia, hyperinsulinemia, hyperlipidemia, and increased adiposity. [594] [595] Glucose-stimulated insulin secretion was blunted, and there was significant insulin resistance. [596] [597] Despite obesity, plasma leptin levels in the Western diet-fed B6 mice were significantly lower than in control mice in the absence of hyperphagia. [594] [598] The weight gain is due primarily to an increase in mesenteric adiposity, which makes this a good model for adult-onset type 2 diabetes. Nagoya-Shibata-Yasuda (NSY) Mice

The NSY mouse shows male-specific, mild IGT with only a minority of the females becoming diabetic. [599] An impairment in beta cell function and obesity are present. These mice do not show the typical islet hyperplasia associated with insulin resistance. TallyHo Mice

The TallyHo mouse also has a male-only development of diabetes associated with beta cell hyperplasia. Both male and

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female TallyHo mice are obese, hyperinsulinemic, and hyperlipidemic, with the males having glucose levels greater than 500 mg/dL.


[600]


Rat Models of NIDDM Zucker Diabetic Fatty (ZDF) Rat

The orthologue of the db mouse, the obese Zucker rat (fa/fa), has a mutation in the leptin receptor that results in significant hyperphagia. [601] The fa mutation is distinct from the mutations in db in that it does not disrupt leptin receptor gene expression and does not affect ligand binding. [601] [602] This mutation results in a constitutive intracellular signaling domain, which may induce a desensitization of the leptin signaling pathways. [603] The selection of the inbred ZDF strain utilized Zucker ( fa/fa) rats that had progressed to a diabetic phenotype. Brother-sister mating resulted in nearly 100% diabetes in the male rats receiving a 5% fat diet. [604] Hyperglycemia begins to develop in males at 7 weeks of age, with serum glucose levels rising to 500 mg/dL by 12 weeks of age. The hyperinsulinemia precedes hyperglycemia with marked islet hyperplasia with dysmorphogenesis, [605] but by 19 weeks of age insulin levels drop concomitantly with islet atrophy, in part because of an imbalance of hyperplasia and apoptosis. [606] The islets of prediabetic ZDF rats secrete significantly more insulin in response to glucose with elevated basal levels of insulin secretion and a leftward shift but a blunted glucose dose-response curve. [607] [608] Islets of prediabetic male ZDF rats also have defects in the normal oscillatory pattern of insulin secretion. [607] In contrast to the male ZDF rat, the female rat has significant insulin resistance but does not become diabetic unless given a proprietary high-fat diet (GMI 13004). The high-fat diet appears to have a direct effect on the beta cell as there is no change in peripheral insulin sensitivity (P. Hansen and C. F. Burant, unpublished). Interestingly, there is a decrease in peripheral triglyceride and FFA levels in the female rat after the institution of the high-fat diet.

[ 609]

The underlying genetic defect that results in beta cell failure in the ZDF rat is unknown. The beta cell number and insulin content are not different from those in homozygous normal animals, but insulin promoter activity is doubled in the ZDF rat. [610] Insulin promoter mapping studies suggest that a critical region in the promoter of the insulin gene is affected. A number of other gene expression differences have been described in ZDF islets, including decreases in the expression of GLUT2 [611] [612] ; increases in glucokinase and hexokinase activity [ 608] ; decreases in mitochondrial metabolism [608] ; accumulation of intraislet lipid and long-chain fatty acyl CoA, which is associated with abnormal beta cell secretion [613] [614] [615] ; and increases in nitric oxide and ceramide accumulation, [616] [617] which is associated with apoptosis. Other gene expression changes are also found in the prediabetic rat islet. [618] Which of these defects are important for the development of the diabetes is not clear. Despite the fixed genetic defect in the male animal that leads to diabetes, this defect interacts with the insulin resistance because treatment with insulin-sensitizing agents can prevent the onset of diabetes in the male and female. [615] [619] These agents are not effective in the male after establishment of diabetes; however, the female rat can respond to thiazolidinediones, even after significant hyperglycemia. Goto-Kakizaki (GK) Rats

The GK inbred rat strain was derived from outbred Wistar rats by selection for IGT. [620] Early in the development of diabetes, there are mild elevations of both glucose and insulin levels in the GK rat, but as the animals age, reduced beta cell mass is evident with markedly diminished insulin stores and abnormal secretory responses to glucose.[621] [622] A number of biochemical defects have been described in the islets of these animals, including decreased energy production, [623] [624] [625] expression of proteins involved in insulin granule movement, [626] and decreased adenylate cyclase activity. [627] Defects in peripheral signaling include decreased maximal and submaximal insulin-stimulated IRS1 tyrosine phosphorylation, IRS1-associated PI 3-kinase activity, and Akt activation in muscle [628] and defective regulation of protein phosphatase-1 (PP-1), PP-2A, and mitogen-activated protein kinase activation by upstream insulin signaling components in adipocytes. [629] Some of these defects may be due to hyperglycemia because they can be reversed by phlorizin-induced normalization of serum glucose. [628] Bureau of Home Economics (BHE/Cdb) Rats

The BHE/Cdb rat is a subline of the parent BHE obtained by selection for hyperglycemia and dyslipidemia without obesity. [630] Glucose-stimulated insulin secretion is markedly diminished in these rats, a trait that is maternally inherited. [631] A significant defect appears to be in the liver, where increased gluconeogenesis and lipogenesis precede the hyperglycemia, which may be due to defects in mitochondrial respiration associated with mitochondrial DNA mutations. [632] [633] Psammomys obesus (Sand Rat)

This is a nutritionally induced obesity model of type 2 diabetes. Genetically, the Sand Rat is in reality a gerbil and the animal usually lives on a low-calorie vegetable diet.[634] When given a high-carbohydrate diet, the Sand Rat rapidly becomes hyperglycemic secondary to weight gain associated with significant insulin resistance [635] and enhanced hepatic glucose production. [636] When a relatively hypocaloric diet is restored, the metabolic syndrome reverts to normoglycemia. A subpopulation of the Sand Rat develops frank beta cell failure and becomes ketotic. Otsuka Long-Evans Tokushima Fatty (OLETF) Rats

The OLETF rat strain was derived from the Long-Evans rat with polyuria, polydipsia, and mild obesity. [637] About 90% of the male animals become diabetic by 1 year of age. Statistical tests have determined that the locus containing the cholecystokinin A receptor is responsible for about 50% of the NIDDM in the OLETF rats. [638] The receptor is disrupted in the OLETF rat because of a 165-bp deletion in exon 1. [639] [640] Genetic segregation analysis has also shown interaction with a second locus, Obd2, which acts in a synergistic fashion to result in NIDDM, and both of these loci are required in homozygous OLETF rats to cause elevated plasma glucose. [641] The role of sex hormones is pronounced in this strain as orchiectomy markedly reduces the incidence of diabetes whereas ovariectomy increases hyperglycemia to 30% in the female. Further, treatment of castrated males with testosterone restores the incidence to 89%. The islets undergo a progressive inflammatory reaction with progressive fibrosis. This reaction is associated with the impairment of beta cell function. [642] Obesity and insulin resistance appear to precede the development of beta cell failure. [643] Studies have also shown that obesity is necessary for the development of NIDDM in OLETF males and that insulin resistance may be closely related to fat deposition in the abdominal cavity. [644] Troglitazone and metformin have been used successfully to treat the diabetes in the OLETF rat, with troglitazone completely preventing the beta cell morphologic and functional deterioration. [645]

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Neonatal Streptozotocin

Two models have been described in which a single dose of the beta cell toxin streptozotocin is given to 2-day-old female Wistar [646] [647] or male Sprague-Dawley rats.[648] [649] These animals have a transient hyperglycemia but develop IGT at 4 to 6 weeks of age. There is an initial reduction of beta cell mass with regeneration resulting in an approximately 50% reduction in adulthood.



MANAGEMENT OF TYPE 2 DIABETES Over the last 10 years, a conceptual transformation in the principles of management of type 2 diabetes has occurred. Fundamentally, there has been a change in the level of concern about diabetes as a public health issue as well as in attitudes toward its treatment. Dramatic advances in the spectrum of pharmacologic agents and monitoring technology available for the treatment of diabetes have made it possible to lower glucose safely to the near-normal range in the majority of patients. Great strides have been made in establishing an evidence base for guidelines regarding glycemic control and efforts to reduce the risk of complications. Both corporate and government health insurance providers have greatly improved the extent to which diabetes equipment and supplies are covered. A comprehensive review of all the subtleties of diabetes management in the 21st century is beyond the scope of this chapter. In the following pages, we deal with the salient features of the epidemiology of the complications of type 2 diabetes, diagnostic strategies, treatment guidelines, lifestyle interventions, and pharmacotherapy before turning briefly to a discussion of preventive measures for type 2 diabetes and its complications. An excellent source of information on these issues that is updated annually is the American Diabetes Association's Clinical Practice Recommendations. It is published as the first supplement to the journal Diabetes Care each January and is available on line at www.diabetes.org by clicking "For Health Care Professionals"; near the end of that document is a listing of technical reviews, which are generally recent, fairly exhaustive treatments of most areas of interest in diabetes care. Scope of the Problem

Type 2 diabetes is estimated to affect some 17 million to 20 million people in the United States. There is an epidemic of diabetes nationwide with a 6% annual growth rate in the prevalence of the disease. Worldwide, the prevalence of diabetes is increasing even faster. This increase is driven by population aging; population growth, particularly among ethnic groups with greater susceptibility to the disease; and dramatic increases in rates of obesity as a consequence of increasingly sedentary lifestyles and greater consumption of simple sugars and high-caloric-density foods. At least in the United States, opportunistic screening for diabetes in high-risk populations is recommended by professional societies and many insurers, resulting in an increase in the proportion of affected individuals diagnosed in this country from approximately one half a decade ago to about two thirds. [650] [651] [652] [653] The morbidity, mortality, and expense associated with diabetes are staggering. In Western society, people with diabetes are three times more likely to be hospitalized than nondiabetic individuals. In the United States, diabetes is the leading cause of blindness and accounts for over 40% of the new cases of end-stage renal disease. The risk of heart disease and stroke is 2 to 4 times higher and the risk of lower extremity amputation is approximately 20 times higher for people with diabetes than for those without. Life expectancy is reduced by approximately 10 years in people with diabetes, and although diabetes is the seventh leading cause of death in the United States, this is clearly an underestimate. Despite the fact that some 70% of people with diabetes die of heart disease and stroke, only approximately 10% have diabetes listed as a contributing cause on death certificates. Tragically, this enormous burden of death and disability has not been reduced by huge health care expenditures. In fact, the epidemic of diabetes is one of the drivers of increasing health care costs, with annual disbursements for people with diabetes approximately three to five times higher per capita than those for individuals without diabetes. In the United States, at least 15% of health care expenditures are related to the treatment of people with diabetes. Nevertheless, whereas rates of coronary artery disease are declining in the United States in general, this is not the case for people with diabetes. However, there is evidence that increased effort to control diabetes and its comorbidities can even reduce costs associated with diabetes and that a public health approach to diabetes can reduce the burden of complications of diabetes. [654] [655] [656] [657] [658] [659] [660]



Screening and Diagnosis

The role of screening to make the diagnosis of diabetes in asymptomatic individuals is an area of substantial controversy. No prospective randomized trials have examined the benefit of such a screening program. On the other hand, it seems self-evident that early diagnosis and intervention have at least the potential to reduce complications in a disease in which 20% to 50% of patients have a complication at the time of diagnosis. The cost-effectiveness of universal approaches to diabetes screening has been called into question. The American Diabetes Association (ADA) recommendations [661] for screening are based on a review that concludes, "Periodic, targeted, and opportunistic screening within the existing health care system seems to offer the greatest yield and likelihood of appropriate follow-up and treatment."[662] The ADA suggests that patients (i.e., screening should be performed only in the context of a routine health care setting) should be screened at 3-year intervals beginning at age 45 and that testing should be considered at an earlier age or be carried out more frequently if diabetes risk factors are present. Those risk factors are listed in Table 29-3 . Most groups recommend FPG as the most practical screening test, although it is recognized that its sensitivity is substantially lower than that of the OGTT. More recent data suggest that alternative screening strategies may have advantages. In a study employing glucose meters to measure random capillary blood glucose, values of 120 mg/dL or higher obtained at random without regard to meals were 75% to 84% sensitive and 86% to 90% specific for detecting diabetes as defined by either FPG or oral glucose tolerance testing. [663] In the future, it is possible that well-validated models will allow us to predict diabetes risk from standard biologic measures such as body mass index, blood pressure, and lipids with greater precision than today. [664] Classically, diabetes has been diagnosed on the basis of prospective epidemiologic data associating circulating glucose levels with the future development of diabetic retinopathy. In 1997, recommendations were made by an expert committee to change the diagnostic criteria for diabetes to improve the sensitivity of FPG for the diagnosis of diabetes. [665] They determined, from a review of several large data sets, that FPG greater than or equal to 126 mg/dL (7.0 mM) identified a population of people with a risk of retinopathy similar to that of those with a 2-hour value in an OGTT of 200 mg/dL or

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higher. In an effort to simplify the OGTT, only the 2-hour plasma glucose after a 75-g oral glucose load needs to be measured for diagnostic purposes. Furthermore, patients with classical symptoms of diabetes in association with a random glucose level of 200 mg/dL or higher also meet diagnostic criteria for diabetes. To avoid misclassification, it is further suggested that patients should meet one of the three diagnostic criteria on at least two separate days before making the diagnosis of diabetes. Because macrovascular disease accounts for the majority of the morbidity and almost all the mortality associated with diabetes and the diagnosis of diabetes is associated with more stringent guidelines for the treatment of comorbidities such as dyslipidemia and hypertension, it seems likely that the diagnostic criteria for diabetes will be lowered again to make the fasting glucose cut points more sensitive. This seems appropriate, as it is clear that glucose levels above normal but below the current thresholds for diabetes are associated with increased cardiovascular risk. A debate unlikely to be answered in the next decade is whether it is acceptable to measure only fasting glucose as an index of glucose intolerance or whether it is cost-effective to use an oral challenge to ascertain fully glucose-related risks. [666] [667] [668] [ 669]



Glucose Treatment Guidelines

Prospective randomized clinical trials have documented improved rates of microvascular complications in patients with type 2 diabetes treated to lower glycemic targets. In the UK Prospective Diabetes Study (UKPDS), [670] patients with newonset diabetes were treated with diet and exercise for 3 months with an average reduction in glycosylated hemoglobin or HbA 1c from approximately 9% to 7% (upper limit of normal 6%). Those with FPG greater than 108 mg/dL (6 mM) were randomly assigned to two treatment policies. In the standard intervention, subjects continued the lifestyle intervention. Pharmacologic therapy was initiated only if the FPG reached 15 mM (270 mg/dL) or the patient became symptomatic. In the more intensive treatment program, all patients were randomly assigned and treated with either sulfonylurea, metformin, or insulin as initial therapy, with the dose increased to try to achieve an FPG less than 108 mg/dL. Combinations of agents were used only if the patients became symptomatic or FPG became greater than 270 mg/dL (15 mM). As a consequence of the design, although the HbA 1c fell initially to about 6%, over the average 10 years of follow-up it rose to approximately 8%. The average HbA 1c in the standard treatment group was approximately 1% higher. The risk of severe hypoglycemia was smallon the order of 1% to 5% per year in the insulin-treated groupand weight gain was modest; both were higher in patients randomly assigned to insulin and lower in those receiving metformin. [671] Associated with this improvement in glycemic control, there was a reduction in the risk of microvascular complications (retinopathy, nephropathy, and neuropathy) in the intensive group. Although there was a trend toward reduced rates of macrovascular events in the more intensively treated group, it did not reach statistical significance. [670] Similar reductions in microvascular events were observed in another trial of entirely different design and much smaller size. In the Kumamoto study, Japanese patients of normal weight with type 2 diabetes treated with insulin were randomly assigned to standard treatment or an intensive program of insulin therapy designed to achieve normal glycemia. The control group maintained HbA 1c values at approximately 9%, whereas the HbA1c in the intensive group was reduced to approximately 7% and the separation maintained for 6 years. Again, there was a modest increased risk of hypoglycemia and weight gain, a reduction in microvascular complications, and a non-statistically TABLE 29-6 -- Glycemic Targets Parameter

Normal

Premeal plasma glucose (mg/dL)

24 hr

20 mg, divided bid

5

Intermediate 1624 hr

3 mg

6 mg bid

3

Shorter

1 mg

8 mg qd

2

Long > 24 hr

Metabolized by liver to weakly active and inactive products, excreted in urine and bile. Mild diuretic activity. Highest risk of hypoglycemia.

