Lung Biology in Health & Disease Volume 170 Disease Markers in Exhaled Breath

DISEASE MARKERS IN EXHALED BREATH Edited by Nándor Marczin Sergei A. Kharitonov Sir Magdi H. Yacoub Peter J. Barnes I...

Author: Nandor Marczin | Sergei Kharitonov | Magdi Yacoub | Peter J. Barnes

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DISEASE MARKERS IN EXHALED BREATH

Edited by

Nándor Marczin Sergei A. Kharitonov Sir Magdi H. Yacoub Peter J. Barnes Imperial College of Science, Technology and Medicine National Heart and Lung Institute London, England

Marcel Dekker, Inc.

New York • Basel

Copyright © 2002 by Marcel Dekker, Inc. All Rights Reserved.

ISBN: 0-8247-0817-2 This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc. 270 Madison Avenue, New York, NY 10016 tel: 212-696-9000; fax: 212-685-4540 Eastern Hemisphere Distribution Marcel Dekker AG Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland tel: 41-61-260-6300; fax: 41-61-260-6333 World Wide Web http:/ /www.dekker.com The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the headquarters address above. Copyright  2003 by Marcel Dekker, Inc. All Rights Reserved. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): 10 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA

LUNG BIOLOGY IN HEALTH AND DISEASE Executive Editor Claude Lenfant Director, National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland

1. Immunologic and Infectious Reactions in the Lung, edited by C. H. Kirkpatrick and H. Y. Reynolds 2. The Biochemical Basis of Pulmonary Function, edited by R. G. Crystal 3. Bioengineering Aspects of the Lung, edited by J. B. West 4. Metabolic Functions of the Lung, edited by Y. S. Bakhle and J. R. Vane 5. Respiratory Defense Mechanisms (in two parts), edited by J. D. Brain, D. F. Proctor, and L. M. Reid 6. Development of the Lung, edited by W. A. Hodson 7. Lung Water and Solute Exchange, edited by N. C. Staub 8. Extrapulmonary Manifestations of Respiratory Disease, edited by E. D. Robin 9. Chronic Obstructive Pulmonary Disease, edited by T. L. Petty 10. Pathogenesis and Therapy of Lung Cancer, edited by C. C. Harris 11. Genetic Determinants of Pulmonary Disease, edited by S. D. Litwin 12. The Lung in the Transition Between Health and Disease, edited by P. T. Macklem and S. Permutt 13. Evolution of Respiratory Processes: A Comparative Approach, edited by S. C. Wood and C. Lenfant 14. Pulmonary Vascular Diseases, edited by K. M. Moser 15. Physiology and Pharmacology of the Airways, edited by J. A. Nadel 16. Diagnostic Techniques in Pulmonary Disease (in two parts), edited by M. A. Sackner 17. Regulation of Breathing (in two parts), edited by T. F. Hornbein 18. Occupational Lung Diseases: Research Approaches and Methods, edited by H. Weill and M. Turner-Warwick 19. Immunopharmacology of the Lung, edited by H. H. Newball 20. Sarcoidosis and Other Granulomatous Diseases of the Lung, edited by B. L. Fanburg 21. Sleep and Breathing, edited by N. A. Saunders and C. E. Sullivan 22. Pneumocystis carinii Pneumonia: Pathogenesis, Diagnosis, and Treatment, edited by L. S. Young 23. Pulmonary Nuclear Medicine: Techniques in Diagnosis of Lung Disease, edited by H. L. Atkins 24. Acute Respiratory Failure, edited by W. M. Zapol and K. J. Falke 25. Gas Mixing and Distribution in the Lung, edited by L. A. Engel and M. Paiva

26. High-Frequency Ventilation in Intensive Care and During Surgery, edited by G. Carlon and W. S. Howland 27. Pulmonary Development: Transition from Intrauterine to Extrauterine Life, edited by G. H. Nelson 28. Chronic Obstructive Pulmonary Disease: Second Edition, edited by T. L. Petty 29. The Thorax (in two parts), edited by C. Roussos and P. T. Macklem 30. The Pleura in Health and Disease, edited by J. Chrétien, J. Bignon, and A. Hirsch 31. Drug Therapy for Asthma: Research and Clinical Practice, edited by J. W. Jenne and S. Murphy 32. Pulmonary Endothelium in Health and Disease, edited by U. S. Ryan 33. The Airways: Neural Control in Health and Disease, edited by M. A. Kaliner and P. J. Barnes 34. Pathophysiology and Treatment of Inhalation Injuries, edited by J. Loke 35. Respiratory Function of the Upper Airway, edited by O. P. Mathew and G. Sant'Ambrogio 36. Chronic Obstructive Pulmonary Disease: A Behavioral Perspective, edited by A. J. McSweeny and I. Grant 37. Biology of Lung Cancer: Diagnosis and Treatment, edited by S. T. Rosen, J. L. Mulshine, F. Cuttitta, and P. G. Abrams 38. Pulmonary Vascular Physiology and Pathophysiology, edited by E. K. Weir and J. T. Reeves 39. Comparative Pulmonary Physiology: Current Concepts, edited by S. C. Wood 40. Respiratory Physiology: An Analytical Approach, edited by H. K. Chang and M. Paiva 41. Lung Cell Biology, edited by D. Massaro 42. Heart–Lung Interactions in Health and Disease, edited by S. M. Scharf and S. S. Cassidy 43. Clinical Epidemiology of Chronic Obstructive Pulmonary Disease, edited by M. J. Hensley and N. A. Saunders 44. Surgical Pathology of Lung Neoplasms, edited by A. M. Marchevsky 45. The Lung in Rheumatic Diseases, edited by G. W. Cannon and G. A. Zimmerman 46. Diagnostic Imaging of the Lung, edited by C. E. Putman 47. Models of Lung Disease: Microscopy and Structural Methods, edited by J. Gil 48. Electron Microscopy of the Lung, edited by D. E. Schraufnagel 49. Asthma: Its Pathology and Treatment, edited by M. A. Kaliner, P. J. Barnes, and C. G. A. Persson 50. Acute Respiratory Failure: Second Edition, edited by W. M. Zapol and F. Lemaire 51. Lung Disease in the Tropics, edited by O. P. Sharma 52. Exercise: Pulmonary Physiology and Pathophysiology, edited by B. J. Whipp and K. Wasserman 53. Developmental Neurobiology of Breathing, edited by G. G. Haddad and J. P. Farber 54. Mediators of Pulmonary Inflammation, edited by M. A. Bray and W. H. Anderson 55. The Airway Epithelium, edited by S. G. Farmer and D. Hay

56. Physiological Adaptations in Vertebrates: Respiration, Circulation, and Metabolism, edited by S. C. Wood, R. E. Weber, A. R. Hargens, and R. W. Millard 57. The Bronchial Circulation, edited by J. Butler 58. Lung Cancer Differentiation: Implications for Diagnosis and Treatment, edited by S. D. Bernal and P. J. Hesketh 59. Pulmonary Complications of Systemic Disease, edited by J. F. Murray 60. Lung Vascular Injury: Molecular and Cellular Response, edited by A. Johnson and T. J. Ferro 61. Cytokines of the Lung, edited by J. Kelley 62. The Mast Cell in Health and Disease, edited by M. A. Kaliner and D. D. Metcalfe 63. Pulmonary Disease in the Elderly Patient, edited by D. A. Mahler 64. Cystic Fibrosis, edited by P. B. Davis 65. Signal Transduction in Lung Cells, edited by J. S. Brody, D. M. Center, and V. A. Tkachuk 66. Tuberculosis: A Comprehensive International Approach, edited by L. B. Reichman and E. S. Hershfield 67. Pharmacology of the Respiratory Tract: Experimental and Clinical Research, edited by K. F. Chung and P. J. Barnes 68. Prevention of Respiratory Diseases, edited by A. Hirsch, M. Goldberg, J.-P. Martin, and R. Masse 69. Pneumocystis carinii Pneumonia: Second Edition, edited by P. D. Walzer 70. Fluid and Solute Transport in the Airspaces of the Lungs, edited by R. M. Effros and H. K. Chang 71. Sleep and Breathing: Second Edition, edited by N. A. Saunders and C. E. Sullivan 72. Airway Secretion: Physiological Bases for the Control of Mucous Hypersecretion, edited by T. Takishima and S. Shimura 73. Sarcoidosis and Other Granulomatous Disorders, edited by D. G. James 74. Epidemiology of Lung Cancer, edited by J. M. Samet 75. Pulmonary Embolism, edited by M. Morpurgo 76. Sports and Exercise Medicine, edited by S. C. Wood and R. C. Roach 77. Endotoxin and the Lungs, edited by K. L. Brigham 78. The Mesothelial Cell and Mesothelioma, edited by M.-C. Jaurand and J. Bignon 79. Regulation of Breathing: Second Edition, edited by J. A. Dempsey and A. I. Pack 80. Pulmonary Fibrosis, edited by S. Hin. Phan and R. S. Thrall 81. Long-Term Oxygen Therapy: Scientific Basis and Clinical Application, edited by W. J. O'Donohue, Jr. 82. Ventral Brainstem Mechanisms and Control of Respiration and Blood Pressure, edited by C. O. Trouth, R. M. Millis, H. F. Kiwull-Schöne, and M. E. Schläfke 83. A History of Breathing Physiology, edited by D. F. Proctor 84. Surfactant Therapy for Lung Disease, edited by B. Robertson and H. W. Taeusch 85. The Thorax: Second Edition, Revised and Expanded (in three parts), edited by C. Roussos

86. Severe Asthma: Pathogenesis and Clinical Management, edited by S. J. Szefler and D. Y. M. Leung 87. Mycobacterium avium–Complex Infection: Progress in Research and Treatment, edited by J. A. Korvick and C. A. Benson 88. Alpha 1–Antitrypsin Deficiency: Biology · Pathogenesis · Clinical Manifestations · Therapy, edited by R. G. Crystal 89. Adhesion Molecules and the Lung, edited by P. A. Ward and J. C. Fantone 90. Respiratory Sensation, edited by L. Adams and A. Guz 91. Pulmonary Rehabilitation, edited by A. P. Fishman 92. Acute Respiratory Failure in Chronic Obstructive Pulmonary Disease, edited by J.-P. Derenne, W. A. Whitelaw, and T. Similowski 93. Environmental Impact on the Airways: From Injury to Repair, edited by J. Chrétien and D. Dusser 94. Inhalation Aerosols: Physical and Biological Basis for Therapy, edited by A. J. Hickey 95. Tissue Oxygen Deprivation: From Molecular to Integrated Function, edited by G. G. Haddad and G. Lister 96. The Genetics of Asthma, edited by S. B. Liggett and D. A. Meyers 97. Inhaled Glucocorticoids in Asthma: Mechanisms and Clinical Actions, edited by R. P. Schleimer, W. W. Busse, and P. M. O’Byrne 98. Nitric Oxide and the Lung, edited by W. M. Zapol and K. D. Bloch 99. Primary Pulmonary Hypertension, edited by L. J. Rubin and S. Rich 100. Lung Growth and Development, edited by J. A. McDonald 101. Parasitic Lung Diseases, edited by A. A. F. Mahmoud 102. Lung Macrophages and Dendritic Cells in Health and Disease, edited by M. F. Lipscomb and S. W. Russell 103. Pulmonary and Cardiac Imaging, edited by C. Chiles and C. E. Putman 104. Gene Therapy for Diseases of the Lung, edited by K. L. Brigham 105. Oxygen, Gene Expression, and Cellular Function, edited by L. Biadasz Clerch and D. J. Massaro 106. Beta2-Agonists in Asthma Treatment, edited by R. Pauwels and P. M. O’Byrne 107. Inhalation Delivery of Therapeutic Peptides and Proteins, edited by A. L. Adjei and P. K. Gupta 108. Asthma in the Elderly, edited by R. A. Barbee and J. W. Bloom 109. Treatment of the Hospitalized Cystic Fibrosis Patient, edited by D. M. Orenstein and R. C. Stern 110. Asthma and Immunological Diseases in Pregnancy and Early Infancy, edited by M. Schatz, R. S. Zeiger, and H. N. Claman 111. Dyspnea, edited by D. A. Mahler 112. Proinflammatory and Antiinflammatory Peptides, edited by S. I. Said 113. Self-Management of Asthma, edited by H. Kotses and A. Harver 114. Eicosanoids, Aspirin, and Asthma, edited by A. Szczeklik, R. J. Gryglewski, and J. R. Vane 115. Fatal Asthma, edited by A. L. Sheffer 116. Pulmonary Edema, edited by M. A. Matthay and D. H. Ingbar 117. Inflammatory Mechanisms in Asthma, edited by S. T. Holgate and W. W. Busse 118. Physiological Basis of Ventilatory Support, edited by J. J. Marini and A. S. Slutsky

119. Human Immunodeficiency Virus and the Lung, edited by M. J. Rosen and J. M. Beck 120. Five-Lipoxygenase Products in Asthma, edited by J. M. Drazen, S.-E. Dahlén, and T. H. Lee 121. Complexity in Structure and Function of the Lung, edited by M. P. Hlastala and H. T. Robertson 122. Biology of Lung Cancer, edited by M. A. Kane and P. A. Bunn, Jr. 123. Rhinitis: Mechanisms and Management, edited by R. M. Naclerio, S. R. Durham, and N. Mygind 124. Lung Tumors: Fundamental Biology and Clinical Management, edited by C. Brambilla and E. Brambilla 125. Interleukin-5: From Molecule to Drug Target for Asthma, edited by C. J. Sanderson 126. Pediatric Asthma, edited by S. Murphy and H. W. Kelly 127. Viral Infections of the Respiratory Tract, edited by R. Dolin and P. F. Wright 128. Air Pollutants and the Respiratory Tract, edited by D. L. Swift and W. M. Foster 129. Gastroesophageal Reflux Disease and Airway Disease, edited by M. R. Stein 130. Exercise-Induced Asthma, edited by E. R. McFadden, Jr. 131. LAM and Other Diseases Characterized by Smooth Muscle Proliferation, edited by J. Moss 132. The Lung at Depth, edited by C. E. G. Lundgren and J. N. Miller 133. Regulation of Sleep and Circadian Rhythms, edited by F. W. Turek and P. C. Zee 134. Anticholinergic Agents in the Upper and Lower Airways, edited by S. L. Spector 135. Control of Breathing in Health and Disease, edited by M. D. Altose and Y. Kawakami 136. Immunotherapy in Asthma, edited by J. Bousquet and H. Yssel 137. Chronic Lung Disease in Early Infancy, edited by R. D. Bland and J. J. Coalson 138. Asthma's Impact on Society: The Social and Economic Burden, edited by K. B. Weiss, A. S. Buist, and S. D. Sullivan 139. New and Exploratory Therapeutic Agents for Asthma, edited by M. Yeadon and Z. Diamant 140. Multimodality Treatment of Lung Cancer, edited by A. T. Skarin 141. Cytokines in Pulmonary Disease: Infection and Inflammation, edited by S. Nelson and T. R. Martin 142. Diagnostic Pulmonary Pathology, edited by P. T. Cagle 143. Particle–Lung Interactions, edited by P. Gehr and J. Heyder 144. Tuberculosis: A Comprehensive International Approach, Second Edition, Revised and Expanded, edited by L. B. Reichman and E. S. Hershfield 145. Combination Therapy for Asthma and Chronic Obstructive Pulmonary Disease, edited by R. J. Martin and M. Kraft 146. Sleep Apnea: Implications in Cardiovascular and Cerebrovascular Disease, edited by T. D. Bradley and J. S. Floras 147. Sleep and Breathing in Children: A Developmental Approach, edited by G. M. Loughlin, J. L. Carroll, and C. L. Marcus

148. Pulmonary and Peripheral Gas Exchange in Health and Disease, edited by J. Roca, R. Rodriguez-Roisen, and P. D. Wagner 149. Lung Surfactants: Basic Science and Clinical Applications, R. H. Notter 150. Nosocomial Pneumonia, edited by W. R. Jarvis 151. Fetal Origins of Cardiovascular and Lung Disease, edited by David J. P. Barker 152. Long-Term Mechanical Ventilation, edited by N. S. Hill 153. Environmental Asthma, edited by R. K. Bush 154. Asthma and Respiratory Infections, edited by D. P. Skoner 155. Airway Remodeling, edited by P. H. Howarth, J. W. Wilson, J. Bousquet, S. Rak, and R. A. Pauwels 156. Genetic Models in Cardiorespiratory Biology, edited by G. G. Haddad and T. Xu 157. Respiratory-Circulatory Interactions in Health and Disease, edited by S. M. Scharf, M. R. Pinsky, and S. Magder 158. Ventilator Management Strategies for Critical Care, edited by N. S. Hill and M. M. Levy 159. Severe Asthma: Pathogenesis and Clinical Management, Second Edition, Revised and Expanded, edited by S. J. Szefler and D. Y. M. Leung 160. Gravity and the Lung: Lessons from Microgravity, edited by G. K. Prisk, M. Paiva, and J. B. West 161. High Altitude: An Exploration of Human Adaptation, edited by T. F. Hornbein and R. B. Schoene 162. Drug Delivery to the Lung, edited by H. Bisgaard, C. O’Callaghan, and G. C. Smaldone 163. Inhaled Steroids in Asthma: Optimizing Effects in the Airways, edited by R. P. Schleimer, P. M. O’Byrne, S. J. Szefler, and R. Brattsand 164. IgE and Anti-IgE Therapy in Asthma and Allergic Disease, edited by R. B. Fick, Jr., and P. M. Jardieu 165. Clinical Management of Chronic Obstructive Pulmonary Disease, edited by T. Similowski, W. A. Whitelaw, and J.-P. Derenne 166. Sleep Apnea: Pathogenesis, Diagnosis, and Treatment, edited by A. I. Pack 167. Biotherapeutic Approaches to Asthma, edited by J. Agosti and A. L. Sheffer 168. Proteoglycans in Lung Disease, edited by H. G. Garg, P. J. Roughley, and C. A. Hales 169. Gene Therapy in Lung Disease, edited by S. M. Albelda 170. Disease Markers in Exhaled Breath, edited by N. Marczin, S. A. Kharitonov, M. H. Yacoub, and P. J. Barnes 171. Sleep-Related Breathing Disorders: Experimental Models and Therapeutic Potential, edited by D. W. Carley and M. Radulovacki 172. Chemokines in the Lung, edited by R. M. Strieter, S. L. Kunkel, and T. J. Standiford 173. Respiratory Control and Disorders in the Newborn, edited by O. P. Mathew 174. The Immunological Basis of Asthma, edited by B. N. Lambrecht, H. C. Hoogsteden, and Z. Diamant

175. Oxygen Sensing: Responses and Adaptation to Hypoxia, edited by S. Lahiri, G. L. Semenza, and N. R. Prabhakar 176. Non-Neoplastic Advanced Lung Disease, edited by J. Maurer

ADDITIONAL VOLUMES IN PREPARATION

Therapeutic Targets in Airway Inflammation, edited by N. T. Eissa and D. Huston Respiratory Infections in Asthma and Allergy, edited by S. Johnston and N. Papadopoulos Acute Respiratory Distress Syndrome, edited by M. A. Matthay Upper and Lower Respiratory Disease, edited by J. Corren, A. Togias, and J. Bousquet Venous Thromboembolism, edited by J. E. Dalen Acute Exacerbations of Chronic Obstructive Pulmonary Disease, edited by N. Siafakas, N. Anthonisen, and D. Georgopolous Lung Volume Reduction Surgery for Emphysema, edited by H. E. Fessler, J. J. Reilly, Jr., and D. J. Sugarbaker

The opinions expressed in these volumes do not necessarily represent the views of the National Institutes of Health.

INTRODUCTION

This volume presents an amazing story that has emerged from the convergence of biological knowledge and application and of technological developments that allow the measurement of molecules. There was a time when the lung was assumed to be a “cooling” organ— for cooling the blood, that is! Subsequently we learned about the exchange of oxygen and carbon dioxide that takes place in the lung. Eventually the impact of cell biology, biochemistry, and molecular biology established the complexity of this organ and, as well, demonstrated the importance and intensity of its metabolic function. At the same time, technological and instrumental developments provided the tools to measure compounds and molecules present in minute quantities. Thus, it is not surprising that scientists have taken advantage of the opportunity offered by the cyclical characteristics of respiration to measure the levels of biological markers and bioproducts in exhaled air. It soon became clear that the level of these compounds can be affected by structural, functional, and pathological changes occurring in the lung, as well as in distant organs. Biomarkers, or surrogate markers, are of great interest, and they are now part of the armamentarium that clinicians can use to evaluate the transition between health and disease, as well as the stage and severity of disease. iii

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Undoubtedly, the seminal work of R. Furchgott, L. Iguarro, F. Murad, and S. Moncada gave a very special, if not unique, focus on nitric oxide as a mediator of many biological processes. Nitric oxide, however, is not the only molecule that can be measured in exhaled breath. This volume gives examples of biomarkers that are now investigated to assess inflammation and other conditions. Markers such as hydrogen peroxide and ecosanoids may be considered, as well as derivatives of carbon monoxide and hydrocarbons. The goal here is not to enumerate all the possibilities, but, as this volume demonstrates so vividly, to show that biology, epidemiology, and medicine have available a powerful tool—biomarkers—that can facilitate investigations, make the practice of medicine better, and thus improve health. Over the years, the Lung Biology in Health and Disease series of monographs has presented many volumes reporting basic science, and many others examining clinical aspects of lung disease. However, this volume is distinctive in that it bridges advances in molecular analyses with clinical situations. Although the field is relatively new, its potential is enormous. This monograph permits clinicians, and eventually their patients, to be exposed to new approaches from which they will all benefit. The editors and authors are all well known and much respected in their fields. I am grateful to them for allowing this series to be the vehicle for the dissemination of their ideas and concepts, and for the evidence provided to support their clinical use. Claude Lenfant, M.D. Bethesda, Maryland

PREFACE

This book is designed to summarize exciting recent developments in a rapidly evolving field of lung biology, which could provide basic scientists and clinicians with novel noninvasive methods to monitor disease activity based on molecular analysis of exhaled breath. Undoubtedly, research activities over the past few decades have resulted in the identification of a number of important biochemical pathways, affording us with a better understanding of the biology of lung diseases. Many of these processes result in bioproducts that are released from tissue to the gas phase and can be detected in the exhaled air. Alternatively, the underlying disease may involve reactions that ultimately produce a naturally occurring component in exhaled breath. There is increasing evidence that nitric oxide (NO), carbon monoxide (CO), oxidative end products, and prostanoids are fascinating examples of novel molecular markers in exhaled air that could be used as rapid and noninvasive diagnostic tools in a variety of lung pathologies and in fact some distant organ dysfunction. Not very surprisingly, a significant number of chapters in this book focus on characteristics of NO in exhaled air. As discussed by Claude Lenfant and Warren Zapol in the introduction to a recent volume in this series, the masterful identification of NO as a principal endogenous mediator precipitated an urge for clinical applications. On the basis of exciting animal experiments, a number of v

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NO-related therapies have been implemented in clinical practice, such as inhibition of NO in septic shock and inhaled NO gas for the treatment of hypoxemic patients. At the same time, many investigators became engaged in studying the characteristics of the endogenous L-arginine-NO pathway in the normal and diseased lung, in both animal models and humans. This activity led to the remarkable discovery of NO in exhaled air by Gustafsson and colleagues in 1991, which launched a decade of considerable progress in understanding how this ‘‘window’’ can be used to reflect production and fluid-phase reactions of NO in lung tissue. We have learned much regarding pitfalls of NO determination and produced recommendations for standardization of NO analysis in exhaled air. Finally, we have completed an initial phase of clinical investigations where one can correlate levels of exhaled NO with certain disease activity and response to medical treatment. Although much of the recent research activity has focused on NO, the diagnostic potential of exhaled breath is by no means limited to NO. There is increasing evidence of another interesting endogenous pathway involving heme oxygenase. The activity of this enzyme leads to the generation of CO, a molecule that exhibits a biological profile similar to that of NO. Recent elegant studies suggest an inducible nature for this pathway, which could be important in limiting stress-induced responses in the lungs. Monitoring CO accumulation in exhaled air appears to be promising, and current investigations suggest that it might provide important information during the course of acute lung injury. Despite the fact that limited and well-controlled oxidative reactions are being increasingly recognized as participants in cellular signal transduction events, there is no doubt that uncontrolled oxidant stress is the underlying mechanism of a wide variety of lung pathologies, including lung injury associated with ischemia-reperfusion and inflammation. It is therefore crucial that researchers and clinicians have appropriate means to detect the presence of untoward oxidant stress, to monitor the extent of injury and response to therapeutic modalities. Intriguing investigations reveal that oxidant stress-induced changes in cellular membranes lead to accumulation of volatile organic compounds that can be detected in exhaled air. Furthermore, hydrogen peroxide, one of the classical mediators of oxidant reactions, and some oxidative products of arachidonic acid metabolism can be collected in condensates of exhaled air to monitor and to quantify the extent of oxidant stress in the lungs. This volume brings together current knowledge of the characteristics of novel biological mediators in exhaled breath. This expertise holds the potential for many important aspects of lung disease to be diagnosed and monitored rapidly, noninvasively, and at the bedside. For these reasons, it is not surprising that these methods are being increasingly used in many centers all around the world. However, there are many pitfalls in these measurements, leading to nonuniformity in the reported data. The only way to avoid artifacts is to understand the basic mechanisms involved in the generation and release from lung tissue to the

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airways of these markers, and their diffusion and distribution characteristics along the airways. Finally, there are important technical aspects of the measurements. These considerations are reflected in the first two main sections of the book. Part One reviews basic physiological aspects of disease mediators in exhaled breath. We begin by reviewing the general importance of the L-arginine-NO pathway as a primary biological mediator system. Belvisi and coauthors provide important information regarding the mechanisms utilized by different cell types to synthesize NO, the molecular and substrate regulation of the major forms of NO synthases, their inhibitors, and effector mechanisms influenced by NO. The next chapter, by Adding and Gustafsson, reflects on the original discovery of exhaled NO and offers the reader an up-to-date review of progress made on fundamentals of the physiology of exhaled NO, with emphasis on regulatory factors such as atmospheric oxygen, stretch, and endogenously formed carbon dioxide and catecholamines. This invaluable chapter culminates in a comprehensive hypothesis regarding regulation of NO synthesis and its physiological implications in less ventilated lung regions. Despite the progress regarding many aspects of the exhaled-NO field, there is still considerable uncertainty as to the precise sources of the NO in expired air. The chapter by Deykin and collaborators reviews the enzymatic formation of NO, summarizes the available data to indicate which of these enzymes contributes NO to the expirate, and then discusses the cellular and anatomical compartments from which the NO so formed contributes to the fraction of expired NO. In contrast, one of the earliest and widely accepted findings regarding concentrations of NO in expired air was the demonstration of flow dependency. This can be extended to other important determinants of gas-phase NO. Among these, ventilation parameters and pulmonary blood flow are major factors in influencing measured NO concentration, which is the topic of the next chapter. Understanding these issues has implications not only for exhaled NO physiology but for standardization of clinical measurements and for data interpretation in various pathological conditions. We then temporarily depart from NO and dedicate two chapters to overviews of the biochemical and physiological regulation of another gaseous molecule, CO. We begin by focusing on heme oxygenase (HO), a remarkable enzyme and highly conserved molecule that seems to be essential for most forms of life. Interestingly, constitutive and inducible isoforms of HO are involved not only in the breakdown of heme but in modulating many cellular and organ responses to oxidative stress and inflammatory stimuli. These issues are reviewed in Chapter 5, by Lee et al., focusing on the modulation of various forms of lung injury and on the potential mechanisms involved in HO-mediated cytoprotection. CO is one of the exciting by-products of HO action, and this extraordinary molecule is the subject of the next chapter, by Otterbein and coworkers. CO has been studied for over 100 years and until the last few years has been touted as a molecule to

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avoid. Its story resembles that of NO in many ways. After many years of consideration as a toxic gaseous molecule, recent studies demonstrate that CO is an intriguing intra- and intercellular regulator of ever-increasing numbers of physiological responses, which exhibit considerable overlap with NO. Otterbein et al. present here, with much personal enthusiasm, an interesting historical perspective on CO research and summarize compelling reasons for a renewed effort to understand the potentially beneficial effects of this interesting molecule. One common scenario in a multitude of lung diseases is the episode of a variable degree of oxidative stress. Originally this was described as a series of relatively simple chemical reactions including oxygen-derived free radicals. It appears that the picture has become much more complicated than originally thought, and the intricate interplay between nitrogen-, oxygen-, and carbon-centered free radicals and oxidizing species requires special attention. Since these reactions are at the core of many lung diseases, we include a section discussing these issues in terms of the physiological aspects. Davis and coauthors have provided a state-of-the-art review of the characteristics, the good and dark sides of reactive nitrogen and oxygen species, and their contribution to experimental and clinical lung injury. Part Two is devoted to methodological aspects and technological issues concerning detection of the proposed disease markers NO, CO, and organic volatile compounds in exhaled air. Kharitonov and Barnes review technical issues and recommendations regarding measurement of NO and CO in the gas phase and techniques to collect breath condensate containing a myriad of potential breath markers. We then discuss methodological aspects of breath analysis involving volatile organic compounds. In Chapter 9, Phillips presents an overview of the advantages and disadvantages of various approaches to collecting, concentrating, and analyzing volatile organic compounds. He also hints at the potential diagnostic value of these markers in several disorders, including cancer, transplant rejection, and heart disease. Part Three focuses on pathological aspects of disease markers. Instead of reviewing each disease individually, we grouped conditions according to the main pathological features of lung disease, such as hypoxia, ischemia-reperfusion, and inflammation. In the hypoxia section, Dweik and Erzurum review recent data on the acute and chronic effects of hypoxia on lung NO production in animals and humans. Chapter 11, by Kharitonov, deals with characteristics of exhaled NO in primary pulmonary hypertension, a condition in which hypoxia is a dominant feature. The common scenario in the next two chapters is ischemia-reperfusion. Due to the delicate structure of the alveolar-capillary unit, its huge surface area available for activated leukocyte–endothelial interactions, and the fact that the lung microvasculature receives the entire cardiac output immediately after reperfusion, the lung is especially vulnerable to this type of injury. In light of these

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considerations, as discussed in the chapter by Ko¨vesi et al., it is quite remarkable that recent studies reveal maintained exhaled NO levels in patients subjected to transient ischemia-reperfusion during cardiac surgery utilizing cardiopulmonary bypass. In contrast, NO completely disappears from the exhaled breath of the majority of lung transplant recipients, suggesting more prominent lung injury with complete lung ischemia. The work by Brown and Risby continues this theme by reviewing mechanisms underlying volatile organic compound production in oxidative stress and by reviewing exciting data regarding monitoring reperfusion injury in a distant organ by analysis of exhaled breath. A major section of this volume is devoted to issues related to inflammation. Kharitonov and Barnes discuss the biology and biochemical markers of asthma, focusing primarily on NO and CO. This is clearly an area in which monitoring of exhaled NO is already in clinical practice. Stitt and Douglas present evidence from animal studies showing that exhaled NO could be used as an early marker of septic shock. Chapter 16, by Schubert et al., takes this further into clinical practice and demonstrates the complexity of the many simultaneous biological processes underlying the development of adult respiratory distress syndrome and the potential of disease markers to help us to understand these events. One of the most difficult medical tasks in caring for lung transplant recipients is the differential diagnosis between acute infection and rejection processes. Novel data are provided by Fisher and colleagues regarding characteristics of NO in these conditions. Cystic fibrosis (CF) occupies a special place in exhaled NO research, because it is one of the few inflammatory disorders that are not accompanied by increased exhaled NO. Kelley and Drumm have done pioneering work in this area, and they summarize characteristics of NO production in CF. We continue our journey of exhaled markers with an important review of what is currently known about the role of NO in autoimmune and rheumatic disorders. Rolla and Caligaris-Cappio examine the fate of exhaled NO in patients with rheumatic disease with and without lung involvement. Finally, the chapter by Demoncheaux and coauthors investigates the contribution of exhaled NO to total body metabolism of nitrogen oxides and how these events are disturbed in liver diseases. In order to assemble the best possible account of this rapidly developing field, we have recruited an expert international faculty from leading groups of investigators. The authors represent a wide range of current activities and sound experience with disease markers in exhaled breath. We hope that this monograph will be an asset for investigators and clinicians who wish to widen their diagnostic and monitoring repertoire both in the laboratory and at the bedside. We share the view that—beyond major technological developments of immense commercial applicability, such as ethanol breath testing and capnography—breath analysis has already provided gratifying potential as a noninvasive diagnostic entity for

x

Preface

monitoring disease severity and responsiveness to medication at a molecular level. Although we continue to debate many important aspects of this field, we also have the common vision that breath analysis upholds its promise as one of the most exciting and innovative aspects of research in lung biology in health and disease. Nańdor Marczin Sergei A. Kharitonov Sir Magdi H. Yacoub Peter J. Barnes

CONTRIBUTORS

L. Christofer Adding, M.D., Ph.D. Division of Physiology, Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden Peter J. Barnes, M.D., D.Sc., F.R.C.P. Professor and Head, Department of Thoracic Medicine, Imperial College of Science, Technology and Medicine, National Heart and Lung Institute, and Royal Brompton Hospital, London, England Maria G. Belvisi, Ph.D. Reader in Respiratory Pharmacology, Respiratory Pharmacology Group, Department of Cardiothoracic Surgery, Imperial College of Science, Technology and Medicine, National Heart and Lung Institute, London, England Robert H. Brown, M.D., M.P.H. Associate Professor, Department of Anesthesiology and Environmental Health Sciences, Johns Hopkins University, Baltimore, Maryland, U.S.A. Federico Caligaris-Cappio, M.D. Professor of Internal Medicine, Department of Oncological Sciences, University of Turin, and Ospedale Mauriziano Umberto I, Turin, Italy xi

xii

Contributors

Jeffrey T. Chapman, M.D. Associate Staff Physician, Department of Allergy, Pulmonary, and Critical Care Medicine, The Cleveland Clinic Foundation, Cleveland, Ohio, U.S.A. Augustine M. K. Choi, M.D. Chief, Division of Pulmonary and Critical Care Medicine, and Professor, Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, U.S.A. Paul A. Corris, F.R.C.P. Professor of Thoracic Medicine, Department of Respiratory Medicine, Freeman Hospital, and University of Newcastle upon Tyne, Newcastle upon Tyne, England Ian C. Davis, Ph.D. Research Instructor, Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A. Eric Demoncheaux, Ph.D. Research Associate, Division of Clinical Sciences (South), Departments of Medicine and Pharmacology, University of Sheffield Medical School, Sheffield, England George T. De Sanctis, Ph.D., F.C.C.P. Head, Respiratory Pharmacology, Aventis Pharmaceuticals, Bridgewater, New Jersey, U.S.A. Anthony De Soyza, M.B.Ch.B., B.M.Sc.(Hons) Department of Respiratory Medicine, Freeman Hospital, and University of Newcastle upon Tyne, Newcastle upon Tyne, England Aaron Deykin, M.D. Medical Director, Pulmonary Function Laboratory, Pulmonary Division, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, U.S.A. James S. Douglas, Ph.D. The John B. Pierce Laboratory, Yale University School of Medicine, New Haven, Connecticut, U.S.A. Jeffrey M. Drazen, M.D. Professor, Department of Medicine, Harvard Medical School, and Brigham & Women’s Hospital, Boston, Massachusetts, U.S.A. Mitchell L. Drumm, Ph.D. Associate Professor, Departments of Pediatrics and Genetics, Case Western Reserve University, Cleveland, Ohio, U.S.A. Raed A. Dweik, M.B., B.S. F.A.C.P., F.C.C.P., F.R.C.P.C. Department of Pulmonary and Critical Care Medicine, Cleveland Clinic Foundation, Cleveland, Ohio, U.S.A.

Contributors

xiii

Serpil C. Erzurum, M.D. Director, Lung Biology Program, Department of Pulmonary and Critical Care Medicine, and Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, U.S.A. Andrew J. Fisher, Ph.D., M.R.C.P. Special Registrar in Respiratory Medicine, Department of Respiratory Medicine, Freeman Hospital, and University of Newcastle upon Tyne, Newcastle upon Tyne, England Klaus Geiger, Prof.Dr. Department of Anesthesiology and Intensive Care Medicine, University Hospital of Freiburg, Freiburg, Germany Dermot Gleeson, M.D., F.R.C.P. Sheffield, England

Liver Unit, Sheffield Teaching Hospital,

Lars E. Gustafsson, M.D., Ph.D. Professor, Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden Judy M. Hickman-Davis, D.V.M., Ph.D. Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A. Tim W. Higenbottam, B.Sc., M.D., M.A., D.Sc., F.R.C.P. Professor, Division of Clinical Sciences (South), Department of Medicine, University of Sheffield Medical School, Sheffield, England Thomas J. Kelley, Ph.D. Assistant Professor, Departments of Pediatrics and Pharmacology, Case Western Reserve University, Cleveland, Ohio, U.S.A. Sergei A. Kharitonov, M.D., Ph.D. Department of Thoracic Medicine, Imperial College of Science, Technology and Medicine, National Heart and Lung Institute, and Royal Brompton Hospital, London, England Lester Kobzik, M.D. Associate Professor, Department of Pathology, Harvard Medical School, and Brigham & Women’s Hospital, Boston, Massachusetts, U.S.A. Tama´s Ko¨vesi, M.D. Department of Anesthesia and Critical Care, Harefield Hospital, Harefield, Middlesex, England Patty J. Lee, M.D. Assistant Professor of Medicine, Critical Care Section, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, U.S.A.

xiv

Contributors

J. Russell Lindsey, D.V.M. Professor, Department of Genomics and Pathobiology, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A. Nańdor Marczin, M.D., Ph.D. Senior Lecturer, Department of Cardiothoracic Surgery, Imperial College of Science, Technology and Medicine, National Heart and Lung Institute, London, and Harefield Hospital, Harefield, Middlesex, England Anthony F. Massaro, M.D. Division of Pulmonary and Critical Care Medicine, Brigham & Women’s Hospital, and Harvard Medical School, Boston, Massachusetts, U.S.A. Sadis Matalon, Ph.D. Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A. Wolfram Miekisch, Ph.D. Department of Anesthesia and Intensive Care Medicine, University of Rostock, and University Hospital of Rostock, Rostock, Germany Jane A. Mitchell, Ph.D. Unit of Critical Care Medicine, Imperial College of Science, Technology and Medicine, National Heart and Lung Institute, London, England Danielle Morse, M.D. Assistant Professor, Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, U.S.A. Leo E. Otterbein, Ph.D. Research Assistant Professor, Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, U.S.A. Michael Phillips, M.D., F.A.C.P., M.R.C.P.(UK) Clinical Professor, Department of Medicine, New York Medical College, Valhalla, New York, and Menssana Research, Inc., Fort Lee, New Jersey, U.S.A. Terence H. Risby, F.R.S.C., C.Chem. Professor, Department of Environmental Health Sciences, Johns Hopkins University, Baltimore, Maryland, U.S.A. Giovanni Rolla, M.D. Associate Professor, University of Turin, and Allergology and Immunology Clinic, Ospedale Mauriziano Umberto I, Turin, Italy David Royston, M.D., F.R.C.A. Consultant in Cardiothoracic Anesthesiology, Department of Anesthesia and Critical Care, Harefield Hospital, Harefield, Middlesex, England

Contributors

xv

Madhu Sasidhar, M.B. Pulmonary Consultant, Department of Internal Medicine, Eastern New Mexico Medical Center, Roswell, New Mexico, U.S.A. Jochen Klaus Schubert, M.D., Dipl.Chem., D.E.A.A. Head Physician, Department of Anesthesia and Intensive Care, University of Rostock, and University Hospital of Rostock, Rostock, Germany Jigme M. Sethi, M.D. Assistant Professor, Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, U.S.A. John T. Stitt, Ph.D. Professor, Department of Cellular and Molecular Physiology and Department of Epidemiology, and Fellow, The John B. Pierce Foundation Laboratory, Yale University School of Medicine, New Haven, Connecticut, U.S.A. I. Gavin Wright, F.C.A. (SA) Consultant Anesthetist, Department of Anesthesia and Critical Care, Harefield Hospital, Harefield, Middlesex, England Sir Magdi H. Yacoub, F.R.S., F.R.C.S. Professor, Department of Cardiothoracic Surgery, Imperial College of Science, Technology and Medicine, National Heart and Lung Institute, London, and Harefield Hospital, Harefield, Middlesex, England

CONTENTS

Introduction Preface Contributors Part One

Claude Lenfant

iii v xi

PHYSIOLOGICAL ASPECTS OF DISEASE MARKERS IN EXHALED GASES

Nitric Oxide 1. Nitric Oxide as a Biological Mediator Maria G. Belvisi, Jane A. Mitchell, and Sir Magdi H. Yacoub I. II. III. IV. V.

Introduction Nitric Oxide Synthesis by Different Cell Types Release of NO by Nerves: Neuronal NOS Release of NO by Endothelial Cells: Endothelial NOS Release of NO by Cells Induced to Express NOS: Inducible NOS

3

3 4 5 7 8 xvii

xviii

Contents VI. Classification of NOS Isoforms VII. Inhibition of NOS by Substrate Analog and Non-Amino Acid Inhibitors: Interactions with Different NOS Isoforms VIII. Effector Mechanisms Utilized by NO IX. Nitric Oxide, Pathophysiological States, and Therapeutic Possibilities X. Summary References

2.

Physiology of Exhaled Nitric Oxide L. Christofer Adding and Lars E. Gustafsson I. II. III. IV. V. VI.

3.

Molecular and Cellular Sources of Exhaled Nitric Oxide Aaron Deykin, Anthony F. Massaro, Lester Kobzik, George T. De Sanctis, and Jeffrey M. Drazen I. II. III. IV. V.

4.

Introduction Measurement Techniques of Exhaled NO Metabolism of Exhaled NO Physiological Factors Regulating Exhaled NO Physiological Role of NO in the Respiratory System Summary References

Introduction to Exhaled NO Enzymatic Source of Exhaled NO Cellular Sources of Exhaled NO Anatomical Sources of Exhaled NO Conclusions References

Determinants of Exhaled Nitric Oxide: Influence of Ventilation and Pulmonary Blood Flow Nańdor Marczin, I. Gavin Wright, and Sir Magdi H. Yacoub I. Introduction II. Influence of Ventilation III. Role of Pulmonary Blood Flow References

10

10 12 17 18 18 29 29 30 35 39 48 52 53

73

73 74 82 82 85 85

91

91 92 105 111

Contents

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Carbon Monoxide 5. Heme Oxygenase-1 in Lung Disease Patty J. Lee, Leo E. Otterbein, Jigme M. Sethi, Madhu Sasidhar, and Augustine M. K. Choi I. II. III. IV. V.

Introduction HO1 and Lung Disease Mechanisms of HO1 HO2 and Lung Disease Conclusions and Future Directions References

6. Carbon Monoxide: A Gaseous Molecule with AntiInflammatory Properties Leo E. Otterbein, Danielle Morse, Jeffrey T. Chapman, and Augustine M. K. Choi I. II. III. IV. V. VI.

Historical Perspective Heme Oxygenase Bilirubin Ferritin Carbon Monoxide Summary and Future Directions References

117

117 119 125 126 126 127

133

133 137 139 139 140 146 149

Markers of Oxidative Damage 7. Role of Reactive Oxygen and Nitrogen Species in Lung Injury Ian C. Davis, Judy M. Hickman-Davis, J. Russell Lindsey, and Sadis Matalon I. Introduction II. Chemistry and Biochemistry of Reactive Oxygen and Nitrogen Species III. Biological Effects of Reactive Oxygen and Nitrogen Species IV. Reactive Species in Acute Lung Injury V. Summary References

159

159 160 164 176 182 182

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Contents

Part Two 8.

Exhaled Nitric Oxide, Carbon Monoxide, and Breath Condensate Sergei A. Kharitonov and Peter J. Barnes I. II. III. IV. V.

9.

METHODOLOGICAL AND TECHNICAL ASPECTS

Introduction Nitric Oxide Carbon Monoxide Exhaled Breath Condensate Future Directions References

Analysis of Volatile Organic Compounds in the Breath Michael Phillips I. II. III. IV.

Part Three

History of Breath Tests Classification of Breath Tests Breath VOC Analysis: The Major Technical Problems Conclusions References

199 199 200 203 207 209 211 219 219 220 221 229 229

PATHOLOGICAL ASPECTS

Hypoxia 10.

Regulation of Nitric Oxide Synthases and Gas-Phase Nitric Oxide by Oxygen Raed A. Dweik and Serpil C. Erzurum I. II. III. IV. V. VI.

11.

Introduction Source(s) of NO in Exhaled Breath Regulation of NO Synthesis by Oxygen Effect of Oxygen on Exhaled NO Regulation of NOS Gene Expression by Oxygen NO as a Mediator of Vascular Response to Oxygen References

Exhaled Markers in Interstitial Lung Disease and Pulmonary Hypertension Sergei A. Kharitonov I. Introduction II. Nitric Oxide

235 235 235 238 239 242 242 243

247 247 247

Contents

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III. Carbon Monoxide IV. Exhaled Breath Condensate References

250 251 253

Ischemia-Reperfusion 12. Exhaled Nitric Oxide in Human Lung IschemiaReperfusion Tama´s Ko¨vesi, David Royston, Sir Magdi H. Yacoub, and Nańdor Marczin I. Introduction: Lung Ischemia-Reperfusion in Cardiothoracic Surgery II. Mechanisms of Ischemia-Reperfusion Injury III. Role of NO in Ischemia-Reperfusion IV. Physiology of Exhaled NO V. Exhaled NO Following CPB and Lung Transplantation VI. Summary References 13. Monitoring Distant Organ Reperfusion Injury by Measurement of Volatile Organic Compounds Robert H. Brown and Terence H. Risby I. II. III. IV. V. VI. VII.

Introduction Breath Collection Breath Analysis Expression of Data Organ Systems Interventions Conclusion and Future Directions References

259

259 260 262 267 268 274 275

281 281 285 291 294 295 301 302 303

Inflammation 14. Exhaled Nitric Oxide, Carbon Monoxide, and Breath Condensate in Inflammatory Lung Disease and Response to Medical Treatment Sergei A. Kharitonov and Peter J. Barnes I. II. III. IV.

Introduction Nitric Oxide Carbon Monoxide Exhaled Breath Condensate

307 307 308 317 320

xxii

Contents V. Future Directions References

15.

Exhaled Gas Disease Markers in Septicemia: A Possible Role for Nitric Oxide in Lung Injury John T. Stitt and James S. Douglas I. II. III. IV. V. VI. VII. VIII.

16.

Introduction Endotoxin Shock Sepsis and Lung Injury Exhaled NO as a Marker of Lung Injury Influence of Endotoxin on Exhaled NO Role of Increased NO in LPS-Induced Lung Injury Conclusions Clinical Implications References

Exhaled Breath Markers in Acute Respiratory Distress Syndrome Jochen Klaus Schubert, Wolfram Miekisch, and Klaus Geiger I. ARDS Overview II. Special Problems of Gas Sampling in Mechanically Ventilated Patients III. Clinical Aspects of Exhaled Volatile Substances IV. Specific Patterns of Exhaled Gases in ARDS V. Conclusions References

17.

Exhaled Nitric Oxide in Lung Transplant Recipients Andrew J. Fisher, Anthony De Soyza, and Paul A. Corris I. Introduction II. Validation of NO Measurement in Lung Transplant Recipients III. Effect of Graft Pathology on Exhaled NO in Lung Transplant IV. Use of Exhaled NO in the Clinical Setting References

18.

Nitric Oxide in Cystic Fibrosis Thomas J. Kelley and Mitchell L. Drumm I. Detection of NO and NO Metabolites in the CF Lung II. Possible Mechanisms of Reduced Exhaled NO in CF

327 328

343 343 344 345 346 346 355 357 358 359

363 363 365 369 372 376 376 381 381 385 387 397 397 403 403 404

Contents

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III. Possible Implications of Reduced Epithelial NOS2 Expression and NO Production in CF Airway Disease IV. Summary References 19. Exhaled Nitric Oxide in Rheumatic Diseases Giovanni Rolla and Federico Caligaris-Cappio I. II. III. IV. V.

Introduction NO and Lymphocyte Function NO and Apoptosis Specific Diseases Conclusions References

20. The Disturbance of Metabolism of Oxides of Nitrogen in Liver Disease: Exhaled Nitric Oxide as a Measure of Severity Eric Demoncheaux, Tim W. Higenbottam, and Dermot Gleeson I. Introduction II. Nitrogen: A Multifaceted Atom of Great Biological Importance III. Whole-Body Turnover of Nitrogen and Related Oxides IV. Intraluminal Bowel Bacterial Turnover of Nitrogen Oxides V. Mechanisms Behind Disturbed Turnover of Oxides of Nitrogen in Liver Cirrhosis VI. Exhaled Nitric Oxide and Liver Diseases VII. Challenges Left for the Future References Author Index Subject Index

408 412 413 421 421 422 423 424 436 436

445 445 446 446 450 451 453 454 455 463 521

Part One PHYSIOLOGICAL ASPECTS OF DISEASE MARKERS IN EXHALED GASES

NITRIC OXIDE

1 Nitric Oxide as a Biological Mediator

MARIA G. BELVISI and JANE A. MITCHELL Imperial College of Science, Technology and Medicine National Heart and Lung Institute London, England

SIR MAGDI H. YACOUB Imperial College of Science, Technology and Medicine National Heart and Lung Institute London and Harefield Hospital Harefield, Middlesex, England

I.

Introduction

Since its discovery as an endothelial-derived relaxant factor (EDRF), nitric oxide (NO) has been demonstrated to play a critical role in numerous physiological processes, as well as in the pathophysiology of many diseases. For example, it is now known that NO is the ubiquitous activator of guanylyl cyclase, resulting in smooth muscle relaxation, platelet reactivity, and central and peripheral neurotransmission. In addition, NO can activate/inhibit a number of other proteins that influence cellular responses. Within a physiological setting, NO release by endothelial cells or nerves contributes to homeostatic processes in every organ system in the body. NO is also released as a primary defense mechanism by immune cells. However, when NO production becomes excessive, its release can contribute to the processes of inflammation and/or cardiovascular dysfunction. The importance of NO as a biological mediator was highlighted by the recent awarding of the Nobel Prize for Physiology and Medicine for description of the interaction between NO and the heme moiety of soluble guanylyl cyclase. The ability of NO to perform its different functions in the body is largely made possible by the presence of multiple isoforms of the enzyme NO synthase 3

4

Belvisi et al.

(NOS), which can be induced, upregulated, or suppressed depending on requirement. Each NOS isoform is encoded by a distinct gene comprising either 26 exons (inducible NOS and endothelial NOS) or 29 exons (neuronal NOS) (1,2). This chapter discusses the relevance of the different isoforms of NOS in the regulation of physiological and pathophysiological events. II. Nitric Oxide Synthesis by Different Cell Types The first examples of the actions of endogenously released NO in mammals were observed in isolated blood vessels. In these studies, activation of the endothelial layer resulted in relaxation of the underlying smooth muscle and an unknown factor, endothelial-derived relaxing factor or EDRF, was identified (3) (Fig 1). The identity of EDRF was not established until 1987, when Palmer and colleagues showed that it was indistinguishable from NO (4). Around this time it was also found that NO was a neurotransmitter (5) used by the inhibitory nonadrenergic, noncholineric nerves (6) and in the central nervous system (7) and that it was an intermediate in the formation of nitrite and nitrate by activated macrophages (8). The fact that these three cellular sources of NO (i.e., endothelial cells, neurons, and inflammatory cells) had been identified was to influence the progress and direction of future biochemical studies of the enzymes that produce it. The

Figure 1 The nitric oxide–cGMP signaling pathway. Activation of endothelial nitric oxide synthase (eNOS) achieved via an increase in intracellular calcium stimulates NO production. Diffusion of NO from the endothelium to the underlying smooth muscle results in activation of soluble guanylyl cyclase (sGC) and the formation of cGMP from GTP. This activates protein kinase G (PKG), which results in a decrease in cytosolic calcium concentration and thus promotes smooth muscle relaxation.

Nitric Oxide as a Biological Mediator Table 1

5

NOS Isoforms

Specificity Molecular mass Cofactor requirement

eNOS 135 kDa Calmodulin-dependent NADPH, FAD, FMN tetrahydrobiopterin

nNOS 155 kDa Calmodulin-dependent NADPH, FAD, FMN tetrahydrobiopterin

Regulation of gene expression

↑ Shear stress, proliferation

↑ Estrogens

↓ TNF-α

Mechanisms involved in regulation of gene expression

Transcription, mRNA stability

Transcription

iNOS 130 kDa Calmodulinindependent NADPH, FAD, FMN tetrahydrobiopterin ↑ TNF-α, IL-1β, cAMP, cGMP, NF-κB, IFN-γ ↓ NO, glucocorticoids, TGF-β Transcription, mRNA stability

characteristics of the three isofoms of NOS that are responsible for NO production are illustrated in Table 1. III. Release of NO by Nerves: Neuronal NOS Despite endothelial cells being the first location identified for NO production, a neuronal source was initially used for characterization and purification of NOS. In 1990, just one year after NO had been identified as a mediator released by rat cerebral tissue, Bredt and Snyder had purified NOS from this tissue (9). This first NOS isoform was called neuronal NOS (nNOS) because of its cellular origin. nNOS is a homodimer with subunits of approximately 150 kDa. It is a soluble protein that requires NADPH, calcium, and calmodulin (9,10) as well as tetrahydrobiopterin (BH 4) (11) for full activity. These characteristics were utilized in a number of variations on the original purification scheme, which include columns packed with 2′5′-ADP sepherose (which binds NADPH-requiring proteins) and affinity columns for calmodulin. For nNOS, NADPH serves as an electron donor, whereas calcium-activated calmodulin binds to the relevant site on the enzyme, producing a conformational change consistent with activation. The nature of the requirement of nNOS for BH 4 is less clear, although it is thought that it may act as a redox reagent, like NADPH (11), and/or to stabilize the NOS protein (12). Antibodies raised by Bredt and Snyder to purified nNOS showed immunohistochemical localization in rat brain in discrete neuronal populations, mainly in the cerebellum and the olfactory bulb, areas associated with roles in hormone

6

Belvisi et al.

release and visualization, respectively. In these neuronal areas, a co-localization with NADPH-diaphorase staining was observed (13). Although the functional relevance of diaphorase is unclear, all the NOS isoforms purified to date posses NADPH-dependent diaphorase activity (13–15). Immunocytochemical and histochemical studies have also shown that nNOS is present in the epithelium of both human bronchi and rat trachea (16), as well as in human skeletal muscle (17). Neuronal cDNA for nNOS was cloned and expressed in human kidney 293 cells (18). The cDNA coded a protein that had structural homology with cytochrome P450 reductase, a hemo-protein with recognition sites for L-arginine, NADPH, FAD, flavin nucleotides, calmodulin, and phosphorylation. In most cases FAD and FMN are so tightly bound to NOS that they are purified along with the protein and so are not required as additional factors. nNOS activity has also been shown to be present in peripheral nonadrenergic, noncholinergic (NANC) inhibitory neurons purified from the rat anococcygeus (19) and the bovine retractor penis muscle (20). NO release by inhibitory NANC nerves is particularly important in human airways, where it serves as a bronchodilator. A. Regulation of nNOS Expression

Although nNOS is a constitutive form of the enzyme, its activity can by modulated by a number of different stimuli (21). nNOS is upregulated at the mRNA or protein level by stimuli including heat, electrical activation, and light (22– 24). A reduction in the expression of nNOS is associated with mediators of sepsis including endotoxin and cytokines (21). nNOS may also be increased as a response to injury after ischemia (25). Indeed, several in vivo studies illustrate a time-dependent increase in nNOS mRNA after hypoxia (26–28). Increased levels of enzyme in these models may be a result in specific hypoxia-induced factors acting on designated response elements in the nNOS gene, as occurs for other similarly regulated response proteins (29). In support of this, sequence consensus for the binding of hypoxia inducible factor-1 has been described on the nNOS gene. In addition to stress, nNOS can be modulated by a number of different chemical agents. Inhibition of glutamatergic transmission increases nNOS expression in cerebral nerves (30). By contrast, increasing endogenous levels of acetylcholine (using a cholinesterase inhibitor) increases nNOS levels in the hippocampus (31). Moreover, nNOS expression is increased by some sex hormones including estradiol and testosterone (32,33) and reduced by corticosterone (34). Furthermore, recent evidence suggests that nNOS expression may be regulated by alternative splicing with the production of numerous mRNA transcripts. This is thought to allow the creation of NOS proteins with differing characteristics (35), but the significance of this observation remains to be determined.

Nitric Oxide as a Biological Mediator

7

IV. Release of NO by Endothelial Cells: Endothelial NOS Endothelial cells from all locations of the circulation express a distinct isoform of NOS named eNOS. However, although eNOS was originally purified and cloned from vascular endothelium, its expression has been reported in cardiomyocytes, blood platelets, and hippocampus neurons. Shear stress and cell proliferation appear to be, quantitatively, the two major regulatory factors for eNOS expression. However, eNOS seems to be mainly regulated by its activity. Stimulation of specific receptors by various agonists (e.g., bradykinin, serotonin, histamine, and thrombin) increases eNOS enzymatic activity at least in part through an increase in intracellular free calcium. eNOS was initially thought to be, like nNOS, a soluble protein (36,37). However, subsequent studies clearly showed that the majority of eNOS resides in the particulate fractions of cells (38,39). The purified particulate eNOS was, however, found to have a number of similarities to nNOS. For instance, eNOS requires calcium, calmodulin, NADPH (40), and BH 4 (41) for full activity. It is also similar in size to nNOS, with a denatured molecular mass of approximately 135 kDa (40). Nevertheless, eNOS and nNOS are the products of separate genes (42). Bovine endothelial cDNA (42) coded a 4.8-kb transcript which gives rise to a protein with an approximate M r of 135 kDa. The amino acid sequence predicted the same regulatory sites and NADPH-dependent diaphorase activity as previously published for the nNOS. Similar results have been published using human umbilical vein endothelial cell cDNA (43), with a predicted M r of 144 kDa. eNOS cDNA, unlike nNOS cDNA, encodes for a N-myristylation site (44), which does not influence catalytic activity but results in the tethering of this isoform to the membrane fraction (44). Phosphorylation represents an important mechanism for posttranslational regulation of various cellular proteins. Phosphorylation of eNOS on serine residues has been reported in endothelial cells in response to bradykinin (45) and to hemodynamic shear stress (46). Phosphorylation of eNOS on tyrosine residues is more controversial. Nonetheless, the biological consequences of eNOS phosphorylation are unclear; however, it has been postulated to play a role in the intracellular translocation of eNOS following agonist-induced activation (47). A. Regulation of eNOS Expression

The mechanisms involved in the regulation of eNOS are still being investigated. However, physical forces of shear and strain increase its expression in endothelial cells in vitro and in vivo (48–50). In addition, a putative shear stress response element has been described in the promoter region of both human and bovine eNOS genes (51,52). Hypoxia downregulates eNOS expression in pulmonary

8

Belvisi et al.

endothelial cells (53) and some reports, but not others, have shown a similar phenomenon in endothelium from systemic vessels (21). Some growth factors increase eNOS expression in endothelial cells. For example, transforming growth factor β increases eNOS mRNA and protein as a result of enhanced promoter activity (54). There is some controversy surrounding the changes in eNOS expression in proliferating cells. For instance, one study has shown that eNOS mRNA and protein are increased in growing versus resting cells. This increased expression of enzyme is thought to be a result of increased mRNA stability (55). By contrast, another group found that eNOS mRNA was actually less stable, resulting in lower levels of enzyme in proliferating cells compared to resting cells (56). These conflicting observations may reflect the complexity of responses produced by NO in different cells and also the variability in responses of cultures at different passages in different laboratories. There are now a number of studies reporting clear effects of different cytokines on the expression of eNOS (21). For example, tumor necrosis factor-α (TNF-α) can downregulate eNOS (21) by destabilizing mRNA, whereas a combination of interferon and endotoxin can upregulate eNOS expression in bovine aortic endothelial cells (57). This is not, however, a consistent observation. In a number of studies, endotoxin administration in vivo resulted in the downregulation of eNOS (58), an effect that may be attributed to increases in endogenous levels of TNF. As is the case for nNOS, sex hormones have been shown to increase levels of eNOS. Indeed, pregnancy and estradiol, but not progesterone or testosterone, increase eNOS mRNA, protein, and activity (59,60). Similar observations have been made in vitro using cultured immortalized endothelial cells. Here estrogen increased eNOS mRNA and activity by increasing the promoter activity via an estrogen-responsive element (61).

V.

Release of NO by Cells Induced to Express NOS: Inducible NOS

During the 1980s, a number of experiments involving the measurement of nitrite/ nitrate excretion by humans and laboratory animals in vivo and by macrophage cell lines in vitro provided a clear link between infection and NO formation (62,63). For instance, lipopolysaccharide (LPS) induces the synthesis of nitrates/ nitrites by macrophages, which was found to be dependent on the presence of L-arginine, and L-citrulline was formed as a by-product (64). Similarly, the cytotoxic ability of LPS-activated macrophages to inhibit mitochondrial respiration, metabolism, and DNA synthesis in tumor cells was found to be L-arginine depen-


9

dent, and associated with the formation of citrulline and nitrite (65). Moreover, analogs of L-arginine in which guanidino nitrogen groups had been substituted were found to inhibit both nitrite formation and the cytotoxic activities of macrophages (65). It is now clear that inflammatory and infective agents “induce” cells to express a distinct form of NOS, inducible (iNOS), and that NO is the active intermediate in nitrite and nitrate production by macrophages. The induction of iNOS has now been demonstrated in most cell types in vitro (66–68) and in all organs of the rat in vivo (69). However, there has been considerable controversy surrounding the relative ease of induction of iNOS in rat and murine tissues compared to human tissues. Nevertheless, there are a number of studies using different cell types which clearly demonstrate that active iNOS is expressed in human tissues (66–68,70). iNOS, unlike its constitutive counterparts, can be regulated by anti-inflammatory steroids such as dexamethasone (71) and is not dependent on free calcium or calmodulin (72). The production of NO therefore occurs only after a lag phase, due to the necessary induction of iNOS protein, and results in the release of relatively large amounts of NO. iNOS was purified first from the cytosol of the mouse macrophage cell line RAW 264.7, activated with LPS and interferon-γ (IFN-γ) (73), and rat peritoneal macrophages activated with LPS. The protein found had an apparent Mr of approximately 130 kDa. The active iNOS appeared as a dimer (approximate M r 250 kDa), requiring NADPH, BH 4, FAD, and FMN, but not exogenous calcium or calmodulin for full activity (73). Macrophage cDNA was cloned and expressed from LPS and IFN-γ-treated RAW 264.7 macrophages (74). The sequenced cDNA codes a protein similar to cNOS isoforms, with a predicted M r of 130 kDa, and binding sites for FAD, FMN, NADPH, and, interestingly, calmodulin (74). Similar results were obtained with cDNA from IFN-γ-stimulated smooth muscle cells (75). Further studies demonstrated that the iNOS contains activated calmodulin which is extremely tightly bound (76), thereby explaining the lack of requirement for exogenous calcium for this isoform. A. Regulation of iNOS Expression

Unlike studies on nNOS and eNOS expression, which display some level of controversy, there is a strong consensus of opinion that iNOS is induced by proinflammatory cytokines and/or endotoxin. Specifically, interleukin-1β, tumor necrosis factor-α, and interferon-γ, alone or in combination, induce iNOS in a wide range of cell types (66–68). Moreover, growth factors such as platelet-derived growth factor inhibit the induction of iNOS (67). The large and increasing number of pro-inflammatory agents demonstrated to induce iNOS and the pathways involved in its induction are beyond the scope of this chapter and are fully discussed in detail elsewhere (66–68).

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Interestingly, the 5′-flanking region of human iNOS possesses around 66% homology to its murine counterpart, although both contain conserved consensus sequences for Nuclear factor-κβ (NF-κβ), IFN-γ-responsive elements, and a TNF-α-responsive element (77). However, the transcriptional control of murine and human iNOS expression is very different. In the murine system, it appears that a proximal 1.6-kb 5′-flanking region contains the necessary promoter sequences to induce full gene expression. However, the corresponding sequence in humans produces little or no gene expression, despite possessing transcription factor consensus sequences. Furthermore, if a 16-kb fragment upstream from the coding sequence is cloned, linked to a luciferase reporter gene, and transfected into human cell lines, it is still insufficient to promote full gene expression (78). Therefore, there is a distinct difference in the requirements for iNOS induction in human compared to mouse, which may explain the difficulty in inducing iNOS gene expression in vitro in humans-compared to rodent cells (79). VI. Classification of NOS Isoforms After the different forms of NOS had been purified, antibodies were raised that recognized nNOS, eNOS, or iNOS. Studies using these antibodies revealed that NOS isoforms were expressed in other cell types. For instance, nNOS is present in epithelial as well as smooth muscle cells of the airway and gut (21). In addition to endothelial cells, eNOS is present in bone cells (21) and neuronal populations in rat brain. Moreover, iNOS is expressed constitutively in certain cells, including those of the macula densor (66). For these reasons the historical classification of eNOS, nNOS, and iNOS has been modified to represent the order of purification of the enzyme. Thus, nNOS becomes type I NOS, iNOS becomes type II NOS, and eNOS becomes type III NOS. However, for the purposes of this chapter the original classification will continue to be used. VII. Inhibition of NOS by Substrate Analog and Non-Amino Acid Inhibitors: Interactions with Different NOS Isoforms In each case the substrate for NO formation by different NOS enzymes is Larginine. The K m for L-arginine differs marginally between enzymes from 1–5 µM. The exact way in which NO and L-citrulline are formed from L-arginine is not fully understood, though a proposed mechanism has been suggested (80,81). The initial step in NO biosynthesis is the conversion of L-arginine to the intermediate N G-hydroxy-L-arginine (80) by substitution onto one of the guanidino nitrogens. In addition, endogenous N G-hydroxy-L-arginine itself is a substrate for the enzyme (82). Less is known of the conversion of N G-hydroxy-L-arginine to L-


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citrulline and NO, apart from a requirement of NADPH. Inhibition of this step by carbon monoxide (81), though, suggests a role for the iron center (Fe 3⫹) of the enzyme. The formation of NO from L-arginine requires a five-electron oxidation, and molecular oxygen is incorporated into both L-citrulline and NO, indicating NOS as a dioxygenase enzyme (83). Analogs of L-arginine in which groups are substituted onto one or more of the guanidino nitrogens have generally proved to be inhibitors of NOS. Moreover, different analogs of L-arginine have varying potencies as inhibitors of eNOS and nNOS versus iNOS. This phenomenon was first described with N G-monomethylL-arginine (L-NMMA) versus N G-nitro-L-arginine (L-NAME). Indeed, LNAME is a more potent inhibitor than L-NMMA of the constitutive forms of NOS (eNOS and nNOS). By contrast, L-NMMA is either more potent than LNAME or of similar potency to L-NAME as an inhibitor of iNOS. Recently, simple non-amino acid compounds have been found to be very potent inhibitors of NOS. Aminoguanidine is a potent NOS inhibitor purported to have some selectivity for iNOS over the constitutive NOS isoforms (84,85). Some of the most potent NOS inhibitors developed are the simple S-alkyl-isothioureas and bis-iso-thioureas, e.g., S-ethyl-iso-thiourea (SEITU), which inhibits nNOS, iNOS, and eNOS with K i values of 29, 19, and 39 nM, respectively (86). Increased selectivity towards iNOS can be achieved in this chemical series by changing the substituent. Mercaptoalkyl-guanidines and substituted N-aryl-isothioureas are also potent inhibitors of the three isoforms, some of them exhibiting high selectivity for the neuronal isoform versus the inducible and endothelial isoforms (87). However, pharmacological use of these iso-thioureas may be limited by their low tissue uptake, in-vivo instability, and toxicity. Continued investigation into this area has led to the discovery of N-(3-(aminomethyl)benzyl) acetamidine (1400W), a slow, tight-binding, and highly selective inhibitor of iNOS (88). 1400W inhibits recombinant iNOS 5000-fold more efficiently than eNOS and iNOS 200-fold more efficiently than nNOS (88). This compound is 50 times more potent against iNOS than against eNOS in rats with endotoxin-induced vascular injury, showing that selectivity can be achieved in vivo (88). Finally, simple alkyl amidines such as 2-iminopiperidines, 2-iminopyrrolidines, and pyrazole-1-carboxamidine are potent iNOS inhibitors (89). NOS isoforms contain a cytochrome P450-type heme. Destruction of the heme after binding is one approach toward NOS inhibition. The inhibitory properties of certain compounds such as the imidazole derivatives (e.g., 1- and 2-phenyl imidazoles) may rely on their ability to coordinate with heme (90). However, these imidazole derivatives inhibit NOS with diverse efficiencies and selectivities. Indazole and other indazole analogs such as 7-nitroindazole (7-NI) have been described as reasonably good NOS inhibitors, with selectivity toward nNOS (91).

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Belvisi et al. VIII. Effector Mechanisms Utilized by NO A. Activation of Guanylyl Cyclase

Organic nitrates such as amyl nitrate or glycerol trinitrate have been used clinically for the treatment of angina pectoris for over 100 years. The effects commonly seen with organic nitrate treatment are flushing, tachycardia, and a fall in blood pressure. All organic nitrates relax vascular and nonvascular smooth muscle via the release of NO and activation of soluble guanylyl cyclase (92), causing an increase in intracellular cGMP (Fig. 1). NO binds reversibly to heme in soluble guanylyl cyclase to form nitrosyl complexes, which activate the enzyme to cause cGMP production. It is now clear that NO formed endogenously by NOS produces many of its effects by activation of guanylyl cyclase. In many cases, cGMP mediates the effects of NO via activation of cytosolic G kinases (93). Much of the evidence linking cGMP-mediated events to G kinase has come from the use of kinase inhibitors, such as cGMP analogs. However, these analogs are selective only for G kinase and have generally been used alongside selective/specific inhibitors of other kinases (e.g., protein kinases A and C) to demonstrate more conclusively the involvement of G kinase in a particular response. Following G-kinase activation there is a reduction in IP 3 generation, which consequently results in inhibition of inositiol phosphate accumulation. Indeed, NO has been shown to reduce inositol phosphate generation in a number of preparations, including blood vessels and platelets (94,95). However, the intermediate steps between G-kinase activation and inositol phosphate inhibition are not clear. It has been suggested that G-kinase activation results in phosphorylation and inhibition of G proteins (96–98). Alternatively, G kinase may modulate the activity of some forms of phospholipase C (99,100). It is not clear whether the putative actions of G kinase on G proteins or phospholipase enzymes is direct or indirect via intermediate candidates, such as the actin-binding protein, VASP, whose phsophorylation correlates well with phospholipase C activity in platelets (101). NO can also exert its inhibitory effects on calcium release via a G-kinase-mediated phosphorylation of the IP 3 receptor. G-kinase-mediated phosphorylation of IP 3 receptors has been demonstrated in smooth muscle and platelets (102–104), but not in all cells. Recently, a role for NO and G kinase in modulating calcium release from ryanodine-sensitive stores has been established. Here, NO mediates the formation of cADP ribose (a metabolite of NAD ⫹), which directly affects ryanodine-sensitive calcium stores. More recently, it has been shown that NO can also directly activate ryanodine-sensitive calcium stores in skeletal (type 1) and cardiac (type 2) tissue by nitrosolating regulatory thiols (105). Release/sequestration from/to intracellular stores and entrance from the


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extracellular environment manage intracellular calcium levels. In addition to the effects of G kinase on movements from intracellular stores, there is also evidence to suggest that NO can modulate calcium exchange with the extracellular environment. For instance, NO has a dual action on store-operated calcium channels. At low levels of NO and cGMP store-operated calcium channels are activated, whereas at high concentrations these channels are inhibited (106). NO can also affect the functioning of second messenger-operated calcium channels, particularly those linked to muscarinic receptors (107–109). In addition, NO, via Gkinase activation, has been shown to activate second messenger-operated calcium channels likened to growth-factor receptors (110,111). It should be remembered that there are some cells in which calcium homeostasis is relatively unaltered by NO (93), an effect which may reflect the lack of G-kinase-mediated pathways in those cells. B. Interactions with Other Enzymes Nitric Oxide Synthase and Cyclo-oxygenase

There is now an increasing list of enzymes which are activated or inhibited by NO (Fig. 2). Indeed, NOS itself can be modulated by NO. NO can inhibit NOS activity directly or as a result of inhibition of the induction of iNOS (112). In

Figure 2 Effects of NO on cellular components. In addition to activation of soluble guanylyl cyclase, NO (either directly or as peroxynitrite, ONOO ⫺) can modulate other proteins, resulting in alterations in cellular function, some of which are shown in this figure.

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addition, NO can stimulate or inhibit cyclo-oxygenase (COX) (112). NO can activate COX by providing either hydroperoxide substrate by formation of peroxynitrite (113), or free-radical-initiator substrate support. The inhibitory effects of NO on COX may, however, be through nitrotyrosylation or interaction with the heme center (112). Alternatively, NO can inhibit the induction of cyclooxygenase protein (114), although the mechanism by which this occurs is unknown. As mentioned previously, NO activates cGMP-dependent kinase, interacts directly with nucleuotides, affects iron homeostasis, and may also, through nitrotyrosylation, inhibit the binding of NF-κβ to DNA (115). Arginases

It is now known that NOS activity can be regulated by the modulation of substrate availability in the presence of arginases. Thus the relative expression levels of these enzymes plus the enzymes involved in the synthesis and transport of arginine across cell membranes may determine local NO production. Arginase is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to urea and L-ornithine. There are two distinct genes coding for two arginase isoforms, AI and AII (116). AI is strongly expressed in the liver, where it functions as a key part of the urea cycle. AII is found in mitochondria of extrahepatic tissues and cells such as the red blood cells, the lactating mammary gland, the kidney, and macrophages. Several agents are known to influence the expression of NOS and arginases. Lipopolysaccharides appear to activate arginases and iNOS genes. As a general rule, cytokines inducing iNOS do not induce arginases and vice versa. For example, cytokines such as IFN-γ produced by the Th1 subset of CD 4⫹ T cells induce iNOS in mouse bone marrow-derived macrophages, whereas Th2-derived cytokines such as the interleukins IL-4, IL-10, and IL-13 are potent inducers of arginases (117). Glucocorticoids are known to inhibit iNOS induction (118). In contrast, AI is induced by glucocorticoids in a delayed manner. Arginase I, previously thought to be restricted to the liver, accounts for high arginase activity at inflammatory sites, where it may limit high-output NO production. Contrasting temporal expression of iNOS and arginase pathways has been observed in inflammation, iNOS being observed at early stages and the arginases at a later stage. A recent study demonstrates that arginase can inhibit neuronal apoptosis induced by multiple stimuli. The protective effects of arginase could not be reproduced by a selection of NOS inhibitors but rather seemed to depend on depletion of L-arginine, resulting in protein synthesis inhibition. These data suggest that an increased understanding of the mechanisms involved in amino acid depletion and therefore the regulation and localization of arginases could lead to the development of novel therapeutic approaches to the control of NO synthesis and the regulation of apoptosis and cell death.


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C. Interactions Between Superoxide Anions and NO: Formation of Peroxynitrite

It is important to recognize that a direct reaction between NO and most biological molecules does not occur, as at low concentrations NO will be removed safely (e.g., through the reaction with hemoglobin). Alternatively, when reactions of NO with other free radicals produced during oxidative stress become predominant, then NO is converted into toxic reactive nitrogen species, e.g., nitrogen dioxide, peroxynitrite, and dinitrogen trioxide. It is now thought that these species mediate the deleterious effects (e.g., DNA mutations, loss of enzyme function, alterations in membrane integrity) of elevated NO production during inflammatory episodes (see Chap. 7). The combination of NO with superoxide anions leads to the detoxification of both, but a hydroxyl radical (a potent oxidant) may be formed as a by-product of the reaction (119). Superoxide anions can also combine with NO to form peroxynitrite, a potent oxidant which can contribute to many of the damaging effects of NO, leaving nitrotyrosylated proteins as a marker (120). The relative effects of NO can therefore change depending on the availability of superoxide, which itself is removed by isoforms of superoxide dismutase (SOD) (121). Thus the level of SOD activity present in tissues is a very important component in the overall effect of NOS activation. It has recently been suggested that NOS activity alone can result in the generation of peroxynitrite. This is most likely to occur at low arginine concentrations, when NOS is capable of producing superoxide anions along with NO (122). Interestingly, the peroxynitrite anion (ONOO ⫺) is relatively stable, but its acid form (ONOOH) decays to nitrate with a half-life of around 1 sec at physiological pH and temperature. However, although peroxynitrite exhibits a half-life of around 1 sec in these conditions, the exact life span of peroxynitrite in vivo is not known. The biological effects of peroxynitrite are due to its reactivity toward a large range of molecules including amino acids such as cysteine, methionine, tyrosine, and tryptophan, nucleic bases, and antioxidants (e.g., phenolics, selenium- and metal-containing compounds, ascorbate, and urate). There is now a body of data suggesting that peroxynitrite is formed in vivo and plays an important role in many different pathophysiological conditions such as inflammatory disorders, cardiovascular diseases, and neurodegenerative diseases. D. Interactions of NO with Thiols

NO signaling is achieved through both cGMP-dependent (as discussed above) and cGMP-independent mechanisms (Fig. 2). An important example of cGMPindependent actions of NO are those achieved by nitrosolation of thiol groups leading to modification of protein function (123). When NO combines with thiol groups, a stable bioactive NO-like moiety can be formed. Such molecules include

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S-nitroso-N-acetylpenicillamine, S-nitrosoglutathione, and S-nitrosocysteine. These modified molecules have been suggested to have similar biological actions as EDRF and NO on smooth muscle preparations (124). However, further studies using traditional bioassay techniques have concluded that this is not the case. A number of other molecules can be polynitrosylated by NO from iNOS induced in murine macrophages in vitro, or in the tracheal secretions of humans being treated with inhaled NO therapy (115,123,125). The various ways in which nitrosylation and polynitrosylation can modify protein structure and function are discussed in detail elsewhere (115,123,125). E.

Mutagenesis of DNA

NO can cause profound effects on living cells by modifying nuclear components directly. Noninherited genetic diseases and cancers involve the spontaneous mutation of DNA. Interactions of NO with isolated DNA, RNA, and nucleotides or nuclear components in intact human cells cause deamination leading to an increased number of mutations (125). The mechanism by this occurs is not completely understood but is thought to involve nitrosylation of nucleotide residues (115,123). F. Direct Toxicity

Large amounts of NO from iNOS have antibacterial, antifungal, and antiviral properties. It is now thought that peroxynitrite, rather than NO itself, is responsible for some of the cytotoxic effects associated with immune cells expressing iNOS. Although the mechanisms involved in NO-mediated cell/pathogen killing are not completely understood, NO has a number of actions that contribute to this property. Binding of NO [or peroxynitrite (126)] to the Fe-S group of aconitase, an important enzyme in the tricarboxylic acid respiration cycle, inactivates this enzyme (127). Aconitase is also an important iron-regulatory protein. These proteins bind to the iron-response elements of RNA, encoding a number of proteins involved in iron homeostasis. Indeed, NO inhibition of aconitase in hepatoma cells increased its binding to the iron-response element and subsequent supression of ferratin synthesis (127). In addition to effects on aconitase activity, NO, or peroxynitrite, can mediate cellular toxicity by (a) inhibition of ribonucleotide reductase, an important rate-limiting enzyme in DNA synthesis, (b) inhibition of mitochondrial electron transport, or (c) damage to DNA. The later mechanism is thought to involve the activation of poly ADP ribose synthase (PARS) (126). Once activated, PARS initiates continual cyclical DNA damage resulting in cellular depletion of ATP and NAD ⫹ and ultimately death (126).


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IX. Nitric Oxide, Pathophysiological States, and Therapeutic Possibilities NO appears to serve a beneficial role in the lung by controlling pulmonary blood flow and ventilation-perfusion matching within the lung and inhibiting platelet adhesion, activation, and aggregation. In addition, NO may have a role in protecting the mucosa and reducing susceptibility to lower respiratory tract infections. Furthermore, NO is the neurotransmitter of inhibitory nonadrenergic, noncholinergic nerves and is responsible for neural bronchodilation of human airway smooth muscle (128). In fact, NO has been demonstrated to possess bronchodilator properties both in asthmatics and in healthy volunteers (129,130). For these reasons it was thought that NO may be of use as a bronchodilator therapy for the treatment of asthma. However, this hope was not supported by experiments demonstrating that less than 50% of patients with asthma bronchodilate in response to NO, and even in the responders the bronchodilation produced was not clinically significant (131). However, inhaled NO is used to treat various cardiopulmonary disorders associated with pulmonary hypertension. In contrast, NO has also been demonstrated to have pro-inflammatory actions in the lung. Interestingly, transbronchial biopsies from asthmatic patients have shown elevated iNOS expression compared to nonasthmatic controls (132). Furthermore, NO has been reported to alter the balance between Th1 and Th2 cell types, leading to the proliferation of Th2 lymphocytes, which produce a cytokine profile that has been associated with the asthma phenotype (133,134). Therefore, it appears that NO has a dual role in the lung, mediating both host protection and injury in inflammatory diseases. This is illustrated through studies using iNOS knockout mice. Consistent with a defensive role for NO mice lacking the iNOS gene have altered immune responses and demonstrate a reduced survival to bacterial and viral infections (135). In contrast, several studies have demonstrated a role for NO in injury. For example, iNOS knockout mice are resistant to endotoxin-induced mortality and end-organ damage after hemorrhagic shock (135). Furthermore, mice lacking iNOS have significantly reduced inflammatory responses to allergen challenge in the lung (136). However, by contrast, other studies have failed to confirm these findings in iNOS knockout mice and suggest that it is nNOS that is the predominant isoform involved in modulating the severity of airway hyperreactivity (137). Asthma is now recognized to be an inflammatory disease of the airways, and the major goal for any potential therapy is to control this inflammatory response. Monitoring this inflammatory response is important so that the disease can be treated optimally. Currently, persistent inflammation is evaluated by bronchial provocation tests in order to assess airway hyperreactivity, biopsies, lavage, and analysis of sputum eosinophilia. Exhaled NO is significantly increased in

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asthmatic airways and is rapidly decreased in asthmatics following steroid treatment (138). The measurement of exhaled NO in exhaled air is noninvasive and simple to perform and as such has potential utility as a test to assess asthmatic inflammation. In summary, there does not appear to be a major role for NO as a therapeutic agent for the treatment of inflammatory diseases of the airways such as asthma. However, the measurement of exhaled NO is a promising way in which to monitor the inflammatory response in asthmatic airways and is a good indicator of steroid responsiveness in a patient. X.

Summary

The synthesis of NO by mammalian cells was once thought to be impossible. However, it is now clear that this simple gas can regulate processes in all bodily organs. Its primary targets seem to be vascular smooth muscle and circulating blood elements in the cardiovascular system, smooth muscle in the airways and the gastrointestinal tract, the central nervous system, and invading pathogens or cancer cells. The functions of NO are partially achieved by a highly developed mechanism for the regulation of its release. Thus, small quanta of NO are formed by calcium activation of the constitutive forms eNOS and nNOS, whereas large cytotoxic amounts of NO are formed by the calcium-independent iNOS. A further layer of regulation is provided for by the different trandsuction mechanisms utilized by NO in different cells. The most important effector pathway for NO is activation of the soluble form of guanylyl cyclase. We now seem to have a wealth of information relating to NO biology in health. However, we are only just beginning to understand how dysfunctions in the L-arginine–NO–cGMP pathway contribute to diseases in humans. It is important to recognize that the physiological consequences of NO produced in the airways are critically dependent on the timing, amount, and sites of synthesis of this reactive molecule, as well as on the chemical milieu of the local environment. A better understanding of the physiological and pathophysiological functions of NO through the use of isoform-selective NOS inhibitors in such diseases will undoubtedly lead to new therapies. References 1. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J 1992; 6:3051–3064. 2. Knowles RG, Moncada S. Nitric oxide synthase in mammals. Biochem J 1994; 298:249–258. 3. Furchgott R, Zawadzki JV. The obligatory role of endothelial cells in relaxation of arterial smooth muscle by acetylcholine. Nature 1980; 288:373–376.


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4. Palmer RJM, Ferrigo AG, Moncada S. Vascular endothelial cells synthesise nitric oxide from L-arginine. Nature 1987; 325:664–666. 5. Garthwaite J. Glutamate, nitric oxide and cell-cell signalling in the nervous system. TINS 1991; 14:61–67. 6. Gillespie, JS. The rat anococcygeus muscle and its response to nerve stimulation and to some drugs. Br J Pharmacol 1972; 45:404–416. 7. Garthwaite J, Charles SL, Chess-Williams R. Endothelium-derived relaxing factor release on activation of NMDA receptors suggest a role as intercellular messenger in the brain. Nature 1988; 336:385–388. 8. Hibbs JB, Taintor RR, Vavrin Z, Rachlin EM. Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem Biophys Res Commun 1988; 157:87– 94. 9. Bredt D, Snyder S. Isolation of nitric oxide synthase, a calmodulin-requiring enzyme. Proc Natl Acad Sci USA 1990; 86:682–685. 10. Schmidt HHHW, Pollock JS, Nakane M, Gorsky LD, Forstermann U, Murad F. Purification of a soluble isoform of guanylyl cyclase-activating factor synthase. Proc Natl Acad Sci USA 1991; 88:865–869. 11. Mayer, B, John M, Bohme E. Purification of a calium/calmodulin-dependent nitric oxide synthase from procine cerebellum. Co-factor role of tetrahydrobiopterin. FEBS Lett 1990; 277:215–219. 12. Giovanelli J, Campos KL, Kaufman S. Tetrahydrobiopterin, a cofactor for rat cerebella nitric oxide synthase, does not function as a reactant in the oxygenation of arginine. Proc Natl Acad Sci USA 1991; 88:7091–7095. 13. Hope BT, Michael GJ, Knigge KM, Vincent SR. Neuronal NADPH diaphorase is a nitric oxide synthase. Proc Natl Acad Sci USA 1991; 88:2811–2814. 14. Mitchell JA, Kohlhass KL, Matsumoto T, Fo¨rstermann U, Warner TD, Murad F. Induction of NADPH dependent diaphorase and NO synthase activity occurs simultaneously in aortic smooth muscle and cultured macrophages. Mol Pharmacol 1992; 41:1163–1168. 15. Lamas S, Marsden PA, Li GK, Tempst P, Michel T. Endothelial nitric oxide synthase: molecular cloning and characterisation of a distinct constitutive enzyme. Proc Natl Acad Sci USA 1992; 89:6348–6352. 16. Kobzik L, BredtDS, Lowenstein CJ, Drazen J, Gaston B, Sugarbaker D, Stamler JS. Nitric oxide synthase in human and rat lung: immunocytochemical and histochemical localisation. Am J Respir Cell Molec Biol 1993; 9:371–377. 17. Nakane M, Schmidt HH, Pollock JS, Forstermann U, Murad F. Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle. FEBS Lett 1993; 316: 175–180. 18. Bredt DS, Hwang PM, Glatt CE, Lowenstein C, Reed RR, Synder SH. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature 1991; 351:714–718. 19. Mitchell JA, Sheng H, Fo¨rstermann U, Murad F. Characterisation of nitric oxide synthase in non-adrenergic-non-cholinergic nerve containing rat anococcygeus. Br J Pharmacol 1991; 104:289–291. 20. Sheng H, Schmidt H, Nakane M, Mitchell JA, Pollock JS, Fo¨rstermann U, Murad F. Characterisation and localisation of nitric oxide synthase in non-adrenergic non-

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2 Physiology of Exhaled Nitric Oxide

L. CHRISTOFER ADDING and LARS E. GUSTAFSSON Karolinska Institute Stockholm, Sweden

I.

Introduction

In the 1970s Ferid Murad and colleagues demonstrated that application of nitric oxide (NO) to bovine airway smooth muscle preparations induced relaxation preceded by increased cGMP levels (1,2). Despite an exogenous origin of NO in these studies, they implied a functional significance of NO in the lung. Not until Furchgott’s discovery of endothelium-derived relaxing factor (3) and its identification as NO (4–6) did the ubiquitous physiological role of endogenous NO emerge (for reviews on NO, see Refs. 7–10). Direct measurements of NO have been one major problem in NO research, due to NO’s rapid oxidation in biological tissues, with a half-time of only seconds, and this explains why it took seven years to identify NO as an endogenous compound. However, several different assays have been used that indirectly reflect NO production—e.g., cGMP, nitrite (NO 2⫺), or citrulline measurements— reflecting the effect of NO, NO metabolism, or NO synthesis, respectively; see Ref. 11. It was not until 1987, when Palmer et al. (6) introduced a chemiluminescence method, originally developed by environmental scientists to detect nitrogen oxides in the atmosphere (12,13), that the first direct measurements of NO re29

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leased from cells could be made. However, liquid specimens from cells or tissues were analyzed, and recovery of NO was dependent on rapid sample acquisition to avoid any oxidation of NO or NO 2⫺, and the method determined the sum of NO and NO 2⫺. In 1991 Gustafsson and colleagues used this method in an attempt to analyze lung perfusate during hypoxic conditions to elucidate the role of NO during hypoxic pulmonary vasoconstriction (HPV). They then considered the possibility that NO, being a gas with a low solubility in aqueous solution, would probably escape into the airspace of the lungs and be detectable in exhaled gas. This hypothesis proved to be right, and direct evidence for the production of NO in humans was thus presented (14–16). The discovery of NO in exhaled gas has not only offered a new approach to study features of NO metabolism directly in vivo, but also exhaled NO measurements seem to be a valuable noninvasive diagnostic tool of airway inflammation (17,18). Over the last decade, over 500 studies in the field of exhaled NO research have been published, with sometimes opposing results leading to conflicting interpretation. This chapter attempts to give the reader an up-to-date review of the fundamentals of the physiology of exhaled NO, with emphasis on regulatory factors such as atmospheric oxygen and endogenously formed carbon dioxide.

II. Measurement Techniques of Exhaled NO A. Identity of Exhaled NO

Measurements of NO in respiratory gas by the chemiluminescence reaction are based on the finding that ozone reacting with NO yields excited NO 2 which emits infrared light, which is directly proportional to the original NO levels, and the light (photons) can be counted by a photomultiplier tube. Many compounds may react with ozone under the emission of light, but when red filters with a cutoff such that only light with wavelength above 600 nm is measured, the method becomes quite specific although not unique to NO. To elicit chemiluminescence, another substance must be volatile and have chemical properties, not likely to occur in biological systems, that will make it react with ozone (11). In the initial study (14), the presence of NO in exhalates of humans and guinea pigs was confirmed by reacting exhaled gas with thioproline and demonstrating the presence of nitrosothioproline by gas chromatography/mass spectrometry (GC-MS). In a later study (16), direct GC-MS on exhaled gas was performed using a cooled gas chromatography column to separate NO from other molecules having a mass of 30 and present in normal atmosphere in significant amounts, e.g., 15,15 N 2 , and the presence of NO in exhalates from humans was confirmed directly. A limitation of this method was that it was not linearly quanti-

Physiology of Exhaled Nitric Oxide

31

tative. Thus, there was a need to evaluate exhaled NO during quantitative chemiluminescence measurements and using physicochemical means to test for its presence. This was initially done in humans and rabbits using a cold trap at ⫺80°C (14). A later study was done in humans, rats, guinea pigs, and rabbits (19). Here the methodology employed was to carry exhaled gas through a cold trap cooled to temperatures where other gases than NO would be expected to be frozen out of exhaled gas, finally taking the temperature down where the NO signal also disappeared. The cooling trap consisted of a copper or stainless steel loop having a configuration allowing countercurrent exchange of heat from afferent to efferent limb to ascertain attainment of desired temperatures. Mixtures of dry ice, acetone, and liquid nitrogen allowed temperatures of the coil down to ⫺196°C. During these experiments it was verified that the chemiluminescence signal disappeared only when coil temperature was brought below the freezing point for NO, giving verification that the chemiluminescence signal derives from exhaled NO. Another support for exhaled NO as the cause for chemiluminescence is testing for the possibility of making it disappear by pharmacological inhibition of its biochemical formation. The chemiluminescence signal of exhalate is inhibited or disappears during administration of NO synthase (NOS) inhibitors of Larginine-derivatives type, and the signal can be partially or fully restored by a surplus of the natural substrate for NO formation, L-arginine (see Tables 1–3 for references). B. Interfering Compounds and Standardization of Techniques

Water vapor, carbon dioxide gas, and nitrous oxide might quench the chemiluminescence signal of NO (20–22). It is therefore advantageous that sampled exhaled gas is led into a dehumidifying tube before entering the NO analyzer, and that in experiments where CO 2 is present in variable amounts a correction of the NO signal for the quenching effect by CO 2 is made. Some studies show that atmospheric NO levels may bias exhaled NO measurements so that exhaled NO increases during days with high NO levels in the ambient air (23,24) whereas others have found no such influence of ambient NO (25). Recent studies suggest that outdoor air pollution per se may increase endogenous exhaled NO in healthy subjects (26,27). To exclude these uncertainties in exhaled NO measurements NO-free inspired air should be used when exhaled NO measurements are performed. A proper charcoal filter, using a sufficiently large filter with the right proportions of length to diameter, can be used to make NO-free air out of ambient air. Although recent data suggest that a charcoal filter does not remove NO from hospital compressed air (28), our own experience shows that this can be achieved (29). With these considerations, pollutant-

Gas emboli i.v. pH (acidosis)

Selective gas-phase vs. vascular hypoxia Alveolar/airway hyperoxia Carbon dioxide inhalation

Chronic airway/alveolar hypoxia

Acute airway/ Alveolar hypoxia

Provocation

iNOS induction

↓ ↓ ↑ ↑ ↑ ↑

Guinea pig Dog Rabbit/dog Rabbit Dog Rat

(155) (111) (14,232) (59) (233) (234)

(106,186) (21,59,125) NO synthase activity is reduced by airway CO 2 /change in pH

↔ ↓

Rat Rabbit

(14,56) (99,125) (57,100,230) (231) (103)

References

(99)

Reduced lung eNOS protein amounts

NO synthase is sensitive to airway/ alveolar PO 2

Proposed mechanism

↓

↓ ↑ ↓

Pig Cat Pig (newborn)

Exh. NO

Rabbit

↓

Rabbit

Species

Table 1 Physiological Factors Regulating Exhaled Endogenous Nitric Oxide in Experimental Animals (↑, increase; ↓, decrease; ↔, unchanged)

32 Adding and Gustafsson

Ethanol (i.v.) Adenosine (i.v.) Angiotensin II i.v. Allergen, Histamine, LTC 4 , inhalation

Endotoxin

Endothelin A-receptor Germ-free

Stretch inhibition (CPETP, Gd i.v.) α 1-Adrenoceptors β-Adrenoceptors (i.v.)

Stretch activation (PEEP, NEEP, CNETP, HFOV)

↑ ↔ ↔ ↑ ↑ ↑ ↓ ↑ ↑ ↑ ⱕ 2 min ↓ ⬎ 10 min

↑ ↓ ↓ ↑

Guinea pig Rabbit/guinea pig Horse Rabbit Rat Rat Pig Pig Rat Dog Rabbit Rabbit Guinea pig Guinea pig

↑

Rabbit

Stimulation of NO synthase via Ca 2⫹-channel activation

1. Stim. of NO synthase via SACa 2⫹-channel activation 2. Reduced pulmonary capillary blood volume

(235) (19) (236) (236,237) (235,238–243) (244) (245) (246) (247) (190,199,248,249)

(126,155) (21,109,126) (161) (90)

(21,52,125)

Physiology of Exhaled Nitric Oxide 33

34


Table 2 Factors Affecting Exhaled Endogenous Nitric Oxide in Healthy Humans (↑, increase; ↓, decrease; ↔, unchanged) Provocation

Oral NO

Acute alveolar hypoxia

↓

Post-moderate altitude stay Circadian rythm Menstrual midcycle Pregnancy Physical exercise

↓ ↔↑ ↑↔ ↔ ↑excretion ↓concentr. ↑excretion ↓concentr. ↑ ↓ ↓ ↓

Hyperventilation Breathhold Cold air inhal./hypothermia Spirometry Cigarette smokers vs. nonsmokers Cessation of smoking Cigarette smoke inhal. Outdoor air pollution Indoor formaldehyde levels Ozone inhal. (acute) Ozone inhal. (chronic) Influenza virus (nasal/vaccination) Asthma; allergen challenge

↑ ↑ ↑ ↑ ↔ ↑ ↑ ↑

Nasal NO ↔ ↔ ↓

References (101,250) (85) (251) (252,253) (254–257) (258) (65,83–87,259–263) (65,83,84,86)

↑ ↓

↔↓

(65,69,71,73,264–271) (272,273) (274) (218,275–280) (281,282) (283,284) (26,27) (285) (286) (287) (288,289) (60,217,218,290)

induced changes in exhaled NO should, in our view, be taken as biological effect measures and not as interference with the determination method. Measurements of exhaled NO in small laboratory animals have usually been performed in mixed expired air. The development of fast NO analyzing methods (response times 100–300 msec) makes it possible to quantify airway peak concentrations of NO, breath by breath, in laboratory animals requiring higher ventilatory frequencies. Such a technique is also preferred in humans, where standardized measurements of exhaled NO are needed (18,30,31). Recently, a new method was described for measurements of exhaled nitric oxide based on conversion of NO to NO 2 , by use of the oxidant chromium trioxide, followed by detection of chemiluminescence in the reaction of NO 2 with an alkaline luminol/H 2 O 2 solution (32). Whether this technique will be applicable to clinical NO measurements is still uncertain.


35

Table 3 Effect of Drugs on Exhaled Endogenous Nitric Oxide in Healthy Humans (↑, increase; ↓, decrease; ↔, unchanged) Provocation L-Arg p.o./i.v./inhal. Nitrovasodilators (i.v.) Dobutamine (i.v.) Corticosteroids (p.o./nasal) Ethanol (p.o.) Ibuprofen (p.o.) Enalapril (ACE inhibitor p.o.) β 2-agonist (inhal.) Prostacyclin (inhal.) Prostaglandin E 2 & F 2α (inhal.) L-NMMA (inhal./i.v.) L-NAME (inhal./nasal) Aminoguanidine (inhal.) α 2-Agonists (nasal) Capsaicin (nasal) Histamine (nasal) Papaverin (nasal)

Oral NO

Nasal NO

References

↑ ↑ ↔ ↔ ↓ ↓ ↑ ↔ ↔ ↓ ↓ ↓ ↔

↑ ↔

(291–295) (296,297) (86) (62,292) (286,298) (299,300) (301) (302) (165–167,303) (304) (305) (89,115,202,298,306–308) (35,162–164,309–312) (309) (162–164) (162) (162) (163,164)

↔

↓

↓↔ ↓ ↔ ↔ ↑

III. Metabolism of Exhaled NO A. Production of NO Enzymatic Sources

The molecular mechanisms of enzymatic production of NO, identification, classification, and regulation of the different isoforms of NO synthases, and their expression profile in the lungs, are described in detail by others in this volume. The isoenzyme responsible for the production of NO in exhaled air is under consideration. Recent evidence suggests that, at least in experimental animals, a major part is synthesized by a calcium-dependent NOS (19). On the other hand, in human airway there is evidence for the involvement of a continuously expressed calcium-independent and corticosteroid-sensitive NOS (33–35). Most likely, pulmonary exhaled NO constitutes the integral of NO that rapidly diffuses into the airway lumen from different NO-producing cell types, e.g., epithelial, endothelial, neuronal, or inflammatory cells, within the lung (33,34,36,37). Nonenzymatic NO Production

Facultative anaerobic bacteria, capable of reducing nitrate to NO 2⫺, are present in the mouth, skin, and gastrointestinal mucosa, where the conditions are acidic.

36


A nonenzymatic reduction of NO 2⫺ to NO has been demonstrated in these organs (38–43). Furthermore, it has been shown that the acidic and highly reduced conditions that occur during myocardial ischemia are sufficient to generate NO from NO 2⫺ (44). In this context it must be mentioned that recent data from our laboratory show that intravenous infusion of sodium nitrite (NO 2⫺) can generate NO in exhaled air with no change in pH (44a). It has also been shown that NO can be generated, by a nonenzymatic reaction, with a mixture of hydrogen peroxide and L- or D-arginine, though the mechanism and the biological significance of this reaction was not elucidated (45). Since exhaled NO both in animals and humans rapidly decreases by administration of NO synthase inhibitors (see Table 3 for references), it is clearly of enzymatic origin, but possible interference from nonenzymatically formed NO must be considered, especially after intake of food, chewing tobacco, etc., rich in nitrite/nitrate. B. Elimination of NO

Regardless of the way of production, the formed NO is a radical molecule with an unpaired electron that reacts rapidly with molecular oxygen or other radical molecules, e.g., superoxide (O 2⫺). This provides the basis for nonenzymatic NO degradation and will, together with the rate of NO production, control the biological action of NO; see Ref. (46). In spite of its extreme reactivity, NO has a relatively long biological half-life and diffusion distance (47). NO released into the blood reacts rapidly with oxygenated hemoglobin (48) to form methemoglobin and nitrate (NO 3⫺). The latter is excreted in the urine or converted to NO 2⫺ by bacteria (see above). Recently, Stamler and colleagues showed that in the lung, where oxygen tension is high, NO can bind to the highly reactive thiol groups on the β subunit of hemoglobin, when oxygen binds to the heme moiety, and that deoxygenation of hemoglobin facilitates the release of NO in the tissues (49). Thus oxygenated hemoglobin might be a functional endogenous NO carrier. This principle could explain the peripheral beneficial effects of NO inhalation on carotid artery smooth muscle proliferation following endothelial damage (50). Since NO is rapidly inactivated by hemoglobin, it is obvious that a major determinant of NO elimination in the airways is the abundance of blood in the pulmonary capillaries. However, changes in pulmonary blood flow within physiological range does not affect exhaled NO, since neither volume loading (51) nor moderate pulmonary artery obstruction (52) had any effect on exhaled NO concentrations. Furthermore, modifying pulmonary blood flow in humans by increased gravity (2g) or head-out water immersion did not change the exhaled NO concentrations (53). However, extreme reductions in pulmonary blood flow, due to severe hypovolemic hemorrhage, increase exhaled NO concentrations (54).


37

Also, in patients with atrial septal defects, closure of the defect decreased pulmonary blood flow, which was associated with a decrease in exhaled NO (55). C. Concentrations of Exhaled NO

The first measurements of exhaled NO in laboratory animals clearly demonstrated a local enzyme-dependent NO production within the lower respiratory tract: the animals were tracheotomized and exhaled NO could still be detected after circulatory arrest but not in the presence of an inhibitor of NO synthase, the enzyme responsible for NO production (see Ref. 15). Another fundamental indication is that in salt buffer-perfused lungs, when blood is no longer present, there is an immediate and more than twofold increase in exhaled NO concentration (19, 56–59). In humans the initial oral measurements of exhaled NO (14) were later attributed to a mixture of NO emanating from the lower respiratory tract, nasally produced NO (35,60,61), and NO from salivary NO 2⫺ and gastric regurgitation (41,42). The relative contribution from the respective sources depends on the measurement technique, especially the exhaled gas sampling procedure. Continuous oral sampling of exhaled NO using, e.g., a nose clamp, may contain up to 90% nasally derived NO (62,63) due to backward diffusion from the nose (64), whereas oral single-breath exhaled NO measurements, during plateau phase, are not affected by NO derived from nasal airways (65–68). Studies of awake humans either intubated (61,63,69,70) or with bronchoscopy (71) have also confirmed the excretion of NO from the lower respiratory tract. To the lower airway NO excretion is added a variable oral, mainly nonenzymatic NO production (43). Nevertheless, the anatomic site and main cell type producing the NO molecules detected in exhaled gas from the lower airways are still a matter of debate. Different volumes of dead-space gas have been sampled, reflecting gas emanating from trachea, bronchi, bronchioles, and respiratory bronchioles, and when analyzed for exhaled NO after breath holding they show significant contribution from distal airways (72,73). However, very low NO concentrations are present in alveolar gas in vivo (65,73–75), due to the rapid scavenging of NO by erythrocytes in the pulmonary capillaries (48) (see also above). However, in salt bufferperfused lungs, end-tidal NO concentrations are very high (19,56–59), probably reflecting alveolar (endothelial or macrophage) NO production that otherwise will be scavenged rapidly by hemoglobin. We have experienced that lung-perfusion experiments using buffered salt solutions, after a “grace period,” inevitably lead to lung edema formation and that exhaled NO concentrations then decrease markedly. The fluid that accumulates in interstitial tissue and alveoli is likely to have large numbers of oxygen radicals that degrade NO. Furthermore, the fluid itself may act as a diffusion barrier. Both mechanisms would reduce the amount of

38


endothelial-derived NO in exhaled air. Along similar lines, Cremona et al. (57) demonstrated a decrease in exhaled NO with increasing weight of lungs perfused with salt solution at high outflow pressure. On the other hand, we have observed increased exhaled NO levels during lung edema formation in vivo, probably due to an increased diffusion barrier and less NO scavenging by erythrocytes (Adding et al., unpublished observations). However, in a recent study by Duplain et al. there was no increase in exhaled NO in subjects who developed high-altitude pulmonary edema (76). This might be explained by a reduced NOS activity due to the relative hypoxic conditions, as will be discussed below (see hypoxia). It is obvious that ventilatory or exhalation flow rates (66,67,77–80) and the pulmonary diffusing capacity of NO into the blood (48,81), the diffusion capacity being 4.5 times that of carbon dioxide (82), influence the concentrations of NO in exhaled gas. This might be of importance when exhaled NO concentration decreases and NO excretion increases during heavy exercise or hyperventilation (65,83–87). This phenomenon has recently been reviewed (88). It might be that this effect is due merely to the relative changes in ventilation, which decrease the NO concentration gradient between the NO producing cells and the capillary blood, thereby decreasing the net diffusion of NO to the blood (81,85). Whether these changes in exhaled NO reflect an increased NO synthesis per se, the postulated mechanisms involving stretch- and catecholamine-induced NO formation (89,90), will be discussed below. D. Endogenous Bioactive NO Concentrations

In general, it is the total dose of NO delivered to the site of action which is likely to be responsible for any biological effect (91). Murad and colleagues found that the half-maximal concentration (apparent K act) of NO required to activate purified soluble guanylate cyclase was in the 1–10-nM range (92). In rabbits, half-maximal reversal of pulmonary vasoconstriction by NO synthase inhibition was obtained by inhalation of approximately 15-nM NO gas (93). Once NO formation is completed in any tissue, regardless of the site of formation, NO diffuses in all directions until it reacts with potential target molecules and is further metabolized. Nitric oxide is lipophilic, has a modest solubility in aqueous solution (similar with oxygen), and therefore, in the respiratory system, NO molecules diffuse readily across epithelial cell membranes and escape into the air spaces of the respiratory tract. However, net diffusion of NO molecules from the site of formation will preferentially occur toward areas where NO concentrations (partial pressures) are lowest. Consequently, provided that NO production and elimination in the respiratory tissue is constant, in time an equilibrium of NO concentration with the surroundings would occur. However, this is evidently not the case in the respiratory system, where the ongoing ventilation and perfusion eliminate NO molecules before a full equilibrium occurs. Therefore,


39

measurements of NO from air sampled in any part of the respiratory tract will not give the absolute tissue concentration of NO, but will merely reflect part of it. Exhaled NO concentrations may reflect pulmonary NO production if ventilatory parameters and NO elimination are controlled. The latter prerequisite concerns the constant scavenging of NO by oxyhemoglobin in erythrocytes, which is dependent on pulmonary and tracheobronchial circulation, and by endogenous oxygen radical activity, e.g., superoxide anions in the lining fluid and tissues of the lung. It is possible to control most of these factors in volume-controlled, ventilated, constant-flow, blood-free, buffer-perfused lungs. Experiments from rabbits in our laboratory show that the mean concentrations of NO increase distally in the tracheobronchial tree, and due to these axial concentration profiles it is likely that lower in the respiratory system, e.g., at the bronchiolar level, where gas sampling is technically impossible, NO concentrations are even higher (94). Nevertheless, the most important question is, can NO concentrations in the parts-per-billion (ppb) range exert biological effects? Lower airway concentrations of NO in the range of 20–30 ppb by volume is equivalent to nanomolar concentrations for a gas at room temperature and 1 atm. However, if these concentrations were auto-inhaled, the theoretical tissue concentration acting on cellular soluble guanylate cyclase would be even lower due to the low solubility of NO. Still, it is evident from the NO inhalation studies that NO concentrations below 30 ppb can influence pulmonary circulation (93,95,96). As mentioned, NO has approximately the same solubility in water as is found for oxygen. Actions of low concentrations of NO are probably favored by its high affinity for heme (e.g., in guanylate cyclase), which is 10,000-fold that of oxygen. NO can in addition, independently of guanylate cyclase activation and cGMP formation, directly activate potassium-channels in pulmonary vascular smooth muscle (97), and also thereby exert pulmonary vasodilatory effects.