Metabolized to inactive metabolities by liver, excreted in urine and bile.

GITS, gastrointestinal therapeutic system. Adapted from Facts and Comparisons, drug information monthly update service. St. Louis, JB Lippincott. of insulin in type 2 diabetes is designed to supplement endogenous production of insulin both in the basal state to modulate hepatic glucose production and in the postprandial state, in which a surge in insulin release normally facilitates glucose clearance into muscle and fat for storage to allow intraprandial metabolism. Currently, the vast majority of insulin used worldwide is of recombinant human origin. The available formulations largely differ in their pharmacokinetics as reviewed in Table 29-12 . Insulin lispro and insulin aspart are rapid-acting insulin analogues that have an onset of action in 5 to 15 minutes, peak activity in approximately 1 hour, and a duration of activity of approximately 4 hours. Regular insulin is approximately half as fast as the rapid-acting analogue with onset in 30 minutes, a peak at 2 to 4 hours, and a duration of action of 6 to 8 or more hours. Intermediate-acting insulinneutral protamine Hagedorn (NPH) and Lenteis approximately twice as slow as regular insulin with an onset of action in 1 to 2 hours, a peak at 4 to 8 hours, and a duration of action of 12 to 16 hours. Ultralente insulin is purportedly long acting, but the pharmacokinetics of human Ultralente are not dramatically different from those of NPH or Lente. Insulin glargine is a novel TABLE 29-12 -- Pharmacology of Insulin Duration (hr) IM or IV Dosing Forms and Modifiers

Insulin

Onset (hr)

Peak (hr)

Lispro, Aspart

0.100.25

0.752.0

3.04.0

No

Regular

0.51.0

1.04.0

4.010

t1/2 IM 20 min t1/2 IV 10 min None

Moderate

NPH

1.03.0

5.07.0

1318

No

Protamine

High

Lente

1.54.0

4.08.0

1320

No

Zinc

High

Ultralente

2.06.0

8.012

1830

No

Zinc

Very High

Glargine

2

No discrete peak

24

No

Insulin analogue, precipitates at neutral pH

Moderate

Insulin analogue, monomeric

Variability in Absorption Minimal

IM, intramuscular; IV, intravenous; NPH, neutral protamine Hagedorn. long-acting insulin analogue with distinctive properties. It provides a flat, peakless profile of activity with a duration of action of more than 24 hours in most patients. Premixed insulin formulations provide greater convenience and accuracy of mixing than those mixed by patients. Premixed formulations available in the United States are 70/30 and 50/50 mixtures of NPH and regular insulin, a 75/25 mixture of lispro insulin in its NPH-like formulation with insulin lispro, and a 70/30 mixture of insulin aspart with its NPH-like congener. Premixed insulin provides a profile of activity as expected from the addition of the activities of its components. Adverse events associated with insulin are well known and include weight gain and hypoglycemia. It is interesting that both fast-acting and long-acting insulin analogues have been shown to provide a modest reduction in hypoglycemia. Insulin allergies are rare, as are chronic skin reactionslipodystrophy and lipohypertrophy. It should be noted that the absolute risk of severe hypoglycemia in patients with type 2 diabetes is relatively small, approximately one third to one tenth as high as in similarly treated patients with type 1 diabetes. This risk can be further minimized with appropriate education of patients and expectant home glucose monitoring at times when

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unrecognized hypoglycemia is most likely to occurmidsleep or during unplanned or strenuous activity. There are rare examples of patients who develop irritation at injection sites (more common but rarely dose limiting with insulin glargine) or allergies. Newer insulin needles cause less discomfort than those previously available because of a finer gauge, shorter length, sharper points, and smoother surfaces. Insulin pen technology makes teaching a patient to take insulin much easier and provides greater convenience and accuracy of dosing. Insulin pump therapy has been used in patients with type 2 diabetes but is not widely accepted as cost-effective in routine use. Even though the vast majority of patients now find insulin therapy much easier and more effective than they had anticipated, there is still substantial resistance to initiating insulin therapy on the part of patients and providers. Practical Aspects of Initiating and Progressively Managing Type 2 Diabetes

A significant challenge in clinical decision making in diabetes is that the increased availability of therapeutic options for antidiabetic therapy is ahead of adequate prospective outcome studies. Currently available clinical trial data have not identified the preferred agents in type 2 diabetes, either as initial therapy or in subsequent care. Each class of drugs and even agents within each class have advantages and limitations, and individual issues may significantly affect the appropriate choice of therapy in particular patients. Table 29-10 (Table Not Available) highlights some of the relative advantages and disadvantages of various agents and classes. A general approach in the absence of any patient-specific factors is suggested in the algorithm presented in Figure 29-20 . A growing body of experience indicates that the use of metformin as initial therapy in combination with diet and exercise can provide impressive lowering of glucose with essentially no risk of hypoglycemia. Because this agent is available as a generic preparation, relative cost is low, and if the response is judged to be inadequate a thiazolidinedione, sulfonylurea, or glinide can be added. It has been proposed that the use of metformin alone or in combination with a thiazolidinedione

Figure 29-20 Treatment algorithm for type 2 diabetes. FPG, fasting plasma glucose; PPG, postprandial plasma glucose.

may lead to a greater reduction in cardiovascular risk than similarly effective (with respect to glycemia) approaches that increase insulin levels. At present, the data are not definitive on this point. Patients with higher levels of glucose (generally FPG > 200 mg/dL) almost always require agents to increase insulin levels. Because insulin, sulfonylureas, and glinides provide much faster improvements in overall control that metformin, glitazones, or AGIs, they are preferred in patients with higher levels of glucose either as monotherapy or as part of initial combined therapy. Starting a patient with a low dose of a glimepiride, glipizide-GITS, or insulin combined with either metformin, glitazone, or AGI is a reasonable initial approach to the poorly controlled condition. In patients who have reasonable control of fasting and preprandial plasma glucose levels (more than 50% of values less than 130 mg/dL) whose overall control as assessed by HbA1c is still higher than desired, monitoring may be either inaccurate or ineffective or postprandial plasma glucose (PPG) levels may be elevated. As it can be more difficult to have patients monitor in the postprandial state, it is important to remember that without specific therapy, almost all patients with type 2 diabetes have elevated PPG. Thus, in such patients, targeting presumed PPG elevations with the use of AGIs, glinides, or rapid-acting insulin analogues can theoretically lower average glucose with a lower risk of weight gain and hypoglycemia than with sulfonylureas or long-acting insulin. The most critical issue in long-term glycemic management is that of continuously reassessing with patients the adequacy of their control, examining glucose monitoring logs and HbA 1c values, and refining treatment regimens to achieve optimal control with the lowest dose of the least number of medications. Most patients in specialty care require two or more drugs to achieve recommended targets. Many patients require three or more (particularly if you consider long-acting and short-acting insulin as two agents). Fortunately, almost all the possible two-drug combinations and many of the three-drug combinations have been examined in modest-sized studies and have been shown to be safe and effective. Generally, it is preferred to add agents if there was an improvement in control with the first agent selected and to continue to add agents as

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needed to achieve goals. Subsequent back-titration to optimize treatment is often possible when glycemic goals are achieved. The selection of initial therapy should be based on mutually (patient and provider) recognized priorities. Increasingly, practitioners are using submaximal doses of agents in combination to increase the ratio of efficacy to adverse effects and in recognition of the potential synergy of sensitizers and secretagogues as well as the value of treatment of postprandial glucose and fasting glucose in combination therapy. When adding insulin in the management of inadequately controlled type 2 diabetes, some practitioners prefer to stop the oral antidiabetic agents and switch to insulin. Most generally continue the oral agents and add an evening dose of insulin. Classically, bedtime NPH insulin and more lately bedtime insulin glargine have been preferred for initiating insulin therapy. In more overweight patients (>120% of ideal body weight), the use of mixed insulin (or premixed insulin) at supper can help clear glucose elevations after the evening meal, generally the largest meal of the day. This works quite well in most patients, although some experience nocturnal hypoglycemia, which is less common with mixtures employing rapidacting insulin analogues. There are data suggesting that glargine given at bedtime can similarly provide for lower morning glucose values with less nocturnal hypoglycemia than NPH insulin, particularly in more overweight patients. Many patients eventually require more complex regimenstwice-daily injections, split-mix insulin, multiple injection regimens, and rarely insulin pump therapy. It should be noted that a minority of patients with type 2 diabetes have a better response to insulin administered in the morning than in the evening. It is important that both patient and health care provider agree on how to reach the goals of therapy. Therefore, biases and concerns of the patient should be addressed when trying to determine which agent should be prescribed. These biases can be elucidated in interviews with patients through discussions of various strategies. Strategies

Minimal Cost Strategy

For a large proportion of patients, particularly those who are elderly, drug costs are an overwhelming issue. Diet and exercise can be extremely effective and almost free. The least expensive drugs for the treatment of diabetes are the sulfonylureas; metformin has become available in generic formulations. Thus, a minimum cost strategy could start with a sulfonylurea and progress to the addition of generic metformin or bedtime or presupper insulin and finally two or more insulin injections per day if necessary. In the Veterans Administration Cooperative Study, excellent control was achieved in the context of a comprehensive program of diabetes education using a combination of daytime sulfonylurea and evening insulin. Although insulin is relatively inexpensive, in high doses (1 U/kg or more) the costs begin to rise, creating a rationale for adding metformin or a thiazolidinedione. It should be noted that most pharmaceutical companies have programs to provide no-cost or low-cost medication to the poor. Many of these are listed with links at www.needymeds.com. Furthermore, for increasing numbers of patients, the major driving force in their drug expenses is the number of prescriptions as each is associated with a copayment, providing a rationale for using combination agents. Minimum Weight Gain Strategy

Weight gain associated with the treatment of diabetes is of concern to most clinicians and is often an overriding issue with patients. A strategy to minimize weight gain would emphasize diet and exercise and would almost certainly employ metformin or an AGI as initial therapy with the addition of the other agent if one was inadequate. As sulfonylureas and repaglinide seem to have a modest weight-sparing effect in combination therapy with insulin, one or the other could be added before insulin administration in such a strategy. As discussed earlier, the weight gain associated with thiazolidinediones, although certainly a cosmetic issue, may not be associated with increased cardiovascular risk. Minimal Injection Strategy

Too many patients are determined to avoid insulin injections at any cost. The minimal injection strategy involves sulfonylureas, metformin, AGIs, and thiazolidinediones, which can be added in any order. Insulin, probably as a bedtime or presupper dose to minimize the inconvenience, would be added only if absolutely necessary. The strategy of using thiazolidinediones early in the course of diabetes in the hope that this may reduce the rate of progressive beta cell dysfunction remains unproved. It is important to try to dispel notions that insulin therapy is difficult, ominous, or fraught with peril by highlighting its efficacy and the great strides that have been made in insulin formulations and delivery devices. Most patients require insulin at some point in their lifetime.

Minimal Insulin Resistance Strategy

The possible atherogenic effects of insulin have been widely touted in the lay press and by marketing programs within the pharmaceutical industry. The relationship between circulating insulin levels and cardiovascular risk in nondiabetic populations is incontrovertible but probably related to the presence of insulin resistance rather than the insulin concentrations per se. Furthermore, in essentially all studies of intensive management with insulin, improved outcomes were observed with insulin treatment. There are no clinical data to suggest that exogenous insulin is associated with adverse side effects or long-term complications beyond its hypoglycemic effects and the associated weight gain. In any case, this strategy is analogous to the minimal injection strategy except that the order of introduction of agents is perhaps important. The thiazolidinediones have the greatest efficacy in reducing insulin resistance, metformin is second, and AGIs are third, with nateglinide associated with more specific stimulation of insulin levels after meals than the other insulin secretagogues, which all increase peripheral insulin levels less than injected insulin. Minimal Effort Strategy

Many patients are capable of making only a minimal effort with regard to their diabetes. Questioning patients about their pill-taking history and their realistic ability to comply with a prescribed frequency of therapy is important. Taking a once-a-day sulfonylurea or thiazolidinedione requires the least effort by the patient. Taking bedtime insulin is actually relatively well accepted by patients to whom this consideration is important. Developing strategies to improve adherence and increase motivation is certainly a long-term goal in this population. Hypoglycemia Avoidance Strategy

This is another important consideration for many patients. The AGIs have been reported in small studies to reduce "reactive" hypoglycemia. Other oral agents could be added in any order with the exception that insulin secretagogues would be added last, their dose minimized, and glyburide avoided. Nateglinide in particular among the secretagogues is associated with an exceptionally low risk of significant hypoglycemia. The

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insulin analogues are associated with a lower risk of hypoglycemia than human insulin. Postprandial Targeting Strategy

Achieving postprandial glucose targets is generally associated with better control than just meeting premeal targets. [705] On the basis of epidemiologic studies, it has been suggested that PPG is more highly correlated with cardiovascular disease risk than fasting glucose levels. Correction for confounding variables such as components of the multiple metabolic syndrome has not been performed, however. Furthermore, there are no outcome studies that have demonstrated the superiority of these approaches in the setting of type 2 diabetes. Control of postprandial glycemia can be achieved only with specific lifestyle efforts and pharmacologic agents, which target postprandial glucose. Postprandial glucose monitoring is helpful in this regard as it reinforces the goals and is the most effective measure to assess the effectiveness of treatment. Techniques that can improve postprandial control include lowering the carbohydrate content of meals, adding fiber, substituting monounsaturated fats for carbohydrates, encouraging physical activity after meals, adding AGIs with meals, and using rapidacting insulin analogues. Nateglinide and repaglinide provide a theoretical advantage in this situation compared with other secretagogues, although formal head-to-head studies have not been completed comparing the glinides with glimepiride and glipizide-GITS.



Prevention of Type 2 Diabetes

The possibility that type 2 diabetes can be prevented in high-risk individuals has been formally tested in a series of large-scale clinical trials. The Da Qing study randomly assigned clinics in an industrial city in China to a dietary intervention, exercise intervention, combined diet and exercise, or no intervention at all. Among the clinics, 577 subjects with IGT were studied. In this study, the interventions were quite modest and conducted largely in group settings. All three interventions led to reductions in the risk of conversion to diabetes of 31% to 46% compared with the control groups. [691] In a Finnish study, a similar number of middle-aged obese subjects with IGT were randomly assigned to a control group that received minimal lifestyle advice or to intensive, individualized instruction on food intake, increased physical activity, and weight reduction. The intensive lifestyle therapy group demonstrated a 58% relative risk reduction compared with the control group in the incidence of diabetes. [692] In the United States, the Diabetes Prevention Program enrolled over 3000 middle-aged, overweight subjects with IGT including substantial representation from high-risk minority groups. The intensive lifestyle group in this study also demonstrated a 58% relative risk reduction in the progression to diabetes.[690] In the Diabetes Prevention Program, there was another arm of the study that evaluated the ability of metformin at 500 mg twice a day to prevent the development of diabetes. It was moderately successful, with a 31% relative reduction in the progression of diabetes, although the benefit seemed to be greater in younger, more overweight, and more hyperglycemic subjects. In other studies not yet fully published, other oral antidiabetic agentstroglitazone in the Troglitazone in the Prevention of Diabetes (TRIPOD) study [690A] and acarbose in the STOP-NIDDM study [690B] have been reported to reduce the risk of developing diabetes. Patients and families as well as health care professionals are excited about the possibilities of preventing the disease. The success of the lifestyle interventions is impressive, demonstrating conclusively that with a variety of techniques it is possible for patients to achieve physiologically relevant changes in body weight. Medications overall had less positive impact than lifestyle intervention, although troglitazone did perform remarkably well in diabetes prevention. The questions that arise from these results are how to screen for people at risk and what intervention should be initiated in those with an interest in prevention. It seems reasonable to screen on the basis of current recommendations as outlined earlier primarily for case finding but also recognizing that patients with abnormal glucose values (fasting greater than 110 mg/dL or IGT with an OGTT) would be ideal candidates for preventive strategies. Certainly, high-risk individuals should be counseled on nutritional approaches to achieve weight loss, instructed to increase physical activity, and observed prospectively to determine whether progression of hyperglycemia has occurred. Treatment for other cardiovascular risk factors should also be considered if they are present. In the absence of outcome studies, it is difficult to recommend drug therapy to prevent diabetes because significant complications are unlikely to develop in the short window of time during which glucose levels increase from a fasting glucose of 110 to 126 mg/dL. An extension phase of the Diabetes Prevention Program that is under way should provide evidence concerning whether prevention or delay in the development of diabetes will prevent death or disability.



Future Directions

The present-day management of type 2 diabetes is significantly more effective and easier for patients than the situation that prevailed even 10 years ago. A better understanding of the barriers to effective diabetes management and how to overcome them would be of great benefit. The epidemic in diabetes and obesity that is under way coupled with the predicted early death and disability that follow threatens to overwhelm our health care system. Practical, cost-effective public health approaches to stem this tide are desperately needed. [706] Novel pharmaceutical agents including glucagon receptor antagonists, inhibitors of gluconeogenic and glycogenolytic pathways, activators of the insulin signaling pathways, modifiers of lipid metabolism, and antiobesity agents are areas of early pharmaceutical development. [707] There is tremendous interest in developing novel PPAR modulators that preserve the glucose-lowering effectiveness of the glitazones, enhance the lipid benefits, and mitigate the effects on fluid retention and weight gain. As these PPAR-active agents are thought to exert their action in the nucleus, there is reason to believe that such a goal is achievable. There are dozens of compounds in early stages of development and several already in phase III trials. The major barrier to success in this arena is the lack of well-validated animal models to predict human responses. [708] Novel methods of insulin delivery similarly have generated a great deal of enthusiasm among patients, particularly techniques to deliver insulin orally or by inhalation. [709] There is considerable controversy in the endocrine community in this regard. On the one hand, it seems unlikely that oral insulin delivery would be efficient enough to treat insulin resistance effectively; on the other hand, any delivery into the portal system could be more effective and perhaps have an improved safety profile because of preferential inhibition of hepatic glucose production. The area of gut hormones offers promise for advances in treatment of type 2 diabetes. Amylin, the second beta cell hormone, is known to act centrally to suppress postprandial glucagon secretion, slow gastric emptying, and increase satiety. Synthetic amylin is in late-phase trials, and although only modestly effective as a glucose-lowering agent, it does seem to be well tolerated and is associated with weight reduction. [710] GLP-1

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is a gut hormone secreted from intestinal cells that has an overlapping but generally more robust profile of action than amylin with the additional effect of preserving functioning beta cell mass. GLP-1 is rapidly degraded in the circulation, and thus inhibitors of the degrading enzyme (dipeptidylpeptidase IV [DP-IV]) as well as DP-IVresistant analogues are being investigated in clinical trials. [711] Additional studies will determine whether glucagon-like peptides provide the next major class of antidiabetic agents.



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Chapter 30 - Type 1 Diabetes Mellitus George S. Eisenbarth Kenneth S. Polonsky John B. Buse

In 1984, Sutherland and co-workers [1] transplanted the tail of the pancreas from nondiabetic identical twins to their twin mates with type 1 diabetes. In contrast to the transplantation of organs such as kidneys, in which the transplants are accepted between identical twins, pancreatic islets but not acinar pancreas were rapidly destroyed.[1] The diabetes of the twin transplant recipients was cured for only a matter of weeks. In retrospect, the results of these transplants were predictable, given the autoimmune nature of type 1A diabetes and similar results in animal models of the disorder. [2] Following this clinical study, type 1 diabetes became one of the most intensively studied autoimmune disorders, and the National Institutes of Health has designated type 1A diabetes a Priority One target for the development of a preventive immunologic vaccine. Knowledge of the immunogenetics and immunopathogenesis of type 1A diabetes is beginning to influence clinical care, [3] greatly influences current clinical research, and will, we hope, lead to disease prevention. [4]



DIFFERENTIAL DIAGNOSIS OF TYPE 1 DIABETES An expert committee of the American Diabetes Association, with its etiologic diagnostic criteria (Table 30-1) , has recommended dividing type 1 diabetes into type 1A (immune-mediated) and type 1B (other forms of diabetes with severe insulin deficiency). [5] At the onset of diabetes, distinguishing type 1A diabetes from type 2 diabetes, let alone type 1B diabetes, is not always a simple task. The best current criterion for diagnosis of type 1A diabetes is the presence of anti-islet autoantibodies measured with highly specific (and reasonably sensitive) autoantibody radioassays. [6] The presence of autoantibodies with assays defined as positive in less than 1 of 100 control subjects (specificity 99%) is reasonably diagnostic of type 1A diabetes. Non-Hispanic white children presenting with diabetes usually have type 1A diabetes, whereas adults older than 40 years usually have type 2 diabetes. [7] More than 90% of such children presenting with diabetes express one of three commonly measured autoantibodies (see later). In contrast, among black or Hispanic American children, almost one half lack any autoantibody. [8] [9] [10] Most of these children appear to have an early age of onset of type 2 diabetes mellitus, and many have attendant risk factors such as obesity and lack human leukocyte antigen (HLA) alleles associated with type 1A diabetes (see later). Imagawa and co-workers [11] described an unusual form of diabetes. The patients had normal hemoglobin A 1c (HbA1c ) despite severe hyperglycemia, suggesting that the diabetes had been present for only a short time. Histologic examination of pancreatic sections demonstrated pancreatitis but no insulitis, and anti-islet autoantibodies were not detected. It is likely that this represents one of the first examples of type 1B diabetes although a fulminant type 1A is possible.

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Condition Type 1A diabetes

TABLE 30-1 -- Differential Diagnosis of Type 1A Diabetes Islet Autoantibodies Genetics Autoantibody positive >90%

30%50% DR3 and DR4

Comments

Children:

90% DR3 or DR4

90% non-Hispanic white type 1A

200) and creatinine (>10) with normoglycemia. The pH and anion gap are usually only mildly abnormal. The treatment is supportive with careful attention to fluid and electrolytes until dialysis can be performed. Rhabdomyolysis is a cause of renal failure in which the anion gap can be significantly elevated and acidosis can be severe. There should be marked elevation of creatine phosphokinase and myoglobin. It should be noted that mild rhabdomyolysis is not uncommon in DKA, but the presence of hyperglycemia and ketonemia leaves no doubt about the primary etiology of the acidosis. [203] 5. Toxic ingestions can be differentiated from DKA by history and laboratory investigation. Salicylate intoxication produces an anion gap metabolic acidosis usually with a respiratory alkalosis. The plasma glucose is normal or low, the osmolality normal, ketones negative, and salicylates can be detected in the urine or blood. It should be noted that salicylates can cause a false-positive glucose determination when using the cupric sulfate method and a false-negative result when using the glucose oxidase reaction. Methanol and ethylene glycol also produce an anion gap metabolic acidosis without hyperglycemia or ketones but need to be kept in mind primarily because they produce an increase in the measured serum osmolality but not in the calculated serum osmolalityan osmolar gap. Their serum levels can also be measured. Isopropyl alcohol does not cause a metabolic acidosis but should be remembered because it is metabolized to acetone, which can produce a positive result in the nitroprusside reaction commonly used for the detection of ketoacids. These intoxications must be appropriately treated. [204] [205] [206] Rare cases of anion gap acidoses have been reported with other ingestions including toluene, iron, hydrogen sulfide, nalidixic acid, papaverine, paraldehyde, strychnine, isoniazid, and outdated tetracycline.

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When DKA is considered, the diagnosis can be made quickly with routine laboratory tests. Blood and urine glucose and ketones can be obtained in minutes with glucose oxidaseimpregnated strips and the nitroprusside reaction, respectively. Osmolarity

The increase in osmolarity that occurs in DKA must be differentiated from the increase in osmolarity seen in hyperosmolar-hyperglycemic nonketotic (diabetic) coma (HHNC). The osmolarity can be measured by freezing point depression or estimated using the following formula: Osmolarity (mOsm/L) = 2 × sodium + glucose/18 + BUN/2.8 + ethanol/4.6 Patients with DKA not uncommonly present with hyperosmolarity and coma. In HHNC, the osmolarity is generally greater than 350 mOsm/L and can exceed 400 mOsm/L. The serum sodium and potassium can be high, normal, or low and do not reflect total-body levels, which are uniformly depleted. The glucose is usually greater than 600 mg/dL, and levels over 1000 mg/dL are quite common. In pure HHNC, there is not a significant metabolic acidosis or anion gap. It should be remembered that patients often present with combinations of the preceding findings. HHNC can involve mild to moderate ketonemia and acidosis. Alcoholic ketoacidosis can contribute to either DKA or HHNC. Lactic acidosis is common in severe DKA and HHNC. Any patient with hyperglycemia greater than 250 mg/dL and an anion gap metabolic acidosis should be treated by the general principles outlined in the following with special consideration of other possible

contributing metabolic acidoses.



Therapy

The optimal management of DKA has been the source of considerable controversy over the past half-century. Only recently have prospective studies of various therapeutic approaches been performed. The guidelines we propose rely heavily on prospective studies of DKA by Kitabchi [207] and co-workers. The general approach is to (1) provide necessary fluids to restore the circulation, (2) treat insulin deficiency with continuous insulin, (3) treat electrolyte disturbances, (4) observe the patient closely and carefully, and (5) search for underlying causes of metabolic decompensation. Fluids

Volume contraction is one of the hallmarks of DKA. It can contribute to acidosis through lactic acid production as well as decreased renal clearance of organic and inorganic acids. It contributes to hyperglycemia by decreasing renal clearance of glucose. If decreased tissue perfusion is significant, it causes insulin resistance by decreasing insulin delivery to the sites of insulin-mediated glucose disposal, namely muscle and adipose tissue, as well as through stimulation of catecholamine and glucocorticoid secretion. Fluid deficits on the order of 5 to 10 L are common in DKA. It should be remembered that the urine produced during the osmotic diuresis of hyperglycemia is approximately half-normal with respect to sodium. Therefore, water deficits are in excess of sodium deficits. Historically, large quantities of isotonic intravenous fluids have been administered rapidly to patients in DKA. For patients with a history of congestive heart failure, chronic or acute renal failure, severe hypotension, or significant pulmonary disease, early invasive hemodynamic monitoring should be considered. When there is physical evidence of dehydrationthat is, hypotension, decreased skin turgor, or dry mucous membranesgenerally administer 1 L of normal saline over the first hour and 200 to 500 mL/hour in subsequent hours until hypotension resolves and adequate circulation is maintained. If hypotension is severe with clinical evidence of hypoperfusion and does not respond to crystalloid, therapy with colloid is considered, often in combination with invasive hemodynamic monitoring. If there is no hypotension and no concern about renal failure, administer 1 L of half-normal saline over the first hour. During that first hour, the laboratory data usually return and can be quite helpful in planning further therapy. Despite the excess of water losses over sodium, the measured sodium is usually low because of osmotic effects of glucose. These osmotic effects can be corrected using a simple formula: Corrected sodium concentration = measured sodium + 0.016(glucose 100) Severe hypertriglyceridemia, which is common in severe diabetes, can cause a false decrease in the serum sodium concentration by approximately 1.0 mEq/L at a serum lipid concentration of 460 mg/dL. [197] An estimated water deficit can be calculated using the corrected sodium: Water deficit in liters = 0.6 (weight in kg) [(sodium/140) 1] Using these formulas, a 70-kg patient with a measured sodium level of 140 mEq/L and a glucose concentration of 1000 mg/dL would have a calculated water deficit of 4.3 L. If the patient is normotensive after the first liter of fluids, it would be reasonable to aim to replace urinary losses with one-half normal saline and also provide approximately one half the water deficit as 5% dextrose over the first 12 to 24 hours (using the preceding example, 2 L) and the remainder over the subsequent 24 hours. The plan for fluid therapy should be continuously reevaluated in light of the clinical and laboratory response of the patient. When the serum glucose reaches 250 to 300 mg/dL, all fluids should contain 5% dextrose and therapy should be aimed at maintaining the serum glucose in that range for 24 hours to allow slow equilibration of osmotically active substances across cell membranes. The primary goal of fluid therapy is to maintain an adequate circulation and secondly to maintain a brisk diuresis. Beyond that, pulmonary edema, hyperchloremic metabolic acidosis, and a rapid fall in the serum osmolality should be avoided by frequent monitoring of the patient, glucose, and electrolytes. It has been demonstrated that fluid administration and subsequent continued osmotic diuresis are responsible for a large portion of the initial decline in glucose during therapy. Insulin