IV. Physiological Factors Regulating Exhaled NO A. Hypoxia and Exhaled NO Production

Molecular oxygen is a substrate for NOS (see above), and it has been shown that oxygen concentrations likely to occur in hypoxic tissue can fall below the Km value for oxygen of type III NOS (98). Furthermore, a similar value of Km was found when plotting a Lineweaver-Burke diagram between alveolar oxygen tension and exhaled NO in rabbits ex vivo (99). Thus oxygen availability is necessary for optimal pulmonary NO production. In accordance, acute hypoxia has been shown to decrease exhaled NO in vivo (14,85,100,101), in perfused lungs (56,57,99,102,103), and in pulmonary endothelial cells (104,105). From dose–

40


response studies of FiO 2 on exhaled NO it is obvious that NO production ceases during extremely severe airway hypoxia (FiO 2 ⬍ 3%) (56,99) (Agvald et al., 2001, submitted for publication). Along similar lines, chronic hypoxia in newborn piglets reduced exhaled NO (103). However, chronic hyperoxia has not been found to increase exhaled NO (106). Nitric oxide and oxygen are two powerful vasodilators in the pulmonary circulation. In fact, NO can reverse pulmonary vasoconstriction induced by O 2 deficiency (HPV) (107,108) and, likewise, oxygen can reverse pulmonary vasoconstriction induced by NO deficiency (93,109). Therefore, the most efficacious vasoconstriction in the lung occurs when oxygen and NO are not present, as is the case for the fetus (whole lung) and in any hypoventilated part of a lung. Thus, exhaled NO production is maintained as long as airway oxygen tensions are high enough (99) to contribute to the oxygenation of blood. In a recent study it was found that the alveolar-to-arterial difference for oxygen was reciprocally correlated with exhaled NO, thus favoring the idea of positive relation between exhaled NO and blood oxygenation efficiency (110). However, in dogs a NO synthase inhibitor given by nebulization unexpectedly improved ventilation perfusion ratio, thereby disputing the role for endogenous NO promoting ventilation perfusion ratio regulation (111). These differences could be due to species differences, since it is known that in dogs vasodilator prostaglandin seem to be more important than NO during basal conditions (112). In humans a clear-cut increase in pulmonary vascular resistance has been demonstrated upon administration of NO synthase inhibitor (113–115). B. Stretch-Related Effects and Exhaled NO

As mentioned above, it has been speculated that an increased flow rate of respiratory gas will induce NO formation in airway epithelial cells, resembling the flow (shear stress)-induced NO production in vascular endothelial cells (65). Electron microscopy studies show that type III NOS is associated with the basal microtubuli membrane of the cilia in bronchial epithelial cells (116), suggesting that epithelial cilia may in some way regulate epithelial NO production. Indeed, there is evidence for increased NO production from airway epithelial cells during increased ciliary activity in response to β-adrenoceptor stimulation (117). A recent study in rabbits showed a significant increase in exhaled NO in response to increased time-weighted tidal volume during pressure-controlled inverse ventilation (118). This study lends support to another even more speculative possibility of a stretch-dependent and breathing-cycle-differentiated formation of airway NO, with a rapid activation of NO production and an increased NO release during inspiration, locally yielding very high NO concentrations in well-ventilated (stretched) distal airways and alveoli. This would mean that we profoundly underestimate lower airway NO concentrations when we measure it in the exhaled


41

gas during an active expiration. A stretch-dependent cellular mechanism in the airways would be in accordance with the mechanically stimulated endothelial NO synthesis encountered under physiological conditions (119–122), and similar stretch-responsive mechanisms influencing NO release have been established in both striated muscle cells and osteoblastic cells of bone (123,124). Our group observed that positive end-expiratory pressure (PEEP) in guinea pigs and rabbits increased lower airway NO formation (52). The mechanism of these PEEP-induced dose-dependent increases of exhaled NO is still not known, but the observation has been confirmed in the blood-perfused rabbit lung (125). The authors of the latter study pointed to the possibility that pulmonary capillaries may become compressed by PEEP and lose their blood, resulting in less scavenging of NO by hemoglobin. The interpretation is likely, especially in situations when high PEEP pressures, which interfere with pulmonary blood flow, are applied (52,126). Nevertheless, the effect of PEEP on exhaled NO is still present in salt-buffer-perfused lungs in situ, with no hemoglobin present (Fig. 1), and therefore reduced capillary blood filling cannot fully subserve as the mechanism involved. The involvement of the vagus nerves (52) and a correlation with changes in functional residual capacity (FRC) (21) (Fig. 2) led to the suggestion that mechanical stretching of lung tissue might be one underlying mechanism modulating exhaled NO in response to PEEP; see (Ref. 15). In addition, we recently demonstrated that the process of high-frequency oscillatory ventilation (HFOV), which is mechanically characterized by small amplitudes and a very

Figure 1 Recording of the effect of PEEP (black bars) on exhaled NO production and insufflation pressure (IP) in an anesthetized and mechanically ventilated rabbit (in vivo, left) and, later in the same animal, during buffer perfusion of the lungs (buffer-perfused lungs, right). Note the higher basal levels of exhaled NO in the perfused lung and that the effect of PEEP on exhaled NO is still pronounced though the blood is no longer present.

42


Figure 2 Change in concentration of NO, measured during plateau phase of effect in trachea of pentobarbital anaesthetized rabbits, as a function of change in FRC (∆FRC, n ⫽ 8). Data derived from experiments using all four ventilatory provocations. First-order linear regression calculated using the least squares method. (From Ref. 21.)

high frequency of tidal variation, significantly increased mean intratracheobronchial NO concentrations and total excretion of NO, in both rabbits in vivo and in the buffer-perfused lung, in comparison with conventional ventilation, suggesting an increase of total pulmonary NO production rather than an increased elimination of NO from the respiratory system. The effect of HFOV on exhaled NO production significantly exceeds the PEEP-related FRC effect during conventional ventilation. Therefore the existence of preferentially high-frequency stretch-responsive mechanisms which regulate pulmonary NO production seems likely, since such a mechanism has been revealed in endothelial cells (127). Such a mechanism in the lung, the cellular basis of which remains to be shown, could explain all observations made in relation to stretch responses on NO homeostasis in the lungs, provided moderate degrees of PEEP and the like are considered. Moreover, it would be in agreement with the suggested role of NO during pulmonary vascular adaptation at birth (128) and the recent findings of pulmonary vascular smooth muscle potassium channels which are sensitive to both stretch and NO (97,129,130).


43

A number of physiological events in the lungs are stimulated by the mechanical forces exerted on the lungs by ventilation. Mechanical ventilation of fetal lambs induces an increase in pulmonary blood flow and a decrease in pulmonary vascular resistance, probably by activating hyperpolarizing potassium channels in smooth muscle (129). Furthermore, normal growth of the fetal lung depends on fetal breathing movements (131), and mechanical forces regulate compensatory lung growth (132). Mechanical strain directly activates fetal lung cell proliferation, cell division, and DNA synthesis (133). Ventilatory stretch also stimulates the formation of pulmonary derived mediators, e.g., cyclooxygenase products (134–137), and intracellular messengers, e.g., inositol triphosphate and calcium (138–140). Specific stretch-activated channels in the endothelial cell membrane convert the mechanical stimuli into intracellular calcium mobilization in the micromolar range (141,142) sufficient to activate the NOS (143). It is likely that an early activation of endothelial potassium channels is involved in shear stressinduced NO production (144). Furthermore, stretch may facilitate calcium flux via voltage-gated L-type Ca 2⫹ channels (145,146). Thus a variety of membrane ion channels may be influenced by mechanical stimulation. Stretch-activated channels have been identified in several pulmonary cells, including alveolar, endothelial, smooth muscle, and airway epithelial cells (147–151). To evaluate the involvement of stretch-activated channels in exhaled NO production, we performed experiments with gadolinium chloride (GdCl 3), an inhibitor of stretch-activated channels, and found that GdCl 3 significantly inhibited basal pulmonary NO producton in guinea pigs and rabbits (109,126). In the latter study GdCl 3 induced a major increase in pulmonary vascular resistance and decreased arterial PO 2 . Interestingly, the effects of GdCl 3 on pulmonary vascular resistance were more pronounced when compared to the effects of an NO synthase inhibitor given in a dose reducing nitric oxide in exhaled air to approximately the same extent as did GdCl 3 (Figs. 3a and 3b). A possible explanation could be that GdCl 3 exerts an inhibitory effect on stretch-activated calcium channels at several sites, thereby preventing the formation not only of NO but also of other important pulmonary vasodilators, e.g., prostacyclin (PGI 2) (135,152), atrial natriuretic peptide (153), or the hitherto somewhat enigmatic endothelialderived hyperpolarizing factor (EDHF) (154). C. Effect of Carbon Dioxide on Exhaled NO Production

Inhaled carbon dioxide (CO 2) has been shown to inhibit formation of NO in exhaled air of rabbits (21), guinea pigs (155), and dogs (111). Furthermore, a specific CO 2-dependent regulatory mechanism on pulmonary NO production could be separated from nonspecific actions elicited by CO 2 inhalation in vivo, e.g., changes in blood pH, sympathetic activation, or pulmonary blood flow, in

44


Figure 3 Comparison of the effects of GdCl 3 30 mg/kg (a: time course) with the effects of L-NAME (b: dose–response) on exhaled NO and pulmonary vascular resistance in artificially ventilated pentobarbital anaesthetized rabbits. (Data from Ref. 109.)

a study with HEPES buffer-perfused lungs (59). During control conditions we also observed higher concentrations of exhaled NO in HEPES buffer solutionperfused lungs (⬃80 ppb), where no CO 2 was added to respiratory gas, than previously reported in bicarbonate buffer solution-perfused lungs (⬃50 ppb), where CO 2 was added to the inspired gas to achieve end-tidal CO 2 readings within the normal range (56,125,156). These observations support the findings that CO 2 exerts an inhibitory effect on pulmonary NO production. Another interesting observation is the rapid and large increase in exhaled NO when blood flow through the pulmonary circulation ceases at the start of buffer solution perfusion. The common opinion is that the increase in exhaled NO is a consequence of decreased scavenging by hemoglobin due to the reduced pulmonary blood flow. However, one has to consider the abolished exchange of carbon dioxide resulting in decreased airway levels of CO 2 as a possible contributing mechanism for the increase in exhaled NO postmortem. Accordingly, inhaled CO 2 can reverse a major part of the increment in exhaled NO seen postmortem (Fig. 4). In another study, the inhibitory effect of CO 2 on exhaled NO was shown to be dependent on the level of stretch (PEEP) applied to the lungs of guinea pigs. At a higher PEEP, when pulmonary NO production increased (as discussed above), CO 2 was more effective as an inhibitor of NO production (Fig. 5) (155). Pulmonary slowly adapting stretch receptors (SAR) and their vagal afferents, mediating the Hering-Breuer reflex, are rapidly inactivated by CO 2 inhalation (157). In fact, the kinetics of the inhibition of SAR by CO 2 (158) resemble the rapid inhibitory effect of CO 2 on pulmonary NO production that we have found (59). A connection between vagal SARs and pulmonary NO production seems


45

Figure 4 Recording of the effect of hemorrhage (arrow indicates start of bleeding), until circulatory arrest and postmortem, on exhaled NO production and end-tidal carbon dioxide (ETCO 2) in an anesthetized and mechanically ventilated rabbit (in vivo). Shown also is the effect of carbon dioxide inhalation (CO 2 , black bars) before and after circulatory collapse. Note the marked effect of CO 2 inhalation even after circulation ceased.

likely, since vagal afferents express neuronal NO synthase (159) and vagotomy attenuated the PEEP-induced increases in exhaled NO (52). Therefore, in addition to a direct effect of CO 2 on NOS, it might be speculated that CO 2 could exert its inhibitory effects on SARs and NO production through a common regulatory mechanism intrinsic to the lung. Local regulation of calcium entry into stretch receptors and NO-producing cells would seem a logical mechanism, whether the stretch receptors and the NO-producing cells are the same type of cells or not. However, the mechanism whereby inhaled CO 2 inhibits pulmonary NO formation is still unknown. Despite a lower increase in FRC, HFOV induced a greater increase in NO concentration and excretion than PEEP (see above), indicating that HFOV might stimulate other airway receptors than SARs. Indeed, diaphragmatic activity can be enhanced in response to HFOV-induced vagal apnea in spontaneously breathing rabbits, but not in response to vagal apnea induced by lung inflation, demonstrating that HFOV not only stimulate SARs but also airway irritant or C-fiber receptors (160). Therefore, HFOV and PEEP might be alternative means of testing whether stretch-dependent mechanisms are prominent in regulating pulmonary NO production in humans. D. Adrenoceptors and Exhaled NO

Stimulation of α-adrenoceptors by nasally nebulized methoxamine, an α 1-adrenergic agonist, reduced nasally exhaled NO in horses (161). Likewise, it has been shown that the nasal decongestant, oxymetazolin (α 1-adrenergic agonist), sig-

46


(a)

(b) Figure 5 (a) Typical recording of basal and PEEP-stimulated (7 cmH 2 O) lower airway exhaled nitric oxide in an anesthetized guinea pig, and the inhibitory effect of inhaled CO 2 (6%). The arrow indicates the start of exposure to 6% CO 2 . (From Ref. 155.) (b) Concentration dependence of the inhibitory effect of inhaled CO 2 on basal levels (solid circles) and on PEEP-induced formation (open circles) of exhaled lower airway nitric oxide in guinea pigs. Values are means ⫾ SEM; n ⫽ 5.* Significant difference (P ⬍ 0.05) between the nitric oxide concentration in exhaled tracheal air during inhalation of CO 2 and the preexposure value; paired t test. (From Ref. 155.)


47

nificantly lowers the concentrations of NO in the nasal cavities of humans (162– 164). It is not clear whether these changes in NO occur in response to a reduced local blood flow and/or reduced temperature in the nasal cavity (164), or if a more direct α-adrenergic regulatory effect on the NO synthase is involved. A reduction in blood flow could possibly limit the supply of L-arginine to NOproducing cells in the nasal cavity. On the other hand, one would expect an increase in exhaled NO with diminished blood flow, since less NO would be scavenged by hemoglobin (48). Inhalation of salbutamol (β 2-adrenoceptor agonist) does not increase exhaled NO in healthy humans (165,166) but may increase exhaled NO in asthmatics taking inhaled glucocorticosteroids (167). A recent in-vitro study of cultured tracheal epithelial cells concluded that isoproterenol, an unselective β-adrenoceptor agonist, could stimulate NO production (117) and that the mechanism involved cAMP-dependent activation of a calcium-independent NO synthase (168). We have found that adrenaline via β 1-adrenoceptors stimulates the release of NO into the airways of anesthetized rabbits (90). In this study, forskolin (an activator of adenylate cyclase), even in concentrations causing profound hypotension and tachycardia, did not stimulate NO production. The lack of effect of an adenylate cyclase stimulator suggests that elevation of cAMP is not a key event in rabbit exhaled NO production. However, administration of the calcium-channel blocker nimodipine reduced the stimulatory effect on NO production in response to β 1adrenoceptor activation. This suggests that the β-adrenoceptor-stimulated NO production involves a calcium-dependent NOS. This is in accordance with the observation that the major part of exhaled NO in rabbits is derived from a calcium-dependent NOS (19). In addition to a pure pharmacological effect of βadrenoceptor-stimulated NO production, we have recently demonstrated a role for endogenous adrenaline released by the adrenals in exhaled NO production and found a strong correlation between plasma concentrations of adrenaline and exhaled NO (169). Taken together, our data suggest that endogenous adrenaline activates pulmonary β 1-adrenoceptors that in turn stimulate calcium influx without the involvement of cAMP. Indeed, in other systems there is evidence for the existence of β 1-adrenoceptors with direct G-protein coupling to calcium channels (170). Thus, β-adrenoceptors via the activated α subunit of the G protein can directly activate the L-type Ca 2⫹ channels in mammalian myocardium (171). Furthermore, cardiac muscle neuronal NOS (nNOS) is located on sarcoplasmatic reticulum (SR) of cardiac myocytes, where it may respond to intracellular Ca 2⫹ concentration and modulate SR Ca 2⫹ ion active transport in the heart (172). In this context it is interesting to note that a general role for β-adrenoceptor-induced NO-dependent arterial vasodilation has been demonstrated in different vascular beds, including the human forearm (173–175). Rapid desensitization of β-adrenoceptors is well known and is believed to

48


result from receptor phosphorylation by G-protein receptor kinase (GRK), protein kinase A (PKA), or tyrosine kinase, followed by uncoupling from the signaling pathway and subsequent internalization of the receptor (176). In our experiments there was a biphasic time course of β-adrenoceptor-stimulated increase in exhaled NO, indicating rapid tachyphylaxis. This was most evident during infusion of high agonist concentrations. This might indicate that the mechanism of tachyphylaxis that we observed in our experiments was due to homologous desensitization, i.e., receptor phosphorylation by a cAMP-insensitive GRK, which is known to occur only at high receptor occupancy (171). V.

Physiological Role of NO in the Respiratory System

The existence of NO in the exhaled air does not necessarily mean that this particular NO plays any important role in the respiratory system. However, the development and use of specific blockers of NO has provided a growing body of evidence that NO has ubiquitous functions in the respiratory system. It is interesting to note that the respiratory mucosa continuously expresses all three types of NO synthases (34) and that some epithelial cells co-express type I and type II NOS (37). These findings most likely reflect the dual role of NO in the airways: physiological regulation and host defense against infection and pollutants (14,177,178). A. Airway Function

NO is a vasodilator in the tracheobronchial circulation and maintains baseline bronchial vascular caliber (179–181). Intravenous administration or inhalation of an NO synthase inhibitor decreases bronchial blood flow (182). The increase in bronchial blood flow in response to acetylcholine infusion is dependent on NO formation, whereas vagal nerve-induced bronchial vasodilation is not mediated via NO (183). In healthy airways, the low continuous mucosal NO production tonically suppresses airway plasma exudation (184–186) and mucus secretion (187), and promotes mucociliary clearance (188). Although NO does not seem to be important in maintaining basal airway tone, NO most certainly plays a major role in counteracting the obstructive effects of bronchoconstrictive mediators and allergens (189–191). Furthermore, NO has been identified as a neurotransmitter of inhibitory nonadrenergic, noncholinergic nerves and is the major bronchodilator pathway in human airways (192). It has been proposed that NO is released with acetylcholine from cholinergic nerves and exerts functional antagonism at the level of airway smooth muscle (193). Accordingly, nNOS is present in cholinergic nerves (192,194). NO can influence ion-channel activity in different pulmonary cells. Endogenous NO produced in human lung epithelial cells promotes flux through chloride


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channels via a cGMP-dependent mechanism (195). Exogenous NO inhibits apical Na ⫹ channels and basolateral Na ⫹-K ⫹-ATPase in alveolar type II cells, suggesting that NO might inhibit reabsorption of alveolar fluid (196,197). In addition, NO donors stimulate cGMP formation in alveolar epithelial cells and thereby inhibit the spontaneous activity in voltage-gated L-type Ca 2⫹ channels (198). Physiological Role of β-Adrenoceptor-Stimulated Pulmonary NO Production

In our study (90), in the presence of propranolol the adrenaline-induced increase in insufflation pressure was more marked. Therefore a β-adrenoceptor-mediated bronchodilator effect balancing the constrictive effects was likely, possibly involving endogenous NO. This would be in agreement with the inhibitory effect of endogenous NO on allergen- and agonist-induced bronchial obstruction found in guinea pig (189,190,199). Prejunctional β 2-adrenoceptors on postganglionic cholinergic nerves inhibit cholinergic neurotransmission in human airways and endogenously released catecholamines inhibit nerve-induced bronchoconstriction via prejunctional β 1-adrenoceptors on postganglionic cholinergic nerves in dog (200). Thus, stimulation of β-adrenoceptors on cholinergic nerve endings might induce NO production or facilitate NO release from these neurons, thereby counteracting the cholinergic effector response. The beneficial effects of β 2-agonists as bronchodilators, acting on airway smooth muscle by elevating cAMP levels, are well known, particularly in asthma. It is interesting to note that the bronchoprotective effect against bronchoconstrictors, e.g., allergen, histamine, etc., but not the bronchodilating effect, of β 2-agonists is reduced in asthmatic patients after regular treatment with short-acting β 2-agonists (201). It is tempting to speculate that part of the β 2-agonist-induced bronchoprotective response is mediated by NO and that tolerance development to this effect is due to diminished NO formation. In theory, this might also explain observed minute effects of β 2-agonist inhalation on exhaled NO production in asthmatic patients (165–167). B. Nitric Oxide and the Pulmonary Circulation

In the human, a number of studies suggest a physiological significance of NO as a tonic vasodilator in the pulmonary circulation (113–115,202), and it has been shown that inhibition of endogenous NO synthesis in rabbits, guinea pigs, rats, pigs, cats, and lambs results in a marked increase in pulmonary vascular resistance (203–205). However, in dogs, vasodilator prostaglandins seem to be more important than NO during basal conditions (112). Nevertheless, NO is invariably of fundamental importance in modulating pulmonary vascular resistance during conditions of increased tone, e.g., during acute hypoxia or adrenergic pulmonary vasoconstriction. Inhibition of NO synthesis markedly potentiates HPV (203,206), suggesting that basally released NO counteracts HPV. There is also

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ample evidence for a tonic production of endothelial-derived NO in the pulmonary circulation. Besides the blood flow-induced NO production, circulating mediators, e.g., bradykinin, serotonin, endothelin-1 and -3, acetylcholine, substance P, ATP, histamine, etc., can induce pulmonary vasodilation through NO release (207), although some of these compounds sometimes are constrictors. Since NOS inhibition leads to increased HPV, it has been suggested that endogenous NO would act to impair optimal ventilation/perfusion matching by dilating most in the more hypoxic pulmonary regions (208). Experiments with NOS inhibition in animals breathing air indicate clearly that the opposite is the case: during NOS inhibition a lowering in PO 2 and desaturation of the blood occurs, and the proposition has been made that endogenous NO is necessary for normal pulmonary oxygenation of the blood (203). If it is considered that NOS inhibition is not equal to administration of a vasoconstrictor, but is in fact the depletion of a vasodilator, this concept becomes rational. Weighing in that this vasodilator, NO, will be produced in highest amounts in well-ventilated (stretched) and well-oxygenated lung regions with relatively less carbon dioxide tensions (Fig. 6), the distinct effect of NOS inhibition on oxygenation seems totally reasonable. C. Transition of Pulmonary Circulation at Birth

The fetal pulmonary circulation is in a constant state of powerful active vasoconstriction, in part due to the relative hypoxic conditions in the uterus. Successful adaptation to extrauterine life necessitates a dramatic fall in fetal pulmonary vas-

Figure 6 Local regulation of nitric oxide production. Nitric oxide is acting in concert with oxygen to dilate pulmonary arteriole in a well-ventilated region. In the poorly ventilated region, carbon dioxide accumulates, airway/alveolar hypoxia occurs, and stretch is minimal. Therefore nitric oxide production decreases and HPV can fully develop, promoting V/Q matching.


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cular resistance to yield the 10-fold increase in pulmonary blood flow and concomitant drop in pulmonary arterial blood pressure at birth. Mechanisms that contribute to this transition are incompletely understood, but include mechanical factors accompanying expansion of the lung (209) and an increase in alveolar oxygen tension and decrease in carbon dioxide tensions (210). NO appears to be of cardinal importance in the transition of pulmonary circulation at birth (128), and chronic inhibition of nitric oxide in utero produces pulmonary hypertension in newborn lambs (211). D. Control of Breathing

Inhibition of endogenous NO production in awake rats (212) and anesthetized rabbits (203) induces hyperventilation. It is possible that endogenous NO produced at supraspinal sites may act as a ventilatory depressant, since infusion of an NO synthesis inhibitor into the cisterna magna of awake dogs increased ventilation, decreased PaCO 2 , and attenuated morphine-induced ventilatory depression (213). In addition to having an inhibitory effect on central respiratory neurons, NO produced in the muscle fibers of the diaphragm might directly inhibit respiratory muscle force (214), and NO is a signalling molecule in peripheral chemoreceptors (214a). E. Host Defense

Macrophages and a number of different cell types within the immune system express the inducible type II NOS and produce very large amounts of NO when activated by pathogenic agents or endogenous cytokines. These large quantities of NO are toxic to viruses, bacteria, fungi, and parasites, e.g., herpes simplex virus, mycobacterium tuberculosis, etc. (215). Most likely NO plays an important role in primary airway host defense, since the respiratory epithelium constitutively expresses an inducible NOS (iNOS) (34) and cytotoxic concentrations of NO can be found in the nose and paranasal sinuses (35). Furthermore, a twofold increase in mortality caused by pneumonia was evident in NO-deficient mice compared to the mortality in control animals (216). F. NO and Airway Inflammation/Asthma

There is increasing evidence that pulmonary NO production is increased in asthma, and that this increased NO production probably reflects the severe airway inflammation in this condition (60,217,218). It is debated whether this high NO production in asthma is advantageous or detrimental (18). Thus, NO may counteract bronchial smooth muscle hyperreactivity (189–191), but large amounts of NO may also be toxic to epithelial cells and thereby increase exudation of plasma (180,185) and stimulate mucus secretion (219). Furthermore, NO favors a Th2 lymphocyte response and promotes eosinophil chemotaxis (193,220), and high

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pulmonary NO levels correlated significantly with high peripheral blood eosinophils in young asthmatic adults (221). G. Autoinhalation of Endogenous Pulmonary NO

The finding that the respiratory airways continuously produce and release NO into the respiratory gas (14) provided a theoretical basis for a physiological correlate to inhaled NO therapy, namely, a continuous autoinhalation of airway NO, as suggested by Gerlach et al. (222). However, the first demonstration of a biological effect of such autoinhalation was from Lundberg et al., who reported that nasally produced NO improved arterial oxygenation in four intubated patients and in six of eight healthy individuals (96,223). They also introduced the term “aerocrine” for this new physiological principle of air-borne mediators and recently showed that open-heart surgery patients respond with a decrease in pulmonary vascular resistance during nasal breathing compared to mouth breathing (224). Measurements of airway NO concentrations in newborns, immediately after caesarean section on intact membranes in quiet uteri, reveal that they have extremely high airway NO levels originating from the nose/sinuses (225), the levels being comparable to therapeutic doses of exogenous NO inhalation, and that this NO is auto-inhaled (226). However, it is not known whether this autoinhalation of NO is important for pulmonary adaptation at birth. Most species, except humans, monkeys, and elephants, have very little upper airway NO formation (227–229). Therefore, if auto-inhalation of NO is a physiologically important principle to regulate lung function, auto-inhalation of NO produced and released within the lower respiratory tract must serve as the alternative in these species. On the other hand, it might be that lower airway NO formation is the primary source of auto-inhaled NO, even in humans, and that upper airway NO formation constitutes a second reserve of auto-inhaled NO. VI. Summary Together these observations suggest that in well-ventilated areas of the lung, where stretch and oxygen tension are optimal, the largest amounts of NO will be synthesized. In poorly ventilated areas of the lung CO 2 accumulates, oxygen concentrations are low, and the small airways and alveoli collapse or exhibit only minor tidal excursion. These conditions would lead to a deficiency in local pulmonary NO production which may enhance HPV and thereby improve ventilation-perfusion matching (Fig. 6). However, further studies are needed to elucidate the role of CO 2 on local NO production in insufficiently ventilated areas. The adaptation of the pulmonary circulation to extrauterine existence requires stretch, oxygen, a reduced carbon dioxide tension, and a surge of catecholamines. These factors may act in concert to massively stimulate pulmonary NO


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production in the perinatal period and thereafter. Newborns have very high levels of NO in their upper airways and this NO, together with oxygen, may be autoinhaled to lower pulmonary vascular resistance and promote oxygenation of the blood, either as a direct effect or as a safeguard if pulmonary NO production fails. In some infants these processes fail to occur and result in persistant pulmonary hypertension of the newborn, a high-mortality condition, which sometimes can be treated with inhalation of exogenous NO. It is of importance to delineate further the exact role of NO in such conditions. Acknowledgments This work was supported by the Swedish Medical Research Council (proj. no. 7919). References 1. Katsuki S, Arnold WP, Murad F. Effects of sodium nitroprusside, nitroglycerin, and sodium azide on levels of cyclic nucleotides and mechanical activity of various tissues. J Cyclic Nucleotide Res 1977; 3:239–247. 2. Murad F, Mittal CK, Arnold WP, Katsuki S, Kimura H. Guanylate cyclase: activation by azide, nitro compounds, nitric oxide, and hydroxyl radical and inhibition by hemoglobin and myoglobin. Adv Cyclic Nucleotide Res 1978; 9:145–158. 3. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980; 288:373–376. 4. Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci USA 1987; 84:9265–9269. 5. Furchgott RF, Khan MT, Jothianandan D. Comparision of endothelium-dependent relaxation and nitric oxide relaxation in rabbit aorta (abstr). Fed Proc 1987; 46: 385. 6. Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987; 327:524–526. 7. Moncada S. Nitric oxide gas: mediator, modulator, and pathophysiologic entity. J Lab Clin Med 1992; 120:187–191. 8. Singh S, Evans TW. Nitric oxide, the biological mediator of the decade: fact or fiction? Eur Respir J 1997; 10:699–707. ¨ ngga˚rd E. Nitric oxide: mediator, murderer, and medicine. Lancet 1994; 343: 9. A 1199–1206. 10. Kuo PC, Schroeder RA. The emerging multifaceted roles of nitric oxide [see comments]. Ann Surg 1995; 221:220–235. 11. Archer S. Measurement of nitric oxide in biological models. FASEB J 1993; 7: 349–360. 12. Fontijin A SA, Ronco RJ. Homogenous chemiluminescent measurement of nitric

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3 Molecular and Cellular Sources of Exhaled Nitric Oxide

AARON DEYKIN, ANTHONY F. MASSARO, LESTER KOBZIK, and JEFFREY M. DRAZEN Brigham & Women’s Hospital and Harvard Medical School Boston, Massachusetts, U.S.A.

GEORGE T. DE SANCTIS Aventis Pharmaceuticals Bridgewater, New Jersey, U.S.A.

I.

Introduction to Exhaled NO

In 1987 it was first determined that nitric oxide (NO) was the previously uncharacterized endothelium-derived relaxing factor (1,2). In the next 13 years it was established that nitric oxide can be produced enzymatically in mammalian tissues and that the NO so formed is a multifunctional biological mediator with many roles in a wide range of physiological systems. Specifically, it is now known that NO is an integral part of nonspecific host defense systems, participates in neurodevelopment and neurotransmission, and regulates various autonomical functions including vascular and bronchial tone and gastrointestinal peristalsis. NO may also both propagate and attenuate systemic inflammatory processes. Using chemiluminescence, Gustafson and colleagues first demonstrated in 1991 that NO could be detected in the exhaled gas of animals and humans (3). Since that time many investigative groups have confirmed this observation and documented that the level of NO in the exhaled gas (F ENO) is influenced by various disease states, experimental manipulations, and measurement techniques. Indeed, the lit73

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erature relating to exhaled NO in human disease appears to be increasing exponentially. However, there is still considerable uncertainty as to the precise sources of the NO which is recovered in the expirate. In the sections which follow, we review the enzymatic formation of NO, summarize the available data to indicate which of these enzymes contributes NO to the expirate, and then discuss the cellular and anatomic compartments from which the NO so formed contributes to the fraction of expired nitric oxide (F ENO). II. Enzymatic Source of Exhaled NO A. Formation of Nitric Oxide by the Nitric Oxide Synthases

In biological systems, NO is formed by the action of one of the isoforms of the enzyme NO synthase (NOS, EC1.13.13.39). In the presence of molecular oxygen and nicotinamide adenine dinucleotide phosphate, these enzymes catalyze the oxidation of the guanido nitrogen moiety of L-arginine, forming L-citrulline and NO (Fig. 1). Cofactors necessary for this reaction include flavin mononucleotide, flavin-adenine dinucleotide, tetrahydrobiopterin, and heme (4–7). To date three isoforms of this enzyme have been identified, and these have been classified in a number of different ways: a system based on their pattern of activity (inducible or constitutively active), one based on calcium requirement (dependent/independent), and another based on their initial cellular site of discovery (neural tissue, inflammatory cells, vascular endothelium). More recently, these enzymes have been cloned and mapped to distinct regions of the human genome, and current nomenclature identifies them as type I, type II, and type III. The relationship among the various designations is outlined in Table 1. Types I and III NOS were originally identified as constitutively present in brain and vascular endothelium. In contrast, type II is normally not expressed (or expressed in very low levels) in most tissues but can be induced in inflammatory, endothelial, epithelial, and smooth muscle cells. B. Differential Regulation of the Nitric Oxide Synthases

The constitutive isoforms, types I and III NOS, require free calcium and calmodulin in order to actively produce NO (8,9). This activity may be regulated by calcium-sensitive protein kinases that have the capacity to phosphorylate the NOS isoenzymes; however, the physiological significance of this event is not known (10). Factors that can influence intracellular calcium concentrations and thus rapidly change NO formation by types I and III NOS include excitatory amino acids, electrical stimulation, bradykinin, leukotrienes, platelet-activating factor, and lipopolysaccharides (4,11). Type I NOS activity is further regulated through the variety of NOS I transcripts which have been described. These different transcripts arise from three different mechanisms: (a) initiation by different transcrip-

Sources of Exhaled Nitric Oxide

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Figure 1 The enzymatic formation of nitric oxide by NOS. (Adapted from Ref. 74 with permission from the American Lung Association.)

Table 1 Characteristics of the Three Nitric Oxide Synthase (NOS) Isoforms Enzyme designation Type I (ncNOS) Type II (iNOS) Type III (ecNOS)

Gene designation

Chromosomal location and gene size (kbp)

NOS1 NOS2 NOS3

12q; ⬎ 100 17cen-q; 37 7q; 21

Cell type where first identified

Regulated by Ca 2⫹ flux

Nerve Macrophage Vascular endothelium

Yes No Yes

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tional units containing alternative promoters, (b) deletions or insertions of cassettes of DNA, and (c) the use of alternative polyadenylation signals (12). In contrast, type II NOS binds calmodulin so avidly that its activity is independent of calcium fluxes within the physiological range and dependent on transcriptional induction by cytokines and other immunological stimuli including tumor necrosis factor-α (TNF-α), interleukin1-β (IL1-β), and interferon-γ (INF-γ) (13–16). Corticosteroids inhibit the transcription of type II NOS messages and decrease type II NOS activity, probably through known nuclear factor-κB (NF-κB) sites in the 5′-flanking region of the inducible NOS (iNOS) gene (17). While there are no clearly established posttranscriptional regulatory mechanisms for type II NOS, additional regulation of this enzyme, as well as of the type I and III isoforms, likely occurs via cytokine induction of GTP-cyclohydrolase, the rate-limiting enzyme in the synthesis of biopterin (18). An interaction between types I and II NOS is supported by the finding that in rat glial cells tonically expressed type I NOS inhibits type II NOS via suppression of NF-κB (19). Posttranslational targeting of NOS via associations with cellular proteins has been demonstrated for types II (with sarcolemmal α 1-syntrophin) (20,21) and III NOS (with caveolin) (22). Detailed reviews of these aspects of NOS regulation have recently been published (12,23–25). C. Distribution of NOS Isoforms Within the Respiratory Tract

The three isoforms of NOS are distributed widely throughout the airways of humans and other mammals, and thus all three isoforms have the potential to contribute NO gas to the expirate. Schmidt et al. used an antibody specific for rat type I NOS to demonstrate the presence of this NOS isoform in rat airway epithelial cells. This finding is consistent with the findings of Kobzik and colleagues, who documented cNOS (i.e., type I or type III) epithelial staining (26,27). Subsequently, other groups have demonstrated low levels of calcium-independent NOS activity (i.e., type II) and patchy type II NOS immunostaining in rat airway epithelium and inflammatory cells (28–30). A similar distribution of NOS within the normal human airway has been documented by several investigative groups, including our own, using immunohistochemical techniques or in-situ hybridization (27,31–36). We used immunohistochemical and histochemical techniques to determine which airway loci had immunoreactivity consistent with type I or type II NOS and the locus of NADPH diaphorase activity in these airways. Fragments of normal human lung from the histologically normal tissue found at the periphery of lung resection specimens or unused lung transplant specimens were examined. An antibody to rat type I NOS was used to characterize type I NOS, while type II NOS was immunolocalized with an antibody raised against the N-terminal 20 residues of the murine type II NOS sequence. Strong immunostaining for type I NOS was observed in


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Figure 2 Immunostaining of normal human airways identifies expression of NOS-1 in nerves (arrows), and NOS-2 and NOS-3 isoforms, as well as nitrotyrosine, within airway epithelial cells. Control antibody shows minimal labeling. (Original magnification: NOS1,400⫻; all other, 150⫻.)

submucosal nerves; immunoreactivity was not observed in smooth muscle or macrophages. In contrast, type II NOS immunoreactivity was observed in epithelial cells from cartilaginous airways (Fig. 2), a finding supported by subsequent work using in-situ hybridization techniques (33,37). We also observed type II NOS in vascular endothelium and in alveolar macrophages obtained from regions of the lung with airway inflammation. Rosbe and colleagues have used similar immunohistochemical techniques to demonstrate strong staining for type II NOS, but no staining with a nonspecific type I-III antibody in human nasal epithelium, an anatomical compartment characterized by high NO levels (38). These findings indicate that within the human airway all three isoforms of NOS are present and can generate NO that may contribute to that recovered in the expirate. D. Contribution of NOS Isoforms to Exhaled NO Immunohistochemical Evidence

In order to determine which of the NOS isoforms is responsible for the physiological production of NO, several investigators have used immunohistochemical methods, enzyme activity assays, and molecular biological techniques to quantify the presence and/or function of the three NOS isoforms under various experimental conditions in animals. Using a guinea pig model, Yan and colleagues have demonstrated that sensitization and subsequent inhalational challenge with

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trimetallic anhydride results in an approximate 10-fold increase in calciumindependent (i.e., type II) NOS activity compared with no change in calciumindependent NOS activity (39). It is important to note that this increase was at least twofold greater in the bronchial tissue fraction than in the lung parenchymal preparation, an indication of the relative anatomical compartmentalization of the increased NOS activity. These results are in contrast to those reported by Mehta et al., who studied the effects of a repeated ovalbumin sensitization and challenge protocol also in guinea pigs (40). In this study, animals were exposed to an ovalbumin aerosol on 2 days separated by 1 week. Fourteen to 17 days later, ovalbumin was instilled intratracheally and F ENO, NOS activity, and NOS RNA expression were measured. The animals developed significant increases in F ENO 3 hr after challenge, the rapid time course of increased F ENO suggesting that the mechanisms responsible for this increase are not likely to require the induction of protein synthesis as would be required to form type II NOS. Notably, these animals had marked histological evidence of pulmonary eosinophilic inflammation, yet no changes in mRNA expression for any of the three NOS isoforms was detected (Fig. 3). Since type II NOS is regulated primarily at the transcriptional level, these data further suggest that the increase in exhaled NO in this setting is not likely to be derived from type II NOS and is instead the product of type I and/or III NOS. Alternatively, allergen-induced alterations in substrate or cofactor availability may result in increased NO production via any combination of the three NOS isoforms. Feder and colleagues similarly failed to detect any changes in type II NOS message or protein after inhalation allergen challenge in a mouse model, although others have shown increased type II NOS staining or calciumindependent NOS activity after allergen inhalation in rats and mice (28,41–43). How applicable these results are to the source of exhaled NO in human disease is uncertain. Hamid and colleagues were the first to report evidence linking expression of type II NOS to asthma, a disease characterized by increased F ENO (44). These authors obtained transbronchial biopsy specimens from 23 individuals with asthma and 20 nonasthmatic control subjects, and stained this material with a specific polyvalent type II NOS antibody. Epithelial type II NOS activity was observed in 22 of the 23 asthmatics; in comparison, only 2 of the 20 control subjects had visible type II NOS staining. These results are consistent with those reported by Liu and co-workers, who measured F ENO and examined NOS staining in lung tissues from patients with cancer (45). Individuals with lung cancer had higher F ENO (16.9 ⫾ 0.9 versus 6.0 ⫾ 0.5) and greater type II NOS staining in alveolar macrophages recovered from bronchoalveolar lavage (BAL) fluid than controls. A causative relationship between alveolar type II NOS expression and F ENO was suggested by the strong positive correlation between levels of type II NOS expression and F ENO (Fig. 4). In addition, examination of tissue samples demonstrated type II NOS expression in bronchial epithelium, alveolar endothe-


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Figure 3 Effect of repeated exposure to saline and antigen (ovalbumin) on glyceraldehyde-3-phosphate dehydrogenase (GADPH)-normalized NOS mRNA expression for type II NOS (open bars), type I NOS (filled bars), and type III NOS (hatched bars) in guinea pigs. Expression of type II NOS was not enhanced in antigen-exposed animals (n ⫽ 29) above levels in control-unexposed (n ⫽ 5) and saline-exposed (n ⫽ 30) animals. Similarly, there were no differences in type I and type III NOS expression in the three groups. Furthermore, expression of mRNA of type I, type II, and type III NOS was not significantly different at any individual time point (i.e., 1, 3, 6, 24, or 72 hr) following aerosol exposure to either saline or antigen. Thus bars for saline and antigen represent time-averaged data for all time points (1–72) from five groups of animals (5–6 animals/time point) for each exposure. (Reproduced from Ref. 40.)

lium, chondrocytes, and mucus glands in the subjects with malignancy, while type II NOS expression was limited to alveolar macrophages in the control subjects. Taken together, these data provide circumstantial evidence that in certain pathological conditions, increased type II NOS expression (and presumably activity) may be responsible for the higher F ENO found in subjects with these conditions. While many authors have embraced the notion that type II NOS is the major source of the higher F ENO encountered in asthma, enthusiasm for this hypothesis must be tempered by human data demonstrating that, with standardized NO collection techniques, inflammatory bronchoprovocation produces increases in F ENO as rapidly as 15 min after challenge (46). This rapid time course is not consistent with type II NOS induction, which would require protein synthesis. Although the NO evolved in the airway after inflammatory challenge could be produced by preexisting type II NOS, these results suggest a critical role for other NOS isoforms in human F ENO.

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Figure 4 The relationship between exhaled NO level and the magnitude of iNOS expression in alveolar macrophages (expressed as mean fluorescence intensity, FI) (r ⫽ 0.73, n ⫽ 76, p ⬍ 0.001). (From Ref. 45.)

Studies in Mice with Targeted Deletions of NOS Genes

Although impossible to perform in humans, studies of animals with deletions of one or more of the genes coding for the various NOS proteins have provided important information regarding the molecular sources of F ENO. De Sanctis and colleagues measured F ENO in tracheally intubated and mechanically ventilated mice with targeted deletion of nos1, the murine gene for type I NOS (47). The targeted deletion was confirmed by polymerase chain reaction (PCR). As shown in Figure 5, animals with nos1 deletions had 40% lower levels of F ENO than the wild-type animals, confirming type I NOS as a significant source of expired NO. A physiological role for the NO produced by type I NOS is further indicated by the finding that the nos1-deficient animals demonstrated decreased methacholine responsiveness. Inhibitor Studies

In order to determine which of the NOS isoforms is responsible for the physiological production of NO under various conditions, several investigators have studied the effects of NOS inhibitors with varying degrees of specificity for the three


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Figure 5 Airway responsiveness and F ENO in wild-type (⫹/⫹) and nos1 knockout (⫺/⫺) mice. Animals were anesthetized and mechanically ventilated with inspired air that was essentially NO-free (⬍1 ppb). We collected expired gas samples in a Mylar bag over 5 min from the expiratory port of the ventilator and analyzed the specimens for NO concentration by chemiluminescence. Airway responsiveness was measured as the log of the effective dose of intravenous methacholine needed to produce a doubling of pulmonary resistance from baseline (ED200 RL). ED 200 R L was higher and FE NO was significantly lower in the ⫺/⫺ animals than in the wild types ( p ⬍ 0.05 for both comparisons). (Adapted from Ref. 47.)

NOS isoforms on exhaled NO. The inhibitors most commonly used to date are derivatives of L-arginine in which the guanidino nitrogen is chemically substituted, thus rendering them resistant to cleavage by the NOS complex. Methyl and nitro substitutions for the guanidino nitrogen yield L-N G-monomethyl arginine (L-N G-MMA) and L-N G-arginine methyl ester (L-NAME), respectively. Additional inhibitors of NOS activity include alkyl amidines; aminoguanidine, a specific inhibitor in type II NOS, is the most commonly used compound in this class (48–51). By the nature of their action, these molecules are predominantly competitive inhibitors of NOS; hence, their competition can be overcome by the addition of excess amounts of L-arginine but not D-arginine. Inhaled L-N G-MMA and L-NAME have been shown to alter the amount of NO in the expired air, an indication that the enzymatic directed synthesis of NO occurs in the airway (52–55). However, since these two agents are relatively nonselective inhibitors of NOS, these data do not definitively indicate which enzymatic source of NO is responsible for the NO detected in the expirate. Inhaled glucocorticoids have also been shown to lower exhaled NO levels in subjects with asthma. As glucocorticoids are potent inhibitors of the induction of type II NOS, these data have been interpreted to indicate that the elevated F ENO encountered in asthma is the result of increased type II NOS activity (53–56). However, since glucocorticoids also inhibit the production of biopterin, a cofactor necessary

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Figure 6 Effect of the inhaled inducible NOS inhibitor aminoguanidine (filled triangles) and 0.9% saline control (filled squares) on exhaled NO in 8 normal subjects (left panel) and 7 patients with asthma (right panel). Mean values ⫾ SEM are shown; *p ⬍ 0.05. (Reproduced from Ref. 52 with permission from the American Lung Association.)

for production of NO by all NOS isoforms, this observation alone is also not definitive evidence for a central role for type II NOS in exhaled NO in general. Nevertheless, data indicating that the reduction in F ENO induced by glucocorticoids is specific to subjects with asthma support a role for type II NOS in the increased F ENO encountered in this condition (52,57). This conclusion is strengthened by the report of Yates and colleagues demonstrating that the nebulized administration of aminoguanidine produces a significant reduction in peak exhaled NO levels in asthmatics, but not in normal subjects (see Fig. 6) (58). III. Cellular Sources of Exhaled NO As reviewed in previous sections, many cell types in the respiratory tract of humans can produce NO. It is likely that all of these cells contribute NO to the final concentration of NO measured in the expirate under various health and disease states. While thus far there are no data that quantitatively assess the fractional contribution of the cellular sources of NO to the expirate, the cellular locations of the NOS isoforms have been studied by several investigative groups. These data are summarized in Table 2. IV. Anatomical Sources of Exhaled NO A. NO Is Synthesized in the Lung

There has been considerable uncertainty about the anatomical source of the NO detected in the expired air. Early concerns that NO is not produced in the lung

Sources of Exhaled Nitric Oxide Table 2

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Cellular Locations of the Three NOS Isoforms

NOS isoform

Cellular location

References

Type I NOS Type II NOS

Bronchial submucosal nerves Bronchial epithelial cells Alveolar macrophages Nasal vascular endothelial cells Nasal ciliated epithelial cells Pulmonary vascular endothelium Bronchial epithelium Nasal epithelium Nasal vascular endothelium

(27) (27,33,37,44) (27,36,45,72) (73) (38,73) (27,32) (32) (73) (73)

Type III NOS

but is transported via the blood to the lung, where it is excreted, are unfounded. Cremona and colleagues have demonstrated that isolated, ventilated, and perfused pig lungs evolve NO in concentrations similar to those detected in vivo, indicating that NO is synthesized in the lung and not carried from extrapulmonary sites (59). Using simultaneous measurements of NO and CO 2 , several investigators have further demonstrated that NO is formed in a region of the lung close to that where CO 2 is formed, possibly in the terminal bronchioles (60,61). B. NO Is Formed in the Lower Airway

In humans, it has been demonstrated that the nasopharynx contains high concentrations of NO (1000 ppb); this observation has led several investigators to hypothesize that the NO in the expirate represents NO produced in the nasal passages that “contaminates” orally exhaled gas (62–66). Another non-airway source of exhaled NO is the gastrointestinal tract, where NOS is found within the gastric mucosa, with additional NO likely formed by the conversion of nitrite to NO in the acidic gastric environment (67). In fact, gas expelled from the gastrointestinal tract has been documented to contain 800–6000 ppb NO (68). While it is certain that a component of the expired NO is derived from extrathoracic sites, studies of NO concentrations in gas derived from the lower airway have provided direct evidence that NO is produced in the lower airway. In one such study, Massaro and colleagues studied F ENO in normal and asthmatic subjects before and after orotracheal intubation (which isolated the lower airway from gas derived from the nasopharynx) (69). After intubation there was a small decrease in F ENO in normal subjects but no significant change in F ENO in subjects with asthma (Fig. 7). The difference between F ENO in normal and asthmatic individuals was preserved at all levels of the lower airway, an indication that the elevated F ENO encountered in this disease state is not due to contamination of the

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Figure 7 The mean NO and CO 2 concentrations of lower respiratory tract gas, sampled at the right lower lobe orifice and end of the endotracheal tube after a 15-sec breath-hold. Concentrations are reported with accompanying SEM. *p ⬍ 0.05 normal versus asthma. (Reproduced from Ref. 69 with permission from the American Lung Association.)

lower airway gas by exogenous sources of NO. In fact, the finding that F ENO does not significantly change after intubation in asthmatics appears to suggest that the fractional contribution to exhaled NO by extrathoracic (i.e., nasal) sources is negligible in this condition. These findings are supported by those reported by Phillips and colleagues, who used balloon occlusion of the upper airway to demonstrate that the majority of orally exhaled gas derives from the lower airway. Nevertheless, currently accepted standards for NO collection and measurement stipulate exhalation against a modest resistance, which produces positive pressure within the oropharynx, elevates the soft palate, and isolates the expired gas from the nasopharynx (64,70). C. Models of NO Production

The interaction of the factors that influence the concentrations of NO encountered in the lower airway has been modeled by Hyde and colleagues (71). These investigators propose that the lower airway NO production rate (V˙ NO) can be determined from the following equation: V˙ NO ⫽ P L (D NO)


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where P L is the partial pressure of NO in the lower airway and D NO is the rate of removal of NO from the airway by lung capillary blood. Validation of this model will require studies of lower airway NO, alveolar ventilation, and pulmonary blood flow under conditions where these parameters are changing, such as exercise, but the model suggests that the central, but lower airways, is a major source of exhaled NO. V.

Conclusions

Nitric oxide measured in the exhaled gas of animals and humans is formed largely in the lower airway. Numerous cell types within the lung contain one or more of the three isoforms of the enzyme nitric oxide synthase and thus have the capacity to produce NO. While traditional thought has emphasized the importance of type II NOS localized within airway epithelial cells, we believe it is likely that under various naturally occurring and experimental conditions all three isoforms and multiple cells contribute to F ENO. Future experiments with truly isoformspecific NOS inhibitors and animal models with deletions of the genes for these NOS isoforms will further clarify the specific contributions of these sources to the NO measured in the expirate. References 1. Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987; 327:524–526. 2. Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci USA 1987; 84:9265–9269. 3. Gustafsson LE, Leone AM, Persson MG, Wiklund NP, Moncada S. Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem Biophys Res Commun 1991; 181:852–857. 4. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J 1992; 6:3051–3064. 5. Forstermann U, Schmidt HH, Pollock JS, Sheng H, Mitchell JA, Warner TD, Nakane M, Murad F. Isoforms of nitric oxide synthase. Characterization and purification from different cell types. Biochem Pharmacol 1991; 42:1849–1857. 6. Pou S, Pou WS, Bredt DS, Snyder SH, Rosen GM. Generation of superoxide by purified brain nitric oxide synthase. J Biol Chem 1992; 267:24173–24176. 7. Mayer B, John M, Bohme E. Purification of a Ca 2⫹ /calmodulin-dependent nitric oxide synthase from porcine cerebellum. Cofactor-role of tetrahydrobiopterin. FEBS Lett 1990; 277:215–219. 8. Lamas S, Marsden PA, Li GK, Tempst P, Michel T. Endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constitutive enzyme isoform. Proc Natl Acad Sci USA 1992; 89:6348–6352.

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9. Bredt DS, Snyder SH. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci USA 1990; 87:682–685. 10. Dinerman JL, Steiner JP, Dawson TM, Dawson V, Snyder SH. Cyclic nucleotide dependent phosphorylation of neuronal nitric oxide synthase inhibits catalytic activity. Neuropharmacology 1994; 33:1245–1251. 11. Larfars G, Lantoine F, Devynck MA, Palmblad J, Gyllenhammar H. Activation of nitric oxide release and oxidative metabolism by leukotrienes B4, C4, and D4 in human polymorphonuclear leukocytes. Blood 1999; 93:1399–1405. 12. Forstermann U, Boissel JP, Kleinert H. Expressional control of the “constitutive” isoforms of nitric oxide synthase (NOS I and NOS III). FASEB J 1998; 12:773– 790. 13. Cho HJ, Xie QW, Calaycay J, Mumford RA, Swiderek KM, Lee TD, Nathan C. Calmodulin is a subunit of nitric oxide synthase from macrophages. J Exp Med 1992; 176:599–604. 14. Stuehr DJ, Cho HJ, Kwon NS, Weise MF, Nathan CF. Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMNcontaining flavoprotein. Proc Natl Acad Sci USA 1991; 88:7773–7777. 15. Ding AH, Nathan CF, Stuehr DJ. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J Immunol 1988; 141: 2407–2412. 16. Denis M. Tumor necrosis factor and granulocyte macrophage-colony stimulating factor stimulate human macrophages to restrict growth of virulent Mycobacterium avium and to kill avirulent M. avium: killing effector mechanism depends on the generation of reactive nitrogen intermediates. J Leukoc Biol 1991; 49:380–387. 17. Xie QW, Kashiwabara Y, Nathan C. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J Biol Chem 1994; 269:4705–4708. 18. Hattori Y, Gross SS. GTP cyclohydrolase I mRNA is induced by LPS in vascular smooth muscle: characterization, sequence and relationship to nitric oxide synthase. Biochem Biophys Res Commun 1993; 195:435–441. 19. Togashi H, Sasaki M, Frohman E, Taira E, Ratan RR, Dawson TM, Dawson VL. Neuronal (type I) nitric oxide synthase regulates nuclear factor kappaB activity and immunologic (type II) nitric oxide synthase expression. Proc Natl Acad Sci USA 1997; 94:2676–2680. 20. Brenman JE, Chao DS, Xia H, Aldape K, Bredt DS. Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy. Cell 1995; 82:743–752. 21. Brenman JE, Chao DS, Gee SH, McGee AW, Craven SE, Santillano DR, Wu Z, Huang F, Xia H, Peters MF, Froehner SC, Bredt DS. Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains. Cell 1996; 84:757–767. 22. Shaul PW, Smart EJ, Robinson LJ, German Z, Yuhanna IS, Ying Y, Anderson RG, Michel T. Acylation targets endothelial nitric-oxide synthase to plasmalemmal caveolae. J Biol Chem 1996; 271:6518–6522. 23. Michel T, Feron O. Nitric oxide synthases: which, where, how, and why? J Clin Invest 1997; 100:2146–2152.


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4 Determinants of Exhaled Nitric Oxide Influence of Ventilation and Pulmonary Blood Flow

´ NDOR MARCZIN and SIR MAGDI H. YACOUB NA Imperial College of Science, Technology and Medicine National Heart and Lung Institute London and Harefield Hospital Harefield, Middlesex, England

I. GAVIN WRIGHT Harefield Hospital Harefield, Middlesex, England

I.

Introduction

The original discovery of nitric oxide (NO) in exhaled breath by Gustaffsson et al. opened new dimensions for clinical applications of NO in the diagnostic assessment of multiple cardiorespiratory interactions in health and disease (1). This promise, however, can only be fulfilled with increased understanding of the mechanisms of generation, cellular sources, fluid-phase reactions, and distribution of NO in the cardiorespiratory system, first under normal conditions and then by analyzing these parameters under specific pathological conditions. Increasing evidence suggests that ventilation characteristics and pulmonary blood flow are primary determinants of the measurable concentrations of gas-phase NO. Since these parameters are continuously changing under normal physiological processes and during disease progression, understanding of these events is obligatory to draw meaningful conclusions and using NO as a diagnostic biological marker. The magnitude of the problem should not be underestimated, and the complexity can be illustrated by two examples. In the first case, we would like to interpret changes in exhaled NO during exercise testing of patients with significant heart failure compared to healthy controls. In the second case, we wish to explain 91

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changes in gas-phase NO levels in ventilated patients presenting with acute reoxygenation injury following lung transplantation compared to patients undergoing routine anesthetic. It is obvious that in both cases changes in ventilation rate, minute volume, air flow characteristics, alveolo-capillary surface area, thoracic blood volume, and pulmonary blood flow all have the potential to significantly and sometimes opposingly affect actual concentrations of NO in the gas phase. This chapter aims to review recent understanding of the influence of ventilation and pulmonary blood flow on measures of exhaled NO. Although we will consider data from animal models, we will examine these variables in spontaneously breathing humans with major emphasis given to clarifying these issues in mechanically ventilated patients with healthy lungs. II. Influence of Ventilation A. Composition of Inhaled Gases Influence of Inhaled NO

Several studies have addressed the possible interference caused by high environmental concentrations of NO or inhaled NO on measurements on interpretation of exhaled NO. Kharitonov et al. demonstrated that inhalation of 800 ppb NO did not change exhaled NO after 15 sec of breath holding in two healthy subjects, suggesting that inspired NO must disappear from the respiratory tract within 15 sec (2). Similar conclusions were reached by Piacentini’s group, who demonstrated that ambient NO concentrations in the range of 0–150 ppb had no significant influence on exhaled NO (3). In contrast, Baraldi et al. found that ambient air breathing containing 3–430 ppb NO resulted in higher exhaled NO concentrations in asthmatic children when compared to breathing NO-free air (4). Although the reasons for these differences remain unclear, it is possible that during tidal breathing in children the higher respiratory rate prevents complete absorption of inhaled NO, which could then contaminate exhaled breath. A special form of NO inhalation which can confound exhaled NO measurements is autoinhalation from nasal and upper airways. Several studies have demonstrated high concentrations of NO in the nasal cavity, which can be inhaled especially during nasal breathing and which can potentially contaminate gasphase NO during exhalation maneuvers (5,6). For this reason the use of some expiratory resistance is recommended in order to increase mouth pressure and to close the soft palate during exhaled NO measurements in spontaneously breathing patients (6,7). This confounding factor and the contribution of NO from upper airways in general is completely eliminated in tracheostomized animals and in intubated and mechanically ventilated patients. Detection of NO in the exhaled breath under these conditions demonstrates contribution of lower airway to exhaled NO.

Determinants of Exhaled NO

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Using this approach, we have studied characteristics of NO waveforms during inspiration and expiration in patients undergoing cardiothoracic surgery ventilated with a mixture of oxygen and compressed medical air containing variable levels of NO. Real-time analysis of NO (Logan Research Ltd., 2000 and 3000 series analysers) reveals a dynamic pattern of NO changes within the respiratory cycle as judged by the phase relationship between NO and CO2 (Fig. 1, middle). However, due to the physical delay between the sequentially positioned NO and CO2 sensors in the analyzer and due to the difference in response times of the sensors (Fig. 1, top), the phase relationship as observed needs to be corrected. When corrected for the 920 msec lag phase between the NO and CO2 signal, the data show that gas-phase NO increases in late expiration, reaching peak levels at end-expiration and declining to zero during inspiration in the absence of inhaled NO (Fig. 1, bottom). In the presence of inhaled NO, the waveform of NO during expiration appears to be unchanged, but there is a superimposed signal with a peak at end inspiration (Fig. 1). These data suggest that exhaled NO generated in the lungs of mechanically ventilated patients can be precisely resolved within the respiratory cycle and distinguished from inhaled NO, enabling intraoperative monitoring of changes in endogenous NO production and excretion by the lungs. Despite this conclusion, for reasons of simplicity and easier data interpretation, we recommend using NO-free air or 100% O2 for clinical measurements of gas-phase NO in mechanically ventilated patients. The described waveform of gas-phase NO in mechanically ventilated animals and patients differs significantly from those obtained in spontaneously breathing patients. The latter is characterized by an initial peak deriving from the upper airways, followed by a plateau, usually below the initial peak, depending on the duration of exhalation and developed mouth pressure. On the basis of the relationship between NO waveform and simultaneoulsy observed CO2 trace, Persson et al. concluded that NO was formed preferentially in the small airways. In mechanically ventilated and intubated patients the early peak is missing, due to exclusion of the upper airways, and the peak of NO appears to be end-tidal in most of the patients. This might suggest more distal origin of NO and greater contribution of terminal bronchi and alveoli to exhaled NO. We believe, however, that this phenomenon might be due to deceleration of expiratory flow rate and thus reduced dilution resulting increased concentration of NO in the gas phase toward the end of expiration. Finally, the plateau phase is generally not observed with normal ventilation settings in mechanically ventilated patients but can be achieved using a breath-holding maneuver (see below). Influence of CO2

The concentration of CO2 in respiratory gases has been reported to influence exhaled NO measurements. Similarly to water vapor, CO2 may interfere with the

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chemiluminescence procedure and quench the NO signal (8). Chemical experiments suggest that there is a 1% decrease in NO reader per volume CO2. Thus, in the normal respiratory range, the technical influence of CO2 is generally negligible on NO chemiluminescence. In addition to quenching, Stromberg et al. reported a significant negative relationship between inspired CO2 concentration and exhaled NO in anesthetized rabbits (9). Similar data were obtained in guinea pigs, showing approximately 25% reduction by 12% carbon dioxide in inhaled air (10). These data suggest that endogeous CO2 might also act as a regulator of NO production in the lung, especially in conditions of permissive hypercapnia of critically ill patients. In order to investigate the human significance of these suggestions, we set out to measure exhaled NO in the same patients at different levels of expired CO2. To do so, we initially hyperventilated patients to achieve a relatively low (3–4%) arterial pCO2, followed by hypoventilation with reduced tidal volume. Expired NO and CO2 were monitored simultaneously as pCO2, and end-tidal CO2 increased until mild hypercapnia was achieved (Fig. 2). During this maneuver, arterial pCO2 increased from 3.9 ⫾ 0.1 to 6.9 ⫾ with a concomitant decrease in pH from 7.49 ⫾ 0.2 to 7.28 ⫾ 0.2 and base excess changed from ⫺0.5 ⫾ 0.6 to ⫺2.8 ⫾ 0.2 in six patients. In these preliminary experiments, peak exhaled NO at the beginning of the hypoventilation period (exhaled CO2 of 3.9 ⫾ 0.2%) was 8.8 ⫾ 2.2 ppb and at the end of the hypoventilation period (exhaled CO2 of 6.2 ⫾ 0.2%) peak NO levels were 9.6 ⫾ 2.3, representing 110 ⫾ 2.3, representing 110 ⫾ 4.1% of baseline values. These data suggest that variation in CO2 concentration from mild hypocapnia to mild hypercapnia is not associated with changes in expired NO in mechanically ventilated patients. The influence of higher CO2 levels, such as seen in permissive hypercapnia, on exhaled NO remains to be established in humans.

Figure 1 (Top) Representative steady state NO and CO2 signals from a reservoir bag containing exhaled breath with sample flow rate of 250 mL/min. Note the lag time and differences in response times of the NO and CO2 analyzer. The lag time between the detection of NO and CO2 signals appears to be 920 msec. (Middle) Real-time analysis of NO and CO2 in mechanically ventilated patients [FiO2 of 1.0, tidal volume of 10 mL/kg and respiratory rate (RR) of 10 breaths/min] undergoing open-heart surgery. Note the dynamic pattern of NO within the respiratory cycle (left-hand panel). Changing ventilation to inhale NO from compressed medical air significantly alters the waveform (right-hand panel). When corrected for the lag phase between the NO and CO2 signal, the data show that in the absence of inhaled NO, gas-phase NO increases in late expiration, reaching peak levels at end-expiration and declining to zero during inspiration (bottom panels). In the presence of inhaled NO, the waveform of NO during expiration appears to be unchanged, but there is a superimposed signal with a peak at end-inspiration.

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Figure 2 Dynamic pattern of exhaled NO and CO2 in mechanically ventilated patients during hypoventilation. While end-tidal CO2 nearly doubled during the observation period, there was no evidence of changes in expired NO concentrations.

B. Parameters and Mode of Ventilation Flow and Minute Volume

One of the most significant earliest observations regarding determinants of exhaled NO was the discovery of marked flow dependency of exhaled NO concentrations in spontaneously breathing subjects (11) and the parallel findings of definite influence of ventilation pattern on exhaled NO trace in mechanically ventilated patients (12,13). A representative trace showing the influence of expiratory flow rate on exhaled NO in a spontaneously breathing subject is shown in Figure 3. As flow decreases, both the initial component representing NO production from large airways and nasal cavity (below a flow rate, which is insufficient to maintain mouth pressure necessary to close the soft palate) and the plateau components of the NO trace representing NO production from lower airway increases. Silkoff described this relationship mathematically for the plateau phase of NO (NO ⫽ 208 ⫻ (flow rate) (⫺0.5995). A significant component of the decrease in NO concentrations can be attributed to dilution of a given amount of NO produced by the airways in the increased volume of ventilation gases as


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Figure 3 Dynamic pattern of exhaled NO, CO2, and air flow (top trace) in a spontaneously breathing patient. Note the influence of low flow rate on peak and plateau-phase NO concentrations.

flow increases. However, the situation appears to be more complicated, since NO excretion (which is the product of NO concentration and minute ventilation) appears to be increased with higher flow rates, both during resting hyperventilation and during excercise. Several explanations have been proposed to explain this phenonemon, including increased NO production by airway epithelial cells in response to increased and tubulent flow and changes in NO distribution between blood and gas phase resulting in reduced uptake by blood and increased clearance of NO via the airways (see below). On the basis of this marked flow dependence of exhaled NO concentration and excretion, Silkoff suggested standardizing exhaled NO measurement and using relatively low flow rates to amplify the NO signal, and he stressed that ANO data must be related to the expiratory flow employed. The task forces of both the European Respiratory Society and the American Thoracic Society have now considered these issues and accepted recommendations for the standarization of exhaled nitric oxide measurements (6,7). We and others similarly noted tidal and minute volume dependence of measured NO concentration and excretion in mechanically ventilated patients (13, 14). Figure 4 demonstrates representative traces showing the remarkable influ-

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ence of ventilatory parameters on exhaled NO traces. Increases in minute ventilation by manipulating either tidal volume (Fig. 4A) or respiratory rate (Fig. 4B) decrease peak exhaled NO. This is, however, associated with a new increase in expired NO amounts. The situation again appears to be somewhat complicated, since these changes in ventilation parameters are associated with marked changes in inspiratory flow rates and in airway and intrathoracic pressures, and as a consequence, expiration takes place with a variable decelerating flow rate. The importance of these considerations can be seen in experiments when minute volume remains constant but flow patterns change with changes in inspiratory–expiratory ratio. Figure 4C depicts a representative experiment showing that at a given respiratory rate (10/min) and tidal volume (10 mL/kg), increasing inspiratory time in CMV ventilation mode reduces peak inspiratory flow and expiratory time with changes in expiratory flow pattern, which is associated with decreased concentration and in fact decreased excretion of NO. These experiments can be extended to increase either expiration or inspiration time to effective breath holding, which also provides exciting insights into some fundamental aspects and controversy of exhaled NO. Breath Holding

Several studies have addressed the influence of breath holding on single breath measurements of exhaled NO in spontaneously breathing patients and found duration-dependent increases. Persson et al. noticed that the first exhalation following a 5–60 sec breath holding consisted of an initial peak followed by a plateau and observed a much greater increase in peak levels and a smaller increase in the plateau levels (15). Their interpretation of these data was that during breath holding the majority of NO was formed in the small airways, with only a minor contribution from the alveoli presumably due to uptake mechanisms. Using bronchoscopic measurements, Dweik et al. also reported increased accumulation of NO during breath holding, but they found that alveolar gases estimated from bronchiolar gases at end-expiration contained near-zero concentrations of NO (16). The significance of some of these observation is somewhat decreased, however, by the observations of Kimberly et al., who found that the increased peak

Figure 4 Influence of ventilation parameters on exhaled NO and CO2 levels. (a) This panel shows the influence of increased minute volume by increasing tidal volume (VT) at constant respiratory rate of 10 breaths/min. (b) This depicts the influence of increased minute volume by increasing respiratory rate at constant tidal volume (5 mL/kg), and (c) shows the effects of altered inspiratory:expiratory ratio (I : E) on flow patterns (top traces), exhaled NO and CO2.

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exhaled NO after breath holding was reduced to one-third after balloon occlusion of the nasopharynx (5). They also found that NO concentration during breath holding increased to a greater extent in the nasopharynx than in the pharynx or trachea, and concluded that the majority of exhaled NO at rest and during breath holding originates in the nasopharynx. In order to clarify some of these issues, we have investigated the influence of breath holding on gas-phase NO levels in intubated and mechanically ventilated patients. With relevance to breath holding, this situation offers several advantages to single breath measurements and to bronchoscopic evaluation in spontaneously breathing patients: (a) the contribution of nasal upper airways to NO production is excluded by the cuff of the endotracheal tube; (b) gas-phase concentrations can be continuously monitored during breath holding and not only interpreted from the first breath following breath holding; (c) breath holding can be performed both at end-inspiration and at end-expiration; and (d) respiratory cycles are controlled by the ventilator setting and are not influenced by variability due to changes in patient effort. Figure 5 shows representative traces of gasphase NO and CO2 during normal ventilation cycles and during breath holding. Figures 5A and 5B demonstrate that both end-expiratory and end-inspiratory breath holding results in rapid accumulation of NO and CO2 in the gas phase. The similar magnitude of the increase in gas-phase NO suggests that the majority of this NO accumulation likely arises from the airways rather than from the alveoli, since these are expected to be collapsed during breath holding in endexpiration and recruited during breath holding in end-inspiration. In addition, the first respiratory maneuver following these two types of breath holding allows us to derive interesting conclusion whether alveolar concentrations of NO are equal to or less than concentrations of NO in the lower airways. Figure 5A shows that, following expiratory breath-hold, the first inspiration delivered by the Servo 900D ventilator of 100% O2 (which contains no NO and CO2), dilute gases accumulated in the airway during breath holding and as a consequence, both CO2 and NO concentrations decrease rapidly during inspiration. The next expiration does not appear to be any different from the expiratory pattern of NO before breath holding, suggesting that all the NO delivered from the airways to the alveoli has been taken up and does not contribute to exhaled NO. In contrast, during the first spontaneous expiration by the patients following end-inspiratory breath holding, alveolar gas rich in CO2-dilute gases accumulated in the airway during breath holding, and as a consequence, CO2 levels appear to rise further (Fig. 5B). If concentrations of NO were higher in the alveoli than in the airways, a similar pattern in CO2 should be seen, and if alveolar NO equals airway NO, the mixing should not change the detected NO concentration. However, it is obvious that NO concentrations fall rapidly during the first exhalation (Fig. 5B), very similarly to dilution of the airway gases with very low concentrations of NO during the inspiratory maneuver by the ventilator following expiratory breath-hold. This provides clear evidence confirming that despite accumulation of high concentra-


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Figure 5 Representative traces exhibiting end-expiratory (a) and end-inspiratory (b) breath-holding maneuvers to evaluate main airway and alveolar concentrations of gaseous NO and CO2 in patients undergoing open-heart surgery. The flow pattern is included for reference (a): downward traces show inspiratory flow rate, whereas upward trace depicts expiratory flow.

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tions of NO in the lower airways, NO concentrations in the alveoli remain very low during breath holding. Since the high diffusion constant of NO would predict a rapid equilibrium of NO concentrations in the gas phase along the whole length of the airways, this phenomenon can only be explained by active removal of NO from the alveoli, presumably by pulmonary blood flow. Thus, our observations in mechanically ventilated patients utilizing two types of breath-holding maneuvers fully support the conclusion of Persson et al. based on their data from single breath measurements and Dweik et al. based on their bronchoscopic data. Ventilation Mode

In light of the above observations it is quite predictable that different ventilatory strategies aimed to provide the same minute volume but through variable combinations of tidal volumes, respiratory rate, inspiratory and expiratory pressures, and flow rates would result in considerable changes in exhaled NO concentrations and excretion. This area is not investigated thoroughly, and much more information is needed to compare exhaled NO measurements during different ventilation modes. In this regard, Forsberg et al. examined the influence of pressurecontrolled inverse ratio ventilation (PCIRV) on exhaled NO, and a recent study by Artlich et al. compared the influence of high-frequency oscillatory ventilation (HFOV) and intermittent mandatory ventilation (IMV) on gas-phase NO in the lower respiratory tract of anaesthetized and tracheotomized healthy rabbits (17,18). During experiments on PCIRV in rabbits, Forsberg et al. found that the total exhaled NO concentration was affected by ventilator setting and increased levels of exhaled NO were observed with increases in time-weighted tidal volume, but not by changing the pattern of inspiratory flow or I: E ratio. In the experiments with HFOV, total NO excretion increased by about twofold during HFOV. This increase was not explained by changes in functional residual capacity. Furthermore, intratracheal mean CO2 and NO concentrations, measured at increasing distance below tracheostomy, increased significantly during IMV and even more during HFOV at every intratracheal location of the sampling catheter. These data provide further evidence that lower airways are the primary source of exhaled NO in this model, and suggest an interesting difference between IMV and HFOV modes of mechanical ventilation. The authors further speculated that increased stretch activation of the respiratory system during HFOV could be the underlying mechanism and that increased gas-phase NO by HFOV might contribute to some of the benefits of HFOV treatment. A similar mechanism has been described for ventilation with positive end-expiratory pressures (PEEP). Influence of PEEP

In search of the mechanisms of airway NO production, Persson et al. investigated the influence of graded PEEP on exhaled NO in anesthetized rabbits receiving


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mechanical ventilation by tracheostomy. In a series of elegant experiments, they demonstrated that introduction of PEEP (2.5–15 cmH2O) elicited dose-dependent and reproducible increments in exhaled NO and in arterial oxygen tension (19). The increase in exhaled NO exhibited a biphasic pattern, with an initial peak followed by a partial reversal during the 4-min period. They concluded that these effects of PEEP were unlikely mediated by PEEP-induced changes in cardiac output and vascular generation of NO. However, bilateral vagotomy reduced the increase in exhaled NO in response to PEEP but did not affect the baseline concentration of NO in exhaled air. The authors postulated a stretch-dependent and vagally influenced mechanism to explain the positive effect of PEEP. Similar data were reported by Carling et al. in an isolated lung preparation of rabbits, in which the individual consequences of PEEP could be further elucidated (20). Although they highlighted the importance of capillary blood volume as a major determinant of exhaled NO (see below), changes in pulmonary blood flow around normal levels had only minor effects on exhaled NO. Thus, they have also concluded that lung distension with alveolar recruitment could be a major mechanism underlying the effects of PEEP and NO. In order to better understand the relationship between lung distension and NO, Stromberg et al. investigated the influence not only of positive but of negative pressures on exhaled NO (9). The animals were enclosed in a chamber and subjected to various modes of positive as well as negative-pressure ventilation, which was adjusted to induce similar changes in functional residual capacity (FRC) with maintained ventilatory rate and tidal volume. Similarly to PEEP during positive-pressure ventilation, negative extrathoracic end-expiratory pressure during negative extrathoracic pressure ventilation produced an increase in NO production. These observations also suggested stretch-related phenomena in the generation of NO in the airways. This was provided by pharmacological experiments in guinea pigs, in which gadolinium chloride profoundly attenuated basal levels of exhaled NO and completely abolished lower-airway NO formation induced by PEEP. These interesting experiments highlight the exciting possibility that stretch-induced cellular calcium influx could play a major role both in basal and PEEP-induced NO production in the lower airways, at least in the guinea pig. These data might have important biochemical, mechanistic, and clinical implications. First, they suggest a link between stretch receptors, calcium entry, and NO release, implying that a calcium-dependent NO synthase would be the molecular source of exhaled NO. Second, the stretch mechanism could explain flow-increased NO production and the above-discussed influence of mechanical ventilation parameters of exhaled NO. Finally, since gadolinium chloride not only attenuated NO production but also induced hypoxemia, the stretch-induced increase in endogenous NO production could be important in maintenance of ventilation perfusion matching and oxygenation. In order to establish the human and clinical significance of these exciting animal data, we set out to investigate the influence of PEEP on exhaled NO in

Figure 6 Dynamic pattern of exhaled NO and CO2 in mechanically ventilated patients subjected to PEEP challenge before (a) and during (b) cardiopulmonary bypass.


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patients undergoing cardiothoracic surgery. As we reported before, PEEP (5–10 cmH2O) produced marked changes on exhaled NO in humans (13). As shown in a representative trace in Figure 6A, application of 10 cmH2O PEEP resulted in an immediate increase in exhaled NO. Although the increase after delivering the first PEEP breath with the Servo 900D ventilator used in this study could be an artifact due to reduced tidal volume (please note altered CO2 trace), increased NO levels were detected after stabilization of tidal volume. This increase was seen in all six patients studied and amounted to a 78 ⫾ 15% increase from prePEEP levels. In contrast to the reported animal studies, PEEP-stimulated exhaled NO returned to baseline levels virtually immediately after cessation of PEEP. These data are equally compatible with alveolar recruitment and reduced pulmonary blood flow as underlyling mechanisms. To clarify the role of pulmonary blood flow, we continued ventilation and applied a similar PEEP maneuver during cardiopulmonary bypass (CPB) and aortic cross-clamping with ventricular fibrillation. Under this condition pulmonary blood flow is greatly reduced and capillaries are expected to contain less blood volume. As shown in Figure 6B, during CPB exhaled CO2 is low, confirming negligible pulmonary blood flow. PEEP, however, appears to induce a similar degree of increase in exhaled NO as before CPB with intact pulmonary blood flow. Taken together, our data suggest that intact pulmonary blood flow is not required for PEEP-induced increases in exhaled NO, and the results are more compatible with airway stretch and recruitment as the primary mechanisms in humans. III. Role of Pulmonary Blood Flow Although there are many potential interactions between pulmonary blood flow and gas-phase NO, animal and recent human studies have tested three major hypotheses regarding bidirectional movement of NO across the alveolocapillary unit: (a) whether exhaled NO originates from systemic sources; (b) whether pulmonary blood flow-mediated release of endothelial NO contributes to exhaled NO; and (c) whether blood and specifically hemoglobin in the pulmonary circulation is responsible for uptake of airway-borne NO and responsible for low alveolar concentrations of NO. A. Local Production or Delivery of NO from Systemic Sources

As reviewed in detail in previous chapters, there is overwhelming evidence for the presence of NO generation in many cell types within the lung, suggesting the possibility that all exhaled NO could be generated in the lung. The question, however, of whether there is a significant contribution of NO delivered from systemic sources by pulmonary blood flow remains an important issue with obvious diagnostic and clinical implications. Early animal studies have provided evi-

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dence that NO detected in exhaled gases was derived primarily from the lung and was not due to delivery from the circulation. This was based on experiments in which NO levels in exhaled gases of rabbits remained unchanged after blood circulation was stopped by intravenous gas administration (1). These data were further supported by observations in isolated and perfused porcine lungs, suggesting that there is enough NO synthetic capacity within the lung itself to account for NO levels in the expired air. We have made similar observations in human lungs during lung transplantation. During en-block heart and lung transplantation utilizing CPB, there is a period when, following bronchial anastomoses, the lungs can be ventilated in the absence of any pulmonary arterial and bronchial arterial blood flow prior to completion of vascular anastomoses. We utilized this setting to investigate the influence of prolonged ischaemia on exhaled NO levels and observed detectable gas-phase NO in the lungs of a number of patients in the absence of blood flow (21). Although these levels are lower than what we find in patients undergoing cardiac surgery, these anecdotal observations provide evidence for the capacity of the lower airways to produce measurable levels of NO in humans in the total absence of pulmonary blood flow. B. Vascular Endothelial or Airway Epithelial Origin of Exhaled NO

Since the original discovery of NO in the exhaled breath there has been a controversy regarding the site of NO production in the lung. Although increasing evidence suggests that NO in the expired air derives primarily from airway sources, published data imply that under certain circumstances vascular-derived NO can also contribute to exhaled NO and that pulmonary endothelial cells, at least in part, could be the source of exhaled NO. Influence of NO Donors on Exhaled NO

The direct release of NO to expired air from administration of a NO donor nitroglycerin (GTN) was first demonstrated by Persson et al. (22). They reported that influsions of GTN induced dose-dependent and biphasic increments in exhaled NO in rabbits, which coincided with reductions in systemic blood pressure. Hussain et al. confirmed this phenomenon in lambs, and we extended these observations to humans (12,23). Similarly to the animal studies, we observed transient, proportionate, and dose-dependent formation and release of NO into exhaled air after administration of GTN to the central circulation of humans. This event coincided with reduction of arterial blood pressure, supporting the hypothesis that release of NO from GTN was responsible for the hypotensive effect. The underlying mechanism likely involves uptake of GTN to vascular endothelial cells and enzymatic metab-


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olism to NO. A portion of NO surviving intracellular fluid-phase reactions might be released into the blood, where it becomes inactivated by hemoglobin. In the systemic circulation, abluminally released NO produces vasorelaxation, whereas in the lung NO diffuses into exhaled air. These mechanisms are less pronounced with sodium nitroprusside, perhaps due to spontaneous release of NO in the blood. These observations on the relationship between nitrovasodilators and exhaled NO provide evidence for direct metabolism of GTN to NO and for the potential of vascular contribution to exhaled NO. Finally, this phenomenon might be useful in elucidating mechamisms and monitoring organic nitrate tolerance and could be a sensitive marker of microvascular lung injury. Contribution of Endothelial Cell-Derived NO to Exhaled NO

In the isolated porcine lung, Cremona et al. observed that stimulation of vascular NO production by the endothelium-dependent vasodilatory acetylcholine increased expired NO associated with its effect on reducing pulmonary vascular resistance (24). Furthermore, increasing PVR by administration of the NO synthase inhibitor L-NAME attenuated expired NO, whereas increasing PVR by the thromboxane analogue had no effect on NO. From these data the authors concluded that in the isolated porcine lung the endothelium could have been the source of expired NO. This issue was further studied in humans by the selective administration of NO synthase inhibitor to the airways or to the vasculature (25). Infusion of the NO synthase inhibitors at a concentration which decreased endothelial production of NO and resulted in increased vascular resistance did not significantly affect exhaled NO levels. In contrast, inhalation of the NO synthase inhibitor, which did not influence endothelial production of NO as judged by unaltered vascular resistance, significantly attenuated exhaled NO. Taken together, these experiments suggest that under normal conditions, airway epithelial cells are a more likely source of expired NO than pulmonary endothelium (25,26). However, under certain conditions, when vascular production of NO is stimulated by endothelium-dependent vasodilators, a portion of NO might diffuse into the alveoli and contribute to exhaled NO. Although it is unlikely that acetylcholine could be a physiological regulator of vascular NO production in the lungs, bradykinin might be important in this respect. Especially following inhibition of angiotension-converting enzyme, which is expressed primarily on the luminal surface of pulmonary microvascular endothelium, bradykinin might accumulate in significant quantities to produce pulmonary vasodilation and increased exhaled NO. In addition, mechanical stimulation of endothelial cells by increased shear stress might also increase exhaled NO via similar mechanisms. This issue might be important in exercise-induced increase in exhaled NO and has been investigated by several groups (27–30).

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There have been two primary hypotheses to explain exercise-induced changes in exhaled NO. It has been proposed that augmented exhaled NO is linked to increases in cardiac output and pulmonary blood flow, causing increased shear stress-mediated increase in endothelial NO generation. Alternatively, exercise-induced hyperventilation and increased or turbulent air flow has been suggested to increase airway epithelial NO production by similar mechanisms. To distinguish between these two possibilities, Phillips et al. investigated the selective influence of dobutamine-induced increased cardiac output and isocapneic hyperventilation on exhaled NO (29). They found that hyperventilation but not dobutamine infusion increased exhaled NO, suggesting that exercise-induced increased exhaled NO output is more closely related to increased ventilation than to changes in pulmonary blood flow. The problem was investigated further by Croix et al., who found no evidence for increases in either vascular or airway production rate of NO during exercise and hyperventilation (30). They suggested that increased NO output in exhaled gases was the result of changes in NO uptake by pulmonary blood flow (see below). C. Regulation of Gas-Phase NO by Hemoglobin

The interaction between hemoglobin and nitric oxide has become conventional wisdom. NO reacts immediately with oxyhemoglobin to form bioinactive nitrate with near-diffusion-limited kinetics. The primary biological consequences of this reaction appears to be removal of NO bioactivity, and this scavenging of NO by hemoglobin has been routinely used as a measure of EDRF activity. However, this does not seem to be absolute, since recent intriguing studies suggest that the chemistry between NO and hemoglobin is far more complex and might involve additional events resulting in some degree of intravascular biostabilization, transport, and delivery of NO to a distant site (31,32). In the lung, Rimar and Gillis utilized inhaled NO and investigated the influence of hemoglobin on the ability of NO to produce vasodilatation in a superfusion type bioassay system (33). Whereas this activity of inhaled NO was present in both the pulmonary vascular bed and the bioassay ring, the effect was diminished after inclusion of hemoglobin. Their interpretation of these data was that hemoglobin rapidly inactivated NO delivered to the pulmonary circulation from the gas phase, and they concluded that this mechanism might explain the selective pulmonary vasodilator nature of inhaled NO. Thus, considering the high diffusibility of NO, the extremely rapid rate of scavenging by hemoglobin, and the rich supply of blood vessels in the lung, hemoglobin is undoubtedly a significant biological sink for endogenous NO. Despite this, some of the NO produced in the airways and at least some of the NO produced in the lung parenchyma escapes hemoglobin scavenging and diffuses


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into the airway and is eliminated via the gas phase. On the basis of these considerations, Dweik et al. concluded that NO levels in the gas phase likely underestimate NO levels in the lung tissue and at pulmonary vascular sites (16). In addition, they suggested that the measured levels in the gas phase reflect in an accurate and qualitative manner the dynamics of NO production and consumption in the lung. We have considered the role of pulmonary blood flow in the determination of gaseous NO concentrations in the human lung. To this end we have measured exhaled NO levels in the airways of patients undergoing open-heart surgery utilizing cardiopulmonary bypass (CPB). We reasoned that upon instrumentation of CPB, when the entire output of the right ventricle is diverted away from the pulmonary circulation, the reduction of pulmonary arterial blood flow should affect capillary blood volume and hence hemoglobin content, which might influence exhaled NO levels. As shown in Figure 7, institution of CPB results in an immediate decline in expired CO2 as a reflection of decreased pulmonary blood flow. One can appreciate that NO exhibits a different pattern and that gaseous concentration of NO appears to increase following CPB. Summary data obtained from six patients before and after the start of CPB reveal a twofold increase in exhaled NO concentration, from 7.4 ppb to 13.7 ppb. These simple experiments demonstrating that gas-phase NO is increased upon sudden reduction in pulmonary arterial blood flow at the start of CPB confirm that pulmonary arterial blood flow is not required for the release of NO to the gas phase. They also indicate that pulmonary blood flow continuously removes gaseous NO accumulated in or delivered to the alveoli from main and upper airways. We believe that these findings together with our observations of increased gaseous NO following breath holding and the demonstration that gaseous NO increases vasodilator tone in the lung microvasculature have important implications to ventilation perfusion matching in the human lung. In Chapter 2, Adding and Gustafsson present an attractive theory of the role of endogenous and autoinhaled NO in regulating ventilation and perfusion matching. They suggest that NO is reduced in poorly ventilated areas of the lung, where stretch and oxygen tension are suboptimal and CO2 accumulates, which may enhance hypoxic pulmonary vasoconstriction (HPV) and thereby improve ventilation–perfusion matching. While we agree with this scenario in the chronic setting, we believe that this theory does not take into account the rapid increase in gaseous concentrations of NO upon sudden interruption of ventilation such as during institution of one-lung ventilation, a procedure routinely performed for thoracic surgery. We believe that under these conditions alveolar collapse and HPV occur with considerable time delay. Similary to breath holding, NO will accumulate during this period in the gas phase in nonventilated lung regions, providing several-fold concentration gradients between well and poorly ventilated areas

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Figure 7 Influence of cardiopulmonary bypass (CPB) on exhaled NO and CO2. Top panel shows representative traces, whereas lower panel shows summary data obtained before and during CPB.

(Fig. 8). The delivery of accumulated NO to the pulmonary microvasculature (as demonstrated in our breath-holding experiments) during incomplete lung collapse might oppose HPV and might play an important role in the development of ventilation–perfusion mismatch, right-to-left pulmonary shunt, and arterial hypoxemia. There is already some experimental support for this hypothesis (34), and the postulation is consistent with everyday experience regarding immediate hypoxemia, which generally improves after full lung collapse following one-lung ventilation. However, we agree with Adding and Gustafsson in that further studies are needed to determine the fate of NO production and consumption in poorly ventilated areas. Given the potential importance of this issue to everyday thoracic anesthesia and the great number of lung pathologies associated with inhomogeneous lung ventilation and perfusion, clarification of this problem would be important progress in lung physiology.


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Figure 8 Influence of one-lung ventilation on characteristics of exhaled NO and CO2. During two-lung ventilation, both lungs contribute to CO2 elimination and exhaled NO appears to be comparable between the left and right lungs. Delivering the entire minute volume to the dependent lung at the onset of one-lung ventilation lowers expired NO and CO2 concentrations in the ventilated lung. Exhaled NO, however, rapidly accumulates in the airways of the nonventilated (nondependent) lung, producing a large NO concentration gradient between the nonventilated and ventilated lungs. This gradient might contribute to ventilation–perfusion mismatch and right-to-left shunt.

References 1. Gustafsson LE, Leone AM, Persson MG, Wiklung NP, Moncada S. Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem Biophys Res Commun 1991; 181(2):852–857. 2 Kharitonov SA, Yates D, Robbins RA, LoganSinclair R, Shinebourne EA, Barnes PJ. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 1994; 343: 133–135. 3. Piacentini GL, Bodini A, Vino L, Zanolla L, Costella S, Vicentini L, et al. Influence of environmental concentrations of NO on the exhaled NO test. Am J Respir Crit Care Med 1998; 158(4):1299–1301. 4. Baraldi E, Azzolin NM, Dario C, Carra S, Ongaro R, Biban P, et al. Effect of atmospheric nitric oxide (NO) on measurements of exhaled NO in asthmatic children. Pediatr Pulmonol 1998; 26(1):30–34. 5. Kimberly B, Nejadnik B, Giraud GD, Holden WE. Nasal contribution to exhaled nitric oxide at rest and during breatholding in humans. Am J Respir Crit Care Med 1996; 153(2):829–836.

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6. Kharitonov S, Alving K, Barnes PJ. Exhaled and nasal nitric oxide measurements: recommendations. The European Respiratory Society Task Force. Eur Respir J 1997; 10(7):1683–1693. 7. Recommendations for standardized procedures for the on-line and off-line measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide in adults and children—1999. This official statement of the American Thoracic Society was adopted by the ATS Board of Directors, July 1999. Am J Respir Crit Care Med 1999; 160(6):2104–2117. 8. van der Mark TW, Kort E, Meijer RJ, Postma DS, Koeter GH. Water vapour and carbon dioxide decrease nitric oxide readings. Eur Respir J 1997; 10:2120–2123. 9. Stromberg S, Lonnqvist PA, Persson MG, Gustafsson LE. Lung distension and carbon dioxide affect pulmonary nitric oxide formation in the anaesthetized rabbit. Acta Physiol Scand 1997; 159(1):59–67. 10. Bannenberg GL, Giammarresi C, Gustafsson LE. Inhaled carbon dioxide inhibits lower airway nitric oxide formation in the guinea pig. Acta Physiol Scand 1997; 160(4):401–405. 11. Silkoff PE, McClean PA, Slutsky AS, Furlott HG, Hoffstein E, Wakita S, et al. Marked flow-dependence of exhaled nitric oxide using a new technique to exclude nasal nitric oxide. Am J Respir Crit Care Med 1997; 155(1):260–267. 12. Marczin N, Riedel B, Royston D, Yacoub M. Intravenous nitrate vasodilators and exhaled nitric oxide. Lancet 1997; 349:1742–1742. 13. Marczin N, Riedel B, Royston D, Yacoub M. Exhaled nitric oxide in patients undergoing cardiothoracic surgery: A new diagnostic tool? In: Matalon S, Sznajder JI, eds. Acute Respiratory Distress Syndrome: Cellular and Molecular Mechanisms and Clinical Management. New York: Plenum, 1998: 365–374. 14. Brett SJ, Evans TW. Measurements of endogenous nitric oxide in the lungs of patients with the acute respiratory distress syndrome. Am J Respir Crit Care Med 1998; 157:993–997. 15. Persson MG, Zetterstrom O, Agrenius V, Ihre E. Gustafsson LE. Single-breath nitric oxide measurments in asthematic patients and smokers. Lancet 1994; 343:146–147. 16. Dweik RA, Laskowski D, Abu-Soud HM, Kaneko F, Hutte R, Stuehr DJ, et al. Nitric oxide synthesis in the lung. Regulation by oxygen through a kinetic mechanism. J Clin Invest 1998; 191(3):660–666. 17. Forsberg S, Ludwigs U, Hedenstierna G. Effects of ventilatory pattern on exhaled nitric oxide in mechanically ventilated rabbits. Acta Anaesthesiol Scand 1999; 43(4): 464–469. 18. Artlich A, Adding C, Agvald P, Persson MG, Lonnqvist PA, Gastafsson LE. Exhaled nitric oxide increases during high frequency oscillatory ventilation in rabbits. Exp Physiol 1999; 84:959–969. 19. Persson MG, Lonnqvist PA, Gustafsson LE. Positive end-expiratory pressure ventilation elicits increases in endogenously formed nitric oxide as detected in air exhaled by rabbits. Anesthesiology 1995; 82(4):969–974. 20. Carlin RE, Ferrario L, Boyd JT, Camporesi EM, McGraw DJ, Hakim TS. Determinants of nitric oxide in exhaled gas in the isolated rabbit lung. Am J Respir Crit Care Med 1997; 155:922–927.


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21. Marczin N, Riedel B, Gal J, Polak J, Yacoub M. Exhaled nitric oxide during lung transplantation. Lancet 1997; 350(9092):1681–1682. 22. Persson MG, Agvald P, Gustafsson LE. Rapid tolerance to formation of authentic NO from nitroglycerin in vivo. Agents Actions Suppl 1995; 45:213–217. 23. Husain M, Adrie C, Ichinose F, Kavosi M, Zapol WM. Exhaled nitric oxide as a marker for organic nitrate tolerance. Circulation 1994; 89:2498–2502. 24. Cremona G, Higenbottam T, Takao M, Hall L, Bower EA. Exhaled nitric oxide in isolated pig lungs. J Appl Physiol 1995; 78(1):59–63. 25. Sartori C, Lepori M, Busch T, Duplain H, Hildebrandt W, Bartsch P, et al., Exhaled nitric oxide does not provide a marker of vascular endothelial function in healthy humans [see comments]. Am J Respir Crit Care Med 1999; 160:879–882. 26. Pietropaoli AP, Perkins PT, Perillo IB, Hyde RW. Exhaled nitric oxide does not provide a marker of vascular endothelial function in healthy humans [letter; comment]. Am J Respir Crit Care Med 2000; 161:2113–2114. 27. Bauer JA, Walk JA, Doran S, Soda D. Endogenous nitric oxide in expired air: Effects of acute exercise in humans. Life Sci 1994; 55:1903–1909. 28. Iwamoto J, Pendergast DR, Suzuki H, Krasney JA. Effect of graded exercise on nitric oxide in expired air in humans. Respir Physiol 1994; 97:333–345. 29. Phillips CR, Giraud GD, Holden WE. Exhaled nitric oxide during exercise: site of release and modulation by ventilation and blood flow. J Appl Physiol 1996; 80: 1865–1871. 30. St Croix CM, Wetter TJ, Pegelow DF, Meyer KC, Dempsey JA. Assessment of nitric oxide formation during exercise. Am J Respir Crit Care Med 1999; 159:1125– 1133. 31. Cannon RO III, Schechter AN, Panza JA, Ognibene FP, Pease-Fye ME, Waclawiw MA, et al. Effects of inhaled nitric oxide on region blood flow are consistent with intravascular nitric oxide delivery. J Clin Invest 2001; 108(2):279–287. 32. Gow AJ, Stamler JS. Reactions between nitric oxide and haemoglobin under physiological conditions. Nature 1998; 391(6663):169–173. 33. Rimar S, Gillis CN. Selective pulmonary vasodilation by inhaled nitric oxide is due to hemoglobin inactivation. Circulation 1993; 88(6):2884–2887. 34. Sprague RS, Thiemermann C, Vane JR. Endogenous endothelium-derived relaxing factor opposes hypoxic pulmonary vasoconstriction and supports blood flow to hypoxic alveoli in anesthetized rabbits. Proc Natl Acad Sci USA 1992; 89(18):8711– 8715.

CARBON MONOXIDE

5 Heme Oxygenase-1 in Lung Disease

PATTY J. LEE Yale University School of Medicine New Haven, Connecticut, U.S.A.

LEO E. OTTERBEIN, JIGME M. SETHI, and AUGUSTINE M. K. CHOI University of Pittsburgh Medical Center Pittsburgh, Pennsylvania, U.S.A.

MADHU SASIDHAR Eastern New Mexico Medical Center Roswell, New Mexico, U.S.A.

I.

Introduction

Heme oxygenase (HO) was first described in 1968 by Raimo Tenhunen as the enzyme responsible for degrading heme, a highly conserved molecule that is essential for most forms of life (1). HO targets the α-methene bridge of the heme ring to catalyze the oxidative breakdown of heme into equimolar amounts of carbon monoxide (CO), biliverdin, and iron (Fe) (Fig. 1). In most mammals, biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Under physiological conditions HO activity is highest in the spleen, where senescent erythrocytes are sequestered and destroyed, but this activity is observed in virtually all organ systems. HO exists as three isozymes, HO1, HO2, and HO3, which are products of three different genes and are summarized in Table 1 (2,3). HO1 gene expression is highly inducible in response to heme, metals, oxidant stress, ultraviolet irradiation, lipopolysaccharides (LPS), and cytokines. HO2 and HO3 are structurally similar and appear to be constitutive. In contrast to both HO1 and HO2, HO3 is 117

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Figure 1 Catalytic reaction of HO. M(methyl): CH 3; V(vinyl): CH C CH 2; P(propionate): CH 2 ECH 2 ECOOH; Fe: iron; CO: carbon monoxide.

a poor heme catalyst and has been postulated to have a regulatory role in hemedependent processes (3). Many facets of HO point to its not yet fully understood biological importance. First, HO is evolutionarily highly conserved (for example, the rat and human HO1 and HO2 are about 90% identical), and despite its ubiquitous nature, no known mutants of the enzyme exist (4,5). Second, HO is present not only in mammals but also in algae, plants, and bacteria, which points to HO’s elemental role in aerobic life (6–9). Third, significant induction of HO1 occurs in response to a diverse spectrum of physiological stresses in a variety of cell and organ systems. Finally, there is accumulating scientific evidence that the induction of HO1 is a cytoprotective response to noxious stimuli, as highlighted below. HO, particularly HO1, has been implicated in a variety of homeostatic as well as biological processes. There is now extensive evidence linking the effects of HO to cytoprotective responses invoked by a variety of physiological stresses including ultraviolet irradiation (UV) (10), heme-mediated renal and vascular injury (11,12), endotoxin (13), hyperoxia (14–16), hypoxia (17), cerebral ischemia (18), and cardiac as well as liver ischemia/reperfusion injury (19,20). Further insights into the functional role of HO1 can be gleaned from the HO1 (⫺/⫺) null mice, which exhibit poor tolerance to oxidative stress (hydrogen peroxide,

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Table 1 Summary of Heme Oxygenase Isozymes HO1

HO2

HO3

Localization

Ubiquitous

Brain and testes

Inducibility Molecular mass (kDa) Number of transcripts Known inducers

High ⬃32–33

Constitutive ⬃36

Ubiquitous, highest in liver, prostate, and kidneys Constitutive ⬃32–33

1 Heme/metalloporphyrins Transition metals Ultraviolet light (UV) Lipopolysaccharides (LPS) Hyperoxia Hypoxia Electrophiles Chemotherapeutic agents Phorbol esters Hydrogen peroxide Sodium arsenite Thiol scavengers Heat shock Prostaglandins Inflammatory cytokines Virus

2 Corticosterone

1 ?

hemin, and endotoxin), chronic inflammation, anemia, and vascular disease (21). More important perhaps, a similar phenotype is also found in human HO1 deficiency, with severe growth retardation, anemia, and extreme sensitivity to oxidants (22). Despite the pleiotropic nature of HO1, a prevailing theme appears to be the induction of HO1 with and protection against oxidant stress. In addition, the potent antioxidant properties of bilirubin and biliverdin (23,24) and the emerging roles of CO (see Chap. 6), all by-products of HO catalysis, further strengthen the hypothesis that HO1 induction is an adaptive response to oxidative stress. Of particular interest, and the main focus of the remainder of the chapter, is the role of HO1 in oxidant-mediated lung injury. II. HO1 and Lung Disease A. Acute Lung Injury Introduction

Oxidative stress, namely, reactive oxygen species (ROS), is a primary mediator of important disease processes including carcinogenesis, atherosclerosis, neuro-

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degenerative disorders, and inflammation. As it is elaborated in detail by Davis et al. in this volume (see Chap. 7), excessive ROS can cause oxidation of nucleic acids, proteins, and lipids. The lungs are a major target for oxidative injury propagated by endogenous ROS generated from the respiratory burst activity of inflammatory cells in a variety of acute and chronic lung diseases or by exogenous oxidants such as supplemental oxygen, environmental pollutants, drugs, and cigarette smoke. ROS play a vital role in inflammatory lung diseases such as adult respiratory distress syndrome (ARDS), fibrosis, asthma, and emphysema (25,26). In addition, patients with ARDS and emphysema are exposed to the additional oxidant burden of supplemental oxygen therapy, which may contribute to further lung injury and eventual respiratory demise. However, aerobic organisms have evolved complex mechanisms to protect against injurious ROS, such as the well-established antioxidant enzyme system (AOE) consisting of manganese superoxide dismutase, copper-zinc oxide dismutase, catalase, and glutathione peroxidase. These enzymes, whose primary function is to scavenge ROS, attenuate the “pro-oxidant” state of the cell and thereby confer cellular homeostasis. It is becoming apparent that other stress-response genes are important in regulating and maintaining cellular viability in the face of oxidants. Heat-shock proteins (HSP), metallothioneins, and transcriptional factors (c-Fos, c-Jun, Egr-1, NF-κB, STAT) likely play critical roles in the early events leading to cellular responses to oxidative stress (27–29). Despite significant progress in delineating the complex regulatory and functional elements involved in oxidative lung injury, a better understanding of the lung’s response to oxidative stress at the molecular level is crucial to understanding the pathogenesis of lung diseases. Role of HO1 in Hyperoxic Lung Injury Regulation of HO1 in Hyperoxic Lung Injury

Hyperoxia (⬎95% O 2) administered to rats produces pathophysiological changes similar to those seen in ARDS and is a well-established model to study oxidantinduced lung injury (14,30). In the rat model of hyperoxic lung injury there is significant lung edema, inflammation, and pleural effusions by 48–60 hr of continuous hyperoxia, with death by 72 hr. Lee et al. have observed increased steadystate HO1 mRNA levels in rat lungs after 48 hr and 64 hr (13-fold) of hyperoxia when compared to normoxic control rats (14). Increased HO1 protein and activity levels accompany this increase in HO1 mRNA. Interestingly, the regulation of HO1 expression is quite different from that of other AOEs. Studies by Clerch and Massaro show that mRNA induction of the antioxidant manganese superoxide dismutase after hyperoxia is not correlated with increased enzyme activity, while the antioxidants copper-zinc superoxide dismutase, catalase, and glutathione peroxidase have no change in mRNA levels


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with a decrease or no change in enzyme activity after hyperoxia (30). This differential regulation of various antioxidant enzymes points to the complex link between gene regulation and expression and underscores the need to explore the specific regulatory mechanisms involved in lung injury models. Of note, there are likely age-related differences in the regulation of HO1 mRNA, given that neonatal rat lungs, as opposed to adult rats, exposed to hyperoxia appear to invoke posttranscriptional mechanisms (31). Immunohistochemical studies of the adult rat lung after hyperoxia showed increased HO1 expression in a variety of cell types, including the bronchoalveolar epithelium, interstitium, and inflammatory cells (14). We confirmed HO1 mRNA and protein induction after hyperoxia in a variety of cell lines such as lung fibroblasts (MRC5, WI-38), pulmonary epithelial cells (A549), peritoneal (RAW 264.7) and alveolar macrophages (MHS, N3838), endothelial cells (2F2B), and primary rat aortic vascular smooth muscle cells (14). We confirmed that the increased HO1 mRNA expression is wholly dependent on increased HO1 gene transcription and not on increased mRNA stability (14). Function of HO1 in Hyperoxic Lung Injury

HO1 upregulation likely confers protection against hyperoxia. Pulmonary alveolar epithelial (A549) cells stably transfected with HO1 complementary DNA (cDNA) demonstrated increased basal levels of HO1 mRNA and enzyme activity (32). These A549 HO1 overexpressors exhibited marked resistance to hyperoxic injury which was reversed by inhibiting HO1 activity with tin protoporphyrin (SnPP), a selective HO1 inhibitor (32). In addition, the HO1 overexpressors had a marked decrease in cell growth that is detailed elsewhere in this chapter. We speculate that HO1 overexpression in pulmonary epithelial cells results in cell growth arrest, which may facilitate cellular protection against hyperoxic insult. Dennery et al. also observed that increased HO1 expression is associated with resistance to hyperoxia and that HO1 likely plays a protective against hyperoxic damage (33). In vivo studies of HO1 in hyperoxic lung injury also support the hypothesis that HO1 is part of a cytoprotective armamentarium. Exogenous administration of HO1 by gene transfer with an adenoviral vector conferred protection in rats exposed to hyperoxia (34). A fragment of the rat HO1 cDNA clone containing the entire coding region was cloned into plasmid pAC-CMVpLpA, and recombinant adenoviruses containing the rat HO1 cDNA fragment Ad5-HO1 were generated by homologous recombination. Increased HO1 protein expression in the bronchiolar epithelium of rats receiving Ad5-HO1 was confirmed by immunohistochemistry (34). Rats that received Ad5-HO1 exhibited marked reduction of pleural effusions (⬎90%), lung edema, hemorrhage, and inflammation after continuous hyperoxia (34). In addition, rats that received Ad5-HO1 showed increased survival in hyperoxic stress when compared to rats receiving control vector (34).

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Taylor et al. also showed that, in vivo, HO1 induction was protective against hyperoxic lung injury (16). Role of HO1 in LPS-Induced Lung Injury Regulation of HO1 in LPS-Induced Lung Injury

Similar to hyperoxia, LPS when administered in vivo produces pathophysiological changes similar to those seen in human ARDS. ROS are thought to mediate, at least in part, LPS-induced lung injury. Camhi and colleagues demonstrated that LPS induces ROS production, which in turn likely transduces signals to activate AP-1 and subsequent HO1 gene transcription (35). LPS causes an initial rise in HO1 steady-state mRNA by 2–4 hr, which peaks at 20 hr and returns to basal levels by 40 hr of LPS treatment in macrophages (36). This HO1 mRNA induction correlates with increased HO1 protein and activity levels. Immunohistochemical staining of rat lungs after LPS treatment shows patterns similar to those in rats exposed to hyperoxia, with particular abundance in alveolar and bronchiolar epithelium and inflammatory cells (36). Function of HO1 in LPS-Induced Lung Injury

Otterbein et al. have shown that HO1 induction in rats plays a key role in protecting against LPS-mediated shock and tissue injury (13). LPS administration in rats produces high levels of HO1 mRNA and enzyme activity (13). Pretreatment of rats with hemoglobin (Hb), a potent inducer of HO1, caused HO1 induction and, more important, provided complete protection against subsequent lethal endotoxemia (13). Hb-treated rats maintained normal mean arterial blood pressures, while control rats experienced complete cardiovascular collapse after a lethal dose of LPS. Hepatic and renal functions, peripheral WBC counts, serum lactate dehydrogenase, and phosphate levels also remained normal after LPS in Hb-treated rats. Additionally, Hb pretreatment caused a marked attenuation (⬎90%) of LPS-induced alveolitis and tumor necrosis factor-α (TNF-α) levels (⬎40%) (37). SnPP, a selective inhibitor of HO, blocked the protective effects of Hb and rendered the rats more susceptible to LPS (13). Role of HO1 in Apoptosis

Apoptosis, or programmed cell death, is increasingly being recognized as an important feature in hyperoxic lung injury models in animals and cells as well as in human ARDS (37–39). Polunovsky et al. demonstrated that fibroblasts and endothelial cells from human ARDS lungs undergo apoptosis (39) and, more recently, pro-apoptotic signals have been detected in the bronchoalveolar lavage fluid of ARDS patients associated with a poor outcome (40). Apoptosis, initially discovered as a crucial component of normal embryological development, is an active form of cell death characterized by nuclear condensation, DNA fragmenta-


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tion, cellular involution and, unlike necrosis, preserves tissue architecture with minimal inflammatory responses (41). ROS have an emerging role as a key modulator of apoptosis and intriguingly, one of the most potent anti-apoptotic genes, bcl-2, has antioxidant properties (42–44). In vivo studies show that hyperoxic stress induces apoptosis in the lungs of mice and rats (45,46). The induction of HO1 in the lungs via exogenous HO1 gene transfer resulted in increased survival to lethal hyperoxia, which correlated with attenuation of oxidant-induced apoptosis (34). The protective effects of exogenous HO1 were duplicated by CO administration (see Chap. 6). In vitro, Petrache et al. have shown that hyperoxia exposure can induce apoptosis, as assessed by DNA laddering, terminal deoxynucleotidyltransferase dUTP nick end labeling, and nucleosomal assays, in murine macrophage cells (RAW 264.7) (47). HO1 induction can protect fibroblasts and endothelial cells from TNF-α-induced apoptosis (48,49). Foresti et al. showed that HO1 induction was associated with a substantial decrease in peroxynitrite-mediated apoptosis in bovine aortic endothelial cells (50). The role of apoptosis in lung injury is undoubtedly fraught with complexities and much is yet unknown, but there is convincing evidence that HO1 is playing an integral role in oxidant-induced apoptosis. B. Roles for HO1 Other Than as an “Antioxidant” and Implications for Other Lung Diseases

The marked induction of HO1 and its cytoprotective effects in two prototypic models of oxidant lung injury, LPS and hyperoxia, are clear from the data thus far. However, it is also clear from the data that the HO1 effects extend beyond its role as an “anti-oxidant” and likely include basic biological processes such as modulating apoptosis, inflammation, and cell growth. This fact is not surprising given the diversity of HO1 inducers, the complex and varied regulatory mechanisms involved in HO1 gene activation, and the multiple by-products generated during HO catalysis, each possessing unique functional characteristics. It is beyond the scope of the current chapter to detail all the biological effects of HO and its by-products. However, we will attempt to highlight some intriguing features of HO and its potential implications for a variety of lung diseases in the remainder of the chapter. Inflammation

There is evidence that HO1 induction modulates the inflammatory response. Both the HO1 (⫺/⫺) null mice and HO1-deficient patient exhibit, among other abnormalities, increased inflammatory profiles including leukocytosis and thrombocytosis (21,22). There was suggestion of anti-inflammatory effects of endogenous and exogenous induction of HO1 in rat models of LPS and hyperoxic injury. Otterbein et al. found that Hb induction of HO1 not only increased survival

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against lethal endotoxemia but also decreased LPS-induced alveolitis and TNFα levels (37). Exogenous administration of Ad5-HO1 also significantly reduced hyperoxia-mediated neutrophil influx, which was associated with a survival benefit (34). HO1 also exerted potent anti-inflammatory effects in an in vivo heart xenotransplant model and completely prevented transplant rejection (51). Allergic Airway Disease

In an ova-albumin model of allergic airway disease, Chapman et al. demonstrated that CO (presumably via HO) attenuates eosinophilic infiltration (52), which may have some applications to human allergen-induced airways disease. A role of HO in human airways disease is highly likely, given that Barnes et al. have detected increased levels of CO in the breath of a variety of lung diseases, including asthma and cystic fibrosis (53,54). Investigators have found a role for HO (especially HO2) via CO as a modulator of synaptic neurotransmission in the lung and airways and a regulator of airway tone (55,56). The details of CO and lung disease are discussed in a subsequent chapter. Proliferation and Cell Cycle

The potential role for HO1 in cell growth has been alluded to by the HO1-overexpressing lung epithelial cells, which demonstrated striking growth arrest in conjunction with increased survival in hyperoxic stress compared to wild-type cells or cells transfected with control DNA (32). The decreased cell proliferation was associated with an increase in the number of cells in G0/G1 phase during the proliferative exponential phase and decreased entry into the S phase as determined by flow cytometric analysis (32). Furthermore, the A549 HO1 overexpressors accumulated at the G2/M phase and failed to progress through the cell cycle with serum stimulation, in contrast to the A549 cells transfected with control DNA. HO1 inhibition with SnPP not only reversed the survival benefits of HO1 overexpression in hyperoxia but also reversed the growth arrest (32). The growth arrest may confer the protective effects of HO1 in epithelial cells exposed to hyperoxic stress, given that inhibiting HO1 reversed the growth retardation as well as the survival benefits of HO1 overexpression (32). The antiproliferative effects of HO1 may also extend into vascular cell types. Morita and Kourembanas (57) have shown in a co-culture system that vascular smooth muscle-derived CO via HO1 exerts a paracrine effect on endothelial cells as it increased endothelial cell cGMP and decreased expression of mitogens such as endothelin-1 and plate-derived growth factor. With adenoviral transfer of HO1 to pulmonary artery smooth muscle cells, there was a decrease in cell growth (58). The antiproliferative effects of HO1 have interesting implications for the role of HO1 in inhibiting hypoxia-induced vascular smooth muscle hypertrophy, which is one of the proposed mechanisms of secondary pulmonary


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hypertension. The anti-apoptotic effects of HO1 induction have already been mentioned above and are further detailed in Chapter 6. III. Mechanisms of HO1 In the face of ample evidence that HO plays an important role in oxidant lung, the question remains as to the precise mechanisms responsible for the actions of HO1. Further insight may be gained by examining the various by-products generated by HO catalysis. A. Iron and Ferritin

HO initiates the release of free iron during the first step of heme degradation and iron is then sequestered into its storage form, ferritin (Fig. 1). The release of free iron and subsequent induction of ferritin poses an interesting dichotomy in understanding the protective effects of HO. On the one hand, free iron can be damaging in its ability to generate the toxic hydroxyl and superoxide radicals via the Fenton and Haber-Weiss reactions. There is also evidence that reduced iron uptake can prevent oxidative stress (59) and that iron in conjunction with hydrogen peroxide can cleave DNA (60). However, on the other hand, HO activity and iron release upregulate ferritin, which has been shown to have cytoprotective effects, either through its iron-binding ability or other, yet uncharacterized, antioxidant effects. The HO-mediated protection against UV irradiation in skin fibroblasts is dependent on ferritin synthesis, as inhibition of ferritin with desferoxamine also abolished the protective effect (61). In cultured endothelial cells, the iron sequestering ability of ferritin appears to be responsible for protection against oxidant-mediated cytolysis (62). Nath et al. also implicated ferritin in HO1-mediated protection during glycerol-induced rhabdomyolysis and renal failure in rats (11). There is suggestion that HO1 and ferritin induction enhance the chemoprotective effects of anticarcinogens (63). However, the beneficial role of HO in LPS-induced injury does not appear to involve ferritin (37). B. Bilirubin/Biliverdin

Bilirubin and biliverdin, metabolites of heme degradation, are also possible candidates for the antioxidant properties of HO. Both are potent radical scavengers, with bilirubin having the ability to scavenge peroxyl radical as efficiently as αtocopherol, the most potent antioxidant of lipid peroxidation (23,24). Stocker et al. demonstrated that bilirubin in micromolar concentrations in vitro is an effective radical scavenger (24). Although the function of biliverdin in mammals is yet unclear, birds, amphibians, and reptiles excrete biliverdin directly and, thus, biliverdin could also act as a hydrophilic antioxidant (24). In neuronal cells, bili-

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rubin, in nanomolar concentrations, is protective against oxidative injury (64). In rat liver ischemia-reperfusion injury, the protective effects of HO are postulated to be through bilirubin (65). C. Carbon Monoxide

Heme degradation via HO is the predominant mode of physiological CO generation. CO, initially thought to be a “waste” product and toxic, is now becoming recognized as an important modulator of vascular tone and cellular messenger (66,67). Analogous to nitric oxide (NO), CO upregulates cGMP via guanylate cyclase, which causes smooth muscle relaxation and platelet aggregation (68). This vasodilatory effect may be important for maintaining tissue perfusion in the lungs during normal and hypoxic states. Lee et al. have shown that aortic vascular smooth muscle cells express high levels of HO1 after hypoxia (69). Increased levels of cGMP after hypoxia require HO activity without any dependence on NO (68). Further details into the potential functions of CO are discussed in Chapter 6. IV. HO2 and Lung Disease HO2 has been detected in canine and rodent GI tracts and human myometrium and is thought to regulate vascular tone and smooth muscle contractility via the effects of CO, a potent vasodilator, smooth muscle relaxant, and putative neural messenger (67,70–72). Further evidence that the functional effects of HO2 appears to be linked to its upregulation of CO is seen in the HO2-null mice, which exhibit various neurophysiological abnormalities (73,74). Little is known about the role that HO2 plays in lung disease. However, when HO2-null mice are exposed to hyperoxia, they exhibit increased oxygen toxicity (75). This increased susceptibility of the HO2-null mice to hyperoxia was postulated to be due to iron accumulation in the lung and supports the hypothesis that HO2 is necessary for iron homeostasis in the lungs. However, there are recent reports of alternative transcripts of HO2 (76) and that HO2 binds heme through an additional heme regulatory motif that is not involved in heme catalysis (77). Therefore, HO2 may have diverse regulatory and functional elements that have yet to be identified. V.

Conclusions and Future Directions

It is clear from the work of many investigators that HO is an integral part of not only physiological, homeostatic balance but also the protective armamentarium against cellular stress. The potentially potent antioxidant properties of HO1 are evident in the various pro-oxidant models used, such as UV irradiation, heme, metals, LPS/endotoxin, and hyperoxia, to name just a few, in which HO induction


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protects against injury and death. We have gained many insights into the regulatory mechanisms and protective effects of HO1 induction in lung disease, specifically acute lung injury, but the potential for extending the studies to other lung diseases such as chronic obstructive disease, asthma, pulmonary hypertension, and cancer is immense. If significant progress is to be made in the understanding and application of HO, further study into the precise molecular mechanisms involved is also critical. The biologically active and versatile by-products of HO activity, ferritin, bilirubin, biliverdin, and CO, are also crucial components of HO and merit intense investigation in order to elucidate the complex roles of HO. There is no doubt that controversy exists as to the degree of beneficial and deleterious effects of HO and its by-products, but this only underscores the importance of further investigations. References 1. Tenhunen R, Marver HS, Schmid R. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc Natl Acad Sci USA 1968; 61:748–755. 2. Cruse I, Maines MD. Evidence suggesting that the two forms of heme oxygenase are products of different genes. J Biol Chem 1988; 263:3348–3353. 3. McCoubrey WKJ, Huang TJ, Maines MD. Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3. Eur J Biochem 1997; 247:725–732. 4. Maines MD, Trakshel GM, Kutty RK. Characterization of two constitutive forms of rat liver microsomal heme oxygenase. Only one molecular species of the enzyme is inducible. J Biol Chem 1986; 261:1323–1328. 5. Maines MD. Heme oxygenase: function, multiplicity, regulatory mechanisms, and clinical applications. FASEB J 1988; 2:2557–2568. 6. Davis SJ. The Arabidopsis thaliana HY1 locus, required for phytochrome-chromophore biosynthesis, encodes a protein related to heme oxygenases. Proc Natl Acad Sci USA 1999; 96:6541–6546. 7. Cornejo J, Willows RD, Beale SI. Phytobilin biosynthesis: cloning and expression of a gene encoding soluble ferredoxin-dependent heme oxygenase from Synechocystis sp. PCC 6803. Plant J 1998; 15:99–107. 8. Richaud C, Zabulon G. The heme oxygenase gene (pbsA) in the red alga Rhodella violacea is discontinuous and transcriptionally activated during iron limitation. Proc Natl Acad Sci USA 1997; 94:11736–11741. 9. Wilks A, Schmitt MP. Expression and characterization of a heme oxygenase (Hmu O) from Corynebacterium diphtheriae. Iron acquisition requires oxidative cleavage of the heme macrocycle. J Biol Chem 1998; 273:837–841. 10. Vile GF, Basu-Modak S, Waltner C, Tyrrell RM. Heme oxygenase 1 mediates an adaptive response to oxidative stress in human skin fibroblasts. Proc Natl Acad Sci USA 1994; 91:2607–2610. 11. Nath DA, Balla G, Vercelotti GM, Balla J, Jacob HS, Levitt MD, Rosenberg ME. Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat. J Clin Invest 1992; 90:267–270.

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6 Carbon Monoxide A Gaseous Molecule with Anti-Inflammatory Properties

LEO E. OTTERBEIN, DANIELLE MORSE, and AUGUSTINE M. K. CHOI University of Pittsburgh Medical Center Pittsburgh, Pennsylvania, U.S.A.

JEFFREY T. CHAPMAN The Cleveland Clinic Foundation Cleveland, Ohio, U.S.A.

I.

Historical Perspective

The first detection of a combustible gas in the blood was made in 1894 by Haldane (1). This gas was supposed by de Saint Martin and Nicloux to be carbon monoxide (2,3). Nicloux and others attempted to show that CO was formed in the body and first asserted that the origins of CO in the body arose via carbohydrate metabolism (4). The proof should have been the determination of the CO in inspired and expired air simultaneously, combined with measurement of carboxyhemoglobin (COHb) in the blood. Unfortunately, these measurements were not possible with the methods available before 1940. With the onset of the industrial revolution and the invention of the combustion engine, it became urgent to work out methods to measure and determine COHb because it was rapidly discerned that CO levels in the atmosphere were dangerous. It was discovered very early that the COHb levels in the blood reflected the CO concentration in the alveolar air determined using rebreathing techniques. It was not until 1949, however, that Sjorstrand and later Coburn discovered that endogenously produced CO arose from the degradation of hemoglobin released from senescing erythrocytes. CO measurements via COHb or by rebreathing techniques in the 1970s were used by clinicians to deter133

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mine the life span of erythrocytes and the rate of heme turnover in their patients. Greater than 75% of CO produced in normal humans arises from erythrocyte turnover generated as a by-product of heme metabolism. In 1969 the source of endogenous CO was discovered. Tenhunen et al. described and characterized heme oxygenase as the enzyme responsible for breaking down heme in the body, demonstrating that heme catalysis resulted in the subsequent release of CO and free iron as by-products. This enzymatic cleavage also resulted in the production of biliverdin, which was subsequently found to be rapidly converted to bilirubin via biliverdin reductase. Certainly the products of heme oxygenase (HO) activity have been observed for decades if not centuries because, unlike most biochemical functions, heme catalysis is colorcoded and readily observable. For instance, a hematoma arising from a blow to the skin is initially black—the color of heme. Over a number of days, the color changes to green—the color of biliverdin—and finally to yellow—the color of bilirubin. Employment of these visual observations can be dated back to the times of Hippocrates, when patients with liver disease presented as hyperbilirubinemic and were recognized because their skin was yellow in color. Similarly, the presence of CO was observable well before there were scientific “instruments” by which to test the atmosphere. With the advent of fire, it is not hard to imagine that primitive people, taking refuge in caves, brought their fire inside and learned rapidly that when some co-dwellers did not survive the night they should next time be sure to extinguish the flames, lest they not awaken to greet the next day. Perhaps they recognized that if they began to turn bright red (from CO binding to hemoglobin), it was time to get outside for some fresh air. And thus the first CO monitor and/or spectrophotometric assay was created (adapted from Penney et al.) (5). While the literature citings over the past century are in heavy favor of CO being only a molecule possessing toxic properties (6,7), it is becoming evident that CO also possesses both physiological and pharmacological properties, including modulation and regulation of vasomotor tone and neurotransmission in the nervous system (8–10). In the brain, regulation of cerebral blood flow is regulated by CO during episodes of epileptic seizures (11). In the central nervous system, CO via HO has also been shown to be involved in neuroendocrine function. It has been reported that CO inhibits the release of hormones such as corticotropin-releasing hormone, arginine vasopressin, and oxytocin while simultaneously stimulating leutinizing hormone-releasing hormone, suggesting that CO may be generated to prevent overexuberant activation of the hypothalamopituitary-adrenal axis (HPA) and inhibition of reproductive processes during stress (12). This has been further supported by Grossman et al., who demonstrated that CO generation in the hypothalamus is able to stimulate cyclooxygenase, producing prostaglandin E2, which is an intermediary in the activation of the HPA (13). Zhou et al. have shown that CO generated in the hippocampus leads to

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long-term potentiation or memory (14). Prabhakar suggested that endogenously generated CO can regulate the ventilatory response to hypoxia by its action on carotid bodies, and perhaps brainstem neurons (15). Exposure to CO has been shown to inhibit hypoxic pulmonary vasoconstriction (HPV) (16). It has been hypothesized that CO generated from the increased activity of HO1 is important in modulation of vascular smooth muscle tone (17,18) and critical in the regulation of blood pressure under stress conditions (19,20). The functional role of the HO1/CO axis has also been demonstrated in other tissue and organ systems, including the liver, where it is involved in the regulation of bile canalicular contractility in hepatocytes, uterine contractions in the pregnant rat, and leukocyte adhesion in venular endothelial cells. Another system where the HO/CO pathway constitutes a regulatory role is in the pancreatic islets of Langerhans, where CO has been shown to stimulate glucagon and insulin release (21–23). The interest in the functional role of this metabolite of heme catalysis is continuing to expand, with ever-increasing data suggesting that, akin to nitric oxide (NO), CO possesses cellular and molecular functions and should not be simply classified as a toxic waste product. Heme oxygenase has been well established as a stress response gene induced by a diverse array of non-heme-induced cellular stresses, including lipopolysaccharide, heat shock, hyperoxia, heavy metals, and UV irradiation. It was hypothesized, based on the premise that HO1 was induced by such stress responses, that CO as a by-product would be generated simultaneously and could be quantitated. Because CO does not undergo further metabolism in the body, its only source of excretion is via exhalation as a gas. The laboratories of Sazaki and Barnes have clearly shown that not only is CO present and measurable in the exhaled air of a normal subject, levels of this gaseous molecule increase correlatively with the severity of numerous inflammatory disease states (24–27). In inflammatory conditions such as asthma, medical intervention such as the use of inhaled glucocorticoids was shown to reduce bronchoconstriction with subsequent correlative decreases in the amount of exhaled CO measured in the breath (28). Exhaled CO may also provide complementary data for assessment of asthma control in children, where exhaled CO has also been shown to increase during an episode of acute asthma (29). Patients with upper respiratory infections have increased levels of CO in the exhaled breath, suggesting that perhaps HO1 possesses an antiviral effect in the airway (30). Because of the noninvasiveness of exhaled breath collection, it is being proposed that increased CO levels may be an early and noninvasive marker of airway and systemic inflammation and disease (31–33). It has been demonstrated that CO modulates histamine release from mast cells in response to allergen as well as the immunological activation of human basophils (34–36). In cases of cystic fibrosis, CO measurements may be clinically useful in the management and monitoring of oxidation and inflammation-mediated lung injury (37). It has been demonstrated that the generation of

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CO directly affects cardiac output, vascular resistance and subsequently blood pressure (38). It has also become evident that CO is involved in pathological conditions including ischemia, endotoxic shock/sepsis, and excitotoxicity, acting as a cytoprotective agent (39,40). Accumulating evidence demonstrates that CO is responsible for numerous cellular and organ functions, certainly more than delineating the toxicity and lethality of CO exposure (41–44). Over the past hundred years CO has demonstrated its lethality again and again. In rigorous examinations in scientific laboratories over the span of a century, its deadly effects have been repeatedly and compellingly demonstrated (45). Yet there are also numerous inconsistencies throughout these research studies. In an extensive review of the literature regarding CO effects on behavior and brain injury, Benignus concludes: Barring the advent of a very well documented and replicable demonstration of COHb with behavioral effects, a physiological understanding of the mechanism of CO effects appears to be the only hope of resolution of the uncertainty of the effects of CO exposure. Too little is known about the effects on average, normal, young, healthy, at rest subjects and much less about cases that do not fit into categories of toxicity and yet are exposed to CO in their normal environment (46).

Benignus goes on to state that “there is reason to believe that research done in the climate of environmental activism during which much of the CO research was performed, an experiment that produced no significant effect when others had already reported such would not have been published. . . . [I]f this were the case and many findings were never published because of no statistical effects, a review of the literature could easily take on a different appearance.” From these conclusions, it could be possible that perhaps CO has been labeled as toxic regardless of concentration and exposure time. Ironically, while CO has been extensively studied for over a century, the literature citings are a mere fraction of what they might otherwise be and it is clearly possible that critical data supporting other “nontoxic” effects of CO may never be known. And so, despite its demonstrable life-ending effects, CO in more recent studies appears to be a player in an innumerable number of life-sustaining organ and cellular activities as described above, dependable, ubiquitous, and inexplicable, a role that has both bemused and dismayed scientists. In the late nineteenth century, Haldane used CO to discover that hemoglobin is the blood protein that carries and delivers oxygen to the tissues (47). And so it is that CO’s ubiquity and reliability are suggestive of something organs or cells require. On the other hand, of course, its toxicity, its lethality, suggests “No,” and demands the opposite inference—that it is, like some virus or bacterium, a malignant, absolutely dispensable, yet stubbornly resilient aberration in the otherwise functional organization of organ and cellular systems. Perhaps it is this enigmatic nature of CO that


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has prompted some investigators, in recent research efforts, to begin to pay more attention to CO, if for no other reason than to explain the mechanisms of its toxicity and, perhaps, to turn its aberrant behavior into something comprehensible. CO insists on being attended to—and understood and appreciated. But that explication requires a context, one that provides the conceptual framework within which the mechanisms of CO’s beneficial effects may be most precisely described. Needless to say, such contextualization necessarily entails more narrowly focused investigations, ones centered on specific well-understood organ/tissue systems, the particular functional features of which can be reasonably extrapolated to other systems. To such contextualization, the following is addressed. Since the late nineteenth century, scientists have known that CO is present in the blood of normal humans and animals. Claude Bernard described carbon monoxide’s affinity for hemoglobin; this initial research became grounds for an essentially universal scientific tenet assigning CO to the category of poisons and toxins, which later included arsenic, nicotine, and opium (48,49). Prior to this time, it was common practice in Europe to pacify infants by holding them over a fire, where COinduced cerebral anoxia could exert its sedating and soothing effects; the fire, of course, provided its own warming and calming influences. In 1968, the source of endogenous CO was discovered through the characterization of the existence of a microsomal enzyme, heme oxygenase, that generated CO via the catabolism of heme. Since this seminal finding, investigations have steadily increased for the purpose of understanding this enzyme, not only as the rate-limiting step in the anabolism of heme, but more recently its involvement in the oxidative stress response.

II. Heme Oxygenase Heme oxygenase, originally defined by Tenhunen, Schmid (50), and others, catalyzes the first and rate-limiting step in the degradation of heme. Via oxidation, HO1 cleaves the b-meso carbon bridge of b-type heme molecules to yield equimolar quantities of biliverdin IXa, carbon monoxide, and free iron. Biliverdin is subsequently converted to bilirubin via the action of biliverdin reductase (Fig. 1). Under physiological conditions, HO activity is greatest in the spleen, where senescent erythrocytes are sequestered and destroyed, but its activity has been observed in all systemic organs. Although heme is the typical HO1 inducer, studies in the late 1980s by Tyrell et al. demonstrated for the first time that HO1 activity could also be stimulated by a variety of nonheme products, including endotoxin, heavy metals, and oxidants such as hydrogen peroxide (51,52). Though these other agents induce HO1, studies indicate that they are unrelated to heme metabolism (53). Common to all these inducers, however, is their capac-

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Figure 1 Heme catabolism pathway.

ity to generate reactive oxygen species (ROS). Thus, these studies demonstrate not only that HO1 functions as an antioxidant, but also that it must exercise a cytoprotective function in cells and tissues exposed to such agents. With the discovery of the enzyme heme oxygenase, however, the sole origin of endogenously generated CO was identified. Heme oxygenase (HO) has since been extensively studied; three different isoforms having subsequently been isolated and identified as HO1, HO2, and HO3. HO2 and HO3 are constitutively expressed primarily in the brain and testes, with HO1 being the only inducible isoform. Many investigators have observed that HO1, earlier thought to be inducible only in the presence of heme, was induced by numerous non-heme-containing agents, including heavy metals, endotoxin, UV irradiation, hypoxia, and hyperoxia, to name a few of an ever-increasing number of agents (54). These landmark findings have sparked increased interest in the investigations of this enzyme, and recently—and perhaps more so—the by-products of its activity. Most interesting, however, is that numerous laboratories have found that if this enzyme is induced prior to an oxidative stress such as hyperoxia, endotoxin, and UV irradiation, the cell and animal are protected from the ensuing insult (55). While these revelations have indeed provoked much research, the mechanism by which HO1 provides protection against injury remains unanswered. In an effort to answer this question, it was hypothesized that carbon monoxide was subserving a unique functional role in the mechanisms that generate the effects seen with induction of HO1 itself. The investigation of CO’s functions in acute lung pathology, including both endogenous and exogenous determinants, is being investigated in models of sepsis, asthma, xenotransplantation, and hyperoxia, all well-established models of acute inflammation and injury. Additionally, studies have demonstrated that, far from being “waste” byproducts of heme metabolism, bilirubin, ferritin, and CO are perhaps products serving a function. In fact, bilirubin, ferritin, and CO are all now accepted as being able to exercise other critical physiological functions.


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III. Bilirubin Bilirubin, for example, has been shown to be a potent antioxidant in the brain, acting to scavenge peroxyl radicals as efficiently as α-tocopherol or vitamin E (56). Bilirubin is the most abundant endogenous antioxidant in mammalian tissues, accounting for the majority of the antioxidant activity of human serum (57). Bilirubin is best known, however, as a potentially toxic agent that accumulates in the serum of neonates, causing jaundice. In high concentrations it deposits in selected brain regions to elicit neurotoxicity associated with kinicterus (58). It is conceivable, however, that neonatal jaundice could also have a protective effect for the infant arriving for the first time into an unsterile environment. Clark et al. have recently shown that bilirubin administration to ischemic heart tissue can provide cytoprotection from postischemic myocardial dysfunction (59).

IV. Ferritin The release of free iron—its two free electrons being capable of generating the vicious hydroxyl radical—through Fenton chemistry with the superoxide radical, is rapidly sequestered into the iron storage protein ferritin (Fig. 1). Such sequestration can itself lower the pro-oxidant state of the cell by removing the free iron (60). Vile and Tyrell showed that ferritin levels increase in the presence of oxidative stress such as UV irradiation (61). This HO1-dependent release of iron also results in upregulation of ferritin, which might provide protection, following irradiation. It is difficult to understand how ferritin can be upregulated fast enough to obviate a rise in free iron which, itself, is a likely candidate for the initial signal for upregulation. In a model of endotoxic shock, when the iron is chelated by the exogenous iron chelator desferoxamine, there is no ferritin induced, and protection is ablated (62). By administering hemoglobin intravenously in the rat kidney, Nath et al., in 1992, showed for the first time that induction of HO1 in vivo protected against oxidant-induced renal failure in rats (63). Using a rat model of rhabdomyolysis, they administered glycerol, which resulted in acute renal failure and eventual death. By administering hemoglobin prior to glycerol injection, renal failure was prevented. Furthermore, when they administered a selective inhibitor of HO1, tin protoporphyrin (Sn-PP), they observed not only that the protective effects of hemoglobin were reversed, but also that in rats receiving Sn-PP only, injuries were exacerbated. In a cardiac xenotransplantation model, Soares et al. show that HO1 confers resistance against cardiac graft rejection in rodents (64), further supporting the cytoprotective functions of HO1. Although the functional significance of HO1 induction is still not perfectly understood in any of these systems, compelling evidence indicates that its func-

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tion entails far more than a simple heme-metabolizing enzyme. Much more intriguing and compelling is the precise mechanism by which HO1 induction/ activity allows for these safeguarding effects. There is strong evidence in the literature to support the emerging paradigm that HO1 plays a vital role in maintaining cellular and tissue homeostasis in various in-vitro and in-vivo models of oxidant-induced injury. Despite these convincing data indicating the protective role of HO1 in oxidant stress, the precise mechanism(s) by which HO1 serves as a potent cytoprotectant remains elusive. While many investigators have speculated as described above that the catalytic by-products of heme catabolism, including bilirubin and ferritin, generated by released iron, may mediate the protective function of HO1 (56,65,66), the evidence remains unresolved. Carbon monoxide (CO), the remaining major end product of heme catalysis by HO, has been implicated as a chemical messenger in neuronal transmission and as a potent vasodilator. However, little is known regarding its functional role in potential cytoprotection against oxidative stress.

V.

Carbon Monoxide

Recent reports by a number of investigators are beginning to show a functional role for CO in models of inflammation. Ndisang et al. showed that CO can modulate the guinea pig mast cell release of histamine, supporting the idea that increases in exhaled CO such as during an asthmatic response is perhaps an effort by the lung to immunomodulate histamine release and subsequent bronchoconstriction. In rodent models of hemorrhagic shock and sepsis, CO has been shown to provide protection against hepatic dysfunction (67). To address the hypothesis that the by-product CO was potentially the mechanism of cytoprotection observed with HO1, in-vivo and in-vitro exposure systems were developed to administer low concentrations of CO exogenously. Subsequent studies unraveled an important function of exogenously administered CO in mammalian systems. Demonstrated in the following is evidence that CO, when administered exogenously at low concentrations, can exert potent anti-inflammatory effects in both in vivo and in vitro models of lipopolysaccharide (LPS)-induced inflammation, hyperoxic lung injury in rodents, allergeninduced inflammation, and cardiac xenotransplantation, which otherwise mimic effects seen with HO1. The concentration of CO (⬍0.03%) used in these studies is one-twentieth of the lethal dose of CO and significantly less than the concentration administered to humans (0.3%), albeit our exposures are continuous and ranged from 1 hr to 4 weeks of exposure. A novel and intriguing aspect of these observations is that CO, even at low concentrations, selectively modulated the pro-inflammatory/anti-inflammatory cascade of cytokines and mediators.


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A. CO and Endotoxic Shock

CO exposure selectively inhibited in a concentration-dependent fashion LPSinduced tumor necrosis factor-α (TNF-α) production and augmented interleukin-10 (IL-10) production measured in the same cells and mice. Surprisingly, these biological effects of CO did not involve the cGMP pathway, which has been implicated in the biological effects of CO in the neuronal and vascular systems, akin to nitric oxide (NO) (68,69). Rather, the anti-inflammatory effects of CO appear to involve the MAP kinase signaling pathway, in particular the p38 MAP kinase pathway. The precise biochemical mechanism by which CO can modulate the MAP kinases is not clear at this time. Based on current knowledge that none of the upstream kinases in the MAPK pathway contain a heme moiety (a common target of CO), and the current observation that CO mediates the anti-inflammatory effects via a cGMP-independent pathway, it is plausible that CO could be modulating the upsteam kinases through an unknown or unidentified intermediate molecule. This speculation is supported by observations that the protective effects of CO require new protein synthesis in TNF-α-induced apoptosis. Our observations demonstrating the CO-induced augmentation of IL-10 production and induction of the p38 MAP kinase are consistent with previous reports that the p38 MAP kinase is critical in regulating IL-10 production (70). There have been a number of reports showing that inhibition of TNF-α independent of augmented IL-10 release can occur following increases in cAMP, decreased intracellular Ca2⫹ or β-adrenoceptor stimulation (71–73). These studies suggest that CO inhibits LPS-induced TNF-α production via an IL-10-independent pathway, by affecting posttranscriptional synthesis of TNF-α. The data raised the immediate question as to how there could be augmentation in the activation of p38—a potent intermediate signal transducer involved in the production of TNF-α following LPS administration (74)—while simultaneously showing decreased production of TNF-α by ELISA. To address this fundamental question we looked at TNF-α mRNA and protein levels. Results showed that CO was regulating LPS-induced TNF-α production posttranscriptionally. Western blot analysis of both cellular protein and secreted protein in the media indicated decreased amounts of TNF-α protein in the presence of normal transcription of the TNF-α gene as seen by Northern blot analysis. Conclusions from these studies are that CO is acting directly on posttranscriptional processes, or a more intriguing possibility is that CO is somehow acting indirectly via the increased activation of p38 in the posttranscriptional regulation. A third possibility is that there exists a delicate balance in the actions of p38; subtle cellular activation (as with LPS alone) stimulates TNF-α synthesis, while hyperstimulation (as seen in the CO/ LPS-treated cells) somehow becomes inhibitory, resulting in posttranscriptional regulation of the TNF-α protein. Taken together, it appears that CO is exerting

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posttranscriptional or secretional regulation of LPS-induced TNF-α expression. The regulation of LPS-induced TNF-α expression by CO exemplifies the accumulating evidence in the literature highlighting the complexity of the molecular regulation of TNF-α (75). Complex transcriptional and translational control of TNF-α expression has also been reported in similar studies (76–78). Studies of mechanism and localization are included as studies to investigate in the future. This study revealed a novel physiological function of the gaseous molecule CO in a model of LPS-induced inflammation. B. CO and Cardiac Xenotransplantation in Rodents

The ability of HO1 and CO to suppress pro-inflammatory responses in macrophage activation may also contribute to the suppression of xenograft rejection, as demonstrated in a recent study by Sato et al. (78a). In this study, the authors demonstrate the beneficial effects of CO in avoiding rejection of a cardiac xenograft, which was similar to their previously published work showing that HO1 provides protection against acute rejection (79). Exposure to 400 ppm for only 2 days following transplant prevented rejection for up to 50 days, whereas air control hearts were rejected in 5–7 days. CO likely mediates these protective effects, in part by modulating platelet function, by causing vasodilatation, and/ or by inhibiting the pro-inflammatory responses of monocytes as described above. Activated monocytes are present in rejecting xenografts, and there are data suggesting that they may well play a role in rejection (80,81). Additionally, the antiinflammatory effects of CO explain how exogenous administration of HO1 by gene transfer or CO inhalation protected against hyperoxia-induced lung injury and inflammation as described below (82,83). Despite the long-standing accepted paradigm that CO in the environment is toxic or even lethal, the data presented here and in the Sato data make clear that CO can serve as a potent anti-inflammatory molecule. It is tempting to speculate that CO might be used therapeutically either by (over)expressing HO1, by inducing HO1 by genetic engineering, or by local CO administration in areas of inflammation. Carbon monoxide might therefore be used therapeutically not only in sepsis and xenotransplantation but in other inflammatory conditions as well. C. CO and Hyperoxia-Induced Lung Injury in Rats

In these studies it was hypothesized that a possible mechanism subserving HO1induced protection is one of its by-products—carbon monoxide (CO). Using low concentrations of CO previously shown to be nontoxic (84), rats were exposed to lethal hyperoxia in the presence of miniscule amounts of CO (similar to that used in the endotoxin and transplantation studies) to determine if protection was similar to that observed with HO1 when administered intratracheally expressed in an adenovirus (82). What was found was intriguing. Exogenous administration


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of low CO concentrations did indeed provide protection against oxidative stress in models of inflammation. It should be noted that the concentration of CO used in these studies, of the order of 50–500 ppm, corresponds to 0.005–0.05% CO, respectively. Since differences in arterial pO2 levels have been implicated in other models of tolerance to hyperoxia (85), the pO2 content was measured in the experimental groups. No difference was observed between rats exposed to hyperoxia and rats exposed to hyperoxia in the presence of a low concentration of CO. The precise mechanism(s) by which CO mediates protection in this model is not clear. The observation that CO attenuated hyperoxia-induced influx of neutrophils into the airways is interesting in that it is well established that neutrophil influx in the bronchoalveolar lavage is of paramount importance in the development of hyperoxia-induced lung injury in in-vivo models and in human patients with ARDS (86–88). Moreover, identical experiments were performed using a second model of oxidant-induced lung injury and inflammation. Lipopolysaccharide administered to rats induces profound neutrophil influx into the airways; however, this neutrophil influx was significantly inhibited in the lungs of rats given LPS and exposed to CO. Willis et al. recently reported that HO1 modulates the inflammatory response in vivo, and a recent report by Soares et al. also showed that HO1 modulates the inflammatory response and apoptosis in vivo in a model of cardiac xenotransplantation (79,89). These findings support a mechanism to explain the anti-inflammatory properties observed with HO1, which have been demonstrated in our laboratories and others (62,64,82,88). However, in this study the use of exogenous CO did not prove directly that it is mimicking endogenous CO, and therefore cannot be compared meaningfully to CO produced during heme metabolism by endogenous HO1. Designing experiment(s) to show that endogenous CO from HO1 actually mediates the protective effect of HO1 in vivo is technically very difficult, perhaps not feasible because current available technology to measure CO in vivo (COHb) is not sensitive enough to detect increased CO levels after HO1 induction. However, observations that exogenous CO could completely ablate or reverse the increased pleural effusion in rats treated concomitantly with a selective inhibitor of HO1, tin protoporphyrin (Sn-PP), suggested that exogenous CO could provide cytoprotective effects even in conditions where endogenous HO activity is completely inhibited. Nevertheless, the marked protection against hyperoxia-induced lung injury by exogenous CO at low concentrations observed here provides another suitable in-vivo model to investigate further the functional physiological role of CO in oxidant-induced lung injury. Furthermore, oxygen exposure results in an increase in programmed cell death or apoptosis in the lung as demonstrated by Mantell et al. (89). Exposure to hyperoxia in the presence of CO showed an inhibition of apoptosis, which may represent an additional mechanism by which CO provides protection against oxidant-induced injury and inflammation. Although the precise physiological

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function of apoptosis in the lung has yet to be established, emerging data strongly suggest that the total lung apoptotic index can serve as a useful marker of lung injury in response to oxidative stress such as hyperoxia (90,91). Soares et al. also showed that HO1 may function as an antiapoptotic molecule in an in-vitro model of TNF-induced apoptosis (62). The known observations that CO can bind avidly to heme moieties such as guanylyl cyclase and thereby increase cGMP, similar to the action of nitric oxide, may provide clues for future studies (92). However, CO could also act via a pathway not involving cGMP, as has recently been described in other invitro models examining HO1 regulation by nitric oxide (93). Presented here is evidence demonstrating that exogenous CO at low concentrations provides protection against hyperoxia-induced lung injury. The concentrations of CO needed to achieve these dramatic protective effects are far less than the known toxic concentrations, and even lower than the concentrations used in pulmonary function tests in humans. While the precise mechanism by which CO exerts its protective effects has yet to be established, the inhibition of neutrophil inflammation and attenuation of total lung apoptotic index represent potential avenues to investigate in the future. This work continues to raise the intriguing possibility of the potential therapeutic use of low concentrations of CO in clinical settings, not only in lung disorders such as ARDS and sepsis but also in a variety of other inflammatory disease states. In a second model of acute lung injury and inflammation in mice, a low concentration of carbon monoxide when mixed with ⬎98% O2 protects the lungs of mice when compared to those exposed to O2 alone. Of obvious concern is the toxicity associated with this gas molecule. The concentration of CO used, however (⬍0.03%), is one-twentieth the lethal dose, and when administered at this concentration mixed with air, produced no untoward effects on the mice. Stuepfel et al. have shown that mice exposed to 500 ppm of CO for up to 2 years showed no untoward effects on multiple physiological or biochemical parameters (94). This concentration is 10-fold lower than that used in the measurement of lung diffusing capacity (DLCO), a standard pulmonary function test used in humans. Here, in a clinically relevant model of ARDS, we show that not only was CO cytoprotective as it reduced markers of lung injury, but also and more importantly, we demonstrate clearly that at this low concentration selective inhibition of hyperoxia-induced TNF-α, IL-1β, and IL-6 occurred at the tissue level. At a more functional level, survival was extended in those animals exposed to O2 and CO. There is increasing evidence that the MAPK are strong regulators of proinflammatory cytokines, in particular the p38 MAPK pathway (95). In the endotoxin model, the MKK3/p38 pathway proved to be an important variable in enabling CO to modulate cytokine production. The data collected in this study showed that CO prevented the increased transcription of TNF-α, IL-1β, and


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IL-6 in response to hyperoxia exposure requiring the MKK3/p38 pathway. It is therefore a formal possibility that modulation of these cytokines is perhaps the mechanism behind the ability of CO to extend the survival of mice exposed to hyperoxia. Numerous studies have shown that if TNF-α and/or IL-1β are inhibited, the inflammatory response is reduced (96,97). In fact, clinical trials are underway to treat sepsis and rheumatoid arthritis by interfering with TNF-α production (98). We took this approach a step further and showed that the ability of CO to modulate these cytokines involves the MAP kinase signaling cascades, in particular the MKK3/p38 MAP kinase pathway. The precise molecular target(s) for CO is not clear at this juncture, but based on our current knowledge that it preferentially binds heme moieties present in numerous proteins, we gain some insight into potential cellular target(s). In this model, CO modulates pro-inflammatory cytokine production through the MAP kinase MKK3/p38, so a plausible cellular target is an upstream intermediate involved in the modulation of these pathways. Obvious targets in the lung would include guanylyl cyclase or NADPH oxidase, both heme-containing enzymes that lie upstream of the p38 MAPK. Inhibition of NADPH oxidase has been shown to protect against oxidant-induced injury (99). For instance, CO could be stimulating production of cGMP in the lung, which has numerous downstream targets; or CO could be inhibiting NADPH oxidase and thereby preventing the increased ROS generated in this model, ROS which have been shown to initiate cytokine expression. Furthermore, the specificity of CO fails to modulate significantly a number of other hyperoxia-induced cytokines such as the TGF-β family members. While some of these are also regulated by the MAP kinases, these cytokines are also dependent on other transcriptional activators, including the Stat and Smad family members (100–102). These studies reveal a novel physiological function of the gaseous molecule CO in a murine model of acute respiratory distress. Despite the long-standing and widely accepted paradigm that CO is toxic and even lethal, these data suggest otherwise. First, we demonstrate here that in a model of acute lung injury, CO at low concentrations is anti-inflammatory. Second, we show that it does so in part through inhibition of pro-inflammatory cytokines. Third, we demonstrate that it somehow modulates these cytokines via the MKK3/p38 MAP kinase axis. A fascinating—if serendipitous—finding is the differences observed in the neutrophils of the wild-type and MKK3(⫺/⫺) mice. Neutrophil extravasation in this and other models has been well described and is believed to exacerbate tissue injury (103). Antineoplastic and antineutrophil antibody studies clearly demonstrate that when the neutrophils are depleted, the injury is diminished (104). In these studies, while only accumulating neutrophils in the airspace was measured, the observation that in the absence of substantial numbers of leukocytes recovered in the BAL in the MKK3(⫺/⫺) mice, these mice have increased susceptibility to the oxidant injury. One could speculate a number of possibilities, including:

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(a) decreased circulating pools; (b) lower numbers of recruitable bone marrow cells, which are thought to be the pool that are subsequently recruited to the tissue; (c) the PMNs have marginated, but have not migrated into the tissue and airspace; or (d) there are altered cytokine signals from the alveolar macrophages and epithelium/endothelium to draw the peripheral white cells into the airspace. Current investigations are underway not only to elucidate these phenomena, but also to use these animals as tools to further our understanding of the role of the neutrophil in tissue injury. It is tempting to speculate that CO might be used therapeutically either by overexpressing HO1, inducing it via genetic engineering, or by local administration of a low concentration of CO in areas of inflammation. Carbon monoxide might prove therapeutic in not only acute respiratory distress syndrome, but also other inflammatory conditions. CO at low concentrations provides cytoprotection and acts as an anti-inflammatory agent. The evidence in support of CO as an anti-inflammatory agent continues in the mouse hyperoxia model with functional potential. TNF-α and IL-1β expressions are once again inhibited, and suggest that the improved survival with decreased lung injury is reflective of this phenomenon. Furthermore, survival in the MKK3-null mice could not be extended with CO, nor did CO have any effect on the same injury markers that were otherwise inhibited in the wild-type mice exposed to O2 in the presence of CO. Interestingly, in whole tissue, message levels of the pro-inflammatory cytokines were decreased by CO, unlike that seen in the RAW 264.7 macrophages, where inhibition occurred posttranscriptionally. This might point to cell type specificity or cell versus tissue effects or even model differences where the milieu is different. Future studies will examine these phenomena.

VI. Summary and Future Directions While heme catalysis liberates three by-products, the only gaseous by-product, carbon monoxide, was examined as a possible mechanism by which HO1 provides beneficial cytoprotective properties. In identical models in which HO1 showed protective effects, CO produced the same effects. What has been observed and described herein is that CO was able not only to mimic these protective effects but also to do so at very low concentrations. At the same time, intracellular targets (MAPK) were identified that could be responsible for the cytoprotective response. These studies also investigated derivative issues related to possible mechanisms by which CO was able to exercise its cytoprotective effects. Obvious research choices included downstream gene products including cytokines, which played important roles in the sequelae of tissue and organ injury that occurred in the models of shock and hyperoxic lung injury employed in these studies. On


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the other hand, upstream molecular targets involved in signal transduction and gene regulation and responsible for the observed downstream effects were examined to understand better the potential mechanism(s) by which CO behaved. To be sure, there are numerous pathways to consider. Certainly, the effects of CO in any given system could also vary. Any heme-containing protein of the thousands that exist could potentially be a target or at least participate indirectly in the cellular response to any given stress. Fortunately, the models of inflammation described in this chapter represent clinically relevant research models. Each provides pertinent insights that advance an understanding of clinical disease because while they diverge in terms of their relative cytotoxic etiologies and tissue injury dynamics, they are closely intertwined at the intracellular level. All generate enormous amounts of reactive oxygen species (ROS) that represent the fundamental underlying mechanisms by which they generate pathology. Ironically, a large percentage of patients with sepsis end up with ARDS, and the only common treatment for these patients is high levels of inspired oxygen. Despite the necessity of such treatment to maintain normal arterial oxygen tensions for delivery to distal tissues, these high levels of inspired oxygen exacerbate the oxidative burden on lung tissue. Understanding the mechanisms of injury and the role of the stress response including that of HO1 will better enable the design of treatment regimens by which to treat both sepsis and ARDS without resorting to the counterproductive oxygenation therapies. In animal studies it is apparent that HO1 is involved in the defense against these syndromes and is therefore a potential therapeutic target. The unanswered question is how and why HO1, an enzyme designed simply to break down heme, is involved in these complex disease states. While the gaseous molecule carbon monoxide has been studied for the past hundred years, those investigations have almost universally focused on its toxic features and effects and CO remains, quite understandably, a molecule to be avoided. Despite those repeated indictments of CO as an exceptionally lethal agent, studies underway are perhaps suggesting otherwise. Put simply, carbon monoxide possesses potent beneficial properties based on the findings presented here, properties that offer potentially enormous benefits in the treatment of sepsis, ARDS, asthma, and transplantation disease states for which no substantial improvements in treatment have occurred over the past 30 years. The mechanisms of CO’s toxicity are in theory well understood—it binds to hemoglobin and causes life-threatening hypoxia, resulting in brain damage and acute cardiovascular collapse. It also binds to cytochrome oxidase and prevents life-sustaining generation of ATP. Among these almost monolithic truths about CO, however, dwell uncertainties. Why is it that when blood is saturated with CO and administered to animals intravenously, they suffer no adverse effects despite the fact that carboxyhemoglobin levels rise to magnitudes that would otherwise create serious if not lethal consequences (105,106)? Why is it that there are reports of CO regulat-

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ing carotid body signaling and neurotransmission? Why is it that HO2 exists constitutively in the body, relentlessly producing CO, releasing it into the bloodstream to regulate vasomotor tone and into the brain to support long-term potentiation or memory (107). Certainly these would seem important functions for survival. And in the end, there is the most fundamental question: if CO is so toxic, why is it constantly generated in the body and, most paradoxical of all, why does it increase in inflammatory states as measured in exhaled breath, creating a hypoxic environment limiting O2 delivery to tissues and interfering with ATP synthesis? Of course, CO may simply be a by-product that, like others, is waste that must be eliminated. But it may indeed possess a functional role in the maintenance of normal cellular homeostasis. It does seem coincidental that, in mice and humans deficient in HO1, there is profound inflammation that supercedes all other abnormalities (108). This inflammation does not appear to occur as a result of infection or other exogenously determined sources; rather, the inflammation exists from birth, ultimately leading to death (of those who are born). In light of these observations, CO via HO1, based on data presented here, appears to be the antidote, as it were, for this dysfunctional inflammatory response, as the data presented suggest. When HO1, and therefore CO, are present, perhaps these molecules lead to homeostatic regulation of the inflammatory response. HO1 is induced via a variety of inflammatory stimuli, some no more stressful than the normal oxidative stress present in all O2-breathing organisms. It is believed that oxidative stress caused by reactive radicals is an essential if not exclusive determinant of the aging process and that it is involved in most if not all disease states. But questions remain. Does CO really subserve these protective effects? Is it the sole mechanism by which HO1 provides cytoprotection? Are bilirubin and ferritin involved? All have been shown to be cytoprotective (53,109). If, for example one titrated the three, could there be a level at which, individually no protective effects occurred but collectively, acting synergistically or additively, effected such protection. Perhaps CO acts as an antioxidant. Weinstock first hypothesized that CO could react with the hydroxyl radical to form CO2 and H⫹ and thereby eliminate a potent free radical. Similarly, many of the reactive oxygen species originate via the mitochondrial cytochrome oxidases and NADPH oxidase present on most cell membranes. Each of these enzymes contains heme moieties, reactive with both O2 and certainly CO. Hence, it is certainly a possibility that CO is acting as a potent antioxidant interfering with the generation of reactive oxygen species. The potent antioxidant enzyme catalase also contains a heme moiety, and perhaps the binding of CO increases its activity levels. Future studies will examine this issue and, if confirmed, help explain most if not all of the cellular and tissue responses. Blockade of radicals has been shown to prevent cytokine release, inhibit apoptosis, and certainly prevent oxygen-medi-


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ated cellular injury such as lipid peroxidation that leads of course to changes in permeability and increases in leukocyte trafficking. Other formal possibilities would include indirect actions of CO on other non-heme-containing proteins or its possible gene-regulating function acting as a regulator of gene expression, a transcription factor generated endogenously via HO1 during a stress response that modulates gene expression responsible for regulation of the downstream cellular responses. One obvious cellular target is the MAPK pathway. It appears that CO, by augmenting p38, could be acting via signaling of downstream events. Perhaps CO via p38 is increasing the expression of other cytoprotective genes. Augmented release of IL-10 suggests as much. Carbon monoxide has been studied for over a hundred years and until the last few years has been touted as a molecule to avoid. During the Industrial Revolution, miners used canaries to detect carbon monoxide levels, assuming that the birds’ higher metabolic rates would predispose them to CO poisoning well before a worker would succumb to such. Shortly thereafter, the Japanese waltzing mouse was used in mines for much the same purpose and reason. It was argued that by the time the bird died or the mouse began to convulse (hence the waltzing), a miner was being exposed only to sublethal CO concentrations and therefore had time to escape. It is ironic that the mouse once used to predict toxicity is now, 100 years later, acting in much the same capacity. Now, however, it offers itself the same, in experimental tests designed to determine whether and by what means carbon monoxide exposure might be beneficial to humans. Presented here, and confirmed by other vascular and neurobiologists, is a compelling reason for a renewed effort to understand this most extraordinary molecule. Perchance in the future, low concentrations of CO will be at the bedside in all hospitals and clinics and might save lives rather then steal them away.

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MARKERS OF OXIDATIVE DAMAGE

7 Role of Reactive Oxygen and Nitrogen Species in Lung Injury

IAN C. DAVIS, JUDY M. HICKMAN-DAVIS, J. RUSSELL LINDSEY, and SADIS MATALON University of Alabama at Birmingham Birmingham, Alabama, U.S.A.

I.

Introduction

The primary function of the lungs is gas exchange. The gas exchange surface is mainly composed of a single thin layer of epithelial cells, the alveolar type I cells. Interspersed among these are larger cuboidal alveolar type II cells, which produce the alveolar lining fluid. This fluid layer must be kept thin, to permit efficient gas exchange. The alveolar epithelium has low permeability to electrolytes and plasma proteins, and actively transports sodium ions away from the lumenal surface, while tight junctions between the epithelial cells provide a highresistance barrier to fluid movement from the interstitium to the alveolar space (1). Pulmonary surfactant also lowers the surface tension of the blood–gas interface (2). Free-ranging phagocytic alveolar macrophages (AMs) are a third cell type found in varying numbers in the extracellular lining fluid on the alveolar surface. These cells patrol the alveolar surface and ingest inspired particulates and invading pathogens (3). The alveolar epithelium is continuously exposed to reactive oxygen and nitrogen species, derived from both endogenous and exogenous sources. Prolonged exposure to these reactive species results in damage to the pulmonary 159

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surfactant system and alveolar epithelium, leading to protein leakage into the alveolar space, pulmonary atelectasis, and hypoxemia. Reactive oxygen species (ROS) are generated as intermediates in mitochondrial electron transport systems and during microsomal metabolism of endogenous cytoplasmic compounds and xenobiotics, such as drugs and environmental pollutants (4). In addition, during the inflammatory response to lung insult, neutrophils, AMs, and other inflammatory cells can generate and release ROS by an NADPH-oxidase-dependent mechanism (5,6). Recently, however, it has been shown that nitric oxide (• NO) also contributes to the alveolar epithelium’s oxidant burden, primarily as a result of formation of reactive oxygen–nitrogen intermediates (7,8). Lung tissues may be exposed to increased concentrations of • NO in inhaled polluted air (9,10), or as a consequence of its overproduction by AMs, or pulmonary epithelial, interstitial, and endothelial cells (see below). Overproduction of • NO and other reactive nitrogen species (RNS) has been implicated in a variety of inflammatory diseases, including acute respiratory distress syndrome (ARDS).

II. Chemistry and Biochemistry of Reactive Oxygen and Nitrogen Species A. Reactive Oxygen Species

At normal oxygen tensions in humans, approximately 98% of oxygen undergoes a four-electron catalytic reduction by mitochondrial cytochrome c oxidase to form water (11). The remaining 2% of oxygen, however, may undergo sequential incomplete reduction by the mitochondrial electron transport chain to form the superoxide anion radical (O 2 •⫺) (12). H 2 O 2 also can be formed by spontaneous, or superoxide dismutase (SOD)-catalyzed dismutation of O 2•⫺ (13). The latter may occur when SOD expression is induced without concomitant upregulation of catalase activity. Neutrophil myeloperoxidase (MPO) can convert H 2 O 2 to hypochlorous acid (HOCl) and chloramines (14). O 2 •⫺ and H 2 O 2 are relatively long-lived compounds in biological systems, and both can enter cells (H 2 O 2 directly crosses cell membranes by simple diffusion, O 2 •⫺ via anion channels) (4). H 2 O 2 is less reactive than O 2 •⫺, but it can exert toxic effects more distally. However, the limited reactivity of O 2 •⫺ and H 2 O 2 with many biological molecules and their low intracellular concentrations (10 pM and 1–100 nM, respectively) has raised questions about their toxicity in vivo. Likewise, the degree to which the MPO/HOCl system contributes to host antimicrobial defenses is still unclear, although microbicidal activity of MPO against Mycobacterium tuberculosis has been demonstrated (15). Moreover, studies with gene “knockout” mice lacking MPO have demonstrated increased susceptibility to pneumonia induced by intratracheal administration of Candida albicans (16).

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A more potent reactive metabolite of O 2 •⫺ that is generated in a variety of biological systems is the hydroxyl radical (• OH) (17). In the Haber-Weiss reaction, O 2 •⫺ directly reduces H 2 O 2 to produce • OH, together with molecular O 2 and hydroxide ion (OH⫺). Alternatively, in the modified Haber-Weiss reaction (the Fenton reaction), O 2 •⫺ can reduce trace metal ions (usually Fe 3⫹, sometimes Cu 2⫹) to form molecular O 2 (18). The reduced form of the metal ion can then react with H 2 O 2 to regenerate an oxidized metal ion, with concomitant production of OH ⫺ and • OH. While there are no known enzymatic scavenging systems for • OH radical in vivo, its reactivity is so high and nonspecific that the site of its reaction with target molecules is confined to within a few molecular radii of the site of its generation. Moreover, because generation of • OH by the Fenton reaction requires the interaction of two different reactive species (O 2•⫺ and H 2 O 2) in the presence of iron, at relatively slow reaction rates, it is unlikely to occur in the normal lung, where most of the iron in the epithelial lining fluid is chelated in an inactive form by transferrin and ceruloplasmin (19). In addition, ascorbate, which is present in the epithelial lining fluid in higher concentrations than O 2 •⫺, also reduces Fe 3⫹, and so can compete with O 2 •⫺. Nevertheless, formation of • OH via the Fenton reaction may still occur in vivo, especially in situations where the intracellular load of free iron is increased (20), or when antioxidant defenses are perturbed (21). Moreover, Beckman et al. have recently described a second pathway for the generation of potential oxidants with the reactivity of • OH in the absence of metal catalysis (see below) (22). The normal lung is protected from the buildup of ROS to toxic concentrations by several antioxidant systems. Lung cells contain three forms of SOD, including CuZnSOD, found mainly in cell cytoplasm but also in peroxisomes; and MnSOD, localized in mitochondria. An extracellular form of SOD (EC-SOD) also has been identified in the lung matrix and is thought to play a major role in the scavenging of extracellular O 2 •⫺ (23). Peroxisomes also contain catalase, which will degrade H 2 O 2 . In addition, lung tissues contain high concentrations of a number of nonenzymatic antioxidants, including vitamin E and reduced glutathione and ascorbate (24). Several factors may exacerbate production of ROS in acute and chronic lung diseases. First, treatment with increased concentrations of oxygen is commonly used to alleviate hypoxemia in patients with lung disease. Exposure of lung cells, subcellular organelles, and tissues to hyperoxia (100% O 2) results in a 10- to 15-fold increase in mitochondrial H 2 O 2 production (25). Second, proinflammatory chemokines and cytokines released by damaged lung cells during inflammatory responses trigger migration of neutrophils into the lungs. These pro-inflammatory stimuli also trigger receptor-mediated activation signals, transduced through protein kinase C and phospholipase C, that lead to translocation of cytosolic components of the NADPH–oxidase complex (gp40 phox , gp47 phox ,

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gp67 phox , and the Rho-family GTPase, Rac2) to a membrane-bound complex (gp91 phox /gp22 phox /Rap1a) that carries cytochrome c. Once activated, the membrane-bound NADPH–oxidase complex generates large quantities of O 2•⫺ (26). Transgenic mice that lack specific components of the NADPH–oxidase complex show increased susceptibility to pulmonary infection with Mycobacterium tuberculosis (27,28) or Aspergillus fumigatus (29). However, the extent to which AMs use this system to generate ROS is not clear. Third, under conditions of ischemia, decreased perfusion, low oxygen tension, or trauma, xanthine dehydrogenase, an innocuous enzyme, acquires oxidase activity and uses xanthine and molecular oxygen to produce partially reduced oxygen species. Indeed, results of several studies suggest that xanthine oxidase may be released from intestine or liver into the circulation and bind to pulmonary endothelium, where it can serve as a locus for the intense production of ROS (30). B. Reactive Nitrogen Species • NO, one of the smallest and most unique biological mediators, is generated by nitric oxide synthase (NOS). • NO synthesis involves the five-electron oxidation of the guanidino nitrogen of L-arginine (31). In this reaction, molecular O 2 and NADPH act as co-substrates, while tetrahydrobiopterin (H 4 B) (32), flavin nucleotides (FMN and FAD), and thiols serve as enzyme co-factors (33). N G-hydroxyL-arginine is formed as a short-lived intermediate and L-citrulline is the by-product (34). • NO is an important mediator of normal physiological effects in a variety of different cell types, including neurons, smooth and smooth and skeletal muscle cells, hepatocytes, neutrophils, macrophages, and epithelial cells. As discussed in detail elsewhere in this volume (see Chaps. 1 and 3), NOS can be broadly classified into three types, based on the source of the enzyme, substrate dependency, and molecular biology: neuronal (nNOS, isoform I), endothelial (eNOS, isoform III), and inducible (iNOS, isoform II) (35). All NOS isoforms are homodimeric heme proteins, with oxygenase and reductase domains in the amino and carboxy termini, respectively (36). These domains are separated by a calcium/calmodulin-binding region. Formation of an active, dimeric enzyme complex also is dependent on H 4 B. The reductase domain is homologous to NADPH–cytochrome P450 (37), and includes binding sites for NADPH, FMN, and FAD. During • NO synthesis, electrons donated by NADPH are transferred via the flavins and calmodulin to the catalytic heme (38). The constitutive forms have different phosphorylation sites, and eNOS has a unique amino-terminal myristylation site (39). Although the three human Nos genes are located on different chromosomes, considerable homology exists among them, suggesting common ancestry, with subsequent gene duplication and transposition. nNOS and eNOS are constitutively expressed in cells as monomers, but their activity is regulated by the availability of calcium/calmodulin within the


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cell cytoplasm. When intracellular calcium concentrations increase in response to stimulation, binding of calmodulin allows dimerization and enzymatic activity (37). These isoforms generate • NO in small quantities for brief periods of time. In contrast to nNOS and eNOS, iNOS protein generally is not expressed constitutively. Rather, transcription of Nos2 in AMs, and possibly neutrophils, is triggered by proinflammatory stimuli, including endorphin-mediated stress (40), oxidative injury (41), depletion of non-ferritin-bound iron (Fe 2⫹) (42), reduced oxygen tension (43), low environmental pH (44), bacterial endotoxin (45) or exotoxins (46), crosslinking of cell-surface CD23 (Fcγ receptor IIb) (47) or CD69 receptors (48), and cytokines (particularly IFN-α/β, IFN-γ, TNF-α, and IL-1β) (reviewed in Ref. 49). IFN-γ also stabilizes Nos2 mRNA. Many anti-inflammatory agents, including glucocorticoids, cytokines (IL-4, IL-8, and IL-10), and growth factors (TGF-β) inhibit Nos2 expression or decrease stability of Nos2 mRNA (50). A number of signal transduction pathways have been implicated in regulation of Nos2 gene transcription, including the Janus kinases, mitogenactivated protein kinases, protein kinase C, phosphatidylinositol-3 kinase, and protein phosphatases (51). Induction of IRF-1 and NF-κB transcription factor activity seems to be central to activation of Nos2 transcription. It is important to note that although iNOS and the NADPH–oxidase system are differentially regulated, they are both induced by similar pro-inflammatory stimuli, and so are likely to be simultaneously active and generating reactive species during an inflammatory response. Once synthesized, iNOS localizes to the cytoplasm and intracellular vesicles (52). iNOS-like activity also has been identified in rat mitochondria (53). Because iNOS tightly binds calcium, its activity is calcium- and calmodulinindependent, permitting sustained catalysis (54). Provided substrate and cofactors are available, iNOS can generate large amounts of • NO for an extended period of time (55,56). Interestingly, in the absence of available L-arginine, iNOS can generate O 2 •⫺ (57,58). iNOS does not appear to play a significant role in homeostasis in the normal animal. iNOS gene “knockout” (Nos2⫺/⫺) mice are born with a normal Mendelian frequency, show no deficits in growth and development, no evidence of pathology related to deletion of the gene, and reproduce normally (59). However, Nos2 gene deletion does appear to result in upregulated pulmonary capillary endothelial transcytosis (60), although it is unclear whether this effect is related to some deficiency of • NO production or is merely an epigenetic effect associated with deletion of the Nos2 gene. Moreover, it appears that the lack of phenotype of Nos2⫺/⫺ mice may be partly a result of redundancy of function between the iNOS and the NADPH–oxidase systems, because transgenic “double-knockout” mice lacking both functional systems (Nos2 ⫺/⫺ /gp91phox ⫺/⫺) have significant defects in normal immunity to enteric commensal microbes (61).

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Potential sources of • NO in the lungs include activated AMs (7), neutrophils (62), alveolar type II cells (63), endothelial cells (64), and airway cells (55). Both nNOS and eNOS have been identified in human lungs. nNOS is localized to nerve terminals that likely contribute to nonadrenergic/noncholinergic airway innervation, and is present in human and rat airway epithelial cells (64). eNOS is localized to pulmonary endothelium and bronchial epithelium (65). Studies have suggested that iNOS is constitutively expressed in human upper airway epithelium (66) and occasional AMs (64), but this may be a result of chronic exposure of these cells to inhaled pollutants and microbes (67). Expression of iNOS in other regions of the normal lung is believed to be minimal. However, iNOS has been immunolocalized to airway cells or human lung tissue obtained from patients with ARDS (C. Sittipunt, et al., submitted for publication), bacterial pneumonia (68), lung cancer (69), pulmonary sarcoidosis (70), idiopathic pulmonary fibrosis (71), and asthma (61). AMs isolated from lungs of patients with tuberculosis (72) or ARDS following sepsis (73) have been shown to express iNOS. These findings raise the possibility that increased amounts of • NO may be released during lung inflammation into the epithelial lining fluid, where it may have both beneficial (antimicrobial) and detrimental (tissue-damaging) effects. III. Biological Effects of Reactive Oxygen and Nitrogen Species A. The Dark Side of • NO •

NO is inactivated upon entering the blood stream because of its rapid and irreversible reaction with oxy-hemoglobin or oxy-myoglobin (74), resulting in formation of nitrate (NO 3⫺ ) and methemoglobin (75): Hb-Fe 2⫹-O 2 ⫹ • NO → Hb-Fe 2⫹-ONOO ⫹ NO 3⫺ Therefore, inhaled • NO has been proposed to be of therapeutic value as a selective pulmonary vasodilator in treatment of bronchopulmonary dysplasia and ARDS (76). However, • NO is a free radical, and therefore can react with other free radicals, either to detoxify them or to create more toxic reactive species (77). Because cytotoxic effects of • NO are nonspecific, they are not limited to invading microbes but also can damage the cells and tissues that produce it (78). Moreover, • NO may contribute to the systemic morbidity of pathological processes through its proposed activity as a peripheral arteriodilator (79), and because it can act as a myocardial depressant (80). Clinical use of • NO may therefore prove to be a double-edged sword. There is now substantial experimental evidence that RNS may be involved in pulmonary epithelial injury in a variety of pathological situations. Induction of immune complex alveolitis in rat lungs results in increased alveolar epithelial


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permeability, which is associated with the presence of elevated concentrations of • NO decomposition products in bronchoalveolar lavage (BAL) fluid (81). Alveolar instillation of the NOS inhibitor L-NMMA ameliorates • NO production and alveolar epithelial injury. Similarly, paraquat-induced (82), and ischemiareperfusion-induced (83), lung injury are both associated with stimulation of • NO synthesis, and are abrogated by NOS inhibitors. Tracheal epithelial cytopathology induced by Bordetella pertussis is associated with induction of • NO synthesis, and is remarkably attenuated by inhibition of NOS (78). Likewise, influenza virus-induced lung pathology in mice results from increased expression of iNOS and increased generation of • NO (84). Administration of NOS inhibitors significantly improves survival of influenza-infected mice. Additional evidence that RNS play a role in pulmonary inflammation is derived from studies utilizing transgenic Nos2⫺/⫺ mice. Lung damage induced by injection of LPS (85), influenza virus infection (86), or hemorrhage and resuscitation (87) is markedly reduced in these mutant mice. Similarly, in an experimental murine model of allergic airway disease, deletion of the Nos2 gene results in a significant decrease in eosinophil infiltration into the lungs (88). Since • NO has an unpaired electron, it can readily react with other free radicals. At high (nonphysiological) concentrations, • NO molecules can react with molecular oxygen to form the highly toxic agent nitrogen dioxide (NO 2). However, when • NO is present at physiological and even pathological concentrations, the low probability of two • NO molecules interacting makes formation of NO 2 unlikely (77). In pathological states, most of the toxic effects of • NO have been attributed instead to its reaction with O 2•⫺ to form peroxynitrite (ONOO ⫺), which is a potent oxidizing and nitrating agent. When both • NO and O 2 •⫺ are present, this reaction occurs extremely rapidly, at a near-diffusion-limited rate (k m approximately 6.7 ⫻ 10 9 M ⫺1 sec ⫺1) (89–91). By trans-isomerization, peroxynitrous acid (ONOOH), the protonated form of ONOO ⫺, also can form nitrogen dioxide (• NO 2) and an intermediate with reactivity equivalent to the • OH radical (22): O 2 •⫺ ⫹ • NO → ONOO ⫺ ⫹ H ⫹ → ONOOH → [• OH ⋅ ⋅ ⋅ • NO 2] Under physiological conditions, a minimum of 25% of ONOO ⫺ will decompose to form [• OH ⋅ ⋅ ⋅ • NO 2], the remainder recombining to form NO 3⫺. In addition, metal ions, such as Fe 3⫹ and Cu 3⫹ (in the active site of SOD) can catalyze heterolysis of ONOO ⫺ to form a nitronium ionlike species (NO 2⫹ ) (92,93). While being highly reactive, its slow rate of decomposition under physiological conditions allows ONOO ⫺ to diffuse for up to several cell diameters (probably as peroxynitrous acid, ONOOH) before becoming inactive (94). Moreover, because of its neutrality and low Stokes’ radius, smooth muscle- and endotheliumderived • NO can easily diffuse across membranes into the alveolar space (95), where it can combine with epithelial cell- or AM-derived O 2 •⫺ to form ONOO ⫺

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in the epithelial lining fluid. Interactions between reactive oxygen and nitrogen species that may be of biological importance are summarized in Figure 1. It can be argued that alveolar cells and the epithelial lining fluid contain a number of antioxidant substances, such as SODs, catalase, reduced glutathione, and urate, which will limit the steady-state concentrations of reactive oxygen and nitrogen species in vivo. Indeed, under normal conditions, intracellular O 2 •⫺

Figure 1 Interactions between reactive oxygen and nitrogen species that may be of biological importance. Superoxide (O 2 •⫺), generated by the mitochondrial electron transport chain or the NADPH–oxidase complex, can dismutate to H 2 O 2 (spontaneously, or in the presence of SOD). H 2 O 2 can oxidize glutathione (GSH). Alternatively, in the presence of neutrophil myeloperoxidase and chloride ions (Cl ⫺), H 2 O 2 is converted to the potent oxidizing agent hypochlorous acid (HOCl). In the presence of free iron (Fe 2⫹ or Fe 3⫹), H 2 O 2 is reduced to the highly reactive hydroxyl radical (• OH). Nitric oxide (• NO), generated by nitric oxide synthase, is rapidly inactivated by interaction with oxy-hemoglobin (HbO 2), generating met-hemoglobin (MetHb) and nitrate (NO 3⫺ ). However, when O 2 •⫺ is present, it reacts rapidly with • NO to form the potent oxidizing and nitrating agents peroxynitrite (ONOO ⫺) and peroxynitrous acid (ONOOH), which can nitrate thiols (RSH), GSH, or tyrosine residues (TYR). In the presence of CO 2 , ONOO ⫺ forms the nitrosoperoxycarbonate anion (ONOOCO 2⫺ ), which may also be a potent nitrating agent. Alternatively, ONOOH can isomerize to form nitrogen dioxide (• NO 2) and an intermediate with reactivity equivalent to • OH. In turn, • NO 2 can react with • NO to form dinitrogen trioxide (N 2 O 3), which is capable of nitrating RSH and amines (RR′NH), and which degrades to nitrite (NO ⫺2). In the presence of HOCl, NO 2⫺ may also nitrate TYR.


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concentrations are kept low (⬍10 pM), and formation of ONOO ⫺ prevented, because eukaryotic cells contain large amounts of SOD (4–10 µM). However, when synthesis of O 2 •⫺ and • NO increase during an inflammatory response, micromolar quantities of • NO can effectively compete with SOD for O 2 •⫺, and ONOO ⫺ is generated. Moreover, because of its high reactivity, ONOO ⫺ can attack biological targets even in the presence of antioxidant substances (96). Production of ONOO ⫺ by human neutrophils (97), rat AMs (92), and bovine aortic endothelial cells (64) has been demonstrated using a luminol-dependent chemiluminescence assay. One of the major cytotoxic effects of RNS is that they can inhibit eukaryotic gene expression by several mechanisms. Firstly, • NO and ONOO ⫺ can induce nucleotide deamination, resulting in abasic sites and DNA strand breaks (98). These trigger activation of the nuclear enzyme poly-ADP-ribosyl transferase (PART) (99). Activated PART catalyzes the attachment of ADP-ribose units to nuclear proteins, resulting in depletion of energy stores and reduced protein synthesis (100,101). Second, stimulated macrophages can produce enough • NO to inhibit activity of the iron–sulfur center of ribonucleotide reductase, the enzyme that converts ribonucleotides to the deoxyribonucleotides necessary for DNA synthesis (102). ONOO ⫺ and high concentrations of • NO can inactivate critical mitochondrial enzymes, such as aconitase, cytochrome c oxidase, and NADH:ubiquinone oxidoreductase, by interacting with the nonheme iron of iron–sulfur centers (103–106). Inhibition of mitochondrial respiration results in dissipation of mitochondrial transmembrane electrochemical proton-motive force, reduced ATP generation, and decreased protein synthesis (107). In addition, • NO can modulate the activity of redox-sensitive transcription factors including nuclear factor-kappa B (NF-κB) and AP-1 components (108). For example, • NO decreases cytokineinduced endothelial cell activation by altering expression of I-κBα (the α isoform of inhibitor of NF-κB) to prevent nuclear translocation of NF-κB. This may block transcription of both vascular cell adhesion molecule (109) and iNOS itself (110). In contrast, in other studies, • NO was shown to enhance gene activity directly, by eliciting nuclear translocation of NF-κB (111) and AP-1 subunits c-fos, and junB (108). Finally, ONOO ⫺ can initiate iron-independent peroxidation of lipids, resulting in damage to cellular membranes (112). Protein thiols are important cellular targets of RNS. Although • NO can react directly with thiol groups, this reaction is kinetically unfavorable and requires the nearby presence of a strong electron acceptor such as Fe 3⫹. In contrast, ONOO ⫺ (and other species such as N 2 O 3 and NO ⫹) can oxidize thiols to form S-nitrosothiols (RS-NO) at high rates. Many transcription factors contain thiol residues in motifs critical for DNA binding (e.g., zinc-finger proteins). These thiols can be modified by ONOO ⫺, and this may inhibit DNA binding and gene transcription (113). Normal function of other biologically important proteins may also be modified by S-nitrosylation. For example, S-nitrosylation of glyceraldehyde-3-phos-

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phate dehydrogenase inhibits its enzymatic activity (99). Similarly, nitrosylation of the neuronal N-methyl D-aspartate (NMDA) receptor results in decreased calcium transport and neuroprotection (114). Alternatively, formation of RS-NO adducts may serve to stabilize • NO, decreasing its cytotoxic potential while maintaining its bioactive properties. Micromolar concentrations of S-nitrosoglutathione (GS-NO) have been detected in normal human BAL fluid ex vivo, and levels are significantly increased in the lungs of patients with pneumonia or during inhalation of 80 ppm • NO (115). It has been proposed that a low level of constitutive iNOS expression in cells of the human respiratory tract may result in formation of RS-NO in the alveolar lining fluid, and that this pool of stored • NO has important physiological functions in the lung (60). In addition to having some degree of microbistatic effect at the level of the respiratory mucosa (see below), RS-NO may regulate pulmonary endothelial and/or epithelial fluid transport, and have important effects on peripheral blood flow. It recently has been demonstrated that when hemoglobin passes through the lung, cysteine 93 of the β chain becomes charged with a nitroso group, possibly derived from alveolar RS-NO (79). Discharge of this group as • NO in peripheral arterioles, in response to changing arterial O 2 tension, may regulate their diameter and resistance to flow. However, other studies have shown that nitrosylation of oxy-hemoglobin increases its affinity for O 2 . This implies that • NO transfer from deoxygenated SNO-hemoglobin in vivo would be limited to regions of extremely low O 2 tension. Furthermore, the kinetics of the transnitrosation reactions between GSH and SNO-hemoglobin are relatively slow, making transfer of • NO from SNO-hemoglobin to GSH less likely as a mechanism to elicit vessel relaxation under conditions of low oxygen tension and over the circulatory lifetime of a given red blood cell. Moreover, the physiological relevance of • NO release in precapillary arterioles is unclear, since it is unlikely that • NO released at this site can diffuse far enough into the relatively thick vessel wall to alter its tone. Another proposed role for RS-NO is in the regulation of apoptotic cell death. Activation of caspase enzymes, which is central to the execution of the apoptotic program, requires proteolytic removal of an N-terminal prodomain, which can be triggered autocatalytically by pro-enzyme dimerization, or by other active caspase molecules (reviewed in Ref. 116). Recently, it has been shown that S-nitrosylation of procaspase-3 also prevents its activation, and that S-nitrosylated procaspase-3 is present in T- and B-cell lines (117). However, there are clear differences in pathways of caspase activation in primary lymphocytes and lymphocyte cell lines (118,119), and it is unclear whether procaspase activation is regulated by S-nitrosylation in primary lymphocytes, or if this phenomenon merely contributes to the capacity of leukemic cell lines to grow in an immortalized fashion.


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Both ONOO ⫺ and No 2⫹ can nitrate phenolic compounds, including proteins containing tyrosine and tryptophan amino acid residues. Due to the relatively higher concentration of CO 2 in plasma (1.2 mM), the majority of ONOO ⫺ generated in biological fluids, such as the epithelial lining fluid, will react with CO 2 to form the nitrosoperoxycarbonate anion (O CN EOOCO 2⫺ ) (120,121). This reaction can enhance the nitrating activity of ONOO ⫺ while at the same time bicarbonate prevents ascorbate and urate from inhibiting ONOO ⫺-induced nitration (120–123). However, it is not clear that this reaction is biologically relevant. Berlett et al. reported that in the absence of CO 2, ONOO ⫺ is an oxidizing but not a nitrating agent (124), while Pfeiffer and Mayer (125) have shown recently that ONOO ⫺ does not nitrate free tyrosine in either the presence or absence of CO 2. Alternatively, physiological levels of CO 2 may enhance nitration by increasing the activity of iNOS, by an as yet uncharacterized mechanism (126). Another possible mechanism for tyrosine nitration involves the interaction of HOCl, the product of neutrophil MPO, with H 2 O 2 , NO 2⫺, and ONOO ⫺, which has been shown to generate reactive intermediates capable of nitrating, chlorinating, and oxidizing a variety of targets, including proteins, with maximum yield at physiological pH (127,128). This reaction may be of in vivo relevance since • NO has been shown to inactivate antioxidant metalloenzymes, such as catalase, and may thereby limit the disproportionation of H 2 O 2. Regardless of mechanism, several studies have provided evidence that nitration reactions occur in vivo during inflammatory processes. 3-Nitrotyrosine residues, products of the addition of a nitro group (NO 2) to the ortho position of the hydroxyl group of tyrosine, are stable end products of RNS-mediated reactions (89). They therefore serve as footprints of RNS action which are readily detectable by immunohistochemistry and ELISA (129). Nitrotyrosine residue formation has been detected in the lungs of infants who died with respiratory failure or ARDS (130), adults with ARDS (130) or idiopathic pulmonary fibrosis (71), and adults who died of hantavirus pulmonary syndrome (Davis et al., unpublished observations). More recently, nitrated ceruloplasmin, transferrin, α 1-protease inhibitor, α 1-anti-chymotrypsin, and β-chain fibrinogen have been detected in the plasma of patients with ARDS (131). Experimentally, nitrotyrosine can be found in the lungs of rats exposed to endotoxin (132) or hyperoxia (130), and in the lungs of mice infected with M. pulmonis (133). Such findings indicate that in vivo injury to the alveolar epithelium and pulmonary surfactant system during pulmonary inflammation, which has previously been attributed to ROS, may be caused instead by reactive oxygen–nitrogen intermediates such as ONOO ⫺ (134,135). Several reports have indicated that protein nitration may lead to loss of function. Nitration of tyrosine residues in human IgG, but not rabbit IgG, abrogated C 1q-binding activity (136). This is consistent with the presence of a tyrosine

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residue at the C 1q receptor-binding site in human but not rabbit IgG. The inactivation of Escherichia coli dUTPase by nitration and the occurrence of a tyrosine residue in a strictly conserved sequence motif suggests the critical importance of this residue for the function of the enzyme (137). Nitration of tyrosine residues in the α 1-protease inhibitor resulted in selective loss of elastase inhibitory activity but not chymotrypsin or trypsin inhibitory activity (138). Tyrosine nitration also inhibits protein phosphorylation by tyrosine kinases in vitro (139), although the in vivo relevance of this finding has not been demonstrated. Likewise, exposure of SP-A to tetranitromethane or ONOO ⫺ led to nitration of a single tyrosine residue in its carbohydrate recognition domain and reduced the ability of SP-A to aggregate lipids and bind to mannose in vitro (135,140,141). Nitrated SP-A also failed to enhance opsonophagocytosis of Pneumocystis carinii by rat AMs, a necessary event in the killing of P. carinii (142). This finding may be of invivo relevance since human AMs have recently been shown to nitrate SP-A (126). It has been proposed that loss of protein function as a result of tyrosine nitration may play a role in the pathogenesis of ARDS. Nitrated proteins, including ceruloplasmin, transferrin, α 1-protease inhibitor, α 1-antichymotrypsin, and βchain fibrinogen, were found in the plasma of patients with ARDS (131) (S. Zhu et al., submitted for publication). As has been shown for SP-A (see above), in vitro exposure to nitrating agents did not alter the activity of α 1-antichymotrypsin, but reduced the ferroxidase activity of ceruloplasmin and the elastase-inhibiting activity of α 1-protease inhibitor, and enhanced the rate of interaction of fibrinogen with thrombin. High levels of nitrated proteins (including SP-A) have also been detected in pulmonary edema fluid of patients with ARDS (C. Sittipunt, et al., submitted for publication; S. Zhu et al., submitted for publication), and levels of tyrosine nitration and chlorination (a marker of neutrophil activation) in BAL fluid were increased after inhaled • NO therapy for ARDS (143). However, the majority of studies in which loss of protein function following nitration has been shown have involved in-vitro exposure of concentrated protein to high levels of nitrating agents. It is not yet clear that levels of protein nitration and chlorination detected in vivo are sufficient to result in significant loss of function. Despite such caveats, there is some experimental evidence to suggest that • NO may damage pulmonary surfactant by nitration in vivo. Exposure of newborn piglets to 100 ppm • NO in 95% O2 for 48 hr resulted in significant injury to the surfactant system (144). Similarly, pulmonary surfactant samples from neonatal lambs exposed to • NO gas (200 ppm) for 6 hr exhibited abnormal surfactant properties and reduced ability to aggregate lipids in vitro (145). Together, these studies indicate that prolonged inhalation of therapeutic • NO by ARDS patients may lead to subacute lung injury, exacerbating pulmonary dysfunction. However, it should be noted that these effects were seen with high concentrations of inhaled • NO, and it is not yet clear that similar effects occur when therapeutic doses of • NO are used.


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B. The Good Side of • NO

Although formation of ONOO ⫺ can result in tissue damage, • NO can ameliorate tissue injury by several mechanisms. • NO binds the heme group of soluble guanylate cyclase, increasing synthesis of cyclic guanosine-3′-5′-monophosphate (cGMP) (146). Effects of cGMP are mediated through cGMP-associated protein kinases (PKGs), which act to lower intracellular calcium (147). Activation of guanylate cyclase by • NO can result in inhibition of platelet and neutrophil adhesion to endothelium, and thereby reduce cell-mediated inflammatory damage (148). It can also result in increased ciliary motility (149) and increased mucin production (150). • NO may directly inhibit activity of the NADPH-oxidase complex (151), while reaction of • NO with any O 2 •⫺ that is generated may protect O 2 •⫺-sensitive target molecules. The reaction with • NO outcompetes SOD kinetically, and forces O 2•⫺ through ONOO ⫺ oxidation and decomposition pathways. As well as reducing steady-state levels of O 2 •⫺, this reaction limits H 2 O 2 buildup, which may be especially important under conditions favoring O 2 •⫺-dependent • OH formation (152). Additionally, • NO can bind to the free coordination sites of heme-bound iron (153), and thereby act indirectly as an iron chelator (154). • NO has also been shown to induce synthesis of the antioxidant glutathione (155), and to react rapidly with tyrosyl radicals (k m-1 ⫾ 0.3 ⫻ 10 9 M ⫺1 sec ⫺1) to limit the extent of nitrotyrosine formation (156). Finally, by annihilating lipid radical species, such as alkoxyl (LO • ) and peroxyl (LOO • ) radicals, • NO can inhibit oxidant-induced membrane and lipoprotein oxidation and terminate chain radical propagation reactions (112). These reactions may be of particular importance, since • NO significantly concentrates in lipophilic cellular compartments (157). However, species resulting from the reaction of • NO with lipid peroxides may themselves be toxic. Several observations have suggested that • NO can protect the lungs from oxidant stress. In buffer-perfused isolated rabbit lungs, inhaled • NO (24 ppm) ameliorated the increase in pulmonary vascular permeability produced by intravascular generation of H 2 O 2, while inhibition of endogenous • NO exacerbated an oxidant-mediated increase in capillary filtration (158). Moreover, treatment of rats with the NOS inhibitor aminoguanidine exacerbated hyperoxia-induced lung injury (159). However, effects of NOS inhibitors may be nonspecific, and results of such studies must be interpreted with some caution. Many studies have provided evidence that RNS may have antimicrobial roles in host defense, during both the innate and adaptive phases of the immune response. They have been most strongly implicated in host defense against intracellular pathogens. However, not all pulmonary pathogens are equally susceptible to the activity of RNS. For example, Nos2 ⫺/⫺ mice are extremely susceptible to death from Chlamydia pneumoniae (160) or Mycobacterium tuberculosis (161) infection, but have normal resistance to infection of Legionella pneumophila

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(162). However, the reasons for these differences in pathogen susceptibility to RNS remain poorly defined. We evaluated the role of RNS in protection of the murine lung from infection with Mycoplasma pulmonis. Infection of mice with this pathogen provides an animal model that reproduces the essential features of human respiratory mycoplasmosis (caused by M. pneumoniae), which is responsible for 20–30% of all pneumonia in the general population of the United States. Mouse strains differ markedly to resistance to M. pulmonis, with C57BL/6 being highly resistant to respiratory infection with this pathogen (163). During the first 72 hr postinfection the numbers of mycoplasmas decrease by more than 83% in the lungs of C57BL/ 6 mice, with maximal mycoplasmacidal activity occurring within 8 hr postinfection. Demonstration of specific antibody in serum, as well as an increase in the number of macrophages, neutrophils, or lymphocytes in the lungs, does not occur until at least 72 hr postinfection (164,165). Thus, nonspecific intrapulmonary killing of M. pulmonis occurs and is most likely mediated by rapidly activated resident AMs. We found that the collectin surfactant protein A (SP-A) bound to mycoplasmas in a concentration- and partially Ca 2⫹ -dependent manner, and significantly enhanced the killing of mycoplasmas in vitro. SP-A probably serves to modulate AM function, rather than acting as a nonspecific opsonin of mycoplasmas. Addition of the iNOS inhibitor, N G-monomethyl-L-arginine (LNMMA), to AM cultures abrogated the SP-A-mediated mycoplasmacidal activity. Concentrations of nitrate and nitrite (NO 2⫺ ) (the decomposition products of • NO) were significantly increased in cultures containing SP-A and decreased in cultures containing L-NMMA (55). Moreover, when resistant C57BL/6 and strain-matched transgenic C57BL Nos2⫺/⫺ were infected with M. pulmonis, the gene knockout mice had significantly greater mycoplasmal growth in the lungs and significantly more severe lung pathology after infection than did control C57BL Nos2 ⫹/⫹ mice (166) (Fig. 2). While • NO is a well-recogized product of microbicidal macrophages, the mechanism(s) by which • NO aids in host defense remain undefined. • NO may have direct microbicidal effects, by reacting with iron or thiol groups on proteins to form iron–nitrosyl complexes and thereby inactivating enzymes important in DNA replication or mitochondrial respiration (see above). In circumstances in which ONOO ⫺ has no apparent effect, • NO is directly microbicidal for some pathogens, including Staphylococcus aureus (167), Leishmania major (168), and Giardia lamblia (169). In contrast, other pathogens, such as Salmonella typhimurium (170), Escherichia coli (171,172) and Rhodococcus equi (173), are killed by ONOO ⫺, but not by • NO alone. Indeed, we found that IFN-γ-activated murine AMs produced • NO (1.1 µM/hr/10 5 AMs) in the presence of SP-A and mycoplasmas and caused a significant decrease in mycoplasmal numbers. However, in the absence of AMs, even the significant amounts of • NO (4–6 µM) produced by PAPANONOate had no effect on mycoplasmal survival, while the combination


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Figure 2 Effect of iNOS deficiency on resistance to M. pulmonis infection in vivo. C57BL Nos2 ⫺/⫺ and control C57BL Nos2 ⫹/⫹ mice were infected intranasally with 1.5 ⫻ 10 7 CFU/mL M. pulmonis. All mice were euthanized at 1, 2, 3, or 7 days p.i., and the mean numbers of CFU (total recoverable mycoplasmas) were determined on whole lung homogenates. *Significant difference between control and experimental groups at each time point, p ⬍ 0.05. Results of quantitative cultures are means ⫾ SE; n ⫽ 18. (Adapted from Ref. 55)

of • NO and O 2•⫺ (generated by SIN-1) was toxic. ONOO ⫺ generation by 1 mM SIN-1 at 37°C was ⬃1 µM/min and caused a significant decrease in mycoplasma CFUs by 20 min, with complete killing by 90 min. Mycoplasmal killing was concentration-dependent, with significant reduction of CFUs occurring only after exposure to ⬃20 µM of ONOO ⫺: 500 µM SIN-1 caused significant mycoplasmal killing by 45 min (22 µM ONOO ⫺) and 200 µM SIN-1 caused significant killing by 90 min (18 µM ONOO ⫺) (Fig. 3). The addition of bovine copper–zinc SOD (Cu,ZnSOD) attenuated SP-A-mediated mycoplasmal killing by activated AMs (Fig. 4). Similarly, in the absence of AMs, inhibition of ⬎ 90% of ONOO ⫺ production by bovine Cu,ZnSOD was protective against the mycoplasmacidal effects of SIN-1. Catalase, however, had no effect on mycoplasma growth, indicating that H 2 O 2 was not important in killing. Likewise, the generation of H 2 O 2 or • OH by xanthine oxidase (in the presence of xanthine and Fe 3⫹) had only a minimal effect on mycoplasmal growth. These data indicate that O 2 •⫺ as well as • NO is necessary for mycoplasma killing and further implicate ONOO ⫺ as the primary bactericidal reactive oxygen–nitrogen metabolite.

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(a)

(b)


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Figure 4 Effect of Cu,ZnSOD on SP-A-mediated killing of M. pulmonis by C57BL AMs. AMs were cultured for 18 hr with 100 U/mL of IFN-γ, washed, and incubated with 1000 U/mL of Cu,ZnSOD at 37°C for 30 min. AMs were treated with SP-A (25 µg/mL) or HEPES (5 mM), infected with 10 10 CFU of M. pulmonis, and incubated at 37°C for 0 and 6 hr. Positive control AM cultures lacking Cu,ZnSOD were processed at the same time. Results of quantitative cultures are means ⫾ SE from a total of 3 experiments with 12–15 data points per group. *Significant difference between control and experimental groups at each time point, p ⬍ 0.05. (Adapted from Ref. 55.)

Figure 3 Effect of reactive oxygen and nitrogen species on mycoplasmal killing in the absence of AMs. M. pulmonis was grown to late log phase, washed to remove serum, and resuspended in 10 mL of 25 mM HEPES buffer, pH 7.4. All experiments were performed in sterile 130-mL centrifuge tubes and agitated constantly in a shaking water bath at 37°C. Aliquots were taken at 0, 20, 45, 60, and 90 min for determination of CFU. (a) HEPES 25 mM, mycoplasmas alone, SIN-1 1 mM, mycoplasmas ⫹ 1 mM SIN-1; SIN-1 200 µM, mycoplasmas ⫹ 200 µM SIN-1; SIN-1C, mycoplasmas ⫹ 1 mM SIN-1C. (b) HEPES 25 mM; mycoplasmas alone; PAPA 100 µM, mycoplasmas ⫹ 100 µM PAPANONOate; Cu,ZnSOD 3000 U/mL, mycoplasmas ⫹ 1 mM SIN-1 ⫹ 3000 U/mL Cu,ZnSOD; Cu,ZnSOD 500 U/mL, mycoplasmas ⫹ 1 mM SIN-1 ⫹ 500 U/mL Cu,ZnSOD. *Significant difference between control and experimental groups at each time point, p ⬍ 0.05. (Adapted from Ref. 55.)

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A number of pathogenic bacteria appear to have developed resistance to killing by ROS and RNS (reviewed in Ref. 174). For instance, M. tuberculosis contains two gene products (NoxR1 and NoxR3) that protect against both ROS and RNS by an undefined mechanism; they are absent from nonpathogenic or opportunistic mycobacteria (175,176). C. Other Effects of • NO

RNS have important immunomodulatory functions that may affect pulmonary host defense and modulate pulmonary inflammation. Clearly, depending on the circumstances and the effect, immunomodulation by RNS may have both beneficial and detrimental effects on the host. As well as inhibiting lymphocyte proliferation, RNS have been shown to modulate activity of a wide range of signal transduction pathways in leukocytes, including ion channels, G proteins, protein kinases, protein phosphatases, and caspases, by mechanisms as diverse as S-nitrosylation, S-glutathionylation, disruption of zinc fingers, or formation of iron– nitrosyl complexes (reviewed in Ref. 49). RNS also influence production of both pro- and anti-inflammatory cytokines in host leukocytes. Of particular importance, native • NO, GS-NO, and ONOO ⫺ have variously been shown to either induce or prevent apoptosis of leukocytes. For example, high concentrations of exogenous • NO are proapoptotic for T cells and macrophages, partly because • NO inhibits degradation of polyubiquitinated p53 by the proteasome (177), but also because • NO can induce increased expression of Fas ligand (CD95L) on these cells (178). In contrast, low-level endogenous generation of • NO appears to be antiapoptotic in macrophages (179,180) and lymphocyte cell lines (117), possibly because S-nitrosylation of procaspase-3 prevents its activation (see above). However, the relevance of these effects to normal function of pulmonary immune cells has not been investigated. IV. Reactive Species in Acute Lung Injury The clinical syndrome of acute lung injury represents a common response of the lung to several insults, including sepsis, endotoxemia, trauma, aspiration, and pneumonia (181). Pulmonary edema is a major component of acute lung injury, and results primarily from increased permeability of the alveolar capillary barrier (182). Many studies have provided evidence that RNS are involved in the development and progression of experimental acute lung injury (1,130,183–190). A. Evidence from In Vitro and Ex Vivo Experiments

Exposure of mouse or rat AMs in vivo or in vitro to diverse proinflammatory stimuli, such as cytokines (IL-1, TNF-α, IFN-γ), lipopolysaccharide (LPS), vari-


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ous pathogens, respirable dusts, or oxidant gases, induces upregulated activity of both iNOS (174) and membrane-bound NADPH oxidase (191), and results in increased elaboration of both • NO and O 2•⫺. Although mitogen-actived human AMs can generate ONOO ⫺ in vitro (7), it remains unclear whether activated AMs, which are present in large numbers in the alveolar lining fluid in inflammatory conditions, but which lack myeloperoxidase, contribute to the nitration and oxidation of proteins detected in the edema fluid (EF) of patients with acute lung injury (192). We therefore assessed whether reactive oxygen–nitrogen intermediates generated by AMs could nitrate and oxidize human SP-A. Exposure of SP-A to LPS-activated rat AMs in the presence of physiological concentrations of CO 2 (1.2 mM) resulted in enhanced SP-A nitration (Fig. 5), and nitration on 3-tyrosine residues (126). Interestingly, in the presence of CO 2, AM iNOS activity was increased, as measured both by higher levels of NO ⫺2 and NO ⫺3 in the medium (Fig. 6) and enhanced conversion of L-[U-14 C] arginine to L-[U-14 C] citrulline (Fig. 7). These findings indicate that physiological quantities of ONOO ⫺, which are likely to be similar to those encountered in vivo during an inflammatory response, can nitrate proteins such as SP-A and that CO 2 increases nitration both by enhancing NOS activity and by allowing formation of more efficient nitrating intermediates such as ONO 2 CO ⫺. Enhanced NOS activity may result partly from formation of the short-lived ONO 2 CO ⫺ adduct itself, which

Figure 5 CO 2-enhanced nitration of SP-A by LPS-activated AMs. SP-A was added to AMs activated with LPS (100 ng/mL) for 6 hr and co-incubated for an additional 30 or 60 min in the absence (⫺) or presence (⫹) of 1.2 mM CO 2. SP-A was immunoprecipitated with a polyclonal rabbit anti-human SP-A antibody, and protein nitration was detected by Western blotting with a polyclonal anti-nitrotyrosine antibody. (Adapted from Ref. 126.)

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Figure 6 Enhancement of AM NO 2⫺ plus NO 3⫺ production by LPS and CO 2. AM activation and CO 2 exposure were performed as in Figure 5. NO 2⫺ plus NO 3⫺ was measured in medium at that time using the Griess reagent. Values are means ⫾ SE; n ⫽ 6 experiments. *p ⬍ 0.01 compared with AMs cultured in the absence of 1.2 mM CO 2 . # p ⬍ 0.01 compared with AMs cultured in the presence of 1.2 mM CO 2 but in the absence of LPS. (Adapted from Ref. 126.)

Figure 7 CO 2 enhances NOS activation by LPS. AM activation and CO 2 exposure were performed as in Figure 5. NOS activity was measured as conversion of L-[U-14 C] arginine to L-[U-14 C] citrulline. Values are means ⫾ SE; n ⫽ 4 experiments. *p ⬍ 0.01 compared with AMs cultured in the absence of 1.2 mM CO 2. #p ⬍ 0.01 compared with AMs cultured in the presence of 1.2 mM CO 2 but in the absence of LPS. (Adapted from Ref. 126.)


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may mitigate the oxidative inactivation of NOS by longer-lived ONOO ⫺ molecules. B. Evidence from Clinical Studies

ARDS is a disease process characterized by diffuse inflammation in the lung parenchyma. NO 2⫺ and NO 3⫺ (NO x) concentrations were significantly higher than normal in the BAL fluid from patients at risk for developing ARDS, as well as those with ARDS, and remained elevated throughout the course of the disease (193). In all cases, the majority of the products detected were in the form of NO 3⫺ (⬎90% NO 3⫺, and ⬍10% NO 2⫺ ). NO x was barely above background in BAL fluid from normal subjects (range 2.5–4.3 µM, median 2.5 µM). In patients at risk for ARDS, NO x concentrations in BAL fluid from days 1 and day 3 after onset of ARDS risk factors (such as multiple trauma, sepsis, or multiple transfusions) was significantly higher than in normal subjects (Fig. 8). Levels of NOx in the epithelial lining fluid of these patients cannot be easily estimated since they are diluted considerably (more than 50-fold) by the BAL fluid. To address this issue, we measured NO x levels in pulmonary edema fluid

Figure 8 NO 2⫺ and NO 3⫺ concentration (NO x) in BAL from normal volunteers (NL), patients at risk for ARDS (RISK), and patients with established ARDS (ARDS) studied at sequential times. The horizontal axis shows the patient group and the day on which the BAL was performed. (n) ⫽ number of subjects in each group. The data are presented as box plots showing the 10th, 25th, 75th, and 90th percentiles and the median. *p ⱕ 0.005 versus normal subjects. (From Ref. 193.)

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(EF ) and plasma samples from patients with ALI/ARDS and, for comparison, in samples from patients with hydrostatic pulmonary edema (192). All of these patients were admitted to the intensive care units at the University of California at San Francisco (UCSF) or San Francisco General Hospital between 1985 and 1998. Pulmonary EF was collected from each patient within 30 min after endotracheal intubation by passing a standard 14 Fr tracheal suction catheter through the endotracheal tube into a wedged position in a distal airway. Pulmonary EF from patients with ALI had significantly higher levels of NO x compared to pulmonary EF from patients with hydrostatic pulmonary edema (108 ⫾ 13 µM versus 66 ⫾ 9 µM; mean ⫾ SEM; p ⬍ 0.05). In addition, patients with shock had higher plasma NO x levels than those without shock (79 ⫾ 11 µM versus 53 ⫾ 12 µM, p ⬍ 0.05). The ratios of NO 2⫺ to NO 3⫺ in 11 edema and 9 plasma samples were 0.01 ⫾ 0.005 versus 0.008 ⫾ 0.004, indicating that more than 90% of NOx was present as nitrate, in agreement with our BAL data (see above). Acidemia and increased anion gap, markers of systemic hypoperfusion, were also associated with twofold higher plasma NO x levels. Reactive oxygen nitrogen intermediates are most likely produced by activated inflammatory cells. Our recent studies show that alveolar macrophages, isolated from the bronchoalveolar lavage of patients with lung transplants, produce very large amounts of • NO when co-incubated with either surfactant protein A (SP-A) or pathogens (194). In contrast, AMs from normal volunteers could not be stimulated to produce nitric oxide. There is also significant evidence for the existence of nitrated and oxidized proteins in the plasma and alveolar spaces of patients with inflammatory diseases. Fore example, Gole et al. (131) reported the presence of nitrated ceruloplasmin, transferrin, α 1-protease inhibitor, α 1-anti-chymotrypsin, and β-chain fibrinogen in the plasma of patients with ALI/ARDS. Significant levels of protein-associated nitrotyrosine (⬃400–500 pmol/mg protein) were also detected in the edema fluid from both ALI/ARDS and hydrostatic edema patients (192) and in the BAL from patients with ARDS (193). These levels of nitrotyrosine are at least one order of magnitude higher than those found in proteins in normal human BAL fluid (28 pmol/mg protein) (195) or normal rat lung tissue (⬃30 pmol/mg protein) (196). Lamb et al. (197) also measured nitrotyrosine content in the BAL fluid of patients with severe ARDS and healthy volunteers using HPLC, although their values were considerably higher than those reported by Sittipunt et al. (193) and Zhu et al. (192). Nitrated pulmonary SP-A was also detected in the EF, but not in the plasma, of patients with ALI, after immunoprecipitation with a specific antibody against this protein (Fig. 9). Although we previously demonstrated that SP-A is nitrated and oxidized in vitro, using LPS-stimulated rat AMs as the source of reactive species (126), this is the first in vivo evidence for nitration of a specific protein


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Figure 9 Nitration of SP-A in pulmonary EF samples from ALI/ARDS patients. SPA was immunoprecipitated and Western blotting used to identify SP-A (A) and nitrotyrosine (B). SP-A was detected in the pulmonary EF but not in the plasma of all patients. E 1 –E 5 , pulmonary EF samples from 5 different ALI/ARDS patients; P 1 –P 3 , plasma samples from 3 different ALI/ARDS patients. C, purified human SP-A from a patient with alveolar proteinosis. Note the lack of nitration in the control sample. (From Ref. 192.)

in the alveolar spaces of human lung. Results of previous in vitro studies indicated that nitrated SP-A loses its ability to enhance the adherence of Pneumocystis carinii to rat AMs (142) and inhibits killing of Mycoplasma pulmonis by mouse AMs (Hickman-Davis et al., unpublished observations). Also, nitration of human SP-A by ONOO ⫺ or tetranitromethane inhibited its lipid aggregation and mannose binding activities (141). Finally, SP-A isolated from the lungs of lambs exposed to high concentrations of inhaled • NO had decreased ability to aggregate lipids (145). Thus, nitration of SP-A may be one of the factors responsible for increased susceptibility of patients with ARDS to nosocomial infections. Interest-

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ingly, despite being present at high concentrations in the epithelial lining fluid of patients with ARDS, albumin was nitrated to a much lesser degree than SP-A (192). V.

Summary

While there is no doubt that reactive oxygen and nitrogen species are of considerable importance in innate immunity, their release may also result in collateral damage to the normal tissues of the lung, which may ultimately compromise both gas exchange and host defense functions. For example, RNS produced by AMs in the presence of physiological CO 2 tensions may induce nitration of SP-A, which may compromise its ability to act as a collectin in vivo. Future studies should determine the functional consequences of reactive species-induced damage for other proteins important either for host defense (e.g., complement components) or for normal lung function (e.g., epithelial sodium channels). However, our data suggest that such studies should take into account aspects of the normal lung environment (such as pH, CO 2 tension) that may affect generation or activity of reactive species, as these environmental factors are crucial to be physiological relevance. Acknowledgments This work was supported by National Institutes of Health grants HL31197 (S.M.), HL51173 (S.M.), RR00149 (J.H.D.), and a grant from the Office of Naval Research (N00014-97-1-0309; S.M.). Dr. Ian Davis is a Parker B. Francis Foundation Fellow. References 1. Matalon S, Egan EA. Effects of 100% O 2 breathing on permeability of alveolar epithelium to solute. J Appl Physiol 1981; 50:859–863. 2. Holm BA, Matalon S. Role of pulmonary surfactant in the development and treatment of adult respiratory distress syndrome. Anesth Analg 1989; 69:805–818. 3. Nicod, LP. Pulmonary defence mechanisms. Respiration 1999; 66:2–11. 4. Fridovich I. Fundamental aspects of reactive oxygen species, or what’s the matter with oxygen? Ann NY Acad Sci 1999; 893:13–18. 5. Kobayashi, T, Seguchi H. Novel insight into current models of NADPH oxidase regulation, assembly and localization in human polymorphonuclear leukocytes. Histol Histopathol 1999; 14:1295–1308. 6. Nauseef WM. The NADPH-dependent oxidase of phagocytes. Proc Assoc Am Physicians 1999; 111:373–382.


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Part Two METHODOLOGICAL AND TECHNICAL ASPECTS

8 Exhaled Nitric Oxide, Carbon Monoxide, and Breath Condensate

SERGEI A. KHARITONOV and PETER J. BARNES Imperial College of Science, Technology and Medicine National Heart and Lung Institute and Royal Brompton Hospital London, England

I.

Introduction

Many lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, cystic fibrosis, and interstitial lung disease, involve chronic inflammation and oxidative stress. Yet these are not measured directly in routine clinical practice because of the difficulties of monitoring inflammation. In asthma, fiber-optic bronchial biopsies have become the “gold standard” for measuring inflammation in the airway wall, but this is an invasive procedure that is not suitable for routine clinical practice and cannot be repeated often. It is also unsuitable for use in children and patients with severe disease. Symptoms may not accurately reflect the extent of underlying inflammation, due to differences in perception and masking by bronchodilators in airway disease. Measurement of airway hyperresponsiveness by histamine or methacholine challenge has been used in asthma as a surrogate marker of inflammation, but interpretation is confounded by the use of bronchodilator therapy. Furthermore, it is difficult to perform this measurement in children and in patients with severe disease. This has led to the use of induced sputum to detect inflammation. This technique is relatively reproducible and allows the quantification of inflammatory cells and mediators (1). However, this technique is somewhat inva199

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sive, as it involves inhalation of hypertonic saline, which may induce coughing and bronchoconstriction, and it is difficult to use in small children. Furthermore, the technique itself induces an inflammatory response, so it is not possible to repeat measurements in less than 24 hr (2). The need to monitor inflammation in the lungs has led to the exploration of exhaled gases and condensates. Noninvasive monitoring may assist in differential diagnosis of pulmonary diseases, assessment of disease severity, and response to treatment. Because these techniques are completely noninvasive, they can be used repeatedly to give information about kinetics, they can be used in patients with severe disease, which has been previously difficult to monitor, and they can be used to monitor disease in children, including infants. Breath analysis is currently a research procedure, but there is increasing evidence that it may have an important place in the diagnosis and management of lung diseases in the future. This will drive the development of cheaper and more convenient analyzers, which can be used in a hospital and later in a family practice setting, then eventually to the development of personal monitoring devices for use by patients. II. Nitric Oxide NO is the most extensively studied exhaled marker, and abnormalities in exhaled NO have been documented in several lung diseases, particularly asthma (3–5). A. Measurement

Expiratory flow, soft palate closure, and dead-space air may all influence exhaled NO levels. Therefore, exhaled NO is usually determined during single-breath exhalations against a resistance (6–8) to prevent contamination with nasal NO (9,10), or using reservoir collection with discarding of the dead space (11). The most commonly used method to measure nasal NO is to sample nasal air directly from one nostril using the intrinsic flow of the chemiluminescence analyzer (12) (Fig. 1) (13). NO is continuously formed in the airways. Mixing during exhalation between the NO produced by the alveoli and the conducting airways explains its flow dependency (10) and accumulation during a breath-hold (14). It is therefore important to register the flow rate if NO is expressed as a concentration. The flow rate recommended in 1997 by a Task Force of the European Respiratory Society is 10–15 L/min or 167–250 mL/s (13). Most authors have used about 100 mL/sec, but a more recent recommendation from the American Thoracic Society suggests 50 mL/sec (15). B. Factors Affecting Exhaled NO Measurements

Exhaled and nasal NO in healthy subjects are independent of age, gender, and lung function (16). There is no evidence for significant diurnal variation (17,18).