Insulin is the mainstay of therapy of DKA because it is essentially an insulin-deficient state. In the past, high doses of insulin (upward of 50 U/hour) were favored. In later studies, low-dose insulin therapy (0.1 U/kg per hour) has been shown to be as effective as higher doses in producing a decrease in serum glucose and clearance of ketones. Furthermore, low-dose therapy results in a reduction in the major morbidity of intensive insulin therapy, namely hypoglycemia and hypokalemia. Studies have also shown that intravenous insulin is significantly more effective than intramuscular or subcutaneous insulin in lowering the ketone body concentration over the first 2 hours of therapy. The subcutaneous route is inappropriate for the critically ill patient because of the possibility of tissue hypoperfusion and slower kinetics of absorption. There are numerous studies that attest to the efficacy of intramuscular therapy in severe DKA. In cases in which there is insufficient nursing monitoring or intravenous access to allow safe intravenous

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administration, intramuscular therapy would be the route of choice. Lastly, it has been shown that a 10-U intravenous insulin priming dose when insulin therapy is started significantly improves the glycemic response to the first hour of therapy. The rationale is to saturate insulin receptors fully before beginning continuous therapy and to avoid the lag time necessary to achieve steady-state insulin levels. When mixing insulin in normal saline, it does not seem to be necessary to add albumin to prevent insulin adsorption to the infusion set. However, the intravenous tubing should be flushed with the insulin infusate before use. In the rare instances in which the glucose does not decrease at least 10% or 50 mg/dL in an hour, the insulin infusion rate should be increased by 50% to 100% and a second bolus of intravenous insulin administered. As the glucose level decreases, it is usually necessary to decrease the rate of infusion. After the glucose reaches approximately 250 mg/dL, it is prudent to decrease the insulin infusion rate and administer dextrose. It usually takes an additional 12 to 24 hours to clear ketones from the circulation after hyperglycemia is controlled. With resolution of ketosis, the rate of infusion approaches the physiologic range of 0.3 to 0.5 U/kg per day. When the decision is made to feed the patient, the patient should be switched from intravenous or intramuscular therapy to subcutaneous therapy. Subcutaneous insulin should be administered before a meal and the insulin drip discontinued approximately 30 minutes later. The glucose should be checked in 2 hours and at least every 4 hours subsequently until a relatively stable insulin regimen is determined. Potassium

Potassium losses during the development of DKA are usually quite high (3 to 10 mEq/kg) and are mediated by shifts to the extracellular space secondary to acidosis and protein catabolism compounded by hyperaldosteronism and osmotic diuresis. Although most patients with DKA or HHNC have normal or even high serum potassium at presentation, the initial therapy with fluids and insulin causes it to fall. Our approach has been to monitor the electrocardiogram (ECG) for signs of hyperkalemia (peaked T wave, QRS widening) initially and to administer potassium if these are absent and the serum potassium is less than 5.5 mEq/L. If the patient is oliguric, we do not administer potassium unless the serum concentration is less than 4 mEq/L or there are ECG signs of hypokalemia (U wave), and even then it is done with extreme caution. With therapy of DKA, the potassium level always falls, usually reaching a nadir after several hours. We usually replace potassium at 10 to 20 mEq/hour, one half as potassium chloride and one half as potassium phosphate, and monitor serum levels at least every 2 hours initially as well as follow ECG morphology. Occasionally, patients with DKA who have had protracted courses with vomiting present with hypokalemia and acidosis and may require 40 to 60 mEq/hour by central line to avoid further decreases in the serum potassium.

Phosphate

Like potassium, phosphate is depleted in patients with DKA. Although patients usually present with elevated serum phosphate, the serum level declines with therapy. No well-documented clinical significance of these findings has been determined and no benefit of phosphate administration has been demonstrated, but most authorities recommend phosphate therapy as before and monitoring for its possible complicationshypocalcemia and hypomagnesemia. Bicarbonate

Serum bicarbonate is always low in DKA, but a true deficit is not present because the ketoacid and lactate anions are metabolized to bicarbonate during therapy. The use of bicarbonate in the therapy of DKA is highly controversial. No benefit of bicarbonate therapy has been demonstrated in clinical trials. In fact, in two trials, hypokalemia was more common in bicarbonate-treated patients. There are theoretical considerations against the use of bicarbonate. Cellular levels of 2,3-diphosphoglycerate are depleted in DKA, causing a shift in the oxyhemoglobin dissociation curve to the left and thus impairing tissue oxygen delivery. Acidemia has the opposite effect, and therefore reversing acidosis acutely could decrease tissue oxygen delivery. In addition, there are in vitro data suggesting that pH is a regulator of cellular lactate metabolism and correction of acidosis could increase lactate production. These observations are of questionable clinical relevance, however. We reserve bicarbonate therapy for use (1) in patients with severe acidosis (pH < 6.9), (2) in the presence of hemodynamic instability if the pH is less than 7.1, or (3) in cases of hyperkalemia with ECG findings. When bicarbonate is used, it should be used sparingly and considered a temporizing measure while definitive therapy with insulin and fluids is under way. Approximately 1 mEq/kg of bicarbonate is administered as a rapid infusion over 10 to 15 minutes with further therapy based on repeated arterial blood gases every 30 to 120 minutes. Potassium therapy should be considered before treatment with bicarbonate as transient hypokalemia is not an uncommon complication of the administration of alkali. Monitoring

It is possible to manage many cases of mild DKA without admission to the intensive care unit, depending on staff availability. We routinely admit patients with DKA to the intensive care unit if they have a pH less than 7.3. If mental status is compromised, prophylactic intubation is considered and nasogastric suctioning is always performed because of frequent ileus and danger of aspiration. If the patient cannot void at will, bladder catheterization is necessary to follow urine output adequately. ECG monitoring is continuous with hourly documentation of QRS intervals as well as T-wave morphology. Initially, serum glucose, electrolytes, BUN, creatinine, calcium, magnesium, phosphate, ketones, lactate, creatine phosphokinase, and liver function tests as well as urinalysis, ECG, upright chest radiograph, complete blood count, and arterial blood gases are obtained. If there is any concern about possible toxic ingestions, toxicology screening is also performed. Subsequently, glucose and electrolytes are measured at least hourly; calcium, magnesium, and phosphate every 2 hours; and BUN, creatinine, and ketones every 6 to 24 hours. It is often not necessary to monitor arterial blood gases routinely because bicarbonate and anion gap are relatively good indices of the response to therapy. Monitoring venous pH has also been shown to reflect acidemia and response to therapy adequately. Usually, frequent blood work is necessary only for the first 12 hours or so. In the severely ill patient with obvious underlying disease, the course is often more protracted and, particularly when venous access is a problem, early consideration should be given to placement of an arterial line. A flow sheet tabulating these findings as well as mental status, vital signs, insulin dose, fluid and electrolytes administered, and urine output allows easy analysis of response to therapy. When the acidosis begins to resolve and the response to therapy becomes predictable, it is reasonable to curtail laboratory use. If cardiovascular status is unclear or troublesome, invasive hemodynamic monitoring is an appropriate guide for fluid

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therapy. The goals should be to achieve hemodynamic stability rapidly and to correct DKA fully in 12 to 36 hours. Search for Underlying Causes

After stabilizing the patient, a careful history and physical examination and a diagnostic strategy should be aimed at determining the precipitating event. In most inner-city practices, the most common cause of DKA is noncompliance with insulin therapy and is usually easily treated. The second most common cause is infection, with viral syndromes, urinary tract infection, pelvic inflammatory disease, and pneumonia predominating. It is often difficult to determine initially whether the patient is infected. Fever can be absent in a significant proportion of patients with diabetic emergencies. The white blood cell count is not uncommonly elevated in the range of 20,000 or higher even in the absence of infection. [208] As a result, cultures should be performed for most patients, and if there is significant concern about infection, empirical broad antibiotic coverage should be considered pending microbiologic findings. Special consideration should be given to ruling out meningitis in the patient with altered mental status. In this regard, most would perform lumbar punctures in all patients with meningismus and in patients with disproportionate mental status changes. If the index of suspicion is lower, gear the antibiotic therapy to cover bacterial meningitis and perform a lumbar puncture if the mental status does not improve quickly with therapy. The cerebrospinal fluid glucose is not particularly useful in determining whether the fluid is infected, and a cerebrospinal fluid glucose level less than 100 mg/dL is unusual when the serum glucose is greater than 250 mg/dL. [209] The relative frequency of sinus infection (particularly Mucor), foot infection, bacterial arthritis, cholecystitis, cellulitis, and necrotizing fascitis should also be considered. Pneumonia can be difficult to diagnose in patients with dehydration because the alveolar edema fluid that shows up as an infiltrate on chest radiographs is often not present but develops along with progressive hypoxia during hydration. To avoid this occurrence, we administer intravenous fluid judiciously to patients we suspect have pneumonia. Pancreatitis and pregnancy are common precipitants and should be especially considered when assessing the abdominal pain that is almost ubiquitous at presentation. Abdominal guarding and tenderness associated with vomiting are common, and rebound is occasionally present. These symptoms and findings usually resolve quickly with therapy in the absence of intra-abdominal pathology. The serum amylase is often elevated without pathologic significance, although lipase is usually more specific. [210] Acute myocardial infarction and stroke as well as thromboembolic phenomena are frequent precipitants and complications of DKA. The more insulin resistant the patient seems to be, the more likely one is to find a precipitating cause. If a precipitating cause is found, treatment is essential if adequate metabolic control is to be achieved.



Complications and Prognosis

It should now be possible to treat almost all cases of DKA successfully. The most troublesome complication is cerebral edema. It is common particularly in children and can be fatal. In most series, specific causes could not be assigned, although aggressive hydration, particularly with hypotonic fluids, may contribute. [211] In 50% of patients who subsequently had a respiratory arrest, there were premonitory symptoms, and despite early intervention only half of them avoided severe or fatal brain damage. Other complications of life-threatening severity that have been reported include the acute respiratory distress syndrome and bronchial mucous plugging. [212] [213] [214] Arterial and venous thromboembolic events are quite common. Standard prophylactic low-dose heparin is certainly reasonable in patients with DKA, but currently no indication exists for full anticoagulation.



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G. Immunogenicity and allergenic potential of animal and human insulins. Diabetes Care 1993; 16:155165.

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RF, Havlik R, Taylor SI, et al. Clinical disorders associated with autoantibodies to the insulin receptor: simulation by passive transfer of immunoglobulins to rats. J Clin Invest 1983; 72:10721080. 178. Engerman

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K, Brinchmann-Hansen O, Hansen KF, et al. Rapid tightening of blood glucose control leads to transient deterioration of retinopathy in insulin dependent diabetes mellitus: the Oslo study. Br Med J (Clin Res Ed) 1985; 290:811815. 191. Cryer

PE. Hypoglycemia-associated autonomic failure in diabetes. Am J Physiol 2001; 281:E1115E1121.

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M. Alcoholism, ketoacidosis, and lactic acidosis. Diabetes Metab Rev 1989; 5:365378.

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J, Andersen PT, Hjorne N, Ditzel J. Nontraumatic rhabdomyolysis during diabetic ketoacidosis. Diabetologia 1986; 29:229234.

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WJ. Cerebrospinal fluid to serum glucose ratios in diabetes mellitus and bacterial meningitis. Am J Med 1981; 71:217220.

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LA, Prakash UB, Colby TV. Pulmonary complications in diabetes mellitus. Mayo Clin Proc 1989; 64:791799.



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Chapter 31 - Complications of Diabetes Mellitus Michael Brownlee Lloyd P. Aiello Eli Friedman Aaron I. Vinik Richard W. Nesto Andrew J. M. Boulton

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BIOCHEMISTRY AND MOLECULAR CELL BIOLOGY All forms of diabetes, both inherited and acquired, are characterized by hyperglycemia, a relative or absolute lack of insulin, and the development of diabetes-specific microvascular pathology in the retina, renal glomerulus, and peripheral nerve. Diabetes is also associated with accelerated atherosclerotic macrovascular disease affecting arteries that supply the heart, brain, and lower extremities. Pathologically, this condition resembles macrovascular disease in nondiabetic patients but is more extensive and progresses more rapidly. As a consequence of its microvascular pathology, diabetes mellitus is now the leading cause of new blindness in people 20 to 74 years of age and the leading cause of end-stage renal disease (ESRD). People with diabetes mellitus are the fastest growing group of renal dialysis and transplant recipients. The life expectancy of patients with diabetic end-stage renal failure is only 3 or 4 years. More than 60% of diabetic patients are affected by neuropathy, which includes distal symmetrical polyneuropathy

Figure 31-1 Relative risks for the development of diabetic complications at different levels of mean hemoglobin A Ic (HbAIc , glycated hemoglobin), obtained from the Diabetes Control and Complications Trial. (Adapted from Skyler J: Diabetic complications: the importance of glucose control. Endocrinol Metab Clin North Am 1996; 25:243254.)