Exhaled NO, CO, and Breath Condensate

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Figure 1 Traces of exhaled NO in normal subject and patient with asthma. (From Ref. 14.)

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Different phases of menstrual cycle may influence exhaled NO (19), as estrogen acutely activates NOS3 in airway epithelial cells (20). Several major factors may change NO levels in normal subjects (Table 1). Intravenous, inhaled, or digested L-arginine, the substrate for NOS, increase exhaled NO levels in normal subjects (21–23). Conversely, nebulized L-NMMA and L-NAME, nonspecific inhibitors of NOS, reduce exhaled NO (7,24) and nasal NO (25,26). Some routinely used tests can transiently reduce exhaled NO— for example, repeated spirometry (27,28), physical exercise (29), and sputum induction (30).

Table 1 Factors Affecting Exhaled and Nasal NO Measurements in Healthy Subjects Increased NO

Decreased NO

Pharmacological Papaverin (25) Oxymetazoline (25,26) Sodium nitroprusside (86); L-arginine NOS inhibitors (24–26,87) (21,88) ACE inhibitors (enalapril) (89) Physiological and procedural Arginine ingestion, nitrite/nitrateRepeated spirometry (27,28) enriched food (23) Acute and transient after forced exhalation (28) Physical exercise (29) Menstrual cycle (19) Sputum induction (30) Body temperature reduction (90) Environmental, occupational Air pollution (NO, ozone) (31) Water vapor, CO 2, nitrous oxide, heptane (95) Occupational hazards: 100% inspired O 2 (96) Fluoride, dust (91) Moderate altitude (97) Ozone, chlorine dioxide (32) Rubber latex (92) Formaldehyde (domestic) exposure (93,32) Electromagnetic field generated by cellular phone (nasal NO) (94) Habitual smoking (34,35) Alcohol ingestion (36,37) Infections URTI (38–40) Abbreviations: URTI, upper respiratory tract infection; ACE, angiotensin-converting enzyme.


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Environmental factors, such as NO, ozone, and chlorine dioxide, are known to increase exhaled NO levels (31–33). Habitual factors, such as smoking (34,35) and alcohol ingestion (36,37), reduce exhaled NO. Upper respiratory infection significantly increases exhaled NO (38,39) and nasal NO (40). III. Carbon Monoxide Carbon monoxide (CO) is a gas that may be formed endogenously and is detectable in exhaled air. A. Source of Exhaled CO

There are three major sources of CO in exhaled air: enzymatic degradation of heme, non-heme-related release (lipid peroxidation, xenobiotics, bacterial), and exogenous CO. The predominant endogenous source of CO (⬃85%) in the body is from the degradation of hemoglobin by the enzyme heme oxygenase (HO), and approximately 15% arises from degradation of myoglobin, catalase, NO synthase, guanylyl cyclase, and cytochromes (41). Several bacteria produce CO (42), but it does not play an appreciable role in the turnover of CO that is inhaled or endogenously produced. Approximately 85% of the CO in the body is bound to hemoglobin in circulating erythrocytes; the remaining 15% is bound to other compounds (i.e., myoglobin) or tissues, and less than 1% is unbound and dissolved in body fluid (43). Approximately 80% of the CO formed from heme degradation is exhaled (44). CO uptake or excretion across the skin is minimal, except in premature infants, and the amount of CO consumed by the tissues is very small (3% of the rate of endogenous CO production) (45). There are several reasons to consider that alveoli are the predominant site of exhaled CO in normal subjects. First, levels of exhaled CO measured at the end of exhalation are similar to those measured via a bronchoscope at the level of main bronchus (46). Second, exhaled CO levels are less flow- or breath-hold dependent than exhaled NO (47), suggesting less airway contribution. Third, maximal CO levels are seen close to the end of exhalation, as for CO 2 . There is also a small proportion of CO derived from the airways, which is higher after allergen challenge measured either via bronchoscope (46) or at the mouth (48). The fact that breathing through the nose increases the CO levels obtained in the exhaled air (49) suggests that nose and paranasal sinuses may also contribute to the CO production of the human airways. Indeed, HO-like immunoreactivity is seen in the respiratory epithelium, in connection with seromucous glands, and in the vascular smooth muscle of the nose (49). B. Heme Oxygenase

CO is a by-product of rate-limited oxidative cleavage of hemoglobin by HO, which exists in three isoforms, HO1, HO2, and HO3. HO2 is constitutively ex-

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pressed in most tissues, whereas HO3 is so far described only in rat (50). HO1 has been identified as the major 32-kDa heat-shock (stress) protein (51). Like other stress proteins, HO1 can be induced by a variety of stimuli, such as proinflammatory cytokines, bacterial toxins, heme, ozone, hyperoxia, hypoxia, reactive oxygen species, and reactive nitrogen species. Both HO1 and HO2 are expressed in human airways and are found in most cell types, with particularly strong immunfluorescence in airway epithelial cells. Heme is converted by HO to biliverdin and thence to bilirubin, with the formation of CO and ferritin. C. Interactions with NO

Like NO, CO is capable of upregulating cGMP via activation of guanylyl cyclase, causing vasodilatation, smooth-muscle relaxation, and platelet disaggregation. The vasodilatory effect of CO may be important in maintaining adequate tissue oxygenation and perfusion in the lung during normal physiology and in hypoxic conditions that result from pulmonary vascular diseases and acute lung injury. It has been suggested that the HO pathway exerts important counterregulatory effects on the NOS pathway and, when blocked, the underlying NOS pathway is unmasked, leading to increased and prolonged release of NO (52). In contrast, exogenously administered or endogenously released NO stimulates HO1 gene expression and CO production in vascular smooth-muscle cells, resulting in a higher resistance to oxidant damage (53). This effect of NO is related to the release of free heme from heme proteins, which are able to transcriptionally upregulate HO1 and lead to their own degradation. CO also directly inhibits NOS2 activity by binding to the heme moiety of the enzyme (54). D. Effect of Oxidative Stress

There is a close link between the reactive oxygen and nitrogen species and CO. Thus, a dose-dependent increase in exhaled CO has been shown following a 1hr exposure to different concentrations of O 2 (55). HO1 activation can be diminished by N-acetylcysteine, a precursor of glutathione with antioxidant properties (56). Both superoxide anions and peroxynitrite can stimulate HO1 activation (57), and subsequent release of CO is an important negative-feedback regulatory mechanism limiting the release of these cytotoxic substances (58). Animals exposed to a low concentration of CO exhibit a marked tolerance of the lungs to lethal concentrations of hyperoxia in vivo (59). The precise mechanisms for this protection are not fully elucidated, but both the degradation of heme (with removal of iron and induction of ferritin) and the generation of bilirubin may be involved. There is evidence that the deleterious effects of reactive oxygen species (ROS), such as superoxide and H 2 O 2, are dependent on the presence of iron. The intracellular pool of free iron can react with both H 2 O 2 and superoxide, giving rise to the OH ⫺ radical via the Fenton reaction.


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The free iron that is not metabolized intracellularly is sequestered in cells as ferritin. Thus, ferritin serves as a reservoir to restrict iron from participating in the Fenton reaction. It has been shown that free iron released from heme by HO may induce ferritin synthesis, and heme-induced HO1 protein also activates ferritin via mRNA expression (60). Furthermore, the metabolite of heme degradation, bilirubin, is itself an effective antioxidant of peroxynitrite-mediated protein oxidation and may be even more effective than vitamin E in preventing lipid peroxidation (61). Moderate overexpression of HO1 improves the resistance of cells to oxygen toxicity (62). However, there is cytotoxicity associated with HO1 overexpression. HO2 may also protect against oxidative stress. HO2 knockout mice are sensitized to hyperoxia-induced oxidative injury, have a higher mortality, and increased lung iron content without increased ferritin, suggesting accumulation of available redox-active iron (63). E. Measurement

Exhaled CO as a marker to assess different diseases (cardiovascular, diabetes, and nephritis) was first described in the USSR in 1972 (64). Over the last 20 years, exhaled CO was measured to identify current and passive smokers, to monitor bilirubin production including hyperbilirubinemia in newborns, and in the assessment of the lung diffusion capacity. CO can be quantified by a number of different techniques. Most of the measurements in humans have been made using electrochemical CO sensors. The sensor is selective, gives reproducible results (65), and is inexpensive. However, these instruments are susceptible to interference from a large number of substances—for example, hydrogen, which is present in exhaled breath and may be increased after glucose ingestion. H 2-insensitive CO sensors, which are now available, are therefore recommended. Exhaled CO can also be measured (at ppb level) by adjustable laser spectrophotometer (55,66), or by a near-infrared CO analyzer (67). Near-infrared instruments are used for continuous monitoring of atmospheric CO, and are fairly sensitive and stable. However, they are larger than electrochemical CO sensors, sensitive to water and CO2 concentrations, and require large sample volumes (68). This may explain the low CO levels detected by these instruments even after a prolonged breath-hold time of 20 sec (67). Gas chromatography is a reference method for CO measurements, but its use is limited to specialized laboratories. End-tidal exhaled CO measurements can be made during a single exhalation and is routine in cooperative adults. It can also be performed easily in children over 5 years of age (69). A method for measuring CO in nasally sampled exhaled air in noncooperative neonates has been developed which involves the

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relatively noninvasive placement of a small catheter into the posterior of the nasopharynx and collection of breath samples either manually or automatically (44). F. Factors Affecting Exhaled CO Measurements

CO exists in the atmosphere as a by-product of incomplete combustion and oxidation of hydrocarbons, and is oxidized to CO 2 by hydroxyl radicals, or eliminated either by soil microorganisms or by stratospheric diffusion. Regional and local levels of CO in ambient air can vary significantly, depending on time of day and the season, wind velocity, industrialization, traffic, and altitude. While some exposure to CO may occur in normal day-to-day life due to environmental pollution, active or passive smoking are the most likely reason for high levels of exhaled CO. Following inhalation, CO displaces oxygen in the erythrocyte to form carboxyhemoglobin (COHb), and has a half-life of about 5–6 hr in this form. A cutoff level of 6 ppm (70) effectively separates nonsmokers from smokers, and

Table 2

Factors Influencing Exhaled CO

Exhaled CO Miscellaneous ↑ Smoking ↑ Airway pollution ↑ Airway obstruction ↑ Hyperbilirubinemia Gender (cyclic variations in women) Race (↑ COHb in Japanese newborns) Disease ↑ Allergen challenge (early and late response) ↑ Asthma (mild–moderate) ↔ Asthma (mild) ↑ Asthma (severe) ↑ Atopy ↑ Asthma in children (persistent asthma) ↑ Allergic rhinitis ↑ COPD (ex-smokers) ↑ Upper respiratory tract infections ↑ Bronchiectasis and lower respiratory tract infections ↑ Interstitial lung disease ↑ CF ↑ Critically ill patients Abbreviations: ↓, decrease; ↑, increase; ↔, no change.

References (70,98) (99,100) (101) (102) (103) (104) (48) (105–107) (108) (108) (109) (69) (110) (111) (69,112) (113,114) (115) (116–119) (120)


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the previously used cutoff of 8 ppm (71) or 10 ppm (72) may be too high. Other individual factors which can markedly affect the amount of CO that a person may inhale are type and location of home and occupation, cooking/heating appliances, and mode of transportation. Many pathological conditions and factors can increase the rate of hemoprotein breakdown and potentially increase the levels of exhaled CO, including anemias, hematomas, and preeclampsia. Nonpathological factors may also increase endogenous CO production, including fasting, dehydration, some drugs (phenobarbitone), and xenobiotic compounds (paint remover) (73) (Table 2). IV. Exhaled Breath Condensate The detection of nonvolatile mediators and inflammatory markers from the respiratory tract involves invasive techniques, such as bronchoalveolar lavage or induced sputum. They cannot be repeated within a short period of time because of their invasiveness, and because the procedures themselves may induce an inflammatory response (2,74). Exhaled breath condensate is collected by cooling or freezing exhaled air and is totally noninvasive. The collection procedure has no influence on airway function or inflammation, and there is accumulating evidence that abnormalities in condensate composition may reflect biochemical changes of airway lining fluid. Several nonvolatile chemicals, including proteins, have now been detected in breath condensates. The first studies identifying surface-active properties, including pulmonary surfactant, of exhaled condensate were published in the USSR in the 1980s (75,76), and since then several inflammatory mediators, oxidants, and ions have been identified in exhaled breath condensates. A. Origin

Potentially, condensate measurements reflect different markers and molecules derived from the mouth (oral cavity and oropharynx), tracheobronchial system, and alveoli, and their proportional contribution has not been yet sufficiently studied. It is assumed that airway surface liquid becomes aerosolized during turbulent airflow, so that the content of the condensate reflect the composition of airway surface liquid, although large molecules may not aerosolize as well as small soluble molecules. A strong correlation between the levels of CO 2 and O2 in exhaled fluid and exhaled breath (77) suggests that aerosol particles exhaled in human breath reflect the composition of the bronchoalveolar extracellular lining fluid. B. Factors Affecting Measurements

Several methods of condensate collection have been described. The most common approach is to ask subject to breathe tidally via a mouthpiece through a

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Figure 2 Exhaled breath condensate: diagram of the aparatus.

nonrebreathing valve in which inspiratory and expiratory air are separated (Fig. 2). During expiration the exhaled air flows through a condenser, which is cooled to 0°C by melting ice (78), or to ⫺20°C by a refrigerated circuit (79), and breath condensate is then collected into a cooled collection vessel. A low temperature may be important for preserving labile markers such as lipid mediators during the collection period, which usually takes 10–15 min to obtain 1–3 mL of condensate. Exhaled condensate may be stored at ⫺70°C and is subsequently analyzed by gas chromatography and/or extraction spectrophotometry, or by immunoassay (ELISA). Salivary contamination may influence the levels of several markers detectable in exhaled breath condensate. Thus, high concentrations of eicosanoids (thromboxane B 2 , LTB-4, PGF 2α), but low levels of PGE 2 and prostacyclin, have been found in saliva of children with acute asthma (80). The presence of high concentrations of nitrite/nitrate from the diet may affect NO-related markers in condensate (81). It is therefore important to minimize and monitor salivary contamination. Subjects should rinse their mouth before collection and keep the mouth dry by periodically swallowing their saliva. Therefore, saliva contamination, measured by amylase concentration of condensate, should be monitored. In most of the studies, amylase has been measured in condensate and no salivary contamination has been detected (79,82,83). Subjects should wear a nose clip in order to collect only mouth-conditioned exhaled air into the collection system. Flushing the nose with helium may help to reduce contamination of exhaled breath with nasal air, containing high levels of NO which potentially may influence the results of NO-related markers (nitrite/nitrate, S-nitrosothiols) (84). Another approach to exclude nasal contamination is to collect condensate during a series of exhalations against a resistance (84). However, it has not yet been shown


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that nasal NO affects measurements in exhaled condensate. The quantity of exhaled condensate is dependent on the ventilation volume per unit time (minute volume), but this does not affect the concentration of mediators (78,85). It is also dependent on exhaled air temperature and humidity (P. Paredi et al., unpublished observation).

V.

Future Directions

A. Standardization of Measurements

Precautions need to be taken to ensure uniformity of measurement between different centers, and physiological and measurement factors are likely to differ between markers. This has most carefully been worked out for exhaled NO, and two international task force meetings have defined standards and procedures for measurement of exhaled NO in adults and children (13,15). Similar standardization methods are now needed for the other exhaled markers currently under investigation. B. Clinical Application

There is a pressing need for the evaluation of these techniques in long-term clinical studies. Whether repeated measurements of exhaled markers will help in the clinical management of lung diseases needs to be determined by longitudinal studies relating exhaled markers to other measurements of asthma control. This is most advanced with measurement of exhaled NO, but it is still uncertain whether routine measurement of exhaled NO will improve the clinical control of asthma in a cost-effective way. None of the exhaled markers is diagnostic for a particular lung disease, apart from the very low nasal and exhaled NO in primary ciliary dyskinesia. Nevertheless, measurement of these markers may aid differential diagnosis of lung diseases. For example, a normal level of exhaled NO in a patient with chronic cough makes the diagnosis of asthma very unlikely. A high level of exhaled NO in an asthmatic patient on inhaled corticosteroids most likely indicates poor compliance with therapy. Exhaled markers may also be used to assess the response to therapies, such as inhaled corticosteroids and novel anti-inflammatory treatments now in development. Some markers may even be used to predict responses to specific treatments. For example, high levels of LTE 4 in exhaled breath condensates may predict a better clinical response to antileukotrienes, and a high level of markers of oxidative stress may indicate a patients who may respond to antioxidant therapy.

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Kharitonov and Barnes C. Measuring Devices

The value of particular markers will depend on the availability of reliable, fast, and inexpensive detector systems. NO chemiluminescence analyzers are currently relatively expensive and are mainly available in academic research laboratories. However, advances in technology have now resulted in smaller devices that are easier to use and cheaper. This will increase the availability of the measurement that will further reduce the price as exhaled NO analyzers become routine lung function measurements. Eventually it may be possible to introduce such analyzers in family practice and even into patients’ homes, so that patients themselves will be able to monitor their own markers and adjust their treatment accordingly. Measurement of some of the other exhaled markers, such as hydrocarbons, is much more difficult using present technology, but it may also be possible to develop much smaller and cheaper detectors that would make this measurement more readily available. Although exhaled breath condensates is an attractive approach that could easily be adapted to home measurements, its value is limited by the fact that complex assays, including ELISAs, fluorimetric assays, and HPLC, are needed to measure the individual chemical markers. In the future these assays may be simplified by the use of strip reagents that give rapid color changes, so that these measurements may be available for clinicians and for patients to use at home. By introduction of the new generation of breath condensate samplers, a perspective use will be possible in each lung clinic, outpatient department of pneumologists, and also by the general practitioner and at home of the patient. Development of various biosensors may offer new diagnostic properties with the advantage to become independent of medical laboratories. Indeed, biosensors for hydrogen peroxide, proteins, urea, pH, DNA chips, electrolytes, and other substances will be available within a few years. First experiments with immunosensors for replacement of ELISA have already been performed. D. New Markers

It is likely that the possibilities for measurement of markers in exhaled breath are far greater than currently realized. It is clear that exhaled breath condensates contain many different molecules, including proteins. In fact, application of proteomics, with high resolution two-dimensional gel electrophoresis and microanalysis of protein spots, may allow the recognition of particular protein patterns in different diseases and may result in the recognition of new diagnostic proteins or therapeutic targets. New and more sensitive assays may also allow the detection of many other markers of inflammation and even specific fingerprints of activation of particular cell types within the respiratory tract, such as eosinophils, neutrophils, epithelial cells, and macrophages. This could have far-reaching potential for the diagnosis and treatment of many airway diseases.


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100. Nightingale JA, Maggs R, Cullinan P, Donnelly LE, Rogers DF, Kinnersley R, Fan CK, Barnes PJ, Ashmore M, Newman-Taylor A. Airway inflammation after controlled exposure to diesel exhaust particulates. Am J Respir Crit Care Med 2000; 162:161–166. 101. Togores B, Bosch M, Agusti AG. The measurement of exhaled carbon monoxide is influenced by airflow obstruction. Eur Respir J 2000; 15:177–180. 102. Stevenson DK, Vreman HJ. Carbon monoxide and bilirubin production in neonates. Pediatrics 1997; 100:252–254. 103. Delivoria-Papadopoulos M, Coburn RF, Forster RE. Cyclic variation of rate of carbon monoxide production in normal women. J Appl Physiol 1974; 36:49– 51. 104. Fischer AF, Nakamura H, Uetani Y, Vreman HJ, Stevenson DK. Comparison of bilirubin production in Japanese and Caucasian infants. J Pediatr Gastroenterol Nutr 1988; 7:27–29. 105. Zayasu K, Sekizawa K, Okinaga S, Yamaya M, Sasaki H. Increased carbon monoxide in exhaled air of asthmatic patients. Am J Respir Crit Care Med 1997; 156: 1140–1143. 106. Horvath I, Donnelly LE, Kiss A, Paredi P, Kharitonov SA, Barnes PJ. Elevated levels of exhaled carbon monoxide are associated with an increased expression of heme oxygenase-1 in airway macrophages in asthma: a new marker of oxidative stress. Thorax 1998; 53:668–672. 107. Yamara M, Sekizawa K, Ishizuka M, Monma M, Sasaki H. Exhaled carbon monoxide levels during treatment of acute asthma. Eur Respir J 1999; 13:757–760. 108. Stirling RG, Lim S, Kharitonov SA, Chung FK, Barnes PJ. Exhaled breath carbon monoxide is minimally elevated in severe but not mild atopic asthma. Am J Respir Crit Care Med 2000; 161:A922. 109. Horvath I, Barnes PJ. Exhaled monoxides in asymptomatic atopic subjects. Clin Exp Allergy 1999; 29:1276–1280. 110. Monma M, Yamaya M, Sekizawa K, Ikeda K, Suzuki N, Kikuchi T, Takasaka T, Sasaki H. Increased carbon monoxide in exhaled air of patients with seasonal allergic rhinitis. Clin Exp Allergy 1999; 29:1537–1541. 111. Culpitt SV, Paredi P, Kharitonov SA, Barnes PJ. Exhaled carbon monoxide is increased in COPD patients regardless of their smoking habit. Am J Respir Crit Care Med 1998; 157:A787. 112. Yamaya M, Sekizawa K, Ishizuka S, Monma M, Mizuta K, Sasaki H. Increased carbon monoxide in exhaled air of subjects with upper respiratory tract infections. Am J Respir Crit Care Med 1998; 158:311–314. 113. Horvath I, Loukides S, Wodehouse T, Kharitonov SA, Cole PJ, Barnes PJ. Elevated levels of exhaled carbon monoxide in bronchiectasis: a new marker of oxidative stress. Thorax 1998; 53:867–870. 114. Biernacki W, Kharitonov SA, Barnes PJ. Carbon monoxide in exhaled air in patients with lower respiratory tract infection. Eur Respir J 1998; 12:345S. 115. Antuni JD, Du Bois AB, Ward S, Cramer DS, Kharitonov SA, Barnes PJ. Exhaled carbon monoxide may be a marker of deterioration of lung function in cryptogenic fibrosing alveolitis and scleroderma. Am J Respir Crit Care Med 1999; 159:A51. 116. Paredi P, Shah PL, Montuschi P, Sullivan P, Hodson ME, Kharitonov SA, Barnes

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Kharitonov and Barnes PJ. Increased carbon monoxide in exhaled air of cystic fibrosis patients. Thorax 1999; 54:917–920. Montuschi P, Kharitonov SA, Ciabattoni G, Corradi M, van Rensen L, Geddes DM, Hodson ME, Barnes PJ. Exhaled 8-isoprostane as a new non-invasive biomarker of oxidative stress in cystic fibrosis. Thorax 2000; 55:205–209. Paredi P, Kharitonov SA, Leak D, Shah PL, Cramer D, Hodson ME, Barnes PJ. Exhaled ethane is elevated in cystic fibrosis and correlates with CO levels and airway obstruction. Am J Respir Crit Care Med 2000; 161:1247–1251. Antuni JD, Kharitonov SA, Hughes D, Hodson ME, Barnes PJ. Increase in exhaled carbon monoxide during exacerbations of cystic fibrosis. Thorax 2000; 55:138– 142. Scharte M, Bone HG, Van Aken H, Meyer J. Increased CO in exhaled air of critically ill patients. Biochem Biophys Res Commun 2000; 267:423–426.

9 Analysis of Volatile Organic Compounds in the Breath

MICHAEL PHILLIPS New York Medical College Valhalla, New York and Menssana Research, Inc. Fort Lee, New Jersey, U.S.A.

I.

History of Breath Tests

Breath tests date from the earliest history of medicine because physicians in ancient times knew that the odor of the breath is altered in some diseases (1). Even today the astute physician uses his or her nose to supplement sight, sound, and touch at the patient’s bedside. Some breath aromas are highly characteristic of disease: patients with diabetic ketoacidosis smell like rotting apples, mainly due to acetonemia. Chronic renal failure causes the breath to smell like stale urine, due to increased levels of dimethylamine and trimethylamine in the blood, and advanced liver failure causes the musty stench of “fetor hepaticus.” A patient with a lung abscess may smell like a sewer because of the proliferation of anaerobic bacteria, and indulgence in tobacco, alcohol, garlic, or curry each leaves its own distinctive olfactory signature in the breath. The era of scientific breath testing dawned in 1784, when Antoine Laurent Lavoisier, the father of modern chemistry, demonstrated that carbon dioxide is excreted in the breath of guinea pigs. This was the first evidence that metabolism of foodstuffs is analogous to the burning of fuel, a discovery which laid the basis of modern biochemistry. It was also the origin of the expression “to be a guinea 219

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pig,” because Lavoisier went on to confirm this finding in human volunteers. Colorimetric assays were developed during the nineteenth century which made it possible to detect volatile organic compounds (VOCs) present in the breath in millimolar (10 ⫺3 M) concentrations. The first breath test for ethanol (still the most common application of breath testing) was developed by a British physician, Francis Anstie, in 1874. He found that breath bubbled through chromic acid turned the solution from red-brown to green if alcohol was present. Nebelthau, in Germany, used an alkaline iodine solution to demonstrate that acetone in the breath of diabetics could also induce a change in color. But the modern era of breath testing did not commence until 1971, when Nobel Prize winner Linus Pauling turned his fertile mind to the problem. He used a cold trap, a tube chilled by dry ice, to freeze out breath VOCs. He then heated the sample and injected it into a gas chromatograph, and found that a sample of normal human breath contained hundreds of different VOCs, most of them in picomolar (10 ⫺12 M) concentrations. Pauling’s historic achievement (amongst his many others) was to provide the first evidence that human breath is a far more complex gas than anyone had previously suspected. He believed that analysis of breath VOCs could open a valuable new window onto human metabolism and illuminate its functions in health and disease. But only in recent years have we begun to achieve this objective (2). Breath testing for endogenous VOCs languished for some years as a mere scientific curiosity for two main reasons. First, it is technically very difficult to analyze breath VOCs present in picomolar concentrations. Second, after overcoming these difficulties, what then? More than 3000 different VOCs have been observed in human breath, and the biochemical significance of most of these compounds is still unknown (3,4). Thus, breath analysis became a solution in search of a problem. This historical background sets the stage for the three main questions which now dominate the scientific study of breath analysis: How should it be done? What do the results mean? And why should we do it? This chapter will focus on the first of these questions, with some incidental comments upon the second and the third.

II. Classification of Breath Tests Breath tests are of two kinds: load and no-load. In a load test, the patient consumes a drug or substrate (which may be labeled with a radionuclide), and its metabolites are subsequently measured in the breath. Load tests are used principally in gastroenterology, for the detection of diseases such as Helicobacter pylori infection and pancreatic insufficiency. No-load tests are confined to measuring

Analysis of Volatile Organic Compounds

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VOCs in the breath without any prior administration of a drug or substrate. This review will deal only with no-load tests. III. Breath VOC Analysis: The Major Technical Problems Technical problems commence at the instant breath exits from the lips, and afflict every step of the analytical process up to and including the final interpretation of the data. There are four main steps in the analytical process: 1. 2. 3. 4.

Capture the breath Concentrate the VOCs in the breath Analyze the concentrated VOCs Compensate for VOCs in background air

A. How to Capture Breath

What could be more intuitively easy than capturing breath? One simply asks a donor to blow into a tube which is attached by appropriate plumbing to a collection device. However, there are difficulties in capturing a sample of breath for subsequent analysis. Resistance to Expiration

It is obviously difficult to breathe out against resistance, especially for the elderly and those with respiratory illness. It is therefore equally obvious that any system for capturing human breath should present the least possible resistance to unimpeded gas flow. Tubing should be wide in bore (e.g., at least 2–3 cm in diameter) and contain little or no obstruction to the free flow of breath, such as valves or water traps. Curiously, there was a long-standing tradition among the pioneer breath researchers (including Lavoisier and Pauling) to design systems with such high internal resistance that only physically fit athletic youths could possibly donate a sample without discomfort. Infection Control

It is also obvious that the breath collection system should not present an infection hazard to the breath donor. A designer of a breath collection system should always plan for the worst-case scenario: What if a breath donor suffers from pulmonary tuberculosis? Could they contaminate the device with infectious organisms which may potentially be transmitted to subsequent breath donors? The device must employ a combination of disposable components and careful design to minimize this potential problem.

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Breath is saturated with water, which promptly condenses within the tubing of the capture system. Breath VOCs may then partition from the gas phase into the aqueous phase. Since water is very abundant and breath VOCs are present in very low concentrations, this partitioning process may deplete the gas phase of VOCs and result in inaccurate low readings in the subsequent assay. In addition, condensed water may be so abundant as to affect the integrity of the collecting system and might also affect the accuracy of an assay. The author has developed a breath collection apparatus (BCA) with heated tubing which inhibits condensation (3,4). This approach is effective, but it introduces additional complexity and expense. Chemical Contamination

Breath VOCs are present in picomolar concentrations, and the highly sensitive assays required to detect them may be readily contaminated by VOCs from other sources. These may include volatile adhesives and plasticizers in disposable components. The breath collection apparatus must therefore be constructed from components which are least likely to contribute VOCs, e.g., stainless steel and inactive plastics such as polycarbonate and Teflon. Disposable plastic tubing of the type employed in anesthetic and ventilator systems should be avoided. Dead-Space Air Dilution

Breath is not a homogeneous gas. At rest, an adult expires approximately 500 mL with each breath, of which the first 150 mL is dead-space air from the upper airways and nasopharynx, and the subsequent 350 mL is alveolar breath from within the lungs. For analytical purposes, dead-space air is useless. Volatile organic compounds in breath and air interchange at the alveolar membrane, so only alveolar breath is of any value for analytical purposes. There are two approaches to this problem, mixed sample collection and alveolar breath collection. Mixed Sample Collection

A mixture of dead space and alveolar breath is collected (5). Advantage. Simplicity: the patient simply inflates the collection system, e.g., a bag or balloon. Disadvantage. Inaccuracy: the sample is diluted by dead space air in which no interchange has occurred. This degree of inaccuracy is not a constant which can be ignored, because the dilution factor varies with the tidal volume, i.e., whether the donor is breathing deeply or shallowly. Alveolar Breath Collection

A breath capture system can be designed with special geometry which ensures that the collected sample is virtually 100% alveolar breath (6). Breath collection


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bags are available with a side port which diverts the first few hundred milliliters of expired breath into an ancillary side bag, so that only alveolar breath enters the main bag (Quintron Instruments Company, Menomonee Falls, WI). The author has developed a breath collection apparatus (BCA) in which breath enters a long tube, the breath reservoir. The geometry of the breath reservoir ensures that dead space air and alveolar breath are separated, so that only alveolar breath is pumped out to the collection system. Advantage. Accuracy: the assayed sample is not diluted by dead-space air. Disadvantage. Complexity and expense. Breath Container Artifact

Probably the simplest and most straightforward way to capture a sample of breath is to ask the donor to blow up a balloon or a bag. This method was employed for breath ethanol testing before digital breathalyzers were available. Typically, samples are collected into a comparatively inert plastic bag (e.g., Tedlar or Teflon), which is then taken to the laboratory for analysis by any desired method (7,8). A more elaborate though conceptually identical approach is to collect breath into a partially evacuated metal cylinder or sphere (9,10). However, use of a container entails a risk of artefactual loss of sample, due to adsorption of VOCs to the walls of the container. There are two approaches to this problem, collecting breath into a container, and collecting breath directly into a trapping system. Collecting Breath into a Container

Advantages 1. The method is simple and straightforward to use. 2. Containers can be reused after cleaning. Disadvantages 1. 2. 3. 4. 5.

Sample can be lost by adsorption to the walls of the container. Containers are often difficult to render chemically clean for reuse. Containers are expensive, particularly if constructed of metal. Inflated containers are bulky and may be difficult to transport. The sample is mixed dead space and alveolar breath, unless a specialized collecting system employed.

Collecting Breath Directly into a Trapping System

Advantages. Avoidance of the disadvantages of containers. Disadvantages. Additional complexity of design, and expense.

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After the breath sample has been captured, it is generally necessary to concentrate the VOCs for assay with laboratory instrumentation. Four methods are in current use: cold trapping, condensate trapping, chemical trapping, and sorbent trapping. Cold Trapping

Cold trapping was the method employed by Pauling et al. to demonstrate VOCs in concentrated breath. Typically, a donor blows through a U-tube which is immersed in a cryogenic fluid (e.g., liquid nitrogen or acetone chilled with dry ice) (11,12). The U-tube may be packed with glass beads which provide a large surface area on which the breath VOCs can condense. The sample is then heated, and the volatilized concentrated VOCs are then analyzed by conventional laboratory methods, e.g., gas chromatography (GC) possibly combined with mass spectroscopy (MS). Advantages. Highly efficient VOC trapping; very effective when it works well. Disadvantages 1. 2. 3. 4.

Difficult to employ outside a laboratory. Icing of water and CO 2 rapidly blocks the U-tube and limits the volume of breath that can be expired through the system. High risk of leakage when sample is heated, because volatilized CO 2 raises pressure in the system. Large quantities of water in the sample may interfere with assay and damage the GC column and detector.

Condensate Trapping

Condensate Trapping is a variant of cold trapping in which the sample is cooled only moderately. A similar system to cold trapping is employed, but the cooling fluid is usually ice water at 0°C. VOCs partition into the water which condenses from the breath, and this sample of condensed water is then analyzed by conventional laboratory methods, e.g., high-performance liquid chromatography (HPLC), or gas chromatography (GC) possibly combined with mass spectroscopy (MS). Advantages. The method is simple and inexpensive. Disadvantages 1. 2.

VOC trapping is inefficient; only a small number of VOCs are collected in low concentrations. Risk of contamination with nonvolatile components of breath, e.g., proteins, in aerosol.


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Chemical Trapping

For chemical trapping, breath is bubbled through a solution which interacts chemically with the analyte of interest. The solution may change color, and/or the sample may be analyzed in the laboratory. This was the basis of Lavoisier’s method for detecting CO 2 in the breath and of the colorimetric methods for ethanol and acetaldehyde described above. These methods are of great antiquity, dating from the eighteenth and nineteenth centuries, and have been largely displaced by more modern techniques. However, modern variants have been described for the detection of carbon disulfide and mercury in breath, as well as for the capture of radiolabeled analytes in load tests for GI diseases such as H. pylori infection. Advantages 1. 2. 3. 4. 5.

Simple and inexpensive. Capture and concentration steps are combined. Convenient for sample collection outside a laboratory. May give color change which can be read by eye without instruments. Convenient for analytes present in high concentrations.

Disadvantages 1. Repertoire limited to a single analyte. 2. Poor sensitivity; cannot detect most endogenous VOCs. 3. High resistance to expiration; may limit use in elderly and in respiratory disease. Sorbent Trapping

In sorbent trapping, breath is passed through a bed of a material such as activated carbon or a specialized resin (e.g., Tenax) which captures the VOCs. The process is reversible, so that the captured VOCs may be subsequently eluted by heating (13) or by chemical stripping with a solvent (14,15). This method of breath VOC analysis has been in use in one form or another for many years, but has recently become the method of choice for many breath researchers. This was an indirect result of federal clean air regulations in the United States; the U.S. Environmental Protection Agency now mandates the monitoring of volatile pollutants in the air. These regulations stimulated manufacturers to develop a new generation of instruments for the capture and analysis of air VOCs. Manufacturers and researchers were pleasantly surprised to discover that this technology also provided a sensitive and convenient new method for the analysis of VOCs in breath. A breath VOC sample is captured on to a sorbent trap, typically a stainless steel tube packed with activated carbon or resin. In the laboratory, the VOCs are

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eluted with an automated thermal desorber, then analyzed by gas chromatography. Advantages 1. 2. 3. 4.

Convenient for sample collection outside a laboratory. Captures a wide variety of VOCs in breath. Highly sensitive; can detect VOCs in picomolar concentration. Traps can be reused several times.

Disadvantages 1. 2. 3. 4. 5.

Traps are too resistant to blow through, so that a specialized pumpassisted breath collecting apparatus is required. Technology is expensive and complicated. Trapping material may be selective, capturing some VOCs efficiently and others inefficiently. Water and CO 2 in breath may interfere with VOC trapping. Cleaning traps for reuse requires scrupulous and time-consuming quality assurance.

C. How to Analyze Breath VOCs Portable Hand-Held Instruments

The most familiar hand-held instruments are the “breathalyzers” for ethanol which have been employed by police forces for several years. These devices generally employ a fuel cell which oxidizes ethanol in breath to acetaldehyde, and the resulting electrical current (which varies with the concentration of ethanol in breath and blood) is displayed digitally. In recent years, newer devices have become available for analysis of other volatiles in breath such as nitric oxide, sulfur derivatives, and carbon monoxide. Advantages 1. 2.

These instruments are very convenient to use and deliver results in seconds. Small sample volume ensures that alveolar breath sample is collected, uncontaminated by dead-space breath.

Disadvantages 1. 2. 3.

Analytical repertoire is usually restricted to a single VOC. Accuracy, precision, and sensitivity are generally poorer than laboratory instruments. Calibration in the field may be difficult.


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Conventional Laboratory Instruments

The instrument of choice in most breath research laboratories is currently gas chromatography. A wide variety of detectors may be employed (e.g., flame ionization detection, flame photometric detection), depending on the analyte of interest. In recent years, mass spectroscopy has become increasingly popular as a universal detector. However, other instruments may potentially be employed for breath VOC analysis. Absorption spectrometry offers the potential of direct analysis of breath VOCs without prior concentration or separation of the sample. Advantages.

High sensitivity, accuracy, and precision.

Disadvantages 1. Large, immobile instruments generally mandate sample analysis in a laboratory. 2. Expensive and complicated. 3. Labor-intensive; require dedicated laboratory staff and frequent extensive maintenance. D. How to Compensate for VOCs in Background Air

As assays of concentrated breath became increasingly sensitive, researchers learned that a sample of normal room air also contains most of the VOCs which are present in the breath (16). Cailleux and Allain (17) reported that pentane could be detected in room air in concentrations not very different from those in breath. This finding initially caused some consternation: it raised the specter that the infant science of breath VOC analysis was not a science at all. Perhaps the phenomenon was nothing more than an artefact of room air contamination? Fortunately, we now know that this is not the case: VOCs are manufactured and cleared in the body, and the composition of inspired air is different from that of expired breath (3). Researchers who analyze VOCs in breath must compensate somehow for the VOCs that are present in background air. There are essentially three options: 1. Ignore the problem. 2. Supply the donor with hydrocarbon-free air to wash out the lungs. 3. Subtract the air background from the breath signal. Ignoring the Problem

The option of ignoring the problem has not been stated frivolously. The overwhelming majority of papers on the topic of breath VOC analysis published in the peer-reviewed literature during the 30 years preceding the year 2000 make no mention at all of this problem.

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Advantages. None. Disadvantages. Breath VOC data are skewed and difficult (or possibly impossible) to interpret. Supplying the Donor with Hydrocarbon-Free Air to Wash out the Lungs

The rationale for supplying the donor with hydrocarbon-free air is that VOCs in breath comprise “signal,” while VOCs in air comprise “noise.” Hence, if the subject inspires hydrocarbon-free air, the VOCs in the expired breath should comprise pure signal uncontaminated by noise, and therefore provide a clear picture of what VOCs are being manufactured in the body. This approach is more theoretical than actual, and has not been validated in a rigorously controlled study. Advantages. In theory, could provide an unambiguous picture of breath VOCs manufactured in the body. Disadvantages 1. 2.

3. 4.

5.

Has not been validated in practice. Commercial “hydrocarbon-free air” is generally not free of hydrocarbons, when an assay sensitive for VOCs in picomolar concentrations is employed. Several VOCs in the body are of exogenous origin, and may require several hours or days to wash out completely (18). The delivery system for delivering air to humans (cylinder valves, flow meter, tubing, and mask) may also introduce contaminant VOCs into the air. Expensive, inconvenient, and time-consuming.

Subtracting the Air Background from the Breath Signal

Another method has been developed by the author (3,4,19,20). In practice, it requires that two samples be collected every time a patient is studied: one of breath and one of room air. Both samples are analyzed in the same way. For each VOC in breath, the alveolar gradient is then calculated as concentration in breath minus concentration in room air. Kinetic analysis has shown that the alveolar gradient of a VOC varies with the rate of synthesis minus the rate of clearance in the body. This approach has generated the surprising finding that approximately half the VOCs in the breath are present in lower concentrations than in room air; i.e., they are actively cleared from the body by metabolism and excretion. Advantages 1. 2.

Provides rational compensation for VOCs in room air. Provides an insight into the kinetics of VOCs in the body.


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Disadvantages 1. Increases the investment of time and money required for analysis of samples. 2. Increases the statistical error of the assay. IV. Conclusions This review has focused exclusively on one question about breath VOC analysis: How should it be done? It should be apparent to the reader that breath VOC analysis is a perfectly feasible undertaking, but it is fraught with a large number of potential difficulties, pitfalls, and sources of error. All of these can be overcome, more or less, as the author has shown over some years of wrestling with these problems. His laboratory employs a number of portable breath collectors which are shared among academic medical centers around the United States and in Europe. Collaborators collect samples from patients enrolled in a variety of clinical studies and sorbent traps are mailed to the laboratory for analysis with automated analytical equipment. This has “democratized” breath testing and made it possible, for the first time, to perform large-scale evaluations of breath VOC analysis. However, as discussed at the beginning of this chapter, there are two other important questions about breath testing: What do the results mean? And why should we do it? Slowly, some answers are emerging. One of the most exciting answers to the question of “What do the results mean?” is that breath VOCs are providing new markers of oxidative stress. Formerly, ethane and pentane were the only known breath markers of oxidative stress (21), but it is now emerging that breath VOCs contain many others. And why should we analyze breath? Clearly, because breath VOC analysis offers the prospect of sensitive and specific new markers of disease. A breath test for lung cancer, for example, could make it possible for physicians to detect the disease in its earliest stages and potentially save or extend many thousands of lives (22,23). The author’s laboratory is currently performing clinical studies of breath testing in several disorders, including breast cancer, heart transplant rejection, and ischemic heart disease. That is the vision which sustains breath testing, and makes it one of the most exciting and innovative areas of biomedical research in the twenty-first century. References 1. Phillips M. Breath tests in medicine. Sci Am 1992; 267:74–79. 2. Cheng WH, Lee WJ. Technology development in breath microanalysis for clinical diagnosis. J Lab Clin Med 1999; 133:218–228.

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3. Phillips M, Herrera J, Krishnan S, Zain M, Greenberg J, Cataneo RN. Variation in volatile organic compounds in the breath of normal humans. J Chromatogr B Biomed Sci Appl 1999; 729:75–88. 4. Phillips M. Method for the collection and assay of volatile organic compounds in breath. Anal Biochem 1997; 247:272–278. 5. Droz PO, Krebs Y, Nicole C, Guillemin M. A direct reading method for chlorinated hydrocarbons in breath. Am Ind Hyg Assoc J 1988; 49:319–324. 6. LeBlanc A, Levesque B, Allaire S. Rapid, sensitive, and noninvasive sampling technique for determination of chloroform in alveolar breath. J Anal Toxicol 1995; 19: 56–57. 7. Hotz P, Hoet P, Lauwerys R, Buchet JP. Development of a method to monitor low molecular mass hydrocarbons in exhaled breath of man: preliminary evaluation of its interest for detecting a lipoperoxidation process in vivo. Clin Chim Acta 1987; 162:303–310. 8. Schoeller DA, Klein PD. A simplified technique for collecting breath CO 2 for isotope ratio mass spectrometry. Biomed Mass Spectrom 1978; 5:29–31. 9. Thomas KW, Pellizzari ED, Cooper SD. A canister-based method for collection and GC/MS analysis of volatile organic compounds in human breath. J Anal Toxicol 1991; 15:54–59. 10. Pleil JD, Lindstrom AB. Measurement of volatile organic compounds in exhaled breath as collected in evacuated electropolished canisters. J Chromatogr B Biomed Appl 1995; 665:271–279. 11. Knutson MD, Viteri FE. Concentrating breath samples using liquid nitrogen: a reliable method for the simultaneous determination of ethane and pentane [published erratum appears in Anal Biochem 1999 May 15; 270(1):186]. Anal Biochem 1996; 242:129–135. 12. Dannecker JR Jr, Shaskan EG, Phillips M. A new highly sensitive assay for breath acetaldehyde: detection of endogenous levels in humans. Anal Biochem 1981; 114: 1–7. 13. Grote C, Pawliszyn J. Solid-phase microextraction for the analysis of human breath. Anal Chem 1997; 69:587–596. 14. Zabiegala B, Namiesnik J, Przyk E, Przyjazny A. Changes in concentration levels of selected VOCs in newly erected and remodelled building in Gdansk. Chemosphere 1999; 39:2035–2046. 15. Rappaport SM, Kure E, Petreas M, Ting D, Woodlee J. A field method for measuring solvent vapors in exhaled air—application to styrene exposure. Scand J Work Environ Health 1991; 17:195–204. 16. Fenske JD, Paulson SE. Human breath emissions of VOCs. J Air Waste Manag Assoc 1999; 49:594–598. 17. Cailleux A, Allain P. Is pentane a normal constituent of human breath? Free Radic Res Commun 1993; 18:323–327. 18. Wallace LA, Nelson WC, Pellizzari ED, Raymer JH. Uptake and decay of volatile organic compounds at environmental concentrations: application of a four-compartment model to a chamber study of five human subjects. J Expo Anal Environ Epidemiol 1997; 7:141–163.


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19. Phillips M, Greenberg J, Sabas M. Alveolar gradient of pentane in normal human breath. Free Radic Res 1994; 20:333–337. 20. Phillips M, Sabas M, Greenberg J. Increased pentane and carbon disulfide in the breath of patients with schizophrenia [published erratum appears in J Clin Pathol 1994 Sep; 47(9):870]. J Clin Pathol 1993; 46:861–864. 21. Kneepkens CM, Lepage G, Roy CC. The potential of the hydrocarbon breath test as a measure of lipid peroxidation [published erratum appears in Free Radic Biol Med 1994 Dec; 17(6):609]. Free Radic Biol Med 1994; 17:127–160. 22. Gordon SM, Szidon JP, Krotoszynski BK, Gibbons RD, O’Neill HJ. Volatile organic compounds in exhaled air from patients with lung cancer. Clin Chem 1985; 31: 1278–1282. 23. Phillips M, Gleeson K, Hughes JM, et al. Volatile organic compounds in breath as markers of lung cancer: a cross-sectional study [see comments]. Lancet 1999; 353: 1930–1933.

Part Three PATHOLOGICAL ASPECTS

HYPOXIA

10 Regulation of Nitric Oxide Synthases and Gas-Phase Nitric Oxide by Oxygen

RAED A. DWEIK and SERPIL C. ERZURUM The Cleveland Clinic Foundation Cleveland, Ohio, U.S.A.

I.

Introduction

Nitric oxide (NO) is endogenously synthesized by nitric oxide synthases (NOS) which convert L-arginine to L-citrulline and NO. Three NOS isoforms (types I, II, and III) have been identified in the human lung (1–5). NOSI and NOSIII are dependent on increases in intracellular calcium for enzyme activation, while NOSII is calcium independent (6). All NOS isoforms require oxygen, NADPH, FAD, FMN, tetrahydrobiopterin, and calmodulin for activity (6,7). In this chapter we will focus on the current knowledge of the crucial role of oxygen in regulating NOS expression and activity and NO levels in the lung. II. Source(s) of NO in Exhaled Breath NO is produced in the human lung, evidenced by NO detectable in the exhaled air of humans (6–8 ppb) and NO metabolites detectable in the airway aspirate and bronchoalveolar lavage fluid from human lungs (2,8). NO is recognized to play key roles in virtually all aspects of lung biology and has been implicated in the pathophysiology of lung diseases (2,9–13). NO in the lung is involved in 235

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pulmonary neurotransmission, host defense and bacteriostasis, airway and vascular smooth-muscle relaxation, pulmonary capillary leak, inflammation, mucociliary clearance, airway mucus secretion, and cytotoxicity (12,13). Cellular sources of NO in the lung include epithelial cells, endothelial cells of pulmonary arteries and veins, inhibitory nonadrenergic noncholinergic neurons, smooth muscle cells, mast cells, mesothelial cells, fibroblasts, neutrophils, lymphocytes, and macrophages (2,3,12,13). All three NOS isoforms are present in the human lung (1,2,7). Specifically, NOSI is located in inhibitory nonadrenergic noncholinergic neurons in the lung, while NOSIII is found in endothelial cells and the brush border of ciliated epithelial cells (1,2,7). NOSII is found in the epithelial cells of the airway. Although NOSII may be induced in several types of cells in response to cytokines, endotoxin, or reactive oxygen species, NOSII is continuously expressed in normal human airway epithelium at basal airway conditions (5). Once produced, NO is freely diffusible and enters target cells, activating soluble guanylate cyclase to produce guanosine 3′,5′-cyclic monophosphate (cGMP), which mediates the majority of NO effects (7). NO also diffuses into the airway and can be measured in the gas phase (8). Potential anatomic sources of NO in exhaled breath include the pulmonary circulation, the lower airways, and the upper airways and paranasal sinuses (7,8,14). NO is formed in high concentrations in the upper respiratory tract (nasopharynx and paranasal sinuses) (14), but several studies have conclusively demonstrated that NO is also produced in the lower respiratory tract (7,8). Studies of gas-phase NO in the airway have been helpful in determining the anatomical source(s) of NO in the lung. During a breath-hold, NO accumulates in bronchiolar gases in an exponential fashion with an initial linear rise, followed by a plateau (Fig. 1). The steady-state NO levels in the bronchiolar gases of the lung are achieved within seconds. Attainment of steady-state levels indicates a constant rate of production balanced by a constant consumption or scavenging of NO (8,15). Since NO is freely diffusible, consumption of NO can occur at different sites within the cell, lung tissue, extracellular fluids, and intravascular compartments. Primary reactions that may consume NO intra- and extracellularly include its reaction with oxygen, superoxide, hemoglobin, another molecule of NO, enzymes containing iron–sulfur centers, heme-containing proteins, and thiol proteins (16). An especially important scavenger of NO in the lung is hemoglobin (8,15–17). NO produced in the lungs may diffuse into the lumen of blood vessels, where most will be trapped by oxy- and deoxyhemoglobin in red blood cells (15,17). The addition of even very small amounts of hemoglobin results in substantial decrease in the steady-state distribution of NO in vitro (15). Furthermore, bronchoscopic measurements of airway NO in humans reveal that bronchiolar but not alveolar gases accumulate NO during a breath-hold (8). During an expiratory breath-hold, bronchiolar gases accumulate NO quickly to a plateau. At the end of expiratory breath-hold, complete exhalation to residual volume causes NO

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Figure 1 Kinetics of NO accumulation in the gas phase of a bronchiole in the normal human lung measured in a segmental bronchus during bronchoscopy. Following a deep inspiration to total lung capacity with inspiratory breath-hold for 8 sec (shaded area 1), the volunteer exhaled until NO levels at the sampling catheter in the bronchiole approached zero, which reflects NO-“free” alveolar gases reaching the level of the catheter. At this point, the individual started breath-hold (shaded area 2). During the expiratory breathhold, bronchiolar gases accumulated NO quickly to a plateau. At the end of expiratory breath-hold, the individual exhaled (indicated by arrow) completely to residual volume. Rapid decrease of NO with exhalation and near-zero NO levels at the end of exhalation indicate that alveolar gases do not accumulate NO. Once the volunteer resumed tidal breathing (marked on the X axis), NO was delivered to the lung in a pulsatile fashion that included NO from the upper (nasal) and lower airways.

levels to decrease rapidly (8). Near-zero NO levels observed at the end of exhalation indicate that alveolar gases do not accumulate NO (Fig. 1). Studies in rabbits also demonstrated that NO levels in exhaled gases remained after blood circulation was stopped by intravenous injection of air or helium (18) or when blood was replaced by a physiological buffer (19). Despite this, at least some of the endogenously produced NO diffuses into the airway and is eliminated via the gas phase. Thus, while NO levels in the gas phase likely underestimate NO levels in the lung tissue and at pulmonary vascular sites, the gas-phase levels reflect in an accurate and qualitative manner the dynamics of NO production and consumption in the lung. The capacity of the blood as a scavenger of NO can be estimated from experiments of isolated perfused lungs (19). Exhaled NO output from bloodperfused lungs is less than half the amount from buffer-perfused lungs (19), attesting to the large scavenging capacity of the blood in the pulmonary circulation. Exhaled NO levels also increase in rabbits with experimental anemia (20). A progressive fall in the hematocrit from 30% to 11% by serial isovolemic hemo-

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dilution in rabbits causes a significant and parallel increase in expired NO levels (20). Considering the high diffusibility of NO, the extremely rapid rate of scavenging by hemoglobin, and the rich supply of blood vessels found in the lung, the pulmonary circulation is undoubtedly a significant biological sink for NO, and not likely to contribute to NO in exhaled breath.

III. Regulation of NO Synthesis by Oxygen All NOS isoforms require the presence of oxygen for activity (6). Although it is recognized that oxygen is a substrate for NOS, its effects on regulation of NOS activity are more complex than simple enzyme–substrate interaction (8,21–25). One mechanism to explain oxygen’s effect on NO levels in the lung is revealed by enzyme kinetic analyses. All NOS are comprised of an oxygenase domain that contains binding domains for iron protoporphyrin IX (heme), tetrahydrobiopterin, and L-arginine, and a reductase domain that contains binding domains for FMN, FAD, and calmodulin (6). The NOS heme iron participates in catalysis by binding oxygen and catalyzing the oxidation of L-arginine (21–25). NOSI activity is dependent on oxygen concentration (K M O 2 400 µM) (21,22). When NO is formed during catalysis, it binds to the heme iron in the catalytic site of NOSI and forms an inactive heme iron–NO complex (19,20). The rate of decay of the complex is subsequently dependent on oxygen concentration to enter the active catalytic cycle (19,20). Studies of purified NOSII activity in vitro as determined by the rate of NADPH consumption demonstrate that NOSII activity is dependent on molecular oxygen concentrations in the physiologically relevant range (K M O 2 135 µM) (8). Although the K M O 2 of NOSII is lower than that of NOSI, NOSII has also been shown to form heme iron–NO complexes. However, studies using scavengers of NO suggest that the mechanism of regulation by NO and oxygen is not similar between the two enzymes (8,26). Recent work utilizing rapid kinetic analysis of the purified NOSIII enzyme in vitro also confirms significant dependence on oxygen (24). The formation of active NOSIII complex is first-order with respect to oxygen, reversible, and follows a simple one-step mechanism. However, decay of the complex is independent of oxygen concentration and occurs via a one- or two-exponential process depending on cofactors or substrate availability (22,24). Based on double-reciprocal plots of NOSIII activity and oxygen concentration in vitro, the apparent NOSIII K M O 2 is strikingly low (4 µM) and consistent with very little heme–NO complex formation in this isoform (24). Taken together, oxygen is less likely relevant to NOSIII enzyme activity in vivo. One proposed mechanism(s) for oxygen regulation of NOS activity is outlined in Figure 2.


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Figure 2 The mechanism of oxygen regulation of NOSI enzyme kinetics. NOS activity during steady state includes an active cycle (A) that generates NO, and an inactive cycle (B) that involves formation and decay of a heme–NO complex. In the active cycle, oxygen binding to ferrous heme (Fe 2⫹) is limiting for enzyme activity. In contrast, resolution of the inactive cycle and entry into active cycle is oxygen-dependent due to effects on the heme–NO complex stability. This includes a reaction between the heme–NO complex and oxygen which results in loss of heme–NO complex. (From Ref. 21.)

IV. Effect of Oxygen on Exhaled NO As predicted from the above enzyme kinetic studies, changes in inspired oxygen concentration lead to significant changes in exhaled NO. NO in exhaled gases from lungs of anesthetized rabbits ventilated with hypoxic gas mixtures are only modestly reduced using 0.14 or 0.10 FiO 2, but decrease markedly using 0.06 FiO 2 (18) (Fig. 3a). In addition, ventilation of an isolated neonatal pig lung with 0.075 FiO 2 rapidly decreases NO in exhaled gases (Fig. 3c) and NO 2⫺ /NO3⫺ in the recirculating perfusate as compared to ventilation with 0.21 FiO 2 (27). Similarly, exhaled NO levels correlate to oxygen levels in the hypoxic range in humans, decreasing as oxygen levels decrease below ambient air (Fig. 3b) (8). Interestingly, the effect of hypoxia on NO levels in the airway is primarily a result of airway and alveolar oxygen tension (FiO 2) rather than vascular oxygen tension (pO 2) (18,19,27–29). In models of isolated perfused lungs (18,19), inspired hypoxic gases result in significant decline in NO output from the lung, while vascular hypoxia has no significant effect on NO lung output. The results are similar whether the lungs are perfused with blood or a physiological buffer. On the other hand, NO metabolites continue to accumulate in the perfusate (29), suggesting a vascular source of NO that may not be affected by oxygen. Differential regulation of lung NO synthesis in response to hypoxia suggests a complex model for NO production in the lung that involves at least two

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different compartments. The airway compartment with predominant NOSII expression in the airway epithelium is capable of rapidly changing NO output depending on inspired oxygen, couples ventilation–perfusion, and thus mediates hypoxic pulmonary vasoconstriction (Fig. 3). In contrast, the vascular compartment predominantly expresses NOSIII, which is less affected by changes in oxygen. NOSIII may be responsible for a continuous low level of NO which modulates the effect of high output NO production by NOSII on the pulmonary circulation. In support of this, genetically engineered NOSIII-null mice have only a slight increase in pulmonary artery pressures (30), but an exaggerated vasoconstrictive response to hypoxia. Furthermore, NOSIII-null murine lungs have significantly higher NOSII expression than the wild-type mice (30).

(a) Figure 3 NO levels are dependent on inspired oxygen levels. (a) Expired NO from anesthetized rabbit decreases during inhalation of a hypoxic (6% FiO 2) gas mixture indicated by the solid horizontal lines. (b) NO measured at the mouth during tidal breathing from a human with varying fractions of inspired oxygen (FiO 2). NO decreases with decreasing FiO 2 . (Inset) Exhaled O 2 and CO 2 levels with varying FiO 2 . (c) In isolated pig lung, expired NO (solid line) drops rapidly in response to hypoxia initiated at 30 min. This decrease is accompanied by an elevation in the lung perfusion pressure (dashed line connecting circles). Increasing oxygen at 60 min reverses these effects. This coupled and immediate response suggests that hypoxic pulmonary vasoconstriction is the result of inhibition of basal NO production. (From Refs. 8, 18, and 27.)


(b)

(c)

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Oxygen concentration in intact tissues ranges from 1 to 150 µM (2,8,31,32), with the highest levels found in the lung. Airway epithelial cells are unique in their exposure to oxygen, since above a thin layer of epithelial lining fluid, the airway cells are exposed directly to air containing 21% oxygen. Based on oxygen solubility and the low differential oxygen gradient between overlying fluid to intracellular endoplasmic reticulum (1–2 µM) (32), the levels of oxygen in airway epithelial cells may actually approach 260 µM. Thus, the K M O 2 determined for NOSII, but not NOSIII or NOSI, is well within the physiological range of oxygen concentrations in lung epithelial cells. Importantly, K M O 2 for NO synthesis in the intact human lung (190 µM) is similar to NOSII K M O 2 in vitro (8). Taken together, these studies support a primary role for NOSII in maintenance of low pulmonary vascular resistance, and in the pulmonary vasoconstrictor response to hypoxia. V.

Regulation of NOS Gene Expression by Oxygen

The immediate effects of short-term changes in oxygen concentration on NOS enzymes activity are likely due to the effects of oxygen on NOS enzyme kinetics. However, prolonged hypoxia can have significant effects on the gene expression of the different NOS isoforms (33,34). These transcriptional effects may vary among species or among organ systems in the same species. While hypoxia produces a progressive decline in constitutive NOS mRNA levels in bovine pulmonary artery endothelial cells (33–35), chronic hypoxia upregulates constitutive NOS expression in rabbit hearts (36) and rat lung pulmonary arteries (37). Chronic hypoxia also increases NOS expression and NOS activity in rat carotid bodies (38). VI. NO as a Mediator of Vascular Response to Oxygen Oxygen is the major physiologic regulator of ventilation perfusion matching in the lung, through vasoconstriction of pulmonary vessels in regions of low ventilation containing low oxygen levels (8,27,39–41). Oxygen regulation of pulmonary vascular tone may be mediated in part by NO (8,27,39–41). Studies in pulmonary endothelial cells, isolated pulmonary vascular rings, isolated perfused lungs, and whole animals support an important role for NO in modulating the pulmonary vascular response to oxygen (8,27,35–41). Due to the free diffusion of NO and the close apposition of airways to medium sized pulmonary vessels which modulate pulmonary vessel tone (42), endogenous NO production in airways proximal to the alveolus may modulate pulmonary vasodilatation. Furthermore, hemoglobin in blood vessels may serve as a natural biological sink for NO, creating a continuous concentration gradient for NO to move toward perivascular myocytes


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and thus regulate blood flow. Taken together, this evidence supports a mechanism in which NOSII mediates vascular response to oxygen in the lung by generating the vasodilator NO at a rate that is proportional to the inspired oxygen concentration throughout the physiologic range. NOSIII in pulmonary vessels may modulate vasoconstrictive response by continuous low-level NO synthesis despite severe hypoxia, but is unlikely to play a significant role in the pulmonary vascular response to hypoxia in the physiological range because of its very low K M O 2 . In summary, NO in exhaled breath is derived primarily from the airway. The pulmonary circulation likely affects exhaled NO levels by acting as a biological sink that consumes NO and creates a gradient toward the pulmonary circulation. Oxygen regulates NOS expression and the enzyme activity. Rapid response of endogenous NO in direct proportion to inspired oxygen strongly supports a role for NOSII as a biochemical oxygen sensor and critical mediator of ventilation– perfusion coupling in the lung. Acknowledgments We thank H. M. Abu-Soud and D. J. Stuehr for helpful discussions. References 1. Kobzik L, Bredt DS, Lowenstein CJ, Drazen J, Gaston B, Sugarbaker D, Stamler JS. Nitric oxide synthase in human and rat lung: immunohistochemical and histochemical localization. Am J Respir Cell Molec Biol 1993; 9:371–377. 2. Gaston B, Drazen JM, Loscalzo J, Stamler JS. The biology of nitrogen oxides in the airways. Am J Respir Crit Care Med 1994; 149:538–551. 3. Guo FH, Uetani K, Haque J, Williams BRG, Dweik RA, Thunnissen FBJM, Calhoun W, Erzurum SC. Interferon-γ and interleukin-4 stimulate prolonged expression of inducible nitric oxide synthase in human airway epithelium through synthesis of soluble mediators. J Clin Invest 1997; 100:829–838. 4. Dweik RA, Guo FH, Uetani K, Erzurum SC. Nitric oxide synthase in the human airway epithelium. Acta Pharmacol Sinica 1997; 18:550–552. 5. Guo FH, De Raeve HR, Rice TW, Stuehr DJ, Thunnissen FBJM, Erzurum SC. Continuous nitric oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo. Proc Natl Acad Sci USA 1995; 92:7809–7813. 6. Stuehr DJ, Griffith OW. Mammalian nitric oxide synthases. Adv Enzymol Relat Areas Molec Biol 1992; 65:287–346. 7. Dweik RA, Erzurum SC. Effects of nitric oxide and cGMP on smooth muscle proliferation. In: Moss J, ed. LAM and other diseases characterized by smooth muscle proliferation. New York: Marcel Dekker, 1999:131:333–349. 8. Dweik RA, Laskowski D, Abu-Soud HM, Kaneko FT, Hutte R, Stuehr DJ, Erzurum SC. Nitric oxide synthesis in the lung: regulation by oxygen through a kinetic mechanism. J Clin Invest 1998; 101:660–666.

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9. Kaneko FT, Arroliga AC, Dweik RA, Comhair SA, Laskowski D, Oppedisano R, Thomassen MJ, Erzurum SC. Correlation of nitric oxide reaction products to severity of pulmonary hypertension. Am J Respir Crit Care Med 1998; 158:917–923. 10. Raychaudhuri B, Dweik R, Connors MJ, Buhrow LT, Malur A, Drazba J, Erzurum SC, Kavuru MS, Thomassen MJ. Nitric oxide blocks NFkB activation in alveolar macrophages in asthma and primary pulmonary hypertension. Am J Respir Cell Molec Biol 1999; 21:311–316. 11. Thomassen, MJ, Raychaudhuri B, Dweik RA, Farver C, Buhrow LT, Malur A, Hammel J, Erzurum SC, Kavuru MS. Effect of segmental allergen challenge on airway nitric oxide, eosinophils, and cytokines in asthmatics. J Allergy Clin Immunol 1999; 104:1174–1182. 12. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J 1992; 6:3051–3064. 13. Schmidt HHHW, Walter U. NO at work. Cell 1994; 78:919–925. 14. Lundberg JON, Farkas-Szallasi T, Weitzberg E, Rinder J, Lidholm J, Anggard A, Hokfelt T, Lundberg JM, Alving K. High nitric oxide production in human paranasal sinuses. Nat Med. 1995; 1:370–373. 15. Lancaster JR Jr. Simulation of the diffusion and reaction of endogenously produced nitric oxide. Proc Natl Acad Sci USA 1994; 91:8137–8141. 16. Wink DA, Hanbauer I, Grisham MB, Laval F, Nims RW, Laval J, Cook J, Pacelli R, Liebmann J, Krishna M, Ford PC, Mitchel JB. Chemical biology of nitric oxide: regulation and protective and toxic mechanisms. Curr Topics Cell Regul 1996; 34: 159–187. 17. Goldstein S, Czapski G. Kinetics of nitric oxide autoxidation in aqueous solutions in the absence and presence of various reductants. The nature of the oxidizing intermediates. J Am Chem Soc 1995; 117:12078–12084. 18. Gustafsson LE, Leone AM, Peersson MG, Wilkund NP, Moncada S. Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem Biophys Res Commun 1991; 181:852–857. 19. Ide H, Nakano H, Ogasa T, Osanai S, Kikuchi K, Iwamoto, J. Regulation of pulmonary circulation by alveolar oxygen tension via airway nitric oxide. J Appl Physiol 1999; 87:1629–1636. 20. Deem S, Hedges RG, McKinney S, Polissar NL, Alberts MK, Swenson ER. Mechanisms of improvement in pulmonary gas exchange during isovolemic hemodilution. J Appl Physiol 1999; 87:132–141. 21. Abu-Soud HM, Rousseau DL, Stuehr DJ. Nitric oxide binding to the heme of neuronal nitric-oxide synthase links its activity to changes in oxygen tension. J Biol Chem 1996;271:32515–32518. 22. Abu-Soud HM, Wang J, Rousseau DL, Fukuto JM, Ignarro L, Stuehr DJ. Neuronal nitric oxide synthase self-inactivates by forming a ferrous-nitrosyl complex during aerobic catalysis. J Biol Chem 1995; 270:22997–23006. 23. Adak S, Wang Q, Stuehr DJ. Molecular basis for hyperactivity in tryptophan 409 mutants of neuronal NO synthase. J Biol Chem 2000; 275:17434–17439. 24. Abu-Soud HM, Ichimori K, Presta A, Stuehr DJ. Electron transfer, oxygen binding, and nitric oxide feedback inhibition in endothelial nitric oxide synthase. J Biol Chem 2000; 275:17349–17357.


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25. Abu-Soud HM, Gachhui R, Raushel FM, Stuehr DJ. The ferrous-dioxy complex of neuronal nitric oxide synthase: divergent effects of L-arginine and tetrahydrobiopterin on its stability. J Biol Chem 1997; 272:17349–17353. 26. Hurshman AR, Marletta MA. Nitric oxide complexes of inducible nitric oxide synthase: spectral characterization and effect on catalytic activity. Biochemistry 1995; 34:5627–5634. 27. Nelin LD, Thomas CJ, Dawson CA. Effect of hypoxia on nitric oxide production in neonatal pig lung. Am J Physiol 1996; 271:H8–H14. 28. Tsujino I, Miyamoto K, Nishimura M, Shinano H, Makita H, Saito S, Nakano T, Kawakami Y. Production of nitric oxide (NO) in intrathoracic airways of normal humans. Am J Respir Crit Care Med 1996; 154:1370–1374. 29. Grimminger F, Spriestersbach R, Weissmann N, Walmrath D, Seeger W. Nitric oxide generation and hypoxic vasoconstriction in buffer-perfused rabbit lungs. J Appl Physiol 1995; 78:1509–1515. 30. Fagan KA, Fouty BW, Tyler RC, Morris KG Jr, Hepler LK, Sato K, LeCras TD, Abman SH, Weinberger HD, Huang PL, McMurtry IF, Rodman DM. The pulmonary circulation of homozygous eNOS-null mice is hyperresponsive to mild hypoxia. J Clin Invest 1999; 103:291–299. 31. Vanderkooi JM, Erecinska M, Silver IA. Oxygen in mammalian tissue: methods of measurement and affinities of various reactions. Am J Physiol 1991; 260:C1131– C1150. 32. Wakita M, Nishimura G, Tamura M. Some characteristics of the fluorescence lifetime of reduced pyridine nucleotides in isolated mitochondria, isolated hepatocytes, and perfused rat liver in situ. J Biochem 1995; 118:1151–1160. 33. Liao JK, Zulueta JL, Yu FS, Peng HB, Cote CG, Hassoun PM. Regulation of bovine endothelial constitutive nitric oxide synthase by oxygen. J Clin Invest 1995; 96: 2661–2666. 34. Melillo G, Musso T, Sica A, Taylor LS, Cox GW, Varesio L. A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promotor. J Exp Med 1995; 182:1683–1693. 35. Phelan MW, Faller VF. Hypoxia decreases constitutive nitric oxide synthase transcript and protein in cultured endothelial cells. J Cell Physiol 1996; 167:469–476. 36. Baker JE, Holman P, Kalyanaraman B, Griffith OW, Pritchard KA Jr. Adaptation to chronic hypoxia confers tolerance to subsequent myocardial ischemia by increased nitric oxide production. Ann NY Acad Sci 1999; 874:236–253. 37. Sato K, Rodman DM, McMurtry IF. Hypoxia inhibits increased ETB receptor-mediated NO synthesis in hypertensive rat lungs. Am J Physiol 1999; 276 (4 Pt 1):L571– L580. 38. Di Giulio C, Grilli A, De Lutiis MA, Di Natale F, Sabatino G, Felaco M. Does chronic hypoxia increase rat carotid body nitric oxide? Compar Biochem Physiol 1998; 120(2):243–247. 39. Voelkel NF. Mechanisms of hypoxic pulmonary vasoconstriction. Am Rev Respir Dis 1986; 133:1186–1195. 40. McQueston JA, Cornfield DN, McMurty IF, Abman SH. Effects of oxygen and endogenous L-arginine on EDRF activity in fetal pulmonary circulation. Am J Physiol 1993; 264:H865–H871.

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41. Cornfield DN, Reeve HL, Tolarova S, Weir EK, Archer S. Oxygen causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel. Proc Natl Acad Sci USA 1996; 93:8089–8094. 42. Sobol BJ, Bottex G, Emirgil C, Gissen H. Gaseous diffusion from alveoli to pulmonary vessels of considerable size. Circ Res 1963; 13:71–79.

11 Exhaled Markers in Interstitial Lung Disease and Pulmonary Hypertension

SERGEI A. KHARITONOV Imperial College of Science, Technology and Medicine National Heart and Lung Institute and Royal Brompton Hospital London, England

I.

Introduction

Exhaled breath analysis allows completely noninvasive monitoring of inflammation and oxidative stress in the respiratory tract in inflammatory lung diseases, including interstitial lung diseases. The technique is simple to perform, may be repeated frequently, and can be applied to children, including neonates, and patients with severe disease in whom more invasive procedures are not possible. Several volatile chemicals can be measured in the breath, and many nonvolatile molecules (mediators, oxidation and nitration products, proteins) may be measured in exhaled breath condensate. Exhaled breath analysis may be used to quantify inflammation and oxidative stress in the respiratory tract, in differential diagnosis of airway diseases, and in the monitoring of therapy. II. Nitric Oxide Various sources of exhaled nitric oxide (NO) are measured in patients with interstitial lung disease and pulmonary hypertension. Nitric oxide synthase 2 (NOS2) may be induced by inflammatory cytokines, endotoxin, and viral infections and 247

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may show increased expression in inflammatory diseases (1–4). In lungs of patients with idiopathic pulmonary fibrosis (IPF), strong expression of nitrotyrosine and NOS was seen in macrophages, neutrophils, and alveolar epithelium, and the active stage of IPF was associated with increased inflammatory and alveolar expression of nitrotyrosine and NOS (5). However, NOS is not the only source of NO in exhaled air, and exhaled NO is not therefore a direct measure of NOS activity in the lower respiratory tract. No reacts with thiol-containing molecules, such as cysteine and glutathione, to form S-nitrosoproteins and S-nitrosothiols (5). Approximately 70–90% of NO is released by S-nitrosothiols, which therefore provide a major source of NO in tissues (6). S-nitrosothiols are potent relaxants of human airways and may play an important role in sequestration, releasing, and transportation of NO to the site of action (5). In addition, NO in exhaled air may also be derived from nitrite protonation to form nitrous acid, which releases NO gas with acidification (7). This pH-related pathway has been implicated in acute asthma, when pH in expired condensate is low (8). A. Interstitial Lung Diseases Systemic Sclerosis

In patients with systemic sclerosis who have developed pulmonary hypertension, there is a reduction in exhaled NO compared to normal subjects and to patients with interstitial lung disease without pulmonary hypertension (Fig. 1) (9,10). This may be due to reduced expression of NOS3 in pulmonary vessels, or a reduction in the pulmonary vascular endothelial surface. However, the presence of NOS3

Figure 1 Exhaled NO in normal individuals and subjects with systemic sclerosis (SSc) with and without evidence of pulmonary hypertension (PHT) compared with controls with no evidence of lung disease. (From Ref. 9.)

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in pulmonary vessels is variable, and it has been found to be either reduced (11– 13), increased (14), variable (15), or unaltered (16). Fibrosing Alveolitis

There is strong expression of nitrotyrosine and NOS2 in macrophages, neutrophils, and alveolar epithelium in lungs of patients with idiopathic pulmonary fibrosis with active inflammation during the early to intermediate stage of the disease (4). This is consistent with elevated levels of exhaled NO in patients with fibrosing alveolitis. Increased exhaled NO levels are associated with disease activity, as assessed by bronchoalveolar lavage (BAL) lymphocyte counts, and are reduced in patients treated with corticosteroids (17). Sarcoidosis

Cytokines, including tumor necrosis factor-α (TNF-α) and interferon-γ (INF-γ) are increased in the pulmonary inflammation of sarcoidosis and there is an up-regulation of NOS2 in respiratory epithelium and granulomata in patients with sarcoidosis (18). The magnitude of exhaled NO rise in sarcoidosis may be related to the disease activity of the disease and is reduced by steroid therapy. This is, perhaps, the reason behind two conflicting observations reporting either elevated (18) or normal (19) exhaled NO in patients with active pulmonary sarcoidosis. B. Pulmonary Hypertension

The pathogenesis of pulmonary hypertension remains poorly understood. Vasoconstriction is likely to be a major factor in the initial stages of the disease, and a reduction in endogenous NO may contribute to the development of pulmonary hypertension. In fact, nebulized epoprostenol increased exhaled NO in patients with pulmonary hypertension, but not in normal control subjects, suggesting that this effect on the hypertensive circulation has an NO-related mechanism (20). In contrast, the angiotensin-converting enzyme (ACE) inhibitor enalapril, used to treat pulmonary hypertension, increases exhaled NO levels in normotensive subjects, but not in patients with systemic hypertension (21). Measurement of exhaled NO, which was associated with lung dysfunction and pulmonary artery pressure, may be an indicator of lung injury in adult patients after cardiopulmonary bypass (22). Biochemical reaction products of NO are inversely correlated with pulmonary artery pressures in patients with primary pulmonary hypertension and with years since diagnosis (23). This may reflect reduced expression of NOS3 in patients with pulmonary hypertension, as reduced NOS3 expression has been reported in patients with primary pulmonary hypertension (11–13). In fact, aerosol-

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ized NOS2 gene transfer increases pulmonary NO production and reduces hypoxic pulmonary hypertension in rats (24), and may be a promising future strategy to target pulmonary vascular disorders. However, interpretation of these low NO levels should be made cautiously and in the context of potential influence of hemoglobin (Hb) on NO. Although stimulation of NO production by pulmonary vascular endothelial cells in response to shear stress has been described, it is not an important determinant of NO production. Low exhaled NO in patients with pulmonary hypertension may be consistent with flow redistribution from alveolar septal capillaries to extra-alveolar vessels and decreased surface area or a direct, stretch-mediated depression of lung epithelial NO production (25), or increased Hb NO scavenging. It may be difficult to use exhaled NO changes as an accurate measure of lung tissue NO production. Recently, it has been shown that modest decreases in basal NO production, the inability to increase NO production, or both may play a role in the altered pulmonary vascular reactivity after cardiopulmonary bypass (26). The decrease in NO may be independent of gene expression. However, other mechanisms for this decrease, such as substrate or cofactor availability, warrant further study.

III. Carbon Monoxide Carbon monoxide (CO) is a by-product of rate-limited oxidative cleavage of hemoglobin by HO, which exists in three isoforms, HO1, HO2, and HO3. HO2 is constitutively expressed in most tissues, whereas HO3 is so far described only in rat (27). Like other stress proteins, HO1 can be induced by a variety of stimuli, such as pro-inflammatory cytokines, bacterial toxins, heme, ozone, hyperoxia, hypoxia, reactive oxygen species, and reactive nitrogen species. A. Interactions with NO

Like NO, CO is capable of upregulating cGMP via activation of guanylyl cyclase, causing vasodilatation, smooth-muscle relaxation, and platelet disaggregation. It has been suggested that the HO pathway exerts important counterregulatory effects on the NOS pathway and, when blocked, the underlying NOS pathway is unmasked, leading to increased and prolonged release of NO (28). In contrast, exogenously administered or endogenously released NO stimulates HO1 gene expression and CO production in vascular smooth muscle cells, resulting in a higher resistance to oxidant damage (29). This effect of NO is related to the release of free heme from heme proteins, which are able to transcriptionally upregulate HO1 and lead to their own degradation. CO also directly inhibits NOS2 activity by binding to the heme moiety of the enzyme (30).


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B. Effect of Oxidative Stress

There is a close link between the reactive oxygen and nitrogen species and CO. Both superoxide anions and peroxynitrite can stimulate HO1 activation (31), and subsequent release of CO is an important negative-feedback regulatory mechanism limiting the release of these cytotoxic substances (32). There is evidence that the deleterious effects of ROS, such as superoxide and H 2 O 2 , are dependent on the presence of iron. The intracellular pool of free iron can react with both H 2 O 2 and superoxide, giving rise to the OH ⫺ radical via the Fenton reaction. The free iron that is not metabolized intracellularly is sequestered in cells as ferritin. Thus, ferritin serves as a reservoir to restrict iron from participating in the Fenton reaction. It has been shown that free iron released from heme by HO may induce ferritin synthesis, and heme-induced HO1 protein also activates ferritin via mRNA expression (33). Furthermore, the metabolite of heme degradation, bilirubin, is itself an effective antioxidant of peroxynitrite-mediated protein oxidation and may be even more effective than vitamin E in preventing lipid peroxidation (34). Moderate overexpression of HO1 improves the resistance of cells to oxygen toxicity (35). However, there is cytotoxicity associated with HO1 overexpression. C. Interstitial Lung Disease

Elevation of exhaled CO is related to lung function deterioration (36) and impaired gas transfer in patients with cryptogenic fibrosing alveolitis and scleroderma (37). Elevated levels of exhaled CO in patients with fibrosing alveolitis are also associated with disease activity as assessed by BAL cell counts (17). This suggests that exhaled CO may be used to monitor disease progression and response to therapy in interstitial lung diseases. IV. Exhaled Breath Condensate Exhaled breath condensate is collected by cooling or freezing exhaled air and is totally noninvasive. The collection procedure has no influence on airway function or inflammation, and there is accumulating evidence that abnormalities in condensate composition may reflect biochemical changes of airway lining fluid. Several nonvolatile chemicals, including proteins, have now been detected in breath condensates. The first studies identifying surface-active properties, including pulmonary surfactant, of exhaled condensate were published in the USSR in the 1980s (38,39), and since then several inflammatory mediators, oxidants, and ions have been identified in exhaled breath condensates. A. Origin

Potentially, condensate measurements reflect different markers and molecules derived from the mouth (oral cavity and oropharynx), tracheobronchial system,

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and alveoli, and their proportional contribution has not been yet sufficiently studied. It is assumed that airway surface liquid becomes aerosolized during turbulent airflow, so that the content of the condensate reflect the composition of airway surface liquid, although large molecules may not aerosolize as well as small soluble molecules. A strong correlation between the levels of CO 2 and O 2 in exhaled fluid and exhaled breath (40) suggests that aerosol particles exhaled in human breath reflect the composition of the bronchoalveolar extracellular lining fluid. B. Eicosanoids

Eicosanoids are potent mediators of inflammation, responsible for vasodilatation/ vasoconstriction, plasma exudation, mucus secretion, bronchoconstriction/bronchodilatation, cough, and inflammatory cell recruitment. They are derived from arachidonic acid and include prostaglandins, thromboxane, isoprostanes, and leukotrienes. Noninvasive exhaled condensate analysis provides an opportunity to assess the eicosanoid profile in lung diseases directly, and may be a better predictor of clinical efficacy of leukotriene antagonists or thromboxane inhibitors in lung disease than urine, serum, or invasive BAL. Isoprostanes

Isoprostanes are a novel class of prostanoids formed by free-radical-catalyzed lipid peroxidation of arachidonic acid (41). They are formed initially esterified in membrane phospholipids, from which they are cleaved by a phospholipase A 2 , circulate in plasma, and are excreted in urine and can be detected in exhaled breath condensate and BAL. Their formation is largely independent of cyclooxygenases-1 (COX-1) and COX-2. They can be detected by ELISA (42,43) and by GC/MS analysis (41). F 2-isoprostanes are the major candidates for clinical measurement of oxidative stress in vivo. Interstitial Lung Disease

Interstitial lung diseases, such as cryptogenic fibrosing alveolitis (CFA) and fibrosing alveolitis associated with systemic sclerosis (FASSc), are characterized by enhanced oxidative stress in both serum (44) and BAL fluid (45). The imbalance between the oxidants and antioxidants is also a prominent feature of sarcoidosis (46). 8-Isoprostane is detectable in BAL fluid of normal subjects and is increased in patients with sarcoidosis, CFA, and FASSc, suggesting a higher level of oxidant stress and greater lung injury in these patients than in sarcoidosis (43). C. NO-Related Products

NO reacts with superoxide to yield peroxynitrite, can be trapped by thiol-containing biomolecules, such as cysteine and glutathione, to form S-nitrosothiols,


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or can be oxidized to nitrate and nitrite (47). Nitrogen intermediates, for example, peroxynitrite, can induce a number of covalent modifications in various biomolecules, such as nitroso- and nitro-adducts. Nitrite and nitrate concentrations were increased in exhaled breath condensate of patients with active pulmonary sarcoidosis (19).

References 1. Barnes PJ, Belvisi MG. Nitric oxide and lung disease. Thorax 1993; 48:1034–1043. 2. Gaston B, Drazen JM, Loscalzo J, Stamler JS. The biology of nitrogen oxides in the airways. Am J Respir Crit Care Med 1994; 149:538–551. 3. Barnes PJ. Transcription factors and inflammatory disease. Hosp Pract 1996; 31: 93–96. 4. Saleh D, Barnes PJ, Giaid A. Increased production of the potent oxidant peroxynitrite in the lungs of patients with idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 1997; 155:1763–1769. 5. Stamler JS, Simon DI, Osborne JA, Mullins ME, Jaraki O, Michel T, Singel DJ, Loscalzo J. S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds. Proc Natl Acad Sci USA 1992; 89:444–448. 6. Sheu FS, Zhu W, Fung PC. Direct observation of trapping and release of NO by glutathione and cysteine with electron paramagnetic resonance spectroscopy. Biophys J 2000; 78:1216–1226. 7. Klebanoff SJ. Reactive nitrogen intermediates and antimicrobial activity: role of nitrite. Free Radic Biol Med 1993; 14:351–360. 8. Hunt JF, Fang K, Malik R, Snyder A, Malhotra N, Platts-Mills TA, Gaston B. Endogenous airway acidification. Implications for asthma pathophysiology. Am J Respir Crit Care Med 2000; 161:694–699. 9. Kharitonov SA, Cailes JB, Black CM, Du Bois RM, Barnes PJ. Decreased nitric oxide in the exhaled air of systemic sclerosis patients with pulmonary hypertension. Thorax 1997; 52:1051–1055. 10. Rolla G, Colagrande P, Scappaticci E, Chiavassa G, Dutto L, Cannizzo S, Bucca C, Morello M, Bergerone S, Bardini D, Zaccagna A, Puiatti P, Fava C, Cortese G. Exhaled nitric oxide in systemic sclerosis: relationships with lung involvement and pulmonary hypertension. J Rheumatol 2000; 27:1693–1698. 11. Giaid A, Saleh D. Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med 1995; 333:214–221. 12. Hislop AA, Springall DR, Oliveira H, Pollock JS, Polak JM, Haworth SG. Endothelial nitric oxide synthase in hypoxic newborn porcine pulmonary vessels. Arch Dis Child Fetal Neonatal 1997; 77:F16–F22. 13. Giaid A. Nitric oxide and endothelin-1 in pulmonary hypertension. Chest 1998; 114: 208S–212S. 14. Black SM, Fineman JR, Steinhorn RH, Bristow J, Soifer SJ. Increased endothelial NOS in lambs with increased pulmonary blood flow and pulmonary hypertension. Am J Physiol 1998; 275:H1643–H1651.

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15. Tyler RC, Muramatsu M, Abman SH, Stelzner TJ, Rodman DM, Bloch KD, McMurty IF. Variable expression of endothelial NO synthase in three forms of rat pulmonary hypertension. Am J Physiol 1999; 276:L297–L303. 16. Everett AD, Le CT, Xue C, Johns RA. eNOS expression is not altered in pulmonary vascular remodeling due to increased pulmonary blood flow. Am J Physiol 1998; 274:L1058–L1065. 17. Paredi P, Kharitonov SA, Loukides S, Pantelidis P, Du Bois RM, Barnes PJ. Exhaled nitric oxide is increased in active fibrosing alveolitis. Chest 1999; 115:1352–1356. 18. Moodley YP, Chetty R, Lalloo UG. Nitric oxide levels in exhaled air and inducible nitric oxide synthase immunolocalization in pulmonary sarcoidosis. Eur Respir J 1999; 14:822–827. 19. O’Donnell DM, Moynihan J, Finlay GA, Keatings VM, O’Connor CM, McLoughlin P, Fitzgerald MX. Exhaled nitric oxide and bronchoalveolar lavage nitrite/nitrate in active pulmonary sarcoidosis. Am J Respir Crit Care Med 1997; 156:1892– 1896. 20. Forrest IA, Small T, Corris PA. Effect of nebulized epoprostenol (prostacyclin) on exhaled nitric oxide in patients with pulmonary hypertension due to congenital heart disease and in normal controls. Clin Sci 1999; 97:99–102. 21. Sumino H, Nakamura T, Kanda T, Sato K, Sakamaki T, Takahashi T, Saito Y, Hoshino J, Kurashina T, Nagai R. Effect of enalapril on exhaled nitric oxide in normotensive and hypertensive subjects. Hypertension 2000; 36:934–940. 22. Ishibe Y, Liu R, Hirosawa J, Kawamura K, Yamasaki K, Saito N. Exhaled nitric oxide level decreases after cardiopulmonary bypass in adult patients. Crit Care Med 2000; 28:3823–3827. 23. Kaneko FT, Arroliga AC, Dweik RA, Comhair SA, Laskowski D, Oppedisano R, Thomassen MJ, Erzurum SC. Biochemical reaction products of nitric oxide as quantitative markers of primary pulmonary hypertension. Am J Respir Crit Care Med 1998; 158:917–923. 24. Budts W, Pokreisz P, Nong Z, Van Pelt N, Gillijns H, Gerard R, Lyons R, Collen D, Bloch KD, Janssens S. Aerosol gene transfer with inducible nitric oxide synthase reduces hypoxic pulmonary hypertension and pulmonary vascular remodeling in rats. Circulation 2000; 102:2880–2885. 25. Berg JT, Deem S, Kerr ME, Swenson ER. Hemoglobin and red blood cells alter the response of expired nitric oxide to mechanical forces. Am J Physiol Heart Circ Physiol 2000; 279:H2947–H2953. 26. McMullan DM, Bekker JM, Parry AJ, Johengen MJ, Kon A, Heidersbach RS, Black SM, Fineman JR. Alterations in endogenous nitric oxide production after cardiopulmonary bypass in lambs with normal and increased pulmonary blood flow. Circulation 2000; 102:III172–III178. 27. McCoubrey WKJ, Huang TJ, Maines MD. Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3. Eur J Biochem 1997; 247:725–732. 28. Chakder S, Rathi S, Ma XL, Rattan S. Hene oxygenase inhibitor zinc protoporphyrin IX causes an activation of nitric oxide synthase in the rabbit internal anal sphincter. J Pharmacol Exp Ther 1996; 277:1376–1382.


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29. Datta PK, Lianos EA. Nitric oxide induces heme oxygenase-1 gene expression in mesangial cells. Kidney Int 1999; 55:1734–1739. 30. Klatt P, Schmidt K, Mayer B. Brain nitric oxide synthase is a haemoprotein. Biochem J 1992; 288:15–17. 31. Foresti R, Clark JE, Green CJ, Motterlini R. Thiol compounds interact with nitric oxide in regulating heme oxygenase-1 induction in endothelial cells. Involvement of superoxide and peroxynitrite anions. J Biol Chem 1997; 272:18411–18417. 32. Foresti R, Sarathchandra P, Clark JE, Green CJ, Motterlini R. Peroxynitrite induces haem oxygenase-1 in vascular endothelial cells: a link to apoptosis. Biochem J 1999; 339:729–736. 33. Camhi SL, Lee P, Choi AM. The oxidative stress response. New Horiz 1995; 3: 170–182. 34. Dailly E, Urien S, Barre J, Reinert P, Tillement JP. Role of bilirubin in the regulation of the total peroxyl radical trapping antioxidant activity of plasma in sickle cell disease. Biochem Biophys Res Commun 1998; 248:303–306. 35. Suttner DM, Sridhar K, Lee CS, Tomura T, Hansen TN, Dennery PA. Protective effects of transient HO-1 overexpression on susceptibility to oxygen toxicity in lung cells. Am J Physiol 1999; 276:L443–L451. 36. Antuni JD, Du Bois AB, Ward S, Cramer DS, Kharitonov SA, Barnes PJ. Exhaled carbon monoxide may be a marker of deterioration of lung function in cryptogenic fibrosing alveolitis and scleroderma. Am J Respir Crit Care Med 1999; 159:A51. 37. Antuni JD, Ward S, Cramer DS, Kharitonov SA, Barnes PJ. Uptake and elimination of exhaled carbon monoxide in patients with interstitial lung disease is related to the degree of impairment of carbon monoxide diffusion capacity. Am J Respir Crit Care Med 1999; 159:A86. 38. Sidorenko GI, Zborovskii EI, Levina DI. Surface-active properties of the exhaled air condensate (a new method of studying lung function). Ter Arkh 1980; 52:65– 68. 39. Kurik MV, Rolik LV, Parkhomenko NV, Tarakhan LI, Savitskaia NV. Physical properties of a condensate of exhaled air in chronic bronchitis patients. Vrach Delo 1987; 37–39. 40. von Pohle WR, Anholm JD, McMillan J. Carbon dioxide and oxygen partial pressure in expiratory water condensate are equivalent to mixed expired carbon dioxide and oxygen. Chest 1992; 101:1601–1604. 41. Morrow JD, Roberts LJ. The isoprostanes: unique bioactive products of lipid peroxidation. Prog Lipid Res 1997; 36:1–21. 42. Montuschi P, Corradi M, Ciabattoni G, Nightingale J, Kharitonov SA, Barnes PJ. Increased 8-isoprostane, a marker of oxidative stress, in exhaled condensate of asthma patients. Am J Respir Crit Care Med 1999; 160:216–220. 43. Montuschi P, Ciabattoni G, Paredi P, Pantelidis P, Du Bois RM, Kharitonov SA, Barnes PJ. 8-Isoprostane as a biomarker of oxidative stress in interstitial lung diseases. Am J Respir Crit Care Med 1998; 158:1524–1527. 44. Jack CI, Jackson MJ, Johnston ID, Hind CR. Serum indicators of free radical activity in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 1996; 153:1918– 1923.

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45. Lenz AG, Costabel U, Maier KL. Oxidized BAL fluid proteins in patients with interstitial lung diseases. Eur Respir J 1996; 9:307–312. 46. Schaberg T, Rau M, Stephan H, Lode H. Increased number of alveolar macrophages expressing surface molecules of the CD11/CD18 family in sarcoidosis and idiopathic pulmonary fibrosis is related to the production of superoxide anions by these cells. Am Rev Respir Dis 1993; 147:1507–1513. 47. Stamler JS. S-nitrosothiols and the bioregulatory actions of nitrogen oxides through reactions with thiol groups. Curr Topics Microbiol Immunol 1995; 196:19–36.

ISCHEMIA-REPERFUSION

12 Exhaled Nitric Oxide in Human Lung Ischemia-Reperfusion

¨ VESI and DAVID ROYSTON ´ S KO TAMA Harefield Hospital Harefield, Middlesex, England

´ NDOR MARCZIN SIR MAGDI H. YACOUB and NA Imperial College of Science, Technology and Medicine National Heart and Lung Institute London and Harefield Hospital Harefield, Middlesex, England

I.

Introduction: Lung Ischemia-Reperfusion in Cardiothoracic Surgery

Modern open-heart surgery utilizing cardiopulmonary bypass (CPB) is predicated on an interesting hierarchical sacrifice as far as vital organs are concerned. Extraordinary procedures are now routinely performed in order to restore congenital and acquired anatomical defects of the heart, and advanced surgical techniques are used to restore blood flow to areas of the ischemic myocardium. From the moment of institution of CPB, the lungs, however, are subjected to severe stresses that result from reduction of pulmonary vascular blood flow. Although the entire output of the right ventricle is directed away from the pulmonary arterial circulation during full CPB, complete lung ischemia is avoided by the preserved bronchial circulation. Interestingly, recent studies question this relatively narrow window of safety provided by the bronchial circulation by showing that bronchial blood flow is dramatically reduced during routine CPB (1). Thus CPB is inevitably associated with a defined period of incomplete lung ischemia, which is followed by reperfusion at the end of CPB. Quite remarkably, the combination of this ischemia-reperfusion (I/R)-induced stress, the systemic inflammatory re259

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sponse caused by the CPB circuit, and the effect of the major surgical stress cause only a slight reduction in pulmonary function in the vast majority of patients following open heart surgery (2,3). According to recent reviews, however, 1–2% of patients appear to develop major pulmonary dysfunction fulfilling the criteria of acute lung injury or ARDS (4–6). Given the fact that open heart surgery is performed on the scale of hundreds of thousands worldwide, lung injury after CPB remains a significant clinical problem, with high morbidity and mortality. It is presumable that ischemia-reperfusion injury contributes to the development of post-CPB lung injury. Complete and prolonged lung ischemia up to several hours is unavoidable during lung transplantation, with dire consequences. Transbronchial biopsies performed after lung transplantation showed characteristic histological features of diffuse alveolar damage in 35% of patients, even when implantation was performed without CPB (7). This is associated with severe graft dysfunction in about 20% of lung transplant recipients, with clinical manifestations of progressive hypoxemia, decreased pulmonary compliance, high-permeability pulmonary edema, and widespread alveolar densities on chest radiographs (8–10). The early lung allograft dysfunction remains the primary cause of early mortality in lung transplantation. Although severe graft dysfunction can be reversible, it is often associated with the need for prolonged mechanical ventilation, intensive care and hospital stay, and compromised recovery among survivals (11). In addition to this morbidity, ischemia-reperfusion injury may also predispose grafts to acute and chronic rejection via upregulation of class II major histocompatibility complex antigens, release of endothelial cell antigens potentially triggering antiendothelial antibody production, and via generation of proinflammatory mediators including cytokines and growth factors (12–14).

II. Mechanisms of Ischemia-Reperfusion Injury A. Role of Microvascular Endothelial and Epithelial Cells

Significant progress has been made in understanding the complex cellular and molecular events that mediate and modulate ischemia-reperfusion lung injury (8,15,16). Much recent evidence indicates that in addition to arteriolar and postcapillary venular alterations, components of the alveolo-capillary unit are the major targets during acute lung injury, with the microvascular endothelium being the most susceptible element. During the injury process, endothelial cells become activated and more permeable, with characteristic loss of their normal function as essential regulators of pulmonary vasoreactivity, intravascular coagulation, and inflammatory response and gas exchange. In addition, the composition, function, and metabolism of pulmonary surfactant produced by alveolar type II epithelial cells are increasingly being recognized as important factors in lung injury (8,17).

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Although type I epithelial cells appear to be more resistant to inflammatory response and injury, and their barrier and metabolic function might remain intact even in high-permeability pulmonary edema, long-term outcome might depend on type I epithelial cell survival. B. Responses of Endothelial Cells to Hypoxia, Ischemia, and Reperfusion

It is now established that human endothelial cells are substantially altered either during hypoxia associated with ischemia or during reestablishment of blood flow and oxygen (reperfusion and reoxygenation), or in response to inflammatory mediators resulting in an activated phenotype (18). This includes changes in the profile of vasoregulatory endothelium-dependent factors, and the expression of activities that initiate and amplify inflammation and coagulation. Prolonged hypoxia can alter membrane properties, disturb the distribution of ions, increase intracellular volume, and impair the cytoskeletal organization of endothelial cells. Hypoxia may lead to severe depletion of energy stores, causing cellular energetic failure. Immediately after reperfusion, there appears to be a burst of oxidant production within the hypoxic endothelial cells. This, together with complement fragment activation on the surface of these cells, causes transient expression of preformed proteins and release of mediators that promote leukocyte–endothelial cell interactions, coagulation, and cytoskeletal rearrangement which might underlie transient increase in permeability (19). Alternatively, this oxidative stress can initiate signal transduction events to activate a delayed transcriptional program of several genes, resulting in the translation and prolonged surface expression of leukocyte adhesion molecules and cytokines that mediate further recruitment of neutrophils to sites of inflammation (20). C. Role of Neutrophil Activation

There has been considerable circumstantial evidence from both animal and clinical studies implicating the neutrophil as a potentially important mediator of the early changes in lung endothelial and epithelial permeability following ischemiareperfusion. The enhanced neutrophil–endothelial interactions might promote microvascular injury by multiple mechanisms. First, activation of neutrophils in the close proximity of endothelial cells might accentuate and prolong oxidative stress, resulting in oxidative stress signaling in endothelial cells to further enhance pro-inflammatory phenotype and sustain cytoskeletal reorganization (21–24). Neutrophils can produce significant quantities of a number of pro-inflammatory cytokines, contributing to cytokine imbalance, potent chemotactic agents that further promote their adherence to the endothelium (25,26). Finally, they can release elastase and other proteases, which might contribute to direct pulmonary cell injury (27,28).

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Despite many studies suggesting the pivotal role of neutrophils in I/R injury, not all investigators have found conclusive evidence for neutrophil involvement. In particular, there are several models in which despite significant decrease in the number of circulating or resident neutrophils, the extent of lung injury remained comparable to that of control situation (29). Thus pulmonary ischemiareperfusion injury remains a complex process whose induction might occur within the hypoxic endothelial cells, with neutrophils playing a major role in amplifying the injury later, during and after reperfusion.

III. Role of NO in Ischemia-Reperfusion Beyond oxygen-centered free radicals and oxidants, nitric oxide appears to play a multifaceted role in ischemia-reperfusion and to modulate the biological effects of reactive oxygen species. As discussed in previous chapters, NO is produced by many cells within the lung and appears to play a critical role in the pathophysiology of the pulmonary vascular bed and airways (30–33). Pulmonary vascular endothelial cells and airway epithelial cells continuously generate NO from the amino acid L-arginine via constitutively active NO synthases (NOS) (34). Recent studies suggest that endothelial cells constitutively express a relatively low-output NO pathway via type III NOS, while airway epithelial cells normally express a high-output NO pathway via type II NOS (iNOS), which could be further induced by inflammatory mediators (35,36). Thus, under normal conditions, there is a considerable release of NO in both the microvasculature and airways to elicit a number of bioactivities through either direct signaling or via a guanylate cyclase- and cGMP-dependent process. In the normal lung these include (a) regulation of pulmonary arteriolar and bronchial tone by relaxing smooth muscle; (b) prevention of platelet aggregation and thrombus formation; (c) modulation of multiple aspects of lung inflammation through attenuation of the adhesive interactions between leukocytes and the endothelial or epithelial cell surface, leukocyte trafficking, and reduction of oxidative stress by effectively scavenging the low intracellular levels of superoxide anion (16). NO production and bioactivity are subjected to great alterations during hypoxia, ischemia, and reperfusion. Enzymatic NO production exhibits a characteristic O 2 dependence and thus hypoxia reduces enzyme activity to synthesize NO. This phenomenon has been demonstrated in both cells in culture and in animal and human lungs (37–40). In addition to basal rates of enzymatic NO production, mechanical forces imposed on the cells by dynamically changing blood and air flow are also important contributors to both microvascular and airway NO production. During ischemia these mechanical stimuli are reduced with a potential effect of decoupling NO synthesis from shear stresses.


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Hypoxia, however, might increase NO generation from nonenzymatic sources. This involves nonenzymatic reduction of inorganic nitrite to NO, a reaction that takes place predominantly during acidic/reducing conditions (41,42). Nonenzymatic NO production has been demonstrated in various organs, including the stomach, on the surface of the skin, in the ischemic heart, and in infected nitrite-containing urine. The potential relevance of this phenomenon to lung pathology has been demonstrated by showing alteration of acid–base balance and increased NO production from nitrite in acidic pH in asthma (43). It has also been suggested that pH changes associated with ischemia can trigger this chemistry in the heart and aorta (41,42). Thus hypoxia and ischemia might alter NO concentrations and bioactivity by multiple and sometimes opposing mechanisms. Nevertheless, animal studies suggest that under conditions of hypoxia and ischemia of the lung, the predominant effect appears to be reduction in NO concentrations. In an orthotopic rat model of lung transplantation, NO release at the surface of the lung was measured directly by a porphyrinic NO microsensor. The study showed that NO levels diminished to one-third of that of control during 6 hr of hypothermic storage in lactated Ringer’s solution (32). In addition to changes in NO availability during ischemia, reperfusion can cause further consumption of NO through interactions with superoxide. In this situation NO undergoes radical–radical reactions with superoxide at neardiffusion-limited rates to yield peroxynitrite, a potent oxidizing agent to lipids, aromatic amino acid residues, protein sulfhydryls, and DNA (44–46). Peroxynitrite has been shown to initiate lipid peroxidation in biological membranes at rates that are 1000-fold higher than for hydrogen peroxide (47). However, NO displays a dual action with lipids: in addition to pro-oxidant characteristics through peroxynitrite-mediated oxidation reactions, it has potent capability to inhibit lipid-radical chain propagation (48). Thus, although NO can serve both as an antioxidant (by inhibiting lipid free radicals) and as an oxidant (by contributing to peroxinitrite formation), both of these reactions will lead to consumption of NO and reduced levels of bioactivity to elicit normal signaling and biological functions in the lung (Fig. 1). These conclusions are supported by experiments in which we evaluated NO bioactivity in the setting of superoxide-mediated oxidative stress to endothelial cells (49). Two experiments were performed. We generated superoxide either by an enzymatic reaction involving xanthine and xanthine oxidase or by activation of neutrophils with phorbol myristate acetate (PMA) during co-culture with endothelial cells. As shown in Fig. 2, insert, both of these reactions yielded graded increase in superoxide anion measured by cytochrome c reduction. Associated with this increased superoxide load, endothelial cell-induced and NO-dependent activation of guanylate cyclase was inhibited in a manner dependent on xanthine oxidase or neutrophil concentrations (Fig. 2). This inhibition was prevented in

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Figure 1 Interactions between reactive oxygen species and nitric oxide (NO). Under normal conditions associated with low levels of oxygen species, NO represents a bioactivity available to elicit numerous biological responses and the normally low levels of oxygen species contribute to certain signaling pathways involved in cellular regulations. During reoxygenation and reperfusion, neutrophils (PMN), endothelial cells (EC), and smooth muscle cells (SM) generate increased amounts of superoxide (OO), hydrogen peroxide (HOOH), and hydroxyl radical (OH). By overwhelming endogenous defense mechanisms and antioxidant capacity, these species attack cellular constituents and lead to protein, DNA, and lipid oxidation. NO, however, can interact via radical–radical reactions either with superoxide to produce peroxynitrite (ONOO) or with lipid radicals to form (LOONO) products. While peroxynitrite formation could lead to further oxidative mechanisms, interaction with lipid radicals usually terminates lipid peroxidation propagation, and thus could be considered an antioxidant mechanism. Note, however, that both reactions consume NO, leading to decreased bioavailability of NO to elicit normal biological signaling.

the presence of superoxide scavengers such as superoxide dismutase and Tiron (not shown), suggesting that the primary reactive species responsible for loss of NO bioactivity was indeed superoxide anion. Interestingly, when we compared sensitivity of the NO pathway to other endothelial functions in this model of leukocyte activation and endothelial dysfunction, loss of NO bioactivity was one of the most sensitive biochemical targets of oxidant stress (49). NO-induced cGMP accumulation was lost earlier and at lower leukocyte concentrations than other cellular responses such as endothelial ectoenzyme function, changes in permeability, and cytotoxicity (Fig. 3). These in-vitro observations are well supported by studies using animal models of oxidative stress. In the above-discussed rat model of lung transplantation, NO release at the surface of the lung plummeted following reperfusion (32). This could be partially recovered by administration of superoxide dismutase, suggesting that superoxide-mediated consumption of NO was responsible for diminished NO.


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Figure 2 Concentration effects of xanthine oxidase (XO) and PMA-activated neutrophils (PMN) on superoxide anion production (measured by cytochrome c reduction, insert) and inhibition of NO-mediated cGMP accumulation in co-cultures of endothelial and smooth muscle cells.

Similarly, available data regarding I/R-induced microvascular dysfunction suggest that I/R induces artioral, capillary, and venular endothelial dysfunction as part of the acute inflammatory response by altering the balance between NO and superoxide within or in the close proximity of endothelial cells (16). The prevailing view is that the relatively low levels of NO that are produced by the hypoxic endothelial cells react with the abundant supply of superoxide, leaving little or no bioactive NO to oppose blood cell–endothelial cell interactions and to maintain optimal tissue blood flow. In addition, the excess accumulation of superoxide that escapes interactions with NO allows for an enhanced generation of hydrogen peroxide through the dismutation reaction, which can rapidly initiate or exacerbate the above-discussed inflammatory state in the microvasculature. Following the acute phase of NO–superoxide interactions, the redox milieu is further complicated by transcriptional induction of iNOS and various antioxidant enzymes (50,51). The resulting reactions will again depend on relative quantities of NO and superoxide and the local redox microenvironment. It is conceivable that in case of continuous ongoing superoxide production, increased NO

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Figure 3 Comparison of neutrophil (PMN)-induced inhibition of NO bioactivity (cGMP accumulation) to other indices of endothelial injury such as inhibition of angiotensinconverting enzyme activity (ACE), increased permeability to albumin, and cytotoxicity on the basis of time and neutrophil requirement to achieve endothelial dysfunction or injury.

synthesis may contribute to further peroxynitrite formation, however increased NO may attenuate the extent of cellular injury through inhibition of apoptosis or may restore endothelial function if concomitant superoxide generation has subsided. All these considerations predict that I/R will be associated with a complicated picture of NOS expression, NO generation, and consumption. Actual NO concentrations will be different according to the dynamically changing cytokine environment, the nature of microvascular and airway inflammation, neutrophil activation, concomitant production of reactive oxygen species, and acidity in the immediate environment of endothelial and airway epithelial cells. Understanding of these reactions and their consequences in lung microvascular or airway damage or protection may provide a more rational basis for new therapeutic strategies toward better preservation of organ viability and function during and following I/R. Given the pivotal importance of all of these fluid-phase reactions in acute lung injury, monitoring changes in the production and bioavailability of NO in the lung would be extremely desirable. However, due to the short half-life of NO and peroxynitrite and the nature of the fluid-phase reactions, the analytical repertoire has been limited to detect stable end products of NO metabolism such as nitrite and nitrate and footprints of peroxynitrite such as nitrotyrosine. The mindful discovery of Gustafsson and colleagues, followed by recent technological developments allowing direct measurements of NO in expired air, however,


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have provided an exciting opportunity to evaluate changes in NO production and consumption in the clinical setting (52).

IV. Physiology of Exhaled NO Although measurements of exhaled NO provide important direct information regarding NO concentrations in the gas phase, interpolation of these data to invivo NO metabolism is far from straightforward. The biological reactions of NO are likely related to local NO concentrations in the fluid phase, which are influenced by many processes including generation rate, fluid-phase reactions such as auto-oxidation, and consumption by a variety of mechanisms including interaction with heme–iron groups, proteins and scavenging by hemoglobin, and interactions with superoxide (37). Since many of these processes appear to be anatomical site-dependent within the lung and they are likely to be differentially altered by dynamically changing pathological processes, it is of crucial importance to consider the implications of the anatomical origin of NO in exhaled air to the molecular pathology of acute lung injury (ALI). As discussed in detail elsewhere in this volume, the anatomical site and the type of cells responsible for the release of NO into the gas phase remains a matter of debate (53). On one hand, there is evidence that under certain conditions vascular mechanisms could contribute to exhaled NO (54). In particular, infusion of endothelium-dependent vasodilators increased exhaled NO in isolated perfused lung models, suggesting that a fraction of microvascular NO may diffuse into the alveolar compartment and contribute to exhaled NO. However, elegant studies by Sartori et al. utilizing an inhaled or infused NO synthase inhibitor suggest that exhaled NO is mostly of airway epithelial rather than of vascular endothelial origin (55). On the basis of these considerations they have concluded that exhaled NO may not be used as a marker for vascular NO production and/or endothelial function in healthy humans. These observations and conclusions provide a solid basis for current promotion to use exhaled NO as a diagnostic tool to monitor inflammatory responses affecting primarily the conducting airways in asthma (56). In contrast, the same considerations indicate major limitations of exhaled NO as a marker of ALI, which is primarily characterized by microvascular and alveolar dysfunction. The major implications are that changes in vascular NO metabolism in ALI likely remain undetectable by exhaled NO measurements and detected changes in exhaled NO would probably reflect altered epithelial NO generation and consumption. We have recently suggested a potential solution to this problem by utilizing exhaled NO responses following intravenous administration of nitroglycerin (GTN), which elicits its biological effect by NO release mediated by thiol-

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dependent enzymatic biotransformation. Shortly after the original observations of Persson et al. in animal models regarding increased exhaled NO levels following vascular metabolism of intravenous nitric oxide donors (57,58), we established and characterized this phenomenon in humans (59,60). We concluded that a fraction of nitroglycerin is metabolized in the pulmonary microvasculature to NO, which then diffuses into the alveolar space, giving rise to exhaled NO. On the basis of these considerations, we have suggested that GTN-induced exhaled NO might be a useful tool to monitor metabolic function of the pulmonary microvasculature.

V.

Exhaled NO Following CPB and Lung Transplantation

At Harefield Hospital, we have studied these issues in the setting of clinical ischemia-reperfusion (61,62). We have measured endogenous NO in the expired air as a means to assess bronchial epithelial function and we have examined GTN-induced exhaled NO indicating vascular and alveolar metabolic function. We have performed these studies in the setting of complete and prolonged lung ischemia and reperfusion during lung transplantation and compared them to those occurring with transient and incomplete lung ischemia during routine open heart surgery for coronary artery bypass grafting (CABG) utilizing CPB. A. Methods

Breath-to-breath measurements of NO concentrations in the lower airways were performed using a real-time, computer-controlled and integrated system (Logan Research, 2000 and 3000 series), as described before (59,61). Inspired and expired samples for analysis of NO and CO 2 were continuously withdrawn directly from the main lower airways through a thin Teflon sampling tube at a flow rate of 150 mL/min. Since detected concentration of exhaled gases depends on both the production rate and ventilation parameters, ventilation was standardized for inspired gas (100% O 2 ), tidal volume (5 mL/kg), respiratory rate (10/min) and inspiratory and expiratory ratio (1: 2). To eliminate the influence of positive end expiratory pressure on gas-phase NO, PEEP was set to zero. Baseline measurements were performed prior to CPB to evaluate endogenous levels of exhaled NO. After the baseline measurements, three increasing boluses of 1, 2, and 3 µg/kg GTN were administered to the patient via the central venous catheter with exhaled NO and hemodynamic response recorded. Between each bolus of GTN a short period of time was allowed for both the hemodynamic and exhaled gas parameters to return to the baseline values. A similar protocol was repeated 1, 3, and 6 hr after CPB. Arterial blood was simultaneously collected for haemoglobin, blood gas, electrolytes, and full blood count analysis.


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B. Results

In all 12 patients undergoing myocardial revascularization involving cardiopulmonary bypass, NO was detectable in the exhaled air before CPB as a characteristic oscillating signal which appeared to increase with expiration as judged by the CO 2 . Figure 4, top, depicts representative NO and CO 2 signals in a cardiac patient during bolus injection of 2 µg/kg of GTN before CPB. Intravenous bolus administration of 1, 2, and 3 µg/kg GTN resulted in a rapid, transient, and dose-depen-

Figure 4 Representative trace of intravenous nitroglycerin (GTN, 2 µg/kg bolus)induced increase in gaseous NO in a mechanically ventilated patient undergoing open heart surgery before CPB (top panel), and summary data of concentration-dependent NO evolution into gas phase following bolus injection of GTN (bottom panel).

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dent increase in exhaled levels of NO (Fig. 4, bottom). The time between administration of GTN boluses and detectable rise in exhaled NO was about 10–12 sec. Associated with the transient increase in exhaled NO following administration of GTN boluses, systolic arterial blood pressure transiently and concomitantly decreased by 17%, 22%, and 27%, and diastolic arterial pressure fell by 17%, 21%, and 21%. Similar changes were observed in PAP responses following administration of GTN. Endogenous exhaled NO levels remained unchanged 1 and 3 hr after CPB in these patients. Although measurements were performed in the intubated patients 6 hr after the operation, at this time point the majority of patients had already made some spontaneous breathing efforts. There were characteristic changes in GTN-induced response in exhaled NO after CPB. The dose-dependent increases in exhaled NO by GTN were significantly smaller at 1 hr and at 3 hr after CPB when compared to levels measured before CPB (Fig. 5). This was true for either peak or mean exhaled NO data and area under the curve. As an internal control, we analyzed the CO 2 output data to ensure that the changes regarding GTN-induced exhaled NO post-CPB were not due to changes in exhalation profiles. This analysis reveals that baseline peak exhaled CO 2 was similar before, 1 hr, and 3 hr after CPB. Similarly, end-tidal

Figure 5 Changes in exhaled NO (area under curve) after intravenous bolus administration of 1, 2, and 3 µg/kg GTN in 12 patients before, 1 hr, and 3 hr after cardiopulmonary bypass (CPB). Note reduced GTN-induced exhaled NO after CPB.


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CO 2 remained the same for each respective GTN bolus before and after CPB (data not shown). There was no characteristic exhaled NO signal, such as is seen with CABG patients in the majority of the lung transplant recipients during the post-CPB period. This may have been due to reperfusion-induced loss of a detectable signal during the ischemia period in two patients, who exhibited characteristic NO signals before reperfusion of their lungs [measured during CPB after completion of airway anastomoses (Fig. 6a)]. In most of the patients, however this was unrelated to reperfusion, since no detectable signals were obtained during ischemia (Fig. 6b). A comparable NO signal to CABG patients was seen in only two of the 10 lung transplant recipients during the postreperfusion period. When considered as a group and compared to exhaled NO levels in CABG patients, both peak expired NO and NO output were lower in lung transplant recipients after reperfusion. GTN-induced increases in exhaled NO were generally absent or appeared very small in lung transplant recipients after reperfusion (Fig. 7, top). Furthermore, total NO output over 30 sec was also profoundly reduced. Interestingly, GTNinduced exhaled NO was attenuated even in those patients whose endogenous exhaled NO was preserved. In addition GTN-induced exhaled NO recovered slowly in the postoperative period (⬎24 hr) despite earlier normalization of endogenous exhaled NO (Fig. 7, bottom). C. Discussion

This series of investigations aimed to clarify the influence of open heart surgery and/or CPB on NO concentration in the expired air. Currently there are contradicting published results showing no change, increase, or decrease in NO concentrations by different groups of investigators using a variety of methodological approaches (63–66). The two principal observations of this part of the study are the unchanged basal concentrations of expired NO during the immediate postoperative period and decreased GTN-induced exhaled NO after surgery. Our interpretation of these data is that basal and GTN-induced exhaled NO represents distinct anatomical compartments and physiological mechanisms contributing to exhaled NO and that these mechanisms are differently affected by CPB and heart surgery. Our conclusion is that in the clinical setting of routine open heart surgery, CPBinduced inflammatory response and ischemia-reperfusion injury do not reach sufficient levels to compromise endogenous NO mechanisms to produce exhaled NO (events that likely reflect airway epithelial processes). Similarly, the pulmonary and systemic hemodynamic response to a challenge with a bolus of GTN is also preserved, yet the characteristic increase in evolution of NO into expired air from GTN (which likely reflects lung microvascular events) is impaired in the early postreperfusion period. This might have clinical implications to heart

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Figure 6 Kinetics of exhaled NO in human heart-lung transplant recipients. Figure depicts representative expired signals from the native lungs of patients with cystic fibrosis (CF, Panel A, Native) and patients with Eisenmenger’s syndrome (Panel B, Native), and from the donor lungs during transplantation. Measurements from the donor lungs were measured prior to, or after reperfusion and 24 hr posttransplantation. Note absence of detectable NO signal in end-stage CF but normal exhaled NO trace from the native lung of the patients with Eisenmenger’s syndrome before explantation. Donor lung in the top panel exhibits a pattern of detectable exhaled NO before reperfusion, followed by reperfusion-associated decrease and recovery in the postoperative period. Donor lung in the lower panel shows diminished expired NO throughout the transplant procedure. Low expired CO 2 before reperfusion is due to full cardiopulmonary bypass.


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Figure 7 Characteristics of endogenous and nitroglycerine (GTN)-induced exhaled NO in lung transplant recipients before explantation of the native lungs and after implantation and reperfusion of the donor lungs. Top panel depicts undetectable endogenous exhaled NO (reflective of airway epithelial NO production) and very small GTN-induced exhaled NO (reflective of vascular events) 30 min following reperfusion. Lower panel depicts time-dependent recovery of these responses. Note the increase in endogenous exhaled NO (control) after reperfusion, whereas GTN-induced exhaled NO is absent up to 24 hr after reperfusion. Compare these to data obtained before explantation of the native lungs suffering from end-stage disease, which shows significant GTN response.

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surgery and CPB-induced pulmonary microvascular injury and support our original idea to further evaluate this response as a bedside test of the metabolic function of the lung. Although routine CPB and open heart surgery is associated with a degree of clinically significant pulmonary dysfunction, this rarely fulfills the criteria of ALI. This might be related to the transient and incomplete nature of ischemia and reperfusion. In contrast, lung transplantation is frequently associated with perioperative ALI, which might be related to prolonged and complete lung ischemia. In accordance with the greater potential to ischemia-reperfusion injury, lung transplantation was associated with a profound loss of GTN metabolism to produce exhaled NO. In addition and in contrast to open heart surgery, we found a variable decrease in endogenous exhaled NO levels. The ability to measure exhaled NO levels during ischemia, reperfusion, and after operation allows the elucidation of distinct mechanisms contributing to loss of exhaled NO. In conclusion, our findings provide additional evidence that even during clinically successful lung transplantation, ischemia-reperfusion injury may reach sufficient levels to routinely compromise vascular mechanisms, at least those responsible for pulmonary metabolism of organic nitrates, and transport and release of NO to the air space. In addition, there is evidence of epithelial dysfunction in releasing NO into the gas phase. In light of recent observations suggesting the critical role of epithelial cells in the resolution of acute lung injury (67,68), exhaled NO might be a useful bedside tool to monitor the onset, extent, and resolution of vascular and epithelial injury and the involvement of the NO pathways.

VI. Summary This review has emphasized recent progress in the direct evaluation of endogenous NO through bedside monitoring of NO concentrations in the expired air of patients subjected to ischemia and reperfusion during cardiothoracic surgery. There has been recent progress in our understanding of the determinants of exhaled NO, the anatomical, cellular, and molecular origins of NO in the expired air. The scientific community has widely accepted that NO levels in the gas phase reflect in an accurate and qualitative manner the dynamics of NO production and consumption in the airways, especially in the microenvironment of epithelial cells. The contribution of vascular compartments to exhaled NO has been debated, and it appears that changes negatively affecting NO metabolism in the microvasculature remain largely undetectable with exhaled NO. We have provided evidence that augmented vascular NO from endogenous metabolism of GTN can be detected in the expired air in humans and have postulated that this phenomenon could be used to assess vascular NO consumption in ALI. In the


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setting of ischemia-reperfusion-related ALI, we have obtained intriguing data, which might have implications to mechanisms, extent, and management of ALI associated with cardiothoracic surgery. Acknowledgments This work has been supported by a Medical Research Council Clinician Scientist Fellowship to Nandor Marczin. Magdi Yacoub is a British Heart Foundation Professor of Cardiothoracic Surgery. The contribution of the Julia Polak Transplant Fund is greatly appreciated. References 1. Schlensak C, Doenst T, Preusser S, Wunderlich M, Kleinschmidt M, Beyersdorf F. Bronchial artery perfusion during cardiopulmonary bypass does not prevent ischemia of the lung in piglets: assessment of bronchial artery blood flow with fluorescent microspheres. Eur J Cardiothorac Surg 2001; 19(3):326–331. 2. Marczin N, Royston D, Yacoub M. Pro: lung transplantation should be routinely performed with cardiopulmonary bypass. J Cardiothorac Vasc Anesth 2000; 14:739– 745. 3. Macnaughton PD, Braude S, Hunter DN, Denison DM, Evans TW. Changes in lung function and pulmonary capillary permeability after cardiopulmonary bypass. Crit Care Med 1992; 20(9):1289–1294. 4. Asimakopoulos G, Smith PL, Ratnatunga CP, Taylor KM. Lung injury and acute respiratory distress syndrome after cardiopulmonary bypass. Ann Thorac Surg 1999; 68:1107–1115. 5. Asimakopoulos G, Taylor KM, Smith PL, Ratnatunga CP. Prevalence of acute respiratory distress syndrome after cardiac surgery. J Thorac Cardiovasc Surg 1999; 117: 620–621. 6. Messent M, Sullivan K, Keogh BF, Morgan CJ, Evans TW. Adult respiratory distress syndrome following cardiopulmonary bypass: incidence and prediction. Anaesthesia 1992; 47:267–268. 7. Gammie JS, Cheul LJ, Pham SM, Keenan RJ, Weyant RJ, Hattler BG, et al. Cardiopulmonary bypass is associated with early allograft dysfunction but not death after double-lung transplantation. J Thorac Cardiovasc Surg 1998; 115:990–997. 8. Novick RJ, Gehman KE, Ali IS, Lee J. Lung preservation: the importance of endothelial and alveolar type II cell integrity. Ann Thorac Surg 1996; 62:302–314. 9. Khan SU, Salloum J, O’Donovan PB, Mascha EJ, Mehta AC, Matthay MA, et al. Acute pulmonary edema after lung transplantation: the pulmonary reimplantation response. Chest 1999; 116:187–194. 10. Kundu S, Herman SJ, Winton TL. Reperfusion edema after lung transplantation: radiographic manifestations. Radiology 1998; 206:75–80. 11. Christie JD, Bavaria JE, Palevsky HI, Litzky L, Blumenthal NP, Kaiser LR, et al. Primary graft failure following lung transplantation. Chest 1998; 114(1):51–60.

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12. Serrick C, Giaid A, Reis A, Shennib H. Prolonged ischemia is associated with more pronounced rejection in the lung allograft. Ann Thorac Surg 1997; 63:202–208. 13. Qayumi AK, Nikbakht-Sangari MN, Godin DV, English JC, Horley KJ, Keown PA et al. The relationship of ischemia-reperfusion injury of transplanted lung and the upregulation of major histocompatibility complex II on host peripheral lymphocytes. J Thorac Cardiovasc Surg 1998; 115:978–989. 14. Mal H, Dehoux M, Sleiman C, Boczkowski J, Leseche G, Pariente R, et al. Early release of proinflammatory cytokines after lung transplantation. Chest 1998; 113: 645–651. 15. Granger DN. Ischemia-reperfusion: mechanisms of microvascular dysfunction and the influence of risk factors for cardiovascular disease. Microcirculation 1999; 6: 167–178. 16. Carden DL, Granger DN. Pathophysiology of ischaemia-reperfusion injury. J Pathol 2000; 190(3):255–266. 17. Freeman BA, Panus PC, Matalon S, Buckley BJ, Baker RR. Oxidant injury to the alveolar epithelium: biochemical and pharmacologic studies. Res Rep Health Eff Inst 1993; 1–30. 18. Verrier ED, Morgan EN. Endothelial response to cardiopulmonary bypass surgery. Ann Thorac Surg 1998; 66:S17–S19. 19. Boyle EM Jr, Pohlman TH, Cornejo CJ, Verrier ED. Endothelial cell injury in cardiovascular surgery: ischemia-reperfusion. Ann Thorac Surg 1996; 62(6):1868–1875. 20. Boyle EM Jr, Canty TG Jr, Morgan EN, Yun W, Pohlman TH, Verrier ED. Treating myocardial ischemia-reperfusion injury by targeting endothelial cell transcription. Ann Thorac Surg 1999; 68(5):1949–1953. 21. Pillai R, Bando K, Schueler S, Zebly M, Reitz BA, Baumgartner WA. Leukocyte depletion results in excellent heart-lung function after 12 hours of storage. Ann Thorac Surg 1990; 50(2):211–214. 22. Bando K, Pillai R, Cameron DE, Brawn JD, Winkelstein JA, Hutchins GM, et al. Leukocyte depletion ameliorates free radical-mediated lung injury after cardiopulmonary bypass. J Thorac Cardiovasc Surg 1990; 99(5):873–877. 23. Eppinger MJ, Deeb GM, Bolling SF, Ward PA. Mediators of ischemia-reperfusion injury of rat lung. Am J Pathol 1997; 150:1773–1784. 24. Eppinger MJ, Jones ML, Deeb GM, Bolling SF, Ward PA. Pattern of injury and the role of neutrophils in reperfusion injury of rat lung. J Surg Res 1995; 58:713– 718. 25. Xing Z, Jordana M, Kirpalani H, Driscoll KE, Schall TJ, Gauldie J. Cytokine expression by neutrophils and macrophages in vivo: endotoxin induces tumor necrosis factor-alpha, macrophage inflammatory protein-2, interleukin-1 beta, and interleukin6 but not RANTES or transforming growth factor-beta 1 mRNA expression in acute lung inflammation. Am J Respir Cell Molec Biol 1994; 10(2):148–153. 26. Kunkel SL, Lukacs N, Strieter RM. Expression and biology of neutrophil and endothelial cell-derived chemokines. Semin Cell Biol 1995; 6(6):327–336. 27. Binns OA, DeLima NF, Buchanan SA, Mauney MC, Cope JT, Thies SD, et al. Neutrophil endopeptidase inhibitor improves pulmonary function during reperfusion after eighteen-hour preservation. J Thorac Cardiovasc Surg 1996; 112(3):607–613. 28. Baird BR, Cheronis JC, Sandhaus RA, Berger EM, White CW, Repine JE. O 2 metab-


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46. Freeman BA, Gutierrez H, Rubbo H. Nitric oxide: a central regulatory species in pulmonary oxidant reactions [editorial; comment]. Am J Physiol 1995; 268:L697– L698. 47. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 1990; 87:1620–1624. 48. O’Donnell VB, Freeman BA. Interactions between nitric oxide and lipid oxidation pathways: implications for vascular disease. Circ Res 2001; 88:12–21. 49. Marczin N, Chen X, Catravas JD. Basal release of EDRF under conditions of oxidant stress. In: Catravas JD, Callow AD, Ryan US, eds. Vascular Endothelium: Physiological Basis of Clinical Problems II. New York: Plenum, 1993:3–16. 50. Liu M, Tremblay L, Cassivi SD, Bai XH, Mourgeon E, Pierre AF, et al. Alterations of nitric oxide synthase expression and activity during rat lung transplantation. Am J Physiol Lung Cell Mol Physiol 2000; 278(5):L1071–L1081. 51. Itano H, Zhang W, Ritter JH, McCarthy TJ, Mohanakumar T, Patterson GA. Adenovirus-mediated gene transfer of human interleukin 10 ameliorates reperfusion injury of rat lung isografts. J Thorac Cardiovasc Surg 2000; 120(5):947–956. 52. Gustafsson LE, Leone AM, Persson MG, Wiklund NP, Moncada S. Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem Biophys Res Commun 1991; 181(2):852–857. 53. Pietropaoli AP, Perkins PT, Perillo IB, Hyde RW. Exhaled nitric oxide does not provide a marker of vascular endothelial function in healthy humans [letter; comment]. Am J Respir Crit Care Med 2000; 161:2113–2114. 54. Cremona G, Higenbottam T, Takao M, Hall L, Bower EA. Exhaled nitric oxide in isolated pig lungs. J Appl Physiol 1995; 78(1):59–63. 55. Sartori C, Lepori M, Busch T, Duplain H, Hildebrandt W, Bartsch P, et al. Exhaled nitric oxide does not provide a marker of vascular endothelial function in healthy humans [see comments]. Am J Respir Crit Care Med 1999; 160:879–882. 56. Kharitonov SA, Barnes PJ. Nitric oxide in exhaled air is a new marker of airway inflammation. Monaldi Arch Chest Dis 1996; 51(6):533–537. 57. Persson MG, Agvald P, Gustafsson LE. Detection of nitric oxide in exhaled air during administration of nitroglycerin in vivo. Br J Pharmacol 1994; 111:825–828. 58. Cederqvist B, Persson MG, Gustafsson LE. Direct demonstration of no formation in vivo from organic nitrites and nitrates, and correlation to effects on blood pressure and to in vitro effects. Biochem Pharmacol 1994; 47:1047–1053. 59. Marczin N, Riedel B, Royston D, Yacoub M. Intravenous nitrate vasodilators and exhaled nitric oxide. Lancet 1997; 349:1742–1742. 60. Marczin N, Riedel B, Royston D, Yacoub M. Exhaled nitric oxide and pulmonary response to iloprost in systemic sclerosis [letter; comment]. Lancet 1998; 352(9125): 405–406. 61. Marczin N, Riedel B, Gal J, Polak J, Yacoub M. Exhaled nitric oxide during lung transplantation [letter]. Lancet 1997; 350(9092):1681–1682. 62. Marczin N, Ryan US, Catravas JD. Effects of oxidant stress on endothelium-derived relaxing factor-induced and nitrovasodilator-induced cGMP accumulation in vascular cells in culture. Circ Res 1992; 70(2):326–340. 63. Beghetti M, Silkoff PE, Caramori M, Holtby HM, Slutsky AS, Adatia I. Decreased


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13 Monitoring Distant Organ Reperfusion Injury by Measurement of Volatile Organic Compounds

ROBERT H. BROWN and TERENCE H. RISBY Johns Hopkins University Baltimore, Maryland, U.S.A.

I.

Introduction

A. Why Breath Analysis Is Desirable

Since medieval times, the analysis of exhaled breath has been used as a noninvasive monitor of mortality. More recently, measurements of volatile organic compounds (VOCs) in exhaled breath such as carbon dioxide and oxygen have greatly improved the safety of medical practice in anesthesia and critical care medicine as a means of assuring appropriate ventilation and verification of endotracheal tube placement. Furthermore, the measurement of exhaled anesthetic gases has improved the safety and reliability of the anesthesia management of the surgical patient. The current medical and physiological practice of assessing organ tissue injury is limited to observing and measuring organ dysfunction, a late-stage and commonly irreversible event. Events such as organ ischemia, when identified as organ dysfunction, are frequently associated with massive tissue destruction, and the use of invasive procedures, such as tissue biopsies, are frequently required to make the diagnosis of organ injury. Therefore, the ability to measure early 281

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signs of tissue injury in a noninvasive manner would help in the prevention and the treatment of ischemic damage and reperfusion injury, as well as other forms of tissue injury. The measurement of compounds in exhaled breath holds many advantages. Foremost, it is noninvasive, allowing easy collection from adults in a wide variety of situations, such as in awake subjects during exercise challenges in a physiology laboratory, as well as in sedated and anesthetized patients who may be intubated in an intensive care unit or in the operating room during a surgical procedure. Measurement of exhaled breath could advance patient care with prompt and timely measurements that could facilitate modification of therapy. Further benefits of breath collection are the ease and inherent mobility of breath collection at the hospital bedside or in the patient’s home. Exhaled breath from children and infants can be easily collected, thereby reducing the potential risk to very low levels of morbidity and mortality associated with necessary invasive procedures. Monitors that sample and analyze exhaled breath in real time are currently available for anesthetic gases, nitrous oxide, carbon dioxide, and nitric oxide. The real-time measurement of exhaled compounds allows immediate identification of early signs of tissue injury. Early detection is critical to implementation of treatment and prevention of permanent organ damage. B. Reperfusion and Lipid Peroxidation

A fundamental consequence of insufficient blood flow to an organ (ischemia) is degradation of ATP (1), evidenced by decreased adenine nucleotide profile. Moreover, periods of ischemia produce impaired cell integrity manifested by enzyme leak, loss of K⫹ and Na⫹ homeostasis, and edema. Mitochondrial function is severely impaired during warm ischemia (2,3) but is relatively preserved by hypothermia (4). After a period of warm or cold ischemia, additional damage occurs at reperfusion due to evolution of oxygen-derived free radicals. It is the combination of ischemic damage and reperfusion injury that governs whether permanent organ damage occurs. One likely mechanism for oxygen-mediated free-radical injury involves the dual transformations of ATP degradation to its lower-energy precursors, eventually to hypoxanthine, and the conversion of xanthine dehydrogenase to xanthine oxidase [D–O conversion (5–7)]. Xanthine oxidase uses oxygen as an acceptor of electrons during oxidation of hypoxanthine and xanthine, producing superoxide. Superoxide may directly donate an electron to reduce a nearby molecule, or it may lead to the formation of the highly reactive hydroxyl radical via the iron-catalyzed Haber-Weiss reaction. Free radicals are evanescent, and their detection in biological settings is usually achieved by monitoring the products of their reactions with biomolecules. These altered constituents reflect damage caused by the free radicals. In the case

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Figure 1 Examples of propagation reactions that may occur due to free-radical-mediated lipid peroxidation by the OH radical. Note the generation of conjugated diene, ethane, malondialdehyde, and other aldehydes.

of interactions with lipids, free radicals initiate chain reactions that result in several possible products that may be used as specific evidence of lipid peroxidation, as diagrammed in Figure 1. The presence of free radicals can also be established by the use of reagents that spin-trap these very reactive species. During the last decade, improved methods for analysis of damage caused by free radicals in a clinical setting have become available in our laboratory (for review, see Ref. 8). These generally involve analysis of the products of lipid peroxidation. Several products of lipid peroxidation have been utilized as biomarkers of free-radical damage (9). Detection of hydrocarbons (i.e., ethane or 1pentane) has the particular advantage in that these molecules appear in exhaled breath within seconds of the release of free radicals into the tissue. Ethane is the better indicator because, unlike 1-pentane, it is not metabolized significantly (10). Other biomarkers of lipid peroxidation include conjugated dienes, and malondialdehyde. Detection of conjugated dienes (11), generated early in the chain reaction triggered by free radicals, provides a quantifiable measurement of events at this early point. However, determination of conjugated dienes is subject to error due to reactions occurring in sampling and processing that may lower their concentration. Detection of malondialdehyde (by the thiobarbituric acid assay) is a commonly employed assay (12). Malondialdehyde is not a specific biomarker that can be used to localize a pulse of free-radical generation in real time, since it may increase gradually over several hours. Moreover, the thiobarbituric acid assay is not specific for malondialdehyde. In our early studies (13) we quantified conjugated dienes and thiobarbituric reactive substances to confirm and validate that breath ethane could be used to monitor in-vivo lipid peroxidation. Determination of the amount of free-radical damage is a critical issue greatly complicated by the multiple reactions that free radicals may undergo and

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the chain reactions that may ensue. The favored propagation reaction will vary depending on activity of various enzymes and endogenous scavengers, the type of lipid in the cell, and the amount of oxygen available. Other molecules (e.g., proteins and DNA) may be damaged by free radicals (14). The relationship between the degree of damage to the different cell components caused by free radicals in different physiological situations is complex. For example, it is possible that lipid peroxidation is a cell protection mechanism, steering the chemical chain reactions away from more damaging pathways by the production of less energetic radicals. Repairable damage to DNA, a very serious consequence of free-radical damage, can now be measured. This approach augments assessment of irreparable damage as determined by tissue histopathology. Hydroxylation of deoxyguanosine (14) can be produced by free radicals in replicating DNA that is not protected by histones. DNA repair enzymes excise the 8-hydroxydeoxyguanosine and the adduct is transported into the extracellular milieu and excreted in the urine (15). Protein damage due to free radicals may be monitored by assaying for reduction of ESH to ESSE, for instance, as occurs with glutathione (16). In addition, certain enzymes, such as glucose 6phosphatase (17) in liver cytosol, are similarly susceptible to inactivation by freeradical attack on the ESH moiety (14). Since free radicals are evanescent, one approach to studying their identity and mechanism of generation is to employ various agents to block or scavenge them (18). These act in different ways. For instance, superoxide dismutase specifically prevents superoxide from forming the very reactive hydroxyl radical (6,7,16), whereas vitamin E (18) interrupts the lipid radical chain reaction depicted in Figure 1. Other such blocking agents are allopurinol (6,7), vitamin C or a derivative (16,19), ubiquinone (16), vitamin A, and catalase, which acts in concert with superoxide dismutase to block hydrogen peroxide (6,7,16,19). Blocking a reaction by any one of these (administering them to produce higher levels than are naturally present, superoxide dismutase being the most specific of the blockers mentioned) supports a role for free radicals in the reaction. Oxygen free radicals are also involved in molecular signaling that results in the recruitment, differentiation, and activation of monocytes. This delayed onset of reperfusion injury may be abrogated by these same blocking agents. Our improved breath-concentrating technique allows quantification of ethane in expired breath that is a reflection of total-body lipid peroxidation. Therefore, the analysis of breath alkanes quantifies the magnitude and timing of freeradical appearance (8). Breath hydrocarbons can be used to evaluate the efficacy of intervention protocols that ameliorate free-radical-mediated reperfusion injury. Finally, quantifying the levels of breath ethane enables reperfusion injury to be studied separately from ischemic damage.


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II. Breath Collection A. Subjects Intubated Subjects

The easiest method of breath collection occurs in patients who are intubated. In these patients, the trachea is secured with a cuffed endotracheal tube, and all inspired and expired ventilation passes from the respirator through the endotracheal tube. Breath can be collected at any point along the path from the end of the endotracheal tube to the exhaust of the respirator. In addition, patients who are intubated usually have their ventilation controlled. Therefore, the minute ventilation as well as the tidal volume and the respiratory rate are known. In the operating room, the end-tidal carbon dioxide is usually measured continuously and used to set the minute ventilation to maintain normal serum carbon dioxide levels in the patient. The maintenance of a constant minute ventilation and a stable end-tidal carbon dioxide allows more precise measurements of exhaled oxidative metabolites. In addition, the fraction of inspired oxygen can be more accurately controlled and other anesthetic gases such as isoflurane can be added in a controlled manner. Nonintubated Subjects

Breath can also be collected from nonintubated subjects. To collect breath from nonintubated subjects, a tight-fitting mask with a good seal around the face is required to prevent loss of exhaled breath through leaks in the mask. Alternatively, the subject is instructed to make a tight seal around a mouthpiece, and nose clips are used to occlude the nares and prevent breathing through the nose. Nonintubated subjects are usually awake, limiting one’s ability to control for minute ventilation, tidal volume, and respiratory rate, although devises such as metronomes and visual coaching can facilitate reproducibility. Moreover, with currently available monitors these parameters can be measured and corrections can be made for changes in frequency and tidal volume that occur during the time breath is collected. Subjects on Cardiopulmonary Bypass

Biomarkers of lipid peroxidation can be collected even when the cardiopulmonary bypass machine is instituted to pump and oxygenate the blood and to remove the carbon dioxide. This machine takes the place of the patient’s heart and lungs. In this situation, the “exhaled” products are collected as the outflow gases from the membrane oxygenator on the cardiopulmonary bypass machine. Since the characteristics of gas exchange for the membrane oxygenator are different from

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the gas-exchange characteristics of the lung, a direct comparison would not be justified. However, comparisons of changes in ethane levels during cardiopulmonary bypass can give insight into reperfusion injury in addition to the changes observed before and after cardiopulmonary bypass (20).

B. Factors Affecting Breath Collection in Intubated Subjects Anesthesia Machine

The modern anesthesia machines have two basic functions: first, the delivery of fresh gas flow of oxygen and defined doses of anesthetic gases; and second, the ventilation of the patient. The most common breathing system used for adults and larger children in operating rooms is a semiclosed breathing system. The important features of this system include a reservoir bag and a CO 2 absorber, which allows for partial rebreathing of exhaled breath. The most widely used semiclosed systems are circle systems, so named for their arrangement of components. To prevent excessive rebreathing, there are two unidirectional valves that force the gases to flow in one direction and pass through the CO 2 absorber each time around the circle system. Moreover, in this system, fresh gas flow rate is not of paramount concern and can even be lower than the patient’s minute ventilation, as long as the supply of oxygen is adequate to meet the oxygen consumption requirements of the subject. An expiratory valve permits escape of gases to a gas-scavenging system. For automated control of breathing, a mechanical ventilator replaces the rebreathing reservoir bag in the circle system. Most important, the motive force for compression of the bellows of the ventilator is externally applied gas pressure of oxygen. The anesthesia machine ventilators are commonly classified according to the mechanism of termination of the inspiratory phase—pressure-cycled, volume-cycled, or time-cycled—but further elaboration of these mechanisms is beyond the scope of this chapter. In addition, anesthesia machine ventilators usually have fewer controls than intensive care ventilators and are limited to tidal volume, respiratory rate and the inspiratory-to-expiratory phase ratio, and positive end-expiratory pressure (PEEP) control. While there are several modern anesthetic vaporizer designs, all vaporizers have certain common features. Modern vaporizers are calibrated, agent specific, and automatically temperature compensated to deliver controlled amounts of anesthetic gases safely to the patient. In addition, the anesthesia machine allows the amount of oxygen to be carefully controlled in the delivered gas mixture. Frequently, 100% oxygen is delivered to the patient during the induction of, and the emergence from, anesthesia. During the maintenance of anesthesia while the surgery is proceeding, the


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fresh gas flow may contain as little as 30% oxygen, with the remainder of the gas composed of nitrous oxide, helium, or air, depending on the surgical case. Nitrous Oxide

Nitrous oxide is a simple gaseous molecule that has anesthetic properties. While nitrous oxide cannot be administered in sufficient concentrations to render a patient completely anesthetized, it is used as an adjuvant with other anesthetics. A major consideration with the use of nitrous oxide during anesthesia is its potential interference with the measurement of breath ethane levels. Most commercially available nitrous oxide contains impurities at concentrations of substantial parts per million that interfere with the gas chromatographic analysis. Therefore, when breath collection is planned on anesthetized patients, nitrous oxide should not be used as part of the anesthesia management. Furthermore, it is necessary to eliminate any residual nitrous oxide from the anesthesia machine prior to breath collection. The two main sources of nitrous oxide in most hospitals in the United States are “wall” nitrous oxide and “bottled” nitrous oxide. The “wall” nitrous oxide is from a central source and is piped throughout the operating room. The “bottled” nitrous oxide is attached to the anesthesia machine as E-cylinder medical gas tanks. Since small amounts of nitrous oxide can continue to flow into the anesthesia machine from the central source, to prevent contamination of the collected breath, disconnection of the central source hoses from the wall to the anesthesia machine is recommended. In addition, the E-cylinders of nitrous oxide should be securely closed. Furthermore, the anesthesia machine should be completely flushed with 100% oxygen at 10 L/min for 20 min prior to acquiring any breath samples, to ensure complete evacuation of any residual nitrous oxide. Fresh Gas Flow Rates

The fresh gas flow rate in clinical settings is usually determined by a number of concerns, including the cost of anesthetic agents, heat and moisture loss from the patient, generation of toxic metabolites from the anesthetic agent, and time delay to changes in gaseous anesthetic concentration. In the current climate of cost reduction in health care, lower fresh gas flows optimize the use of anesthetic gas and decrease the total anesthesia costs. A lower fresh gas flow allows greater rebreathing of anesthetic gases and subsequently less exhausted anesthetic gases. In addition, since the fresh gas is cool relative to normal human body temperature and dry relative to normal humidity in the air, lower fresh gas flow has less cooling and drying effects on the patient’s airways. Fresh gas flows as low as 600 mL/min are now commonly used with some of the newer, more expensive anesthetic agents.

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For the breath collection, signal from breath biomarkers to noise optimization is essential. The higher the fresh gas flow, the lower is the concentration of ethane in the collected breath. We have found that a fresh gas flow of 1 L/min is a good compromise for optimizing breath collection and patient care. Any changes in fresh gas flow during the surgery should be noted and used in the subsequent calculation of breath ethane concentration. Type and Concentration of Anesthetic Gases

In theory, the choice of anesthetic gas is irrelevant to breath ethane collection. However, in practice, several anesthetic gases have characteristics that make them less then optimal for use during breath collection studies. Sevoflurane reacts with soda lime, a common carbon dioxide absorbent, and produces a potentially nephrotoxic compound, fluoromethyl-2,2-difluoro-1-(trifluoromethyl) vinyl ether, known as “compound A” (21). Therefore, the recommended fresh gas flow for sevoflurane is greater than 2 L/min, which causes dilution of the breath ethane concentration. Desflurane, another new inhalational anesthetic, can be used at very low fresh gas flow (⬇ 600 mL/min). However, the clinically relevant concentration of desflurane (6–8%) is high relative to the other inhalational anesthetics (1–2%). Therefore, at very low fresh gas flow, desflurane becomes a significant portion of the collected gases and interfers with the collection efficiency of ethane using adsorbent traps. The older inhalational anesthetics, halothane, ethrane, and isoflurane, can all be delivered with low fresh gas flow (1 L/min) and at clinically relevant concentrations that do not interfere with the measurement of breath ethane levels. However, halothane should not be used for breath ethane measurements because a significant portion of this anesthetic gas is metabolized in the liver, and these metabolites have the potential to initiate lipid peroxidation. Intravenous Anesthetics

More recently, total intravenous anesthesia has become popular for outpatient procedures. The advantages of these agents are the speed of induction and recovery from general anesthesia. Breath ethane can also be collected from patients whose anesthesia is solely by intravenous administration. Carbon Dioxide Absorbers

Several products are used in a semiclosed circle system to adsorb carbon dioxide, the most common being soda lime and baralyme. While both substances absorb carbon dioxide and some other volatile organic compounds such as acidic molecules and molecules with low volatilities, soda lime does not absorb ethane (22). Therefore, even during low flow of fresh gas, when a large amount of the exhaled


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breath passes through the soda lime, breath ethane can still be measured accurately. Medical Gas Purity

An additional concern is the purity of the medical gases delivered to the patient. Impurities can affect the measurement of exhaled oxidative metabolites. The purity of oxygen delivered as a medical gas is 99.7% to 99.9% pure. Moreover, the purity of the bulk liquid oxygen (99.9%) delivered to hospitals is higher than that found in the smaller cylinders which are in the gaseous state (99.7–99.9%). However, impurities may be introduced during delivery throughout the hospital center from the bulk liquid oxygen supply, and these impurities may interfere in the subsequent analysis of the breath. C. Location of Sampling

As mentioned above, collection of exhaled breath can be made at several points along the breathing circuit of the anesthesia machine. Initially, to prevent any impediment to patient care, we chose the exhaust valve at the back of the anesthesia machine, which was the farthest point from the patient, to sample breath. While adequate measurements could be obtained in this manner, the volume of gas in the expiratory circuit and the distance from the patient required that a higher fresh gas flow be used. Current anesthesia practice includes the use of several respiratory gas monitors, some of which sample exhaled and inhaled gas to measure end-tidal CO 2 and anesthetic agent concentrations. These monitors withdraw gas from the anesthesia circuit continuously at approximately 100 mL/min. Initially, we collected the exhaust gases from the back of a respiratory gas monitor (Ohmeda model 5250) so that we could sample at a site closer to the patient. Unfortunately, internal filters in this respiratory gas monitor absorbed all the exhaled ethane prior to release through the monitor’s exhaust port. Currently, we sample exhaled breath from the proximal connection site of the sampling tubing of the respiratory gas monitor. A three-way stopcock is placed in-line at the connector attached to the end of the endotracheal tube. This modification allows breath to be collected directly from the end of the endotracheal tube without risk to the patient and enables a breath profile to be generated that is more reflective of the actual composition of the breath. D. Timing of Collections

The two major decisions that must be reached regarding breath collection are the exact time points at which to collect the breath and the duration of the collection.

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The increase in exhaled ethane levels occurs almost immediately after the occurrence of the reperfusion injury and is of relatively brief duration, of the order of 0–5 min (22). Therefore, the timing of the breath collection is critical. Since there is a background level of ethane, it is necessary to have baseline measurements of ethane when the patient is stable for comparison purposes. After induction of anesthesia, when the patient is hemodynamically stable, a series of samples can be collected for the measurement of baseline ethane levels. The peak breath ethane level usually occurs immediately after reperfusion of the organs, and therefore it is desirable to collect a series of breath samples around this time point. As we have previously shown, if reperfusion is partial, the peak ethane level can be attenuated or delayed (22). Furthermore, to elucidate the extent of the reperfusion injury, both a peak measurement and an estimate of the duration of the response are necessary. Therefore, multiple breath collections should be made after reperfusion of the organs. Depending on the rate and extent of the reperfusion, the breath ethane levels can remain elevated for as long as 60 min (23). The number of samples that can be collected in this period is limited by the time it takes to collect each sample. Multiple samples collected for shorter time intervals will allow better precision of the peak ethane levels. While longer collection time samples will give an average measure of ethane levels, any rapid changes will be masked. Initially samples of exhaled breath were collected over 4 min (⬎8 L) in five-layer bonded polymeric gas sampling bags (polyethylene, polyamide, aluminum foil, polyvinylidene chloride, and polyester). These inert sampling bags do not adsorb gases and the gas samples remain stable, with respect to hydrocarbon levels, for at least 1 week. More recently, we have demonstrated that gas samples can be collected on glass thermal desorption tubes packed with sequential beds of a graphitized carbon and a carbon molecular sieve. Collection is performed at a constant flow rate of 10 mL/min for 3 min. This collection coupled with two-stage thermal desorption gas chromatography provides a superior way to collect and analyze breath for studies aimed at quantifying reperfusion injury. Gas samples are stable on thermal desorption tubes for at least 3 months. E.

Confounders

Electrocautery

Increased ethane production can occur as a result of the use of electrocautery. This potential interference has been demonstrated in a swine skin incision model. The ethane levels increased with the use of electrocautery (20) and remain elevated for approximately 10 min after use. Therefore, in general, during breath ethane levels collection, and specifically during critical portions of the reperfusion events, electrocautery should be avoided when it is surgically feasible. This problem is particularly apparent during sternotomy during cardiac surgery.


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Changes in Surgical Progress

Other factors that are difficult to control and can affect the timing and quality of the breath collection are changes in the surgical procedure. While surgical procedures are carefully planned, changes often occur during the procedure that necessitate changes in breath collection or negate the data from future analysis. For example, converting from a laperoscopic nephrectomy to an open procedure changes the dynamics of splanchnic blood flow. Or multiple unclamping and reclamping of the hepatic artery during liver transplantion can affect the profile of lipid peroxidation signal due to reperfusion. Therefore, with discrete breath collection time points, and strict adherence to collection protocol, changes in surgical plan can negate a whole subject data set. III. Breath Analysis Exhaled breath contains as many as 400 different molecules that originate from cellular processes that are occurring in the body or as contaminants in the inspiratory gas. The composition of breath consists of mainly (99.99⫹%) nitrogen, oxygen, water vapor, and carbon dioxide. The remaining molecules are present at concentrations at parts-per-million or parts-per-billion levels. The focus of this chapter is the use of breath hydrocarbons to quantify the stable end products of lipid peroxidation that originate from oxygen free-radical-mediated reperfusion injury. Breath hydrocarbons are present in exhaled breath at concentrations at the parts-per-billion level. Currently, there are no monitors commercially available that are able to detect hydrocarbons in breath directly. Therefore, methods for quantifying breath hydrocarbons must be based on general analytical chemistry techniques such as chromatographic or spectroscopic methods. Gas chromatography is the ideal method for breath analysis, since it allows this complex sample to be separated into its components. However, this analytical method requires that the sample be introduced as a discrete sample over a brief finite period of time. Additionally, the volume of the sample introduced must be significantly less than the volume flow of mobile phase through the column. These limitations and the concentrations of the molecules of interest require that breath samples be concentrated prior to separation and quantification. A. Breath Concentration

The goal of breath concentration is to separate the molecules of interest in breath from the bulk matrix. Since the major components of breath are nitrogen, oxygen, water vapor, and carbon dioxide, various approaches have been used to perform this bulk separation. It is relatively easy to separate oxygen and nitrogen from

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breath since these molecules will not be removed when the breath is cooled to liquid-nitrogen temperatures. However, this cryogenic temperature will also condense water vapor and carbon dioxide, and these abundant molecules can block the passage of breath through a cryogenic trap. Therefore, the amount of water vapor and carbon dioxide in the breath sample will limit the volume of breath that can be concentrated. Various adsorbents have been investigated as a method for concentrating the molecules of interest in breath. Adsorbents increase the available surface area for the capture of breath molecules and as the activity of the adsorbent surface increases, the temperature at which breath concentration can be performed increases. Currently, adsorbents are being developed that can concentrate breath at ambient temperatures (8). In the future, one may be able to select an adsorbent to maximize the concentration of a specific analyte of interest. The next step after concentration is the removal of the collected breath molecules from the trap and their introduction into the gas chromatograph, which is usually performed by thermal desorption. Clearly, if the breath molecules are concentrated at room temperature, a much higher temperature will be required to thermally desorb the collected molecules than if the breath is concentrated at subambient temperatures. Various off-line thermal desorption systems have been reported. With these systems the thermally desorbed concentrated breath sample is injected into the gas chromatograph. Automated two-stage thermal desorption systems coupled directly to gas chromatographs are now available. These systems allow breath to be concentrated off-line, and the tube containing the adsorbent is inserted into the instrument for subsequent analysis. A recent review (8) presents a more detailed discussion on the various approaches that can be used to collect and concentrate breath hydrocarbons. B. Gas Chromatographic Analysis

The requirements for gas chromatographic analysis are dependent on the breath concentration step and the concentrations of the analyte molecules. Gas–solid chromatography has typically been used for gas analysis, but the concentration of water vapor in breath will deactivate gas–solid column packing materials such as silica or alumina. Deactivation of these column packing materials will cause retention data to be reduced, with the result that the resolution of breath molecules is difficult. The use of column packing materials that are not water sensitive is also possible, but these polymeric adsorbents often have poor temperature characteristics, making breath profiling difficult. Currently, wall-coated or porous-layer fused-silica open tubular columns provide the optimum method for separating concentrated breath samples. A more detailed discussion of the column requirements for the separation of breath hydrocarbons has recently been published (8).


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Figure 2 Capillary gas chromatogram of a representative breath sample collected and analyzed using a thermal desorption tube.

An example of a breath sample collected from a human subject after intubation is shown in Figure 2. C. Confirmation of Breath Biomarkers

Preliminary identification of breath hydrocarbons can be made on the basis of comparison of the retention volume for the unknown molecule with those of known standards. Additionally, these preliminary identifications can be confirmed by the addition of standards to the sample of breath. Confirmation of this identity should be made using a secondary method of identification such as electron impact mass spectrometry. The problem of misidentification of breath molecules is particularly relevant to breath analysis. For example, isoprene, the most abundant breath C 5 hydrocarbon, was frequently misidentified as 1-pentane in many published reports. This technical error would not have occurred if the identity of the hydrocarbon had been confirmed with mass spectrometry (24).

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Brown and Risby IV. Expression of Data

The first step in the quantification of breath hydrocarbons is the determination of their response factors. Response factors can be determined from the slopes of calibration curves for known concentrations of hydrocarbons. The concentration of the breath hydrocarbons can be determined from the response factors. The concentrations of breath hydrocarbons in concentrated breath should fall within the linear portions of the calibration curves. The concentrations determined from the calibration curves are then adjusted for the breath concentration step in order to calculate the concentrations of the hydrocarbons in the original breath sample. A. Concentration

The traditional way to express data for the compositions of gas mixtures is volume or moles per unit volume (typical concentrations of molecules in breath fall within the ranges parts per million to parts per billion or nmol/L to pmol/L). Many difficulties can result from expressing the composition of breath samples in terms of these units, since the concentration of breath molecules will change with different breathing patterns. The composition of breath will change dramatically if a subject holds his or her breath for different periods of times and then exhales. Similarly, the concentrations of breath molecules will change with minute ventilation. This intrasubject variability makes intersubject comparisons tenuous. Moreover, attempting to make clinical assessments or interventions based on the analyses of serial breath samples for a single subject collected during different breathing patterns may be impossible. This difficulty is relevant to studies aimed at quantifying reperfusion in the clinical setting. However, conversion of concentration units to generation rates allows intra-and intersubject comparisons to be performed. B. Generation Rates

Current practice suggests that breath concentration data should be converted to generation rates by quantifying minute ventilation. This information should then be corrected for body weight or body surface area. Generation rates are expressed in moles/unit time/unit body weight or moles/unit time/unit body surface area (typical generation rates fall within the ranges nmol/min/kg to pmol/min/kg, or nmol/min/m 2 to pmol/min/m 2 ). This method for reporting data allows intersubject comparisons to be made. Moreover, generation rates enables breath from pediatric subjects to be compared to breath collected from adult study subjects (22).

Monitoring Reperfusion Injury by Measurement of VOCs V.

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Organ Systems

Although lipid peroxidation is initiated when any ischemic organ system is reperfused, we have focused the majority of our studies to date on two specific organ systems, the liver and the heart. These organs were chosen initially for several reasons. First, with currently available analytical methods, we anticipated that organs with considerable tissue mass must undergo ischemia for significant periods of time followed by reperfusion in order to produce measurable increases in the concentration of breath ethane levels above baseline. Second, these organs were selected with clinical considerations in mind. In all cases, planned surgical interventions on these organ systems involved controlled and predictable ischemia and reperfusion, which facilitated breath collection. Finally, ischemia and reperfusion injury to these organ systems can contribute significantly to patient morbidity and mortality. Therefore, measurement of reperfusion injury is a necessary first step to initiate future therapeutic interventions. We also performed preliminary studies in other organ systems, the pancreas, and the gut, to investigate the methodological limitations to the quantification of reperfusion injury. A. Liver

Studies aimed at relating the magnitude of reperfusion injury to liver dysfunction were performed in a juvenile porcine model of warm hepatic ischemia (13). This experimental protocol was developed since it modeled the ischemic damage that may incur during implantation of a transplanted graft. A similar surgical technique was employed in this animal model as those used in human liver transplantation, including inhalational anesthesia and venovenous bypass of the liver except that no organ was transplanted. Prior to the 2-hr period of warm ischemia the pigs were infused with heparin and cannulas were placed in the gastro-duodenal artery and a branch of the portal vein to facilitate infusion of pharmacological agents to block reperfusion injury. Breath, blood, bile, and liver samples were collected throughout the surgical procedure. These samples were used to quantify reperfusion injury (breath ethane and conjugated dienes), liver function/injury (serum aspartate aminotransferase, alanine aminotransferase, bilirubin, ammonia, plasma factor VII, indocyanine green clearance, bile output), and oxidative damage in liver tissue [heat shock protein-72 (25)]. Blood and bile samples were collected daily for 3 days postoperatively and assayed for levels of serum AST, ALT, bilirubin, ammonia, plasma factor VII, ICG clearance, and bile production. At day 3 the pigs were euthanized humanely and liver tissue samples were collected and assayed for levels of HSP-72. Eight pigs were used in this study, four controls, and four that received boluses of recombinant human superoxide dismutase (SOD) 5 min prior to reper-

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fusion of the warm ischemic liver followed by 15 min infusion of the same SOD. SOD catalytically converts superoxide to hydrogen peroxide. For the control pigs the time course of oxygen free-radical-mediated reperfusion injury, as measured by time profiles for the concentrations of breath ethane and serum conjugated dienes, reached a maximum 10–15 min after reperfusion. Based on these results, reperfusion of the warm ischemic porcine liver appeared to occur more slowly than the reperfusion of a preserved transplanted liver (see below). Reperfusion injury as evidenced by extensive lipid peroxidation was demonstrated to be completely abrogated when the pigs were treated with SOD at reperfusion, and this reduction was statistically significant ( p ⬍ 0.01). Concomitant to this reduction in reperfusion injury was statistically improved liver function and reduced hepato-cellular injury in the SOD-treated pigs compared to the control pigs ( p ⬍ 0.05). Moreover, this pharmacological intervention suggests that the oxidant-mediated component of reperfusion injury was produced by radicals derived from superoxide anion and that this damage could be ameliorated selectively. It was remarkable that reperfusion injury was blocked so significantly, since there was no evidence to support the transport of recombinant human SOD across cell membranes. Our first clinical studies with breath ethane collection were designed to quantify reperfusion of ischemic tissue in orthotopic liver transplantation in humans (22). This surgical procedure was chosen initially because we hypothesized that transplantation of this large, well-perfused organ should provide the maximum signal from the peroxidation of lipids at reperfusion. Additionally, the liver is known to contain large amounts of xanthine oxidoreductase, which has been implicated in the mechanism of reperfusion injury. A detailed description of the harvest, preservation, and implantation of human orthotopic liver transplants falls outside the scope of this chapter, therefore only the relevant factors will be discussed. The donor liver can be ischemic for up to 20 hr prior to revascularization and reperfusion in the recipient. The surgical procedure in the liver recipient involves dissection and removal of the diseased liver, followed by placement of the patient on venovenous bypass (except for pediatric patients). Before completion of the portal vein anastomosis, the preservation solution is flushed from the liver by infusion of iced Ringers lactate solution. Blood is allowed to flow through the mesenteric venous system to remove stagnant blood and clots. Anastomosis of the hepatic artery is performed after the liver has been reperfused with portal blood and any bleeding controlled. The time course of the generation of oxygen free radicals at reperfusion and the rate of excretion of the volatile products of lipid peroxidation from ischemic tissue into exhaled breath were previously unknown. In our initial studies, we were uncertain of the times to collect the samples of breath to quantify reperfusion injury. Therefore, multiple 2-min breath samples were collected sequentially starting when the patient’s trachea was intubated until the end of the surgical


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operation when the skin was closed. In general, the concentration of breath ethane remained constant during the initial tissue dissection, although it did drop slightly during the anhepatic phase. The most significant change in breath ethane occurred immediately after reestablishing portal blood flow. There was a threefold increase in the level of breath ethane in the sample collected 1–3 min after portal blood flow to the ischemic graft was initiated (Fig. 3). The portal vein supplies approximately 70% of the oxygenated blood to the liver. This rapid rise in concentration of breath ethane decreased during the subsequent 25 min, and just before the hepatic artery anastomosis was completed, ethane returned to baseline levels. No significant change in breath ethane was observed when the hepatic artery was unclamped. The statistically significant ( p ⬍ 0.001) increase in concentration of breath ethane immediately after the portal blood flow was restored to the liver was observed in 7 of the 8 patients studied. For one patient who experienced hemodynamic instability during the operation and for whom it was necessary to increase the portal blood flow slowly and intermittently, the increase in breath ethane was found to increase more slowly and peak 15 min after portal vein reperfusion (similar to the time course to the porcine warm ischemic model). Parenthetically, it appears that the time profile for the rise in breath ethane corre-

Figure 3 Reperfusion of ischemic tissues in humans during different surgical procedures: (a & b) data collected during human cardiopulmonary bypass, time points 0–5 min after reperfusion, (a) p ⬍ 0.05, n ⫽ 8, compared to baseline, (b) p ⬍ 0.02, n ⫽ 8, compared to baseline; (c) data collected during human liver transplantation, time points 1–3 min after portal vein opened, (c) p ⬍ 0.001, n ⫽ 8, compared to baseline; (d) data collected during human supraceliac aortic cross-clamping, time points 16–25 min after reperfusion, (d) p ⬍ 0.05, n ⫽ 6, compared to baseline. Statistical analyses were performed with ANOVA, data are means ⫾ SE.

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lated to the time course for the change in color of the transplanted liver from gray to bright red. In preliminary studies (26) this change in color appears to be associated with increased levels in blood carboxyhemoglobin after reperfusion, signifying that the enzyme heme oxygenase-1 may be induced during the period of hepatic ischemia. The patient demographics of this group of patients ranged in age from 2 to 56 years, preservation times from 8 to 17 hr, and venovenous bypass time from 0 (pediatric patient, 2 years old) to 135 min. One patient had primary nonfunction of the graft and underwent a second transplant within 48 hr. All patients had 100% 30-day survival. Attempts were made to confirm the breath ethane data with blood biomarkers of lipid peroxidation by the collection of blood samples pre- and postreperfusion to determine the concentrations of conjugated dienes and malondialdehyde. However, the patients received significant amounts of blood or blood products during the procedure due to preexisting conditions, and these transfusions confounded the measurement of the blood biomarkers of lipid peroxidation. These initial studies demonstrated that reperfusion injury occurred during orthotopic liver transplantation in humans despite the fact that the preservation solution used during retrieval and storage of the transplanted organ (University of Wisconsin preservation solution, ViaSpan) contains allopurinol. Additionally, boluses of mannitol were given intravenously as standard practice prior to unclamping the portal vein. Allopurinol blocks d-to-o conversion of xanthine oxyreductase, and mannitol is a hydroxyl-radical scavenger. Comparable evidence of oxygen free-radical-mediated reperfusion injury was confirmed in a porcine model of liver transplantation (27). Human liver transplantation is complex and involves many technical and clinical variables relating to the harvest of the donor liver, the status and anatomy of the recipient, and the storage, preservation, and complexity of implantation of the orthotopic graft. All these variables contribute to the clinical outcome of this procedure and make it difficult to investigate therapeutic interventions to block or reduce contributions due to reperfusion injury and thereby improve clinical outcome. Therefore we examined a series of patients (n ⫽ 7) undergoing surgical repair of abdominal aneurysms (23), since this procedure includes many features common to human liver transplantation, such as warm ischemia and venovenous bypass. These patients required cross-clamping of the aorta above the celiac axis for 18 to 50 min in order to perform the aortic revascularization. Successive samples of breath were collected before aorta cross-clamp, during aorta cross-clamp, and for 60 min after the aorta cross-clamp was removed. Although all patients were administered mannitol (12.5 g) prior to cross-clamping the aorta, statistically significant increases in ethane ( p ⬍ 0.05) were observed in 6 of the patients (Fig. 3). For the patient whose aorta was cross-clamped for only 18 min, no increase in ethane was observed. Attempts were made to relate


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the time of cross-clamping to the magnitude of the change in ethane and/or clinical outcome, but the number of patients, potential differences in collateral blood flow, and their preexisting conditions precluded these correlations. B. Heart

Human orthotopic heart transplantation is another surgical procedure in which reperfusion injury may play a significant role in the clinical outcome of the graft. This injury was studied using three different approaches. The first involved an anesthetized closed-chest dog model in which the left anterior descending coronary artery was occluded with a balloon catheter for a period of 90 min (28). After this period of ischemia, the balloon was deflated and blood flow was restored. Breath samples were collected twice during the period of occlusion and over successive time intervals after reperfusion (0 to 4, 5 to 9, 10 to 14, and 15 to 19 min). The concentration of breath ethane was found to increase after reperfusion, and this increase reached a maximum during the time period 5–9 min postocclusion. This model was repeated with the exception that, prior to the deflation of the balloon catheter, a solution of a manganese superoxide dismutase mimetic was infused into the occluded coronary artery. The introduction of this selective reagent to superoxide completely blocked the generation of breath ethane when blood was restored to the coronary artery. Companion studies with magnetic resonance imaging showed that area of myocardial injury was reduced by this treatment that blocked the oxidant-mediated component of reperfusion injury. These studies demonstrated that breath ethane could detect injury to selected regions of the myocardium and could be used to monitor therapeutic intervention. Additional studies are planned to investigate whether breath ethane levels can be used to evaluate the efficacy of coronary artery angioplasty therapy. The second approach involved the collection of breath samples from patients undergoing cardiac surgery to bypass blocked coronary arteries or to repair heart defects [n ⫽ 8 (20)]. In all these patients, a cardiopulmonary bypass machine was used to perfuse the patient with blood during the time when the heart was not functioning as a pump. During the time that cardiac surgery was being performed, a membrane oxygenator, an integral component of the cardiopulmonary bypass machine, was functioning as a gas exchanger to oxygenate the blood and remove carbon dioxide. Breath samples were collected from the patient when the lungs were ventilated and from the outflow port of membrane oxygenator when the patient was on cardiopulmonary bypass. Conceptually, we knew there were two occasions when reperfusion of ischemic tissue would occur. The first would occur during bypass when the aorta was unclamped and blood flow was restored to the myocardium. The second would occur when the patient was removed from cardiopulmonary bypass and normal physiology was restored and blood flows to the extramyocardial tissue.

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Clearly, these two events involved significantly different amounts of reperfused ischemic tissues. Moreover, the rates and extents of the exchange of ethane in the human lungs versus in the membrane oxygenator would be different. Successive 2-min samples of gas were collected throughout this surgical procedure. Additionally, various technical and clinical parameters related to surgery were recorded. Evidence of reperfusion injury, as manifested by an increased concentration of ethane, was observed within the first 2 min after the aorta was unclamped ( p ⬍ 0.05, Fig. 3). Furthermore, a greater change in the concentration of ethane was observed within the first 2 min after the patient was removed from cardiopulmonary bypass ( p ⬍ 0.05, Fig. 3). In addition, the increase in ethane concentration after cardiopulmonary bypass continued for the next sample collected 5 min later. Linear regression models were generated to relate the magnitudes of the changes in ethane with the various technical and clinical parameters. The change in ethane concentration after the aorta was unclamped was found to be significantly associated with impaired cardiac function. The patients who were less acidotic and who maintained their levels of hemoglobin throughout the surgery had greater increases in ethane after removal from cardiopulmonary bypass. No attempts were made to block reperfusion injury by the use of pharmacological agents that block superoxide, although a solution of mannitol (25 g) was added during cardiopulmonary bypass for all patients. In a similar fashion, evidence of reperfusion injury was observed when we examined human orthotopic heart transplantation. The only difference in breath hydrocarbon concentrations observed between orthotopic heart transplant and coronary bypass surgery was that the magnitude of change in ethane was greater when the aorta of the graft was unclamped (29). This result was anticipated, since the transplanted graft was subjected to cold and warm ischemia for longer periods of time. C. Pancreas

Preliminary studies were undertaken to investigate further any limitations in the methodology to quantify reperfusion injury. For these studies, a porcine model of pancreas transplantation was investigated. Although reperfusion of this small organ did produce increases in ethane compared to baseline when blood flow was restored, the increases in ethane did not reach statistical significance [n ⫽ 6 (29)]. In addition, the results suggest that the acute pancreatitis often observed post-pancreas transplantation may be mediated by reactive oxygen species. D. Gut

Recently we have studied gut ischemia in patients who underwent laperoscopic surgery for removal of a donor kidney (30). During this surgical procedure, the peritoneum is inflated with carbon dioxide to a pressure of 20 mmHg. This ab-


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dominal inflation allows excellent surgical exposure of the donor kidney along with the renal artery, vein, and the ureter. The artery, vein, and ureter can be ligated laperoscopically, and the kidney can be removed through a small incision in the anterior abdominal wall. Immediately prior to removal of the kidney, the peritoneum is deflated and the donor kidney is removed. The goal of this study was to investigate whether inflation of the peritoneum produced ischemia of the gut. Moreover, if the gut was ischemic, could deflation of the peritoneum result in quantifiable reperfusion injury at the time of removal of the donated kidney. Of the 12 patients studied, 8 patients underwent the laproscopic surgery successfully; in the remaining 4 patients, the surgery was converted to an open procedure for surgical reasons. Breath samples were collected when the patients were stabilized after the trachea was intubated, during the period of inflation of the peritoneum (total duration 2–3 hr), and at 5-min intervals after the peritoneum was deflated. No significant changes in breath ethane were observed at deflation of the peritoneum (reperfusion). This negative study suggests that either the gut is not made ischemic by inflation of the peritoneum or that the amount of gut made ischemic was not sufficient to produce quantifiable changes in ethane when the peritoneum was deflated. Based on our earlier studies on repair of complex aneurysms (23) and the pioneering studies on feline intestinal ischemia by Grangier and colleagues (5), we believe that this negative study suggest that inflation of the peritoneum does not produce tissue ischemia in the gut.

VI. Interventions When normal blood flow to a tissue is interrupted, there is a change in cellular physiology in response to this stress. The rate and extent of this change is dependent on the tissue type and its oxygen consumption requirements. There are many effects caused by the reduction in cellular oxygen, for instance, the degradation of ATP, alteration of redox couples, or the modification of enzymes such as the proteolysis of xanthine dehydrogenase. All these processes are designed to protect the cell during the period of anoxia. Concomitant with this response to oxygen depletion are changes due to the lack of blood flow, such as substrate depletion, and the buildup of metabolites leading to cellular acidosis. If subsequently blood flow is restored to this anoxic tissue, a second “hit” of injury is produced via a number of mechanisms including the production of reactive oxygen species. Pharmacological intervention to ameliorate reperfusion injury can be performed by a variety of mechanisms. For instance, superoxide dismutase or its manganese mimetic can be introduced to catalyze the conversion of the superoxide anion to its less reactive product, hydrogen peroxide. Hydrogen peroxide can be catalytically detoxified to water and oxygen by treatment with catalase, peroxidase, or glutathione peroxidase. Other approaches to minimize the concentrations of hy-

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droxyl radicals are to selectively block the Fenton reaction by the introduction of iron chelators such as desferrioxamine or to introduce allopurinol to selectively block d-to-o conversion of xanthine oxyreductase. Interventions by the introduction of any of the potential pharmacologic agents will block the formation of the very reactive species, the hydroxyl radical, and thereby prevent an important contributor to reperfusion injury. Alternative approaches to block reactive oxygen-mediated reperfusion injury are to interrupt ensuing free-radical chain reactions by the addition of vitamin E, vitamin C, ubiquinone, vitamin A, or mannitol. Breath ethane is a valuable tool to evaluate the efficacies of any these interventions if elevated breath ethane can be linked to other indices of tissue injury. In our studies to date, elevated breath ethane has been linked to evidence of liver dysfunction and myocardial injury in various animal models. Oxygen free radicals are also involved in molecular signaling resulting in the recruitment, differentiation, and activation of monocytes. Administration of pharmacological agents to block or scavenge the free-radical messenger can attenuate this delayed reperfusion injury. Currently, it is unknown whether breath ethane has sufficient sensitivity to detect changes in this delayed form of reperfusion injury.

VII. Conclusion and Future Directions These initial studies demonstrated that increased levels of breath ethane are quantifiable shortly after reperfusion of ischemic organs in both humans and animals. Moreover, in animal models it has been demonstrated that interventions to prevent oxygen radical-mediated reperfusion injury also minimized tissue damage. Therefore, interventions have potential applications in clinical situations where periods of ischemia occur. The limiting factor in administration of these pharmacological interventions is the ability to detect the onset of injury. Breath biomarkers have the potential to detect this injury. As with other exhaled breath biomarkers, such as carbon dioxide and nitric oxide, the next step for biomarkers of ischemia-reperfusion injury is real-time measurement so that treatment can be instituted in a timely fashion. Ischemiareperfusion injury is the major component of diseases such as stroke and myocardial infarction, which are leading causes of morbidity and mortality in the developed world. With the future development of real-time noninvasive measures of breath biomarkers of ischemia-reperfusion injury, treatment could be detected, initiated, and modified rapidly even before the patient arrives at the hospital. In the hospital setting, real-time noninvasive measurement would improve the assessment of aggressive treatment regimens such as thrombolytic agents and angioplasty. Similarly, prevention of reperfusion injury during organ transplantation could improve the outcome and thereby ensure maximum use of available


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organs. These potential therapeutic interventions will only be achieved by the development of real-time monitors for breath hydrocarbons. Acknowledgments We wish to acknowledge our many clinical colleagues who have collaborated with us during the course of these studies. The generous gift of automatic thermal desorption systems, capillary gas chromatograph, and data system by the PerkinElmer Corporation is gratefully acknowledged. This work was partially supported by funds from the National Institutes of Health (PO1HL56091). We also thank Dr. W. Michael Foster and Dr. Shelley S. Sehnert for their advice and help in this research and also suggestions made during the preparation of this chapter. References 1. Farkouh E, Daniel A, Beaudoin J, MacLean L. Predictive value of liver biochemistry in acute hepatic ischemia. Surg Gynecol Obstet 1971; 132:832–838. 2. Ohkawa M, Clemens M, Chaudry I. Studies on the mechanism of beneficial effects of ATP-MgCl 2 following hepatic ischemia. Am J Physiol 1983; 244:R695–R702. 3. Chaudry I, Ohkawa M, Clemens M. Improved mitochondrial function following ischemia and reflow by ATP-MgCl 2 . Am J Physiol 1984; 246:R799–R804. 4. Cho P, Miescher E, Clemens M. Calcium-free reperfusion prevents mitochondrial calcium accumulation but exacerbates injury. Circ Shock 1990; 32:43–53. 5. Granger D, Rutili G, McCord J. Superoxide radicals in feline intestinal ischemia. Gastroenterology 1981; 81:22–29. 6. Parks D, Bulkley G, Granger D, Hamilton S, McCord J. Ischemic injury in the cat small intestine: role of superoxide radicals. Gastroenterology 1982; 82:9–15. 7. Reilly P, Schiller H, Bulkley G. The pharmacologic approach to tissue injury mediated by free radicals and other reactive oxygen metabolites. Am J Surg 1991; 161: 488–503. 8. Risby TH, Sehnert SS. Clinical applications of breath biomarkers of oxidative stress status. Free Rad Biol Med 1999; 27:1182–1192. 9. Smith M, Thor H, Hortzell P. The measurement of lipid peroxidation in isolated hepatocytes. Biochem Pharmacol 1982; 31:19–26. 10. Burk R, Ludden T, Lane J. Pentane clearance from inspired air by the rat: dependence on the liver. Gastroenterology 1982; 84:138–142. 11. Poli G, Gravela E. Lipid peroxidation in isolated hepatocytes. In: Free Radicals, Lipid Peroxidation and Cancer. McBrien DCH, Slater TF, eds. London: Academic, 1982, pp 215–241. 12. Reiter R, Burk R. Effect of oxygen tension on the generation of alkanes and malondialdehyde by peroxidizing rat liver microsomes. Biochem Pharmacol 1987; 36:925–929. 13. Kazui M, Andreoni KA, Norris EJ, Klein AS, Burdick JF, Beattie C, Sehnert SS, Bell WR, Bulkley GB, Risby T. Breath ethane: a specific indicator of free radical-

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17. 18. 19.

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24. 25.

26. 27. 28.

29. 30.

Brown and Risby mediated lipid peroxidation following reperfusion of the ischemic liver. Free Rad Biol Med 1992; 13:509–515. Kasai H, Crain P, Kuchino Y, Nishimura S, Ootsuyama A, Tanooka H. Formation of 8-hydroxyguanine moiety in cellular DNA by agents producing oxygen radicals and evidence for its repair. Carcinogen 1986; 7:1849–1851. Shigenaga M, Gimeno C, Ames B. Urinary 8-hydroxy-2′-deoxyguanosine as a biological marker of in vivo oxidative DNA damage. Proc Natl Acad Sci USA 1989; 86:9697–9701. Korthuis R, Granger D. Ischemia-reperfusion injury: role of oxygen-derived free radicals. In: Physiology of Oxygen Radicals. Taylor AE, Matalon S, Ward PA, eds. Bethesda, MD: American Physiological Society, 1986, pp 217–249. Ross D, Moldeus P. Antioxidant defense systems and oxidative stress. In Vigo-Pelfrey C, ed. Membrane Lipid Oxidation. Boca Raton, FL: CRC Press, 1990:152–170. Kappus H. Oxidative Stress. Orlando, FL: Academic, 1985. Grisham M, McCord J. Chemistry and cytotoxicity of reaction oxygen metabolites. In: Physiology of Oxygen Radicals. Bethesda, MD: American Physiological Society, 1986, pp 1–18. Andreoni KA, Kazui M, Cameron DE, Nyham D, Sehnert SS, Rohde CA, Risby TH. Ethane: a marker of lipid peroxidation during cardiopulmonary bypass in humans. Free Rad Biol Med 1999; 26:439–455. Kharasch ED, Jubert C. Compound A uptake and metabolism to mercapturic acids and 3,3,3-trifluoro-2-fluoromethoxypropanoic acid during low-flow sevoflurane anesthesia: biomarkers for exposure, risk assessment, and interspecies comparison. Anesthesiology 1999; 91:1267–1278. Risby TH, Maley W, Scott RPW, Bulkley GB, Kazui M, Sehnert SS, Schwarz KB, Potter J, Mezey E, Klein AS, Colombani P, Fair J, Merritt WT, Beattie C, Mitchell MC, Williams GM, Perler BA, Donham RT, Burdick JF. Evidence for free radicalmediated lipid peroxidation at reperfusion of human orthotopic liver transplants. Surgery 1994; 115:94–101. Kazui M, Andreoni KA, William GM, Perler BA, Bulkley G, Beattie C, Donham R, Sehnert S, Burdick J, Risby T. Viceral lipid peroxidation occurs at reperfusion after supraceliac aortic crossclamping. J Vasc Surg 1994; 19:473–477. Kohlmuller D, Kochen W. Is n-pentane really an index of lipid peroxidation in humans and animals? A methodologic reevaluation. Anal Biochem 1993; 210:268–276. Schoeniger LO, Andreoni KA, Ott GR, Risby TH, Bulkley GB, Udelsman R, Burdick JF, Buchman TG. Induction of heat shock gene expression in the post-schemic liver is dependent upon superoxide generation at reperfusion. Gastroenterology 1994; 106:177–184. Sehnert SS, Kazui M, Risby TH. Unpublished results, 1996. Maley W, Taguchi Y, Potter J, Bulkley G, Burdick J. Evidence for damage due to free radicals at reperfusion in porcine liver transplants. Transplant Proc 1989; 21:1316. Wu KC, Rochitte CE, Gerber B, Honda T, Riley D, Salvemini D, Risby TH, Zweier JL, Becker LC, Lima JAC. Antioxidant therapy reduces post-reperfusion infarct size progression by contrast-enhanced MRI. Circulation 1999; 100:I797. Andreoni KA, Kazui M, Burdick JF, Risby TH. Unpublished results, 1996. Brown RH, Ratner L, Kavoussi L, Risby TH. Unpublished results, 1999.

INFLAMMATION

14 Exhaled Nitric Oxide, Carbon Monoxide, and Breath Condensate in Inflammatory Lung Disease and Response to Medical Treatment

SERGEI A. KHARITONOV and PETER J. BARNES Imperial College of Science, Technology and Medicine National Heart and Lung Institute and Royal Brompton Hospital London, England

I.

Introduction

There has recently been an explosion of interest in the analysis of breath constituents as a way of monitoring inflammation and oxidative stress in the lungs. Although most studies have focused on exhaled nitric oxide (NO), recently several other volatile gases (carbon monoxide, ethane, pentane) have also been used. In addition, several endogenous substances (inflammatory mediators, cytokines, oxidants) may be detected in expired breath condensates, opening up new perspectives for exhaled breath analysis. Many lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, cystic fibrosis, and interstitial lung disease, involve chronic inflammation and oxidative stress. Yet these are not measured directly in routine clinical practice because of the difficulties in monitoring inflammation. Noninvasive monitoring may assist in differential diagnosis of pulmonary diseases, assessment of disease severity, and response to treatment. Because these techniques are completely noninvasive, they can be used repeatedly to give information about kinetics, they can be used in patients with severe disease, who have previously been difficult to monitor, and they can be used to monitor disease in children, including infants. Breath analysis is currently a research procedure, but 307

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there is increasing evidence that it may have an important place in the diagnosis and management of lung diseases in the future. This will drive the development of cheaper and more convenient analyzers, which can be used in a hospital and later in a family practice setting, then eventually to the development of personal monitoring devices for use by patients. II. Nitric Oxide NO is the most extensively studied exhaled marker, and abnormalities in exhaled NO have been documented in several lung diseases (1), particularly asthma (2–4). A. Asthma

Increased levels of exhaled NO have been widely documented in patients with asthma (5) (Fig. 1) (6,7). The increased levels of exhaled NO in asthma have a predominant lower airway origin (8,9) and are most likely due to activation of NO synthase 2 (NOS2) in airway epithelial and inflammatory cells (10,11). However, there may be a small contribution from NO synthase 1 (NOS1), as polymorphisms of the NOS1 gene are correlated with exhaled NO (12), may be further elevated by NO substrate L-arginine (13).

Figure 1 Exhaled NO in normal subject and patient with asthma. (From Ref. 8.)

Exhaled Breath Constituents in Inflammatory Lung Disease

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Diagnosis and Epidemiology

An elevation of exhaled NO is not specific for asthma, but an increased level may be useful in differentiating asthma from other causes of chronic cough (14). The diagnostic value of exhaled NO measurements to differentiate between healthy subjects with or without respiratory symptoms and patients with confirmed asthma has been recently analyzed by Dupont et al. (15) with 90% specificity and 95% positive predictive value when exhaled NO ⬎ 15 ppb is used as a cutoff for asthma. Exhaled and nasal NO may be used to identify subjects with atopy, because nonatopic asthmatics have normal exhaled NO (16). There is a strong association between elevated exhaled and nasal NO and skin prick test scores, total IgE (17), and blood eosinophilia (18) in mild asthma. Elevated nasal NO is also related to the size of skin test reactivity in asymptomatic asthmatic subjects (19). This may denote “subclinical” airway inflammation. Airway responsiveness measurements (PC 20 ) in this “high-risk” group make the combination of exhaled NO and PC 20 a more specific test for allergic asthma. This has recently been demonstrated in a study of over 8000 adolescents in Norway (20). Because of the noninvasive character and practicality of exhaled and nasal NO measurements, they may be used cost effectively for screening of large populations. Atopy and exposure to pro-inflammatory stimuli: Exhaled NO is elevated in allergic/atopic adults and children (21–23). It is further increased as a result of allergen exposure, such as during the late-phase response to allergen challenge (24,25), during the grass pollen season (26), or during exposure to indoor allergens (27,28). In house dust mite (HDM)-sensitive subjects the wheal size for HDM correlates with exhaled and nasal NO levels (19). Both adults (21) and children (23) with atopic asthma have higher levels of exhaled NO than patients with nonatopic asthma, even without airway hyperresponsiveness (29). Exhaled NO may represent a useful biomarker of individual exposure to air pollutants, as even healthy subjects may have elevated exhaled NO levels on days with high outdoor air pollution (30,31). This may reflect an airway inflammatory response to ozone and nitrogen dioxide (32). Asthma Monitoring

Exhaled NO has been used to monitor the effect of anti-inflammatory treatment in asthma (4,33) and asthma exacerbations, both spontaneous (34) and induced by steroid reduction (35,36). There is a lack of long-term serial studies of exhaled NO, together with other markers of airway inflammation in sputum and exhaled condensate, lung function, and symptoms. Exhaled NO behaves as a “rapid response” marker, which is extremely sensitive to steroid treatment, as it may be significantly reduced even after 6 hr following a single treatment with a nebulized

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(a)

(b) Figure 2 Effect of corticosteroids on exhaled NO in asthma. (a from Refs. 33, 37; b from Ref. 42.)

corticosteroid steroid (37), or within 2–3 days after inhaled corticosteroids (33), reaching maximal effect after 2–4 weeks of treatment (33,35,38–42) (Fig. 2a). An important issue in asthma management is to prevent overtreatment of patients with steroids. The high sensitivity of exhaled NO to corticosteroid treatment is an advantage, as higher doses of inhaled steroids are not necessary to improve asthma control, e.g., in mild persistent asthma. We have demonstrated a dose-dependent reduction in exhaled NO and improvement in asthma symptoms in mild asthmatics following treatment with low doses of inhaled corticosteroids (42) (Fig. 2b), whereas the reduction in sputum eosinophils and similar improvement in symptoms was observed only after the higher dose of steroids (39). This


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suggests that exhaled NO levels may be too sensitive to determine whether inflammation is adequately controlled. Although exhaled NO levels are normal in patients with mild and moderate asthma (6), increased levels have been observed in patients with severe asthma, despite treatment with oral corticosteroids (43,44). Individual NO values, like individual peak expiratory flows, should be established and monitored, and when the levels are above or below a certain reference level, steroid treatment should be either reduced or increased. A considerable advantage of exhaled NO is that NO levels may increase before any significant changes in other parameters, such as lung function and sputum eosinophils, and may therefore serve as an early warning of loss of control (2). Disease Severity

Treatment with inhaled corticosteroids reduces exhaled NO levels and therefore exhaled NO cannot be related directly to asthma severity. Exhaled NO levels are almost three times higher in children with recent symptoms compared with symptom-free subjects (45), and are further elevated during the asthma attack in both adults (46) and children (47,48). In fact, the levels of NO in children with acute severe asthma (48) are more than twofold higher than in children with less severe wheezing exacerbations and almost fourfold higher than in children with first-time wheeze (47). A reduction in exhaled NO (by 65% after 5 days of corticosteroid therapy) is accompanied by clinical and FEV1 improvement from asthma exacerbations in children (49), and NO has been a more sensitive marker of asthma activity than serum ECP or soluble interleukin-2 receptors (50). Higher exhaled NO levels are related to asthma symptoms and β 2-agonist use in patients with difficult severe asthma (43). Exhaled NO is increased in patients who remain symptomatic despite oral steroids and who have a relative steroid resistance, and may therefore be useful to quantify steroid resistance in asthma. Relationship to Other Markers of Asthma

The traditional means of monitoring asthma have limitations. Lung function and PC 20 measurements are not directly related to airway inflammation, have little room for improvement in mild asthma (FEV1 ), and are affected by bronchodilators. Both parameters are slow to change and are not able to distinguish the effect of different doses of steroids. Exhaled NO in patients with asthma is correlated with sputum eosinophils (39,51), and methacholine reactivity (52,53), as well as peak flow variability (35,38). However, the relationship between exhaled NO and airway inflammation is still uncertain, and in smaller studies no significant relationship is seen between

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exhaled NO and eosinophils in bronchial biopsies or bronchoalveolar lavage (38,54). This may indicate that increased exhaled NO reflects some but not all aspects of airway inflammation, and further work is needed to determine how it relates to some other markers of airway inflammation. Corticosteroids

Systemic corticosteroids have no effect on exhaled NO in normal subjects, but decrease its levels in patients with asthma (34,55). Oral dexamethasone (4 mg/ day for 2 days) similarly has no effect on exhaled NO or serum concentrations of interferon-γ and interleukin(IL)-1β in normal subjects (56) (Table 1). A large dose (1 mg/kg/day for 5 days) of oral prednisolone normalized exhaled NO in infants and young children with wheezing exacerbations (47), whereas the same dose in more severe asthmatic children only shifted their exhaled NO down to the levels of mild-to-moderate asthma in spite of the improvement in lung function (48). A cumulative dose of methylprednisolone (180–500 mg) caused 36% reduction within 50 hr in the majority of severe adult patients with acute asthma (34), and a combination of oral prednisolone and inhaled steroids reduced exhaled NO by 65% in children with acute asthma (49). Recently, it has been shown that NO levels correlate with the percentage improvement in FEV1 from baseline to the poststeroid (30 mg prednisolone/day for 14 days), postbronchodilator value. A NO level of ⬎10 ppb at baseline has

Table 1 Effect of Corticosteroids on Exhaled NO Drugs Normal subjects *Pred 30 mg/day, 3 days Asthma patients *BUD 1600 µg/day (mild) *BUD 100 µg/day (mild) BUD 400 µg/day *Pred 30 mg/day, 3 days (mild) Pred ⫹ IS (severe) Pred 1 mg/kg, 5 days (severe) Pred 1 mg/kg, 5 days (moderate) FP 1000 µg/day, 4 weeks *BUD 100 µg/day (mild) BUD 400 µg/day

Magnitude

Time after IS treatment

No effect ↓ 30% ↓ 29% ↓ 50% ↓ 22% ↓ 40% ↓ 46% ↓ 52% ↓ 76% No change ↓ 26%

Reference (55)

7 days 28 days 28 days 72 h 48 h 5 days 5 days 2 weeks 3 days 3 days

(33) (39) (55) (34) (48) (47) (40) (42)

BUD, budesonide; FP, fluticasone propionate; Pred, prednisolone; IS, inhaled steroids; ↓, decrease; ↑, increase; *, placebo-controlled randomized trial.


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a positive predictive value of 83% for an improvement in FEV1 of ⱖ15%, and therefore may be useful in predicting the response to a trial of oral steroid in asthma (57). Inhaled corticosteroids reduce exhaled NO in asthmatic patients (33), and this effect is dose related (39,42). The effect of inhaled steroids on exhaled NO is very rapid and may occur within 6 hr after a single high dose (8 mg) of budesonide (Pulmicort Respules  ) in symptomatic moderate asthma (37). Recently, it has been shown that the onset of action of inhaled BUD on exhaled NO and the time to reach the maximal reduction were also dose dependent (42). A gradual reduction in exhaled NO is seen during the first week of regular treatment (33,41,42), with maximal effect between 3 (33,40) and 4 (38,39) weeks. Corticosteroids may reduce exhaled NO by directly inhibiting the induction of NOS2 (58) or by suppressing the pro-inflammatory cytokines that induce NOS2. There is inhibition of NOS2 immunoreactivity with inhaled corticosteroid treatment in asthmatic patients and a parallel reduction in immunoreactivity for nitrotyrosine, which may reflect local production of peroxynitrite from an interaction of NO and superoxide anions (11). β 2-Agonists

Neither short-acting (33,48,59–61) nor long-acting (48,59,60,62) β 2-agonists reduce exhaled NO. This is consistent with the fact that they do not have any anti-inflammatory effects in asthma. There may even be a short-term increase in exhaled NO after β 2-agonists, which may be due to opening up of airways with higher local NO concentrations (63). Anti-leukotrienes

The leukotriene receptor antagonist pranlukast inhibits the rise in exhaled NO when the dose of inhaled corticosteroids is reduced (64), and montelukast rapidly reduces exhaled NO by 15–30% in children with asthma (65,66). Interleukin inhibitors (nebulized IL-4 receptors) have been able to reduce exhaled NO in patients with moderate asthma (67). This effect of anti-leukotrienes on exhaled NO may be due to a reduction of inflammatory cytokines and their induction of NOS2. NOS Inhibitors

Nebulized L-NMMA and L-NAME, which are nonselective inhibitors of NOS, both reduce exhaled NO in asthmatic patients, although this is not accompanied by any changes in lung function (55,68). Aminoguanidine, a more selective inhibitor of NOS2, reduces exhaled NO in asthmatic patients, but has little effect in normal subjects, indicating that NOS2 is an important source of the increased exhaled NO in asthma (69).

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Kharitonov and Barnes Prostaglandins and Other Drugs

Prostaglandin (PG)E 2 downregulates NOS2 expression (70), and inhaled PGE 2 and PGF2α decrease exhaled NO in normal and asthmatic subjects (71). Immunosuppressive drugs, cyclosporin and rapamycin, can inhibit NOS2 expression (72), suggesting that exhaled NO can be used to monitor their effect. Ibuprofen, a COX inhibitor, reduced elevated levels of exhaled NO in normal subjects after i.v. administration of endotoxin (73). A low dose of theophylline had no effect on exhaled NO levels in asthmatic patients (74). B. COPD

Exhaled NO levels in stable COPD (75–77) and chronic bronchitis (78) are lower than in either smoking or nonsmoking asthmatics (79) and are not different from normal subjects. This reduction in exhaled NO is due to the effect of tobacco smoking, which downregulates NOS (80) and reduces exhaled NO (75). In addition to the effects of cigarette smoking, a relatively low value of exhaled NO in COPD may reflect more peripheral inflammation than in asthma, low NOS2 expression (77) and increased oxidative stress that may consume NO in the formation of peroxynitrite (81). Patients with unstable COPD, however, have high NO levels compared with stable smokers or ex-smokers with COPD (82), which may be explained by increased neutrophilic inflammation and oxidant/antioxidant imbalance. Eosinophils that are capable of expressing NOS2 and producing NO are present in exacerbations of COPD (83). Acidosis, which is frequently associated with exacerbations of COPD, may increase the release of NO (84). C. Bronchiectasis

An increase in exhaled NO is found in bronchiectasis and the increase in NO is related to the extent of disease as measured by a computerized tomography score (85). As in asthma, the elevation of exhaled NO is not seen in patients treated with inhaled corticosteroids. This suggests that exhaled NO in bronchiectasis may reflect active inflammation in the lower airways and may be used to monitor disease activity. This is supported by increased NOS2 expression in lungs from patients with bronchiectasis (86). However, in another study, exhaled NO levels were not elevated compared to normal subjects in clinically stable patients with bronchiectasis, and it was suggested that NO is either trapped in viscous airway secretions, or removed by reaction with reactive oxygen species (87). D. Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD), including Kartagener’s syndrome, is a genetic disease characterized by defective motility of cilia, in which the levels of exhaled


Figure 3

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Exhaled and nasal NO in primary ciliary dyskinesia (PCD). (From Ref. 89.)

and NO are very low compared to normal subjects (88) (Fig. 3) (89–91). Such low values of exhaled and nasal NO are not seen in any other condition and are therefore of diagnostic value. Measurement of exhaled NO might be used as a screening procedure to detect PCD among patients with recurrent chest infections or male infertility due to immotile spermatozoa. NO plays an important role in bactericidal activity in the lungs, sodium and chloride transport in nasal epithelium, and ciliary beating (92), so that a lack of endogenous NO production might contribute to the characteristic recurrent chest infections in PCD patients. Treatment with NO donor L-arginine increased nasal NO and also improved mucociliary transport in PCD patients (89). The mechanism for such a low NO production by nasal and airway epithelia in PCD is unknown, but might be linked to genetic abnormalities in NOS2 gene expression as in cystic fibrosis. E. Interstitial Lung Diseases Systemic Sclerosis

Systemic sclerosis is reviewed in detail elsewhere in this volume. Briefly, there is a reduction in exhaled NO in patients with systemic sclerosis who have developed pulmonary hypertension compared to normal subjects and to patients with interstitial lung disease without pulmonary hypertension (93). This may be due to reduced expression of NOS3 in pulmonary vessels, or a reduction in the pulmonary vascular endothelial surface. However, presence of NOS3 in pulmonary vessels is variable and it has been found either reduced (94–96), or increased (97), or variable (98), or unaltered (99). Fibrosing Alveolitis

There is strong expression of nitrotyrosine and NOS2 in macrophages, neutrophils, and alveolar epithelium in lungs of patients with idiopathic pulmonary

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fibrosis with active inflammation during the early to intermediate stage of the disease (100). This is consistent with elevated levels of exhaled NO in patients with fibrosing alveolitis. Increased exhaled NO levels are associated with disease activity, as assessed by BAL lymphocyte counts, and are reduced in patients treated with corticosteroids (101). Sarcoidosis

Cytokines, including TNF-α and interferon-γ, are increased in the pulmonary inflammation of sarcoidosis and there is an upregulation of NOS2 in respiratory epithelium and granulomata in patients with sarcoidosis (102). The magnitude of exhaled NO rise in sarcoidosis may be related to the disease activity of the disease and is reduced by steroid therapy. This is, perhaps, the reason behind two conflicting observations reporting either elevated (102) or normal (103) exhaled NO in patients with active pulmonary sarcoidosis. F. Infections

NO may play an important role in nonspecific host defense against bacterial, viral, and fungal infections. One of the general mechanisms of antimicrobial defenses involving NO is S-nitrosylation by NO of cysteine proteases, which are critical for virulence or replication of many viruses, bacteria, and parasites. The low endogenous NO production, resulting in low exhaled and nasal NO levels, may contribute to recurrent chest infections in PCD and CD patients, as discussed above. Low nasal NO is associated with colonization of the upper respiratory tract with Staphylococcus aureus in active Wegener’s granulomatosis (104). Viral Infections

Exhaled, but not nasal NO, is elevated during viral infections in adults and children (105,106). Exhaled NO is also increased in experimental human influenza (107) and rhinovirus infection (108). The increase in NO production during viral infection is likely to be protective, as NO inhibits virus replication either by inhibiting viral RNA synthesis, or/and by S-nitrosylation of the cysteine proteases that are critical for virulence and replication of viruses (109). Viral infection may also induce the expression of NOS2 via activation of NF-κB and other transcription factors (110). Exhaled NO in HIV-positive individuals is less than in control subjects (111), and NO synthesis is further depressed in terminally ill HIV patients (112), suggesting that low NO may indicate a mechanism of impaired host defense in HIV infection. This may be explained by an inhibitory role of the HIV type 1 regulatory protein Tat on NOS2 activity, as shown in a murine macrophage cell line (113).


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Tuberculosis

NO plays an important role in resistance to Mycobacterium tuberculosis infection, and exposure of extracellular M. tuberculosis to ⬍100 ppm of NO for a short period (⬍24 hr) results in microbial killing (114). Elevated exhaled NO and NOS2 expression in alveolar macrophages are found in patients with active TB and are reduced with antituberculous therapy (115). Bacterial Infections

Nitrate concentrations are significantly higher in BAL in immunosuppressed children with pneumonia than in normal control subjects (116), and elevated exhaled NO levels are found in patients with lower respiratory tract inflammation and chronic bronchitis (78). G. Chronic Cough

Not all forms of airway inflammation are accompanied by increased levels of exhaled NO. Patients with chronic cough that is not attributable to asthma have lower NO values as compared with healthy volunteers and patients with asthma (14,53), including those with cough due to gastroesophageal reflux (117). Measurement of exhaled NO may therefore be a useful screening procedure for patients with chronic cough and would readily identify those patients with cough due to asthma (14). III. Carbon Monoxide Carbon monoxide (CO) is a gas that may be formed endogenously and is detectable in exhaled air. A. Asthma

Elevated levels of exhaled CO have been reported in stable asthma (118,119), with normal levels in patients treated with inhaled corticosteroids (119). The increased levels in stable asthma are likely to be due to increased heme oxygenase 1 (HO1) expression, which is seen in alveolar macrophages in induced sputum of patients with asthma. There is also an increase in the concentration of bilirubin in induced sputum, indicting increased HO1 activity. Further evidence that exhaled CO increases may reflect HO activity is the demonstration that inhaled hemin, which is a substrate for HO, results in a significant increase in exhaled CO concentration in normal and asthmatic subjects. Increased levels of exhaled CO are seen in acute exacerbations of asthma, and are reduced after treatment with oral corticosteroids (120). Significantly elevated CO levels are found in

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patients with severe asthma (121), including patients treated with 30 mg of prednisolone for 2 weeks (122). In view of the simplicity of CO measurements and the portability of CO analyzers, exhaled CO may be useful in noninvasive monitoring of pediatric asthma. For example, children with persistent asthma despite treatment with steroids, which reduce their NO levels, have significantly higher exhaled CO compared with those with infrequent episodic asthma (123). B. COPD

A major limitation of exhaled CO in COPD is the marked effects of cigarette smoking, which masks any increase that may occur due to the disease process. There is no difference in exhaled CO in patients with chronic bronchitis (without airflow obstruction) when compared with normal subjects (124). However, exhaled CO levels are elevated in ex-smoking COPD patients (125), suggesting ongoing oxidative stress or inflammation. HO is induced in fibroblasts exposed to cigarette smoke (126). There is an increase in exhaled CO during acute exacerbations of COPD, with a decline after recovery (127). C. Bronchiectasis

Exhaled CO levels are elevated in patients with bronchiectasis, regardless of whether they are treated with inhaled corticosteroids or not (128). D. Cystic Fibrosis

In contrast to NO, exhaled CO levels were markedly elevated in stable CF patients (129–131) and increase further during exacerbations (Fig. 4a) and reduced with antibacterial treatment (Fig. 4b) (132). It may suggest that exhaled CO is not only a marker of oxidative stress/inflammation in CF, but also a marker of disease severity. The level of exhaled CO is not affected significantly by treatment with inhaled corticosteroids treatment (129–131). This is consistent with the fact that inhaled corticosteroids do not appear to benefit CF patients symptomatically (133). E.

Interstitial Lung Disease

Elevation of exhaled CO is related to lung function deterioration (134) and impaired gas transfer in patients with cryptogenic fibrosing alveolitis and scleroderma (135). Elevated levels of exhaled CO in patients with fibrosing alveolitis are also associated with disease activity as assessed by BAL cell counts (101). This suggests that exhaled CO may be used to monitor disease progression and response to therapy in interstitial lung diseases.


(a)

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(b)

Figure 4 Exhaled CO in cystic fibrosis (CF): (a) disease severity; (b) effect of antimicrobial treatment. (From Ref. 132.)

F. Allergic Rhinitis

Stable levels of CO are recorded during continuous sampling from one nostril during normal breathing through the mouth in normal subjects (136). Sampling through a drainage tube inserted into the maxillary sinus reveals CO levels comparable to the levels obtained by sampling through the nose. In patients with allergic rhinitis, exhaled CO is increased during the pollen season and returns to normal values after the season (137). The levels of exhaled CO are significantly higher in patients with symptoms than in those without. However, there is no difference between nasal and exhaled samples, suggesting that the increase is derived from the lower respiratory tract. We did not measure any nasal CO production in either normal or asthmatic subjects (138). G. Infections

HO1 is induced by many infectious agents, and HO1 may provide protection to cells against attack by infectious agents. Upper respiratory tract viral infections may induce the expression of HO1, resulting in increased exhaled CO in adults (139) and children (123). Elevated exhaled CO levels might provide an early warning signal for an acute infective episode, which may lead to exacerbation of asthma and COPD. Elevated levels of CO have been measured in patients in general practice with lower respiratory tract infection, which were significantly reduced after 5 days of treatment with antibiotics (127).

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Kharitonov and Barnes H. Other Conditions

Critically ill patients have a significantly higher CO concentration in exhaled air as well as total CO production compared to healthy controls (140). Interestingly, the levels of exhaled CO in these patients are similar to the levels seen in severe asthma and may be a reflection of systemic rather than local oxidative stress. Exhaled CO levels are also increased in diabetes and the level is significantly related to the level of hyperglycemia (141). The mechanism is unclear, but hyperglycemia and oxidative stress in uncontrolled diabetes may activate HO1. IV. Exhaled Breath Condensate The detection of nonvolatile mediators and inflammatory markers from the respiratory tract involves invasive techniques, such as bronchoalveolar lavage or induced sputum. They cannot be repeated within a short period of time because of their invasiveness, and because the procedures themselves may induce an inflammatory response (142,143). Exhaled breath condensate is collected by cooling or freezing exhaled air and is totally noninvasive. The collection procedure has no influence on airway function or inflammation, and there is accumulating evidence that abnormalities in condensate composition may reflect biochemical changes of airway lining fluid. Several nonvolatile chemicals, including proteins, have now been detected in breath condensates. The first studies identifying surface-active properties, including pulmonary surfactant, of exhaled condensate were published in the USSR in the 1980s (144,145), and since then several inflammatory mediators, oxidants, and ions have been identified in exhaled breath condensates. A. Hydrogen Peroxide

Activation of inflammatory cells, including neutrophils, macrophages, and eosinophils, result in an increased production of O 2⫺ which by undergoing spontaneous or enzyme-catalyzed dismutation lead to formation of H 2 O 2 . As H 2 O 2 is less reactive than other reactive oxygen species, it has the propensity to cross biological membranes and enter other compartments (146). Because it is volatile, increased H 2 O 2 in the airway equilibrates with air (147). Compared with the cellular antioxidant scavenging systems, the extracellular space and airways have significantly less ability to scavenge reactive oxygen species (148,149). Catalase is the major enzyme involved in removing H 2 O 2 and is present in low concentrations in the respiratory tract. Thus exhaled H 2 O 2 may be a good marker of oxidative stress in the lungs.


321

Asthma

H 2 O 2 has been detected in exhaled condensate in healthy adults and children, with increased concentrations in asthma (147,150–152). There is no correlation between the levels of exhaled H 2 O 2 and age, gender, or lung function in healthy children (152). However, exhaled H 2 O 2 concentration is related to the number of sputum eosinophils and airway hyperresponsiveness in asthma of different severity, and is elevated in severe unstable asthmatics, although exhaled NO is significantly reduced by treatment with corticosteroids (151). COPD

Cigarette smoking causes an influx of neutrophils and other inflammatory cells into the lower airways, and fivefold higher levels of H 2 O 2 have been found in exhaled breath condensate of smokers than in nonsmokers (153). Levels of exhaled H 2 O 2 are increased compared to normal subjects in patients with stable COPD and are further increased during exacerbations (154,155). Cigarette smoking is by far the most common cause of COPD, but only 10–20% of smokers develop symptomatic COPD. No significant differences have been found between H 2 O 2 levels in current smokers with COPD and COPD subjects who have never smoked, and there is no correlation between expired H 2 O 2 concentration and daily cigarette consumption (155). Thus oxidative stress is a characteristic feature of COPD and presumably related to airway inflammation, and cannot be explained entirely by the oxidants present in tobacco smoke. Other Lung Diseases

Increased H 2 O 2 levels in exhaled breath condensate have been found in adult respiratory distress syndrome (ARDS) (156,157) and bronchiectasis (158), indicating increased oxidative stress in these conditions. B. Eicosanoids

Eicosanoids are potent mediators of inflammation responsible for vasodilatation/ vasoconstriction, plasma exudation, mucus secretion, bronchoconstriction/bronchodilatation, cough, and inflammatory cell recruitment. They are derived from arachidonic acid and include prostaglandins, thromboxane, isoprostanes, and leukotrienes. C. Prostanoids

There is an increased expression of COX-2, which forms prostaglandins and thromboxane in asthma and COPD (159) and CF (160). Most prostaglandins and

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thromboxane have pro-inflammatory properties, but others, for example PGE 2 and PGI 2 , are anti-inflammatory (161). In contrast, TxB 2 is increased in asthma but not detectable in normal subjects or in patients with COPD (Montuschi et al., unpublished observation). Exhaled thromboxanes may predict more accurately than urinary levels those patients who may benefit from a thromboxane receptor antagonist in asthma (162). D. Leukotrienes

Leukotrienes (LTs), a family of lipid mediators derived from arachidonic acid via the 5-lipoxygenase pathway, are potent constrictor and pro-inflammatory mediators that contribute to pathophysiology of asthma. The cysteinyl-leukotrienes (cys-LTs) LTC 4 , LTD 4 , and LTE 4 are generated predominantly by mast cells and eosinophils, and are able to contract airway smooth muscle, cause plasma exudation, and stimulate mucus secretion, as well as recruiting eosinophils (163). By contrast, LTB 4 has potent chemotactic activity toward neutrophils (164). Detectable levels of LTB 4 , C 4 , D 4 , E 4 , and F4 have been reported in exhaled condensate of asthmatic and normal subjects (165). In mild asthmatic patients, LTE 4 , LTC 4 , and LTD 4 levels in exhaled condensate are increased during the late asthmatic response to allergen challenge (166). Steroid withdrawal in moderate asthma leads to worsening of asthma and is associated with a significant increase in the concentration of LTB 4 , LTE 4 , LTC 4 , and LTD 4 in exhaled condensate (166) (Fig. 5a). LTE 4 is increased in exhaled condensate in patients with asthma, but not those with COPD (Montuschi et al., unpublished observation). LTB 4 concentrations are increased in exhaled breath condensate of patients with COPD and to a lesser extent in asthma (Montuschi et al., unpublished observations). E.