(DSPN), mononeuropathies, and a variety of autonomic neuropathies causing erectile dysfunction, urinary incontinence, gastroparesis, and nocturnal diarrhea. Accelerated lower extremity arterial disease in conjunction with neuropathy makes diabetes mellitus account for 50% of all nontraumatic amputations in the United States. The risk of cardiovascular complications is increased by twofold to sixfold in subjects with diabetes. Overall, life expectancy is about 7 to 10 years shorter than for people without diabetes mellitus because of increased mortality from diabetic complications. [1] Large, prospective clinical studies show a strong relationship between glycemia and diabetic microvascular complications in both type 1 and type 2 diabetes mellitus.[2] [3] There is a continuous, though not linear, relationship between level of glycemia and the risk of development and progression of these complications (Fig. 31-1) .[4] [5] Hyperglycemia and the dyslipidemia induced by insulin resistance both appear to play important roles in the pathogenesis of macrovascular complications.[6] [7] [8] [9] [10] SHARED PATHOPHYSIOLOGIC FEATURES OF MICROVASCULAR COMPLICATIONS In the retina, glomerulus, and vasa nervorum, diabetes-specific microvascular disease is characterized by similar pathophysiologic features. Requirement for Intracellular Hyperglycemia

Clinical and animal model data indicate that chronic hyperglycemia is the central initiating factor for all types of diabetic microvascular disease. Duration and magnitude of hyperglycemia are both strongly correlated with the extent and rate of progression of diabetic microvascular disease. In the Diabetes Control and Complications Trial (DCCT), for example, type 1 diabetic patients whose intensive insulin therapy resulted in hemoglobin A 1c (Hb A1c ) levels 2% lower than those receiving conventional insulin therapy had a 76% lower incidence of retinopathy, a 54% lower incidence of nephropathy, and a 60% reduction in neuropathy. [2] [3]

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Although all diabetic cells are exposed to elevated levels of plasma glucose, hyperglycemic damage is limited to those cell types (e.g., endothelial cells) that develop intracellular hyperglycemia. Endothelial cells develop intracellular hyperglycemia because, unlike many other cells, they cannot down-regulate glucose transport when exposed to extracellular hyperglycemia. As illustrated in Figure 31-2 , vascular smooth muscle cells, which are not damaged by hyperglycemia, show an inverse relationship between extracellular glucose concentration and subsequent rate of glucose transport measured as 2-deoxyglucose uptake (Fig. 31-2A) . In contrast, vascular endothelial cells show no significant change in subsequent rate of glucose transport after exposure to elevated glucose concentrations (Fig. 31-2B) .[11] That intracellular hyperglycemia is necessary and sufficient for the development of diabetic pathology is further demonstrated by the fact that overexpression of the GLUT1 glucose transporter in mesangial cells cultured in a normal glucose milieu mimics the diabetic phenotype, inducing the same increases in collagen type IV, collagen type I, and fibronectin gene expression as diabetic hyperglycemia (Fig. 31-3) .[12]



Abnormal Endothelial Cell Function

Early in the course of diabetes mellitus, before structural changes are evident, hyperglycemia causes abnormalities in blood flow and vascular permeability in the retina, glomerulus, and peripheral nerve vasa nervorum. [13] [14] The increase in blood flow and intracapillary pressure is thought to reflect hyperglycemia-induced decreased nitric oxide (NO) production on the efferent side of capillary beds, and possibly an increased sensitivity to angiotensin II. As a consequence of increased intracapillary pressure and endothelial cell dysfunction, retinal capillaries exhibit increased leakage of fluorescein and glomerular capillaries have an elevated albumin excretion rate (AER). Comparable changes occur in the vasa vasorum of peripheral nerve. Early in the course of diabetes, increased permeability is reversible; as time progresses, however, it becomes irreversible.

Figure 31-2 Lack of down-regulation of glucose transport in cells affected by diabetic complications. Upper, 2-deoxyglucose (2DG) uptake in vascular smooth muscle cells preexposed to either 1.2, 5.5, or 22 mM glucose. Lower, 2DG uptake in bovine endothelial cells preexposed to either 1.2, 5.5, or 22 mM glucose. (From Kaiser N, Feener EP, Boukobza-Vardi N, et al. Differential regulation of glucose transport and transporters by glucose in vascular endothelial and smooth muscle cells. Diabetes 1993; 42:8089.)

Figure 31-3 Overexpression of GLUT1 in mesangial cells cultured in normal glucose mimics the diabetic phenotype. Mesangial cells transfected with either LacZ (MCLacZ)- or GLUT1 (MCGT1)-expressing constructs were cultured in 5-mM glucose, and the amount of the indicated matrix components secreted was determined. (From Heilig CW, Concepcion LA, Riser BL, et al. Overexpression of glucose transporters in rat mesangial cells cultured in a normal glucose milieu mimics the diabetic phenotype. J Clin Invest 1995; 96:18021814.)



Increased Vessel Wall Protein Accumulation

The common pathophysiologic feature of diabetic microvascular disease is progressive narrowing and eventual occlusion of vascular lumina, which results in inadequate perfusion and function of the affected tissues. Early hyperglycemia-induced microvascular hypertension and increased vascular permeability contribute to irreversible microvessel occlusion by three processes: The first is an abnormal leakage of periodic acidSchiff (PAS)-positive, carbohydrate-containing plasma proteins, which are deposited in the capillary wall and which may stimulate perivascular cells such as pericytes and mesangial cells to elaborate growth factors and extracellular matrix. The second is extravasation of growth factors, such as transforming growth factor 1 (TGF- 1 ), which directly stimulates overproduction of extracellular matrix components, [15] and may induce apoptosis in certain complication-relevant cell types. The third is hypertension-induced stimulation of pathologic gene expression by endothelial cells and supporting cells, which include glut-1 glucose transporters, growth factors, growth factor receptors, extracellular matrix components, and adhesion molecules that can activate circulating leukocytes. [16] The observation that unilateral reduction in the severity of diabetic microvascular disease occurs on the side with ophthalmic or renal artery stenosis is consistent with this concept. [17] [18]



Microvascular Cell Loss and Vessel Occlusion

The progressive narrowing and occlusion of diabetic microvascular lumina are also accompanied by microvascular cell loss. In the retina, diabetes mellitus induces programmed cell death of Müller cells and ganglion cells, [19] pericytes, and endothelial cells. [20] In the glomerulus, declining renal function is associated with widespread capillary occlusion and podocyte loss, but the mechanisms underlying glomerular cell loss are not yet known. In the vasa nervorum, endothelial cell and pericyte degeneration occur, [21] and these microvascular changes

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Figure 31-4 Development of retinopathy during posthyperglycemic normoglycemia ("hyperglycemic memory"). Quantitation of retinal microaneurysms and acellular capillaries in normal dogs, dogs with poor glycemic control for 5 years, dogs with good glycemic control for 5 years, dogs with poor glycemic control for 2.5 years (P G a ), and the same dogs after a subsequent 2.5 years of good glycemic control (P G b ). (Adapted from Engerman RL, Kern TS. Progression of incipient diabetic retinopathy during good glycemic control. Diabetes 1987; 36:808812.)

appear to precede the development of diabetic peripheral neuropathy. [22] The multifocal distribution of axonal degeneration in diabetes supports a causal role for microvascular occlusion, but hyperglycemia-induced decreases in neurotrophins may contribute by preventing normal axonal repair and regeneration. [23]



Development of Microvascular Complications During Posthyperglycemic Euglycemia ("Hyperglycemic Memory")

Another common feature of diabetic microvascular disease has been termed hyperglycemic memory, or the persistence or progression of hyperglycemia-induced microvascular alterations during subsequent periods of normal glucose homeostasis. The most striking example of this phenomenon is the development of severe retinopathy in histologically normal eyes of diabetic dogs that occurred entirely during a 2.5-year period of normalized blood glucose that followed 2.5 years of hyperglycemia (Fig. 31-4) . [24] Normal dogs were compared to diabetic dogs with either poor control for 5 years, good control for 5 years, or poor control for 2.5 years (P Ga ) followed by good control for the next 2.5 years (P G b ). Hb A1 values for both the good control group and the P G b group were identical to the normal group. Hyperglycemia-induced increases in selected matrix gene transcription also persist for weeks after restoration of normoglycemia in vivo, and a less pronounced, but qualitatively similar, prolongation of hyperglycemia-induced increase in selected matrix gene transcription occurs in cultured endothelial cells. [25] Data from the DCCT study suggest that hyperglycemic

Figure 31-5 Cumulative incidence of further progression of retinopathy 4 years after the end of the Diabetes Control and Complications Trial. Median glycosylated hemoglobin was 8.2% for the conventional therapy group and 7.9% for the intensive therapy group. EDIC, Epidemiology of Diabetes Interventions and Complications [Research Group]. (From Retinopathy and nephropathy in patients with type 1 diabetes four years after a trial of intensive therapy. The Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Research Group. N Engl J Med 2000; 342:381389.)

memory occurs in patients. In the secondary-intervention cohort, there was no difference in the incidence of sustained progression of retinopathy for the first 3 years, no difference in development of clinical albuminuria for 4 years, and no difference in the rate of change in creatinine clearance during the entire study. For neuropathy, the sural nerve sensory conduction velocity did not differ between the groups for 4 years, and intensive therapy did not slow the rate of decline of autonomic function at all. [2] [26] [27] [28] Even more strikingly, the effects of former intensive and conventional therapy on the occurrence and severity of retinopathy and nephropathy were shown to persist for 4 years after the DCCT, despite nearly identical glycosylated hemoglobin values during the 4-year follow-up (8.2% versus 7.9%, respectively) (Fig. 31-5) . [29] Together, these observations from animal and clinical studies imply that hyperglycemia induces prolonged and sometimes irreversible changes in long-lived intracellular molecules that persist and cause continued pathologic function in the absence of continued hyperglycemia.



Genetic Determinants of Susceptibility to Microvascular Complications

Clinicians have long observed that different patients with similar duration and degree of hyperglycemia differed markedly in their susceptibility to microvascular complications. Such observations suggested that genetic differences existed that affected the pathways by which hyperglycemia damaged microvascular cells. The leveling of risk of overt proteinuria after 30 years' duration of type 1 diabetes at 27% is evidence that only a subset of patients are susceptible to development of diabetic nephropathy. [30]

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Figure 31-6 Familial clustering of diabetic nephropathy. Prevalence of diabetic nephropathy in two studies of diabetic siblings of probands with or without diabetic nephropathy. (Adapted from Seaquist ER, Goetz FC, Rich S, Barbosa J. Familial clustering of diabetic kidney disease: evidence for genetic susceptibility to diabetic nephropathy. N Engl J Med 1989; 320:11611165; and Quinn M, Angelico MC, Warram JH, Krolewski AS. Familial factors determine the development of diabetic nephropathy in patients with IDDM. Diabetologia 1996; 39:940945.)

A role for genetic determinant of susceptibility to diabetic nephropathy is most strongly supported by the demonstration of familial clustering of diabetic nephropathy. In two studies of families with two or more siblings having type 1 diabetes, if one diabetic sibling had advanced diabetic nephropathy, the other diabetic sibling had a nephropathy risk of 83% or 72%; in contrast, the risk was only 17% or 22% if the index patient did not have diabetic nephropathy (Fig. 31-6) .[31] [32] For retinopathy, the DCCT reported familial clustering as well, with an odds ratio of 5.4 for the risk of severe retinopathy in diabetic relatives of positive versus negative subjects from the conventional treatment group. [33] Numerous associations have been made between various genetic polymorphisms and the risk of various diabetic complications. Examples include the 5' insulin gene polymorphism,[34] the G2m23+ immunoglobulin allotype, [35] angiotensin-converting enzyme (ACE) insertion/deletion polymorphisms, [36] [37] HLA-DQB1*0201/0302 alleles, [38] polymorphisms of the aldose reductase gene, [39] and a polymorphic CCTTT (n) repeat of NO synthetase (NOS) 2A. [40] In all of these studies, there is no indication that the polymorphic gene actually plays a functional role rather than simply being in linkage disequilibrium with the locus encoding the unidentified relevant genes.



PATHOPHYSIOLOGIC FEATURES OF MACROVASCULAR COMPLICATIONS Unlike microvascular disease, which occurs only in patients with diabetes mellitus, macrovascular disease resembles that in subjects without diabetes. However, subjects with diabetes have more rapidly progressive and extensive cardiovascular disease (CVD), with a greater incidence of multivessel disease and a greater number of diseased vessel segments than nondiabetic persons. [41] Although dyslipidemia and hypertension occur with great frequency in type 2 diabetic populations, there is still excess risk in diabetic subjects after adjusting for these other risk factors. [42] [43] Diabetes itself may confer 75% to 90% of the excess risk of coronary disease in these diabetic subjects, and it enhances the deleterious effects of the other major cardiovascular risk factors (Fig. 31-7) .[44] [45] In subjects with or without diabetes, atherosclerosis begins with endothelial dysfunction or injury. [46] These endothelial changes or injury induce the secretion of chemokines such as monocyte chemoattractant protein 1 (MCP-1), increase the expression of endothelial adhesion molecules for leucocytes and platelets, and enhance permeability to lipoproteins and other plasma constituents. As detailed in Chapter 34 , this leads to recruitment of monocyte-macrophages to the subendothelial space and to the infiltration of plasma LDL, which binds to arterial proteoglycan. The retained LDL then undergoes oxidation and is taken up by macrophages. Activated macrophages and other leukocytes, as well as adherent aggregated platelets, stimulate smooth muscle cell proliferation and elaboration of extracellular matrix, culminating in the formation of a complex lesion filled with prothrombotic material contained by a fibrin cap. Rupture of this fibrin cap by matrix metalloproteinases causes thrombus formation and arterial occlusion. [47] [48] [49] Because macrovascular disease also occurs in nondiabetic subjects, diabetes is thought to accelerate the process by increasing endothelial cell dysfunction and by exacerbating dyslipidemia. The pathogenesis of endothelial cell dysfunction in diabetic arteries appears to involve both insulin resistance and hyperglycemia. In vitro studies suggest that insulin has both antiatherogenic and proatherogenic effects (Fig. 31-8) .[50] [51] One major antiatherogenic effect is the stimulation of endothelial NO production. NO released from endothelial cells is a potent inhibitor of platelet aggregation and adhesion to the vascular wall. Endothelial NO also controls the expression of genes involved in atherogenesis. It decreases expression of the chemoattractant protein MCP-1, and of surface adhesion molecules such as CD11/CD18, P-selection, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). Endothelial cell NO also reduces vascular permeability and decreases the rate of oxidation of low-density lipoprotein (LDL) to its proatherogenic form. Finally, endothelial cell NO inhibits proliferation of vascular smooth muscle cells. [52] Two major proatherogenic effects of insulin are the potentiation of platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell (VSMC) proliferation and the stimulation of VSMC plasminogen activator inhibitor 1 (PAI-1) production. [53] [54] Since insulin-induced NO production is mediated by the insulin receptor substrate PI3 kinase signal transduction pathway, while the effects on smooth muscle cells are mediated by the ras raf mekk map kinase signal transduction pathway, [50] [51] it has been proposed that pathway-selective insulin resistance in arterial cells may contribute to diabetic atherosclerosis. Recently, evidence of such selective vascular resistance to insulin has been demonstrated in the obese zucker rat. [55] Hyperglycemia also inhibits arterial endothelial NO production, both in vivo and in vitro. [56] [57] [58] [59] Similarly, hyperglycemia potentiates PDGF-induced VSMC proliferation and stimulates endothelial cell PAI-1 production. [60] [58] In addition, hyperglycemia has a variety of other proatherogenic effects on endothelial