Isoprostanes

Isoprostanes are a novel class of prostanoids formed by free radical-catalyzed lipid peroxidation of arachidonic acid (167). They are formed initially esterified in membrane phospholipids, from which they are cleaved by a phospholipase A 2 , circulate in plasma, and are excreted in urine and can be detected in exhaled breath condensate and BAL. They are stable compounds, detectable in all normal biological fluids and tissues (168), and their formation is increased by systemic oxidative stress, for example, in patients with diabetes (169), or ARDS (170). F2-isoprostanes are reduced by antioxidants, for example, by alpha-lipoic acid in normal subjects (171). They are not simply markers of lipid peroxidation but also possess biological activity, and could be mediators of the cellular effects of oxidant stress and a reflection of complex interactions between the RNS and ROS. Indeed, peroxynitrite is capable of activating biosynthesis of endoperoxide synthase and thromboxanes in inflammatory cells (172), and oxidizing arachi-


323

(a)

(b) Figure 5 Exhaled breath condensate: (a) exhaled nitrotyrosine and leukotrienes before and after steroid withdrawal in patients with moderate asthma (166); (b) exhaled 8-isoprostane in asthma. (From Ref. 174.)

donic acid to form F2-isoprostanes. The most prevalent isoprostane in humans is 8-epi-PGF2α , also known as 8-isoprostane. Asthma

F2-isoprostanes are increased in BAL fluid of asthmatic patients and further increased after allergen challenge (173). Levels of 8-isoprostane are approximately doubled in mild asthma compared with normal subjects, and increased by about

324


threefold in those with severe asthma, regardless of their treatment with corticosteroids (174) (Figure 5B). The relationship to asthma severity is a useful aspect of this marker, in contrast to exhaled NO. The relative lack of effect of corticosteroids on exhaled 8-isoprostane has been confirmed in a placebo-controlled study with two different doses of inhaled steroids (42). This provides evidence that inhaled corticosteroids may not be very effective in reducing oxidative stress. Exhaled isoprostanes may a better means of reflecting disease activity than exhaled NO. COPD

Urinary levels of isoprostanes, in particular 8-isoprostane, are increased in COPD, and decline in patients with acute exacerbation as their clinical condition improves (175). Aspirin treatment failed to decrease urinary levels of isoprostanes, whereas TxB 2 were significantly reduced, confirming that cyclo-oxygenases are not involved in their formation. The concentration of 8-isoprostane in exhaled condensate is also increased in normal cigarette smokers (176), but to a much greater extent in COPD patients (177). Interestingly, exhaled 8-isoprostane is increased to a similar extent in COPD patients who are ex-smokers as in smoking COPD patients, indicating that the exhaled isoprostanes in COPD are largely derived from oxidative stress from airway inflammation, rather than from cigarette smoking. Cystic Fibrosis

CF is characterized by marked oxidative stress in the airways (178) and elevated levels of 8-isoprostane have been detected in plasma (179). Concentrations of 8-isoprostane in the breath condensate of patients with stable CF are increased about three-fold compared with normal subjects (130). Interstitial Lung Disease

Interstitial lung diseases, such as cryptogenic fibrosing alveolitis (CFA) and fibrosing alveolitis associated with systemic sclerosis (FASSc), are characterized by enhanced oxidative stress in both serum (180) and BAL fluid (181). The imbalance between the oxidants and antioxidants is also a prominent feature of sarcoidosis (182). 8-Isoprostane is detectable in BAL fluid of normal subjects and is increased in patients with sarcoidosis, CFA, and FASSc, suggesting a higher level of oxidant stress and greater lung injury in these patients than in sarcoidosis (183). F. NO-Related Products

NO reacts with superoxide to yield peroxynitrite, can be trapped by thiolcontaining biomolecules, such as cysteine and glutathione, to form S-nitrosothi-


325

ols, or can be oxidized to nitrate and nitrite (184). Nitrogen intermediates, for example, peroxynitrite, can induce a number of covalent modifications in various biomolecules, such as nitroso- and nitro-adducts. One such modification yields 3-nitrotyrosine, and detection of this adduct in proteins is now commonly used as a diagnostic tool to identify involvement of NO-derived oxidants in many disease states (185). The balance between nitrite/nitrate, S-nitrosothiols, and nitrotyrosine in lung epithelial lining fluids, as reflected by exhaled breath condensate, gives insight into NO synthesis and short- and long-term changes in NO production. There are several methods, apart from the immunoassays, available for nitrite/nitrate and S-nitrosothiol quantification. They include adsorptive stripping voltammetry (186), electrochemical (187), fluorimetric, and colorimetric measurements (188,189). There is also a method that allows the separation of the thiols from their S-nitrosylated derivatives using capillary zone electrophoresis (190). Asthma

High levels of nitrite have been found in exhaled breath condensate of asthmatic patients, especially during acute exacerbations (191). A deficiency in S-nitrosothiols has been demonstrated in tracheal lining fluid in asthmatic children with respiratory failure (192), suggesting that the levels of S-nitrosothiols, which are endogenous bronchodilators, may normally counteract increased airway tone in asthma. The levels of S-nitrosothiols in exhaled breath condensate are reduced after 3 weeks of treatment with a higher (400 µg daily) but not a low dose (100 µg daily) of inhaled budesonide (42). In contrast, there is a rapid and dose-dependent reduction in nitrite/nitrate in exhaled breath condensate in the same mild asthmatics, suggesting that nitrite/nitrate are more sensitive to anti-inflammatory treatment. Increased levels of nitrotyrosine in exhaled breath condensate are associated with worsening of asthma symptoms and deterioration of lung function during inhaled steroid withdrawal in moderate asthma (166), suggesting that nitrotyrosine may be a predictor of asthma deterioration. COPD

Habitual smokers have unusually high antioxidant concentrations in the epithelial lining fluid and higher resistance to oxidative pulmonary damage. NO can be trapped in the epithelial lining fluid of the respiratory tract in the form of Snitrosothiols or peroxynitrite and released thereafter, leading to transient elevation of exhaled NO after smoking a cigarette (193). Chronic oxidative stress presented to the lung by cigarette smoke may decrease the availability of thiol compounds and may increase decomposition of nitrosothiols, explaining elevated levels of S-nitrosothiols in exhaled condensate in healthy smokers, which are related to smoking history (194). Levels of exhaled nitrite/nitrate are increased

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in COPD (unpublished observation). A significant negative correlation between FEV1 and the amount of nitrotyrosine formation has been demonstrated in patients with COPD, but not in those with asthma and normal subjects (195), suggesting that NO produced in the airways is consumed by its reaction with superoxide anion and/or peroxidase-dependent mechanisms and reactive nitrogen species play an important role in the pathobiology of the airway inflammatory and obstructive process in COPD. Cystic Fibrosis

Elevated levels of nitrite and nitrate have been found in exhaled condensate (196) and sputum of patients with CF during both stable periods and exacerbations (197). In children with CF and normal lung function, however, the nitrite/nitrate in BAL were normal and concentrations of S-nitrosothiols were reduced (198). In contrast, elevated levels of nitrite and S-nitrosothiols are found in exhaled breath condensate of adult patients with more severe CF (199). Myeloperoxidase, a heme enzyme of neutrophils that uses H 2 O 2 to oxidize chloride to hypochlorous acid, is capable of catalyzing nitration of tyrosine, providing an alternative to peroxynitrite in the formation of 3-nitrotyrosine (200). At sites of neutrophilic inflammation, myeloperoxidase will nitrate proteins because the co-substrate tyrosine will be available to facilitate the reaction (200). Patients with stable CF have significantly higher levels of nitrotyrosine in exhaled breath condensate than normal subjects (201). This suggests that nitration of proteins by myeloperoxidase may be a major source of nitrotyrosine in patients with CF who have a very low NO production. Other Lung Diseases

Nitrite and nitrate concentrations were increased in exhaled breath condensate of patients with active pulmonary sarcoidosis (103). G. Electrolytes

Increased airway fluid osmolality in the lower airways as a result of exercise may activate mast cells and cause subsequent bronchoconstriction in a subset of asthmatics. A deficiency in magnesium and an elevation in calcium concentrations in exhaled breath condensate have been reported in atopic asthma (202), although a histamine-induced decrease in plasma magnesium levels occurs regardless of the diagnosis of asthma (203). We have recently demonstrated that exhaled Na⫹ and Cl⫺ are elevated in exhaled condensates of patients with CF and correlate with the sweat test and the disease severity (Balint et al., unpublished observation). Recently, a strong negative correlation between sputum Cl⫺ concentrations and exhaled NO has been demonstrated in patients with PCD (204), sug-


327

gesting that airway mucociliary clearance impairment might be monitored by exhaled/nasal NO and exhaled Cl⫺ levels. H. Hydrogen Ions

An acidic microenvironment upregulates NOS2 in macrophages through the activation of NF-κB (205), making NO release moderately pH dependent (206). Elevated levels of lactic acid have been found in exhaled condensate in patients with acute bronchitis (207), and a low pH of exhaled condensate is reported in patients with acute asthma (84). Exhaled pH is free of salivary, nasal, and gastric contamination and is not influenced by either airflow obstruction or by inhaled albuterol, but is increased by corticosteroid therapy. I. Proteins and Cytokines

Measurement and identification of proteins in exhaled condensate is controversial. It has been reported that the amount of protein in the breath condensate of 8 healthy individuals was from 4 µg to 1.4 mg, originating from the nasopharynx, oropharynx, and lower airways (208). The same group has also reported the presence of IL-1β, soluble IL-2 receptor protein, IL-6, and TNF-α in exhaled breath condensate of patients with a variety of respiratory conditions (208). Recently, higher concentrations of total protein in exhaled condensate have been found in young smokers compared to nonsmokers, while the levels of IL-1β and TNF-α were not different (209). We have found that IL-8 levels in exhaled condensate are mildly elevated in stable CF, but are more than doubled in unstable CF patients compared with normal subjects (Balint et al., unpublished observations). V.

Future Directions

Exhaled breath analysis has enormous potential as a noninvasive means of monitoring airways and inflammation, oxidative stress, and other conditions (for example, metabolic disorders, bacterial and viral infections) (Table 2). The technique Table 2 Changes in Exhaled Gases in Lung Disease Asthma

Nitric oxide Carbon monoxide Ethane

COPD

CF

stable

unstable

stable

unstable

stable

unstable

Bronch

ILD

PCD

↑↑↑ ↑

↑↑↑↑ ↑↑

↔ ↑

↑ ↑↑

↓ ↑↑

↓ ↑↑↑

↑ ↑↑

↑ ?

↓↓ ↑

↑↑

?

↑↑

?

↑↑

↑↑↑

?

?

?

CF, cystic fibrosis; bronch, bronchiectasis; ILD, interstitial lung disease; PCD, primary ciliary dyskinesia; ↑, increase; ↓, decrease; ↔, no change; ?, not yet known.

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is simple for patients to perform and may be applied in neonates and in patients with severe disease. Because the techniques are noninvasive, it is possible to make repeated measurements without disturbing the system, in contrast to the invasive procedures currently used.

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184. Stamler JS. S-nitrosothiols and the bioregulatory actions of nitrogen oxides through reactions with thiol groups. Curr Top Microbiol Immunol 1995; 196:19–36. 185. van Der VA, Eiserich JP, Shigenaga MK, Cross CE. Reactive nitrogen species and tyrosine nitration in the respiratory tract. Epiphenomena or a pathobiologic mechanism of disease? Am J Respir Crit Care Med 1999; 160:1–9. 186. Vukomanovic DV, Hussain A, Zoutman DE, Marks GS, Brien JF, Nakatsu K. Analysis of nanomolar S-nitrosothiol concentrations in physiological media. J Pharmacol Toxicol Meth 1998; 39:235–240. 187. Hou Y, Wang J, Arias F, Echegoyen L, Wang PG. Electrochemical studies of Snitrosothiols. Bioorg Med Chem Lett 1998; 8:3065–3070. 188. Wink DA, Kim S, Coffin D, Cook JC, Vodovotz Y, Chistodoulou D, Jourd’heuil D, Grisham MB. Detection of S-nitrosothiols by fluorometric and colorimetric methods. Meth Enzymol 1999; 301:201–211. 189. Kostka P, Park JK. Fluorometric detection of S-nitrosothiols. Meth Enzymol 1999; 301:227–235. 190. Stamler JS, Loscalzo J. Capillary zone electrophoretic detection of biological thiols and their S-nitrosated derivates. Ann Chem 1992; 64:779–785. 191. Hunt J, Byrns RE, Ignarro LJ, Gaston B. Condensed expirate nitrite as a home marker for acute asthma. Lancet 1995; 346:1235–1236. 192. Gaston B, Sears S, Woods J, Hunt J, Ponaman M, McMahon T, Stamler JS. Bronchodilator S-nitrosothiol deficiency in asthmatic respiratory failure. Lancet 1998; 351:1317–1319. 193. Chambers DC, Tunnicliffe WS, Ayres JG. Acute inhalation of cigarette smoke increases lower respiratory tract nitric oxide concentrations. Thorax 1998; 53:677– 679. 194. Corradi M, Kharitonov SA, Donnelly LE, Montuschi P, Pesci A, Barnes PJ. Elevated levels of nitrosothiols in breath condensate of healthy smokers. Am J Respir Crit Care Med 2000; 161:A857. 195. Ichinose M, Sugiura H, Yamagata S, Koarai A, Shirato K. Increase in reactive nitrogen species production in chronic obstructive pulmonary disease airways. Am J Respir Crit Care Med 2000; 162:701–706. 196. Ho LP, Innes JA, Greening AP. Nitrite levels in breath condensate of patients with cystic fibrosis is elevated in contrast to exhaled nitric oxide. Thorax 1998; 53:680– 684. 197. Linnane SJ, Keatings VM, Costello CM, Moynihan JB, O’Connor CM, Fitzgerald MX, McLoughlin P. Total sputum nitrate plus nitrite is raised during acute pulmonary infection in cystic fibrosis. Am J Respir Crit Care Med 1998; 158:207–212. 198. Grasemann H, Gaston B, Fang K, Paul K, Ratjen F. Decreased levels of nitrosothiols in the lower airways of patients with cystic fibrosis and normal pulmonary function. J Pediatr 1999; 135:770–772. 199. Corradi M, Montuschi P, Donnelly LE, Hodson ME, Kharitonov SA, Barnes PJ. Nitrosothiols and nitrite in exhaled breath condensate of patients with cystic fibrosis. Am J Respir Crit Care Med 1999; 159:A682. 200. van Dalen CJ, Winterbourn CC, Senthilmohan R, Kettle AJ. Nitrite as a substrate and inhibitor of myeloperoxidase. Implications for nitration and hypochlorous acid production at sites of inflammation. J Biol Chem 2000; 275:11638–11644.


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15 Exhaled Gas Disease Markers in Septicemia A Possible Role for Nitric Oxide in Lung Injury

JOHN T. STITT and JAMES S. DOUGLAS Yale University School of Medicine New Haven, Connecticut, U.S.A.

I.

Introduction

The measurement of exhaled gas compositions has proved to be an invaluable noninvasive technique for the detection of pathophysiological changes in the lung. Common techniques that assess lung volumes (multiple breath washouts, helium dilution), gas extraction, and pulmonary capillary blood flow (carbon monoxide diffusing capacity, measurement of ventilation/perfusion ratios) all rely on the collection and measurement of exhaled gas (1). However, most of these methods rely on the determination of changes in the composition of inspired gas mixtures that result from their inhalation. With the development of improved analytical techniques by the early 1970s, it became possible to detect and assay very low concentrations of endogenously generated exhaled gases, and these observations raised the possibility of using endogenous gas production as an indicator of lung injury. The utility of such measurements was demonstrated by Reily et al. (2), who showed that exhaled ethane could be used as a surrogate in the detection of the peroxidation of unsaturated lipids in the liver. Subsequent toxicological studies suggest that the measurements of the endogenously generated gases ethane or pentane can serve as sensitive indicators of lipid peroxidation (3). With the recent reports that nitric oxide (NO) can be detected in exhaled air, 343

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the possibility that exhaled breath contains additional markers that may serve as surrogate measurements of lung injury has received renewed attention (4). These more recent studies have examined the exhalation of gaseous substances such as NO and CO as well as compounds in breath condensates such as hydrogen peroxide and isoprostanes (5,6). Not surprisingly, some measurements have been made in patients with adult respiratory distress syndrome (ARDS), bronchiectasis, and asthma; however, the evolving complexity of these clinical conditions has made the interpretation of such results problematic (4,7–10). In the case of NO, for example, the measured exhaled concentration of NO actually represents “net” production (i.e., the difference between NO produced and NO removed). Since either or both of these parameters might change during evolving injury, methods that evaluate both NO production and NO extraction need to be employed if we are to understand the relationship between exhaled NO levels and the underlying pathology that causes them. In this chapter we describe a rat model of endotoxemia that augurs the events that lead to the development of acute lung injury (ALI), by measuring the appearance of NO in exhaled air after intravenous injection of an endotoxin, lipopolysaccharide (LPS). We propose that these results are relevant to the condition of septicemia and the subsequent development of ALI and ARDS in humans.

II. Endotoxin Shock Endotoxin affects the systemic circulation by causing the profound hypotension that characterizes “septic shock”; this is more correctly termed the systemic inflammatory response syndrome (SIRS), and it often leads to disseminated intravascular coagulation, multiple organ dysfunction (MOD), and eventually to death. Symptoms of systemic shock due to endotoxin or sepsis are usually manifest as decreasing systemic vascular resistance with a compensatory increase in cardiac output, leading eventually to a low mean arterial pressure, venous pooling, and myocardial (left ventricular) dysfunction (11). Later complications will often include disseminated intravascular coagulation and hepatic, renal, and gastrointestinal ischemia, lactacidosis, and other metabolic disorders, eventually causing multiple organ failure and death (11–14). Neutrophil margination and activation is an early event, and initiation of the complement and cytokine cascades appear essential in the production of hypotensive shock syndrome (15). Endotoxin elicits the production of the “early” acute-phase cytokines, TNF-α, IL-1, IL-6, and IL-8 from sites such as blood monocytes and liver Kuppfer cells, and this is the most important event in the vascular shock syndrome (16–22). Recent studies have shown that not only are most cardiovascular signs and symptoms of SIRS produced by TNF-α infusions in animal models but, perhaps more important, the severity and lethality of LPS-induced SIRS are abrogated in mice

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that are pretreated with an anti-TNF-α monoclonal antibody (23,24). There is still debate as to whether TNF-α acts directly to induce these effects or whether it triggers secondary mediators which reduce vascular resistance, cause myocardial dysfunction, and increase vascular permeability (16,17,24,25). In the case of reduced vascular resistance, there is strong evidence that an endothelial cell derived factor (NO) is the most important mediator of the vascular impairment that is observed in endotoxic shock (26–28). It has been shown that inhibition of the L-arginine:NO pathway in vitro restores vascular responsiveness of vessels taken from endotoxin-treated rats (29). Infusions of inducible nitric oxide synthase (iNOS) inhibitors restore both systemic vascular resistance and vascular responsiveness to pressor stimuli, and this recovery can be reversed by the infusion of L-arginine (28). Patients in septic shock have abnormally high levels of urinary nitrate excretion coupled with extremely low levels of plasma arginine, indicating that iNOS is greatly upregulated during endotoxemia (30,31). However, treatment with NOS inhibitors does not appear to improve overall outcome in endotoxic patents (7).

III. Sepsis and Lung Injury The lung is a primary target organ of sepsis. Lung injury may vary from minor changes in respiratory function to major respiratory failure with increased microvascular permeability, pulmonary edema, and an impaired ability to maintain oxygen saturation (18,32,33). Trauma patients who develop sepsis have ⬃40% incidence of lung injury, and in ⬎20% of these patients, respiratory dysfunction is often the first symptom of a distant-focus sepsis (34,35). The predominant cause of ARDS is nonpulmonary sepsis and ARDS is often a prelude to multiple organ failure (MOF) and death (36–38). Mortality rates with ARDS still remain at 50%–60%, even today (39). Early acute lung injury, induced by endotoxins, is characterized by the sequestration and aggregation of neutrophils within the lung vasculature (40) and an initial period of pulmonary hypertension which subsides after about 2 hr. It is then followed by a period of increased pulmonary capillary permeability, which lasts for several hours or days. Depending on the severity of the neutrophil aggregation, activation, and diapedesis, a later-phase injury is manifested as pulmonary edema and by fibroblast proliferation that results in interstitial and intra-alveolar fibrosis. Areas of the lung may become relatively acellular, being replaced with fibrous tissue, and they manifest an impairment of gas exchange. This is compounded by ventilatory abnormalities, which may be the result of the normally protective mechanism of hypoxic pulmonary vasoconstriction, that minimizes V/Q mismatch leading to pulmonary hypertension and resulting in hypoxemia and cardiac hyperdynamia (34,36,41). At this point, diagnosable ARDS becomes apparent and in many cases it is too late

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to treat (34,36,42). It should be noted that septicemia is only one of the several causes of ARDS and that a similar pathology can be initiated by pneumonia, tissue trauma, and gastric aspiration injury. However, systemic sepsis is still the major cause of ARDS today, and development of ARDS without a neutrophil leukocytosis in the lungs is much less common (33). Ever since the recognition of ARDS as a syndrome (36), pulmonologists have sought an early marker of its impending pathology (33); since it is generally accepted that only by the very early use of specific anti-inflammatory therapies to prevent or modify the consequences of acute lung injury can one improve outcome and lower the mortality that attends ARDS (13,43). Recently, we have made observations on the acute effects of intravenously administered LPS on the rat lung, which we believe might provide just such an early marker for identifying the onset of septicemic ALI and ARDS. IV. Exhaled NO as a Marker of Lung Injury While it has been known for some time that in humans, NO gas is normally produced by the large and small conducting airways and to an even larger extent by the upper respiratory (nasopharyngeal) passages, and that NO can be detected largely in exhaled dead-space gas during the respiratory cycle, all evidence points to the alveoli as being net absorbers of NO rather than producers of NO gas (44– 46). It has also been proposed that lung airway inflammatory conditions, such as bronchiectasis or asthma, can exacerbate this NO production in the conducting airways and can increase the level of NO detected in the expired air (4,7,8,10). Experiments in which LPS was instilled into rat lungs via the trachea have been described before, and they have even been reported to produce increases in the level of NOS expression in a variety of airway cells and in resident alveolar macrophages. This induction of NOS took a period of 6–24 hr to occur (47). Quite recently, however, we observed that rats, which normally have little or NO gas in their expired air, responded to intravenous injections of small amounts of LPS endotoxin by exhaling large amounts of NO within 60 min of such LPS treatment (46). The rapidity of this response, together with its large magnitude (⬎250 ppb), prompted us to investigate the phenomenon further, in order to determine its location, the mechanisms that induced it, and its possible relationship to acute lung injury. V.

Influence of Endotoxin on Exhaled NO

A. LPS-Induced Exhaled NO Originates in the Lung

Figure 1a shows the time course of the NO exhaled from the lungs after endotoxin treatment in rats over a range of LPS from 1 µg/kg to 1 mg/kg, i.v. NO was first detected at just less than 60 min after the injection, and it gradually rose to

Figure 1 (a) Time course of NO appearance in exhaled air of rats treated with doses of LPS over the range 1 µg/kg to 1 mg/kg, i.v. Alveolar lung [NO] is expressed in parts per billion (ppb). (b) A log dose–response curve constructed for peak lung [NO] in ppb, using the data shown in Figure 1a. Values are expressed as the mean ⫾ SE, and n ⫽ 6 for each dose of LPS. (From Ref. 46.)

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a peak during the next 2 hr. The NO level then plateaued and gradually declined over the ensuing 3 hr, which was as long as we observed the phenomenon. The lung [NO] shown in Figure 1a are corrected to alveolar levels from the mixed expired values measured on-line. In control rats, that were injected with 0.9% saline and monitored for 6 hr, lung [NO] values never exceeded 3 ppb throughout the experiments. Figure 1b illustrates the dose–response relationship between peak lung [NO] and endotoxin over the dose range 1 µg/kg to 1 mg/kg, i.v. The threshold for detecting NO appears to be around 1 µg/kg, and there is a logarithmic relationship between peak lung [NO] and LPS dose over the range of doses investigated. Figure 2 illustrates that the NO gas contained in the exhaled air of LPStreated rats originated from within the alveolar compartment of the lungs, since it can be seen that the end-tidal levels exceeded those of the mixed-expired levels by approximately the same ratio as the dead-space to the total tidal volume used to ventilate the animals. Furthermore, Figure 2 also demonstrates that this production of NO is dependent on the level of inspired O 2 , since a brief period of ventilation using pure N 2 resulted in immediate and almost complete disappearance of the NO from the expired gas. This suggested that the source of the NO was in the lungs themselves, rather than coming from more distant systemic sites

Figure 2 Record of the mixed expired lung [NO] from a rat after an LPS injection (1 mg/kg, i.v.), illustrating that end-tidal lung [NO] (arrows) exceeds that of mixed-expired lung [NO]. At 4 hr the rat is ventilated with pure N 2 for a period of 2 min, and it can be seen that lung [NO] rapidly falls to zero.


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which were “unloading” NO into the venous blood and hence to the lungs— much in the manner that peripherally produced CO 2 is unloaded by the lungs from venous blood. However, given that LPS is well known to induce iNOS upregulation throughout the body vasculature and that this NO production is known to be the cause of the profound hypotension of endotoxic shock, it was necessary to determine whether the venous return was the source of exhaled NO. This was done by inducing NO exhalation in rats and, when production had reached a plateau, circulation was arrested by inducing cardiac arrest with xylocaine (i.v.) while ventilation of the animals continued. The results, shown in Figure 3, clearly demonstrate that the NO has its origin within the lung itself and, indeed, the pulmonary circulation acts as a sink that removes a large portion of the NO produced by the lungs, rather than delivering NO to the lungs. When LPS-induced NO levels in expired air had reached their plateau levels after 4 hr, cardiac arrest and

Figure 3 The effect of complete circulatory arrest on the mixed-expired lung [NO] of rats treated with 1 mg/kg LPS i.v. at time zero. Arrest was performed at time 240 min. Also shown is the normal progression of lung [NO] without circulatory arrest (control) and the effect of circulatory arrest on control animals that were sham injected with 0.9% saline at time zero (control arrested). Values are mean ⫾ SE, and n ⫽ 5 for the LPStreated arrested group. All values of LPS-treated arrested lung [NO] after 240 min are significantly different from controls (p ⬍ 0.01). (From Ref. 46.)

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the concomitant cessation of pulmonary blood flow resulted in an increase in lung mixed expired NO concentration (lung [NO] me ) from 170 ppb to 615 ppb (⬎250%). This means that when the lung is being perfused, only a small portion of the NO produced within the lungs actually reaches the alveolar compartment to be exhaled, while the larger portion of the NO production is removed from the lung by pulmonary blood flow. Circulatory arrest performed on non-LPS-treated rats did not result in NO production at any point during the 6-hr duration of the experiments. This controlled for the effects of inducing cardiac arrest on the rats per se. B. Mechanisms of LPS-Induced Increases in Exhaled NO

Having established that the source of the NO exhalation in response endotoxemia was the lungs themselves, we then set about determining the mechanism by which this NO production was induced by endotoxin. Two obvious possibilities existed: either the LPS had a direct effect on lung elements such as the alveolar epithelium, macrophages, or endothelium; and/or it acted through intermediaries such as the blood neutrophils, which are well known to aggregate and adhere within the pulmonary circulation after LPS treatment. Rats were injected with LPS (1 mg/kg, i.v.) and after 3 hr, when NO production had reached plateau levels, bronchoalveolar lavages were performed and the total number and distribution of leukocytes were determined and compared to control non-LPS-injected rats. These results are illustrated in Table 1. There were no significant differences in either the total number of leukocytes present or in the relative distribution of macrophages, lymphocytes, and polymorphonucleocytes within the airways of the two groups at this time. Furthermore, the histological picture of a rat lung removed at 3 hr after LPS treatment compared to a control rat lung (Fig. 4) revealed a pronounced congestion within the alveolar wall capillaries, which con-

Table 1 Effect of Endotoxemia on the Bronchoalveolar Lavage Contents of the Lungs of Control Rats and of Rats Treated with LPS (1 mg/kg i.v.) 3 hr Earlier (n ⫽ 4 for each of the control and LPStreated groups; none of the LPS values were significantly different from control values) BAL parameter Total cell count ⫻ 10 6 cells/lung Viability Alveolar macrophages Alveolar lymphocytes Alveolar polymorphonucleocytes

Control 3.68 96.2 91.6 6.8 1.80

⫾ ⫾ ⫾ ⫾ ⫾

0.15 0.47% 2.20% 2.6% 0.93%

LPS-treated 3.25 92.7 91.3 5.6 3.25

⫾ ⫾ ⫾ ⫾ ⫾

1.08 ns 1.31% ns 1.80% ns 0.80% ns 1.04% ns


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Figure 4 (A) shows the lung of a control animal, while (B) shows a lung of a rat treated with LPS, 1 mg/kg i.v., 3 hr earlier. Slides were stained with hematoxylin and counterstained with eosin. Original magnification ⫻400.

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tained neutrophils that had aggregated there in response to LPS treatment. While not conclusive, these results seemed to indicate the probable site of action of the LPS was the pulmonary vasculature rather than the alveolar air space and that lung neutrophils were involved in the response. The possibilities that LPS was acting on the lung directly and/or that lung neutrophil aggregation was involved in the response were then investigated by repeating the LPS experiments on rats that had been made neutropenic by prior treatment with a microtubular poison vinblastine. Rats were injected with 0.75 mg/kg, i.v., with vinblastine sulfate, which induced stasis in neutrophil production. Since the average circulating life of neutrophils is ⬃36 hr, a complete neutropenia could be induced within 96 hr using this method (48). The effect of LPS injection on NO exhalation was determined in control rats, rats pretreated with vinblastine 24 hr earlier, and rats pretreated with vinblastine 4 days previously. Circulating leukocyte counts, taken from peripheral blood prior to the injection of the LPS, were also determined in each of the three groups of animals used. The results of these experiments are illustrated in Figure 5, which shows both lung NO after LPS injection and the circulating leukocytes counts, taken prior to LPS treatment. It can be seen that in both the control and vinblastine ⫹1-day rats, there were identical responses. Neutrophil and other white cell counts were similar in both groups, and the production of NO by the lungs in response to LPS was not significantly different. This indicates that the vinblastine treatment per se did not inhibit the production of NO. However, when a period of 4 days was allowed to elapse after the vinblastine treatment, so that a complete neutropenia was induced, these rats were totally refractory to LPS treatment and NO was not produced. This indicated two things: that the effects of LPS were not directly on the cellular elements of the lung; and that neutrophils were essential for the production of NO by the lungs in response to the LPS treatment. However, these results did not indicate the cellular site of the NO production in response to LPS treatment, which could have been from the neutrophils themselves, the lung vascular endothelium as a result of the aggregated neutrophil interactions, or both. As noted earlier, aggregation and adhesion of neutrophils within the lung vasculature in response to circulating endotoxins is both a recognized and a well-

Figure 5 The upper panel illustrates lung [NO] exhaled by rats injected with LPS (1 mg/kg, i.v.) under three different conditions. These are: control LPS-injected rats (filled symbols), LPS-injected rats at ⫹1 day after vinblastine treatment, and rats LPS-injected at ⫹4 days after vinblastine treatment (open symbols). The lower panel shows the corresponding circulating neutrophil (hatched columns) and other white cell counts (open columns) obtained from the three groups of rats, prior to LPS injection. Values are mean ⫾ SE, and n ⫽ 5 for each of the three treatments.


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documented phenomenon (15,40). It is believed to be a precursor in the eventual acute lung injury that is frequently associated with endotoxemia (48) and is thought to precipitate the subsequent development of ARDS, the more serious and often fatal sequela to endotoxemia (15). The initial events surrounding this neutrophil sequestration in the lungs are, by and large, “silent,” since there are no overt symptoms, other than a mild transient pulmonary arterial hypertension and dyspnea (49). Indeed, neutrophil sequestration usually can only be demonstrated directly in experimental animals, either by prelabeling the neutrophil population with a marker such as indium ( 49 In) and then scanning the chest after endotoxin has been injected intravenously (50), or by measuring the total neutrophil accumulation in the lung tissue by assaying a neutrophil-specific enzyme such as myeloperoxidase (MPO) before and after endotoxin treatment (51). However, the subsequent “activation,” and the acute-phase responses of these lungsequestered, adherent neutrophils, lead to the production of free radicals (e.g., superoxide), lysosomal and proteolytic enzymes that culminate with the diapedesis of the neutrophils into the alveoli and the production of an acute lung injury, many hours or even days later (49). We hypothesized that the exhalation of NO by the lungs of LPS-treated rats described above may be caused directly by the aggregation and adhesion of blood neutrophils to the endothelium of the pulmonary vasculature, in response to LPS (15). If this is true, then the pretreatment of rats with an antibody directed against the primary rat neutrophil adhesion molecule L-selectin may be expected to reduce or ameliorate the accumulation of neutrophils within the lung vasculature after LPS treatment and to abate the appearance of exhaled NO. Rats were injected with 400 µg/kg, i.v., of a monoclonal antibody directed against L-selectin (LECAM), 30 min prior to the injection of 100 µg/kg, i.v., of LPS. An index of neutrophil accumulation, occurring within the lungs after LPS, was obtained by removing them after 3 hr, and assaying the amount MPO in the disrupted whole lung tissue. Since MPO is not found in lung tissues, the amount of MPO determined in the supernatant fraction of the disrupted lung tissue, using a kinetic spectroophotometric assay, is an index of the total mass of neutrophils present in the lungs (51). A nonspecific IgG directed against keyhole limpet hemocyanin (KLH) was used as a control for the specificity of the LECAM antibody. Furthermore, control and ⫹4-day vinblastine-treated rats were assayed in the same manner. The results of such experiments are shown in Figure 6. It can be seen that neither the untreated control rats nor vinblastine-treated rats injected with LPS produced any NO, and their lungs contained only small amounts of MPO. Rats that were treated with LPS had an exhaled lung [NO] me of about 125 ppb and lung MPO activity of about 80 units/g lung tissue after 3 hr. The rats pretreated with LECAM antibody, on the other hand, exhibited a reduction in both exhaled lung [NO] me to about 52 ppb and lung MPO content to about 30


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Figure 6 Mixed-expired lung [NO] (open columns) and whole-lung tissue myeoloperoxidase content (filled columns) in control, LPS-injected rats, neutropenic LPS-injected rats, LPS-injected rats pretreated with Mab to LECAM, and LPS-injected rats pretreated with Mab to KLH. The dose of LPS, when employed, was 100 µg/kg, i.v. Values were determined at 3 hr after the treatment and are mean ⫾ SE (n ⫽ 5 for each treatment).

units/g lung tissue, whereas the rats treated with the KLH IgG were not significantly different from control, untreated LPS-injected rats. Therefore, we concluded that the neutrophil adhesion molecule LECAM played an essential role in both the aggregation of neutrophils within the lung circulation and the production of NO by the lung in response to LPS treatment. This tended to link the aggregation and adhesion of neutrophils within the lung in response to LPS to the subsequent production and exhalation of NO by the lungs. VI. Role of Increased NO in LPS-Induced Lung Injury Finally, we wondered whether NO might have a salutary effect on the lungs and whether the production of NO in response to LPS in some manner minimized

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the acute lung injury produced by LPS that would otherwise occur in its absence. To this end we repeated the above experiments except that we induced an inhibition of lung NO production by treating the animals with the NOS inhibitor N-methyl-L-arginine (L-NAME) at 50 mg/kg, i.v., both prior to and again, 1 hr after the injection of 100 µg/kg, i.v., LPS. In this manner lung NO production was suppressed for the entire 3-hr period after the LPS injection. The lungs were then removed and assayed for MPO activity as before. As a control, L-NAME was also administered in the same manner to a group of non-LPS-injected rats. Conversely, we also made rats breathe therapeutic doses of NO (20 ppm) administered via the intake port of the ventilator immediately prior to and for a period of 3 hr after the injection of LPS. In this manner we could ascertain whether NO affected the subsequent accumulation of neutrophils in the lungs and/or the level of endogenous NO exhalation that occurred at 3 hr after the LPS treatment. Figure 7 shows the results obtained from these experiments. The first two pairs of columns show both lung [NO] and lung MPO content in control and LPS-injected rats, respectively. The next columns illustrate that inhibition of NO production

Figure 7 Mixed-expired lung [NO] (open columns) and whole-lung tissue myeoloperoxidase content (filled columns) in control, LPS-injected rats, control L-NAME-treated rats, LPS-injected rats treated with L-NAME, and LPS-injected rats that were ventilated with NO (20 ppm) for 3 hr after LPS injection. The dose of LPS, when employed, was 100 µg/kg, i.v. Values were determined at 3 hr after the treatment and are the mean ⫾ SE (n ⫽ 5 for each treatment).


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by L-NAME produced a small but significant increase in the amount of neutrophils in the lung, even in the non-LPS-injected group of rats. LPS-injected rats which received L-NAME did not produce any NO, and their lung neutrophil content was slightly higher than in LPS-injected rats. Finally, rats that were administered the therapeutic inhalation dose of NO had both significantly lower exhaled [NO] me after the exogenous NO was removed, and their lung neutrophil content was 50% lower than that found in LPS-injected rats. We interpret these results as demonstrating that inhibition of endogenous NO production exacerbates the aggregation of neutrophils within the pulmonary circulation, in both control and LPS-injected rats, whereas the administration of exogenous NO gas by inhalation reduces both the aggregation of neutrophils within the lungs and also the subsequent exhalation of endogenously produced NO by the lungs in response to LPS injection.

VII. Conclusions Based on the foregoing results and our earlier description of the sequelae of endotoxemia in the pulmonary circulation in rats, we propose the following progression of events, which we believe accounts for many of the previously published observations on endotoxemia and for the results that we obtained using the rat LPS model. Furthermore, we propose that the appearance of NO in the exhaled gas from the lungs, shortly after injection of endotoxin into the circulation, serves as an early marker for the subsequent development of ALI and ARDS that invariably attend endotoxemia. Sequestration and adhesion of circulating neutrophils is a widespread vascular response to endotoxemia. However, there are three important reasons why so many more neutrophils aggregate within the pulmonary circulation as opposed to the systemic circulation, and why the injurious effects of endotoxin are often first apparent in the lung when sepsis occurs. First, neutrophil adhesion is greatly enhanced within the lungs because of the uniformly small diameter of the lung capillaries (⬃5.5 µm) with respect to the larger diameter of neutrophils (⬃7 µm). Second, the entire blood volume passes through the lungs about once every minute; it is believed that at any given time ⬃60% of the circulating neutrophils in the blood are passing through the pulmonary circulation. Finally, sepsis usually induces a rapid mobilization of noncirculating neutrophils, causing large numbers of these cells to be disgorged from bone marrow and other tissue reserves into the blood, increasing the circulating neutrophil population and thereby putting more neutrophils into the pulmonary circulation. The production of NO by endothelial cells is a generalized response that accompanies LPS-induced neutrophil margination onto the vascular endothelium.

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Within the systemic circulation, the localized vasodilatory effects of NO are manifest as a reduction in total peripheral resistance—this leads to hypotension and “vascular shock” and the NO production is evident as an increase in reactive nitrogen intermediates (RNIs) such as nitrites, nitrates, and nitrosothiols in the plasma. In the pulmonary circulation, however, a portion of this NO production appears as NO gas exhaled in the alveolar air, because proportionally many more neutrophils aggregate within the lungs, producing greater concentrations of endothelial-derived NO, and the unique architecture of the alveolar wall acts as a “window” on the gas exchange that is occurring within the pulmonary vasculature. Thus, some of this NO enters into the alveolar air as NO gas. Furthermore, due to the very early onset of this NO exhalation, we have reason to believe that constitutive eNOS present in lung capillary endothelial cells may act as a “facilitator” for the passage of neutrophils along the lung capillaries, even in health, as well as being the source of the adherent neutrophil-induced NO production that is caused by LPS injections. Later induction of iNOS after several hours may, in fact, be a deleterious development, which leads to the SIRS pathophysiology of endotoxemia and to the enhancement of lung injury. NO production by the endothelia in response to neutrophil adherence and margination ameliorates both the inflammatory and injury responses that are caused by neutrophil adhesion and activation induced by the LPS. Both are more evident in the pulmonary circulation because of the greater concentrations of neutrophils there. We hypothesize that there are three possible mechanisms by which NO could act protectively within the lung. First, it may inhibit the mechanisms by which the neutrophils adhere to the vascular endothelial walls. Indeed, it may be that NO plays such a physiological role in the passage of neutrophils through the constraining pulmonary capillaries normally, even in the absence of LPS. Second, by inducing pulmonary vasodilatation and augmenting lung blood flow, it might facilitate the passage of neutrophils through the pulmonary vasculature, thereby reducing the propensity for neutrophils to sequestrate within the lungs. Third, NO overrides normal pulmonary hypoxic vasoconstrictor reflexes that divert blood flow away from areas of poor ventilation, where alveolar damage has occurred. While this may do little to alleviate hypoxemia, and is even reported to exacerbate right-to-left shunting, it would alleviate the resulting pulmonary hypertension and reduce the afterload on the right heart that is often caused by lung injury, and thus, it would tend to reduce any attendant hypertensive pulmonary edema.

VIII. Clinical Implications It has been reported and is widely believed that patients suffering from ARDS do not exhibit any increases in the level of NO in their exhaled gases. Indeed,


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levels of exhaled NO in ARDS patients have been reported to be even lower than those measured in control subjects (9). How, then, do our current observations on NO exhalation by endotoxemic rats relate to the clinical observations that are made in the condition of ARDS? We believe that this discrepancy can be readily explained by the time at which the observations are made. We propose that the reason lung [NO] peaks and starts to decline after 3 hr may be due to conversion of NO into peroxynitrite by the superoxide radicals that are produced by the “activated” neutrophils—a process that takes some time to develop. If so, then an accumulation of nitrotyrosine in lung tissues would be expected, since the peroxynitrite will react with protein tyrosine residues in the lung to produce high nitrotyrosine levels. Indeed, it has been reported that high levels of in-vivo peroxynitrite production are found in the lungs of ALI patients (52), and more recently, Cross et al. (53) have proposed that peroxynitrite production within the lung might be a major pathway of NO metabolism which leads to the production of lung injury in ARDS patients. Thus, it is our belief that the absence of NO in the exhaled air of patients who have developed ARDS does not necessarily mean that they are not generating NO, only that the NO that they are producing within their lungs is being converted to peroxynitrite, a species that cannot be detected in their exhaled air. However, it is our contention that if the exhaled NO levels were monitored in patients that were at risk for the development of sepsis and/or ARDS, they would exhibit increased levels of NO in their exhaled air during the very early stage of ARDS, were it to develop—that is, when the neutrophil aggregation is actually occurring within the lungs and before these sequestered neutrophils become “activated” and begin to produce large amounts of superoxide radicals and lysosomal enzymes which result in subsequent injury to the lung tissues. We believe it is essential that clinical trials be undertaken to monitor the exhaled NO levels in patients who are at risk for the development of septicemia and/or ALI/ARDS, to determine when exhaled NO levels elevate prior to the onset of any subsequent pathology and whether such elevations could be used “predict” the impending lung injury. References 1. Comroe JH, Forster RE, DuBois AB, Briscoe WA, Fisher AB. The Lung; Physiologic Basis of Pulmonary Function Tests. 3d ed. Chicago: YearBook Medical Publishers, 1986:1–295. 2. Reily CA, Cohen G, Leiberman M. Ethane evolution: A new index of lipid peroxidation. Science 1974; 183:208–210. 3. Foster WM, Jiang L, Stetkiewicz PT, Risby TH. Breath isoprene: temporal changes in respiratory output after exposure to ozone. J Appl Physiol 1996; 80:706–710. 4. Barnes PJ, Kharitinov SA. Exhaled nitric oxide: a lung function test. Thorax 1996; 51:233–237.

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5. Keitzmann D, Kahl R, Muller M, Burchardi H, Kettler D. Hydrogen peroxide in expired breath condensate of patients with acute respiratory failure and with ARDS. Int Care Med 1993; 19:78–81. 6. Montuschi P, Corradi M, Ciabattoni G, Nightingale J, Kharitinov SA, Barnes PJ. Increased 8-isoprostane, a marker of oxidative stress, in exhaled condensate of asthma patients. Am J Respir Crit Care Med 1999; 160:216–220. 7. Gaston B, Drazen JM, Loscalzo J, Stamler JS. The biology of nitrogen oxides in the airways. Am J Respir Crit Care Med 1994; 149:538–551. 8. Kharitinov SA, Wells AU, O’Connor BJ, Hansell DM, Cole PJ, Barnes PJ. Elevated levels of nitric oxide in bronchiectasis. Am J Respir Crit Care Med 1995; 151:1889– 1893. 9. Brett SJ, Evans TW. Measurement of endogenous nitric oxide in the lungs of patients with acute respiratory distress syndrome. Am J Respir Crit Care Med 1997; 156: 993–997. 10. Ho LP, Innes JA, Greening AP. Exhaled nitric oxide is not elevated in the inflammatory airways of cystic fibrosis and bronchiectasis. Eur J Respir 1998; 12:1290–1294. 11. Hale HS, Robinson JA, Loeb HS, Gunnar RM. Pathophysiology of endotoxin shock in man. In: Procter RA, ed. Handbook of Endotoxin, Vol. 4: Clinical Aspects of Endotoxin Shock. Amsterdam: Elsevier, 1986:185–237. 12. Border JR, Hassett JM. Multiple systems organ failure: history, pathophysiology, prevention and support. In Clowes GHA, ed. Trauma, sepsis, and shock—The Physiological Basis of Therapy. New York: Marcel Dekker, 1988:335–356. 13. Donner RL, Elin RJ, Henson SM, Wesley RA, Riley JM, Parillo JE. Endotoxemia in human septic shock. Chest 1991; 99:169–175. 14. Murry JF. Acute pulmonary injury in sepsis. In Root RK, Sande MA, eds. Septic Shock. New York: Churchill Livingstone, 1981:105–115. 15. Haslett C, Worthen GS, Giclas PC, Morrison DC, Henson JE, Henson PM. The pulmonary vascular sequestration of neutrophils in endotoxemia is initiated by an effect of endotoxin on the neutrophil in the rabbit. Am Rev Respir Dis 1987; 136: 9–18. 16. Beutler B, Krochin N, Milsark IW, Leudke C, Cerami A. Control of cachetin (tumor necrosis factor) synthesis: mechanisms of endotoxin resistance. Science 1986; 232: 977–980. 17. Beutler B, Cerami A. Tumor necrosis factor-alpha. Annu Rev Immunol 1989; 7: 635–670. 18. Dinarello CA. Interleukin-1 and its biologically related cytokines. Adv Immunol 1989; 44:153–205. 19. Dinarello CA. Interleukin-1. Blood 1991; 77:1627–1678. 20. Morrison DC, Ryan JL. Endotoxin and disease mechanisms. Ann Rev Med 1987; 38:417–440. 21. Okusawa S, Gelfand JA, Ikejima T, Connolly RJ, Dinarello CA. Interleukin-1 induces a shock-like state in rabbits. J Clin Invest 1988; 81:1162–1169. 22. Tracy KJ, Beutler B, Lowry SF, Merryweather J, Wolpe S, Milsark IW, Hariri RJ, Fahey TJ, Zentella A, Albert JD, Shires GT, Cerami A. Shock and tissue injury induced by recombinant human cachectin. Science 1986; 234:470–472. 23. Beutler B, Milsark IW, Cerami A. Passive immunization against cachetin/tumor


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necrosis factor protects mice from lethal effect of endotoxin. Science 1985; 229: 869–871. Valcek VJ, Lee TH. Tumor necrosis factor-alpha. J Biol Chem 1991; 266:7313– 7335. Tracy KJ. TNF in the biology of septic shock syndrome. Circ Shock 1991; 35:123– 137. Cobb JP, Natanson C, Hoffmann WD, Lodato RD, Banks S. N-amino-L-arginine, an inhibitor of nitric oxide synthase, raises vascular resistance, but increases mortality rates in awake canines challenged with endotoxin. J Exp Med 1992; 176:1175– 1182. Kilbourn RG, Gross SS, Jubran A, Adams J, Griffith OW, Levi R, Lodato RF. Nmethyl-L-arginine inhibits tumor necrosis factor-induced hypotension: implications for the involvement of nitric oxide. Proc Natl Acad Sci USA 1990; 87:3629–3635. Thiemermann C, Vane J. Inhibition of nitric oxide synthesis reduces the hypotension induced by bacterial lipopolysaccharides in the rat in vivo. Eur J Pharmacol 1990; 18:259–265. Fleming I, Julou-Schaeffer GJ, Geay GA, Parrat JR, Stoclet J-C. Evidence that an l-arginine/nitric oxide dependent elevation of tissue cyclic GMP content is involved in depression of vascular reactivity by endotoxin. Br J Pharmacol 1991; 103:1047– 1052. Freund A, Atamian S, Holroyde J, Fischer JE. Plasma amino acids as predictors of the severity and outcome of sepsis. Ann Surg 1979; 190:571–580. Wagner DA, Young VR, Tannenbaum SR. Mammalian nitrate biosynthesis: incorporation of 15 NH 3 into nitrate is enhanced by endotoxin treatment. Proc Natl Acad Sci USA 1983; 80:4518–4527. Brigham KL, Meyrick B. Endotoxin and lung injury. Am Rev Respir Dis 1986; 133: 913–946. Pittet JF, Machersie RC, Martin TR, Matthay MA. Biological markers of acute lung injury: prognostic and pathogenetic significance. Am J Respir Crit Care Med 1997; 155:1187–1205. Demling RH. Cardiopulmonary dysfunction from sepsis: diagnosis and treatment. In Procter RA, ed. Handbook of Endotoxin, Vol. 4, Clinical Aspects of Endotoxin Shock. Amsterdam: Elsevier, 1986:185–237. Vito DR, Weisel R, Hechtman H. Sepsis presenting as acute respiratory insufficiency. Surg Gynecol Obstet 1974; 138:99–106. Ashbaugh DG, Bigelow DB, Petty TL, Levine BE. Acute respiratory distress in adults. Lancet 1967; ii:319–326. Bone RC. Let’s agree on terminology: definitions of sepsis. Crit Care Med 1991; 19:973–976. Bone RC. The pathogenesis of sepsis. Ann Intern Med 1991; 115:457–469. Simpson R, Hechtman HB. Pulmonary injury following sepsis. In Levin J, van Deventer SJH, van der Poll T, Sturk A, eds. Bacterial Endotoxins: Basic Science to Anti-sepsis Strategies. New York: Wiley-Liss, 1994:265–275. Worthen GS, Haslett C, Rees RS, Gumbay RS, Henson JE, Henson PM. Neutrophil mediated pulmonary vascular injury. Am Rev Respir Dis 1987; 136:19–28. Dantzker DR, Brook CJ, Dehart P, Lynch JP, Weg JG. Ventilation-perfusion distri-

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16 Exhaled Breath Markers in Acute Respiratory Distress Syndrome

JOCHEN KLAUS SCHUBERT and WOLFRAM MIEKISCH University of Rostock and University Hospital of Rostock Rostock, Germany

I.

KLAUS GEIGER University Hospital of Freiburg Freiburg, Germany

ARDS Overview

A. Definition According to the Consensus Conference on ARDS

First described by Ashbough et al. (1) in 1967, the acute respiratory distress syndrome (ARDS) represents a uniform response of the lung to direct or indirect injury. According to the American–European consensus conference it is characterized by an acute onset of respiratory failure, a PaO 2 /FiO 2 ⬍ 200 mmHg regardless of positive end expiratory pressure (PEEP), bilateral infiltrates on frontal chest radiograph, and pulmonary artery wedge pressure (PCWP) ⱕ 18 mmHg or lack of clinical evidence of left atrial hypertension (2). The incidence is about 75 cases/100,000 population, representing 150,000 cases annually in the United States (3). Various clinical conditions may precipitate the onset of ARDS. ARDS may arise from mechanical injury to lung parenchyma, from bacterial, viral, or mycotic pulmonary infection, or from inhalation of toxic gases such as nitrous oxide (NO 2 ), hydrochloric acid (HCl), or sulfuric oxide (SO 2 ). Indirect lung injury may be elicited by multiple trauma without lung injury, by sepsis, by ischemia-reperfusion, or by mass transfusion. 363

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Schubert et al. B. Underlying Diseases, Pathophysiology, and Pathology of ARDS

Many ARDS patients need mechanical ventilation to avoid hypoxemia or excessive hypercarbia. Mechanical ventilation aims at “buying” time for the resolution of the disease, but paradoxically, mechanical ventilation itself can provoke lung injury in different ways. High inspiratory oxygen concentrations may promote inflammatory reactions and fibrosis. Barotrauma (4) is produced by the high inspiratory pressures often necessary to assure sufficient ventilation. As a consequence, lung injury is worsened or pneumothorax may occur. Even at low inspiratory pressures the lungs may be subject to direct injury through shear forces caused by elevated tidal volumes (5). There is convincing experimental evidence (6–8) that ARDS can be induced by mechanical ventilation using large tidal volumes at low inspiratory pressures. What recently has been named “biotrauma” (9) is lung injury caused by mediators and reactive oxygen species liberated through the impact of mechanical ventilation. Thus, the pathophysiology of ARDS is closely related to inflammatory responses including cellular and humoral components. The early stage of ARDS is characterized by neutrophil sequestration into the lung. Activation of these neutrophils causes liberation of oxygen-derived free radicals and numerous mediators. Increased concentrations of superoxide, hydrogen peroxide, or hypochlorous acid (10) have been found in the blood of ARDS patients. Inflammatory mediators such as complement fragments, thromboxane, leukotrienes, proteases, cytokines, or platelet-activating factor (PAF) are variably present in both patients at risk for as well as those with ARDS. Studies of circulating inflammatory cells suggest that there are differences between major cell categories (i.e., lymphocytes and neutrophils) in response to systemic inflammation. In addition, subpopulations of these cells seem to have very different roles (11). Recent work has shown that at the same time as hydrogen peroxide, serum catalase, manganese-superoxide dismutase, and ceruloplasmine are increased, glutathione is decreased. Findings such as these suggest that the balance between oxidants and antioxidant capacity must be important (12). Reactive oxygen species and inflammatory mediators released from neutrophils have a direct effect on endothelial and alveolar cells and may simultaneously precipitate a general inflammatory response (SIRS ⫽ systemic inflammatory response syndrome) in the body. These remote effects contribute significantly to the mortality of ARDS. In fact, many patients die because of multiple organ failure (MOF) rather than of respiratory failure itself. Direct injury to endothelial and alveolar cells results in increased alveolar–capillary permeability, lung edema, pulmonary hypertension, and respiratory failure. Hypoxic pulmonary vasoconstriction is deranged and shunt fraction is consecutively elevated, contributing to refractory hypoxemia. During later stages of ARDS, fibrosis of the lung tissue determines the

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course and outcome of the disease. Despite new methods in treatment such as inhalation of NO, instillation of surfactant, ventilation in prone position, or extracorporeal membrane oxygenation (ECMO), mortality of ARDS remains high. Nevertheless, those patients who survive have the chance to recover totally, since changes in lung tissue occurring during ARDS are basically reversible. C. Important Clinical Aspects of ARDS

Many of the mechanisms in the pathogenesis of ARDS have not yet been elucidated. As a result, a specific therapy of ARDS is lacking. Actual therapy consists of controlling the underlying disease, avoiding hypoxemia by means of lungprotective ventilatory support, and supportive care. Diagnostic parameters indicating risk or onset of the disease and characterizing the course of ARDS are lacking. The association between lung injury and the systemic inflammatory response (SIRS) very often triggered by an ARDS is not completely understood. Specific prognostic criteria are still to be defined. In ARDS, inflammatory processes take place near or in the alveoli. Elevated concentrations of volatile substances generated through the effects of radicals or cytokines on cellular structures should, therefore, be found in the alveolar gas. If these substances can be identified and linked to a specific event in the course of ARDS, analysis of exhaled air will provide valuable information for diagnosis, treatment, control, and research in ARDS.

II. Special Problems of Gas Sampling in Mechanically Ventilated Patients A. Substance Origins: Inspiratory and Expiratory Substance Concentrations

Since most ARDS patients depend on ventilatory support, sampling of gas is muddled by the complexity of the gas delivery (gas supply, ventilator, humidifier, and tubing) and the patient’s respiratory system. Substances generated within the patient have to be separated from those arising from the gas delivery system. Since exhaled air consists of alveolar and dead-space gas and substance concentrations of interest fall in the range of 10⫺12 to 10⫺9 mol/L, approximation of alveolar concentrations from mixed-expired gas may be considerably influenced by errors due to dilution and contamination from dead-space gas. In mechanically ventilated patients dead space even comprises parts of the respiratory tubing, which may contain additional contaminants. This contamination is the reason that concentrations of some substances of clinical interest in dead-space gas are higher than in alveolar gas (13).

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Figure 1 Schematic drawing of the CO 2-controlled sampling device. 1 ⫽ patient. 2 ⫽ endotracheal tube. 3, 4 ⫽ respiratory tubing. 5 ⫽ ventilator. 6 ⫽ CO 2 mainstream sensor. 7 ⫽ stopcock. 8 ⫽ electrical two-way valve. 9 ⫽ CO 2 analyzer. 10 ⫽ electronic processing unit. 11 ⫽ trap. 12 ⫽ alveolar sampling (“expiratory”) position. 13 ⫽ mixed-inspiratory and dead-space sampling (“inspiratory”) position. 14 ⫽ roller pump. The sensor (cuvette) of a fast-responding infrared absorption mainstream CO 2 analyzer (930, Siemens-Elema, Solna, Sweden, 6) was inserted between the Y-piece of the respiratory circuit and the patient. A stopcock (7) was mounted through a bore into the cuvette to prevent leakage of respiratory gas when the sampling system was not connected. The stopcock joined the cuvette with the electrically operated two-way valve (LFYA, The LEE Company, Westbrook, CT) having an internal dead space of 88 µL (8). Adsorption traps (62) containing 80 mg of activated charcoal (Analyt, Mu¨llheim, Germany) were mounted onto the two outlets of the valve. By means of a Y-piece the traps were connected to a roller pump (model 7553-75, Cole-Parmer Instruments Co., Niles, IL, USA), working at a constant flow of 200 mL/min (14). Depending on the position of the valve, respiratory gas passed exclusively through one of the adsorption traps (11). The analog output of the CO 2 analyzer (9) was fed into the processing unit (10), which controlled the switching of the valve depending on the actual CO 2 concentration. All parts of the apparatus coming into contact with respiratory gas were made of chemically inert material such as polytetrafluoroethylene (PTFE).


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B. Alveolar Gas Sampling Versus Mixed Expiratory Sampling

In order to analyze the relationship between chemical composition of exhaled gas and patients’ clinical conditions, substance concentrations in alveolar gas have to be determined, because only alveolar concentrations reflect concentrations in blood. We therefore developed a sampling method using the expired CO 2 to separate dead-space gas from alveolar gas (Fig. 1) in mechanically ventilated patients (14). From the ratio of end-tidal/arterial CO 2 partial pressures, estimates of arterial tracer gas partial pressures can be derived. Arterial blood concentrations could then be derived from blood/gas partition coefficients or solubility: Pet tracer gas/Part tracer gas ⬇ Pet CO 2 /Part CO 2 ⇒ Part tracer gas ⬇ Pet tracer gas (Part CO 2 /Pet CO 2 ) C blood tracer gas ⬇ Pet tracer gas ∗ (Part CO 2 /Pet CO 2 ) ∗ k solubility (Henry’s law) or C blood tracer gas ⬇ C et tracer gas ∗ (Part CO 2 /Pet CO 2 ) ∗ k blood/gas where Part ⫽ arterial partial pressure, Pet ⫽ end-tidal partial pressure, k solubility ⫽ solubility coefficient, and k blood/gas ⫽ blood-gas partition coefficient. Since any substance detected in the gas sample may either come from the patient or from the delivery system, it is necessary to sample from two different sites of the respiratory circuit and to determine the inspiratory (C In ) and expiratory

Figure 2 Schematic drawing of the sampling devices mounted into the respiratory circuit showing T-pieces at the different sampling sites (1 and 2 in the inspiratory limb, 3 and 4 in the expiratory limb), the trap, the roller pump, and the volume-measuring device.

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Table 1 Ratio of Concentrations of Constituents of Exhaled Human Gas Substance Acetone 2,3-Dimethylbutane 2,4-Dimethylpentane n-Hexane Isoflurane Isoprene Methanol 2-Methylbutane n-Pentane

(C Ex ⫺ C In )/C Ex 0.90 0.50 0.47 ⫺6.2 0.96 0.90 0.16 ⫺0.88 0.54

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.15 0.06 0.18 5.0 0.15 0.03 0.07 0.56 0.08

n 45 32 23 41 31 39 49 35 40

Values are means ⫾ SE. C Ex ⫽ expiratory, C In ⫽ inspiratory concentration, n ⫽ number of measurements. A Q value between 0.9 and 1 represents a low inspiratory substance concentration (C Ex ⱖ 10 ∗ C In ) and is therefore indicative of a substance coming from within the patient. A Q value between 0.5 and 0.9 represents an expiratory substance concentration considerably higher than the inspiratory concentration (2 ∗ C In ⱕ C Ex ⬍ 10 ∗ C In ), which means that the substance emanates predominantly from the patient. A Q value ⬍ 0.5 represents expiratory substrate concentration comparable to (0 ⱕ Q ⬍ 0.5) or less than the inspiratory concentration (Q ⬍ 0), and is therefore indicative of a substance given off by the delivery system. Source: Ref. 13.

(C Ex ) substance concentrations (Figs. 1 and 2, Table 1). Pentane concentrations, for example, have to be corrected for background levels (C Ex ⫺ C In ). Because of low substance concentrations and an excess of water vapor, acetone, and halogenated compounds, analysis is difficult, and a standardized sampling method has yet to be established. Chemical analysis of exhaled gases has been performed in various ways (15–29). Most studies were done in spontaneously breathing patients or volunteers (15,16,18–20,22–29). There are only a few studies in mechanically ventilated patients (13,30). C. Analytical Procedures

Volatile substances were collected and concentrated by adsorption onto activated charcoal (31,32), organic polymers (Tenax), or inorganic fibers. The preconcentrating effect of the adsorption procedures is about 1500-fold. Precision and reproducibility of the preconcentration process have been evaluated in several stud-


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Figure 3 Autosampling device for thermodesorption of Tenax traps and subsequent gas chromatographic separation and mass spectrometric detection.

ies (30,31,33). Relative standard deviations were ⱕ10%; correlation coefficients for calibration curves were always found to be greater than 0.95. Substances were desorbed from the activated charcoal by means of microwave energy (31), and from Tenax and from inorganic fibers by means of thermodesorption (Fig. 3). After gas chromatographic separation, substances were detected by flame ionization (FID) and identified by mass spectrometry (Fig. 4). Substance concentrations were obtained from calibration curves. III. Clinical Aspects of Exhaled Volatile Substances A. Alkanes: Ethane, Pentane (Lipid Peroxidation)

More than 100 volatile substances in concentrations of 10⫺6 to 10⫺12 mol/L have been found in human exhaled air. Alkanes such as n-pentane and ethane are believed to be markers of lipid peroxidation (20–23) and have been demonstrated in a variety of pathological conditions. Elevated breath pentane concentrations have been found in myocardial infarction (25), in heart failure (24), and in patients or animals with inflammatory bowel disease (34,35). Breath pentane, apparently being linked to oxidative stress, also increased with age (18). Breath ethane was increased after ischemia/reperfusion (17), alcohol abuse (19), and in patients with chronic obstructive pulmonary disease (36). Pentane arises from peroxidation of ω-6 polyunsaturated fatty acids (37), ethane from peroxidation of ω-3 fatty acids (e.g., linolenic acid) (38).

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Figure 4 Chromatograms of inspired (1) and expired air (2) from a mechanically ventilated patient suffering from ARDS (microwave desorption, FID). IS ⫽ internal standard.

B. Alkenes: Isoprene (Cholesterol Metabolism)

Isoprene (2-methylbutadiene-1,3) is always present in human breath, and is thought to be formed along the mevalonic pathway of cholesterol synthesis (26). An important reaction in the cholesterol biosynthesis is the formation of mevalonate from acetic acid. This rate-limiting step of sterol synthesis is catalyzed by hydroxymethylglutaryl(HMG)-CoA. Mevalonate is then converted in the cytosol to isopentenyl pyrophospate, which undergoes isomerization to dimethylally pyrophosphate (DMPP) (39). Proceeding through a carbonium-ion intermediate, DMPP is rapidly converted to isoprene via an acid-catalyzed elimination reaction in rat liver cytosole (40). It is not known, however, whether this nonenzymatic reaction accounts for isoprene formation under physiological conditions. Certain plants also produce isoprene from DMPP. In these plants, the reaction is catalyzed by a Mg 2⫹-containing enzyme (41). A similar, still unknown, enzyme may catalyze the conversion of DMPP to isoprene in mammalian tissue. This enzyme may be the Mg 2⫹-dependent isopentenyl pyrophosphate isomerase, which catalyzes


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the interconversion of isopentenyl pyrophosphate and DMPP. This isomerization reaction proceeds through the same carbonium-ion intermediate (42) as occurs in the acid-catalyzed nonenzymatic conversion of DMPP to isoprene (43). Isoprene is formed by loss of a single proton from this carbonium ion. The parallel decrease in isoprene secretion and sterol synthesis caused by acute or chronic lovastatin administration suggests that breath isoprene is derived from the cholesterol synthesis pathway in humans in vivo (26). There is experimental evidence that isoprene exhalation may be related to oxidative damage to fluid lining of the lung (27) and the body (28). Surprisingly, isoprene concentrations in the breath of laboratory animals are considerably lower than in human breath (44). C. Aldehydes: Hexanal, Nonenal (Peroxidation)

Direct assessment of oxidative stress to the lung is an important aspect of ARDS research. Hydrogen peroxide (H 2 O 2 ) represents a classical marker of oxidant stress, which can be collected and determined in breath condensate (61). Aldehydes such as 4-hydroxy-2-nonenal (46) or malonedialdehyde (47) have been associated with lipid peroxidation. Malondialdehyde (MDA) and thiobarbituric acid-reactive substances (TBARS), the sum of all substances reacting with thiobarbituric acid, are nonvolatile lipid peroxidation products that have to be determined in plasma. Increased levels of MDA have previously been demonstrated in several pathological conditions such as diabetes (48), ischemic heart disease (49), or in patients under hemodialysis (50), and have been interpreted as a sign of increased lipid peroxidation. D. Ketones: Acetone (Dextrose Metabolism)

Acetone (propylketone) is formed by decarboxylation of acetoacetate which derives from lipolysis or lipid peroxidation. As acetone is produced by spontaneous decarboxylation of acetoacetate, it is impossible to quantitate the fraction that arises from lipid peroxidation. High concentrations of acetone are found in uncontrolled diabetes mellitus (45). E. Sulfur-Containing Compounds: Dimethylsulfide (Bacteria), CS 2 (Liver)

Sulfur-containing compounds such as dimethylsulfide may serve as markers of impaired liver function in humans (51,52). The role of CS 2 that has been found in humans as well as in experimental animals has yet to be determined (53). Bacteria or fungi are capable of generating some of the volatile substances found in breath (54–56).

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Schubert et al. F. Isoprostanes (Inflammation)

Recently, a series of structurally unique prostaglandin F2-like compounds (F2isoprostanes) have been discovered. They are produced in vivo in humans from arachidonic acid primarily, if not exclusively, through a nonenzymatic process of lipid peroxidation catalyzed by the action of oxygen free radicals on cell membranes. Quantification of F2-isoprostanes has been suggested to represent an important advance in our ability to assess oxidant status in vivo in humans, and they have been found to be more specific and precise markers of oxidant stress in several conditions such as cigarette smoking and hypercholesterolemia. Human pulmonary tissue has been shown to produce isoprostanes in response to inflammatory challenge, and plasma levels of 8-isoprostane have been reported to correlate with outcome in patients with ARDS (57). G. Nitric Oxide

Increased NO exhalation has been found in inflammatory processes of the airways (58). Exhaled NO concentrations have to be determined by chemiluminescence. NO and superoxide can combine to form peroxynitrite (59,60) a potent oxidant capable of damaging cell structures by peroxidation. Peroxynitrite readily nitrates phenolic rings, including tyrosine. In addition to the above markers, additional compounds have received attention in the exhaled breath field. Volatile anesthetics such as isoflurane are exhaled for a long period after exposure during anaesthesia (62). Benzene is not related to metabolism but represents a widespread environmental pollutant (29). IV. Specific Patterns of Exhaled Gases in ARDS Mechanically ventilated patients with ARDS were compared to patients without ARDS (13,30). Due to the admission policy of the ICU, analysis of the exhaled air did not always coincide with the very beginning of ARDS. A. Metabolic Markers (Acetone)

Acetone production rates tended to be higher in ARDS patients. The finding might be due to an increased metabolism (15) caused by a stress response to mechanical ventilation, but high variations of the measured values make clinical interpretation difficult. B. Markers of Cholesterol Metabolism (Isoprene)

In our studies, ARDS patients produced over 50% less isoprene than those without ARDS (Tables 2–4). Decreasing isoprene concentrations indicate a reduction

Exhaled Breath Markers in ARDS Table 2 ARDS

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Substance Elimination (nmol/m 2 /min) in the Presence and Absence of non-ARDS

ARDS

Variable

Median (95% CI)

N

Median (95% CI)

n

p

Acetone Pentane Isoprene

119 (52–270) 5.1 (1.4–18.6) 21.8 (13.9–41.4)

14 18 18

149 (113–485) 4.15 (3.7–9.3) 9.8 (8.2–21.6)

18 19 19

0.25 0.37 0.04

Values are medians and 95% confidence intervals (95% CI). n ⫽ number of patients. Source: Ref. 13.

of cholesterol synthesis, possibly resulting in an impairment of membrane repair capacities. Foster (27) observed an increase in isoprene production 19 hr after exposure to ozone. An activation of cholesterol synthesis at the onset of repair processes following oxidative damage to fluid linings in the lung is assumed to be the underlying mechanism. Mendis (28) reported an increase in isoprene production in patients with an acute myocardial infarction. They considered an interrelationship between isoprene production and activation of neutrophils. At first glance, these results seem to be contradictory, but since there was a similar reduction in isoprene production in patients who developed pneumonia (13), decreasing isoprene concentrations in exhaled air may specifically signal an exhaustion of membrane repair capacities during inflammatory processes in the lung. This hypothesis does not preclude that exhaled isoprene concentrations may be elevated in the very beginning of acute lung injury (ALI) as a result of neutrophil activation and membrane repair processes. Later, isoprene concentrations may fall because of reduction in neutrophil activation and some impairment of cholesterol metabolism.

Table 3 Concentrations of Acetone, Pentane, and Isoprene in the Exhaled Gas of Mechanically Ventilated Patients

Acetone (nmol/L) Pentane (nmol/L) Isoprene (nmol/L)

ARDS (n ⫽ 22) Median (95% CI)

Head injury (n ⫽ 20) Median (95% CI)

34 (25–122) 0.75 (0.03–12) 1.3 (1.1–3.4)

35 (3.5–181) 0.42 (0.30–0.86) 5.6 (4.1–6.4)

ARDS ⫽ acute respiratory distress syndrome, 95% CI ⫽ 95% confidence interval. Source: Ref. 30.

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Table 4 Concentrations of Nonvolatile Peroxidation Markers and Exhaled Breath Markers in Healthy Volunteers, in Patients with Head Injury, and in Patients with and at Risk for ARDS

Substance

Volunteers median 95% CI/n

Head injury median 95% CI/n

ARDS median 95% CI/n

At risk median 95% CI/n

MDA (ng/mL)

0.35 0.18–0.57/9

0.27 0.22–0.42/16

0.52 0.34–0.76/12

0.55 0.44–0.78/21

TBARS (ng/mL)

0.55 0.47–1.09/9

0.72 0.64–0.95/16

1.32 0.73–1.82/12

1.52 1.13–2.21/21

Acetone (nmol/L)

33.2 7.9–51.5/4

45.0 18.4–82/6/10

57.3 0.0–283/8

24.1 21.2–85.7/22

Isoprene (nmol/L)

5.78 3.52–8.45/10

6.02 4.45–9.87/12

2.34 0.71–4.82/7

6.25 5.8–7.9/25

n-Pentane (nmol/L)

0.12 0.10–0.16/10

0.18 0.00–1.07/11

0.51 0.13–1.52/8

0.32 0.24–0.59/9

Values are medians and 95% confidence intervals (95% CI). n ⫽ number of patients.

C. Markers of Lipid Peroxidation (Ethane and Pentane)

Since pentane is believed to be a marker of lipid peroxidation, elevated exhaled pentane concentrations are expected in ARDS. However, no difference in pentane production could be observed between ARDS and non-ARDS patients in our first studies (13,30). This finding may be due to the fact that the analysis of air was not performed at the very beginning of ALI/ARDS, when inflammatory activity is highest. Furthermore, patients in the non-ARDS group, with a wide range of diagnoses, were not completely free of inflammation. In a group of patients who developed pneumonia during their ICU stay, it could be shown that pentane concentrations did increase at the very beginning of an inflammatory process (13). In order to get a better understanding of the different lipid peroxidation markers, exhaled breath markers and nonvolatile peroxidation markers were determined in healthy volunteers, in patients with and at risk for ARDS, and in patients with head injury (Table 4). Malondialdehyde (MDA) and thiobarbituric acid-reactive substances (TBARS) were chosen as nonvolatile serum markers. MDA and TBARS concentrations were highest in the ARDS group and in patients at risk to develop ARDS, i.e., in patients with sepsis, multiple trauma, or following major abdominal or cardiovascular surgery. Increased MDA and TBARS concentrations in these patients reflect increased lipid peroxidation through reactive oxygen species liberated, for instance, by activated nucleophiles (66,67).


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MDA concentrations in the ARDS group were slightly lower than in the at-risk group and showed wide variation. This is once more due to the fact that the degree of inflammation in ARDS depends on the stage of the disease (68) and that patients were not examined at the very beginning of ARDS, when inflammatory activity is highest. Nevertheless, lipid peroxidation was significantly increased in these patients when compared with healthy volunteers or with patients with cranio-facial trauma without lung injury. Pentane concentrations were also lowest in the healthy volunteer and the head-injury groups and highest in the ARDS group. For the same reasons as for MDA, wide variation of concentrations occurred in ARDS patients. The number of patients was too small to decide whether the differences between pentane concentrations in the ARDS and the atrisk group was due to principal differences in lipid peroxidation or to statistical variation. Since the distribution of exhaled pentane concentrations was very similar to that of MDA and TBARS, one may conclude that increased pentane concentrations in patients with and at risk for ARDS are in fact due to increased lipid peroxidation in these patients. The different behavior of MDA/TBARS and npentane in ARDS patients with and those at risk for ARDS (Table 4) may indicate that MDA/TBARS and n-pentane reflect different aspects of lipid peroxidation. D. Markers of Inflammation (Isoprostanes)

Hydrogen peroxide levels in expired-air condensate were increased in patients with respiratory failure and were highest in ARDS patients (61). In a recent study, expired isoprostane levels were quantified in breath condensate of patients with or at risk for the development of ARDS (57). This compound was detected in the condensate of exhaled breath of these patients at greater than two standard deviations above the mean of the normal group. The results suggest that exhaled isoprostanes might provide a novel, noninvasive method to potentially quantify lung specific oxidant stress and evidence that lipid peroxidation does occur in patients with ARDS. E. Exhaled NO

Exhaled NO concentrations are elevated in patients with inflammation of the airways (58,63). Inflammatory insults induce an increase in the expression of the inducible isoform of nitric oxide synthase (iNOS) in pulmonary tissue (64). Since inflammatory processes play an important role in ARDS, elevated exhaled NO concentrations are expected in those patients. Surprisingly, Brett et al. (65) found lower exhaled NO concentrations in ARDS patients when compared to mechanically ventilated patients without lung injury. Changes in diffusion capacity of NO or formation of peroxynitrite from NO and superoxide are considered the cause of this finding. The question arises, however, why does not peroxynitrite formation take place in airway inflammation as occurs in asthma or bronchitis,

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in which elevated exhaled NO concentrations are found? It is more likely that, just as in the case of isoprene, the lower NO concentrations in ARDS are caused by decreased NO production during later stages of the disease. Brett et al. did not address this issue, as they focused on fully developed ARDS. There is limited evidence that there might be a specific pattern of exhaled volatile substances in ARDS, and that the profile of these substances may change during the course of the disease. The most marked change is likely to occur when acute inflammation progresses to fibrosis. Changes in isoprene concentrations probably reflect alteration in cholesterol metabolism during membrane repair or neutrophil activation, elevated NO concentrations are the result of increased iNOS expression, and changes in pentane concentrations mirror lipid peroxidation through oxygen derived radicals. Analysis of these substances, therefore, provides useful information on very different aspects of inflammatory activities during ARDS. In addition, exhaled isoprene seems to be a specific marker of impaired cholesterol metabolism during inflammatory processes in the lung. Further insight into the complex pathophysiology of ARDS can be gained through analysis of substances such as isoprostanes and hydrogen peroxide, which seem less dependent on the stage of the disease. V.

Conclusions

Exhaled volatile substances in ARDS can be linked to different pathways of inflammation and to membrane metabolism. As concentrations of such markers show a specific pattern along the course of ARDS, diagnosis of different stages of the disease should be possible by means of breath analysis. Future progress of analytical techniques and a better understanding of the relationship between expired substance concentrations and clinical status will certainly help to improve diagnosis, to stimulate basic research, and to guide and evaluate therapy for ARDS. References 1. Ashbaugh DG, Biglow DB, Petty TL, et al. Acute respiratory distress syndrome in adults. Lancet 1967; ii:319–323. 2. Bernard GR, Artigas A, Brigham KL, Carlet J, Falke K, Hudson L, et al. The American-European Consensus Conference on ARDS. Definitions, mechanisms, relevant outcomes, and clinical trial coordination. Am J Respir Crit Care Med 1994; 149:818–824. 3. American Lung Program. Respiratory diseases. Task force report on problems, research approaches, needs. The Lung Program. National Heart and Lung Institute. Washington, DC: U.S. Government Printing Office, 1972, DHEW Publication No. (NIH) 73-432, pp. 165 -180.


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4. Kolobow T, Moretti MP, Fumagalli R, Mascheroni D, Prato P, Chen V, Joris M. Severe impairment in lung function induced by high peak airway pressure during mechanical ventilation: an experimental study. Am Rev Respir Dis 1987; 135:312– 315. 5. Dreyfuss D, Soler P, Basset G, Saumon G. High inflation pressure pulmonary edema. Respective effects of high airway pressure, high tidal volume and positive endexpiratory pressure. Am Rev Respir Dis 1988; 137:1159–1164. 6. Chess PR, Toia L, Finkelstein JN. Mechanical strain-induced proliferation and signaling in pulmonary epithelial H441 cells. Am J Physiol Lung Cell Mol Physiol 2000; 279(1):L43–L51. 7. Gronski T Jr, Lum E, Campbell J, Shapiro SD. A murine model of volutrauma: potential contribution of inflammatory cell proteases to lung injury. Chest 1999; 116(1 suppl):28S. 8. Finfer S, Rocker G. Alveolar overdistension is an important mechanism of persistent lung damage following severe protracted ARDS. Anaesth Intensive Care 1996; 24(5):569–573. 9. Slutsky AS, Tremblay LN. Multiple system organ failure: is mechanical ventilation a contributing factor? Am J Respir Crit Care Med 1998; 157:1721–1725. 10. Tate RM, Regine JE. Neutrophils and the adult respiratory distress syndrome. Am Rev Respir Dis 1983; 125:552–559. 11. Weiss SJ. Tissue destruction by neutrophils. N Engl J Med 1989; 320:365–376. 12. Brigham KL. Role of free radicals in lung injury. Chest 1986; 89:859–863. 13. Schubert JK, Mu¨ller WPE, Benzing A, Geiger K. Gas chromatographic analysis of expired air in mechanically ventilated patients. Intensive Care Med 1998; 24:415– 421. 14. Schubert JK, Spittler K-H, Braun G, Geiger K, Guttmann J. CO 2-controlled sampling of alveolar gas in mechanically ventilated patients. J Appl Physiol 2001; 90:486– 492. 15. Kneepkens CMF, Ferreira C, Lepage C, Roy CC. The hydrocarbon breath test in the study of lipid peroxidation; principles and practice. Clin Invest Med 1992; 15: 163–186. 16. Phillips M, Greenberg J. Method for the collection and analysis of volatile compounds in the breath. J Chromatogr 1991; 564:242–249. 17. Kazui M, Andreoni KA, Norris EJ, Klein AS, Burdick JF, Beattie C, et al. Breath ethane: a specific indicator of free-radical-mediated lipid peroxidation following reperfusion of the ischemic liver. Free Radic Biol Med 1992; 13:509–515. 18. Mendis S, Sobotka PA, Euler DE. Pentane and isoprene in expired air from humans: gas chromatographic analysis of single breath. Clin Chem 1994; 40:1485–1488. 19. Letteron P, Duchatelle V, Berson A, Fromenty B, Fisch C, Degott C. Increased ethane exhalation, an in vivo index of lipid peroxidation, in alcohol-abusers. Gut 1993; 34:409–414. 20. Phillips M, Greenberg J. Ion-trap detection of volatile organic compounds in alveolar breath. Clin Chem 1992; 38:60–65. 21. Van Gossum A, Decuyper J. Breath alkanes as an index of lipid peroxidation. Eur Respir J 1989; 2:787–791. 22. Van-Rij AM, Wade CR. In vivo lipid peroxidation in man as measured by the respi-

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17 Exhaled Nitric Oxide in Human Lung Transplant Recipients

ANDREW J. FISHER, ANTHONY DE SOYZA, and PAUL A. CORRIS Freeman Hospital and University of Newcastle upon Tyne Newcastle upon Tyne, England

I.

Introduction

A. Background to Transplantation

Advances in medical care of patients with respiratory disease have expanded to include pulmonary transplantation for patients with end-stage lung disease. Clinical lung transplantation has evolved over 20 years, becoming a reality after the widespread introduction of Cyclosporin A as the principal immunosuppressant. The International Society for Heart and Lung Transplantation (ISHLT) registry now records over 8000 pulmonary transplant operations performed worldwide (1). Four distinct operations are performed by pulmonary transplant surgeons, comprising single lung transplantation, sequential single lung transplantation, heart-lung transplantation, and more recently, lobar donation either from cadaveric or living donors. It is of note that single lung transplants are performed on patients with nonseptic pulmonary conditions in which remaining contralateral native lung is left in situ. The other operative types are indicated for patients with bronchiectasis (including cystic fibrosis), pulmonary hypertension, and congenital heart disease. 381

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Postimplantation graft dysfunction can occur due to widely differing etiologies, including acute and chronic vascular rejection, acute and chronic airway rejection, as well as infections of viral, fungal, and bacterial origin. These processes can be asymptomatic until advanced, and as a result many transplant centers have established a surveillance program of bronchoscopy and biopsy at regular intervals (2). In addition, many centers advocate daily measurement of home spirometry by recipients, with patients advised to report to the center if there is a sustained fall in FEV1 of 20% or greater. Acute graft dysfunction can occur very early post-transplant and can be due to many processes including lung vascular injury, acute rejection, or infection. These may be recognized by arterial oxygen desaturation, pulmonary infiltrates on radiographic examination, declining pulmonary function, and pyrexia. These single clinical measurements, however, do not distinguish between rejection and infection. Allograft infections may occur either as a result of donortransmitted infection or secondary to prolonged intubation, and are common. Acute vascular rejection affects up to 40% of recipients in the first year (3), and though rarely fatal, it is associated with morbidity in the immediate phase and contributes to long-term morbidity by increasing the risk of the development of chronic rejection. An airway-centered rejection recognized histologically as lymphocytic bronchitis (LB) presents more insidiously with wheeze, and commonly is associated with a less marked deterioration in the FEV1 as compared to acute vascular rejection. Up to 30% of patients following single lung transplantation have been reported as developing LB (3). Airway biopsies are characterized by a small airways inflammation with a predominant (CD-8⫹ve) T-cell inflammatory infiltrate of airway epithelium and submucosa (4,5). Infections are common in this group of patients and can be of bacterial, viral, or fungal origins. Common bacterial infections include gram-negative organisms such as Pseudomonas aeruginosa, especially in cystic fibrosis patients whose upper airway remains colonized. Candida is commonly isolated in early post-transplant bronchoalveolar lavage; however, prompt treatment with the antifungal fluconazole leads to this organism being a rare cause of disease. Aspergillus species, however, are a common cause of post-transplant fungal infections, although a wide difference in incidence is noted relating to the local environment. Centers with high rates of Aspergillus infections are prophylactic itraconazole or nebulized amphotericin. Cytomegalovirus (CMV) pneumonitis was a common and potentially fatal illness in sero-negative recipients exposed to a sero-positive donor organ. Many units now offer oral prophylaxis to such patients in the form of the antiretroviral drug ganciclovir, which delays the presentation of CMV disease to a time when the recipient is less immunosuppressed.

Exhaled NO in Human Lung Transplant Recipients

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Acute infection, lymphocytic bronchitis, and acute vascular rejection may coexist in an affected recipient. Cryptogenic organizing pneumonia is a common and ill-understood phenomenon affecting the allograft (6). It can occur in isolation or in conjunction with other causes of graft dysfunction as discussed above. It tends to respond to oral corticosteroids and has been linked to an increased risk of chronic rejection (7). Chronic allograft rejection remains the most important long-term complication of lung transplantation, affecting up to 50% of 5-year survivors (8). The disease process is associated with the development of airflow obstruction leading to a fall in FEV1 and low-volume expiratory flow (FEF25-75). It is defined histologically by the presence of obliterative bronchiolitis (OB) or functionally by progressive irreversible airflow obstruction, when it is called bronchiolitis obliterans syndrome (BOS) (9). BOS is classified by the International Society of Heart Lung Transplantation guidelines into four grades based on a comparison between the FEV1 at least 3 months after transplantation and the mean of the recipient’s best two posttransplant FEV1 taken 1 month apart (Table 1). The progressive and irreversible nature of OB is associated with central airway bronchiectasis, recurrent infections, and ultimately death (9). Autopsy specimens of affected grafts show a proliferation of myofibroblasts obliterating the small airways with mononuclear cell infiltrates (10,11). There are few good animal models of chronic pulmonary rejection, and most risk factors have been identified by retrospective analysis on human recipients. Risk factors include infection, organizing pneumonia (7), and acute allograft rejection, including both airway-centered and vascular rejection (12). Airway-centered rejection has also been identified as a risk factor for chronic rejection in a rat tracheal model (13). Current strategies aimed at controlling OB have been disappointing, with few patients regaining their previous level of function and most demonstrating a reduction in functional decline (10). Early identification and prompt treatment of

Table 1 Bronchiolitis Obliterans Syndrome; ISHLT Working Party Gradings BOS grade

Percent of best post-transplant FEV1

0 1 2 3 Source: Adapted from Ref. 4.

80%⫹ 66–79% 51–65% ⬍50%

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any precursor lesion of OB may therefore prove a more worthwhile target for treatment. A noninvasive surveillance program that could detect and identify the cause of early graft dysfunction when the recipient is asymptomatic could allow prompt treatment without the need for hazardous procedures such as transbronchial biopsy. Such a system would need to be reproducible, reliable, financially acceptable, and well tolerated. C. Background to NO in Allograft Dysfunction

Inhaled nitric oxide has been used in human pulmonary transplantation as a bridge to transplantation (14) and as a therapeutic agent for patients suffering from early graft dysfunction (15). This is, however, outside the scope of this chapter and will not be discussed further. Readers are directed to two recent reviews covering these topics (16,17). Interest in NO and pulmonary allograft dysfunction has been provoked by the findings in other human solid organ transplants such as liver (18) and cardiac transplantation (19), where elevated products of NO metabolism have been associated with graft dysfunction, particularly rejection. The majority of this work has been based on serum NO breakdown products or inducible NO synthase (iNOS) mRNA expression in tissue specimens. In human cardiac transplantation the rejection grade based on endomyocardial biopsy correlated with serum nitrite concentrations, leading the authors to conclude that such assays may have a diagnostic role in the future (19). Dysfunction of the lung allograft, however, has the unique advantage that analysis of exhaled breath may reflect underlying tissue pathology. Studies supporting the role of endothelial NO (eNO) in asthma have prompted the transplant community to assess the role of eNO in assessing lung allograft dysfunction. It is conceivable that NO production in pulmonary transplantation may be bicompartmental. Airway inflammation may be associated with raised eNO and serum nitrites associated with vascular or parenchymal rejection as in other solid organs. Much of the work regarding nitric oxide and pulmonary transplant rejection has used rat (20–22) and canine models (23,24), where there are obvious advantages of controlled experiments as compared to human transplantation. Investigators have studied allograft dysfunction, serum markers of NO metabolism, and the effects of iNOS inhibition in experimental lung transplantation with the rat lung model. The selective inhibitor of iNOS, aminoguanidine, has been shown in rat models to reduce both functional and histological changes associated with experimental rejection (22,25). Caution, however, must be raised in the extrapolation of animal data to humans until the comparative biology of NO and the source of exhaled NO is


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established. Other aspects of pulmonary transplantation do exhibit an interspecies difference; for example, groups investigating canine (26) and rat (27) lung transplant models have noted interspecies differences in bronchoalveolar cell profiles. As leukocytes are a source of nitric oxide in humans (28), this variation among species may prevent the extrapolation of results derived from animal models directly to the field of human transplantation. A second note of caution must be raised if nitric oxide metabolism does have a bi-compartmental distribution between vascular endothelium and airway epithelium.

II. Validation of NO Measurement in Lung Transplant Recipients If exhaled NO levels are to provide a potentially useful tool in the diagnosis of graft dysfunction in lung transplant recipients, it is essential that the method of NO measurement used is valid within this population. The method must be repeatable, reproducible, and reflect NO production from the lower airways. Furthermore, the measurement technique must be well tolerated and valid in recipients with varying degrees of lung function. A. The Use of eNO in Human Lung Transplantation

We will review published data regarding eNO in the field of human pulmonary transplantation critically and highlight relevant findings in other transplantation fields including both human and animal data as necessary. The biology of eNO has been discussed elsewhere in this volume. Recent data suggest that in stable lung transplant recipients there appears to be little cNOS expression in airway epithelium, and iNOS expression in airway epithelium correlates closely with levels of exhaled NO (29). The source of eNO in allograft pathology remains to be identified, but two potential sources include allograft epithelial cells and infiltrating host leukocytes, both of which are capable of iNOS upregulation in humans (30) and rats (31,32). The single-breath method of eNO measurement has been used in many asthma studies and forms the basis of the European Respiratory Society guidelines (33). In our center, we have evaluated this method in lung transplant recipients using end-exhaled NO plateau at the point where exhaled CO2 reaches its plateau and the mouth pressure remains above 5 cm H2O. We found that the method provides a repeatable measurement of eNO and correlates highly with NO measured directly in the graft bronchus at bronchoscopy over a range of lung function (Fig. 1) (34). Furthermore, the technique is reproducible from day to day in stable recipients (Fig. 2) (34). These data suggest that eNO is potentially

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Figure 1 Correlation of eNO (ppb) with NO sampled from graft bronchus at bronchoscopy. (From Ref. 34.)

a useful surrogate marker of graft lower airway NO production in pulmonary transplant recipients. When NO is measured by this technique in recipients with stable lung function and without any clinical symptoms of graft dysfunction, eNO concentrations remain stable, even over a 1-month period, with a coefficient of reproducibility of ⬍3 ppb NO (34). Measurements of exhaled nitric oxide (eNO) in recipients is well tolerated and can be performed within the first post-transplant week. Patients with advanced OB with poor lung function, even when the FEV1 approaches 1 L, can perform eNO measurements. In accordance with current guidelines, we perform eNO measurements in a seated recipient using nose clips. The mean of the two closest eNO concentrations (out of three measurements that are within 10% variance) is used in accordance with guidelines. Further work confirming the validity of eNO as a marker of lower airway NO metabolism has come from Melbourne, Australia. Work assessing eNO and bronchoalveolar lavage nitrite levels in 20 lung transplant recipients has shown a high correlation between the two measurements ( p ⫽ 0.001, r ⫽ 0.74) (35). This group has subsequently shown that in transplant recipients eNO levels reflect bronchial epithelial iNOS, the inducible isoform of nitric oxide synthase, rather than submucosal iNOS. Expression of the constitutive isoform cNOS appears to


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Figure 2 Bland-Altman plot of the repeatability of eNO concentrations in stable recipients. Visits 1 and 2 separated by a median of 42 days. (From Ref. 34.)

be downregulated with increasing iNOS expression. This suggests that epithelial iNOS is the major source of eNO in stable lung transplant recipients (29). Thus eNO measurements in lung transplant recipients fulfill the initial criteria for a screening tool of acceptability and reproducibility. These measurements reflect lower airway (graft) NO production. To be of any potential clinical use a screening tool may also have discriminatory capabilities, allowing the clinician to diagnose causes of graft dysfunction. Strategies to test such a discriminatory role for eNO in pulmonary transplantation can be cross-sectional or longitudinal and are discussed below.

III. Effect of Graft Pathology on Exhaled NO in Lung Transplant A. Cross-Sectional Measurements

In order to determine the usefulness of exhaled NO measurement in the identification of graft pathology in the lung transplant recipient population, it is important to identify any confounding recipient factors relating to the underlying disease process leading to the need for lung transplantation. It has been previously

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reported that exhaled NO levels vary in different lung diseases and therefore it is essential that the underlying pulmonary pathology does not influence the NO level after transplantation. Furthermore, the level of immunosuppression could potentially affect the exhaled NO level recorded in recipients. Finally, the type of lung transplant performed, e.g., single or bilateral, may be expected to contribute to the size of the NO signal, as any graft pathology would be limited to the allograft in a single lung transplant recipient. Between June 1987 and May 1997, the Freeman Hospital Cardiopulmonary Transplant Unit had performed 216 lung transplants in 212 recipients. In a cross-sectional study of 104 recipients (45 male, 59 female) eNO concentrations were recorded. Measurements of eNO were made immediately prior to each surveillance biopsy and at routine clinic review. This allowed accurate correlation of biopsy data and lavage cultures to eNO measurements. The recipients had undergone different lung transplant operations: 42 had undergone single lung transplantation, 38 bilateral sequential lung transplantation, and 24 heart-lung transplantation. Transplant recipients received standard immunosuppression comprising a combination of oral prednisolone, azathioprin, and cyclosporin. The indications for transplantation fell into four categories: 22 patients had obstructive lung disease, 22 had interstitial lung disease, 38 had suppurative lung disease, and 22 had pulmonary vascular disease. We found no evidence that the pretransplantation indication or the transplant operation type affected the post-transplant exhaled NO concentration. Those with single or bilateral or heart-lung transplants had comparable levels. The dose of oral corticosteroid and the serum cyclosporin level did not correlate with the exhaled eNO concentration, and these agents do not appear to affect levels of exhaled NO. The results of this cross-sectional analysis of 104 transplant recipients provided useful insight into the potential use of exhaled NO measurements as a marker of graft dysfunction in this group of patients (Fig. 3) (36). NO concentrations were elevated in recipients with lymphocytic bronchiolitis as compared to those who were clinically well, in accordance with the results obtained in other conditions characterized by airway inflammation such as asthma (37,38). The increased NO production was associated with an inflammatory upregulation of the enzyme inducible nitric oxide synthase (iNOS) in epithelial cells. Immunolocalization studies have been carried out to confirm this (Fig. 4). There were no significant increases in eNO concentrations recorded from recipients with acute vascular rejection without evidence of lymphocytic bronchiolitis noted, and this corroborates the findings of a smaller study from Toronto (39). This suggests that perivascular inflammation with an associated upregulation of iNOS in the endothelium is not sufficient to cause an elevation in exhaled NO if the airway epithelium is not involved. In recipients with isolated acute vascular rejection, the exhaled NO level was determined before any augmentation


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(a)

(b) Figure 3 Cross-sectional measurements of eNO in 104 lung transplant recipients by diagnostic category (a) excluding obliterative bronchiolitis (b) by bronchiolitis obliterans grading. (From Ref. 36.)

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Figure 4 Airway biopsy of a lung transplant recipient with lymphocytic bronchitis (LB). Immuno-histochemistry for iNOS (inducible nitric oxide synthase) was performed using standard techniques.

to their immunosuppression was commenced. The lack of a rise in eNO concentration associated with the acute vascular rejection was therefore not due to the effect of increased immunosuppressant treatment. Several studies using animal models have shown increased levels of circulating nitrates in association with acute rejection of pulmonary and other allografts (20,23,40). These findings suggest that the effect of upregulated iNOS activity in the endothelium is found mainly in the vascular compartment. Of particular interest is the finding of raised eNO concentrations found in patients with obliterative bronchiolitis who have early disease on functional criteria, i.e., BOS grade 1. This elevation is lost in the more advanced stages of the disease, BOS grades 2 and 3. From histopathology studies of obliterative bronchiolitis it is recognized that the disease has an inflammatory phase which is followed by a fibrotic scarring stage. In early disease it is possible that the inflammatory activity in the airways leads to increased iNOS expression and a subsequent elevation in exhaled NO concentrations, which is reduced when the inflammatory phase is replaced by fibrosis due to myofibroblast proliferation. There is some evidence that iNOS is expressed in the epithelium of patients with early obliterative bronchiolitis (28,41), but this requires further evaluation using iNOS immunolocalization techniques in patients with different BOS grades.


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Untreated lymphocytic bronchiolitis is believed to predispose the development of obliterative bronchiolitis (12,42). The prompt detection of lymphocytic bronchiolitis and its effective treatment may therefore prevent the progression to irreversible airway damage recognized as obliterative bronchiolitis. The results of this study suggest that exhaled NO measurements may have a role in the identification of airway inflammation causing graft dysfunction while it is still reversible. B. Longitudinal Measurements

As all studies previously have been cross-sectional, we aimed to assess the value of serial exhaled NO measurements in an individual recipient by performing a prospective longitudinal study. Eighteen lung transplant recipients were followed from the time of transplantation for 6 months. Exhaled NO concentrations were measured at routine hospital visits and at each presentation with an acute change in symptoms or lung function. Graft biopsies and lavage were performed routinely at 1, 3, and 6 months, and whenever recipients presented acutely. A baseline NO was determined for each patient (mean of three consecutive measurements performed while clinically stable) deviations of ⬎3 ppb NO from baseline were then identified. The median (range) number of NO measurements performed for each recipient was 7 (3–12), baseline NO was mean (range) 5.4 (2.1–18.9) ppb. The sensitivity of the test (1-False negative rate) was 63%, and the specificity (1-False positive rate) was 93% for detecting inflammatory airway pathology. These findings suggest that an increase in exhaled NO concentration of 3 ppb or greater from an individual’s baseline provides a rapid, noninvasive screening test for inflammatory graft dysfunction due to allogenic or infective bronchitis. We suggest that any rise in eNO greater than 3 ppb should be considered carefully for investigation with bronchoalveolar lavage and transbronchial biopsy, provided there is not an obvious clinical cause such as a viral upper respiratory tract infection. Acute Rejection

Acute vascular rejection is a common early phenomenon and may be associated with the development of infiltrates on chest radiographs and a deterioration in lung function (43). The process can also be asymptomatic, however, and is not infrequently diagnosed at routine surveillance biopsy (2). Uncontrolled early acute vascular rejection has been identified as a risk factor for OB (12,44). A stronger association with OB has been noted if the acute vascular rejection occurs after 47 days. Acute vascular rejection is graded according to the degree of leukocyte perivascular infiltrates, from grade A0, no significant abnormality, to grade A4 or severe rejection, where diffuse perivascular rejection with pneumocyte damage,

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necrosis, or vasculitis is present (4). The infiltrating T cells in acute rejection have been recently shown to proliferate actively within the graft. Grade A2 (characterized by frequent perivascular infiltrates around venule or arterioles) or higher are in general treated. Management strategies include increasing doses of cyclosporin, steriod augmentation, and/or changing to newer agents such as tacrolimus and mycophenolate. Isolated acute vascular rejection in our hands and at the University of Leuven, Belgium, and the Alfred Hospital, Australia, has not been associated with a rise in eNO concentrations (G. M. Verleden, 1999, personal communication; E. Gabbay, 1999, personal communication). Of the published data, Silkoff et al. suggest that acute vascular rejection was associated with a rise in eNO. There remains a concern that coexistent airway inflammation or LB was not specifically excluded. Some of the patients included in the acute rejection group did not have biopsy data and were included based on clinical grounds. Results from rat studies are of some relevance to human transplantation, where data suggest there is an association between acute vascular rejection and raised products of nitric oxide metabolism in the serum (21,22,32). Furthermore, inhibition of iNOS with aminoguanidine reduced experimental rejection caused by immunosuppressant withdrawal (22,25). This suggests that NO may actually cause tissue damage in this setting as opposed to acting as a surrogate marker of graft dysfunction. A raised eNO in this rat model noted in one study to be highly associated with acute vascular rejection although the exclusion of coexistent airway inflammation was not clear (45). Further studies looking at eNO in acute vascular rejection with and without coexistent lymphocytic bronchitis in rat models are required. Chronic Rejection

Early BOS or its risk factors may prove the most treatable state, as later stages of BOS are associated with established airway fibrosis and damage. Data from other groups support the hypothesis that OB is associated with raised eNO concentration, but the distinction between BOS grades and differential NO concentrations has not been investigated elsewhere (46–48). In more advanced BOS, where there is often proximal bronchiectasis, lower respiratory tract infections are common, especially with Pseudomonas aeruginosa. In our cross-sectional study we have been able to demonstrate that eNO concentrations are high in BOS 1 and that BOS 3 patients have eNO concentrations similar to healthy post-transplant patients (36). The patient caseload is not well described in the other published series with regard to BOS gradings (46–48). Two possible explanations for the raised eNO in all BOS patients in these studies exist: either the patients were similar to our BOS group 1/group 2 patients, or they were BOS 3 patients with concurrent infections. It is difficult to definitively exclude respiratory infections


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in this group, as BAL is hazardous and sputum sampling even with induction regimes is difficult. Our data may support the hypothesis that the brunt of inflammation is found in early BOS, whereas the latter stages are more associated with myofibroblast proliferation in the absence of inflammation. One bronchoscopic study did distinguish BOS patients from those with bacterial infection and the study could still demonstrate increased expression of iNOS, the inducible isoform of nitric oxide synthase, in biopsies in BOS (49). Human biopsy data suggests that there is both an epithelial- and leukocyte-derived component of eNO with iNOS and peroxynitrite co-localizing to small airways and infiltrating leukocytes in OB (28,30). The Melbourne group has shown that the aqueous NO derivative, NO2(⫺) within lavage fluid (BAL) in 14 OB patients correlated with BAL % neutrophils r 2 ⫽ 0.9, p ⬍ 0.01, but no correlation was seen in 8 stable lung transplant recipients (50). Lavage NO2(⫺) was significantly higher in BOS patients as compared to nontransplant controls but not significantly different to stable recipients. In BOS subjects, but not stable recipients, BAL ferritin was significantly related to BAL albumin r(s) ⫽ 0.8, p ⫽ 0.05. These relationships led to the hypothesis that the allograft could be subjected to iron-generated oxidative stress which could be compounded by NO and neutrophil reactive oxygen species particularly in established BOS (50). The elevated BAL albumin in BOS patients may suggest a microvascular leak leading to local iron excess, thus further promoting airway damage and possibly airway remodelling. Infection

Relatively little published data are available for the pulmonary transplant population with regard to the role of eNO in monitoring for infection and its treatment. In our cross-sectional study, bacterial or viral infection of the allograft was associated with a rise in eNO. Other transplant groups report similar changes in eNO, including the Melbourne transplant group (46) and the Leuven transplant group (G. M. Verleden, 1999, personal communication), reflecting international data regarding eNO and pulmonary infections in nontransplant patients (51). In a study from the Melbourne group the rise in eNO in 4 patients (associated with unspecified infection) was associated with a concurrent rise in iNOS staining in bronchial biopsies (46). Silkoff et al., however, were unable to demonstrate a rise in eNO associated with infection in a recently published series (48). No obvious methodological differences or patient group disparity has been noted to explain this discrepancy. The clinical distinction, however, between infection and acute vascular rejection without a complete biopsy data set and also variable times elapsed between bronchoscopy and eNO measurement in Silkoff et al.’s study may in part explain the differences. Nontransplant human respiratory tract infections, e.g., in bronchiectatic patients, are associated with an elevation in Eno (49), and data

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from canine models of pulmonary transplantation also show an increase in nitric oxide metabolites and iNOS mRNA associated with infection (24). It therefore seems plausible that bacterial pulmonary infections in human transplantation are associated with raised eNO, in concordance with our findings. We have instituted a Cytomegalovirus (CMV) prophylaxis program using oral ganciclovir and are fortunate to have low rates of symptomatic CMV infections. Of 4 patients who required intravenous ganciclovir, no statistically significant rise in eNO concentration was noted, median eNO concentration prediagnosis 12.8 ppb (range 4.8–17) and 12.5 (4.5–17.8) ppb during the clinical infection and 12.1 ppb (5.35–15.9) after treatment and clinically well. Patients with CMV infection were included in Silkoff et al.’s study and did not appear to have eNO concentrations different from stable recipients (48). Other Causes of Graft Dysfunction

We have not been able to demonstrate a rise in eNO in patients with cryptogenic organizing pneumonia (COP), which is a relatively common cause of graft dysfunction (6). Previous reports suggest that there is an association with a raised eNO and post-transplant lymphoproliferative disease (PTLD) (48). We have 4 lung transplant recipients at our center with PTLD affecting the allograft and who have had regular NO measurements. These patients all showed an improvement after intervention with either simple immunosuppressant reduction or chemotherapy. The median eNO prior to diagnosis of PTLD was 8.25 ppb (range 3.3–21.9) and at diagnosis was 9.4 ppb (range 4.57–17.1), p ⬎ 0.05. Despite the small numbers with PTLD, there is no clear association with increased eNO concentration in our recipient population. Lymphocytic Bronchitis

The rates of progression of LB to OB in a previously reported study were high, reaching 65% (52). In the cross-sectional study we demonstrated that the eNO concentration associated with LB was high. As LB is a common condition in lung transplant recipients (3) and has been previously reported to be refractory to systemic augmentation of immunosuppression (52), we were interested to assess the potential treatment with topical inhaled steroids for this condition. The airway inflammation seen in LB, as in asthma, appears to be associated with a rise in eNO. Other similarities include the presence of intraepithelial lymphocytes in both conditions. Intervention Studies in LB

We hypothesized that inhaled steroids could reverse the airway inflammation caused by LB and the associated declines in FEV1 (and in FEF25-75). In a prospective study we placed 14 patients with isolated LB on Budesonide, 800 µg b.d. via turbuhaler for at least 1 month (53). As all patients undergoing follow-


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up at our center have eNO, FEF 25–75, and FEV1 prior to each biopsy, we were able to compare these indices for three time points: prediagnosis, at diagnosis, and after treatment. No placebo arm was included in the study. The 14 recipients who developed isolated LB during the study had eNO concentrations performed prior to the diagnosis of LB (9 had 2 or more premorbid eNO concentrations). This allowed changes associated with the diagnosis of LB to be compared to each patient’s own baseline. Patients with isolated LB demonstrated a significant increase in eNO concentrations at the time of diagnosis compared to when well (Fig. 5). Median (range) eNO concentration (ppb) when well was 4.33 (1.0– 10.76), rising at diagnosis to 10.9 (4.6–48), p ⫽ 0.001. The median rise in eNO concentration was 4.75 (range 0.17–34 ppb). The recipients with isolated LB had NO concentrations performed at review after treatment with inhaled steroids for at least 1 month (range 1–2.5 months). They demonstrated a fall in eNO concentrations back to baseline levels, 4.85 (2.6–15.9), p ⫽ 0.002 (Fig. 5). Levels of eNO prior to the diagnosis of LB and after treatment were not statistically different, p ⫽ 0.19. No significant differences in cyclosporin trough levels or oral prednisolone doses were noted when comparing prediagnosis levels and those at diagnosis (p ⫽ 0.4). Median cyclosporin level at diagnosis of LB was 280 ng/ mL and prednisolone dose 10.5 mg, which was not significantly different from prediagnosis or post-treatment levels (p ⬎ 0.05). Deterioration in lung function was associated with the diagnosis of LB. A fall from baseline FEV1 (L) 2.28 (1.36–4.45) to 1.91 (0.92–4.55) was noted ( p ⫽ 0.004). After treatment with inhaled steroids a significant increase in FEV1 (L) from diagnosis of LB was noted 2.32 (1.06–4.61), p ⫽ 0.02 (Fig. 5). There was no statistical difference in FEV1 prediagnosis to post-treatment, p ⫽ 0.79. Tests of small airway function, using FEF 25-75, displayed a similar pattern, with a fall in FEF from 2.25 L (0.84–8.12) to 1.71 L (0.46–5.69) associated with the diagnosis of LB. After treatment there was an increase in FEF 25–75, although this did not return to baseline 1.73 (0.66–6.76), p ⫽ 0.08 (53). These data correlate with previous data showing that a fall in lung function is associated with the diagnosis of LB (52). Further, reports have suggested that LB is refractory to systemic corticosteroid treatment (52). We have been able to demonstrate that the changes in eNO concentration and FEV1 are returned to baseline with inhaled steroids. A similar pattern was seen for the marker of small airway function FEF25–75, with improvement after inhaled cortocosteriods. This improvement did not, however, return to baseline, which may simply be a reflection of the poorer reproducibility of the flow volume maneuver. The median rise in eNO concentration associated with the diagnosis of LB was greater than 4 ppb, which is in agreement with our previous reproducibility data suggesting that a rise of greater than 3 ppb is outside assay variability (36). Long-term follow-up of these patients is being undertaken to assess the long-term effects of inhaled steroid on rates of progression to chronic rejection.

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(a)

(b) Figure 5 The effect of the development of lymphocytic bronchitis and subsequent treatment with inhaled Budesonide, 800 µg b.d. on (a) exhaled nitric oxide (eNO) parts per billion (ppb); (b) forced expiratory volume 1 sec (FEV1, L).


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Graft Inflammation Summary

Using the single-breath technique, eNO concentrations are stable in a clinically well recipient over a period of up to 1 month. Rises in eNO above 3 ppb above a patient’s baseline are outside assay repeatability using our technique. We have been unable to demonstrate that eNO concentrations are increased with acute vascular rejection. Our cross-sectional and intervention studies, however, suggest that acute airway rejection, lymphocytic bronchiolitis, is associated with rises in eNO concentrations similar to those induced by graft infections. Cryptogenic organizing pneumonia in our experience has not been associated with raised eNO concentrations. Chronic rejection is associated with a raised eNO, although we have only demonstrated this change in early chronic rejection or BOS 1.

IV. Use of Exhaled NO in the Clinical Setting Measurement of NO in exhaled breath has provided a valuable research tool for investigation of the pathophysiology of many different diseases. However, an established clinical use for this noninvasive test, allowing both early diagnosis and monitoring response to treatment, has been lacking. In lung transplant recipients a unique opportunity exists in a well-defined group of patients where pathology arising from a range of different etiologies is relevant to graft function both at present and in the future. In our hands the technique of eNO measurement has been reproducible, acceptable, and correlates with graft eNO production. Furthermore, we have established that a rise in eNO concentration above 3 ppb is outside assay variability and is a sensitive marker of underlying graft pathology. This simple noninvasive test can now be applied to further our knowledge and management of graft dysfunction. Longitudinal studies are required to further the diagnostic use of a change in serial exhaled NO measurements in an individual recipient and its predictive value for the subsequent development of obliterative bronchiolitis. The use of exhaled NO levels as a surrogate marker of airway inflammation in the graft to indicate response to treatment is under further study. Our further work will aim to determine the role of serial exhaled NO levels in the clinical management of lung transplant recipients, correlating eNO with pro-inflammatory markers and the progression to obliterative bronchiolitis.

References 1. Hosenpud JD, Bennett LE, Keck BM, Fiol B, Boucek MM, Novick RJ. The Registry of the International Society for Heart and Lung Transplantation: fifteenth official report—1998. J Heart Lung Transplant 1998; 17:656–668.

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2. Trulock EP. Flexible bronchoscopy in lung transplantation. Clin Chest Med 1999; 20:77–87. 3. Foerster A, Bjortuft O, Geiran O, Rollag H, Leivestad T, Froysaker T. Single lung transplantation. Morphological surveillance by transbronchial biopsy. Apmis 1993; 101:455–466. 4. Yousem SA, Berry GJ, Cagle PT, et al. Revision of the 1990 working formulation for the classification of pulmonary allograft rejection: Lung Rejection Study Group. J Heart Lung Transplant 1996; 15:1–15. 5. Yousem SA. Lymphocytic bronchitis/bronchiolitis in lung allograft recipients. Am J Surg Pathol 1993; 17:491–496. 6. Gabbay E, Dark JH, Ashcroft T, Corris PA. Cryptogenic organizing pneumonia is an important cause of graft dysfunction and should be included in the classification of pulmonary allograft rejection. J Heart Lung Transplant 1998; 17:230–231. 7. Milne DS, Gascoigne AD, Ashcroft T, Sviland L, Malcolm AJ, Corris PA. Organizing pneumonia following pulmonary transplantation and the development of obliterative bronchiolitis. Transplantation 1994; 57:1757–1762. 8. Chaparro C, Scavuzzo M, Winton T, Keshavjee S, Kesten S. Status of lung transplant recipients surviving beyond five years. J Heart Lung Transplant 1997; 16:511– 516. 9. Cooper JD, Billingham M, Egan T, et al. A working formulation for the standardization of nomenclature and for clinical staging of chronic dysfunction in lung allografts. International Society for Heart and Lung Transplantation. J Heart Lung Transplant 1993; 12:713–716. 10. Kelly KH, Hertz MI. Obliterative bronchiolitis. Clin Chest Med 1997; 18:319–338. 11. Milne DS, Gascoigne A, Wilkes J, et al. The immunohistopathology of obliterative bronchiolitis following lung transplantation. Transplantation 1992; 54:748–750. 12. Girgis RE, Tu I, Berry GJ, et al. Risk factors for the development of obliterative bronchiolitis after lung transplantation. J Heart Lung Transplant 1996; 15:1200– 1208. 13. Boehler AC, Kesten D, Slutsky S, Liu AS, Keshavjee SM. Lymphocytic airway infiltration as a precursor to fibrous proliferation in a rat model of bronchiolitis. Transplantation 1997; 64:311–317. 14. Snell GI, Salamonsen RF, Bergin P, Esmore DS, Khan S, Williams TJ. Inhaled nitric oxide used as a bridge to heart-lung transplantation in a patient with end-stage pulmonary hypertension. Am J Respir Crit Care Med 1995; 151:1263–1266. 15. Adatia I, Lillehei C, Arnold JH, et al. Inhaled nitric oxide in the treatment of postoperative graft dysfunction after lung transplantation. Ann Thoracic Surg 1994; 57: 1311–1318. 16. Adatia I, Wessel DL. Therapeutic use of inhaled nitric oxide. Curr Opin Pediatr 1994; 6:583–590. 17. Meyer KC, Love RB, Zimmerman JJ. The therapeutic potential of nitric oxide in lung transplantation. Chest 1998; 113:1360–1371. 18. Ioannidis I, Hellinger A, Dehmlow C, et al. Evidence for increased nitric oxide production after liver transplantation in humans. Transplantation 1995; 59:1293– 1297. 19. Benvenuti C, Bories PN, Loisance D. Increased serum nitrate concentration in car-


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diac transplant patients. A marker for acute allograft cellular rejection. Transplantation 1996; 61:745–749. Worral NK, Boasquevisque CH, Misko TP, Sullivan PM, Ferguson TB Jr, Patterson GA. Inducible nitric oxide synthase is expressed during experimental acute lung allograft rejection. J Heart Lung Transplant 1997; 16:334–339. Shiraishi T, DeMeester SR, Worrall NK, et al. Inhibition of inducible nitric oxide synthase ameliorates rat lung allograft rejection. J Thoracic Cardiovasc Surg 1995; 110:1449–1459; discussion 1460. Shiraishi T, Chen B, Okabayashi K, et al. Inhibition of inducible nitric oxide synthase prolongs rat lung allograft survival. Thoracic Cardiovasc Surgeon 1997; 45: 78–82. Wiklund L, Lewis DH, Sjoquist PO, et al. Increased levels of circulating nitrates and impaired endothelium-mediated vasodilation suggest multiple roles of nitric oxide during acute rejection of pulmonary allografts. J Heart Lung Transplant 1997; 16:517–523. Wang X, Lewis DA, Kim HK, et al. Alterations in mRNA for inducible and endothelial nitric oxide synthase and plasma nitric oxide with rejection and/or infection of allotransplanted lungs. Transplantation 1998; 66:567–572. Worrall NK, Boasquevisque CH, Botney MD, et al. Inhibition of inducible nitric oxide synthase ameliorates functional and histological changes of acute lung allograft rejection. Transplantation 1997; 63:1095–1101. Whitelaw MN, Gigli P, Pepper JR. Broncho-alveolar lavage in lung transplantation. Thoracic Cardiovasc Surgeon 1983; 31:139–141. Pepper JR, Parfett GJ, Reader JA, Kirby JA. Lung transplantation in the rat: cellular mechanisms of allograft rejection. Transplant Proc 1989; 21:470–472. Mason NA, Springall DR, Pomerance A, Evans TJ, Yacoub MH, Polak JM. Expression of inducible nitric oxide synthase and formation of peroxynitrite in posttransplant obliterative bronchiolitis. J Heart Lung Transplant 1998; 17:710–714. Gabbay E, Walters EH, Orsida B, Whitford H, Ward C, Kotsimbos TC, Snell GI, Williams TJ. In stable lung transplant recipients exhaled nitric oxide levels positively correlate with airway neutrophilia and bronchial epithelial iNOS. Am J Respir Crit Care Med 1999; 160:2093–2099. McDermott CD, Gavita SM, Shennib H, Giaid A. Immunohistochemical localization of nitric oxide synthase and the oxidant peroxynitrite in lung transplant recipients with obliterative bronchiolitis. Transplantation 1997; 64:270–274. Utsumi T, Mizuta T, Fujii Y, et al. Nitric oxide production by bronchoalveolar cells during allograft rejection in the rat. Transplantation 1999; 67:1622–1626. Steinmuller C, Steinhoff G, Bauer D, et al. Analysis of leukocyte activation during acute rejection of pulmonary allografts in noninfected and cytomegalovirus-infected rats. J Leuk Biol 1997; 61:40–49. Kharitonov S, Alving K, Barnes PJ. Exhaled and nasal nitric oxide measurements: recommendations. The European Respiratory Society Task Force. Eur Respir J 1997; 10:1683–1693. Gabbay E, Fisher AJ, Small T, Leonard AJ, Corris PA. Exhaled single-breath nitric oxide measurements are reproducible, repeatable and reflect levels of nitric oxide found in the lower airways. Eur Respir J 1998; 11:467–472.

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35. Ward RJ, Gabbay E, Taylor J, Ward C, Whitford H, Kotsimbos T, Snell G, Williams TJ, Walters EH. BAL nitrite levels are directly correlated to exhaled nitric oxide levels. Am J Respir Crit Care Med 1999; 159:A277. 36. Fisher AJ, Gabbay E, Small T, Doig S, Dark JH, Corris PA. Cross sectional study of exhaled nitric oxide levels following lung transplantation. Thorax 1998; 53:454– 458. 37. Barnes PJ. Nitric oxide and airway disease. Ann Med 1995; 27:389–393. 38. Kharitonov SA, Yates DH, Barnes PJ. Inhaled glucocorticoids decrease nitric oxide in exhaled air of asthmatic patients. Am J Respir Crit Care Med 1996; 153:454– 457. 39. Caramori M, Silkoff P, McLean P, Chaparro C, Keshavjee S, Kesten S, Hutcheon M, Slutsky AS, Zamel N. Exhaled nitric oxide (eNO) in lung transplantation: the correlation with bronchoscopy. Am J Respir Crit Care Med 1997; 155:A270. 40. Goto M, Yamaguchi Y, Ichiguchi O, et al. Phenotype and localization of macrophages expressing inducible nitric oxide synthase in rat hepatic allograft rejection. Transplantation 1997; 64:303–310. 41. Giaid A, Corris PA, Chikhani N, Shennib H. Expression of nitric oxide synthase in lung transplant recipients with bronchiolitis obliterans. Eur Respir J 1995; 58:550S. 42. Husain AN, Siddiqui MT, Holmes EW, et al. Analysis of risk factors for the development of bronchiolitis obliterans syndrome. Am J Respir Crit Care Med 1999; 159: 829–833. 43. King-Biggs MB. Acute pulmonary allograft rejection. Mechanisms, diagnosis, and management. Clin Chest Med 1997; 18:301–310. 44. Yousem SA, Dauber JA, Keenan R, Paradis IL, Zeevi A, Griffith BP. Does histologic acute rejection in lung allografts predict the development of bronchiolitis obliterans? Transplantation 1991; 52:306–309. 45. Mizuta T, Fujii Y, Minami M, et al. Increased nitric oxide levels in exhaled air of rat lung allografts. J Thoracic Cardiovasc Surg 1997; 113:830–835. 46. Gabbay E, Orsida B, Walters EH, et al. Post-lung transplant bronchiolitis obliterans syndrome (BOS) is characterized by increased exhaled nitric oxide and epithelial inducible nitric oxide synthase. Am J Respir Crit Care Med 2000; 162(6):2182– 2187. 47. Verleden GM, Dupont L, Lamont J, et al. Is there a role for measuring exhaled nitric oxide in lung transplant recipients with chronic rejection? J Heart Lung Transplant 1998; 17:231–232. 48. Silkoff PE, Caramori M, Tremblay L, et al. Exhaled nitric oxide in human lung transplantation. A noninvasive marker of acute rejection. Am J Respir Crit Care Med 1998; 157:1822–1828. 49. Gabbay E, Walters EH, Orsida B, et al. Post-lung transplant bronchiolitis obliterans syndrome (BOS) is characterized by increased exhaled nitric oxide levels and epithelial inducible nitric oxide synthase. Am J Respir Crit Care Med 2000; 162(6):2182– 2187. 50. Reid D, Snell G, Ward C, et al. Iron overload and nitric oxide-derived oxidative stress following lung transplantation. J Heart Lung Transplant 2001; 20(8):840–849. 51. Kharitonov SA, Wells AU, O’Connor BJ, Cole PJ, Hansell DM, Logan-Sinclair RB,


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18 Nitric Oxide in Cystic Fibrosis

THOMAS J. KELLEY and MITCHELL L. DRUMM Case Western Reserve University Cleveland, Ohio, U.S.A.

Nitric oxide (NO) is a multifaceted molecule that is involved in numerous pathways relevant to normal and diseased airway function. The role of NO in cystic fibrosis (CF) airway disease is particularly interesting given a number of what could be considered counterintuitive findings regarding the production, metabolism, and function of NO in the CF lung. How NO, or the apparent lack of NO, influences or participates in CF airway disease and possible mechanisms that may explain the puzzling observations regarding CF-related NO production will be discussed. I.

Detection of NO and NO Metabolites in the CF Lung

CF airway disease is characterized by chronic bacterial infection and hyperinflammatory responses (1). Given these conditions, one would expect increased NO production due to expression of the inducible form of nitric oxide synthase (NOS2). Consistent with the normal role of NO in the inflammatory process, exhaled NO levels are typically used as a primary marker of airway inflammation in several airway diseases, particularly asthma. Exhaled NO levels show a strong correlation to inflammation in asthma and are a useful measure of treatment effec403

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tiveness (2–4). However, despite chronic inflammation, the same correlation between disease progression and exhaled NO levels does not exist in CF. Two papers simultaneously reported that exhaled levels of NO in patients with CF were not elevated compared to non-CF controls and that nasal NO concentrations in CF patients were decreased when compared to healthy controls (5,6). Several reports have subsequently been published that support these findings (7–11). Concentrations of S-nitrosothiols (SNOs) have also recently been found to be reduced in CF airways (12). SNOs are important to airway function because they act as stable NO donors, allowing NO to be delivered via respiration to the lower airways or to influence cell signaling events in inflammatory cells. Diminished concentrations of SNOs in CF airways, particularly S-nitrosylated glutathione derivatives, may be in part due to an apparent inability of mutant cystic fibrosis transmembrane conductance regulator (CFTR) to transport glutathione across the airway epithelium into the lumen (13,14). Limited concentrations of SNOs in CF airways may also be indicative of a lack of NO production in this environment, however, the same report indicates that CF airways contain normal levels of NO metabolites nitrite and nitrate (12). In contrast to data consistently showing decreased levels of SNOs and exhaled NO from CF airways, many reports have demonstrated increased levels of the NO metabolites nitrite and nitrate in samples of sputum, saliva, and breath condensate from CF patients (15–18). These findings suggest that NO production takes place in the CF airway but the NO is retained in the chronically suppurative CF lung, resulting in the observed decrease in levels of exhaled NO. Given the utility of gauging inflammation by measuring airway NO, a more careful examination of the possible mechanisms behind these incongruous results is necessary.

II. Possible Mechanisms of Reduced Exhaled NO in CF A. Airway NO Reactivity and Metabolism

The environment of the airway lumen is significantly altered in the CF lung compared to a nondiseased lung (1). Poor mucociliary clearance, altered ion and fluid transport resulting in a dehydrated and viscous mucus layer, and chronic bacterial colonization characterize a suppurative airway that provides ample opportunity for NO to be retained and metabolized to nitrite and nitrate. The altered composition of the mucus layer has been postulated to retain generated NO, thus reducing the level of NO in exhaled breath. Additionally, many bacterial strains produce NO reductase as a means of energy conservation and respiration in primarily anaerobic conditions (19–22). Chronic bacterial colonization in CF lungs may result in the accelerated reduction of NO through bacterial denitrification pathways, again potentially accounting for the observation of reduced exhaled NO. Oxidative stress is also characteristic of the CF lung, in part due to increased

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infiltration of polymorphonuclear neutrophils (PMNs), which release high levels of superoxide. NO interacts quickly with reactive oxygen species (ROS) as a defense against oxidative change. One postulated mechanism to explain lowered levels of exhaled NO in the CF lung is that higher levels of superoxide generated by infiltrating PMNs quickly interact with NO, decreasing free gas-phase NO (23). To test this hypothesis, researchers added PMNs producing superoxide to cultures of murine lung epithelial cells (LA-4) stimulated to produce NO. As PMNs were added to the cells, NO levels measured in the headspace above the cultures was correspondingly reduced. No changes in NOS2 protein or message levels were observed, indicating that conversion of NO to peroxynitrite and nitrate decreased NO concentration in the headspace (23). Clearly, increased reactivity and metabolism of generated NO in the CF lung is a consideration in identifying mechanisms responsible for the puzzling observation of reduced exhaled NO in an inflammatory disease like CF. B. Impaired NO Production in CF Epithelial Cells

Another factor that needs to be considered when examining reduced levels of exhaled NO in CF, however, is the ability of the CF airways to produce NO. One study examined the possibility of reduced NO production compared to increased NO retention by giving CF and non-CF patients a 250-µg intravenous bolus of the NO donor glyceril trinitrate (GTN) and then measuring the increase in gas-phase exhaled NO (24). The relative increase in gas-phase NO levels was comparable in both groups (36.7 ⫾ 4 ppb CF; 48.7 ⫾ 4 ppb non-CF), indicating that there is not an excessive loss of NO due to retention in the CF lung. These data suggest that impaired NO synthesis may be responsible for reduced exhaled NO observed in CF. In support of this hypothesis, two studies have identified a significant reduction in the expression levels of the inducible form of nitric oxide synthase (NOS2) in bronchial and tracheal epithelium from human CF subjects compared to non-CF controls and in the airway epithelium from murine models of CF (25,26). Consistent with this finding is the observation that NO production is apparently reduced in the lungs of CF mice (26). Meng et al. have shown not only the reduction of NOS2 protein in primary CF human bronchial epithelial cells, but also the reduction of NOS2 message levels (25). Steagall et al. demonstrate that the loss of NOS2 message and protein is dependent on the presence of functional CFTR (27). Cultured cells lacking functional CFTR chloride transport due to the overexpression of the regulatory domain of CFTR have reduced NOS2 mRNA levels and diminished NO production compared to matched mocktransfected controls. Consistent with that observation is that NOS2 expression in the epithelial cells of the ileum of mice lacking CFTR expression (cftr ⫺/⫺) is restored by the expression of human CFTR specifically in the intestines by the use of fatty acid-binding protein (FABP) promoter. The nasal epithelium of these

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mice does not express human CFTR and does not have NOS2 expression restored. Clearly, three is a CF-related reduction of epithelial NOS2 expression contributing to reduced NO production which needs to be considered when interpreting the lack of exhaled NO in CF. Although the expression of NOS2 is reduced in CF epithelium, two reports do find increases in NOS activity in CF cells using in-vitro assay systems (28,29). These findings may reflect increases in either NOS1 or NOS3 isoforms in CF cells, but most reports find that overall NO production is still reduced in CF epithelium. This hypothesis is consistent with reports that show NOS2 is the predominant source of epithelial NO production, particularly in the airways (30–34). C. Altered Regulation of NOS2 Expression in CF Epithelial Cells

Decreased expression of NOS2 in CF airway epithelial cells is a surprising finding in such an inflammatory disease. Similar findings of reduced expression of RANTES (regulated on activation, normal T-cell expressed and secreted) in CF epithelial cells in response to TNF-α and/or IFN-γ suggest that cell signaling processes necessary for the proper regulation of these inflammatory proteins are intrinsically altered in CF epithelium (35). Improper regulation of these inflammatory pathways may account for some of the secondary symptoms of CF. Schwiebert et al. suggest that alterations in the proper regulation of the NF-κB pathway are involved in the lack of RANTES expression in CF epithelial cells (35). Other pathways that should be examined are those known to influence NOS2 expression, such as the transforming growth factor-β1 (TGF-β1)-dependent signaling and phosphatidylinositol-3 (PI-3) kinase pathways. TGF-β1 action is known to be a negative effector of NOS2 expression as demonstrated in several reports (36–39). Mice lacking TGF-β1 expression have been shown to spontaneously express increased levels of NOS2, further demonstrating the role of TGF-β1 as a negative regulator of NOS2 expression (40). Although there is less agreement about the role of PI-3 kinase in NO regulation, there are several reports that demonstrate PI-3 kinase is a potent negative regulator of NOS2 expression (41–44). The components of the IFN-γ pathway also need to be examined when looking at the reduced expression of NOS2 in CF epithelial cells (Fig. 1). A factor necessary for the full expression of NOS2 is the interferon regulatory factor-1 (IRF-1) (45–47). IRF-1, like NOS2, is dependent on the activity of the signal transducer and activator of transcription 1 (Stat1) (48–51). Alterations of the Stat1-dependent pathways resulting in diminished expression of IRF-1 have been shown in airway and intestinal epithelium from mouse models of CF (52). The reduced expression of NOS2 in CF is a clue to more general changes in cell signaling regulation brought about by the loss of CFTR activity, and understanding these alterations will greatly clarify our understanding of CF disease.


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Figure 1 Regulation of NOS2 expression. Full expression of NOS2 requires both NF-κB and IFN-γ-regulated pathways. Agents such as lipopolysaccharide (LPS), IL-1β, and TNF-α stimulate the activation of NF-κB, which acts synergistically with the active form of the signal transducer and activator of transcription 1 (Stat1) to drive the expression of NOS2. Dimers containing the phosphorylated form of Stat1 (pStat1) bind to the interferon-γ-activated site (GAS) along with the binding of active dimers to NF-κB sites stimulate the expression of the interferon regulatory factor-1 (IRF-1). IRF-1 then binds to an interferon regulatory element (IRE) within the NOS2 promoter along with NF-κB dimers and pStat1 to drive NOS2 expression. One study indicates not only reductions in NOS2 levels of CF epithelium, but reductions in IRF-1 expression levels as well (52). These data suggest that possible alterations in NF-κB and IFN-γ signaling pathways may be present in epithelial cells lacking normal CFTR function.

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Kelley and Drumm III. Possible Implications of Reduced Epithelial NOS2 Expression and NO Production in CF Airway Disease A. Nitric Oxide as a Cell Signaling Agent

NO has been shown to influence several cell signaling pathways, the most studied of which is the cGMP-mediated pathway. NO binds to a heme group within the soluble form of guanylate cyclase, causing a conformational change and stimulating the generation of cGMP from GTP (53). This pathway is relevant to CF since cGMP-dependent pathways have been implicated in the regulation of amiloridesensitive sodium absorption (Fig. 2). Hyperabsorption of sodium is a characteris-

Figure 2 Nitric oxide-mediated regulation of epithelial ion transport. The production of cGMP via soluble guanylate cyclase (GC-s) can positively regulate transepithelial chloride transport via both protein kinase A (PKA) and protein kinase G (PKG) pathways. Amiloride-sensitive sodium absorption is also known to be negatively regulated by cGMP through PKG, although it is not known whether this is a direct effect of PKG phosphorylation or an indirect effect due to the phosphorylation of associated proteins. The proposed net effect of increased epithelial NO production on ion transport would be an increase in chloride transport and a decrease in sodium absorption resulting in a transient and localized inhibition of fluid absorption from the lumen. This process may play a role in mucociliary clearance and innate defense against bacterial pathogens. Improper regulation of these pathways, as well as reduced NO production, are characteristic of CF airway epithelium. The ⫹ and ⫺ symbols indicate positive or negative regulatory effects on protein function, respectively.


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tic of CF that is yet to be completely explained. It has been shown that the addition of cGMP or the addition of guanylate cyclase agonists reduces amiloride-sensitive sodium absorption in CF, suggesting that a lack of cGMP production may contribute to this aspect of CF (54). Also, NO has been implicated in the simulation of transepithelial chloride transport (55–57). Multiple reports implicate cGMP and cGMP-dependent pathways in the activation of CFTR, either by the inhibition of phosphodiesterases (58,59), direct cGMP binding to CFTR (60), or through the PKG-mediated phosphorylation of CFTR (61). NO has also been implicated in the stimulation of non-CFTR-mediated chloride transport. In A549 cells, NO donors were shown to stimulate whole-cell chloride currents that were CFTR-independent (62). Chloride-free responses in the nasal transepithelial potential difference (TEPD) assay in mice were shown to be stimulated by NO. NOS2 ⫺/⫺ mice were found to lack the chloride-free response though they possess normal CFTR activity, while wild-type mice of the same genotype had a robust response (63). These data show that though chloride-free responses in the nasal TEPD assay are CFTR-dependent at some level, they are also stimulated by NO. Other pathways are also influenced by NO. The regulation of NF-κB has obvious implications for an inflammatory disease such as CF. One report suggests that NF-κB is constitutively active in CF epithelial cells, suggesting that a basal negative regulatory mechanism is absent (64). The effect of NO on the NF-κB pathway is somewhat unclear. Several reports indicate that NO added exogenously can itself stimulate NF-κB translocation and activation (65). However, this may not be the most relevant test of the role of NO in vivo. Other studies demonstrate that pretreatment of cells with NO inhibit the ability of TNF-α or other cytokines to stimulate NF-κB 966,67). One study demonstrates the importance of endogenous NO on the stability of the NF-κB inhibitor IκB (68). The addition of NO synthase inhibitors leads to increased phosphorylation and degradation of IκB, implicating a basal role for NO in modulating the NF-κB response. These data may indicate a dual role for NO in the inflammatory process. Exogenously produced higher levels of NO may stimulate NF-κB and the inflammatory response, while lower levels of endogenous NO may play an inhibitory role meant to keep basal NF-κB activity under control. However, few of these studies concerning NO regulation of NF-κB have been performed in airway epithelial cells. The regulation of these processes in the airway epithelium needs to be understood to have a more complete picture of the inflammatory process in CF. B. Effects of Nitric Oxide on Cytokine Production and Inflammatory Responses

Closely related to the regulation of NF-κB activity by NO is the regulation of inflammatory cytokine production. Not surprisingly, these studies parallel the

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studies performed on NF-κB activity. Exogenously added high levels of NO can stimulate IL-8 production, while endogenous NO inhibits IL-8 production stimulated by other cytokines (69,70). One of the more convincing and CFrelevant studies examined neutrophil recruitment and IL-8 production in guinea pig airways in response to TNF-α. Pretreatment with the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) significantly reduced the level of IL-8 production and neutrophil recruitment (71). These data show that endogenous NO likely plays a significant role in balancing the inflammatory response. Similarly, other studies demonstrate the ability of NO to reduce neutrophil infiltration into airways in response to inflammatory stimuli (72,73). Although exogenously added NO apparently causes inflammation in various systems, the in vivo role of endogenous NO appears to be as much protective as it is damaging. One property of NO that may play a role in determining whether it acts as a protective agent is its ability to act both as an antioxidant and as a mediator of oxidative stress. In the right levels, NO reacts readily with ROS, reducing the damage inflicted on airway tissues and blocking the ability of ROS to stimulate inflammatory responses. However, additions of NO at high concentrations will act as an oxidative stress and result in damage and inflammation. This effect can likely be an in vivo factor as well, since prolonged elevation of NO production has been implicated in a variety of pathogenic processes. In a mouse model of adult respiratory distress syndrome (ARDS) utilizing C57BL/6 mice, mice challenged with LPS and formyl-norleucyl-phenylalanine (FNLP) showed significant increases in airway NO production compared to mice injected with saline solution (74). Inhibition of NO production during LPS and FNLP challenge resulted in remarkably increased pulmonary edema, increased TNF-α production, and increased levels of tissue damage. These findings are consistent with work that demonstrates that endogenous nitric oxide inhibits LPSinduced TNF-α production by macrophages (75), and with clinical trials in which ARDS patients receiving inhaled NO showed significantly decreased levels of IL-6 and IL-8 (76). It was concluded from this study that inhaled NO would reduce lung inflammation as well as improve arterial oxygenation. The reduction of epithelial NO production in CF is a potentially powerful observation that can be utilized to more fully understand the role of endogenous NO in airway defenses and in turn lead to a better understanding of disease processes in CF. C. Nitric Oxide as an Antibacterial Agent

The most devastating aspect of CF airway disease is the presence of severe and chronic bacterial infections with common pathogens. Exciting theories have recently been postulated to hopefully explain the origin and persistence of these infections. Two theories that have been postulated to explain the multitude of


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symptoms are the “high salt” and the “dehydration” theories. The “high salt” theory proposes that a lack of chloride absorption through CFTR leads to higher salt concentrations in epithelial lining fluid (ELF), which disables antimicrobial peptides and leads to chronic infection (77,78). The “dehydration” theory contends that a lack of chloride secretion coupled with hyperabsorption of sodium leads to ELF dehydration, reducing mucociliary clearance due to thickened mucus leading to infection (79–81). Our contention is that, in addition to various aspects of these two models that likely lead to infection, reduced NO levels contribute to the initial susceptibility to infection in the airways since NO is a powerful first-line innate defense mechanism. NO has been implicated as a bactericidal agent in several systems, particularly as an agent produced from macrophages in response to infection (82–84). NO has also been shown to be a significant mediator of murine resistance to airway infection by Pseudomonas aeruginosa (85). Injection of the normally infection-resistant BALB/c mice with the nitric oxide synthase (NOS) inhibitor aminoguanidine severely decreased their ability to control the growth of P. aeruginosa. Aminoguanidine had no effect, however, on the infection-susceptible DBA/2 mice, suggesting that reduced NO production in the DBA/2 mice is a factor in their increased susceptibility to P. aeruginosa infection. This model is consistent with the recent observation that exhaled NO levels are reduced in patients with CF despite chronic bacterial infection (8,86). It is possible that the lack of NO production in CF patients is contributing to increased susceptibility to bacterial infection as was seen in the mouse models. In the airway lumen, the two primary sources of NO in response to bacterial infection are airway epithelial cells and macrophages. Diffusion of NO out of endothelial cells may also contribute to total lumenal NO concentrations. It is generally assumed that macrophage-derived NO and other cytokines are responsible for host bactericidal activity. However, Saito et al. have demonstrated that NO produced from L-arginine-treated macrophages is insufficient to kill various strains of bacteria, including P. aeruginosa, placed in culture with the stimulated macrophages (87). These data imply that a second source of NO may be needed in the host response to defend against bacterial infection. Clearly, epithelial production of NO is a candidate for that source. In human airway epithelial cells, NOS2 has been shown to be induced by LPS and other bacterial challenges, but also to be continuously expressed and provide a constant source of epithelial NO production (34). Guo et al. concluded that airway epithelial cells serve as important inflammatory cells functioning in host defense through NO-dependent mechanisms. This contention is supported by a recent report that studied the oxidative killing of the CF pathogen Burkholderia cepacia. The authors demonstrate that B. capacia is extremely susceptible to oxidative killing by peroxynitrite which is formed by the interaction between NO and peroxide (88). Reduced epithelial NO likely prevents the adequate formation of peroxynitrite with neutrophilderived peroxide and leaves the airways susceptible to bacterial infection.

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Kelley and Drumm IV. Summary

The role of NO in CF airway disease is a complicated subject open to debate (Fig. 3). There is convincing evidence that the lack of exhaled NO observed in CF is due to increased retention by thick mucus layers and increased conversion to peroxynitrite, nitrite, and nitrate due to increased production of superoxide and other ROS as a result of increased PMN infiltration into the CF lung (15– 18,23). However, the CF lung is characterized by poorly cleared, thick, and dehydrated mucus (1). Measurements demonstrating increased levels of NO products such as nitrate in samples of CF sputum may reflect a buildup of reactive products in the mucus over time due to poor clearance rates and as such may not be an accurate indication of increased NO production. This hypothesis is supported by a study that shows little difference in short-term retention of gas-phase NO in the CF lung after giving CF patients and control patients a bolus of the NOdonor GTN (24). These data support the idea that NO production is reduced in the CF lung resulting in decreased levels of exhaled NO.

Figure 3 Proposed functions of epithelium-derived NO. NO has been proposed to be involved in several airway epithelial functions. Most notable with respect to CF airway disease are the roles of an endogenous negative regulator of NF-κB and cytokine production, a regulator of transepithelial ion and fluid transport, a protective mechanism against oxidative stress, and as a mediator of innate antimicrobial defense. Each of these functions is involved in the pathogenesis of CF airway disease.


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The hypothesis that NO production in the CF lung is diminished is supported by observations that the expression of the NO synthase NOS2 is reduced at both the protein and mRNA levels in CF epithelial cells (25,26). Although there are indications that the expression of other isoforms of NOS may be increased, direct measurements of NO production by CF epithelial cells demonstrate a decrease in overall NO synthesis (26). These data are consistent with reports that NOS2 is the primary source of both constitutive and induced NO production by airway epithelial cells (30–34). If NOS2 is normally an integral part of airway epithelial cell function, one must postulate as to what the effects of reduced epithelial NOS2 expression and NO production would be. It is here that an intriguing picture of CF airway disease begins to be illuminated. CF airways are characterized by an increase in amiloride-sensitive, transepithelial sodium absorption, chronic inflammation, and chronic bacterial infection (1). NO is an important factor in each of these processes. It has been demonstrated that sodium absorption can be inhibited in mouse models of CF by the addition of NO via a cGMP-dependent mechanism (53,54). The magnitude of CF-related sodium hyperabsorption is likely to be influenced by reduced NO/cGMP signaling. There is also ample evidence that endogenous NO synthesis plays a modulating role in the NF-κB-dependent inflammatory pathways (66–69). Reduced NO production could lead to an overly active inflammatory process which is seen in CF. Finally, NO as peroxynitrite is a potent antimicrobial agent (88). Reduced epithelial NO production could lead to a diminished innate defense against bacterial challenge. The role of NO as a potent innate epithelial defense against infection has been demonstrated in intestinal epithelium by describing NO-mediated protection against the protozoan Giardia lamblia (89). The study of NO, the regulation of its production, and its normal role in inflammation and lung defense is clarifying various aspects of CF lung disease. Although surprising and seemingly disparate observations regarding NO in CF have characterized the field thus far, elucidation of NO-dependent cell signaling pathways and regulation of cellular events will deepen our understanding of lung function and lead to possible sites of therapeutic intervention for CF airway disease. References 1. Welsh MJ, Tsui LC, Boat TF, Beaudet AL. Cystic fibrosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Disease. 7th ed. New York: McGraw-Hill, 1995:3799–3863. 2. Jatakanon A, Kharitonov S, Lim S, Barnes PJ. Effect of differing doses of inhaled budesonide on markers of airway inflammation in patients with mild asthma. Thorax 1999; 54:108–114. 3. Stirling RG, Kharitonov SA, Campbell D, Robinson DS, Durham SR, Chung KF,

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19 Exhaled Nitric Oxide in Rheumatic Diseases

GIOVANNI ROLLA and FEDERICO CALIGARIS-CAPPIO University of Turin and Ospedale Mauriziano Umberto I Turin, Italy

I.

Introduction

Experimental and clinical observations have been accumulated in the last few years pointing to excessive nitric oxide (NO) production during the course of many rheumatic diseases. The consequences of NO production in these disorders are manifold and might be either beneficial or detrimental. The recent finding of increased production of NO within the respiratory tract that allows its quantification in the exhaled air may represent an easy-to-obtain marker of inflammation in some rheumatic diseases. The clinical value and importance of this strategy is currently under intense evaluation. This chapter reviews the studies pertinent to exhaled NO in systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and Sjo¨gren syndrome (SS). Despite the fact that there are no published data available on exhaled NO in rheumatoid arthritis (RA), the potential role of excessive NO production in this disease will also be discussed. NO, secreted at high levels by activated macrophages and neutrophils, is one of the major cytotoxic effector molecules in the defense against parasites, fungi, protozoa, helminths, and mycobacteria (1). Evidence is accumulating that NO, besides its action as immune defense molecule, also has an immunoregulatory role. The role NO might play in rheumatic diseases appears to be dual, pro421

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Figure 1 Possible pathogenetic role of increased NO production in autoimmune diseases. Intracellular antigens may be released as a result of direct cytotoxicity and increased apoptosis in target organs. A decrease in the apoptotic rate of autoreactive lymphocytes, combined with an expansion of CD4 Th-2 lymphocytes, leads to B-cell activation and autoantibody production. The immunocomplexes may eventually upregulate NO synthase in macrophages and in endothelial cells with resulting increased NO production.

inflammatory and anti-inflammatory, depending on the amount produced, the site of production, and the local environment. Anti-inflammatory activity is displayed by the small quantity of NO released under physiological conditions by the vascular endothelium, which regulates the relaxation of adjacent smooth muscle and protects against the adhesion of leukocytes and platelets to the blood vessel wall (2). On the other hand, the much larger amount of NO released by cells in response to cytokines can damage host tissues. A dual effect of NO on lymphocyte function and on apoptosis might be relevant to the pathogenesis and clinical course of rheumatic diseases (Fig. 1). II. NO and Lymphocyte Function In murine models NO has an important effect on T-helper (Th-1/Th-2) lymphocyte balance. It has been demonstrated that murine Th-1 clones produce NO following activation and that NO inhibits the cytokine production of Th-1, but not of Th-2 clones upon T-cell receptor activation (3). An imbalanced T-cell

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immune response seems to be important for the pathogenesis of atopic as well as autoimmune diseases. Correction of Th-1/Th-2 balance has in fact led to the prophylaxis and therapy of these diseases in various models of autoimmunity (4). Bauer et al. (5), however, reported that cytokine production by purified T-cell subpopulations (Th-1 and Th-2) was equally impaired by NO donors, suggesting that NO did not preferentially inhibit Th-1 cytokine secretion of activated human T cells in vitro. On the contrary, Hogaboam et al. 96) provided evidence that increased NO exerts a critical regulatory role during antigen-specific pulmonary granuloma formation in murine model. Mice treated with NG-nitro-L-arginine methylester (L-NAME), an inhibitor of NO synthesis, developed lung lesions significantly larger than the granuloma lesions measured in control mice. The enlarged granuloma of L-NAME-treated mice contained markedly greater numbers of neutrophils and eosinophils and produced significantly higher levels of IL-4 and IL-10 and smaller amounts of IL-12 and IFN-γ than control mice. According to these findings, NO regulates the size and cellular composition of the Th-1-type lung granuloma, possibly through its effects on the cytokine profile associated with this lesion. In Wegener’s granulomatosis, a necrotizing granulomatous vasculitis involving the upper and lower respiratory tracts, Haubitz et al. (7) found that pulmonary NO excretion did not differ significantly from that of healthy controls. In the same subjects the NO excretion rate in nasally sampled gas was significantly reduced in patients with active disease as compared to healthy controls and patients in remission. The reduced NO concentration may well compromise host defense in the upper airways, thus contributing to the colonization with Staphylococcus aureus and further promoting the progression of Wegener’s granulomatosis.

III. NO and Apoptosis Apoptosis is another important mechanism through which NO may contribute to the pathogenesis of rheumatic diseases. An increase in apoptosis rate due to the release of cytoplasmic and/or nuclear antigens (e.g., Rp/La, dsDNA) may play a central role in promoting and exacerbating autoimmune diseases, such as SLE. A decrease in apoptosis may also play an important immunoregulatory role. A decrease in eosinophil apoptosis may be crucial in eosinophil-mediated diseases, such as bronchial asthma and Churg-Strauss vasculitis. Furthermore, defective apoptosis may cause survival of autoreactive T-cell clones, which are normally deleted from the host repertoire. NO has been shown to display both pro-apoptotic and anti-apoptotic actions. Several cell types undergo apoptosis in response to NO or peroxynitrite, including macrophages (8), thymocytes (9), and pancreatic islets (10). The induc-

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tion of apoptosis often requires exposure to high levels of NO. The pro-apoptotic effect of NO might result from the formation of peroxynitrite able to induce apoptotic DNA fragmentation and p53-dependent apoptosis (11). At lower concentrations NO has been reported to inhibit apoptosis in human B lymphocytes (12), splenocytes (13), and eosinophils (14). NO has also been shown to inhibit apoptosis induced by many different stimuli, including TNF-α or Fas (15), either through the low levels of NO generated by endothelial NO synthase (eNOS) in TNF-induced apoptosis (16), or via the high levels generated by the overexpression of inducible NO synthase (iNOS) in LPS-induced apoptosis (17). Although different mechanisms might be involved in the anti-apoptotic actions of NO, inhibition of caspase activity by S-nitrosylation is one of the best characterized mechanisms for the inhibition of apoptosis by NO (18). IV. Specific Diseases A. Systemic Lupus Erythematosus Animal Models

The inbred MRL-lpr/lpr mouse is a recognized model for human SLE that spontaneously develops an autoimmune disease characterized by DNA antibodies, arthritis, nephritis, and vasculitis (19). This mouse model of lupus-like disease is accompanied by increased urinary excretion of nitrite, enhanced macrophage NO production, and increased immunoreactive NO synthase in spleen and renal tissue. In the same murine model, the oral administration of the NOS inhibitor L-nG monomethyl arginine (L-NMMA), after the onset of disease, reduced proteinuria, and the addition of dietary arginine restriction also significantly improved renal pathology scores (20). On the other hand, recent studies showed that MRL-lpr/lpr iNOS knockout mice were not protected from the development of significant nephritis and arthritis (21). In the latter model, however, the severity of vasculitis was decreased, supporting a role for iNOS in vascular inflammation. Human SLE

SLE is a disease of unknown etiology in which tissues and cells are damaged by pathogenic autoantibodies and immune complexes. The disease is generally multisystemic, even if at onset SLE may involve only one organ, and additional manifestations occur over time. Most patients experience exacerbations interspersed with periods of relative quiescence. Belmont et al. (22) have demonstrated that SLE is characterized by an increased production of NO, as reflected by elevation of serum nitrites in 46 patients with SLE compared to controls. The same authors showed that patients with active disease had higher levels of serum nitrites compared with those with inactive disease. Serum nitrite levels corre-


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lated with disease activity, which was evaluated by SLE disease activity index (SLEDAI) (23), and with level of antibodies to dsDNA. Endothelial and keratinocyte expression of iNOS were significantly elevated in SLE patients compared with controls, and were higher in patients with active disease as compared to those with inactive disease. Similar results have been obtained by Gilkeson and colleagues (24), who reported that the correlation between serum nitrites and SLEDAI was due to the high values of serum nitrites observed in patients with active nephritis. Both nitrite and citrulline serum levels have been shown to be significantly higher in patients with SLE than in controls by Wanchu and colleagues (25), who found the highest values in the patients with the highest score of disease activity. In 15 patients with cerebral SLE the cerebrospinal fluid levels of nitrite/ nitrate were significantly correlated with the severity of neurological symptoms (26). This observation led the authors to hypothesize a new possibility for the pathogenesis of cerebral SLE and to conclude that the analysis of NO2⫺ /NO3⫺ in cerebrospinal fluid may prove to be of value in monitoring the activity of the disease. In contrast with the studies reporting a positive relationship between disease activity and increased NO production is the observation of GonzalesCrespo and colleagues (27), who studied prospectively the relationship of serum and urinary nitrate levels with SLE activity. To this end, 50 patients were followed for 2 years, by measuring SLEDAI, serum, and urinary nitrate levels every 4 months. The analysis of serial measurements did not reveal a significant relationship between the changes in SLEDAI and the changes in serum and urinary nitrate levels. However, nitrate measurement may be influenced by a number of variables, including diet, infections, and creatinine clearance. Importantly, 12 of the patients examined had infections and 12 had active nephritis during the follow-up. Exhaled NO

Higher than normal expired NO concentrations have been reported in SLE patients (28,29). Both peak and mean concentration of NO were increased in exhaled air compared to controls, and the peak NO concentration was related directly to ECLAM activity score (r ⫽ 0.42, p ⬍ 0.05) (Fig. 2). No patient had a major respiratory problem (pleurisy, pneumonia, radiological evidence of interstitial lung disease), but more than half of the patients had a significant decrease in lung diffusing capacity and in the values of MEF25, a test that reflects small airway function. In addition, a significant inverse correlation was observed between eNO and MEF25 (r ⫽ ⫺0.44, p ⬍ 0.05). Even in the absence of overt respiratory disease, the studied patients had a high prevalence of functional lung impairment, in agreement with literature data. A mixed pattern of functional lung damage, characterized by mild restrictive and diffusive abnormality, along with

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Figure 2 Relationship between exhaled NO (in ppb) and disease activity score (ECLAM) in patients with SLE. (From Ref. 28.)

a decrease of MEF25, is commonly reported in SLE (30,31). Respiratory function abnormalities in patients with SLE have been related to inflammatory reactions. Alveolar lymphocytosis has been correlated with lung diffusion decrease (32), while peribronchial and bronchial wall inflammation, together with interstitial mononuclear cell infiltrate, have been suggested as the pathological basis for small airway obstruction (30,33). The inverse correlation between eNO and MEF25 (28) may support the hypothesis that respiratory tract inflammation causes increased eNO in patients with SLE, but direct investigation (i.e., BAL, lung biopsies) is needed to clarify this point. Origin and Significance of NO in SLE

The cellular origin of increased production of NO in SLE and the role of NO in the pathogenesis of the disease remain important questions. One of the emerging concepts is the widespread activation of the vascular endothelium in active SLE. Activated endothelial cells may release excessive amounts of NO, correlated with evidence of increased disease activity, based on SLEDAI, anti-DNA levels, and C3a levels (22). Activated endothelial cells have also been shown to increase the expression of adhesion molecules such as E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1), which


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might contribute to tissue injury in the disease (34). Increased NOS expression has been shown in keratinocytes of patients with SLE, where the increased NO production could contribute to inflammatory skin lesions characteristic of the disease (22). Moreover, increased keratinocyte NO production could promote apoptosis (35), leading to the release of cytoplasmic and/or nuclear antigens (e.g., Ro/La, dsDNA) that maintain the disease process (36). Increased NO levels may also contribute to the increased rate of lymphocyte apoptosis, which has been observed in SLE and in other autoimmune diseases (37). It is likely that during the immune-activated state which characterizes active SLE, iNOS is induced in many other cells, including cells of the respiratory tract, such as smooth muscle cells, bronchial epithelial cells, and alveolar macrophages. Immune stimuli that may directly upregulate iNOS are cytokines such as IL-1, TNF-α, and IL-6. Even if data on plasma levels of TNF-α in SLE are conflicting (38), IL-6 is believed to play a central role in the pathophysiology of SLE (39). The increase in NO production may contribute to maintain the imbalance between Th-1 and Th-2 cytokines, which has been shown to play a key role in the development of several autoimmune diseases (40). In patients with SLE, serum levels of Th-2 cytokines, such as IL-4, IL-6, and IL-10, are elevated, while a decrease in production of Th-1 cytokines, including IL-2 and IFN-γ, is observed (41). Thus, SLE is frequently considered a disease where Th-2 predominates, even if this notion is not confirmed (40,42). Moreover, other immune stimuli which may account for endothelial cell activation in SLE include immune complexes, complement components, and antiphospholipid antibodies (43). The correlation between exhaled NO and ECLAM score of disease activity (28) may thus depend either on respiratory tract inflammation or on inflammatory cytokines and other circulating factors released in sites other than the respiratory system. In conclusion, NO production is increased in patients with SLE proportionally to the activity of the disease. The ultimate clinical significance of this observation is presently undefined. The measurement of NO in the exhaled air is the simplest and more rapid test which may document the increased NO production in SLE. Whether eNO measurement is merely one of the many available nonspecific markers of inflammation or whether it may help in the follow-up of patients, particularly those with lung involvement, remains to be assessed. B. Systemic Sclerosis

Systemic sclerosis is a multisystemic disease characterized by Raynaud phenomenon, vascular lesions, and widespread fibrosis of the skin and visceral organs. The accumulation of inflammatory mononuclear cells is frequently detected in early cutaneous and visceral SSc lesions (44), followed by excessive collagen deposition. Rodent fibroblasts have been shown to produce NO after stimulation with cytokines and LPS (45), suggesting that NO produced by fibroblasts may

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play a role in regulating the process of inflammation and wound healing (46). Wang et al. (47) have shown that human dermal fibroblasts also produce NO and express both cNOS and iNOS mRNA. Yamamoto et al. (48) found that serum of patients with SSc contains increased amounts of nitrites compared with controls. By using immunohistochemical techniques, the same authors found positive staining for iNOS in infiltrating mononuclear cells and in fibroblasts of patients with SSc, while fibroblasts in normal skin showed only faint staining. Inducible NOS messenger RNA expression was detected in scleroderma-derived culture fibroblasts, while negative expression was detected on cultured normal fibroblasts. These results suggest that SSc fibroblasts are already activated by exposure to inflammatory cytokines. Indeed, patients with SSc exhibit a complex cytokine profile with stimulated production of various cytokines including IL-1, TNF-α, platelet-derived growth factor (PDGF), and TGF-β. While IL-1 and TNF-α increase NO production in a wide variety of cells (49), both PDGF and TGF-β decrease NO production by inhibiting iNOS gene expression and by suppressing IL-1-induced NO production in macrophages by posttranscriptional mechanism (50). Peripheral blood mononuclear cells from patients with SSc have been shown to produce higher levels of NO spontaneously and in response to IL-1β stimulation (51). In conclusion, the majority of studies show that scleroderma mononuclear cells and fibroblasts may escape the downregulation of NO production, and the resulting excessive NO or its reaction products may be cytotoxic to host cells. Exhaled NO

Pulmonary involvement is common in SSc and pulmonary hypertension is the most frequent cause of death (52). More than 30% of SSc patients may develop pulmonary hypertension (53), either in association with extensive interstitial lung disease or as a consequence of prominent vascular lesions of the pulmonary arterioles. Kharitonov et al. (54) found that peak eNO levels were significantly higher in 23 patients with SSc than in normal controls. They also found that the six patients with pulmonary hypertension had eNO values significantly lower than SSc patients without pulmonary hypertension and normal controls. There was no relationship between eNO and lung function tests, while a negative correlation was observed between PaO2 and peak eNO in patients with SSc without pulmonary hypertension. An increased mean concentration of NO in exhaled air, collected for 30 s during quiet breathing at rest, was observed in 14 nonsmoking patients with SSc by Fajac et al. (55), in comparison with normal controls. No correlation was detected between eNO measurements and either the age of the patients or the disease duration. No significant effects of pulmonary function tests or current


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therapy on eNO measurements were observed. The four patients with pulmonary hypertension (three of them with mild hypertension, PAP ⬍40 mmHg) were not found to have lower eNO values in comparison with the other patients without pulmonary hypertension. In 47 patients with SSc (19 with limited and 28 with diffuse form of disease), higher levels of eNO (plateau value) than in controls were observed (56). Patients with lung involvement (defined on the basis of a CT score for interstitial lung disease ⬎5, associated with a TLC or VC ⬍80% of predicted) had the highest value of eNO, while patients with isolated decreased DLCO and patients without lung involvement had mean eNO not significantly different from controls. In agreement with previous observation by Kharitonov et al. (54), eNO was significantly lower in the 16 patients with pulmonary hypertension than in patients without pulmonary hypertension, and there was a significant inverse correlation between PAP and eNO in all the patients (r ⫽ ⫺0.53, p ⫽ 0.004, Fig. 3). Exhaled NO was not correlated with age, disease duration, form of disease (limited or diffuse), or current therapy. In 17 patients with fibrosing alveolitis associated with SSc (FASSc), Paredi et al. (57) found eNO levels higher than in controls, and the 8 patients who had

Figure 3 Pulmonary artery pressure (PAP) in relation to exhaled NO concentration in patients with systemic sclerosis (n ⫽ 28). (From Ref. 56.)

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active BAL (defined as either lymphocyte ⬎14%, neutrophils ⬎4%, or eosinophils ⬎3%) had significantly higher NO levels than patients who had inactive BAL. There was a significant correlation between eNO and lymphocyte cell count in patients with FASSc (r ⫽ 0.58, p ⬍ 0.05). Origin and Significance of eNO in SSc

All studies concerning eNO measurement in SSc patients report values higher than in controls. The inflammatory process underlying interstitial lung disease in SSc is the most likely explanation for the increased NO production by the respiratory tract. Fibrosing alveolitis is sustained by the production of cytokines, such as IL-1β and TNF-α (58), which upregulate iNOS in a wide variety of cells (macrophages, neutrophils, fibroblasts, epithelial cells), all potentially contributing to the increase of NO concentration in the exhaled air. In idiopathic pulmonary fibrosis, where the interstitial lung disease is very similar to that seen in SSc, an increase in the expression of iNOS was observed in macrophages, neutrophils, and alveolar epithelial cells of patients with early to intermediate stages of fibrosis (59). In agreement with this report, Paredi et al. (57) observed that eNO is related to activity of BAL characterized by the number of lymphocytes, neutrophils, or eosinophils. Excess NO production might have cytotoxic effects, particularly in the presence of superoxide anion, when peroxynitrite, an even more toxic molecule, is generated. Increased production of peroxynitrite has been demonstrated in idiopathic pulmonary fibrosis (59). Moreover, increased production of NO may imbalance T-cell immune response in favor of Th-2 T-cell clones on T-cell receptor activation (60). In patients with SSc, the production of type 2 cytokine mRNA by CD8⫹ T cells has been shown to be related to a significant decline in lung function over time (61). On the other hand, patients with pulmonary hypertension associated with SSc have been shown to have normal or low values of exhaled NO (54,56,62). In SSc, pulmonary hypertension may develop either in association with extensive pulmonary fibrosis or as a consequence of prominent vascular lesions of the pulmonary arterioles with minor parenchymal fibrosis. In patients with advanced pulmonary fibrosis and pulmonary hypertension the vascular endothelium of the pulmonary arterioles showed reduced or absent expression of iNOS (59). This finding could partially explain the low levels of eNO found in patients with SSc and pulmonary hypertension associated with extensive lung fibrosis. Damage of endothelial cells either primary or linked to substances circulating in the serum of patients with SSc (63) may explain the low levels of exhaled NO in patients with pulmonary hypertension not associated with interstitial lung disease. These patients have been shown to have eNO values even lower than patients with pulmonary hypertension associated with parenchymal lung involvement (56). The diminished production of NO could favor the unopposed action of


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Figure 4 Relationship between the increase in exhaled NO after i.v. L-arginine administration and the decrease of pulmonary artery pressure in 6 patients with systemic sclerosis and pulmonary hypertension. (Elaborated from Ref. 65.)

vasoconstrictor substances such as endothelin-1 and could contribute to proliferative changes and obstruction in the pulmonary arteries (64). The capability to increase NO production by the respiratory system after L-arginine administration has been proposed as a test of endothelial integrity, which could anticipate the response of pulmonary artery pressure to vasodilator drugs in patients with SSc (62,65) (Fig. 4). In conclusion, the measurement of exhaled NO may be a useful noninvasive marker of activity in patients with SSc and interstitial lung disease as well as a useful test to identify SSc patients with pulmonary hypertension. C. Sjo¨gren Syndrome

Sjo¨gren syndrome is a chronic, slowly progressive, polysystemic disease, characterized by lymphocyte infiltration of the exocrine glands, which results in diminished or absent glandular secretion and mucosal dryness, particularly of the mouth and eyes (sicca syndrome). SS can occur alone (primary SS) or in association with other rheumatic diseases such as RA, SLE, and SSc (66). NO and Sicca Syndrome

Although it has been proposed that xerostomia is due to the structural destruction of acinar cells in SS salivary glands, there is a poor correlation between the focal salivary gland involvement and the greatly diminished salivary flow in SS patients. This discrepancy suggests that focal periductal inflammation alters the

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vasoneural regulation of the vascular, motor, and secretory components of normal salivary flow. In support of this hypothesis, evidence of a diminished innervation of salivary glands has been provided (67). In addition to adrenergic and cholinergic nerves, the nonadrenergic, noncholinergic system plays a role in normal and abnormal regulation of salivary glands. More recently, NO has been measured in human saliva (68), demonstrating that it is released from salivary glands rather than derived from the activity of oral bacteria. NO may influence parasympathetic vasodilation and salivary flow by regulating peptide release and a second messenger system for both VIP and acetylcholine, as reported in pigs and rats (69). Physiologically, NO is produced in normal salivary glands mainly by constitutively expressed neural and endothelial NOS isoforms, and may contribute to the many events that regulate vascular reactivity, salivary flow, and neuropeptide release. Increased concentration of nitrite levels and output have been reported in SS patients compared with healthy control subjects (70). The increased salivary NO production in SS patients has been found to be mediated by iNOS upregulation, induced by cytokines, such as TNF-α, IL-1, and IFN-γ, which are produced by the salivary gland epithelial cells and/or activated T lymphocytes (71,72). Inflammatory NO production reaches high cytotoxic concentrations for extended time periods. This appears to be a different situation from the physiological salivary NO production, which is coordinated in time and by site and is regulated by the well-functioning normal neurovascular system of normal salivary glands. Another mechanism which may lead to increased salivary NO production in SS patients has been suggested by Perez Leiros and colleagues (73), who presented evidence that circulating antibodies of patients with SS can activate NO signaling in submandibular glands by interacting with muscarinic acetylcholine receptors. In conclusion, salivary NO production is greatly increased in SS patients and may contribute to the diminished salivary flow, characteristic of sicca syndrome, either through cytotoxic mechanisms and increased rate of apoptosis (74,75) or through functional impairment of secretion. Exhaled NO

Pulmonary involvement has been reported both in primary and secondary SS, the most common respiratory symptoms being chronic dry cough and dyspnea (76). Evidence of airway obstruction has been shown in patients with SS by several authors (77–79), and bronchial hyperresponsiveness to methacholine has been observed in 60% of the patients. An increased production of exhaled NO has been reported in patients with SS compared to healthy controls by the Swedish BHR study group (80). Exhaled NO was correlated to age and serum human neutrophil lipocalin, a newly recognized secretory protein of the secondary granules of neutrophil granulocytes (81).


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Exhaled NO was not found to be correlated with bronchial responsiveness, respiratory symptoms, or lung function. The mechanisms underlying the increased exhaled NO in SS patients were not investigated. The elevated NO levels may derive from inflammatory cells in the bronchial mucosa and/or from epithelial cells or macrophages activated by cytokines released from lymphocytes. An increased number of CD4⫹ T lymphocytes has been shown in the bronchial mucosa of patients with SS (82). The possibility of a contribution to exhaled NO from nonenzymatically formed NO derived from nitrites in saliva cannot be excluded in SS patients (83). D. Rheumatoid Arthritis

Even though data on exhaled NO in patients with RA are presently lacking, the importance of NO in the development of RA is increasingly recognized. NO is thought to be a critical mediator of the inflammatory cascade, which leads to synovial proliferation and mononuclear cell infiltration—characteristics of involved joints in RA. Animal Studies

Adjuvant-induced arthritis is a murine model of human RA, whose symptoms are preceded by elevated production of nitrates and nitrites. Ialenti et al. (84) demonstrated elevated nitrite generation by peritoneal macrophages collected from rats with adjuvant arthritis compared with controls; nitrite generation and the severity of arthritis was exacerbated by L-arginine (the source for NO production) and suppressed by L-NAME (an inhibitor of NO synthesis). A significant, more than threefold increase of urinary nitrate excretion was found in rats 20 days after induction of adjuvant arthritis compared with nonarthritic rats (85), and L-NAME decreased NO biosynthesis, paw swelling, and histopathological changes in ankle joints (86). These data suggest that in adjuvant arthritis endogenous NO formation is clearly enhanced and relates to joint inflammation. In streptococcal cell wall-induced arthritis of rats, NO production has been shown to be elevated in the inflamed joints, and administration of an inhibitor of NO synthesis profoundly reduced the synovial inflammation and tissue damage (87). These studies implicate the NO pathway in the pathogenesis of inflammatory arthritis and demonstrate the capability of a NOS inhibitor to modulate the disease. Human Studies NO Metabolites

Farrel et al. (88) first reported an increased concentration of nitrite in synovial fluid and in serum of patients with RA, compared to serum nitrite concentration

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of healthy controls. Nitrite concentration in synovial fluid was higher than in serum samples. This finding is consistent with the presence of NO-generating cells, such as fibroblasts, chondrocytes, macrophages, endothelial cells, and granulocytes within the inflamed synovium. There is no explanation for the high serum levels of nitrites in RA. Widespread synovial inflammation might increase serum nitrite by equilibration with the vascular compartment, but this may not entirely account for the whole serum nitrite concentration. A possible source of increased nitrite is the systemic vasculature, where the induction of NO synthesis may occur, as an effect of circulating cytokines (89). Stichtenoth et al. (90) found the urinary nitrate excretion of 10 patients with RA to be 2.7-fold greater ( p ⬍ 0.001) than that of healthy controls. After 2 weeks of therapy with prednisolone (0.5 mg/kg), when inflammatory activity (as indicated by C-reactive protein, ESR, swelled joint count, early morning stiffness) was significantly reduced, urinary nitrate excretion decreased significantly, by 28%. To avoid the influence of dietary intake of nitrates, Grabowski et al. (91) developed a simple method for assessing NO production, which consists of the nitrate :creatinine ratio in morning samples of urine following an overnight fast. They found that urinary nitrate: creatinine ratio was significantly elevated in patients with RA—average three-fold elevated over controls. By chemiluminescence analysis following nitrite reduction, Ueki et al. (92) found that the serum concentration of NO was significantly higher in patients with RA as compared with patients with osteoarthritis and healthy subjects. Furthermore, the serum concentration of NO was significantly higher in patients with active RA than in patients with inactive disease, and serum NO levels were significantly correlated with the duration of morning stiffness, the number of tender or swollen joints, CRP, serum TNF-α, and IL-6 levels. These results suggest that increased NO synthesis may reflect a pathogenic immune response in patients with RA. NO can form adducts with molecules containing sulfhydryl functional groups to yield S-nitrosothiols (93). Indeed, most plasma NO is present as Snitrosothiols, predominantly S-nitrosoproteins (S-NP). Nitrosylated derivatives are much more stable than NO itself and retain its biological properties. Serum S-NP concentrations have been found to be significantly higher in patients with RA than in normal controls, concentrations being higher in synovial fluid than in serum (94). Fourteen patients received pulse methylprednisolone (MP) treatment (1 g i.v. on three consecutive days) for RA flare, and in these patients S-NP levels decreased significantly the day after pulse MP therapy, in parallel with the clinical improvement. The fall of S-NP concentration after steroid treatment was small but statistically significant, consistent with the observation that NO production in human joint-derived cells, such as synovial fibroblasts and chondrocytes, seems to be relatively insensitive to inhibition by corticosteroids (95). In addition to nitrite and S-NP levels, increased levels of nitrotyrosine, a


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product formed by the reaction of peroxynitrite with tyrosine residues, have also been shown in serum and synovial fluid from arthritis patients (96). NO Synthase Activity

McInnes et al. (97) provided evidence for spontaneous NO production by human synovial tissue from RA patients, which may be further upregulated by bacterial superantigens (staphylococcal enterotoxin B). The predominant cellular source of NO was shown to be the synovial fibroblasts and, to a lesser extent, cells positive for nonspecific esterase and CD68 and thus presumably tissue macrophages. Peripheral mononuclear blood cells (PMBC) from RA patients had increased NOS activity and increased iNOS antigen content compared to those from normal subjects, and responded to interferon-γ with increased NOS expression and nitrite/nitrate production in vitro (98). NOS activity of freshly isolated blood mononuclear cells correlated significantly with disease activity, as assessed by tender and swollen joint counts. These findings demonstrate that patients with RA have systemic activation of iNOS expression, and that the degree of activation correlates with disease activity. Clinical trials of TNF-α-neutralizing antibodies or a soluble recombinant TNF-α-receptor fusion protein (p75) in RA patients demonstrate that TNF-α blockade leads to reduction in disease activity (99,100). Since TNF-α can promote iNOS expression and increased NO production, Perkins et al. (101) investigated whether a chimeric monoclonal antibody against TNF-α might decrease iNOS protein expression and NOS enzyme activity in PBMC from RA patients. They found that the elevated levels of baseline iNOS protein and NOS enzyme activity in PBMC from RA patients were significantly reduced by the anti-TNF-α treatment. Changes in NOS activity following treatment correlated significantly with changes in the number of tender joints. These findings clearly indicate that the anti-inflammatory affects of anti-TNF-α treatment may be mediated by a reduction of NO overproduction. Role of NO in Rheumatoid Arthritis

The evidence that NO plays a proinflammatory role in RA may be summarized as follows. 1. Elevated NO levels in the serum and synovial fluid of patients with RA have been found. 2. Increased production in synovial tissue and overexpression of iNOS antigen and NOS activity of PBMC have been shown in RA patients. 3. The production of NO and the NOS activity has been shown to be related to clinical activity of the disease. 4. Therapeutic interventions (corticosteroids, anti-TNF-α) which de-

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Rolla and Caligaris-Cappio crease clinical activity of the disease also decrease NO production and NOS activity.

NO from synovial fibroblasts has been suggested to enhance pro-inflammatory cytokine production by macrophages, which in turn may upregulate iNOS expression, thereby generating a positive feedback loop (97). Additionally, NO may upregulate matrix metalloprotease production (102) and is implicated in IL-1β-mediated inhibition of proteoglycan synthesis (103). Recently, an IL-17dependent production of NO has been reported in synovial tissue (104), where IL-17 is produced by activated T cells (105). On the other hand, NO levels measured in synovial culture are sufficient to suppress T-cell proliferation and contribute to synovial T-lymphocyte hyporesponsiveness (106). In addition, physiological amounts of NO produced by endothelial cells have been shown to reduce adhesion and emigration of granulocytes (107), and a chondroprotective role for endogenous NO in bovine cartilage has recently been proposed (108). Notwithstanding all this information, the net effect of NO production in human arthritis remains unclear. V.

Conclusions

NO overproduction seems to occur frequently in rheumatic diseases, either within target organs, as in the inflamed synovia of RA, or at the systemic level, as in SLE, SSc, and probably RA itself. The role NO may play in rheumatic diseases might include many key aspects: tissue damage, lymphocyte function, and apoptosis. Exhaled NO may reflect both the systemic involvement of disease, as in SLE, where its concentration has been shown to be related to disease activity, and the pulmonary involvement, as in SS and SSc. In this latter disease the inverse correlation between exhaled NO and pulmonary artery pressure might have pathogenetic as well as therapeutic implications. The measurement of exhaled NO has proved to be an interesting tool to investigate inflammation in autoimmune diseases, even though its clinical significance remains to be defined. Further investigations will establish whether exhaled NO measurement is merely one of the many available nonspecific markers of inflammation or whether it may help in the follow-up of patients, particularly those with lung involvement. References 1. Fang FC. Perspective series: first host/pathogen interactions. Mechanisms of nitric oxide related antimicrobial activity. J Clin Invest 1997; 99:2818–2825. 2. Stamler JS. Nitric oxide in the cardiovascular system. Overview. Coronary Artery Dis 1999; 10:273–276.


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20 The Disturbance of Metabolism of Oxides of Nitrogen in Liver Disease Exhaled Nitric Oxide as a Measure of Severity

ERIC DEMONCHEAUX and TIM W. HIGENBOTTAM

DERMOT GLEESON

University of Sheffield Medical School Sheffield, England

Sheffield Teaching Hospital Sheffield, England

I.

Introduction

The discovery of nitric oxide (NO) and the L-arginine–citrulline pathway has led to important advances in biological sciences. It sparked an explosion of interest in the biochemistry, pharmacology, physiology, and metabolism of gaseous NO. Research has shown impairment of NO production in a variety of diseases such as disorders of the systemic and pulmonary circulations, cystic fibrosis, diabetes, and stroke, while in asthma and septic shock NO production is increased (1–5). A hyperdynamic circulation is often seen in liver diseases and is an important cause of morbidity and death (6,7). This hyperdynamic circulation has been attributed to an excess endogenous production of NO (8). In this chapter we give a brief overview of the mammalian nitrogen balance, with particular emphasis on the turnover of endogenous and exogenous oxides of nitrogen. We then present some evidence of impairment of this turnover in liver diseases and its relationship with exhaled nitric oxide.

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Demoncheaux et al. II. Nitrogen: A Multifaceted Atom of Great Biological Importance

The chemistry of nitrogen within the body is extremely varied. Nitrogen is unique among the elements in forming seven molecular oxides, all of which are thermodynamically unstable with respect to decomposition to nitrogen (N 2 ) and oxygen (O 2 ). Nitrogen atoms in nitrogen oxides and oxoacids have oxidation numbers ranging from ⫺3 and ⫹5. If no reductants are present or if no catalysis takes place, such as by trace metals or enzymes, most of the nitrogen oxides are irreversibly oxidized to nitrate (NO 3⫺), the most stable aqueous oxide of nitrogen under the conditions that prevail on Earth. In the environment, through enzymatic catalysis, N 2 is reduced to ammonium (NH 4⫹), NH 4⫹ is oxidized to NO 3⫺, and NO 3⫺ is reduced to N 2 , forming a biological cycle which provides an explanation for the steady state in atmospheric N 2 obtained. Nitrogen is an important compound in the biochemistry of all species. A complex nitrogen turnover exists in mammals.

III. Whole-Body Turnover of Nitrogen and Related Oxides A. Mammalian Nitrogen Cycle

The supply of nitrogen to mammals is ultimately limited by the need for soil bacteria and plants to convert N 2 to a form that can be assimilated by humans and animals. Humans can only synthesize half of the 20 amino acids essential for life and therefore depend on food containing amino acids and proteins together with “fixed” forms of nitrogen to provide the necessary elements to synthesize the essential amino acids. Normal healthy adults are generally in nitrogen balance, with intake and excretion being well matched. Amino acids are stored but not excreted as such. Amino groups are removed by transamination or oxidative deamination. The ammonium ion which results from these reactions is removed from the circulation mainly through the liver, where it enters the Krebs cycle and is converted into urea, which is excreted in the urine by the kidneys. The concentration of urea in the plasma of blood is thus kept to a minimum (9). Healthy humans consume about 300 g of carbohydrate, 100 g of fat, and 100 g of protein and excrete about 16.5 g of nitrogen daily. Of the excreted nitrogen, 95% is excreted by the kidneys and 5% in the feces. A considerable quantity of the body’s NH 4⫹ is not from the diet directly but is formed by intestinal bacteria from dietary protein and from urea present in fluids secreted in the gastrointestinal tract (10). This NH 4⫹ is absorbed from the intestine into the portal venous blood, which is known to contain higher levels

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of ammonia than systemic blood (9). Ammonium is also formed by the kidney tubules, mainly from glutamine, where it acts as hydrogen-ion acceptor. Circulating NH 4⫹ can be incorporated in proteins by amination of non-nitrogenous residues (11). Ammonia exists in two forms in solution, NH 4⫹ and free ammonia (NH 3 ). It has been estimated that at physiological pH, 1–3% of the total ammonia is present in the free form. Interestingly, this ammonia can be measured in the expired air (10). The lungs therefore contribute to excretion of NH 3 . Hyperammonemia is commonly found in liver cirrhosis (12), which is associated with increased exhaled breath levels of NH 3 (13). Although the nitrogen mammalian balance is now well described, a paradox has puzzled investigators for some time. As early as 1916 there were observations on the production of NO 3⫺ by tissue (14). This was originally believed to be a result of intestinal microbial metabolism, although this was to prove only partially true. B. Endogenous Source of Nitric Oxide

Nitric oxide is synthesized from molecular oxygen and the guanidino amino group from L-arginine forming citrulline, on a 1:1 molar ratio, by a family of enzymes called nitric oxide synthases (NOS) (EC 1.14.13.39). There are three distinct isoforms of NOS, the product of single-copy genes represented on separate chromosomes (15). One isoform, inducible or iNOS (NOS II), is independent of Ca 2⫹ for activity and can be induced by a number of cytokines. It can be expressed in most cell types. The isoform first described in endothelial cells, eNOS (NOS III), is Ca 2⫹ dependent and is expressed in smooth muscle cells and nerves as well as endothelial cells. Finally, neural or nNOS (NOS I) is also Ca 2⫹ dependent and was first described in neurons but has now been described in epithelial and endothelial cells. NOS I and III require intracellular Ca 2⫹ to form the Ca 2⫹ /calmodulin complex and subsequent enzyme activation. Co-factors are flavin adenosine dinucleotide (FAD), flavin adenosine mononucleotide (FMN), and tetrahydrobiopterin (TH 4 ) (16). While there are to date no reports implicating FMN, nicotinamide adenine dinucleotide phosphate (NADPH), and FAD as important control mechanisms for NOS, TH 4 has been reported to mediate NOS activity (17). Clinically, administration of NOS inhibitors produces increases in mean arterial blood pressure and decreases in regional blood flow in humans, indicating that NO is basally produced and contributes to vascular tone (18). Basal release of NO from endothelial cells may be of the order of 1 nM (19), while it can be as high as 1–10 µM when stimulated (2,15). These values are to be taken with caution, as it is difficult to determine NO concentrations accurately in physiological settings. This will be discussed later in this chapter.

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Demoncheaux et al. C. Exogenous Nitrogen Oxides Contribute Significantly to Whole-Body Nitrogen Oxides Turnover

Endogenously formed nitrogen oxides are diluted in a large pool of circulating oxides of nitrogen which originate from sources outside the body. While it has been estimated that approximately 40 mg/day of endogenous NO are formed in the average person (20), total dietary intake of NO 2⫺ and NO 3⫺ range from 4 to 5 and from 50 to 70 mg/day per person, respectively (21). Inhaled oxides of nitrogen may also be of some significance in altering their circulating levels (22). Cigarette smoke may also contribute in the smoking population, as cigarettes contain large amounts of NO and may also alter circulating levels of oxides of nitrogen (23,24). All these sources of oxides of nitrogen are interfering factors in the measurement of whole-body NO turnover and have limited the clinical utility of circulating oxides of nitrogen and exhaled levels of NO. These measurements should be really determined after a low-NO 2⫺ and -NO 3⫺ diet for at least 2 days before the experiments (25). There is an extensive literature available on environmental exposure to atmospheric NO x (NO and nitrogen dioxide, NO 2 ) as well as NO 2⫺ and NO 3⫺, due to their potential harmful effects. Until recently, NO 3⫺ was perceived as a purely harmful dietary component, which causes infantile methemoglobinemia, carcinogenesis, and possibly even teratogenesis. This is thought to be due to conversion of NO 3⫺ by facultative bacteria to NO 2⫺, which, when entering the systemic circulation, can convert oxyhemoglobin to methemoglobin. Epidemiological studies, however, have failed to substantiate this (26–28). D. Metabolic Fate of Nitric Oxide

Nitric oxide half-life in blood is as short as 5 ⫻ 10⫺4 sec (29), and it is currently thought that hemoglobin is NO’s main physiological sink (25,30) (Fig. 1). Exogenous uptake, endogenous synthesis, and metabolic losses are part of mammals’ nitrogen oxides metabolism. Approximately 60% of an ingested dose of 15 NO 3⫺ is recovered as 15 NO 3⫺ in urine. About 2–4% is excreted as 15 NH4⫹ and urea (31). When inhaled, about 90–97% of NO is retained in the lungs, of which approximately 75% is excreted as NO 3⫺ (32). Whole-body metabolism of L-arginine has been well studied over the years (33). On a normal diet, a human will be exposed to 50–80 mg/kg per day of arginine (34). Of this, 1.8–2.1% is converted to NO 3⫺ in urine when given intragastrically; this falls to approximately 0.6% when given intravenously (34). Sixteen percent of dietary arginine is converted to NO 3⫺ (34,35). L-Arginine therefore also contributes to circulating oxides of nitrogen. Very little is yet known on the exact metabolic fate of NO and related


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Figure 1 Schematic of whole-body nitric oxide turnover. Endogenously formed oxides of nitrogen are diluted in a large pool of circulating oxides of nitrogen originating from both exogenous sources and gut bacterial metabolism. Their ultimate fate is excretion through the kidneys, mostly as nitrate but also as urea and ammonia. The lung, with its extensive surface area for gas exchange, is considered an excretory organ for both ammonia and nitric oxide and most probably for nitrite and nitrate.

species. Early work by Yoshida et al. (36) demonstrated that when given intraperitoneally in rats, a single dose of NO 2⫺ is converted to NO 3⫺ (approx. 50%), urea (approx. 30%), and the rest to unidentified compounds. For NO 3⫺, only 5% is recovered as urea. This indicates that NO 2⫺ and NO 3⫺ have different metabolisms. Current wisdom assumes that NO cannot be transported distal to its source of synthesis. Nitric oxide is quickly removed from the blood, being rapidly oxidized to NO 2⫺ and NO 3⫺. Both NO 2⫺ and NO 3⫺ have been thought to have only

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minor biological significance. The different metabolic pathways for these two anions and the discovery of new metabolic products in biological fluids has led to this view being challenged. E.

Naturally Occurring NO Adducts Are Present in Biological Fluids

Stamler et al. (37) proposed that the free sulfydryl group in albumin could be nitrosated and that S-nitrosoalbumin does occur in human plasma in relatively high concentrations (38). This raised the notion that there is/are physiological NO carrier(s) capable of transporting NO from sites of endogenous production to distant regions of the body. The ability of S-nitrosothiols (RSNO) to transnitrosate other free thiols has been proposed as the main transport mechanism for NO in physiological conditions, possibly regulating affinity sites in enzymes or proteins (39). Recently, a free sulfydryl group on hemoglobin has been proposed as a systemic carrier for NO. A change in protein configuration, possible through deoxygenation, would release NO to various sites in the body (40,41). Other nitrosylated species, such as dinitrosyl–iron complexes, can also be found in biological fluids (42). Nitrite anions are important circulating NO donors and are found in biological fluids. Dietary supplement of NO 2⫺ anions is a systemic vasodilator (43). Nitrite anions release NO, through a process called disproportionation which is dependent on pH (44). This has been confirmed in vitro (45,46). Tissue NO 2⫺ is an important source of NO in myocardial ischemia as the tissue pH falls (47). We have confirmed that solutions of nitrite contain nitric oxide (48). In healthy humans, concentration of free NO in plasma is thought to be 1–5 nM (37), RSNO 0.5–1 µM (37), NO 2⫺ and NO 3⫺ 0.5–15 and 1–100 µM (49). The impact of bacterial metabolism on circulating levels of NOrelated species and on whole-body nitrogen oxides turnover is unknown.

IV. Intraluminal Bowel Bacterial Turnover of Nitrogen Oxides In the gastrointestinal tract, the bacterial flora contribute to oxides of nitrogen metabolism, oxidizing organic nitrogen or NH 4⫹ to NO 3⫺ and NO 2⫺ or reducing NO 2⫺ and NO 3⫺ to gaseous products (50). They are then absorbed through the intestinal wall and enter an entero-hepato cycle (51). Nitrate can be cycled back to the stomach when secreted by the salivary glands. There it can undergo further reduction to NO 2⫺ and NO by anaerobic bacteria colonizing the oral cavities (26). The low pH in the stomach favors formation of nitrous acid (HNO 2 ) and NO from NO 2⫺, which can react with amines to form carcinogenic nitrosamines (52,53). Fecal elimination of oxides of nitrogen is negligible (14).

Metabolism of Oxides of Nitrogen in Liver Disease V.

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Mechanisms Behind Disturbed Turnover of Oxides of Nitrogen in Liver Cirrhosis

A. The Hyperdynamic Circulation

A hyperdynamic circulation is often seen in liver disease and is an important cause of morbidity and death (6). It is characterized by a high cardiac output, low systemic vascular resistance, and low pulmonary vascular resistance (PVR) is present in 30–50% of patients with cirrhosis and animals model of portal hypertension (54,55). Its clinical manifestations are portal hypertension, fluid retention, renal dysfunction, spider naevi, and hepatic encephalopathy. Increased endogenous production of NO has been proposed as an important determinant of the hyperdynamic circulation of the cirrhotic patient (8), and there are many observations linking excess NO production to the hyperdynamic circulation (7,56). The conventional view is that endotoxin and cytokines, as for sepsis, are responsible for the hyperdynamic circulation (8). However, experimentally there is no evidence of expression of mRNA for NOS II in systemic vessels, either in biliary cirrhosis (57) or portal hypertension (58). By contrast, in lypopolysaccharide (LPS)-treated animals and forms of cirrhosis associated with peritonitis, there is overexpression of NOS II (59,60). Endogenous production of NO is not confined to NOS II but can result from NOS I and III. Some have argued that NOS III is able to contribute to the excess NO levels through increased activity (61). However, the evidence from experimental and human cirrhosis is conflicting as to the role of NOS III (55). B. Impaired Turnover of Nitrogen Oxides

Circulating oxides of nitrogen are higher in patients with cirrhosis, especially those with ascites or renal failure, than in controls (62). These differences persisted after correction for differences in renal function between cirrhotic and control subjects (63). Neither Campillo et al. (62) nor Garner et al. (63) measured plasma NO 2⫺ and NO 3⫺ separately, and to date none of the other naturally occurring NO adducts has been measured in liver cirrhosis. Barak et al. (64) did attempt to do so, but the precision of their methodology did not allow for distinction between plasma NO 2⫺ levels within their study subjects. We and others have found that the dosage of plasma NO 2⫺ anions still remains a challenging task (65–67). C. Role of the Stomach

There is evidence of a shift in gastric juice pH from acidic to alkaline in some patients with liver cirrhosis (68). An increase in gastric pH may allow harmful

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bacteria to colonize the small intestine and could be partly responsible for bacterial overgrowth often seen in cirrhotic patients (68–70). Bacterial counts from the stomach and small intestine are very low, in contrast with the large intestine, where an enormous population of symbiotic bacteria flourish. Stasis of the small intestine also contributes, in cirrhosis, to bacterial overgrowth. Patients with advanced liver cirrhosis are malnourished and receive antibiotic therapy to control superimposed infections (71,72). D. Role of the Gut

The nonabsorbable antibiotic collistin reduces circulating oxides of nitrogen levels in patients with decompensated cirrhosis (63). This indicates that bacterial overgrowth is an important determinant in whole-body nitrogen oxides turnover. Bacterial sources of NO 2⫺ and NO 3⫺ may be of importance when considering that a normal healthy human being is made of 10 13 cells and is host to 10 14 bacterial cells, a large proportion of which are anaerobic (73,74). Many attempts have been made to identify the composition of human microflora organisms, which remain elusive as to exact composition. E.

Role of the Liver

The liver plays a pivotal role in whole-body NO turnover. In addition to removing circulating NH 4⫹ via the Krebs cycle, liver cytochromes and catalase (EC 1.11.1.6) are known to interact directly with NO and NO 2⫺ (75,76). It is therefore tempting to speculate that one of the liver functions is to detoxify the blood of NO 2⫺ by converting it to the less reactive NO 3⫺. Functional intrahepatic shunting is a common feature of cirrhosis. As a result, portal blood bypasses the liver and enters the systemic circulation directly. This situation may also be aggravated by a decrease in hepatocellular function, where blood flows through the liver without been metabolized. Thus, shunts are common in cirrhosis and may be important in the pathogenesis of the hyperdynamic circulation. Interestingly, increase in portosystemic shunting, such as when induced therapeutically as treatment of portal hypertension by transjugular intrahepatic portosystemic shunts (TIPS), worsens the hyperdynamic circulation seen in some patients (77) and may be associated with an increase in circulating levels of oxides of nitrogen (78). The importance of the porto-systemic shunt is further supported by the observed fall in plasma oxides of nitrogen when the shunt is closed (79). F. Pulmonary Vascular Disorders

The liver exerts a critical influence on regulation of pulmonary vascular tone and angiogenesis. Hepatoportal systemic shunts allow substances cleared by the liver


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Figure 2 Pulmonary vascular complications may be due to development of porto-systemic shunt that allows substances normally cleared by the liver to reach the lungs.

to gain access to the pulmonary circulation. There are pulmonary vascular complications of chronic liver failure and portal hypertension, which range from hepatopulmonary syndrome (HPS), characterized by intrapulmonary vascular dilatation, to pulmonary hypertension (55) (Fig. 2). Their pathophysiological mechanisms are unknown. VI. Exhaled Nitric Oxide and Liver Diseases A. The Lungs as Secretory and Excretory Organs for Nitrogen Oxides

The finding that inhaled nitric oxide (iNO) acts as a selective pulmonary vasodilator (80) led to the idea that a ventilatory exchange of NO could occur within the lung, and that endogenously produced NO could leave the body via the exhaled breath. Exhaled NO (eNO) can be measured in animals (81) and humans (82). The lungs, like other organs, have a complex network of NO-producing cells (3). They differ in having an aqueous layer covering the lung epithelial surface, which has an enormous surface area in contact with air (83). As a gas, NO will partition across the gas–liquid interface. Unlike other organs, as a result of measurements of NO in the exhaled air, the output of NO from various NOScontaining cells of the lungs can be assessed directly. We have shown previously that eNO in the expired air of isolated lungs is related to pulmonary vascular resistance (84). We have also shown that stimulated release of NO is reduced in human lungs with end-stage chronic obstructive lung disease (COLD) and severe pulmonary hypertension (SPH) (5). Finally, we have shown that eNO was not reduced in primary hypertension (85), although sources other than the pulmonary vasculature may have affected the results. B. Hyperdynamic Circulation and Exhaled Nitric Oxide

Nitric oxide contributes to the normal low pulmonary vascular tone (86). Exhaled NO levels are normal in compensated cirrhosis but elevated in severe cirrhosis

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(87). They are also elevated in the hepatopulmonary syndrome, where advanced liver disease is associated with a hyperdynamic circulation (88). In one of our patients, eNO levels went back to normal after liver transplantation (88). In an animal model of liver cirrhosis, chronic inhibition of lung NO production prevents the development of hepatopulmonary syndrome and associated hemodynamic alterations (89). Thus, increase in lung endogenous NO production may be responsible for some of the increase in eNO levels. C. Possible Source of Exhaled Nitric Oxide

While an increase in NOS activity may be responsible for some of the elevated eNO in liver cirrhosis (89), other sources may also be sought. Although we have shown that a naturally occuring NO donor, NO 2⫺, affects eNO levels and decreases pulmonary vascular resistance (PVR) in isolated porcine lungs (90), the physiological importance of this phenomenon is still unclear (91). In diseases where plasma oxides of nitrogen are elevated, eNO may be decreased (92). Until more reliable tests are available to determine plasma NO 2⫺, NO 3⫺, and RSNOs separately with sufficient precision, the impact of these circulating factors on the amount of NO excreted by the lungs will be difficult to gauge. Increase in circulating oxides of nitrogen may also yield to an increase in eNO from salivary NO formation by bacterial reduction of NO 3⫺ in the mouth (93). The contribution of the epithelial lining fluid itself to eNO is also unknown. Nitrite, nitrate, and nitrosothiols can be detected in bronchoalveolar lavage fluids in humans (94). It may be possible that patients with cirrhosis have an epithelial lining fluid pH imbalance leading to an increase conversion of NO 3⫺, NO 2⫺, or RSNO to NO. In asthmatics, inflammation is associated with marked increase in eNO, most probably due to increased expression of NOS II (95,96). However, it is possible that increase in oxidant stress or a decrease in epithelial lining fluid pH may further enhance eNO concentration (97,98). Finally, the observation that intravenous infusion of NO donors induces changes in eNO concentrations (99–101) further underlines the need to study in more detail the processes involved in the regulation of NO gas-exchange properties in the lungs. VII. Challenges Left for the Future Due to its involvement in variety of diseases, whole-body NO turnover has become important to assess clinically. Measurements of plasma and urine NO 2⫺ and NO 3⫺ have some merit but suffer from a lack of specificity due to the variety of confounding factors associated with their measurements. Exhaled nitric oxide values are promising, and attempts have been made to standardize the existing methods (102). Perhaps the use of laser magnetic resonance (LMR) spectroscopy


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(103), which can differentiate between isotopes of nitrogen, will offer complementary information on the exact origin of exhaled nitric oxide. Very little is yet known about NO’s exact metabolic route, but new tools are now becoming available to carry out studies. The measurement of wholebody NO turnover is made specific by the use of safe and stable isotopes of arginine ([ 15 N] 2-arginine). Transfer of the 15 N-guanido label of arginine to urinary 15 N-nitrate allows a more specific measure of whole-body NO turnover (104). Patients with essential hypertension, where impairment of endogenous NO production has been implicated, have a lower conversion of [ 15 N] 2-arginine to urinary 15 N-nitrate than control subjects (105). The reverse is seen in patients with infective gastroenteritis (106). It is important to note that less than 1% of a single dose of arginine is recovered in the urine as NO 3⫺ in humans. This poses problems in interpreting the exact physiological meaning of this measurement, which perhaps should be done in conjunction with other tests. A recent report using conversion of [ 15 N] 2-arginine to 15 N-citrulline as an in vivo marker of whole body NO production also looks promising (107). Measurement of reactive nitrogen oxide species in biological fluids is still a challenge for the analyst. Improvement of existing technology and the use of labeled compounds will certainly help unravel the complexity and interactions between nitrogen oxides in biological conditions. These steps will be necessary before tests of clinical rigor can be put in place in clinical practices. More effort is needed to understand the exact fate of NO in biological fluids, and recent technological breakthroughs are encouraging. The value of exhaled nitric oxide measurements as a measure of severity of liver diseases is still controversial. Combining the measurements of exhaled and circulating levels of nitrogen oxides and related species with molecular tools to probe for the involvement of nitric oxide synthase in animal models of liver diseases may offer some help in understanding their complex relationships and physiological roles. References 1. Dinh-Xuan AT, Higenbottam TW, Clelland CA, Pepke-Zaba J, Cremona G, Butt AY, Large SR, Wells FC, Wallwork J. Impairment of endothelium-dependent pulmonary-artery relaxation in chronic obstructive lung-disease. N Engl J Med 1991; 324:1539–1547. 2. Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 1993; 329:2002–2012. 3. Higenbottam TW. Nitric oxide and the lung. In: Lightman S, ed. Horizons in Medicine. Vol. 7. London: Blackwell Science, 1996:203–224. 4. Marriott H, Higenbottam TW. The role of nitric oxide in respiratory disease. Schweiz Med Wochenschr 1997; 127:709–714.

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AUTHOR INDEX

Italic numbers give the page on which the complete reference is listed.

A Aarden LA, 145, 155 Aarts LP, 321, 338 Abcarian H, 369, 378 Abdul-Hussain MN, 33, 67 Abe S, 322, 338 Abeles RH, 371, 379 Abelli L, 48, 64 Aben KK, 321, 338 Abman SH, 42, 60, 240, 242, 245, 249, 254, 315, 334 Abraham NG, 118, 128 Abrams J, 432, 441 Abramson D, 314, 332 Abramson S, 427, 439 Abramson SB, 314, 332, 424, 426, 427, 436, 438, 439, 443 Absood A, 165, 187 Abu-Soud HM, 97, 109, 112, 162, 163, 184, 235, 236, 237, 238, 239, 240, 242, 243, 244, 245, 262, 277 Accavitti MA, 169, 190 Acevedo CH, 126, 131 Adachi H, 34, 68 Adak S, 238, 244 Adams J, 345, 361

Adams LB, 162, 184 Adatia I, 271, 278, 384, 398 Adcock IM, 163, 185, 321, 338 Adding C, 39, 58, 102, 112 Adding LC, 32, 33, 37, 38, 39, 44, 47, 56, 58, 59, 63 Aderka D, 427, 439 Adisesh A, 309, 329 Adisesh LA, 309, 329 Adler KB, 51, 66, 160, 164, 171, 177, 183, 192 Adrie C, 36, 56, 106, 113 Agellon LB, 409, 417 Agertoft L, 313, 332 Agosti JM, 313, 332 Agrenius V, 51, 66, 97, 112, 308, 328 Agusti AG, 206, 217 Agvald P, 32, 33, 37, 38, 39, 44, 47, 56, 58, 63, 68, 102, 106, 112, 113, 268, 278 Ahmed A, 126, 131 Ajuebor MN, 410, 418 Akahoshi M, 427, 439 Akaike T, 165, 187 Akley NJ, 160, 164, 177, 183 al Assaad AS, 78, 88 Al-Mehdi A, 176, 194

463

464 Al-Mehdi AB, 165, 187 Al-Nakkash L, 409, 417 Alam J, 118, 120, 121, 122, 123, 128, 129, 130, 138, 142, 144, 152, 154, 155, 204, 214 Alam JA, 126, 131 Albert J, 35, 40, 49, 60 Albert JD, 345, 360 Alberts MK, 237, 238, 244 Albina JE, 423, 433, 437, 442 Aldape K, 76, 86 Alderdice CA, 428, 440 Alexander A, 12, 25, 424, 438 Alexander G, 33, 68 Alexander RW, 7, 21 Ali IS, 260, 275 Allain P, 227, 230, 371, 379 Allaire S, 222, 230 Allegra L, 203, 206, 213, 309, 329 Allemann Y, 38, 57 Allen C, 317, 335 Allen CM Jr, 371, 379 Allen JB, 433, 442 Allen JS, 167, 188 Allmers H, 202, 216 Alm P, 6, 20, 135, 150 Alms WJ, 430, 440 Aluwalia M, 144, 155 Alvarez A, 424, 437 Alving K, 32, 34, 35, 36, 37, 39, 47, 48, 51, 52, 55, 56, 58, 62, 63, 66, 69, 70, 83, 84, 89, 92, 112, 205, 208, 214, 215, 236, 244, 263, 277, 308, 311, 315, 328, 331, 334, 385, 399, 404, 414, 433, 442 Amano H, 427, 439 Amber IJ, 16, 26 American Lung Program, 363, 376 Amersi F, 118, 128 Ames B, 284, 304 Ames BN, 119, 125, 128, 128, 139, 140, 148, 152, 156 Amin A, 424, 426, 427, 438 Amin AR, 314, 332, 427, 436, 439, 443 Amirkhanian JD, 170, 191 Anand A, 76, 87

Author Index Anden T, 34, 68 Anderson BO, 354, 362 Anderson GM, 411, 418 Anderson PG, 169, 190 Anderson RGW, 162, 184 Anderson TJ, 40, 49, 60 Andersson G, 37, 56, 83, 89 Andersson JA, 203, 206, 213, 319, 336 Andersson M, 425, 438 Ando D, 432, 441 Andon NA, 43, 61 Andonegui G, 171, 192 Andreoni KA, 283, 284, 286, 290, 295, 298, 299, 300, 301, 303, 304, 368, 369, 377 Andrew PJ, 410, 413, 418 Andrews AW, 167, 188 Andrianopoulos G, 369, 378 Anggard A, 35, 37, 47, 55, 62, 236, 244 Anggard E, 29, 53 Anglo DD, 7, 21 Anholm JD, 207, 215, 252, 255 Anno PR, 12, 25 Anrather J, 123, 124, 130, 142, 143, 153, 154 Ansarin K, 47, 49, 63, 202, 212, 309, 328 Antczak A, 321, 337, 338 Antoni C, 145, 155, 435, 443 Antuni JD, 135, 151, 206, 217, 218, 251, 255, 318, 319, 336 Anvari M, 317, 335 Aoki K, 126, 131 Aoyagi K, 36, 55 Apple F, 125, 131, 139, 153 Arai S, 354, 362 Arana R, 432, 441 Aratani Y, 160, 164, 177, 183 Archer S, 29, 30, 53, 242, 246 Archer SL, 37, 42, 43, 49, 57, 61, 65 Arenas J, 425, 438 Arias F, 325, 340 Arijamaa O, 43, 62 Arinobu Y, 427, 439 Armaganidis A, 375, 380

Author Index

465

Armstead WM, 47, 63 Arnelle D, 168, 189 Arnold JH, 384, 398 Arnold WP, 12, 24, 29, 53 Arroliga AC, 235, 244, 249, 254, 409, 413, 418 Arruda MA, 164, 186, 411, 419 Artigas A, 363, 376 Artlich A, 33, 38, 39, 47, 52, 58, 63, 66, 102, 112, 311, 331 Asada A, 39, 59, 202, 216 Asano K, 35, 48, 55, 76, 78, 87, 164, 186, 262, 277, 424, 437 Ashbaugh DG, 345, 346, 361, 363, 376 Ashcroft T, 383, 394, 398 Ashihara T, 164, 187 Ashmore M, 206, 217 Ashton DS, 11, 24 Asimakopoulos G, 260, 275 Aslander M, 426, 438 Asmussen K, 431, 441 Assreuy J, 172, 193 Astudillo R, 52, 66 Atamas SP, 430, 440 Atamian S, 345, 361 Attur MG, 314, 332, 436, 443 Aubrey M, 428, 440 Audano G, 431, 441 Aust AE, 161, 183 Aust SD, 160, 164, 177, 183 Autunes E, 51, 66 Awolesi MA, 41, 47, 60 Axxolin NM, 404, 414 Aymerich MS, 163, 184 Ayres JG, 34, 70, 325, 340 Azzolin N, 309, 329 Azzolin NM, 31, 54, 92, 111, 200, 211, 311, 312, 313, 331

B Baader SL, 6, 20 Babior BM, 162, 184 Babu BR, 11, 24 Bach FH, 123, 124, 130, 142, 143, 153, 154

Baer JW, 352, 362 Baer PA, 428, 440 Bagetta G, 6, 20 Bai XH, 265, 278 Baile EM, 48, 64 Bailey A, 207, 215 Baird BR, 261, 276 Bake B, 34, 70, 203, 212 Baker C, 180, 195 Baker JE, 242, 245 Baker JR, 169, 191, 372, 380 Baker RR, 176, 194, 260, 276 Balatti I, 424, 437 Baldwin SR, 321, 338 Balfour-Lynn I, 318, 336 Balfour-Lynn IM, 404, 414 Balint B, 326, 341 Balla G, 118, 125, 127, 131, 139, 153 Balla J, 118, 125, 127, 131, 139, 140, 142, 153, 154 Balligand JL, 164, 187 Bambery P, 425, 438 Bando K, 261, 276 Banfi E, 160, 164, 177, 183 Bankhurst AD, 435, 443 Banks S, 345, 361 Banks SM, 35, 71, 314, 333 Bannenberg GL, 32, 33, 39, 41, 43, 44, 46, 59, 60, 62 Bao C, 406, 416 Baraldi E, 31, 54, 92, 111, 200, 211, 309, 329, 404, 414 Barber CM, 7, 21 Barber SA, 406, 416 Barbera JA, 34, 35, 70, 72, 313, 332 Barbinova L, 202, 216 Barbul A, 428, 439 Bardini D, 248, 253, 429, 430, 440 Baretta ED, 207, 215 Barja-Fidalgo C, 164, 186, 411, 419 Barlow J, 76, 87, 164, 186, 314, 333 Barnard GR, 176, 194 Barnard JW, 39, 49, 59 Barnes NC, 321, 338 Barnes PJ, 17, 18, 26, 27, 30, 33, 34, 35, 37, 39, 47, 48, 49, 49, 50, 51, 51,

466 [Barnes PJ] 54, 55, 56, 59, 63, 64, 65, 66, 69, 70, 71, 72, 76, 81, 83, 84, 87, 88, 89, 92, 111, 112, 124, 130, 135, 151, 160, 163, 164, 177, 183, 185, 186, 200, 202, 203, 205, 206, 207, 208, 209, 211, 212, 213, 214, 215, 216, 217, 218, 248, 249, 251, 252, 253, 254, 255, 262, 267, 277, 278, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 325, 326, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 344, 346, 359, 360, 369, 375, 378, 380, 385, 388, 393, 399, 400, 404, 406, 413, 414, 415, 428, 429, 430, 440 Barnes S, 167, 171, 189 Baron M, 428, 440 Baronti R, 135, 151 Barrazone C, 123, 129 Barre J, 205, 214, 251, 255 Barros RC, 51, 65 Bartens C, 374, 380 Bartley J, 309, 329 Bartley J, 200, 212 Barton CH, 316, 335 Bartsch P, 49, 65, 107, 113, 267, 278 Basclain KA, 76, 77, 87 Basford RE, 172, 193 Basili S, 324, 339 Basiouny KF, 169, 177, 178, 180, 190 Bassenge E, 7, 21 Basset G, 364, 377 Bast A, 321, 338 Basu-Modak S, 118, 127 Bates JN, 16, 26 Battistini E, 309, 329 Baud L, 163, 185, 327, 341 Bauer D, 385, 392, 399 Bauer H, 423, 437 Bauer JA, 38, 58, 107, 113 Bauer M, 140, 153 Baumgartner WA, 261, 276 Baur X, 202, 216 Bavaria JE, 260, 275

Author Index Baxter LM, 203, 206, 213 Beale SI, 118, 127 Beasjours S, 6, 20 Beattie C, 283, 288, 290, 294, 295, 296, 298, 301, 303, 304, 368, 369, 377 Beaudet AL, 403, 404, 412, 413 Beaudoin J, 282, 303 Becher G, 209, 216, 322, 338 Becherel P, 163, 185 Beck E, 322, 338 Beck J, 404, 414 Becker LC, 47, 63, 300, 304 Beckman JS, 15, 26, 160, 161, 164, 165, 167, 169, 172, 176, 177, 183, 187, 188, 189, 190, 191, 193, 263, 277, 278, 359, 362, 372, 380 Beckman TW, 15, 26, 161, 183, 263, 278 Beehler CJ, 352, 362 Beghetti M, 271, 278 Beilin LJ, 322, 339 Bein M, 428, 440 Bekker JM, 37, 56, 250, 254 Bel EH, 310, 312, 330, 404, 414 Belardinelli R, 34, 68 Belghit C, 375, 380 Bell WR, 283, 295, 303 Bellan JA, 49, 65 Bellet S, 147, 155 Bellocq A, 163, 185, 327, 341 Bellofiore S, 33, 48, 51, 64 Belloni P, 434, 442 Belmont HM, 424, 426, 427, 438, 439 Belvisi M, 406, 415 Belvisi MG, 17, 26, 48, 51, 64, 248, 253 Ben Baouli A, 371, 379 Ben-Tabou S, 43, 61 Bengoechea-Alonso MT, 163, 184 Benignus VA, 136, 152 Benjamin N, 36, 55, 83, 84, 89, 434, 442 Bennett LE, 381, 397 Bensard DD, 354, 362 Benson PF, 176, 194 Benvenuti C, 384, 398

Author Index Benzing A, 365, 368, 372, 373, 374, 377 Beraldi E, 311, 312, 313, 331 Berg JT, 250, 254 Berger EM, 261, 276 Bergerone S, 248, 253, 429, 430, 440 Bergin P, 384, 398 Berisha HI, 165, 187 Berk B, 7, 21 Berk PD, 203, 213 Berka V, 163, 186 Berlett BS, 168, 190 Bernard C, 137, 152 Bernard GR, 176, 194, 363, 376 Bernareggi M, 32, 48, 51, 64 Berni LA, 135, 151 Berry E, 43, 62 Berry GJ, 383, 391, 398 Berson A, 368, 369, 377 Bertero MT, 425, 426, 427, 430, 431, 438, 440 Berti F, 32, 48, 64 Bertrand F, 163, 185, 327, 341 Besse TJ, 368, 370, 371, 378 Betyia ED, 370, 378 Beutler B, 142, 154, 344, 345, 360 Bevan AM, 309, 330 Beyersdorf F, 259, 275 Bhalla CN, 123, 129 Bhiladvala M, 52, 66 Bialasiewicz P, 321, 337, 338 Bialecki RA, 43, 61 Biban P, 31, 54, 92, 111 Bibbs-Erwin J, 172, 193 Bibbs L, 141, 153 Bicknell RJ, 6, 20 Biernacki W, 135, 151, 206, 217, 318, 319, 320, 336, 337 Bigelow DB, 345, 346, 361 Biggs TE, 316, 335 Biglow DB, 363, 376 Bijman J, 409, 417 Billiar TR, 9, 10, 16, 23, 26, 32, 67, 165, 167, 168, 188, 424, 437 Billingham M, 383, 398 Binding N, 31, 54, 202, 216

467 Binns OA, 261, 276 Birnbaum A, 369, 378 Birnbaum AJ, 368, 378 Birnbaumer L, 47, 63 Bisgaard H, 313, 332 Bishop AE, 405, 413, 415 Bitterman PB, 122, 129 Bjermer L, 309, 329 Bjorne H, 263, 277 Bjornsson E, 309, 329, 432, 442 Bjortuft O, 382, 394, 398 Black CM, 248, 253, 315, 334, 428, 440 Black HR, 320, 321, 337 Black SM, 6, 20, 37, 56, 249, 250, 253, 254, 315, 334 Blake DR, 433, 442 Blake KV, 38, 57 Blalock DK, 172, 193 Blaschke TF, 203, 213 Blaser K, 424, 437 Blaustein MP, 39, 42, 59 Bloch CD, 7, 21 Bloch K, 76, 87 Bloch KD, 7, 21, 249, 250, 254, 315, 334 Block ER, 314, 333 Block KD, 36, 56 Block KJ, 17, 26 Block LH, 262, 277 Blomqvist H, 39, 59 Blumenthal NP, 260, 275 Boasquevisque CH, 384, 390, 392, 399 Boat TF, 403, 404, 412, 413 Boczkowski J, 260, 276 Bode-Boger SM, 433, 442 Bodini A, 31, 54, 92, 111, 202, 212, 309, 329 Bodis S, 432, 441 Bodman ME, 352, 362 Boechat N, 164, 187 Boehler AC, 383, 398 Bogdan C, 163, 185, 428, 440 Boger RH, 433, 442 Bohme E, 43, 61, 74, 85 Boissel JP, 6, 20, 76, 86

468 Boitano S, 43, 61 Bolling SF, 261, 262, 276, 277 Boman G, 309, 329, 432, 442 Bombardier C, 425, 438 Bonaventura J, 36, 56 Bond JH, 203, 213 Bone HG, 135, 151, 206, 218, 320, 337 Bone RC, 345, 361 Bonecini-Almeida MDG, 164, 187 Boner AL, 31, 54, 202, 212, 309, 329 Bonneau M, 375, 380 Bonomo A, 423, 437 Bonoventura J, 164, 168, 187 Booth G, 135, 150 Bootsma H, 426, 438 Borda ES, 432, 441 Border JR, 344, 360 Borderie D, 434, 442 Borean A, 12, 25 Boreham J, 207, 215 Borelli V, 160, 164, 177, 183 Bories PN, 384, 398 Borisenko LV, 327, 341 Borish LC, 313, 332 Borland C, 33, 37, 57, 68, 83, 89, 346, 362 Borland CD, 38, 58 Borland JAA, 9, 23 Borm A, 317, 335 Borner C, 123, 129 Bosca L, 406, 416, 424, 437 Bosch M, 206, 217 Boss GR, 167, 189 Bossy-Wetze E, 123, 129 Botkin SJ, 40, 60 Botney MD, 384, 392, 399 Bottex G, 242, 246 Botting RM, 14, 26 Boucek MM, 381, 397 Boucher R, 411, 419 Boucher RC, 411, 418, 419 Boule M, 411, 419 Bouley G, 142, 144, 154, 155 Boumba D, 432, 441 Bousquet J, 17, 27, 308, 328

Author Index Boustani MR, 171, 192 Boveris A, 160, 164, 167, 177, 183, 188 Bower EA, 32, 38, 39, 56, 82, 89, 107, 113, 267, 278 Boxer LA, 321, 338 Boyd JT, 32, 33, 41, 44, 60, 103, 112 Boyle EM Jr, 261, 276 Bradley E Jr, 162, 184 Brady KG, 405, 409, 415, 417 Branco LG, 51, 65 Brand MP, 409, 417 Brandholtz L, 171, 192 Brandtzaeg P, 346, 362 Brannon TS, 6, 20, 35, 55 Bratton DL, 313, 332 Braude S, 260, 275 Braun D, 424, 437 Braun G, 367, 377 Braun JN, 346, 362 Braun JS, 163, 185 Braun V, 125, 130 Brause JE, 163, 186 Brawn JD, 261, 276 Bredt DS, 6, 9, 20, 23, 34, 47, 51, 55, 63, 65, 74, 76, 85, 86, 87, 162, 163, 164, 184, 186, 235, 236, 243, 346, 362 Breedveld FC, 145, 155 Breiteneder H, 35, 71 Brenman JE, 6, 20, 76, 86 Brennan F, 427, 439 Brennan FM, 141, 153 Brett SJ, 97, 112, 271, 279, 344, 359, 360, 375, 380 Brieland, 172, 192 Brien JF, 325, 340 Brigham KL, 176, 194, 345, 361, 363, 364, 376, 377 Briles DE, 172, 192 Briscoe WA, 343, 359 Bristow J, 37, 56, 249, 253, 315, 334 Brogan R, 36, 55 Brogan TV, 32, 39, 43, 59 Brook CJ, 345, 361 Brooks A, 163, 185

Author Index Brooks SL, 81, 88 Brottman MD, 368, 378 Brouard S, 142, 154 Brown AM, 47, 63 Brown CR, 163, 185 Brown H, 406, 416 Brown JM, 354, 362 Brown LA, 49, 64 Brown MC, 32, 39, 40, 59 Brown NJ, 52, 66, 309, 329 Brown RH, 301, 304 Brown T, 142, 154 Broxmeyer HE, 436, 443 Bruce AC, 180, 194 Brundin L, 425, 438 Brune B, 15, 26, 167, 168, 176, 188, 193 Brunelli L, 172, 193 Brunet FJJ, 375, 380 Brunkhorst R, 316, 335, 423, 437 Bruno G, 314, 332 Brussino L, 425, 426, 427, 430, 431, 438, 440 Bry K, 161, 183 Bryan R, 409, 417 Bryan TW, 405, 412, 415 Bryden K, 34, 69 Bryk R, 11, 24 Bucca C, 248, 253, 425, 426, 427, 429, 430, 431, 438, 440, 441 Buchanan SA, 261, 276 Buchanan WW, 427, 439 Buchet JP, 223, 230 Buchman TG, 295, 304 Buckley BJ, 260, 276 Bucknell KA, 51, 66, 411, 419 Budts WS, 250, 254 Buelow R, 118, 128 Buga GM, 29, 53, 73, 85 Buhrow L, 409, 413, 418 Buhrow LT, 235, 244 Bulkley G, 282, 284, 290, 298, 299, 301, 303, 304 Bulkley GB, 283, 288, 290, 294, 295, 296, 303, 304