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Figure 31-7 Adjusted death rates by number of cardiovascular disease (CVD) risk factors for diabetic and nondiabetic men. Subjects are participants from the Multiple Risk Factor Intervention Trial (MRFIT) study; risk factors are hypercho-lesterolemia, hypertension, and cigarette smoking. (From Stamler J, Vaccaro O, Neaton JD, Wentworth D. Diabetes, other risk factors, and 12-year cardiovascular mortality for men screened in the Multiple Risk Factor Intervention Trial. Diabetes Care 1993; 2:434444.)

cells, platelets, and monocyte/macrophages. These include increased expression of MCP-1, [61] up-regulation of adhesion molecules such as ICAM-1 and VCAM-1, [62] [63] [64] potentiation of collagen-induced platelet activation, [65] and increased secretion of collagen type IV and fibronectin. [66] [67] Both insulin resistance and hyperglycemia have been implicated in the pathogenesis of diabetic dyslipidemia as well. Insulin resistance is associated with a characteristic lipoprotein profile that includes a high very-low-density lipoprotein (VLDL), a low high-density lipoprotein (HDL), and small, dense LDL. Both low HDL and small, dense LDL are each independent risk factors for macrovascular disease. This profile arises as a direct result of increased net free fatty acid (FFA) release by insulin resistant adipocytes (Fig. 31-9) . [68] Increased FFA flux into hepatocytes stimulates VLDL secretion. In the presence

Figure 31-8 Schematic summary of proatherosclerotic and antiather-osclerotic actions of insulin on vascular cells. See text for abbreviations. (Adapted from King G, Brownlee M. The cellular and molecular mechanisms of diabetic complications. Endocrinol Metab Clin North Am 1996; 2:255270; and Hsueh WA, Law RE. Cardiovascular risk continuum: implications of insulin resistance and diabetes. Am J Med 1998; 105:4S14S.)

of cholesteryl ester transfer protein, excess VLDL transfers significant amounts of triglyceride to both HDL and LDL while depleting HDL and LDL of cholesteryl ester. The resultant triglyceride-enriched HDL carries less cholesteryl ester for reverse cholesterol transport to the liver, and loss of Apo1A-1, from these particles reduces the total concentration of HDL available for reverse cholesterol transport. The triglyceride-enriched, cholesteryl esterdepleted LDL is smaller and denser than normal LDL, allowing it to penetrate the vessel wall and be oxidized more easily. Hyperglycemia appears to contribute to diabetic dyslipidemia by causing delayed clearance of postprandial lipoproteins, resulting in elevated levels of atherogenic cholesterol-enriched remnant particles. [9] This remnant clearance defect is caused by a hyperglycemia-induced reduction in expression of the heparan sulfate proteoglycan perlecan on hepatocytes. Perlecan interaction with apoB-48containing lipoprotein remnant particles is necessary for efficient uptake by the LDL receptor-related protein. The importance of hyperlipidemia in the pathogenesis of diabetic macrovascular disease in patients with type 2 diabetes is underscored by recent studies validating that effective treatment of hyperlipidemia in such patients substantially reduces their risk of CVD. [7] [8] The importance of hyperglycemia in the pathogenesis of diabetic macrovascular disease is suggested by the observation that carotid wall thickness is increased in persons with established diabetes but not in persons with impaired glucose tolerance. [6] The United Kingdom Prospective Diabetes Study (UKPDS) identified hyperglycemia as an important risk factor for macrovascular disease in type 2 diabetes, and numerous correlational studies show that hyperglycemia is a continuous risk factor for macrovascular disease. [69] [70] [71] [72] [73] Similarly, glycohemoglobin A 1 is an independent risk factor for CVD [74] in type 1 diabetes. The relative importance of hyperglycemia in type 1 patients is suggested by the 41% reduction in macrovascular

disease (P = .06) observed in the intensive therapy group of the DCCT. [2]



MECHANISMS OF HYPERGLYCEMIA-INDUCED DAMAGE Four major hypotheses about how hyperglycemia causes diabetic complications have generated a large amount of data as

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Figure 31-9 Schematic summary relating insulin resistance (IR) to the characteristic dyslipidemia of type 2 diabetes mellitus. IR at the adipocyte results in increased free fatty acid (FFA) release. Increased FFA flux stimulates very-low-density lipoprotein (VLDL) secretion, causing hypertriglyceridemia (TG). VLDL stimulates a reciprocal exchange of TG to cholesteryl ester (CE) from both high-density lipoprotein (HDL) and low-density lipoprotein (LDL), catalyzed by CE transfer protein (CETP). TG-enriched HDL dissociates from ApoA-1, leaving less HDL for reverse cholesterol transport. TG-enriched LDL serves as a substrate for lipases that convert it to atherogenic small, dense LDL particles (SD LDL). (From Ginsberg HN. Insulin resistance and cardiovascular disease. J Clin Invest 2000; 106:453458.)

well as several clinical trials based on specific inhibitors of these mechanisms. Until recently, there was no unifying hypothesis linking these four mechanisms together, nor was there an obvious connection between any of these mechanisms, each of which responds quickly to normalization of hyperglycemia, and the phenomenon of hyperglycemic memory (see earlier). Increased Polyol Pathway Flux Aldose Reductase Function

Aldose reductase (alditol:NAD(P) + 1-oxidoreductase, EC 1.1.1.21) is a cytosolic, monomeric oxidoreductase that catalyzes the NADPH-dependent reduction of a wide variety of carbonyl compounds including glucose. Triphosphopyridine nucleotide, reduced form of NADP (NADPH) is the cofactor in both this reaction and in the regeneration of glutathione by glutathione reductase. Aldose reductase has a low affinity (high Michaelis constant [K m ]) for glucose, and at the normal glucose concentrations found in nondiabetic patients, metabolism of glucose by this pathway constitutes a small percentage of total glucose utilization. In a hyperglycemic environment,

Figure 31-10 Aldose reductase and the polyol pathway. Aldose reductase reduces reactive oxygen species (ROS)-generated toxic aldehydes to inactive alcohols, and glucose to sorbitol, using triphosphopyridine nucleotide, reduced form of NADP (NADPH) as a cofactor. In cells where aldose reductase activity is sufficient to deplete reduced glutathione (GSH), oxidative stress would be augmented. Sorbitol dehydrogenase (SDH) oxidizes sorbitol to fructose using nicotinamide-adenine dinucleotide (NAD + ) as a cofactor. GSSG, oxidized glutathione.

however, increased intracellular glucose results in increased enzymatic conversion to the polyalcohol sorbitol, with concomitant decreases in NADPH. In the polyol pathway, sorbitol is oxidized to fructose by the enzyme sorbitol dehydrogenase, with NAD + reduced to NADH (Fig. 31-10) . Biochemical Consequences of Increased Polyol Pathway Flux

A number of mechanisms have been proposed to explain the potential detrimental effects of hyperglycemia-induced increases in polyol pathway flux. These include sorbitol-induced osmotic stress, decreased Na + /K+ ATPase activity, increased cytosolic NADH/NAD + , and decreased cytosolic NADPH. Sorbitol does not diffuse easily across cell membranes, and it was originally suggested that this resulted in osmotic damage to microvascular cells. However, sorbitol concentrations measured in diabetic vessels and nerves are far too low to cause osmotic damage. Another early suggestion was that increased flux through the polyol pathway decreased Na + /K+ ATPase activity. Although this was originally thought to be mediated by polyol-pathwaylinked decreases in phosphatidylinositol synthesis, it has been shown to result from activation of protein kinase C (PKC) (see later). Hyperglycemia-induced activation of PKC increases cytosolic phospholipase A 2 activity, which increases the production of two inhibitors of Na + /K+ ATPase, arachidonate and prostaglandin E 2 (PGE2 ). [75] More recently, it has been proposed that oxidation of sorbitol by NAD + increases the cytosolic ratio of NADH/NAD + , thereby inhibiting activity of the enzyme glyceraldehyde-3-phosphate dehydrogenase and increasing concentrations of triose phosphate. [76] Elevated triose phosphate concentrations could increase formation of both methylglyoxal, a precursor of advanced glycation end products (AGEs), and diacylglycerol (DAG) (via -glycerol-3-phosphate), thus activating PKC (discussed in subsequent sections). Although increased NADH production is supported by the observation that hyperglycemia increases both lactate concentration and the lactate/pyruvate ratio, there is no direct evidence that the concentrations of NADH and NAD + , as opposed to NADH and NAD+ flux, are altered. In endothelial cells, where aldose reductase activity is low, increased NADH production may also reflect hyperglycemia-induced increased flux through glycolysis [77] and through the glucuronic acid pathway. [78] Other evidence presented in support of this hypothesis includes the observation that administration of pyruvate can prevent

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diabetes-related endothelial dysfunction in some systems. However, the observed effects of pyruvate on microvascular function may reflect its potent antioxidant properties rather than effects on the NADH/NAD + ratio, because reactive oxygen species (ROS) also partially inhibit glyceraldehyde-3-phosphate dehydrogenase and increase glyceraldehyde-3-phosphate levels. [79] [80] The source of hyperglycemia-induced ROS is discussed later in this section. It has also been proposed that reduction of glucose to sorbitol by NADPH consumes the cofactor NADPH. Because NADPH is required for regenerating reduced glutathione (GSH), this could induce or exacerbate intracellular oxidative stress. Less reduced glutathione has in fact been found in the lens of transgenic mice that overexpress aldose reductase, and this is the most likely mechanism by which increased flux through the polyol pathway has deleterious consequences. [81] Hyperglycemia-induced inhibition of glucose-6-phosphate dehydrogenase, the major source of NADPH regeneration, may further reduce NADPH concentration in some vascular cells or neuronal cells. [82]



Advanced Glycation End-Product Inhibitors

The hydrazine compound aminoguanidine was the first AGE inhibitor discovered, [101] and its effect on diabetic pathology has been investigated in the retina, kidney, nerve, and artery. In the rat retina, diabetes causes a 19-fold increase in the number of acellular capillaries. Aminoguanidine treatment of diabetics prevented excess AGE accumulation and reduced the number of acellular capillaries by 80%. Diabetes-induced pericyte dropout also was markedly reduced by aminoguanidine treatments.[83] Similar results have been obtained in animal models of diabetic kidney disease. [203] [204] [205] Diabetes increased AGEs in the renal glomerulus, and aminoguanidine treatment prevented this diabetes-induced increase. Untreated diabetic animals developed albuminuria that averaged 30 mg every 24 hours for 32 weeks. This was more than a 10-fold increase above control levels. In aminoguanidine-treated diabetic rats, the level of AER was reduced nearly 90%. [75] Untreated diabetic animals also developed the characteristic structural feature of human diabetic nephropathy (i.e., increased fractional mesangial volume). When diabetic animals were treated with aminoguanidine, the increase was completely prevented. A structurally unrelated AGE inhibitor, OPB-9195, also prevented the development and progression of experimental diabetic nephropathy by blocking type IV collagen overproduction and normalizing the expression of TGF-. [206] [207] In the peripheral nerve of diabetic rats, both motor nerve and sensory NCVs are decreased after 8 weeks of diabetes. [208] Nerve action potential amplitude is decreased by 37% and peripheral nerve blood flow is decreased by 57% after 24 weeks of diabetes. [209] Aminoguanidine treatment prevented each of these abnormalities of diabetic peripheral nerve function. [208] [209] In a large randomized, double-blind, placebo-controlled, multicenter trial of aminoguanidine in type 1 diabetic patients with overt nephropathy, aminoguanidine lowered total urinary protein and slowed progression of nephropathy, over and above the effects of existing optimal care. In addition, aminoguanidine reduced the progression of diabetic retinopathy (defined as an increase by three or more steps in the Early Treatment Diabetic Retinopathy Study [ETDRS] scale). [210] [211]



Activation of Protein Kinase C Mechanism of Hyperglycemia-Induced Protein Kinase C Activation

PKCs are a family of at least 11 isoforms, 9 of which are activated by the lipid second-messenger DAG. Intracellular hyperglycemia increases DAG content in cultured microvascular cells and in the retina and renal glomeruli of diabetic animals. [144] [145] [146] Intracellular hyperglycemia appears to increase DAG content primarily by increasing its de novo synthesis from the glycolytic intermediate glyceraldehyde-3-phosphate via reduction to glycerol-3-phosphate and stepwise acylation. [145] [147] Increased de novo synthesis of DAG activates PKC both in cultured vascular cells [146] [148] [149] [150] and in retina and glomeruli of diabetic animals. [145] [146] [148] Increased DAG primarily activates the and isoforms of PKC, but increases in other isoforms have also been found, such as PKC- and PKC-epsilon isoforms in the retina [151] and PKC- and PKC- in the glomerulus [152] [153] of diabetic rats. Consequences of Hyperglycemia-Induced Protein Kinase C Activation

In early experimental diabetes, activation of PKC- isoforms has been shown to mediate retinal and renal blood flow abnormalities, [154] perhaps by depressing NO production and increasing endothelin-1 activity (Fig. 31-13) . Abnormal activation of PKC has been implicated in the decreased glomerular production of NO induced by experimental diabetes [155] and in the decreased smooth muscle cell NO production induced by hyperglycemia. [156] PKC activation also inhibits insulin-stimulated expression of endothelial nitric oxide synthase (eNOS) messenger RNA (mRNA) in cultured endothelial cells. [157] Hyperglycemia increases endothelin 1-stimulated mitogen-activated protein kinase activity in glomerular mesangial cells by activating PKC isoforms. [158] The increased endothelial cell permeability induced by high glucose in cultured cells is mediated by activation of PKC-, however. [159] Activation of PKC by elevated glucose levels also induces expression of the permeability-enhancing factor VEGF in smooth muscle cells. [160] In addition to affecting hyperglycemia-induced abnormalities of blood flow and permeability, activation of PKC contributes to increased microvascular matrix protein accumulation by inducing the expression of TGF- 1 , fibronectin, and 1 (IV) collagen in both cultured mesangial cells [161] [162] and in the glomeruli of diabetic rats. [163] This effect appears to be mediated through PKC's inhibition of NO production. [164] Hyperglycemia-induced expression of laminin C1 in cultured mesangial cells is independent of PKC activation, however. [165] Hyperglycemia-induced activation of PKC has also been implicated in the overexpression of the fibrinolytic inhibitor PAI-1[166] and in the activation of the pleiotrophic transcription factor NF-B in cultured endothelial cells and vascular smooth muscle cells. [167] [168]



Increased Hexosamine Pathway Flux

A fourth hypothesis about how hyperglycemia causes diabetic complications has recently been formulated, [169] [170] [171] [172] in which glucose is shunted into the hexosamine pathway (Fig. 31-14) . In this pathway, fructose-6-phosphate is diverted from glycolysis to provide substrates for reactions that require UDP-N-acetylglucosamine, such as proteoglycan synthesis and the formation of O-linked glycoproteins. Inhibition of the rate-limiting enzyme in the conversion of glucose to glucosamine, glutamine:fructose-6-phosphate amidotransferase, blocks hyperglycemia-induced increases in the transcription of both TGF- [169] and TGF- 1 .[170] This pathway has previously been shown to play an important role in hyperglycemia-induced and fat-induced insulin resistance. [173] [174] [175]

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Figure 31-14 Schematic representation of the hexosamine pathway. The glycolytic intermediate fructose-6-phosphate (Fruc-6-P) is converted to glucosamine-6-phosphate (Glc-6-P) by the enzyme glutamine:fructose 6-phosphate amidotransferase (GFAT). Increased donation of N-Acetylglucosamine moieties to serine and threonine residues of transcription factors such as Sp1 increases production of such complication-promoting factors as PAI-1 and TGF- 1 . See text for additional abbreviations. (Adapted from Du XL, Edelstein D, Rossetti L, et al. Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc Natl Acad Sci USA 2000; 97:1222212226.)

The mechanism by which increased flux through the hexosamine pathway mediates hyperglycemia-induced increases in gene transcription has not been clear, but the observation that Sp1 sites regulate hyperglycemia-induced activation of the PAI-1 promoter in vascular smooth muscle cells [176] suggests that covalent modification of Sp1 by N-acetylglucosamine may explain the link between hexosamine pathway activation and hyperglycemia-induced changes in gene transcription. Virtually every RNA polymerase II transcription factor examined has been found to be 0-GlcNacylated, [177] and the glycosylated form of Sp1 appears to be more transcriptionally active than the deglycosylated form of the protein. [178] A four-fold increase in Sp1 0-GlcNacylation caused by inhibition of the enzyme 0-GlcNac--N-acetylglucosaminidase resulted in a reciprocal 30% decrease in its level of serine/threonine phosphorylation, supporting the concept that 0-GlcNacylation and phosphorylation compete to modify the same sites on this protein. [179] GlcNac modification of Sp1 may regulate other glucose-responsive genes in addition to TGF- 1 and PAI-1. Glucose-responsive transcription is regulated by Sp1 sites in the acetyl-CoA carboxylase gene, the rate-limiting enzyme for fatty acid synthesis, for example, and it appears that post-translational modification of Sp1 is responsible for this effect. [180] [181] Because virtually every RNA polymerase II transcription factor examined has been found to be O-GlcNacylated, [177] it is possible that reciprocal modification by O-GlcNacylation and phosphorylation of transcription factors other than Sp1 may function as a more generalized mechanism for regulating glucose-responsive gene transcription. In addition to transcription factors, many other nuclear and cytoplasmic proteins are dynamically modified by O-GlcNAc moieties and may exhibit reciprocal modification by phosphorylation in a manner analogous to Sp1. [177] Thus, activation of the hexosamine pathway by hyperglycemia may result in many changes in both gene expression and in protein function that together contribute to the pathogenesis of diabetic complications.



DIFFERENT PATHOGENIC MECHANISMS REFLECT A SINGLE HYPERGLYCEMIA-INDUCED PROCESS Although specific inhibitors of aldose reductase activity, AGE formation, and PKC activation each ameliorate various diabetes-induced abnormalities in animal models, there has been no apparent common element linking the four mechanisms of hyperglycemia-induced damage discussed in the preceding section. [154] [182] [183] [184] [185] It has also been conceptually difficult to explain the phenomenon of hyperglycemic memory (discussed in an earlier section) as a consequence of four processes that quickly normalize when euglycemia is restored. These issues have now been resolved by the recent discovery that each of the four different pathogenic mechanisms reflects a single hyperglycemia-induced process: overproduction of superoxide by the mitochondrial electron transport chain. [77] [186] Hyperglycemia increases ROS production inside cultured bovine aortic endothelial cells. [187] To understand how this occurs, a brief overview of glucose metabolism is helpful. Intracellular glucose oxidation begins with glycolysis in the cytoplasm, which generates NADH and pyruvate. Cytoplasmic NADH can donate reducing equivalents to the mitochondrial electron transport chain via two shuttle systems, or it can reduce pyruvate to lactate, which exits the cell to provide substrate for hepatic gluconeogenesis. Pyruvate can also be transported into the mitochondria, where it is oxidized by the tricarboxylic acid (TCA) cycle to produce carbon dioxide (CO2 ), water (H2 O), four molecules of NADH, and one molecule of FADH 2 . Mitochondrial NADH and FADH 2 provide energy for adenosine triphosphate (ATP) production via oxidative phosphorylation by the electron transport chain. Electron flow through the mitochondrial electron transport chain is carried out by four inner membrane-associated enzyme complexes, plus cytochrome- c and the mobile carrier ubiquinone. [188] NADH derived from both cytosolic glucose oxidation and mitochondrial TCA cycle activity donates electrons to NADH:ubiquinone oxidoreductase (Complex I). Complex I ultimately transfers its electrons to ubiquinone. Ubiquinone can also be reduced by electrons donated from several FADH 2 -containing dehydrogenases, including succinate:ubiquinone oxidoreductase (Complex II) and glycerol-3-phosphate dehydrogenase. Electrons from reduced ubiquinone are then transferred to ubiquinol:cytochrome c oxidoreductase (Complex III) by the ubisemiquinone radical-generating Q cycle. [189] Electron transport then proceeds through cytochrome-c, cytochrome-c oxidase (Complex IV), and finally, molecular oxygen. Electron transfer through Complexes I, III, and IV generates a proton gradient that drives ATP synthase (Complex V). When the electrochemical potential difference generated by this proton gradient is high, the life of superoxide-generating electron transport intermediates such as ubisemiquinone is prolonged. There appears to be a threshold value above which superoxide production is markedly increased (Fig. 31-15) .[190] Using inhibitors of both the shuttle that transfers cytosolic

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Figure 31-15 Production of superoxide by the mitochondrial electron transport chain. Increased hyperglycemia-derived electron donors from the tricarboxylic acid cycle (NADH and FADH 2 ) generate a high mitochondrial membrane potential (µH + ) by pumping protons across the mitochondrial inner membrane. This inhibits electron transport at complex III and increases the half-life of free radical intermediates of coenzyme Q, which reduce O 2 to superoxide. See text for abbreviations. (From Boss O, Hagen T, Lowell BB. Uncoupling proteins 2 and 3: potential regulators of mitochondrial energy metabolism. Diabetes 2000; 49:143156.)

NADH into mitochondria, and the transporter that transfers cytosolic pyruvate into the mitochondria, the TCA cycle was shown to be the source of hyperglycemia-induced ROS in endothelial cells. Overexpression of uncoupling protein 1 (UCP-1), a specific protein uncoupler of oxidative phosphorylation capable of collapsing the proton electrochemical gradient, [191] also prevented the effect of hyperglycemia. These results demonstrate that hyperglycemia-induced intracellular ROS are produced by the proton electrochemical gradient generated by the mitochondrial electron transport chain. Overexpression of manganese superoxide dismutase, the mitochondrial form of this antioxidant enzyme, [192] also prevented the effect of hyperglycemia. This result demonstrates that superoxide is the reactive oxygen radical produced by this mechanism. The effect of hyperglycemia-induced mitochondrial superoxide overproduction on polyol pathway flux was evaluated after first determining that sorbitol in these cells was exclusively derived from aldose reductase activity. Sorbitol levels were 2.6-fold higher than baseline (5-mM glucose) when endothelial cells were incubated in 30-mM glucose (Fig. 31-16) . Hyperglycemia-induced sorbitol accumulation was completely prevented by UCP-1 and superoxide dismutase (Mn-SOD) (see Fig. 31-16) , indicating that mitochondrial superoxide overproduction stimulates aldose reductase activity. This effect appears to reflect the well-described reversible inhibition of glyceraldehyde-3-phosphate dehydrogenase by ROS, [80] [186] which increases glyceraldehyde-3-phosphate levels and the levels of proximal glycolytic metabolites, including glucose (Fig. 31-17) . Next, the effect of hyperglycemia-induced mitochondrial superoxide overproduction on intracellular AGE formation was determined. In bovine aortic endothelial cells, hyperglycemia increases intracellular AGEs primarily, if not exclusively, by increasing the formation of AGE-forming methylglyoxal. [96] Therefore, the effect of UCP-1 and Mn-SOD on hyperglycemia-induced formation of intracellular methylglyoxal-derived AGEs was examined (see Fig. 31-16) . Each of these agents completely prevented hyperglycemia-induced formation of intracellular AGEs (see Fig. 31-16) , indicating that mitochondrial superoxide initiates intracellular AGE formation. Because methylglyoxal is formed by fragmentation of glyceraldehyde-3-phosphate, this dependency on increased mitochondrial superoxide production also likely reflects increased glyceraldehyde-3-phosphate levels due to inhibition of glyceraldehyde-3-phosphate dehydrogenase by ROS (see Fig. 31-17) . The effect of UCP-1 and Mn-SOD on hyperglycemia-induced activation of PKC was also evaluated (see Fig. 31-16) . Each of these agents completely inhibited PKC activation, suggesting that mitochondrial superoxide overproduction initiates the hyperglycemia-induced de novo synthesis of DAG that activates PKC. [151] Most likely this too reflects increased glyceraldehyde-3-phosphate levels due to inhibition of glyceraldehyde-3-phosphate dehydrogenase by ROS (see Fig. 31-17) . Finally, the effect of hyperglycemia-induced mitochondrial superoxide overproduction on the hexosamine pathway was determined. [186] Hyperglycemia induced an increase in hexosamine pathway activity that was completely prevented by UCP-1, Mn-SOD, and azaserine, an inhibitor of the rate-limiting enzyme in the hexosamine pathway. Hyperglycemia-induced activation of the redox-sensitive pleiotropic transcription factor NF-B was also prevented by inhibition of mitochondrial superoxide overproduction.[77]



A POSSIBLE MOLECULAR BASIS FOR HYPERGLYCEMIC MEMORY In contrast to the four known hyperglycemia-inducible abnormalities of intracellular metabolism, hyperglycemia-induced mitochondrial superoxide production may provide an explanation for the development of complications during posthyperglycemic normoglycemia (hyperglycemic memory). Hyperglycemia-induced increases in superoxide would not only increase aldose reductase activity, AGE formation, PKC activity, and hexosamine pathway activity but may also induce mutations in mitochondrial DNA (mtDNA).[193] Mitochondria are more vulnerable to mutation because mtDNA contains virtually no introns, lacks protective histones, and has no effective DNA repair mechanism. [194] [195] [196] mtDNA has a 10- to 20-fold higher mutation rate than nuclear DNA. [197] [198] Defective electron transport complex subunits encoded by mutated mtDNA would eventually cause increased superoxide production at physiologic concentrations of glucose, with resultant continued activation of the four pathways despite the absence of hyperglycemia.

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Figure 31-16 Effect of agents that alter mitochondrial electron transport chain function on the three main pathways of hyperglycemic damage. A, Hyperglycemia-induced protein kinase C (PKC) activation. B, Intracellular advanced glycation end-product (AGE) formation. C, Sorbitol accumulation. Cells were incubated in 5-mM glucose, 30-mM glucose alone, and 30-mM glucose plus either agents that uncouple oxidative phosphorylation and reduce the high mitochondrial membrane potential (TTFA, CCCP, UCP-1), or dismutate superoxide (Mn-SOD). See text for additional abbreviations. (From Nishikawa T, Edelstein D, Du XL, et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 2000; 404:787790.)



PROSPECTS FOR PHARMACOLOGIC INTERVENTION Aldose Reductase Inhibitors

In vivo studies of polyol pathway inhibition have yielded promising results with neuropathy but disappointing results in other target tissues of diabetic complications. During the course of a 5-year study, nerve conduction velocity (NCV) progressively decreased in untreated diabetic dogs, whereas this decrease was prevented by treatment with an aldose reductase inhibitor (ARI). [182] Positive effects of ARIs on human diabetic neuropathy have been reported. [199] [200] In contrast, aldose reductase inhibition failed to prevent retinopathy in the 5-year study in dogs, nor did it prevent capillary basement membrane thickening in the retina, kidney, or muscles. [201] A 3-year human trial also failed to show any effect on diabetic retinopathy. [202]



Advanced Glycation End-Product Inhibitors

The hydrazine compound aminoguanidine was the first AGE inhibitor discovered, [101] and its effect on diabetic pathology has been investigated in the retina, kidney, nerve, and artery. In the rat retina, diabetes causes a 19-fold increase in the number of acellular capillaries. Aminoguanidine treatment of diabetics prevented excess AGE accumulation and reduced the number of acellular capillaries by 80%. Diabetes-induced pericyte dropout also was markedly reduced by aminoguanidine treatments.[83] Similar results have been obtained in animal models of diabetic kidney disease. [203] [204] [205] Diabetes increased AGEs in the renal glomerulus, and aminoguanidine treatment prevented this diabetes-induced increase. Untreated diabetic animals developed albuminuria that averaged 30 mg every 24 hours for 32 weeks. This was more than a 10-fold increase above control levels. In aminoguanidine-treated diabetic rats, the level of AER was reduced nearly 90%. [75] Untreated diabetic animals also developed the characteristic structural feature of human diabetic nephropathy (i.e., increased fractional mesangial volume). When diabetic animals were treated with aminoguanidine, the increase was completely prevented. A structurally unrelated AGE inhibitor, OPB-9195, also prevented the development and progression of experimental diabetic nephropathy by blocking type IV collagen overproduction and normalizing the expression of TGF-. [206] [207] In the peripheral nerve of diabetic rats, both motor nerve and sensory NCVs are decreased after 8 weeks of diabetes. [208] Nerve action potential amplitude is decreased by 37% and peripheral nerve blood flow is decreased by 57% after 24 weeks of diabetes. [209] Aminoguanidine treatment prevented each of these abnormalities of diabetic peripheral nerve function. [208] [209] In a large randomized, double-blind, placebo-controlled, multicenter trial of aminoguanidine in type 1 diabetic patients with overt nephropathy, aminoguanidine lowered total urinary protein and slowed progression of nephropathy, over and above the effects of existing optimal care. In addition, aminoguanidine reduced the progression of diabetic retinopathy (defined as an increase by three or more steps in the Early Treatment Diabetic Retinopathy Study [ETDRS] scale). [210] [211]



Protein Kinase C Inhibitors

The recent development of a isoformspecific PKC inhibitor has allowed in vivo studies to go forward, because the

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Figure 31-17 Potential mechanism by which hyperglycemia-induced mitochondrial superoxide overproduction activates four pathways of hyperglycemic damage. Excess superoxide partially inhibits the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, thereby diverting upstream metabolites from glycolysis into pathways of glucose overutilization. This results in increased flux of triose phosphate to diacylglycerol (DAG), an activator of protein kinase C (PKC), and to methylglyoxal, the major intracellular advanced glycation end-product (AGE) precursor. Increased flux of fructose-6-phosphate to UDP- N-acetylglucosamine increases modification of proteins by hexosamine, and increased glucose flux through the polyol pathway consumes NADPH and depletes GSH. See text for additional abbreviations.

toxicity of non-selective PKC inhibitors precludes their use. LY333531 inhibits PKC- 1 and PKC-2 with a half-maximal inhibitory constant (IC 50 ) that is at least 50-fold less than for other PKC isoforms. [154] Treatment with LY333531 significantly reduced PKC activity in the retina and renal glomeruli of diabetic animals. Concomitantly, LY333531 treatment significantly reduced diabetes-induced increases in retinal mean circulation time, normalized diabetes-induced increases in glomerular filtration rate (GFR), and partially corrected urinary AER. Treatment of db/db mice with LY333531 for a longer period also ameliorated accelerated glomerular mesangial expansion. [212] Clinical trials of LY333531 in human diabetic patients are currently in progress.



Future Drug Targets

The recent discovery that each of the four different pathogenic mechanisms discussed in this section reflect a single hyperglycemia-induced process [77] [186] suggests that interrupting the overproduction of superoxide by the mitochondrial electron transport chain would normalize polyol pathway flux, AGE formation, PKC activation, hexosamine pathway flux, and NF-B activation. Novel compounds that act as superoxide dismutase/catalase mimetics already exist, [213] [214] [215] and these compounds have been shown to normalize hyperglycemia-induced mitochondrial superoxide overproduction. [186] These and the other agents described in this section may have unique clinical efficacy in preventing the development and progression of diabetic complications.



RETINOPATHY, MACULAR EDEMA, AND OTHER OCULAR COMPLICATIONS* Diabetic retinopathy is a well-characterized, sight-threatening, chronic microvascular complication that eventually afflicts virtually all patients with diabetes mellitus. Diabetic retinopathy is characterized by gradually progressive alterations in the retinal microvasculature, leading to areas of retinal nonperfusion, increased vasopermeability, and pathologic intraocular proliferation of retinal vessels. The complications associated with the increased vasopermeability, termed macular edema, and uncontrolled neovascularization, termed proliferative diabetic retinopathy (PDR), can result in severe and permanent visual loss. Despite decades of research, there is presently no known means of preventing diabetic retinopathy and, despite effective therapies, diabetic retinopathy remains the leading cause of new-onset blindness in working-aged Americans. [218] With appropriate

[218]

*Portions of this section draw on, among others, (1) Principles and Practices of Ophthalmology: The Harvard System (Aiello LP, et al. Ocular Complications of Diabetes, 2nd ed., WB Saunders, 2000); (2) Diabetic Retinopathy: Technical Review (Aiello LP, et al. American Diabetes Association. Diabetes Care 1998; 21:143156); and (3) Diabetic Retinopathy (Aiello LP, Cavellerano J. Diabetic retinopathy. In Johnstone MT, Veves A [eds]. Contemporary Cardiology. Diabetes and Cardiovascular Disease. Humana Press, 2001, pp 385398).

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medical and ophthalmologic care, however, more than 90% of visual loss resulting from diabetic retinopathy can be prevented.

[219]

Thus, until a cure for diabetes is discovered, the primary clinical care emphasis for the prevention of vision loss is appropriately directed at the early identification, accurate classification, and timely treatment of retinopathy. Emphasis must also be placed on ensuring compliant life-long routine ophthalmologic follow-up of the diabetic patient and optimization of associated systemic disorders. EPIDEMIOLOGY AND IMPACT Sixteen million Americans have diabetes mellitus, but only half are aware that they have the disease. [218] [220] Diabetic retinopathy is the leading cause of new cases of legal blindness among Americans between the ages of 20 and 74 years. [221] There is a higher risk of more frequent and severe ocular complications in type 1 diabetes.[222] Approximately 25% of patients with type 1 diabetes have retinopathy after 5 years, with this figure increasing to 60% and 80% after 10 and 15 years, respectively. However, because there are more adult-onset cases than juvenile-onset cases, type 2 disease accounts for a higher proportion of patients with visual loss. The most threatening form of retinopathy (PDR) is present in approximately 25% of type 1 patients with diabetes of 15 years' duration. [223] An estimated 700,000 persons have PDR, 130,000 with high-risk PDR, 500,000 with macular edema, and 325,000 with clinically significant macular edema (CSME) in the United States. [224] [225] [226] [227] [228] An estimated 63,000 cases of PDR, 29,000 high-risk PDR, 80,000 macular edema, 56,000 CSME, and 5000 new cases of legal blindness occur each year as a result of diabetic retinopathy. [224] [225] Blindness has been estimated to be 25 times more common in persons with diabetes than in those without the disease. [229] [230] Estimates of the medical and economic impact of retinopathy-associated morbidity have been performed using computer simulations that incorporate clinical trial and cost reimbursement data to model effects of applying accepted evaluation and treatment techniques to patients with type 1 and type 2 diabetic retinopathy. [226] [228] [231] [232] [233] [ 234] [ 235] [ 236] [ 237] [ 238] [ 239] The models predict that in the absence of good glycemic control, 72% of patients with type 1 diabetes will develop PDR requiring panretinal photocoagulation (PRP) over their lifetime and that 42% will develop macular edema. [231] If patients with type 1 diabetes receive currently suggested treatment, there is a predicted cost of $966 per person-year of vision saved from PDR and $1120 per person-year of central acuity saved from macular edema as of 1990. Indeed, current estimates are that only 60% of patients in need of retinopathy treatment are receiving appropriate ophthalmic care. [240] If all patients with both type 1 and type 2 diabetes were to receive care according to currently suggested guidelines, annual savings of $624 million and 173,540 person-years of sight would be realized. [226] [232] The DCCT showed that both the rate of development of any retinopathy as well as the rate of retinopathy progression once it was present were significantly reduced after 3 years of intensive insulin therapy, [241] an effect maintained even 4 years after conclusion of the study. [26] [29] [242] Applying DCCT intensive insulin therapy to all persons in the United States with insulin-dependent diabetes mellitus would result in a gain of 920,000 person-years of sight, [243] although the costs of intensive therapy are three times that of conventional therapy. [244]



PATHOPHYSIOLOGY A detailed discussion of the pathophysiologic mechanisms underlying diabetic retinopathy and other diabetes-related complications has been presented earlier in this chapter. The earliest histologic effects of diabetes mellitus in the eye include loss of retinal vascular pericytes (supporting cells for retinal endothelial cells), thickening of vascular endothelium basement membrane, and alterations in retinal blood flow (Fig. 31-18) . [24] [245] [246] [247] [248] [249] [250] With increasing loss of retinal pericytes, the retinal vessel wall develops outpouchings (microaneurysms) and becomes fragile. Clinically, microaneurysms and small retinal hemorrhages may not always be readily distinguishable and are evaluated together as "hemorrhages and microaneurysms" (Fig. 31-19A) (Figure Not Available) (see also Color Plate). Rheologic changes occur in diabetic retinopathy resulting from increased platelet aggregation, integrin-mediated leukocyte adhesion, and endothelial damage. [251] [252] [253] Disruption of the blood retinal barrier may ensue, characterized by increased vascular permeability. [254] [255] The subsequent leakage of blood and serum from the retinal vessels results in retinal hemorrhages, retinal edema, and hard exudates (Fig. 31-19A and C) (Figure Not Available) . Moderate visual loss follows if the fovea is affected by the leakage. [239] With time, increasing sclerosis and endothelial cell loss lead to narrowing of the retinal vessels, which decreases vascular perfusion and may ultimately lead to obliteration of the capillaries and small vessels (Fig. 31-19 B) (Figure Not Available) . The resulting retinal ischemia is a potent inducer of angiogenic growth factors. Several angiogenic growth factors have been isolated from eyes with diabetic retinopathy, including IGFs, bFGF, hepatocyte growth factor (HGF), and VEGF. [256] [257] [258] [259] These factors promote the development of new vessel growth and retinal vascular permeability. [ 260] [ 261] [ 262] [263] [264] Indeed, inhibition of molecules such as VEGF and their signaling pathways can suppress the development of retinal neovascularization and retinal vascular permeability. [261] [265] [266] [267] [268] [269] New vessels tend to grow in regions of strong vitreous adhesion to the retina, such as at the optic disc and major vascular arcades (Fig. 31-19 D and E) (Figure Not Available) . The posterior vitreous face also serves as a scaffold for pathologic neovascularization, and the new vessels commonly arise at the junctions between perfused and nonperfused retina. When the retina is severely ischemic, the concentration of angiogenic growth factors may reach sufficient concentration in the anterior chamber to cause abnormal new vessel proliferation on the iris and the anterior chamber angle. [258] [270] Uncontrolled anterior segment neovascularization may result in rubeotic glaucoma because the fibrovascular proliferation in the angle of the eye causes blockage of aqueous outflow through the trabecular meshwork. [271] Proliferating new vessels in diabetic retinopathy have a tendency to bleed, which results in preretinal and vitreous hemorrhages (VHs) (Fig. 31-19 E and F) (Figure Not Available) . Although the presence of a large amount of blood in the preretinal space or vitreous cavity per se is not damaging to the retina, these intraocular hemorrhages often cause prolonged visual loss by blocking the visual axis. Membranes on the retinal surface can be induced by blood and result in wrinkling and traction on the retina. Although all retinal neovascularization eventually becomes quiescent, as with most scarring processes there is progressive fibrosis of the new vessel complexes that is associated with contraction. However, in the eye, such forces may exert traction on the retina, leading to tractional retinal detachment and retinal tears that may result in severe and permanent visual loss if left untreated (Fig. 31-19 G and H) (Figure Not Available) . In short, causes of visual loss from complications of diabetes mellitus include retinal ischemia involving the fovea, macular

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Figure 31-18 Diabetic retinopathy pathogenesis flow chart. The schematic flow chart represents the major preclinical and clinical findings associated with the full spectrum of diabetic retinopathy and macular edema. VEGF, vascular endothelial growth factor.

edema at or near the fovea, preretinal or vitreous hemorrhages, retinal detachment, and neovascular glaucoma. Visual loss may also result from more indirect effects of disease progression in diabetic patients, such as retinal vessel occlusion, accelerated atherosclerotic disease, and embolic phenomena.



CLINICAL FEATURES Risk Factors

Duration of diabetes is closely associated with the onset and severity of diabetic retinopathy. Diabetic retinopathy is rare in prepubescent patients with type 1 diabetes, but nearly all patients with type 1 diabetes and more than 60% of patients with type 2 diabetes develop some degree of retinopathy after 20 years. [218] [223] [272] In patients with type 2 diabetes, approximately 20% have retinopathy at the time of diabetes diagnosis and most have some degree of retinopathy over subsequent decades. Diabetic retinopathy is the most frequent cause of new-onset blindness among American adults aged 20 to 74 years. In the Wisconsin Epidemiologic Study of Diabetic Retinopathy, approximately 4% of patients younger than 30 years of age at diagnosis and nearly 2% of patients older than 30 years of age at diagnosis were legally blind. In the younger-onset group, 86% of blindness was attributable to diabetic retinopathy. In the older-onset group, where other eye diseases were also common, 33% of the cases of legal blindness were due to diabetic retinopathy. [223] [272] Lack of glycemic control is another significant risk factor for the onset and progression of diabetic retinopathy. The DCCT demonstrated a clear relationship between hyperglycemia and diabetic microvascular complications, including retinopathy in 1441 patients with type 1 diabetes. [26] [27] [273] [274] [275] In patients monitored 4 to 9 years, the DCCT showed that intensive insulin therapy reduced or prevented the development of retinopathy

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Figure 31-19 (Figure Not Available) Clinical features of diabetic retinopathy: some typical findings in human diabetic retinopathy. A, Findings in severe nonproliferative diabetic retinopathy, including microaneurysms (Ma), venous beading (VB), and intraretinal microvascular abnormalities (IRMA). B, Fluorescein angiogram showing marked capillary nonperfusion. C, Clinically significant macular edema with retinal thickening and hard exudates involving the fovea. D, Extensive neovascularization of the optic disc (NVD). This is high-risk proliferative diabetic retinopathy. E, Neovascularization elsewhere (NVE) and two small vitreous hemorrhages (VH). F, Extensive vitreous hemorrhage arising from severe neovascularization of the disc (NVD). G, Severe fibrovascular proliferation surrounding the fovea. H, Traction retinal detachment from extensive fibrovascular proliferation. I, Panretinal (scatter) laser photocoagulation. The macula and fovea and optic disc are not treated to preserve central vision. Laser burns are evident as white retinal lesions. (A to I, Adapted from Aiello LP. Eye complications of diabetes. In Korenman SG, Kahn CR [eds]. Atlas of Clinical Endocrinology. Vol 2: Diabetes. Philadelphia, Blackwell Scientific, 1999.)

by 27% as compared with conventional therapy. Additionally, intensive insulin therapy reduced the progression of diabetic retinopathy by 34% to 76% and had a substantial beneficial effect over the entire range of retinopathy severity. This improvement was achieved with an average 10% reduction in Hb A 1c from 8% to 7.2%. These results underscore that although intensive therapy does not prevent retinopathy completely, it reduces the risk of retinopathy onset and progression. Renal disease, as manifested by microalbuminuria and proteinuria, is yet another significant risk factor for onset and progression of diabetic retinopathy. [276] [277] Hypertension is associated with PDR and is an established risk factor for the development of macular edema. [278] Additionally, elevated serum lipid levels are associated with extravasated

Williams Textbook of Endocrinology

Williams Textbook of Endocrinology

Williams Textbook of Endocrinology