469 Bulpitt KJ, 435, 443 Bult H, 427, 439 Burchardi H, 344, 346, 360, 371, 375, 380 Burdick J, 290, 298, 299, 301, 304 Burdick JF, 283, 288, 290, 294, 295, 296, 300, 303, 304, 368, 369, 377 Burge DJ, 435, 443 Burk R, 283, 303 Burke V, 322, 339 Burnett AL, 126, 131 Burns KD, 408, 413, 417 Busch T, 34, 49, 52, 65, 66, 68, 107, 113, 267, 278, 311, 316, 331, 335, 423, 437 Busconi L, 7, 21 Bush A, 37, 57, 315, 334 Buskila D, 428, 440 Busse R, 7, 9, 21, 23, 41, 47, 60 Busset C, 37, 57 Busset CM, 34, 68, 202, 212 Busuttil RW, 118, 128 Butler AR, 16, 26 Butler J, 171, 192 Buttery LDK, 9, 23 Buyon J, 427, 439 Byrnes CA, 37, 57 Byrns RE, 29, 53, 73, 85, 325, 340

C Cabeen W, 428, 440 Cai J, 123, 129 Cailes JB, 248, 253, 315, 334, 428, 440 Cailleux A, 227, 230, 371, 379 Calaycay J, 9, 23, 76, 86, 163, 164, 185, 187 Calaycay JR, 11, 24 Calcagni P, 314, 333 Caldarera CM, 167, 189 Calhoun W, 235, 236, 243, 313, 331, 406, 413, 415 Caligaris-Cappio F, 425, 426, 427, 430, 431, 438, 440, 441 Callow AD, 264, 278

470 Callsen D, 176, 193 Cameron DE, 261, 276, 284, 286, 290, 299, 304 Camhi SL, 120, 122, 128, 129, 205, 214, 251, 255 Campagna A, 176, 194 Campbell A, 406, 416 Campbell D, 311, 330, 404, 413 Campbell G, 172, 193 Campbell J, 364, 377 Camporesi EM, 32, 33, 36, 39, 40, 41, 44, 56, 59, 60, 67, 103, 112 Cannella B, 423, 437 Canning BJ, 124, 130 Cannizzo S, 248, 253, 429, 430, 440 Cannon C, 425, 438 Cannon RO, 108, 113 Cantin AM, 161, 183 Canty TG Jr, 261, 276 Capasso F, 409, 417 Caramori M, 47, 49, 63, 202, 212, 271, 278, 310, 312, 330, 388, 392, 393, 394, 400 Cardell LO, 124, 130, 203, 206, 213, 319, 336 Carden DL, 260, 262, 265, 276 Carlet J, 363, 376 Carlin E, 32, 33, 41, 44, 60 Carlin RE, 36, 56, 103, 112 Carlson M, 432, 442 Carnuccio R, 314, 332, 410, 418 Caron D, 425, 438 Caron E, 411, 419 Carpenter CT, 322, 339, 372, 375, 379 Carr K, 262, 277 Carra S, 31, 54, 92, 111, 309, 329 Carraway MS, 118, 122, 128 Carreras MC, 167, 171, 188, 192 Carroll S, 34, 68 Carstrom K, 35, 71 Cartner SC, 172, 192 Caruso JE, 32, 67 Carvalho P, 48, 64 Casas I, 33, 45, 62 Cassell GH, 172, 192, 193 Cassin S, 51, 65, 170, 181, 191

Author Index Cassina AM, 167, 189 Cassivi SD, 265, 278 Casteneda J, 135, 150 Castranova V, 409, 413, 418 Castrillo A, 406, 416 Castro L, 167, 189 Cataneo RN, 220, 227, 228, 230 Catravas JD, 264, 268, 278 Cattaruzza M, 163, 185 Catz SD, 167, 188 Cavallini L, 12, 25 Cebula TA, 167, 188 Cederbaum SD, 16, 26 Cederqvist B, 35, 71, 268, 278 Cerami A, 142, 154, 344, 345, 360 Cernacek P, 33, 67 Cervin A, 202, 216 Cervos J, 6, 20 Chabin RM, 11, 24 Chabot F, 15, 26 Chagnon FP, 84, 89 Chakder S, 204, 214, 250, 254 Chalmers GW, 313, 331 Chamberlain A, 346, 362 Chambers DC, 34, 70, 325, 340 Chanez P, 17, 27, 78, 88, 308, 328 Chang CH, 13, 24, 425, 438 Chang LY, 161, 183 Chang R, 49, 50, 65 Chao CC, 161, 183 Chao DS, 6, 20, 76, 86 Chao TJ, 33, 67 Chaparro C, 383, 388, 398, 400 Chapman JT, 124, 130 Chapman KR, 37, 38, 47, 49, 57, 63, 200, 202, 211, 212, 309, 310, 312, 328, 330 Chapman RW, 78, 88 Charan NB, 48, 64 Charbonneau F, 40, 49, 60 Charles IG, 17, 27, 430, 440 Charlotte EE, 411, 419 Chartrain N, 163, 186 Chartrain NA, 10, 23 Chatfield BA, 42, 60 Chatkin J, 47, 49, 63, 202, 212

Author Index Chatkin JM, 309, 328 Chaudhuri G, 29, 53, 73, 85 Chaudry I, 282, 303 Chee C, 168, 189 Chee CB, 35, 48, 55, 76, 87, 164, 186, 262, 277 Chelser L, 406, 416 Chen B, 122, 129, 384, 392, 399 Chen F, 409, 413, 418 Chen GH, 51, 66, 411, 419 Chen HI, 33, 67 Chen HS, 168, 189 Chen J, 15, 26, 161, 165, 169, 183, 188, 190, 263, 278 Chen JH, 165, 187 Chen Q, 172, 193 Chen S, 371, 379 Chen TC, 78, 88, 164, 186 Chen V, 364, 377 Chen X, 264, 278 Chen XJ, 49, 64 Chen YQ, 406, 416 Chen Z, 34, 68, 202, 212, 216, 406, 413, 415 Cheng J, 126, 131 Cheng S, 170, 181, 191 Cheng WH, 220, 229 Chensue SW, 423, 437 Cheronis JC, 261, 276 Cherqui G, 163, 185, 327, 341 Chess PR, 364, 377 Chester AH, 9, 23 Chetty R, 164, 186, 249, 254, 316, 334 Cheul LJ, 260, 275 Chew SL, 134, 150 Chi EY, 122, 129 Chiavassa G, 248, 253, 429, 430, 440 Chiesara E, 32, 48, 64 Chikhani N, 390, 400 Chin BY, 120, 122, 123, 126, 129, 130, 131, 139, 143, 144, 153, 155 Ching KF, 314, 333 Chirpaz-Oddou MF, 34, 38, 58 Chistodoulou D, 325, 340 Chiyotani A, 40, 47, 60, 63 Cho HJ, 9, 23, 76, 86, 163, 185

471 Cho P, 282, 303 Chock PB, 170, 191 Choe N, 180, 195 Choe NH, 51, 66, 171, 192 Choi AM, 251, 255 Choi AMK, 118, 120, 121, 122, 123, 124, 126, 128, 129, 130, 131, 135, 138, 139, 142, 143, 144, 150, 152, 153, 154, 155, 204, 205, 214 Choi I, 169, 190 Choi M, 176, 193 Choi ME, 123, 130 Choi YB, 168, 189 Chollet-Martin S, 410, 418 Chopp M, 6, 20 Chowdhury NC, 262, 263, 264, 277 Chowienczyk PJ, 47, 63 Chrisp C, 172, 192 Christensen PJ, 177, 194 Christian JL, 145, 155 Christiani D, 76, 87 Christie JD, 260, 275 Christman BW, 322, 339, 372, 375, 379 Christon JA, 34, 68, 372, 379 Christou H, 118, 128, 134, 150 Chuchalin AG, 205, 214 Chung FK, 200, 201, 203, 206, 208, 211, 213, 215, 217, 308, 318, 321, 328, 336, 337 Chung KF, 34, 35, 48, 64, 69, 70, 71, 81, 84, 88, 89, 202, 212, 308, 309, 310, 311, 312, 314, 317, 328, 330, 331, 332, 335, 404, 413 Ciabattoni G, 206, 208, 209, 215, 218, 252, 255, 318, 323, 324, 336, 339, 344, 360 Ciaccia A, 314, 333, 432, 441 Cifone MG, 163, 185 Clancy RM, 427, 436, 439, 443 Clark JE, 123, 130, 135, 139, 150, 153, 204, 214, 251, 255 Claussen L, 313, 332 Clemens M, 282, 303 Clemens MG, 140, 153 Clementi E, 12, 13, 24, 25, 25 Clements NC Jr, 369, 378

472 Clements PhJ, 428, 440 Clerch LB, 121, 129, 143, 154 Coassin M, 12, 25 Cobb JP, 345, 361 Coburn RF, 136, 152, 203, 206, 213, 217 Cockrill BA, 17, 26, 143, 154 Coers W, 34, 70, 314, 333 Coffey JW, 433, 442 Coffin D, 325, 340 Cogny M, 371, 379 Cohen G, 343, 359 Coito AJ, 118, 128 Colagrande P, 248, 253, 425, 426, 427, 429, 430, 431, 438, 440, 441 Colasanti M, 167, 189 Cole P, 315, 334 Cole PJ, 135, 151, 202, 208, 215, 216, 314, 315, 318, 321, 333, 334, 336, 338, 344, 346, 360, 393, 400 Coleman HA, 43, 62 Colivn RB, 142, 143, 153, 154 Collen D, 250, 254 Collins CE, 324, 339 Collins JV, 324, 339 Collison HA, 203, 213 Colombani P, 288, 290, 294, 296, 304 Colombari E, 136, 151 Colucci WS, 43, 61 Colvin RB, 123, 124, 130 Comhair SA, 235, 244, 249, 254, 313, 331 Comroe JH, 343, 359 Comtois A, 51, 65 Cong-Khanh H, 368, 378 Conners MJ, 235, 244 Connolly RJ, 344, 360 Connor JR, 11, 24 Connor MP, 81, 88 Connors MJ, 409, 413, 418 Constantopoulos SH, 432, 441 Converso M, 425, 426, 427, 438 Cook CD, 51, 65 Cook J, 236, 244 Cook JA, 171, 172, 192, 193 Cook JC, 325, 340

Author Index Cook JL, 121, 129, 142, 144, 154, 155 Cooke JP, 43, 61 Coonrod JD, 161, 183 Cooper AM, 162, 184 Cooper CJ, 40, 49, 60 Cooper JD, 383, 398 Cooper SD, 223, 230 Cope AP, 427, 439 Cope JT, 261, 276 Copland M, 434, 442 Corasania MT, 6, 20 Corbett JA, 164, 165, 187, 406, 416, 423, 437 Corbin JD, 12, 25 Cordova C, 324, 339 Cornejo CJ, 261, 276 Cornejo J, 118, 127 Cornfield DN, 42, 43, 61, 242, 242, 245, 246 Cornwell TL, 171, 191 Corradi M, 31, 54, 206, 208, 209, 215, 218, 252, 255, 310, 312, 313, 318, 324, 325, 325, 326, 330, 336, 339, 340, 344, 360 Corraliza IM, 16, 26 Corridi M, 323, 324, 339 Corris PA, 35, 71, 249, 254, 383, 385, 386, 388, 390, 392, 394, 398, 399, 400, 401 Corson M, 7, 21 Cortese G, 248, 253, 429, 430, 440 Costa A, 134, 150 Costabel U, 252, 256, 324, 339 Costella S, 31, 54, 92, 111, 202, 212, 309, 329 Costello CM, 326, 340, 404, 412, 414 Cote CG, 242, 245 Cotton CU, 409, 413, 417 Couderc R, 375, 380 Coulston E, 34, 69, 202, 212 Cox GW, 163, 185, 242, 245 Cox NR, 172, 193 Cox Y, 37, 57, 83, 89 Cracco A, 200, 211 Crain P, 284, 304 Cramer D, 200, 211, 318, 336, 369, 378

Author Index

473

Cramer DS, 206, 217, 251, 255, 318, 319, 336 Cramm R, 404, 415 Crapo JD, 120, 128, 161, 183, 184, 320, 337 Craven SE, 76, 86 Crawley Y, 78, 88 Creager MA, 39, 40, 49, 59, 60 Cremona G, 32, 33, 38, 39, 56, 68, 82, 89, 107, 113, 267, 278 Crews BC, 14, 25, 322, 339 Crierlaard JM, 410, 418 Croft KD, 322, 339 Cross AH, 81, 88 Cross CE, 167, 169, 171, 188, 190, 192, 314, 325, 333, 340, 359, 362 Crow JP, 49, 64, 167, 169, 172, 177, 178, 180, 189, 190, 193, 194 Cruse I, 117, 127 Crystal RG, 161, 183 Csizmadia E, 123, 124, 130, 142, 143, 153, 154 Cucchiaro G, 32, 39, 40, 59 Cui S, 423, 437 Cui T, 313, 332 Cullinan P, 206, 217 Culpitt SV, 206, 217, 314, 318, 333, 336 Cunha FQ, 164, 172, 186, 193, 410, 411, 418, 419 Cunningham DA, 9, 23 Cuomo A, 31, 54 Curran RD, 167, 188 Currie KE, 39, 49, 59 Currie MG, 11, 24 Custovic A, 309, 329 Cxizmadia E, 142, 154 Czapski G, 165, 188, 236, 244 Czeschinski PA, 31, 54, 202, 216

D D’Acguisto F, 314, 332, 410, 418 Dahia PL, 134, 150 Dailly E, 205, 214, 251, 255 Dall’Ava-Santucci J, 47, 49, 63, 313, 332, 428, 440

Daniel A, 282, 303 Dannecker JR Jr, 224, 230 Danner RL, 35, 71, 314, 333 Dantzker DR, 345, 361 Danziger LH, 327, 341 Dario C, 31, 54, 92, 111, 309, 311, 312, 313, 329, 331, 404, 414 Dark JH, 383, 388, 392, 394, 398, 400 Darley-Usmar V, 167, 188 Darley-Usmar VM, 167, 189 Darnell JE, 406, 417 Darrah PA, 172, 193 Datta P, 33, 67 Datta PK, 204, 214, 250, 255 Dauber JA, 391, 400 Daucher JW, 32, 39, 40, 59 Daughton D, 34, 70 Davidson MK, 172, 193 Davidson PJ, 425, 426, 427, 438 Davies N, 167, 189 Davies RJ, 309, 330 Davis CW, 411, 419 Davis D, 145, 155 Davis G, 180, 194 Davis IC, 180, 194 Davis JK, 172, 193 Davis JM, 170, 176, 191, 194 Davis SJ, 118, 127 Dawes GS, 51, 65 Dawes M, 47, 63 Dawson CA, 32, 39, 59, 239, 240, 242, 245, 262, 277 Dawson TM, 47, 63, 74, 76, 86 Dawson V, 74, 86 Day BJ, 161, 183 Day CE, 374, 380 de Andrade JA, 180, 194 De Caterina R, 167, 189 de Gouw HW, 316, 335 de Groot H, 317, 335, 404, 412, 414 De Groote MA, 172, 193 de Jonge HR, 409, 417 de Jongste JC, 321, 337, 404, 414 De La Cruz J, 425, 438 De Li Y, 314, 333 De Lutiis MA, 242, 245

474 de’ Munari E, 31, 54 De Nuci G, 51, 66 De Raeve HR, 34, 48, 51, 55, 77, 87, 235, 236, 243, 262, 277, 406, 411, 413, 415 De Saint-Martin L, 133, 149 De Sanctis GT, 17, 27, 80, 88 De Soyza A, 394, 401 de Vera ME, 10, 23 Deans RJ, 427, 439 Debatin KM, 168, 189 Debre P, 163, 185 Deby GP, 410, 418 Decuyper J, 368, 369, 377 Deeb GM, 261, 262, 276, 277 Deem S, 32, 67, 237, 238, 244, 250, 254 Degott C, 368, 369, 377 DeGraff W, 172, 193 Dehart P, 345, 361 Dehmlow C, 384, 398 Dehoux M, 260, 276 Dekhuijzen PN, 321, 338 Dekker I, 321, 338 Delaire M, 34, 38, 58, 69 Delaney CA, 167, 188 Delen FM, 318, 336 DeLima NF, 261, 276 Delivoria-Papadopoulos M, 206, 217 DeMarco V, 170, 181, 191 DeMaria R, 163, 185 DeMaster EG, 368, 370, 371, 378 Demedts MG, 34, 70, 309, 311, 314, 329, 331, 333 DeMeester SR, 384, 392, 399 Demers LM, 409, 413, 418 Demirakca S, 404, 414 Demling RH, 345, 346, 361 Demoncheaux E, 33, 45, 62, 68 Demoncheaux EA, 35, 72 Dempsey JA, 34, 38, 39, 58, 107, 113 Deneris ES, 370, 371, 378, 379 Denicola A, 168, 190 Denis M, 76, 86, 404, 410, 412, 414, 418 Denison DM, 260, 275

Author Index Dennery PA, 118, 121, 126, 128, 129, 131, 205, 214, 215, 251, 255 Deodhar SD, 425, 438 DeRaeve HR, 164, 186 Dergal E, 147, 155 Dermand J, 52, 66, 309, 329 DeRoberts D, 34, 69, 202, 216 Derynck R, 406, 416 DeSanctis J, 411, 419 Dessanges JF, 47, 49, 63, 313, 332, 428, 440 Detore GR, 406, 416 Devalia JL, 309, 330 Devall LJ, 321, 338 Devaraj S, 322, 339 Devine JO, 47, 63 Devlin RB, 180, 194 Devoy M, 34, 69 Devynck MA, 74, 86, 322, 338 Deykin A, 34, 69, 79, 88, 202, 212, 308, 328 Dezateux C, 318, 336 Dhainaut JF, 375, 380 Di Giulio C, 242, 245 Di Natale F, 242, 245 Di Pietro LA, 428, 440 Di Rosa M, 314, 332, 410, 418, 433, 442 Di SA, 314, 333 Di Silvio M, 167, 168, 188 Diaz-Guerra MJ, 406, 416 Dibbert B, 424, 437 Dick EC, 316, 335 Diefenbach A, 163, 185 Dietl P, 49, 65 Dietzschold B, 165, 187 Dillon WC, 37, 57 DiMango E, 409, 417 DiMaria GU, 33, 48, 51, 64 Dimberger E, 35, 38, 58 Dimmeler S, 424, 437 Dinarello CA, 344, 345, 360 Dinarevic S, 37, 57 Dinauer M, 163, 186 Dinauer MC, 162, 184 Dinerman JL, 51, 66, 74, 86

Author Index Ding A, 406, 416 Ding AH, 76, 86 Dingle P, 34, 70, 202, 216 Dinh-Xuan AT, 47, 49, 63, 313, 332, 428, 440 Dinwiddie R, 404, 414 Dirksen ER, 43, 61, 62 Dirnberger E, 35, 71 DiSilvio M, 16, 26 Dittman WA, 10, 23 Do BQ, 369, 378 Dobbs LG, 43, 62, 171, 192 Docker SP, 7, 21 Doctrow SR, 176, 194 Dodd HC, 207, 215 Doenst T, 259, 275 Doerschuk CM, 162, 184 Dohlman AW, 320, 321, 337 Doig S, 388, 392, 400 Dolgodvorov AF, 326, 341 Donati YR, 123, 129 Donham KJ, 314, 333 Donham R, 290, 298, 301, 304 Donham RT, 288, 290, 294, 296, 304 Donnelly LE, 206, 208, 215, 217, 310, 312, 313, 317, 321, 324, 325, 325, 326, 330, 335, 337, 340, 341 Donner RL, 344, 346, 360 Doran S, 38, 58, 107, 113 Dore S, 125, 131 Dornand J, 411, 419 Dotsch J, 404, 414 Dotsenko EK, 327, 341 Dougall H, 36, 55, 83, 84, 89 Douglas JS, 33, 37, 57, 67, 346, 347, 348, 349, 362 Downard PJ, 136, 151 Downes MJ, 29, 53 Doyle MP, 164, 187 Drabkin DL, 147, 155 Drake AF, 77, 87 Dramond SL, 7, 21 Drapier JC, 16, 26 Drazba J, 235, 244, 409, 413, 418 Drazen J, 6, 19, 34, 35, 48, 55, 76, 87, 164, 186, 235, 236, 243, 346, 362

475 Drazen JM, 17, 27, 33, 34, 37, 48, 57, 63, 68, 69, 78, 84, 87, 88, 89, 164, 168, 186, 189, 200, 202, 207, 211, 212, 235, 236, 242, 243, 248, 253, 262, 277, 308, 309, 311, 312, 328, 330, 331, 344, 345, 346, 360 Dreher KL, 160, 164, 177, 183 Dreyfuss D, 364, 377 Drinker PA, 51, 65 Driscoll KE, 261, 276 Droz PO, 222, 230 Druml W, 374, 380 Drumm ML, 405, 409, 413, 415, 417 Drummond RS, 202, 212 Du Bois AB, 33, 37, 57, 67, 206, 217, 251, 255, 318, 319, 336, 343, 346, 347, 348, 349, 359, 362 Du Bois RM, 248, 249, 251, 252, 253, 254, 255, 315, 316, 318, 324, 334, 339, 428, 429, 430, 440 Duane WC, 368, 370, 371, 378 Duchatelle V, 368, 369, 377 Duckworth LJ, 38, 57 Duffy JY, 32, 66 Dugas B, 163, 185 Dulin K, 205, 214 Dunams TM, 167, 188 Duncan C, 36, 55, 83, 84, 89 Dunstan DW, 322, 339 Duplain H, 38, 49, 57, 65, 107, 113, 267, 278 Dupont L, 392, 400 Dupont LJ, 34, 70, 309, 311, 314, 329, 331, 333 Duran D, 168, 190 Durham SK, 160, 164, 169, 177, 183, 190 Durham SR, 311, 330, 404, 413 Durieux ME, 7, 21 Duszyk M, 49, 64, 406, 409, 415, 417 Dutto L, 248, 253, 429, 430, 431, 440, 441 Dutz JP, 428, 440 DuVall MD, 49, 64 Dweik R, 235, 244, 409, 413, 418

476

Author Index

Dweik RA, 97, 109, 112, 235, 236, 237, 238, 239, 240, 242, 243, 244, 249, 254, 262, 277, 313, 331, 406, 413, 415 Dworski R, 323, 339 Dye JA, 160, 164, 177, 183 Dykhuizen R, 434, 442 Dzau VJ, 43, 61 Dziedzic D, 172, 193

E Earle RH, 426, 438 Easterbrook-Smith SB, 169, 191 Eaton DC, 49, 64 Eaton JW, 125, 131, 139, 140, 153 Eccles R, 203, 213, 316, 335 Echegoyen L, 325, 340 Eckmann L, 413, 419 Edixhoven M, 409, 417 Edmonds JF, 43, 62 Edwards MW, 29, 53 Efthimiadis A, 199, 211, 317, 335 Egan EA, 159, 176, 182 Egan T, 383, 398 Egashira K, 406, 416 Eger,EI II, 372, 380 Egli M, 38, 57 Eguchi K, 434, 442 Ehrlich WH, 147, 155 Ehrt S, 176, 193 Eichler HG, 34, 35, 38, 39, 58, 59, 68, 71 Eichmann K, 16, 26 Eidsath A, 35, 71, 314, 333 Eigenthaler M, 12, 25 Einarsson O, 316, 335 Eiserich JP, 169, 171, 190, 192, 314, 325, 333, 340, 359, 362 Eissa NT, 313, 331 Eke BC, 6, 20 Ekekezie I, 31, 54 Ekindjian OG, 434, 442 Eklund KK, 432, 441 Ekstrom P, 135, 150

El Dwairi Q, 33, 51, 65, 67 Elespuru RK, 167, 188 Elias JA, 124, 130, 316, 335 Elin RJ, 344, 346, 360 Elliott MJ, 145, 155, 435, 443 Elmer HL, 405, 406, 409, 415, 417 Elsey TS, 29, 53 Elsing M, 34, 70 Elzakker BG, 31, 34, 54, 203, 212, 309, 330 Emel’ianov AV, 326, 341 Emirgil C, 242, 246 Endo T, 33, 68 Engelmann H, 427, 439 England AJ, 434, 442 Engleberg C, 172, 192 English JC, 260, 276 Epperlein M, 172, 193 Eppinger MJ, 261, 276 Erbas D, 34, 68, 202, 216 Erecinska M, 242, 245 Erjefalt I, 48, 64 Erjefalt JS, 48, 64 Ernst P, 76, 87, 308, 321, 328, 338 Erzurum SC, 34, 48, 51, 55, 77, 87, 164, 186, 235, 236, 237, 238, 239, 240, 242, 243, 244, 249, 254, 262, 277, 313, 331, 406, 409, 411, 413, 415, 418 Esmore DS, 384, 398 Esteban-Loos I, 369, 378 Esteban M, 427, 439 Estell K, 406, 415 Esterly JR, 426, 438 Eterradossi J, 34, 38, 58 Etoh T, 430, 440 Euler DE, 368, 369, 371, 373, 377, 378, 379 Evans CD, 368, 370, 371, 378 Evans CH, 433, 436, 442, 443 Evans D, 34, 69 Evans DJ, 200, 201, 211, 308, 309, 328, 329 Evans TG, 316, 335 Evans TJ, 385, 390, 393, 399, 405, 413, 415

Author Index

477

Evans TW, 15, 26, 29, 53, 81, 88, 97, 112, 170, 180, 191, 195, 260, 271, 275, 279, 344, 354, 359, 360, 362, 375, 380 Everett AD, 249, 254, 315, 334 Exon DJ, 135, 150

F Fabbri LM, 314, 333, 432, 433, 441, 442 Fabian AJ, 17, 27 Fackler J, 168, 189 Fagan KA, 240, 245 Fahey TJ, 345, 360 Fair J, 288, 290, 294, 296, 304 Fajac I, 47, 49, 63, 313, 332, 428, 440 Falke K, 316, 335, 363, 376, 423, 437 Falke KJ, 34, 39, 49, 52, 58, 65, 66, 68, 83, 89, 164, 187, 311, 331 Fall R, 370, 371, 379 Faller DV, 262, 277 Faller VF, 242, 245 Fan Chung K, 34, 70 Fan CK, 206, 217 Fanburg BL, 171, 192 Fang F, 163, 186 Fang FC, 172, 176, 177, 193, 421, 436 Fang K, 248, 253, 263, 277, 314, 326, 333, 340, 404, 414 Fanta C, 81, 88, 200, 207, 211, 309, 311, 312, 330, 331 Fantone J, 172, 192 Faragher B, 309, 329 Faraone S, 35, 72 Fardeau M, 136, 152 Farkas-Szallasi T, 35, 37, 55, 236, 244 Farkouh E, 282, 303 Farmer DG, 118, 128 Farrell AJ, 433, 442 Farrugia G, 126, 131 Farrukh IS, 171, 192 Farver C, 235, 244 Fauler J, 434, 442 Fava C, 248, 253, 429, 430, 440

Favre-Juvin A, 34, 38, 58, 69 Feder LS, 78, 88 Fedoseev GB, 326, 341 Fehsel K, 423, 437 Fein AF, 122, 129 Felaco M, 242, 245 Feldmann M, 141, 145, 153, 155, 427, 435, 439, 443 Felix JA, 43, 61 Feng G, 430, 440 Feng GH, 17, 27 Fenske JD, 227, 230 Feppetti P, 33, 48, 51, 64 Fercher AF, 35, 71 Ferguson EA, 203, 213, 316, 335 Ferguson TB Jr, 384, 390, 399 Fergusson W, 200, 212, 309, 329 Fernades PD, 172, 193 Feron O, 7, 21, 76, 86 Ferrari FK, 167, 188 Ferrario L, 32, 33, 41, 44, 60, 103, 112 Ferreira C, 368, 372, 377 Ferreira HH, 51, 66 Ferreira SH, 410, 418 Ferrige AG, 29, 53, 73, 85 Ferris CD, 125, 131, 134, 150 Fessler HE, 122, 129, 139, 143, 144, 153 Feste A, 170, 191 Festuccia C, 163, 185 Field M, 435, 436, 443 Fielding P, 427, 439 Fierro IM, 164, 186, 411, 419 Fike CD, 32, 39, 40, 59 Filipe PM, 371, 379 Filla MB, 163, 185 Finazzi A, 6, 20 Findl O, 35, 71 Fineman JR, 37, 49, 50, 51, 56, 65, 249, 250, 253, 254, 315, 334 Finfer S, 364, 377 Fink G, 432, 441 Fink MP, 176, 194 Finkbeiner WE, 274, 279 Finkel B, 169, 190 Finkelstein JN, 364, 377

478 Finlay GA, 249, 253, 254, 316, 326, 334 Finn D, 78, 88 Fiol B, 381, 397 Fisch C, 368, 369, 377 Fischer A, 124, 130 Fischer AF, 206, 217 Fischer BM, 51, 66, 160, 164, 171, 177, 183, 192 Fischer JE, 345, 361 Fischer LG, 33, 67 Fischer M, 374, 380 Fischer N, 411, 418 Fischer PH, 31, 34, 54, 309, 330 Fisher AA, 7, 21 Fisher AB, 165, 176, 187, 194, 343, 359 Fisher AJ, 385, 386, 388, 392, 394, 399, 400, 401 Fisher JH, 406, 416 Fisher L, 323, 339 Fisher TN, 207, 215 Fitzgerald GA, 324, 339 Fitzgerald MX, 249, 253, 254, 316, 326, 334, 340, 404, 412, 414 Flavahan NA, 135, 150 Fleischmann RM, 435, 443 Fleming I, 41, 47, 60, 345, 361 Fletcher A, 309, 329 Fletcher DS, 163, 186 Flick MR, 176, 194 Flore P, 34, 38, 58, 69 Floreani AA, 34, 69, 203, 213, 314, 333 Flores CA, 51, 66 Flower RJ, 410, 418 Foerster A, 382, 394, 398 Foey AD, 141, 153 Fok KF, 11, 24 Folkerts G, 48, 51, 64 Folkesson HG, 176, 194 Fontijin A, 29, 53 Ford PC, 236, 244 Foresti R, 123, 130, 135, 139, 150, 153, 204, 214, 251, 255 Forman HJ, 404, 414 Fornhem C, 48, 63 Forrest IA, 35, 71, 249, 254

Author Index Forsberg S, 40, 60, 102, 112 Forsling ML, 134, 150 Forst S, 271, 279 Forster RE, 38, 57, 84, 89, 206, 217, 343, 359 Forstermann U, 6, 7, 19, 20, 21, 74, 76, 85, 86, 87, 164, 186, 314, 333 Foster EV, 206, 216 Foster MW, 368, 371, 373, 378 Foster PS, 17, 27, 165, 188 Foster WM, 343, 359 Fouqueray B, 163, 185, 327, 341 Foust RF III, 169, 190 Fouty BW, 240, 245 Fowler M, 11, 24 Fox RB, 145, 155 Fox RI, 432, 435, 441, 443 Foxwell BM, 141, 153 Frampton MW, 34, 69 Francis, 11, 24 Francis H, 309, 329 Francis PL, 30, 54 Francoeur C, 404, 410, 412, 414, 418 Frank AA, 162, 184 Frank PI, 309, 329 Frank TL, 309, 329 Frankel EN, 369, 378 Franklin P, 34, 70, 202, 216 Frasier LM, 34, 69 Fratacci MD, 39, 59 Frederick R, 143, 154 Freeman B, 36, 55 Freeman BA, 15, 26, 161, 167, 168, 169, 171, 183, 184, 189, 190, 260, 263, 276, 277, 278, 314, 320, 333, 337 Freemont AJ, 427, 439 Fregonese B, 309, 329 Freitas JP, 371, 379 Freitas MS, 164, 186, 411, 419 French PJ, 409, 417 Freund A, 345, 361 Friberg SG, 33, 48, 51, 64 Frick LW, 11, 24 Fridovich I, 160, 164, 167, 177, 182, 183, 189 Fridrich P, 374, 380

Author Index

479

Friederich C, 12, 25 Friedman N, 172, 193 Friedrich B, 404, 415 Friedrich F, 49, 65 Friesen C, 168, 189 Fritz H, 374, 380 Frohman E, 76, 86 Frolich JC, 423, 433, 434, 437, 442 Fromenty B, 368, 369, 377 Frostell C, 31, 35, 37, 38, 39, 40, 49, 52, 54, 56, 57, 59, 60, 66, 83, 89 Frostell CG, 17, 27 Froysaker T, 382, 394, 398 Fuglsang G, 313, 332 Fujii Y, 33, 67, 385, 392, 399, 400 Fujimura M, 406, 416 Fukuta T, 136, 152 Fukuto JM, 238, 244 Fulda S, 168, 189 Fultz MJ, 163, 185 Fumagalli R, 364, 377 Fung PC, 248, 253, 327, 341 Furchgott RF, 29, 41, 47, 53, 60 Furfine ES, 11, 24, 162, 184 Furlott HG, 37, 38, 57, 84, 89, 96, 112, 200, 207, 211 Furst D, 428, 440 Furukawa K, 84, 89

G Gabbay E, 383, 385, 386, 387, 388, 392, 393, 394, 398, 399, 400, 406, 413, 415 Gachhui R, 238, 245 Gagne GD, 76, 87 Gai P, 135, 151 Gaine SP, 135, 150 Gal J, 37, 56, 106, 113, 268, 278 Galbraith TA, 271, 279 Galliani C, 169, 176, 190, 372, 380 Gamberale R, 171, 192 Gammie JS, 260, 275 Gan JC, 170, 191 Ganz P, 40, 49, 60 Gao G, 163, 185 Gao J, 406, 416

Gao JJ, 163, 185 Gao L, 404, 414 Gao Y, 43, 61 Gardiner R, 171, 192 Gardner CR, 169, 190 Garewal S, 369, 378 Garey KW, 327, 341 Garg ML, 324, 339 Garlepp MJ, 76, 77, 87 Garnier P, 47, 49, 63, 313, 332 Garrison L, 313, 332 Garrison RN, 136, 151 Garting SS, 35, 71 Garvey EP, 11, 24, 162, 184 Gascoigne A, 383, 398 Gascoigne AD, 383, 398 Gassmann M 6,20 Gaston B, 34, 35, 48, 48, 55, 63, 76, 81, 84, 87, 88, 89, 164, 168, 186, 189, 200, 207, 211, 235, 236, 242, 243, 248, 253, 262, 277, 309, 311, 312, 314, 325, 326, 330, 331, 333, 340, 344, 345, 346, 360, 362, 404, 414 Gatecel C, 410, 418 Gatzy J, 411, 419 Gatzy JT, 411, 418, 419 Gauldie J, 261, 276 Gaustad P, 346, 362 Gautam S 6,20 Gavita SM, 385, 393, 399 Geay GA, 345, 361 Gebel S, 318, 336 Geddes DM, 206, 218, 318, 324, 336 Gee SH, 76, 86 Geffner JR, 171, 192 Gehman KE, 260, 275 Geigel EJ, 38, 57, 84, 89 Geiger K, 365, 367, 368, 369, 372, 373, 374, 377, 378 Geiger KK, 140, 153 Geiran O, 382, 394, 398 Geiser AG, 406, 416 Geiser T, 177, 180, 181, 182, 194 Gelfand JA, 344, 360 Gelinder S, 39, 52, 58 Geller DA, 10, 16, 23, 26, 424, 437

480 Gelman S, 162, 184 Gelmont D, 371, 379 Genaro AM, 424, 437 Geng J, 164, 187 George SC, 34, 38, 57, 69 Geppert TD, 142, 154 Gerard C, 35, 48, 55, 76, 87, 164, 186, 262, 277 Gerard R, 250, 254 Gerber B, 300, 304 Gerlach H, 34, 39, 52, 58, 66, 68, 83, 89, 316, 335, 423, 437 Gerlach M, 316, 335, 423, 437 German Z, 34, 68, 76, 86, 162, 184, 202, 212 Geroulanos S, 34, 69 Gerritsen ME, 118, 128 Ghahary A, 428, 439 Ghobrial RM, 118, 128 Ghosh S, 238, 244 Giaid A, 76, 78, 84, 87, 88, 89, 164, 186, 248, 249, 253, 260, 276, 308, 315, 316, 321, 328, 334, 338, 385, 390, 393, 399, 400, 430, 440 Giammarresi C, 32, 33, 43, 46, 62 Gibbons RD, 229, 231 Gibbs-Erwin J, 169, 190 Gibbs L, 126, 131 Giclas PC, 344, 360 Gifford MA, 162, 184 Gigli P, 385, 399 Gigliotti D, 171, 192 Gigliotti Rothfuchs AC, 171, 192 Gilbey T, 310, 311, 312, 313, 330 Gilker C, 162, 184 Gilkeson G, 424, 425, 438 Gilkeson GS, 424, 435, 438, 443 Gillijns H, 250, 254 Gillin FD, 413, 419 Gillis CN, 36, 37, 38, 56, 108, 113 Gimbrone MA Jr, 167, 189 Gimeno C, 284, 304 Giordano E, 167, 189 Giordano M, 171, 192 Giorno R, 424, 426, 427, 438, 439 Giraldo C, 314, 333

Author Index Giraud C, 375, 380 Giraud GD, 34, 35, 38, 47, 58, 62, 63, 69, 83, 84, 89, 92, 107, 111, 113, 202, 212, 316, 335 Girgis RE, 383, 391, 398 Girn J, 144, 155 Gissen H, 242, 246 Giulivi C, 163, 185 Gladman DD, 425, 438 Glatt CE, 126, 131, 140, 141, 153, 162, 163, 184 Glatt CS, 9, 23 Glazer AN, 119, 125, 128, 128, 139, 140, 148, 152, 156 Gleeson K, 229, 231 Glockzin S, 176, 193 Goda N, 135, 151 Godard P, 17, 27, 308, 328 Godin DV, 260, 276 Godin PJ, 35, 71, 314, 333 Godwin JE, 320, 337 Goel A, 424, 426, 427, 438 Goldbaum LR, 147, 155 Goldberg P, 33, 67, 165, 187 Golden JA, 274, 279 Golden M, 36, 55, 83, 84, 89 Goldman D, 425, 438 Goldman MJ, 411, 418 Goldman WE, 164, 165, 187 Goldstein J, 6, 20 Goldstein S, 165, 188, 236, 244 Gole MD, 169, 190 Goller NL, 160, 164, 169, 177, 183, 190 Gomez FP, 34, 35, 70, 72, 313, 332 Gomez-Reino JJ, 425, 438 Goncharova VA, 327, 341 Gong H, 428, 440 Gonzales A, 135, 150 Gonzalez C, 135, 150 Gonzalez PK, 176, 194 Gonzales-Crespo MR, 425, 438 Goodman RB, 179, 180, 194 Gopinathan V, 139, 140, 152 Gordon SM, 229, 231 Gortner L, 311, 331

Author Index Gosselin D, 411, 419 Goter Robinson CJ, 430, 440 Goto M, 390, 400 Gougerot-Pocioalo MA, 410, 418 Gourley GR, 139, 152 Gouya G, 35, 71 Gow A, 168, 190 Gow AJ, 108, 113, 168, 189 Grabowski PS, 434, 442 Graf P, 36, 55 Granger D, 172, 193, 282, 284, 301, 303, 304 Granger DL, 16, 26, 424, 435, 437, 443 Granger DN, 171, 191, 260, 262, 265, 276, 436, 443 Granit R, 171, 192 Grasemann H, 17, 27, 35, 71, 308, 317, 326, 328, 335, 340, 404, 411, 412, 414, 419 Grasseli U, 35, 71 Graycar J, 406, 416 Grayck EN, 39, 59 Green CJ, 123, 130, 135, 139, 150, 153, 204, 214, 251, 255 Green DR, 123, 129 Green IC, 167, 188 Green J, 411, 419 Green MH, 167, 188 Green S, 36, 55 Greenberg J, 220, 227, 228, 230, 231, 368, 369, 371, 377, 379 Greening AP, 208, 209, 215, 309, 313, 314, 326, 329, 332, 333, 340, 344, 346, 360, 404, 412, 414 Greenland T, 430, 440 Greenwood MT, 51, 65 Greis KD, 170, 191 Grennan DM, 427, 439 Grey ST, 123, 124, 130, 143, 153 Gribin BJ, 11, 24 Griese M, 404, 414 Griffith BP, 391, 400 Griffith OW, 11, 24, 162, 184, 235, 236, 238, 242, 243, 245, 345, 361 Griffith TM, 42, 60 Griffiths MJ, 81, 88

481 Grigor MR, 16, 26 Grilli A, 242, 245 Grimbert F, 34, 38, 58 Grimminger F, 32, 37, 39, 40, 44, 56, 62, 239, 245 Grimwood K, 324, 339 Grisham M, 284, 304 Grisham MB, 236, 244, 325, 340, 405, 406, 412, 415, 416 Groen H, 426, 438 Gronski T Jr, 364, 377 Gross A, 411, 419 Gross M, 426, 438 Gross SS, 76, 86, 345, 361, 428, 439 Grossman AB, 134, 150 Grote C, 225, 230 Grubb BB, 411, 419 Gruenert DC, 405, 413, 415 Grum CM, 321, 338 Grunberg K, 316, 335 Grunewald C, 35, 71 Grunke M, 427, 439 Gryglewski RJ, 43, 61 Guastella D, 125, 131 Gudbjornsson B, 432, 441, 442 Guenard H, 346, 362 Guerra R, 16, 26 Guerra Rodrigo F, 371, 379 Guggenbach M, 34, 69 Guillemin M, 222, 230 Guleva LI, 326, 341 Gultekin E, 315, 334 Gumbay RS, 345, 361 Gunawarden D, 78, 88 Gunnar RM, 344, 360 Gunzburg S, 176, 193 Guo FH, 34, 48, 51, 55, 77, 87, 164, 186, 235, 236, 243, 262, 277, 313, 331, 406, 411, 413, 415 Guo Y, 6, 20, 33, 49, 51, 64, 65, 67 Gura V, 432, 441 Gustafsson LE, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 48, 49, 50, 51, 51, 52, 53, 54, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 71, 73, 83, 85, 89, 91, 95,

482

Author Index

[Gustafsson LE] 97, 102, 103, 106, 111, 112, 113, 200, 203, 207, 211, 213, 237, 239, 240, 244, 267, 268, 278, 308, 309, 328 Gutierrez C, 47, 49, 63, 202, 212, 309, 310, 312, 328, 330 Gutierrez H, 263, 277, 278 Gutteridge JM, 170, 180, 191, 195 Gutteridge JMC, 15, 26, 371, 379 Guttman J, 367, 369, 377, 378 Gutzki F-M, 433, 442 Guynn TP, 262, 277 Guzel NA, 34, 68, 202, 216 Gwaltney JJ, 316, 335 Gyi KM, 405, 413, 415 Gyimesi E, 432, 441 Gyllenhammar H, 74, 86, 322, 338 Gyurech D, 34, 69

H Habib MP, 369, 378 Habib S, 405, 413, 415 Hack CE, 145, 155 Haddad IY, 169, 170, 176, 181, 190, 191, 194, 372, 380 Haendeler J, 424, 437 Hafizi S, 9, 23 Hagberg S, 34, 70, 203, 212 Hagen TJ, 11, 24 Hahnenkamp K, 33, 67 Hahtola P, 34, 70 Hakim TS, 32, 33, 36, 39, 40, 41, 44, 56, 59, 60, 67, 103, 112 Halbrugge M, 12, 25 Haldane J, 133, 149 Haldane JBS, 136, 152 Hale HS, 344, 360 Haley KJ, 78, 87 Hall AV, 8, 21 Hall L, 32, 38, 39, 56, 82, 89, 107, 113, 267, 278 Hall SL, 42, 60, 122, 129 Hallam TJ, 43, 61

Hallgren R, 432, 441 Halliwell B, 167, 169, 171, 188, 190, 192, 314, 333, 435, 443 Hallman M, 161, 183 Halpern O, 79, 88 Halversen S, 346, 362 Hamada K, 17, 27 Hameister WM, 425, 426, 427, 438 Hamid Q, 17, 27, 78, 88, 308, 328 Hamilton S, 282, 284, 303 Hamilton TP, 165, 188 Hammel J, 235, 244 Hammerle H, 208, 215, 327, 341 Hampl V, 37, 57 Hamre R, 202, 216 Han J, 141, 142, 153, 154 Han JH, 142, 154 Han RN, 43, 61 Han W, 314, 333 Han Z, 125, 130 Hanazawa T, 202, 203, 213, 216, 322, 323, 325, 326, 338, 341 Hanbauer I, 171, 192, 236, 244 Hannaford P, 309, 329 Hanrahan JW, 404, 414 Hanse TN, 118, 128 Hansell DM, 314, 333, 344, 346, 360, 393, 400 Hansen DW Jr, 11, 24 Hansen M, 43, 61, 205, 214 Hansen TN, 251, 255 Hansson GK, 78, 87 Haque J, 235, 236, 243 Haque SJ, 406, 413, 415 Harada RN, 145, 155 Haraguchi Y, 39, 59, 202, 216 Harant H, 410, 413, 418 Harbeck R, 311, 331 Haregewoin A, 432, 441 Harel S, 171, 192 Hargreave FE, 199, 211, 317, 335 Harinder BS, 369, 378 Hariri RJ, 345, 360 Harken AH, 354, 362 Harmon MF, 11, 24 Harris M, 47, 63, 202, 212

Author Index Harrison D, 7, 21 Harrison DG, 7, 16, 21, 26, 84, 89 Harrison J, 165, 188 Harrison JG, 165, 188 Harrison JK, 7, 21 Hartig E, 404, 415 Hartsfield CL, 144, 155 Hasegawa S, 33, 68 Hashimoto H, 427, 439 Hashimoto M, 322, 338 Hashimoto S, 164, 187 Hasko G, 141, 153 Haslett C, 344, 345, 360, 361 Hassett JM, 344, 360 Hassoun PM, 171, 192, 242, 245 Hataishi R, 313, 332 Hattler BG, 260, 275 Hattori H, 13, 24 Hattori Y, 76, 86 Haubitz M, 316, 335, 423, 437 Hauselmann HJ, 436, 443 Hausladen A, 167, 168, 189 Hawkins RD, 135, 148, 150, 155 Haworth SG, 249, 253, 315, 334 Hayashi S, 135, 150 Hayden F, 34, 70, 203, 213, 316, 335 Haydn WE, 406, 413, 415 He XY, 436, 443 Heales SJ, 409, 417 Heard SO, 321, 338 Heath L, 172, 192 Heberstreit H, 424, 437 Hechtman H, 345, 361 Hechtman HB, 345, 361 Hecker M, 163, 185 Hedenstierna G, 34, 37, 38, 39, 40, 57, 59, 60, 68, 102, 112 Hedenstrom H, 309, 329, 432, 441, 442 Hedges RG, 32, 39, 43, 59, 67, 237, 238, 244 Hedqvist P, 33, 48, 49, 50, 51, 64, 65 Heffner JE, 320, 337 Hegbrant J, 371, 379 Hegner D, 161, 183 Heidersbach RS, 250, 254

483 Heiss LN, 164, 165, 187 Heitmeier Mr, 406, 416 Heller M, 7, 21 Hellgren J, 34, 70 Hellinger A, 384, 398 Hemenway DR, 180, 195 Hendenstierma G, 17, 27 Hendenstrom H, 17, 27 Heng HH, 8, 21 Henkart PA, 176, 193 Henke C, 122, 129 Henle ES, 125, 130 Henningsson R, 135, 150 Henriksen AH, 309, 329 Henriksson L, 34, 70, 203, 212 Henrotin YE, 410, 418 Henry P, 430, 440 Henry RL, 324, 339 Henschen M, 311, 331 Henson JE, 344, 345, 360, 361 Henson PM, 344, 345, 360, 361 Henson SM, 344, 346, 360 Hentze MW, 163, 185 Hepler LK, 240, 245 Herbison AE, 6, 20 Herman AG, 427, 439 Herman SJ, 260, 275 Hermans PWM, 321, 337 Hernvann A, 434, 442 Herrera J, 220, 227, 228, 230 Herrmann M, 427, 439 Hertkorn C, 169, 190 Hertz M.I., 383, 398 Herulf M, 263, 277 Hess DT, 168, 189 Hester LD, 125, 131 Hetsko ML, 413, 419 Hevel JM, 165, 187 Hewat VN, 206, 216 Hibbs JB, 16, 26 Hibbs JBJ, 316, 335 Hickey WF, 81, 88 Hickman-Davis J, 169, 172, 190, 193 Hickman-Davis JM, 163, 164, 172, 173, 175, 180, 185, 194 Hieronymos T, 427, 439

484 Higenbottam T, 32, 33, 35, 37, 38, 39, 45, 56, 57, 62, 72, 82, 83, 89, 107, 113, 267, 278, 346, 362 Higenbottam TW, 33, 38, 58, 68 Higgenbottam T, 48, 64 Higgs EA, 162, 164, 184, 187, 262, 277 Hildebrandt J, 34, 68 Hildebrandt W, 49, 65, 107, 113, 267, 278 Hill CS, 145, 155 Hill GE, 34, 69, 203, 213, 271, 279, 314, 333 Hill JR, 423, 437 Hilliquin P, 434, 442 Hind CR, 252, 255, 324, 339 Hints TH, 7, 21 Hirakata H, 427, 439 Hirata K, 33, 34, 68, 69 Hirata M, 13, 24 Hirata Y, 409, 413, 418 Hirayama A, 36, 55 Hirosawa J, 249, 254, 271, 279 Hirsch DJ, 126, 131, 140, 141, 153 Hislop AA, 249, 253, 315, 334 Hjelm M, 34, 70, 202, 212 Hjemdahl P, 35, 40, 49, 60 Hlastala MP, 32, 39, 43, 59 Ho CF, 35, 71 Ho LO, 208, 209, 215 Ho LP, 309, 313, 314, 326, 329, 332, 333, 340, 344, 346, 360, 404, 412, 414 Hoare GS, 9, 23 Hobbs AJ, 38, 58 Hodara R, 167, 189 Hodson M, 405, 413, 415 Hodson ME, 124, 130, 135, 151, 206, 217, 218, 318, 324, 326, 336, 340, 404, 414 Hoeffel JM, 176, 194 Hoekstra JW, 164, 187 Hoet P, 223, 230 Hoffman BB, 48, 63 Hoffman EP, 10, 23

Author Index Hoffman RA, 433, 442 Hoffmann G, 49, 65 Hoffmann SP, 32, 33, 67 Hoffmann WD, 345, 361 Hoffstein E, 37, 38, 57, 84, 89, 96, 112, 200, 211 Hofmann F, 13, 25 Hogaboam CM, 423, 437 Hogan SP, 17, 27, 165, 188 Hogman M, 17, 27, 37, 38, 57, 309, 329, 432, 442 Hohenadl C, 316, 335 Hoidal JR, 171, 192 Hokfelt T, 35, 37, 55, 236, 244 Holbrook NJ, 120, 128, 143, 154 Holden WE, 34, 35, 38, 47, 58, 62, 63, 69, 83, 84, 89, 92, 107, 111, 113, 202, 212, 316, 318, 335, 336 Hole B, 140, 153 Holgate S, 308, 328 Holgate ST, 17, 27 Holland SM, 162, 184 Holm BA, 159, 176, 182, 372, 380 Holman P, 242, 245 Holmes EW, 391, 400 Holroyde J, 345, 361 Holtby HM, 271, 278 Holz O, 207, 215, 320, 337 Holzer P, 34, 69 Hom G, 163, 186 Honda T, 300, 304 Hondalus MK, 172, 193 Hood JS, 49, 65 Hop WC, 404, 414 Hop WCJ, 321, 337 Hope BT, 6, 19 Horio F, 126, 131 Horiuchi T, 427, 439 Horley KJ, 260, 276 Hornik V, 427, 439 Horowitz S, 122, 123, 129, 130, 136, 144, 152, 155, 170, 176, 191, 194 Horstman DJ, 33, 67 Hortelano S, 424, 437 Hortzell P, 283, 303

Author Index Horvath I, 34, 70, 135, 151, 202, 203, 206, 208, 212, 213, 215, 217, 308, 309, 315, 317, 318, 321, 328, 329, 334, 335, 336, 337, 338 Hosenpud JD, 381, 397 Hoshino J, 34, 35, 69, 71, 202, 216, 249, 254 Hosko MJ, 207, 215 Hotz P, 223, 230 Hou Y, 325, 340 Howarth P, 17, 27, 34, 69, 78, 88 Howarth PH, 308, 328 Howie AD, 427, 439 Hoyland J, 427, 439 Hristova M, 169, 190, 314, 333 Hsieh CM, 118, 128 Hsu K, 33, 67 Hsu MF, 145, 155 Hu P, 169, 176, 190, 372, 380 Hu Y, 126, 131 Hua C, 34, 68, 372, 379 Huang F, 430, 440 Huang FP, 17, 27, 435, 436, 443 Huang PL, 17, 27, 240, 245 Huang TJ, 117, 118, 126, 127, 131, 204, 213, 250, 254 Huang YC, 39, 59 Hubbard RC, 161, 183 Huber H, 423, 437 Hubscher O, 432, 441 Huckett L, 6, 20 Hudson L, 363, 376 Hudson LD, 143, 154, 179, 180, 194 Huez G, 142, 154 Huffnagle G, 172, 192 Huffnagle GB, 423, 437 Hughes D, 135, 151, 206, 218, 318, 336 Hughes JM, 229, 231 Huie RE, 165, 188 Hukkanen M, 432, 441 Hull J, 324, 339 Huls G, 404, 414 Hultqvist-Bengtsson U, 371, 379 Hultqvist M, 371, 379 Hunt J, 325, 340

485 Hunt JF, 248, 253, 263, 277, 314, 333 Hunter D, 34, 68 Hunter DN, 260, 275 Hurley M, 172, 192 Hurshman AR, 238, 245 Hurst JK, 168, 190 Husain AN, 391, 400 Husain M, 106, 113, 238, 244 Huso DL, 47, 63 Hussack P, 199, 211 Hussain A, 325, 340 Hussain SN, 33, 51, 65, 67, 165, 187 Hussain SNA, 6, 20 Hutcheon M, 388, 400 Hutcheson IR, 42, 60 Hutchins GM, 261, 276 Hutchinson N, 163, 186 Hutchinson NI, 10, 11, 23, 24 Hutte R, 97, 109, 112, 235, 236, 237, 238, 239, 240, 242, 243, 262, 277 Hwang KH, 410, 418 Hwang PL, 80, 88 Hwang PM, 6, 19, 162, 163, 184 Hyde RW, 34, 38, 57, 69, 84, 89, 107, 113, 267, 278

I Iakovlev AA, 208, 215 Ialenti A, 433, 442 Ibrado J, 123, 129 Ichiguchi O, 390, 400 Ichimori K, 238, 244 Ichinose F, 106, 113 Ichinose M, 326, 340 Ide H, 32, 39, 40, 59, 237, 239, 244 Idle M, 207, 215 Idriss S, 167, 189 Igararhi A, 430, 440 Igarasshi N, 119, 128, 128, 148, 156 Iglesia R, 35, 72, 313, 332 Ignarro L, 238, 244 Ignarro LJ, 11, 23, 29, 38, 53, 58, 73, 85, 171, 191, 325, 340 Ihorst G, 311, 331

486 Ihre E, 51, 66, 97, 112, 308, 328 Iijima H, 33, 68 Ijiri S, 165, 187 Ikebe T, 13, 24 Ikeda K, 319, 337 Ikeda K, 135, 151 Ikejima T, 344, 360 Imada M, 34, 69 Imlay JA, 160, 164, 177, 183 Inacker O, 208, 215, 327, 341 Inada Y, 368, 369, 378 Inamdar N, 118, 120, 121, 128 Inari K, 136, 152 Ingbar DH, 122, 129 Ingi T, 126, 131 Innes J, 313, 332 Innes JA, 208, 209, 215, 309, 314, 326, 329, 333, 340, 344, 346, 360, 404, 412, 414 Inoue M, 39, 59, 202, 216 Intaglietta M, 204, 214 Invernizzi G, 320, 337 Ioannidis I, 317, 335, 384, 398, 404, 412, 414 Iozunii K, 430, 440 Ischiropoulos H, 134, 149, 150, 160, 164, 165, 167, 168, 169, 172, 177, 183, 187, 188, 189, 190, 191, 193, 372, 380 Ishibashi Y, 430, 440 Ishibe Y, 249, 254, 271, 279 Ishigaki M, 32, 67 Ishikawa K, 137, 152 Ishimaru H, 136, 152 Ishimura Y, 135, 150, 151 Ishizuka M, 135, 151, 206, 217, 317, 336 Ishizuka S, 319, 337 Israel E, 34, 69, 79, 88, 202, 212, 308, 328 Itano H, 265, 278 Iuvone T, 314, 332, 410, 418 Iwamoto J, 32, 34, 39, 40, 58, 59, 69, 107, 113, 237, 239, 244 Iyer NV, 126, 131

Author Index Izumi K, 424, 437 Izzo AA, 409, 417

J Jablowski M, 165, 188 Jack CI, 252, 255, 324, 339 Jackson CG, 435, 443 Jackson MJ, 252, 255, 324, 339 Jackson RM, 180, 194 Jacksonroberts L, 323, 339 Jacob HJ, 36, 56 Jacob HS, 118, 125, 127, 131, 139, 140, 153 Jacobs RA, 134, 150 Jacobson HN, 51, 65 Jaffrey SR, 126, 131, 134, 150 Jain B, 48, 64, 171, 191, 315, 334 Jain L, 49, 64 Jakobsson J, 37, 56, 83, 89 James A, 124, 130, 135, 151, 205, 206, 215, 318, 319, 336 James AJ, 315, 334 James N, 7, 21 Jang D, 436, 443 Janson C, 309, 329, 432, 442 Janssens S, 250, 254 Janssens SP, 7, 21 Jansson O, 34, 70 Jaraki O, 248, 253, 434, 442 Jarrott B, 45, 62 Jarvis MJ, 206, 216 Jatakanon A, 124, 130, 135, 151, 205, 206, 215, 309, 310, 311, 312, 313, 318, 319, 330, 331, 336, 404, 413 Jau J, 33, 67 Jeczkowski K, 321, 337 Jenkins HS, 309, 330 Jenkinson CP, 16, 26 Jennings B, 121, 129 Jerome GM, 11, 24 Jia L, 36, 37, 38, 56, 164, 168, 187 Jialal I, 322, 339 Jiang BH, 126, 131 Jiang L, 343, 359, 368, 371, 373, 378

Author Index Jiang NF, 160, 164, 177, 183 Jiang Q, 168, 190 Jiao A, 17, 27, 80, 88 Jilma B, 34, 35, 38, 58, 68, 71 Jinkins PA, 405, 412, 415 Jobsis Q, 321, 337 Jochum M, 374, 380 Joe EK, 164, 187 Johengen MJ, 250, 254 John M, 74, 85, 310, 311, 312, 313, 330 Johns DG, 126, 131 Johns RA, 39, 40, 59, 60, 164, 186, 249, 254, 314, 315, 333, 334 Johnson DC, 17, 26 Johnson RA, 136, 151 Johnson SR, 48, 64 Johnston ID, 252, 255, 324, 339 Jonas M, 122, 129 Jonas S, 311, 331 Jones AD, 169, 190, 314, 333 Jones CJ, 427, 439 Jones DP, 123, 129 Jones GJ, 33, 68 Jones H, 78, 88 Jones KL, 405, 412, 415 Jones ML, 261, 276 Jones R, 39, 59 Jonsson B, 31, 52, 54, 66 Jordan M, 78, 88 Jordan W, 161, 183 Jordana M, 261, 276 Jorens PG, 427, 439 Jorgensen S, 207, 215 Joris M, 364, 377 Jorres RA, 34, 38, 57, 69, 207, 215, 320, 337 Joshi MS, 171, 192 Joshi PC, 76, 87 Josis Q, 404, 414 Jothianandan D, 29, 53 Jourd’heuil D, 325, 340 Jubert C, 288, 304 Jubran A, 345, 361 Julou-Schaeffer GJ, 345, 361 Jung T, 34, 68, 423, 437

487 K Kachel DL, 170, 181, 191 Kacmarek RM, 17, 26 Kadowaki M, 409, 417 Kadowitz PJ, 49, 65 Kagami M, 432, 441 Kagan E, 180, 195 Kagnoff MF, 413, 419 Kahan A, 428, 440 Kahl R, 344, 346, 360, 371, 375, 380 Kaiser LR, 260, 275 Kalaria RK, 6, 20 Kalden JR, 145, 155, 435, 443 Kallenberg CGM, 426, 438 Kalogeropoulos N, 326, 341 Kalyanaraman B, 167, 171, 189, 242, 245 Kalzen H, 38, 39, 40, 58 Kamada D, 317, 335 Kameyama T, 136, 152 Kamm S, 13, 25 Kamosinska B, 49, 64, 409, 417 Kanazawa H, 33, 34, 68, 69 Kanda T, 35, 71, 202, 216, 249, 254 Kane LS, 168, 189 Kaneda H, 119, 128, 128, 148, 156 Kaneko F, 97, 109, 112, 262, 277 Kaneko FT, 235, 236, 237, 238, 239, 240, 242, 243, 244, 249, 254 Kaneko H, 427, 439 Kaneko S, 322, 338 Kang HI, 432, 441 Kankaanranta H, 34, 70 Kanner J, 171, 192 Kantrow SP, 39, 59 Kapiotis S, 35, 38, 58, 71 Kaplan R, 136, 152 Kaplan SS, 172, 193 Kaplowitz MR, 32, 39, 40, 59 Kappas A, 118, 128 Kappus H, 284, 304 Karadag B, 315, 334 Kari UP, 411, 418 Karlsson G, 34, 70

488 Karp PH, 411, 418 Karupiah G, 17, 27, 165, 187, 188 Kasai H, 284, 304 Kashiwabara Y, 76, 86 Kasielski M, 321, 338 Kasprzak KS, 167, 188 Kass RM, 394, 401 Kastner J, 34, 35, 39, 59, 68, 71 Katagata Y, 137, 152 Katajama I, 428, 440 Katayama I, 428, 440 Kato H, 118, 128 Kato I, 406, 416 Katoh A, 136, 152 Katsuki S, 12, 24, 29, 53 Katsuyama K, 409, 413, 418 Kauffman HF, 34, 70, 314, 333 Kaur H, 139, 153, 167, 188, 435, 443 Kavosi M, 106, 113 Kavoussi L, 301, 304 Kavuru MS, 235, 244, 409, 413, 418 Kawakami T, 313, 332 Kawakami Y, 34, 37, 57, 239, 245 Kawamura K, 249, 254, 271, 279 Kay AB, 322, 323, 325, 338 Kazui M, 283, 284, 286, 288, 290, 294, 295, 296, 298, 299, 300, 301, 303, 304, 368, 369, 377 Kazzaz JA, 122, 129, 143, 154 Ke B, 118, 128 Keatings V, 34, 69, 203, 213, 314, 333 Keatings VM, 249, 253, 254, 316, 326, 334, 340, 404, 412, 414 Kechner NE, 33, 67 Keck BM, 381, 397 Keenan R, 391, 400 Keenan RJ, 260, 275 Keirulf P, 346, 362 Keitzmann D, 344, 346, 360 Keller A, 314, 333 Keller E, 43, 61 Kelley PM, 37, 57 Kelley TJ, 405, 406, 409, 413, 415, 417 Kelly C, 314, 333 Kelly DR, 359, 362 Kelly KH, 383, 398

Author Index Kelly RA, 164, 187 Kelly TJ, 409, 413, 417 Kelly TM, 11, 24 Kensler TW, 125, 131 Keogh BF, 260, 275 Keown PA, 260, 276 Kerger H, 204, 214 Kermarreck N, 410, 418 Kern RM, 16, 26 Kerr ME, 250, 254 Kerwin JF, 11, 23 Keshavjee S, 383, 388, 398, 400 Keshavjee SM, 383, 398 Keske U, 52, 66 Kesten D, 383, 398 Kesten S, 383, 388, 398, 400 Ketai LH, 321, 338 Kettle AJ, 160, 164, 177, 183, 326, 340 Kettler D, 344, 346, 360, 371, 375, 380 Keyse SM, 137, 138, 152 Khan MT, 29, 53 Khan S, 384, 398 Khan SU, 260, 275 Kharasch ED, 288, 304 Kharitonov S, 385, 399, 404, 413 Kharitonov SA, 18, 27, 30, 34, 35, 37, 47, 49, 51, 54, 55, 56, 63, 66, 68, 69, 70, 71, 72, 81, 82, 83, 84, 88, 89, 92, 111, 112, 124, 130, 135, 144, 151, 155, 200, 202, 203, 205, 206, 207, 208, 209, 211, 212, 213, 214, 215, 216, 217, 218, 248, 249, 251, 252, 253, 254, 255, 267, 278, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 325, 326, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 344, 346, 359, 360, 369, 375, 378, 380, 388, 393, 400, 404, 413, 414, 428, 429, 430, 440 Khawaja AM, 48, 64 Kheiter A, 176, 194 Kheradmand F, 176, 194 Khostant I, 428, 440 Khullar M, 425, 438

Author Index Kieff E, 424, 437 Kiener RA, 122, 129 Kietzmann D, 371, 375, 380 Kiff RJ, 11, 24 Kikuchi K, 32, 39, 40, 59, 237, 239, 244 Kikuchi T, 135, 151, 206, 217, 319, 337 Kilbourn RG, 345, 361, 434, 442 Kim AM, 123, 129 Kim HK, 384, 399 Kim KJ, 404, 414 Kim S, 325, 340 Kim YK, 43, 62 Kim YM, 12, 25, 424, 437 Kimberly B, 34, 69, 83, 84, 89, 92, 111 Kimura H, 12, 24, 29, 53 Kimura S, 354, 362 King-Biggs MB, 391, 400 Kinnersky R, 206, 217 Kinninmonth A, 435, 436, 443 Kinnula VL, 120, 128 Kirby JA, 385, 399 Kirk M, 167, 171, 189 Kirkman JM, 352, 362 Kirpalani H, 261, 276 Kirsch EA, 34, 68, 202, 212 Kirschmann V, 202, 216 Kirsten AM, 34, 69 Kirsten D, 34, 69 Kispert PH, 167, 188 Kiss A, 206, 208, 215, 217, 317, 321, 335, 337 Kissoon N, 38, 57 Kita D, 81, 88, 200, 207, 211, 309, 311, 312, 330, 331 Kitajima M, 135, 150 Kitamoto S, 406, 416 Kitamura Y, 164, 187 Kitterman JA, 43, 61 Kiuritpnsky A, 17, 26 Klatt P, 204, 214, 250, 255 Klebanoff SJ, 248, 253 Klein AS, 283, 288, 290, 294, 295, 296, 303, 304, 368, 369, 377 Klein PD, 223, 230 Kleinert H, 6, 20

489 Kleinschmidt M, 259, 275 Klienert H, 76, 86 Klinan DM, 427, 439 Kline WO, 6, 20 Klinenberg JR, 427, 439 Klinghofer V, 76, 87, 164, 186, 314, 333 Kluck RM, 123, 129 Kneepkens CM, 229, 231 Kneepkens CMF, 368, 372, 377 Knigge KM, 6, 19 Knight JM, 39, 59 Knorr M, 34, 52, 66, 68, 83, 89 Knowles MR, 411, 418, 419 Knowles RG, 11, 24, 81, 88 Knutson MD, 224, 230 Koarai A, 326, 340 Kobayashi A, 164, 187 Kobayashi H, 313, 332 Kobayashi T, 160, 182 Kobayashi Y, 34, 69 Kobzik L, 6, 17, 19, 27, 37, 57, 76, 80, 83, 87, 88, 89, 118, 128, 164, 186, 235, 236, 243, 308, 328 Kobzik LD, 346, 362 Koch KM, 316, 335, 423, 437 Koch WH, 167, 188 Kochen W, 293, 304 Koesling D, 144, 155 Koeter GH, 31, 34, 54, 68, 70, 95, 112, 200, 211 Kohl J, 45, 62 Kohler N, 140, 153 Kohlhass KL, 6, 9, 19, 23 Kohlmuller D, 293, 304 Kohsaka T, 427, 439 Kohse KP, 13, 24 Kokoszka J, 369, 378 Kolb-Bachofen V, 423, 437 Kolbe J, 200, 212, 309, 329 Kollberg H, 404, 414 Koller EA, 45, 62 Kolls JK, 118, 121, 128, 129, 142, 154 Kolobow T, 364, 377 Komlavilas P, 12, 25 Kompa A, 47, 48, 63

490 Kon A, 250, 254 Kondo M, 36, 40, 47, 55, 60, 63 Kong SK, 170, 191 Kongerud J, 202, 216 Konietzko N, 35, 71 Konno K, 40, 47, 60, 63 Konttinen YT, 432, 441 Kooguchi K, 164, 187 Kooy NW, 164, 186, 263, 277, 359, 362 Koppenol WH, 15, 26, 164, 165, 187 Korsmeyer SJ, 168, 190 Kort E, 31, 54, 95, 112 Korthuis R, 284, 304 Kostka P, 325, 340 Kostoglou-Athanassiou I, 134, 150 Kotsimbos T, 386, 400 Kotsimbos TC, 385, 387, 399, 406, 413, 415 Koty PP, 10, 23 Kourembanas S, 118, 124, 126, 128, 130, 131, 134, 140, 141, 150, 153 Kovacs EJ, 428, 440 Kovesdi I, 424, 437 Kowase M, 406, 416 Koyama A, 36, 55 Koyama H, 160, 164, 177, 183 Koyanagi M, 406, 416 Kozarich JW, 11, 24 Kozma F, 136, 151 Kradin R, 76, 87 Krahenbuhi JL, 162, 184 Kramer M, 394, 401 Krammer PH, 168, 189, 190 Krasney JA, 34, 36, 37, 38, 56, 57, 58, 69, 84, 89, 107, 113, 202, 216 Krebs Y, 222, 230 Krejcy K, 34, 35, 68, 71 Kremer JM, 435, 443 Kriegsfeld LJ, 126, 131 Krishna M, 236, 244 Krishna MC, 171, 172, 192, 193 Krishnan S, 220, 227, 228, 230 Kristof AS, 165, 187 Krochin N, 142, 154, 344, 345, 360 Kroes AC, 316, 335

Author Index Kroesbergen A, 404, 414 Krol M, 321, 337 Kroncke KD, 423, 437 Krotoszynski BK, 229, 231 Krstew E, 45, 62 Krunkosky TM, 160, 164, 177, 183 Kubes P, 171, 191 Kubes PM, 436, 443 Kubo-Inoue M, 406, 416 Kuchino Y, 284, 304 Kuehr J, 311, 331 Kuhl PG, 404, 414 Kuhlen R, 34, 68 Kuhnel A, 427, 439 Kuitert LM, 321, 338 Kula P, 321, 337 Kulik TJ, 43, 61 Kumar GK, 6, 20 Kumlien G, 35, 71 Kundu S, 260, 275 Kunkel SL, 261, 276, 423, 437 Kuo HP, 35, 48, 51, 63, 71, 78, 84, 88, 89, 164, 186, 317, 335, 410, 418 Kuo PC, 29, 53 Kupiec-Weglinski JW, 118, 128 Kuppusamy P, 125, 131, 263, 277 Kura F, 160, 164, 177, 183 Kuramoto H, 409, 417 Kurashina T, 34, 35, 69, 71, 202, 216, 249, 254 Kure E, 225, 230 Kurik MV, 207, 215, 251, 255, 320, 337 Kurmanowska Z, 321, 337 Kurokawa K, 322, 338 Kurthara N, 34, 69 Kutty RK, 118, 127 Kuwahara A, 409, 417 Kuylenstierna R, 37, 56, 83, 84, 89, 315, 334 Kuzma J, 371, 379 Kvieukova I, 6, 20 Kwon G, 423, 437 Kwon NS, 9, 23, 76, 86, 162, 163, 167, 184, 186, 188 Kwon O, 163, 185 Kyrle PA, 35, 71

Author Index

491 L

La Corte R, 432, 433, 441, 442 Labasse AH, 410, 418 LaCourse R, 171, 192 Laine M, 43, 62 Laitinen JT, 135, 148, 150, 155 Lalloo UG, 164, 186, 249, 254, 316, 334 Lamas S, 6, 19, 74, 85 Lamb N, 180, 195 Lamb NJ, 170, 191, 371, 379 Lamont J, 392, 400 Lancaster JR, 16, 26 Lancaster JR Jr, 164, 165, 172, 187, 188, 193, 236, 244 Lancaster JRJ, 171, 192 Lander HM, 167, 189 Landino LM, 14, 25, 322, 339 Landry ML, 316, 335 Landzberg MJ, 40, 49, 60 Lane J, 283, 303 Lang T, 435, 443 Lange M, 435, 443 Langenberg A, 209, 216 Langevitz P, 428, 440 Langford TD, 413, 419 Langton PD, 43, 61 Lanken PN, 169, 190 Lanore T, 375, 380 Lansman JB, 43, 61 Lantoine F, 74, 86, 322, 338 Lanyon LE, 41, 60 Lanz MJ, 311, 312, 313, 331, 332 Lapa ESJ, 164, 187 Lapetina EG, 167, 168, 188 Lapresle J, 136, 152 Larfars G, 74, 86, 322, 338 Larkin S, 14, 25, 406, 415 Laskin DL, 160, 164, 169, 177, 183, 186, 190 Laskin JD, 160, 164, 169, 177, 183, 186, 190 Laskowski D, 97, 109, 112, 235, 236, 237, 238, 239, 240, 242, 243, 244, 249, 254, 262, 277

Lassi K, 34, 70 Lassman CR, 118, 128 Laszlo F, 11, 24 Latta S, 7, 21 Latzin P, 404, 414 Lau KS, 35, 41, 47, 55, 60, 406, 413, 415 Laubach V, 165, 187 Laughner E, 134, 150 Laurent F, 413, 419 Laurito CE, 51, 65 Lauro GM, 167, 189 Lausier J, 124, 130 Lauwerys R, 223, 230 Laval F, 171, 192, 236, 244 Laval J, 236, 244 Lavallee M, 47, 63 Lavergne E, 41, 47, 60 Laverty A, 404, 414 Lavrova OV, 326, 341 Lavrovsky Y, 118, 128 Law S, 318, 336 Lawrence AJ, 45, 62 Layne MD, 118, 128 Le CT, 249, 254, 315, 334 Leach CL, 425, 426, 427, 438 Leak D, 206, 218, 318, 336, 369, 378 LeBlanc A, 222, 230 Lebovitz HE, 371, 379 Leckie MJ, 203, 206, 213, 309, 329 LeCras TD, 240, 245 Lee CS, 118, 126, 128, 131, 205, 214, 251, 255 Lee J, 260, 275 Lee JD, 141, 153 Lee JS, 36, 56 Lee KH, 32, 67 Lee ME, 118, 126, 128, 131, 140, 141, 153 Lee P, 205, 214, 251, 255, 428, 440 Lee PJ, 118, 120, 121, 126, 128, 129, 131, 138, 152 Lee S, 11, 24 Lee SB, 427, 439 Lee TD, 76, 86, 163, 185 Lee TH, 345, 361

492 Lee TM, 9, 23 Lee WJ, 220, 229 Leeb B, 145, 155 Lefevre G, 375, 380 Leff AR, 322, 338 Leff JA, 352, 362, 374, 380 Lefkowitz RJ, 48, 63 LeGendre M, 172, 192 Lehtimaki L, 34, 70 Lei SZ, 168, 189 Leiberman M, 343, 359 Leifert C, 36, 55 Leighton SB, 35, 71, 314, 333 Leise KL, 48, 64, 315, 334 Leivstad T, 382, 394, 398 Leja FL, 371, 379 Lenz AG, 252, 256, 324, 339 Leonard AJ, 385, 386, 399 Leone AM, 11, 24, 29, 30, 33, 35, 37, 39, 47, 48, 52, 53, 54, 73, 85, 91, 111, 200, 207, 211, 237, 239, 240, 244, 267, 278 Leonenko IN, 205, 214 Lepage C, 368, 372, 377 Lepage G, 229, 231 Lepori M, 38, 49, 57, 65, 107, 113, 267, 278 Leroux T, 430, 440 Leseche G, 260, 276 Letterio JJ, 406, 416 Letteron P, 368, 369, 377 Leudke C, 142, 154, 344, 345, 360 Leung BP, 435, 436, 443 Leung DY, 311, 312, 331 Leung S, 406, 417 Levartovsky D, 314, 332, 424, 426, 427, 438 Levere RD, 118, 128 Levesque B, 222, 230 Levi R, 345, 361 Levina DI, 207, 215, 251, 255, 320, 337 Levine A, 423, 437 Levine AS, 203, 213 Levine BE, 345, 361 Levine DM, 167, 189 Levine RL, 168, 190

Author Index Levinson H, 51, 65 Levitt MD, 118, 127, 203, 213 Levo Y, 427, 439 Levy RD, 33, 36, 56, 67 Lewandowski K, 52, 66, 311, 331 Lewandowski M, 52, 66 Lewey FH, 147, 155 Lewis DA, 384, 399 Lewis DH, 384, 390, 399 Lexer F, 35, 71 Li F, 168, 189 Li GK, 6, 7, 19, 21, 74, 85 Li H, 51, 66, 171, 192 Li J, 424, 437 Li LL, 162, 184 Li X, 406, 417 Li XC, 135, 148, 150, 155 Lianos EA, 204, 214, 250, 255 Liao H, 262, 263, 264, 277 Liao JK, 167, 189, 242, 245 Libby P, 167, 189 Lidholm J, 35, 37, 55, 236, 244 Lie JT, 427, 439 Liebmann J, 236, 244 Lieu MW, 33, 67 Liew F, 435, 436, 443 Liew FY, 17, 27, 172, 193, 322, 437 Light B, 317, 335 Light DB, 12, 25 Liles WC, 122, 129 Lillehei C, 384, 398 Lilly CM, 33, 35, 37, 48, 55, 57, 68, 76, 78, 83, 87, 89, 164, 186, 262, 277, 308, 328 Lim S, 76, 87, 203, 206, 208, 213, 215, 217, 308, 309, 310, 311, 312, 313, 314, 318, 321, 328, 330, 331, 333, 336, 337, 404, 413 Lima CS, 51, 66 Lima JAC, 300, 304 Lin CN, 145, 155 Lin HC, 78, 84, 88, 89, 164, 186, 317, 335, 410, 418 Lin RF, 81, 88 Lin Y, 123, 124, 130, 142, 143, 153, 154

Author Index Lincoln TM, 12, 25, 171, 191 Lindberg S, 202, 216 Linden T, 371, 379 Lindholm T, 371, 379 Lindley IJ, 410, 413, 418 Lindley KJ, 409, 417 Lindqvist M, 35, 40, 49, 60 Lindsey JR, 163, 164, 169, 172, 173, 175, 185, 190, 192, 193 Lindstrom AB, 223, 230 Lingaas-Holmen T, 309, 329 Link F, 16, 26 Linker-Israeli M, 427, 439 Linnane SJ, 326, 340, 404, 412, 414 Linsdell P, 404, 414 Lipton SA, 168, 189 Liska J, 52, 66 Little SA, 313, 331 Litzky L, 260, 275 Liu AS, 383, 398 Liu CY, 35, 71, 78, 84, 88, 89, 164, 186, 317, 335 Liu K, 123, 129 Liu L, 168, 189 Liu M, 43, 61, 62, 265, 278 Liu PS, 13, 25 Liu R, 249, 254, 271, 279 Liu S, 48, 51, 63 Liu SF, 49, 50, 65, 76, 87, 375, 380 Liu X, 171, 192 Liu Y, 134, 150 Lizonova A, 424, 437 Ljungkvist G, 34, 70, 203, 212 Llesuy S, 140, 153 Lobo M, 34, 70, 203, 213, 316, 335 Lockhart A, 47, 49, 63, 313, 332 Lodato RD, 345, 361 Lodato RF, 345, 361 Lode H, 252, 256, 324, 339 Loeb AL, 41, 47, 60 Loeb HS, 344, 360 Logan-Sinclair R, 18, 27, 34, 51, 66, 68, 92, 111, 200, 202, 207, 211, 212, 308, 311, 328, 375, 380 Logan-Sinclair RB, 314, 333, 393, 400 Loh E, 39, 49, 59

493 Lohbrunner H, 52, 66 Lohmann SM, 409, 417 Loisance D, 384, 398 Loland L, 313, 332 Long R, 317, 335 Longrois D, 164, 187 Longtine K, 321, 338 Lonnqvist PA, 31, 32, 33, 36, 39, 41, 43, 45, 52, 54, 56, 58, 66 Lonqvist PA, 95, 102, 103, 112 Lopez F, 164, 187 Lopez-Lopez JR, 135, 150 Lopez-Moratalla N, 163, 184 Lopez-Zabalza MJ, 163, 184 LopezGostra JJ, 6, 20 Lorenz H-M, 427, 439 Loscalzo J, 16, 26, 36, 43, 48, 56, 61, 63, 84, 89, 168, 189, 235, 236, 242, 243, 248, 253, 325, 340, 344, 345, 346, 360, 434, 442 Lotvall JO, 78, 87 Lou YP, 124, 130 Loukides S, 135, 151, 200, 202, 206, 208, 211, 215, 216, 217, 249, 251, 254, 314, 315, 316, 318, 321, 326, 333, 334, 336, 338, 341, 429, 430, 440 Love RB, 384, 398 Loveless MO, 316, 335 Lowe JE, 167, 188 Lowe VC, 122, 129, 139, 143, 144, 153 Lowenstein C, 6, 19, 162, 163, 184 Lowenstein CJ, 6, 9, 16, 19, 20, 23, 26, 34, 51, 55, 66, 76, 87, 164, 186, 235, 236, 243, 316, 335, 346, 362, 406, 416 Lowenstein JM, 316, 335 Lowry SF, 345, 360 Lu LC, 410, 418 Lu Y, 409, 413, 418 Lubec B, 34, 70, 202, 212 Lubec G, 34, 70, 202, 212 Lucan H, 35, 71 Lucht WD, 176, 194 Lucia MS, 406, 416 Luckman SM, 6, 20

494

Author Index

Ludden T, 283, 303 Ludviksdottir D, 309, 329 Ludviksdottir DI, 432, 442 Ludwigs U, 40, 60, 102, 112 Lukacs N, 261, 276 Lukacs NW, 423, 437 Lum E, 364, 377 Lum M, 121, 129 Lund MB, 202, 216 Lundberg I, 425, 438 Lundberg JM, 32, 34, 35, 37, 39, 47, 48, 51, 52, 55, 56, 58, 59, 62, 63, 66, 69, 70, 71, 83, 84, 89, 236, 244, 308, 315, 328, 334, 432, 441 Lundberg JO, 34, 35, 36, 37, 39, 52, 55, 58, 66, 69, 70, 83, 84, 89, 200, 211, 263, 277, 315, 334, 404, 414 Lundberg JON, 35, 47, 62, 205, 208, 214, 215, 236, 244, 433, 442 Lundquist I, 135, 150 Luo Y, 125, 130 Lymar SV, 168, 190 Lynch JP, 345, 361 Lynch KR, 7, 21 Lyons R, 250, 254

M Ma N, 126, 131 Ma XL, 204, 214, 250, 254 MacAllister RJ, 81, 88 MacCormack D, 39, 59 Macfarlane JD, 145, 155 Machersie RC, 345, 346, 361 Machtey I, 432, 441 MacLean JA, 17, 27 MacLean L, 282, 303 MacLeod KJ, 313, 331 MacMicking J, 163, 185 MacMicking JD, 163, 165, 171, 186, 187, 192 Macnaughton PD, 260, 275 MacNaul KL, 11, 24 Macpherson H, 434, 442 Madhusudanaraju S, 11, 24

Maeda H, 165, 187 Maeda N, 160, 164, 177, 183 Maemura K, 118, 128 Maeshima H, 137, 152 Maestrelli P, 314, 333, 433, 442 Magde D, 144, 155 Magder S, 33, 36, 56, 67 Maggs R, 206, 217 Magnussen H, 34, 69, 207, 215, 320, 337 Mahadevan V, 371, 379 Mahalingam S, 165, 187 Maier KL, 252, 256, 324, 339 Maines MD, 117, 118, 126, 127, 128, 131, 204, 213, 250, 254 Maini RN, 145, 155, 427, 435, 439, 443 Mains MD, 6, 20 Majori M, 31, 54 Maki T, 164, 187 Makino N, 135, 150 Makita H, 34, 37, 57, 239, 245 Mal H, 260, 276 Malcolm AJ, 383, 398 Malcolm S, 169, 190 Maley W, 288, 290, 294, 296, 298, 299, 304 Malfoy B, 176, 194 Malhotra N, 248, 253, 263, 277, 314, 333 Malik R, 248, 253, 263, 277, 314, 333 Maloney EK, 171, 192 Maltby NH, 39, 59 Malur A, 235, 244, 409, 413, 418 Man SF, 49, 64, 409, 417 Mancuso C, 134, 150 Manfra D, 78, 88 Manger B, 427, 439 Maniscalco M, 35, 72 Manke HG, 208, 215, 327, 341 Mann DA, 316, 335 Mann GE, 135, 150 Mannaioni PF, 135, 151 Mannick JB, 168, 189, 424, 437 Manning J, 163, 186 Manning PT, 11, 24

Author Index Mantell LL, 121, 122, 123, 129, 130, 142, 144, 154, 155, 204, 214 Manthrope R, 431, 441 Mapp CE, 314, 333, 433, 442 Maragos CM, 167, 188 Marangon K, 322, 339 Marathia K, 34, 69 Marcer G, 309, 329 Marchevsky A, 394, 401 Marczin N, 35, 37, 56, 71, 96, 97, 105, 106, 112, 113, 260, 264, 268, 275, 278, 405, 412, 415 Marczynski B, 202, 216 Margraf LR, 35, 55, 406, 413, 415 Marilena G, 136, 151 Marinii JJ, 354, 362 Marino M, 163, 186 Mariotto S, 167, 189 Markewitz BA, 405, 412, 415 Marklund SL, 161, 183 Marks GB, 52, 66, 309, 329 Marks GS, 325, 340 Marletta MA, 10, 23, 162, 165, 177, 184, 187, 194, 238, 245 Marlin DJ, 33, 45, 62 Marnett LJ, 14, 25, 322, 339 Marsden PA, 6, 8, 19, 21, 74, 85 Marshall PA, 15, 26, 161, 183, 263, 278 Marshall S, 11, 24 Martasek P, 162, 184 Marti HH, 6, 20 Martin EB, 42, 43, 61 Martin JC, 165, 188 Martin LD, 160, 164, 177, 183 Martin-Mola E, 425, 438 Martin-Sanz P, 406, 416 Martin TR, 122, 129, 143, 154, 179, 180, 194, 345, 346, 361 Martin U, 34, 69 Martin WJ, 170, 181, 191 Martinez C, 424, 437 Martinez Palli G, 34, 70 Marumo F, 409, 413, 418 Maruyama T, 432, 441 Marver HS, 117, 127, 137, 152

495 Mascha EJ, 260, 275 Mascheroni D, 364, 377 Mascolo N, 409, 417 Masini E, 135, 151 Mason NA, 385, 390, 393, 399 Mason SN, 10, 23, 424, 437 Massaro AF, 34, 37, 57, 69, 79, 81, 83, 84, 88, 89, 200, 202, 207, 211, 212, 308, 309, 311, 312, 328, 330, 331 Massaro DJ, 121, 129, 143, 154 Massias L, 375, 380 Masters BS, 163, 186 Masters BSS, 162, 184 Masuda H, 34, 69 Matalon S, 49, 64, 159, 162, 163, 164, 169, 170, 172, 173, 175, 176, 177, 178, 179, 180, 181, 182, 182, 184, 185, 190, 191, 193, 194, 195, 260, 276, 372, 380 Matches C, 13, 25 Mateo RB, 423, 437 Matheson PJ, 136, 151 Matouk C, 411, 419 Matsamura R, 432, 441 Matsuda M, 32, 67 Matsui H, 411, 419 Matsumoto K, 135, 150 Matsumoto S, 44, 62 Matsumoto T, 6, 19 Mattes J, 311, 331 Matthay MA, 176, 177, 180, 181, 182, 194, 260, 274, 275, 279, 345, 346, 361 Matthews D, 162, 184 Matulla B, 35, 38, 58 Matute-Bello G, 122, 129 Maunder RJ, 143, 154 Mauney MC, 261, 276 Mayer B, 43, 61, 74, 85, 168, 190, 204, 214, 250, 255 Mayoral V, 33, 68 Maziak W, 311, 314, 331, 333 Mazzi P, 309, 329 McBrien D, 283, 303 McCall MN, 169, 191

496 McCarthy TJ, 265, 278 McCartney-Francis N, 433, 442 McCauley E, 11, 24 McClean P, 47, 49, 63, 84, 89, 96, 112, 202, 212, 309, 328 McClean PA, 37, 38, 57, 200, 207, 211, 310, 312, 330 McCord J, 282, 284, 301, 303, 304 McCord JM, 167, 189, 352, 362, 374, 380 McCormick DR, 311, 331 McCoubrey W, 126, 131 McCoubrey WKJ, 117, 118, 127, 204, 213, 250, 254 McDaniel ML, 423, 437 McDermott CD, 385, 393, 399 McDonagh AF, 119, 125, 128, 128, 139, 140, 152 McDonald B, 167, 168, 188 McElroy MC, 171, 192 McGee AW, 76, 86 McGrath JL, 35, 72 McGraw DJ, 32, 33, 36, 41, 44, 56, 60, 67, 103, 112 McGriffin DC, 180, 194 McInnes IB, 435, 436, 443 McIntyre IA, 436, 443 McKenzie DC, 34, 38, 58 McKenzie H, 36, 55 McKinney S, 32, 39, 43, 59, 237, 238, 244 McKnight GM, 202, 212 McLean P, 388, 400 McLoughlin P, 249, 253, 254, 316, 326, 334, 340, 404, 412, 414 McMahon T, 325, 340 McMahon TJ, 49, 65 McMillan A, 316, 335, 406, 416 McMillan J, 207, 215, 252, 255 McMillan K, 162, 184 McMullan DM, 250, 254 McMurtry IF, 42, 60, 240, 242, 245, 249, 254, 315, 334 McNamara JL, 32, 66 McQueston JA, 242, 245 McSharry C, 313, 331

Author Index Mead JF, 370, 371, 378, 379 Meah S, 312, 314, 331, 333 Medeiros MV, 51, 66 Mee TR, 316, 335 Mehao G, 6, 20 Mehta AC, 260, 275 Mehta S, 17, 27, 33, 36, 37, 56, 57, 68, 78, 80, 83, 87, 88, 89, 308, 328 Meijer RJ, 31, 54, 95, 112 Meinkoth JL, 167, 189 Melcher A, 35, 40, 49, 60 Meldolesi H, 12, 25 Melillo G, 163, 185, 242, 245 Melinek J, 118, 128 Melkova Z, 427, 439 Menconi MJ, 176, 194 Mendis S, 368, 369, 371, 373, 377, 378, 379 Menegazzi M, 167, 189 Meng Q-H, 405, 413, 415 Menkes CJ, 428, 434, 440, 442 Mensik C, 34, 68 Merritt TA, 170, 176, 191, 194 Merritt WT, 288, 290, 294, 296, 304 Merryman PF, 436, 443 Merryweather J, 345, 360 Meschter C, 433, 442 Messent M, 81, 88, 260, 275 Messmer UK, 15, 26 Metha S, 33, 67 Metnitz PGH, 374, 380 Metz S, 11, 24 Meuwese-Arends MT, 371, 379 Meyer J, 135, 151, 206, 218, 320, 337 Meyer KC, 34, 38, 39, 58, 107, 113, 384, 398 Meyer KL, 423, 437 Meyers K, 433, 442 Meyrick B, 345, 361 Mezey E, 288, 290, 294, 296, 304 Miao QX, 168, 189 Michael GJ, 6, 19 Michael JR, 171, 192 Michel BA, 436, 443 Michel T, 6, 7, 19, 21, 35, 55, 74, 76, 85, 86, 162, 184, 248, 253, 434, 442

Author Index Michler E, 411, 419 Michler RE, 262, 263, 264, 277 Middleton ET, 206, 215 Midtvedt T, 30, 33, 35, 37, 47, 54 Miekisch W, 368, 369, 372, 373, 374, 378 Miescher E, 282, 303 Mikkelsen K, 207, 215 MilbergJA, 143, 154 Milczuk HA, 47, 63, 202, 212 Milla PJ, 409, 417 Millatmal T, 34, 70 Miller MF, 76, 87 Miller MS, 171, 192 Miller NJ, 139, 140, 152 Miller SM, 126, 131 Milligan SA, 405, 406, 412, 415, 416 Millington DS, 39, 59 Mills PC, 33, 45, 62 Mills TM, 6, 20 Milne DS, 383, 398 Milner AD, 139, 140, 152 Miloso M, 12, 25 Milsark IW, 142, 154, 344, 345, 360 Mims JW, 77, 87 Minami M, 392, 400 Ming Z, 47, 63 Minor RL, 16, 26 Mirabella C, 135, 151 Misko TP, 81, 88, 384, 390, 399 Misra M, 167, 188 Mister RL, 309, 330 Mistretta A, 33, 48, 51, 64 Misukonis MA, 10, 23, 424, 435, 437, 438, 443 Mitchell J, 271, 279 Mitchell JA, 6, 7, 9, 11, 14, 15, 19, 21, 23, 25, 26, 48, 51, 64, 74, 85, 406, 415 Mitchell JB, 171, 172, 192, 193, 236, 244 Mitchell MC, 288, 290, 294, 296, 304 Mitsufuji H, 313, 332 Mittal CK, 12, 24, 29, 53 Miyahara K, 409, 413, 418 Miyake S, 434, 442

497 Miyamoto K, 34, 37, 57, 239, 245 Miyazaki H, 171, 192 Miyazaki N, 322, 338 Miyazawa N, 313, 332 Mizel DE, 433, 442 Mizukami H, 171, 192 Mizuta K, 319, 337 Mizuta T, 385, 392, 399, 400 Mochida M, 34, 69 Modelska K, 171, 192 Modin A, 263, 277, 432, 441 Modoell M, 16, 26 Mohanakumar T, 265, 278 Mohr S, 15, 26 Mohsenin V, 37, 57 Moilanen E, 34, 70 Molad Y, 427, 439 Moldeus P, 284, 304 Molina YVL, 167, 168, 188 Moncada S, 7, 9, 11, 17, 21, 23, 24, 27, 29, 30, 37, 39, 48, 49, 50, 52, 53, 54, 65, 73, 81, 85, 88, 91, 111, 162, 163, 164, 172, 184, 185, 187, 193, 200, 207, 211, 237, 239, 240, 244, 262, 267, 277, 278, 430, 433, 440, 442 Mongovin S, 122, 129 Monitzer B, 35, 71 Monma M, 135, 151, 206, 217, 317, 319, 336, 337 Monsallier JF, 375, 380 Montecot C, 134, 150 Montuschi P, 124, 130, 135, 151, 206, 208, 209, 215, 217, 218, 252, 255, 310, 312, 313, 318, 323, 324, 325, 325, 326, 330, 336, 339, 340, 344, 360, 404, 414 Moodley YP, 164, 186, 249, 254, 316, 334 Moody A, 200, 212, 309, 329 Moore AR, 143, 154 Moore FA, 374, 380 Moore P, 37, 56 Moore TM, 39, 49, 59 Moore WM, 11, 24 Morales TI, 436, 443 Moran F, 427, 439

498 Morcos E, 425, 438 Morello M, 248, 253, 429, 430, 440 Moretti MP, 364, 377 Morgan CJ, 260, 275 Morgan EN, 261, 276 Morgan K, 405, 413, 415 Morgan MJ, 126, 131 Morganroth ML, 262, 277 Morgenstern DE, 162, 184 Mori M, 6, 20 Mori TA, 322, 339 Morice AH, 206, 215 Morimoto S, 427, 439 Morin FC, 51, 65, 170, 191 Morita S, 368, 369, 378 Morita T, 124, 126, 130, 131, 134, 140, 141, 150, 153 Mormile M, 35, 72 Mornex JF, 430, 440 Morris BJ, 409, 417 Morris KG Jr, 240, 245 Morris NH, 34, 68 Morris SM Jr, 10, 23 Morrison DC, 344, 360, 406, 416 Morrison JFJ, 309, 329 Morrow JD, 14, 25, 252, 255, 322, 323, 338, 339 Mosgoller W, 262, 277 Moshage H, 34, 70, 314, 333 Mossalayi MD, 163, 185 Mosser DM, 172, 193 Mott JC, 51, 65 Motterlini R, 123, 130, 135, 139, 150, 153, 204, 214, 251, 255 Mourgeon E, 265, 278 Moutsopoulos HM, 432, 441 Moynihan J, 249, 253, 254, 316, 326, 334 Moynihan JB, 326, 340, 404, 412, 414 Mozalevskii AF, 208, 215 Muallem S, 13, 25 Mucke M, 207, 215, 320, 337 Mudgett JS, 10, 23, 171, 192, 424, 438 Mullen JB, 33, 67 Muller M, 344, 346, 360, 371, 375, 380 Muller T, 318, 336

Author Index Muller W, 17, 27, 31, 54, 202, 216, 430, 440 Muller WPE, 365, 368, 369, 372, 373, 374, 377, 378 Mulligan MS, 165, 187 Mullins ME, 168, 189, 248, 253, 434, 442 Mulsch A, 7, 9, 21, 23 Mumford R, 164, 187, 435, 436, 443 Mumford RA, 9, 23, 76, 86, 163, 185 Muphy TJ, 7, 21 Murad F, 6, 7, 9, 11, 12, 13, 19, 21, 23, 24, 29, 38, 53, 58, 76, 87 Muramatsu M, 249, 254, 315, 334 Murase K, 136, 152 Murashko VA, 205, 214 Murphy AW, 34, 70, 203, 213, 316, 335 Murphy SP, 38, 57 Murphy T, 12, 25 Murphy WJ, 163, 185, 406, 416 Murray JJ, 323, 339 Murray PJ, 163, 185 Murray T, 76, 87 Murrell GAC, 436, 443 Murry JF, 344, 346, 360, 362 Musso T, 163, 185, 242, 245 Mustafa ME, 44, 62 Myers PR, 16, 26 Myles C, 170, 179, 180, 181, 191, 194

N Nabeshima T, 136, 152 Nadel JA, 33, 48, 51, 64, 124, 130 Nagai A, 326, 341 Nagai R, 35, 71, 202, 216, 249, 254, 312, 331 Nagano S, 427, 439 Nagase S, 36, 55 Nairn AC, 409, 417 Naka Y, 262, 263, 264, 277 Nakajima H, 126, 131 Nakamura H, 206, 217 Nakamura T, 34, 35, 69, 71, 82, 89, 202, 216, 249, 254, 312, 331

Author Index Nakane M, 6, 7, 11, 19, 21, 23, 76, 87 Nakano H, 32, 39, 40, 59, 237, 239, 244 Nakano M, 411, 419 Nakano T, 34, 37, 57, 239, 245 Nakashima H, 427, 439 Nakashima S, 13, 24 Nakata J, 326, 341 Nakatsu K, 325, 340 Nakayama DK, 16, 26 Nakayama T, 145, 155 Namiesnik J, 225, 230 Nandi J, 32, 67 Naqvi S, 314, 332 Narula P, 176, 194 Naruse K, 43, 61 Nascimento-DaSilva V, 164, 186, 411, 419 Natanson C, 345, 361 Nath DA, 118, 127 Nath K, 125, 131, 139, 153 Nath KA, 139, 140, 153 Nathan C, 9, 23, 74, 76, 85, 87, 163, 164, 168, 176, 185, 186, 187, 193, 235, 236, 244, 428, 440 Nathan CF, 76, 86, 162, 165, 167, 171, 184, 187, 188, 189, 192, 406, 416, 433, 442 Nauseef WM, 160, 182 Navarra P, 134, 150 Navarro JA, 425, 438 Navas JP, 7, 21 Ndisang JE, 135, 151 Nebenzahl H, 133, 149 Nehls M, 424, 437 Nejadnik B, 34, 69, 83, 84, 89, 92, 111 Nelin LD, 32, 39, 40, 59, 239, 240, 242, 245, 262, 277 Nelson DP, 32, 66 Nelson HS, 313, 332 Nelson RL, 369, 378 Nelson WC, 228, 230 Nemecek-Marshal M, 371, 379 Nemeth ZH, 141, 153 Nerem R, 7, 21 Nerem RM, 7, 21

499 Neubauer G, 322, 338 Neuhauser MM, 327, 341 Neumann C, 423, 437 Newman-Taylor A, 206, 217 Newman-Taylor AJ, 309, 329 Newmeyer DD, 123, 129 Newton R, 321, 338 Ng C, 34, 70 Nguyen BL, 12, 25 Nguyen PH, 34, 68 Nicholson S, 163, 164, 186, 187 Nicloux M, 133, 149 Nicod LP, 159, 176, 182 Nicod P, 38, 49, 57, 65 Nicolaides KH, 34, 68 Nicole C, 222, 230 Niedermeyer ME, 176, 194 Nielsen VG, 162, 184 Nieszpaur-Los M, 35, 71 Nightingale J, 252, 255, 323, 324, 339, 344, 360 Nightingale JA, 34, 70, 200, 203, 206, 207, 208, 209, 211, 212, 215, 217, 320, 337 Niho Y, 427, 439 Niida Y, 119, 128, 128, 148, 156 Niiro H, 427, 439 Nijkamp FP, 48, 51, 64 Nikbakht-Sangari MN, 260, 276 Nikberg II, 205, 214 Nilanjana M, 118, 128 Nilsson F, 207, 215 Nilsson P, 371, 379 Nilsson-Thorell C, 371, 379 Nims RW, 236, 244 Nisen PA, 6, 20 Nishida K, 7, 21 Nishimura G, 242, 245 Nishimura JS, 162, 184 Nishimura K, 326, 341 Nishimura M, 34, 37, 57, 239, 245 Nishimura S, 284, 304 Nishioka K, 428, 440 Nistic G, 12, 25 Nistico G, 6, 12, 13, 20, 25, 25 Niu G, 122, 129

500

Author Index

Nobuhiro I, 9, 23 Noguchi S, 176, 193 Noguchi T, 32, 67 Noguchi Y, 165, 187 Nohl H, 161, 183 Nolkrantz K, 34, 70 Nomura A, 33, 68 Nonaka S, 34, 69 Nong Z, 250, 254 Nordvall SL, 37, 56, 83, 84, 89, 315, 334, 404, 414 Norman M, 31, 54 Noronha-Dutra A, 172, 193 Norregaard J, 207, 215 Norris EJ, 283, 295, 303, 368, 369, 377 North AJ, 6, 20, 35, 55 North RJ, 171, 192 North SL, 161, 183 Novak R, 163, 185 Novick RJ, 260, 275, 381, 397 Novikova NP, 208, 215 Novogrodsky A, 167, 189 Nowak D, 321, 337, 338 Nozawa K, 427, 439 Nozawa Y, 13, 24 Nunokawa Y, 9, 23 Nurnberg B, 13, 25 Nussinovitch, 43, 61 Nussler AK, 10, 16, 23, 26 Nyberg F, 6, 20 Nyham D, 284, 286, 290, 299, 304 Nyman G, 52, 66 Nyman OP, 170, 191 Nyui S, 160, 164, 177, 183

O Oates JA, 323, 339 Oates JC, 424, 438 Oates T, 312, 331 O’Brien GD, 206, 216 O’Connor BJ, 34, 35, 69, 72, 200, 201, 211, 308, 309, 310, 311, 312, 313, 314, 328, 329, 330, 333, 344, 346, 360, 393, 400 O’Connor CM, 249, 253, 254, 316, 326, 334, 340, 404, 412, 414

Oddou MF, 34, 69 O’Donnell CA, 172, 193 O’Donnell DM, 249, 253, 254, 316, 326, 334 O’Donnell VB, 263, 278 O’Donovan PB, 260, 275 O’Driscoll F, 36, 55, 83, 84, 89 Ogasa T, 32, 39, 40, 59, 237, 239, 244 Ogita K, 136, 152 Ogletree ML, 176, 194 Ognibene FP, 108, 113 Ohgami E, 427, 439 Ohinishi ST, 169, 190 Ohkawa M, 282, 303 Ohtuska H, 136, 152 Ohyama Y, 34, 69 Oj JA, 313, 332 Okabayashi K, 384, 392, 399 Okamoto H, 406, 416 Okamura H, 44, 62 Okano Y, 13, 24 Okazaki H, 32, 67 Okinaga S, 206, 217, 317, 335 Oksne PI, 202, 216 Okusawa S, 344, 360 Oldfield W, 322, 323, 325, 338 Oler A, 32, 33, 67 Olin AC, 34, 70, 203, 212 Oliveira H, 249, 253, 315, 334 Olivenstein R, 321, 338 Oliver B, 314, 333 Olopade CO, 34, 68, 372, 379 O’Loughlin EV, 324, 339 Olson DM, 43, 61 Olsson T, 425, 438 Olszowka AJ, 38, 57, 84, 89 Ondrula D, 369, 378 O’Neal KD, 406, 417 O’Neill CA, 167, 188 O’Neill HJ, 229, 231 Ongaro R, 31, 54, 92, 111, 309, 311, 312, 329, 331, 404, 414 Ono Z, 34, 69, 82, 89, 312, 331 Onozuka K, 411, 419 Ootsuyama A, 284, 304 Oplinger JA, 11, 24 Oppedisano R, 235, 244, 249, 254

Author Index

501

Opperhuizen A, 31, 34, 54, 203, 212, 309, 330 Oppliger L, 436, 443 O’Reilly P, 180, 194 Orellano T, 147, 155 Oriani G, 32, 48, 64 Orme IM, 162, 184 Ormerod A, 36, 55 Orr LM, 35, 72 Orsida B, 385, 387, 392, 393, 399, 400, 406, 413, 415 Osanai S, 32, 39, 40, 59, 237, 239, 244 Osawa Y, 172, 176, 192, 193 Osborne JA, 434, 442 Osborne ML, 318, 336 Osbourne JA, 248, 253 Otsuka T, 427, 439 Ott GR, 295, 304 Otterbein L, 118, 120, 121, 122, 128, 129, 135, 139, 142, 143, 144, 150, 153, 154, 155 Otterbein LE, 123, 124, 130, 142, 154, 204, 214 Otterbein SL, 139, 143, 144, 153 Oury TD, 161, 183 Owens MW, 405, 406, 412, 415, 416 Oxholm P, 431, 441 Oz MC, 262, 263, 264, 277 Ozeri-Chen T, 427, 439

P Pacelli R, 172, 193, 236, 244 Packer L, 322, 339 Padmaja S, 165, 188 Paganini N, 311, 312, 331, 404, 414 Page CP, 48, 64 Pahan K, 406, 416 Paik J, 163, 186, 428, 440 Paine R3, 177, 194 Paith JE, 11, 24 Pakbaz H, 165, 187 Palaia TA, 122, 129 Paler-Martinez A, 263, 277 Palevsky HI, 260, 275 Palm JP, 36, 55 Palmblad J, 74, 86, 322, 338

Palmer RM, 29, 53, 73, 85, 162, 164, 184, 187 Palmer RMJ, 7, 9, 11, 21, 23, 24, 262, 277, 433, 442 Pan ZH, 168, 189 Panagou P, 326, 341 Panahian N, 118, 128 Pandol SJ, 13, 25 Pannen BH, 140, 153 Pantelidis P, 249, 251, 252, 254, 255, 316, 318, 324, 334, 339, 429, 430, 440 Panus PC, 260, 276 Panza JA, 108, 113 Panza N, 311, 312, 331, 404, 414 Paoletti AM, 6, 20 Papadimitriou CS, 432, 441 Papatheodorou G, 326, 341 Papi A, 432, 441 Papiris SA, 433, 442 Pappert D, 39, 52, 58, 66, 83, 89 Paradis IL, 391, 400 Parameswaran K, 199, 211, 317, 335 Pare PD, 48, 64, 202, 205, 214, 216 Paredi P, 124, 130, 135, 151, 200, 203, 206, 211, 213, 217, 218, 249, 251, 252, 254, 255, 309, 316, 317, 318, 320, 324, 329, 334, 335, 336, 337, 339, 369, 378, 404, 414, 429, 430, 440 Parent R, 47, 63 Parfett GJ, 385, 399 Pargament GA, 167, 188 Pariente R, 260, 276 Parillo JE, 344, 346, 360 Park JK, 325, 340 Park SH, 161, 183 Parker RF, 172, 193 Parkhomenko NV, 207, 215, 251, 255, 320, 337 Parkington HC, 43, 62 Parks D, 282, 284, 303 Parks DA, 162, 184 Parmely TJ, 406, 416 Parrat JR, 345, 361 Parry AJ, 250, 254 Parry SL, 141, 153

502 Parsons PA, 374, 380 Pataki G, 169, 176, 190, 372, 380 Patel K, 314, 332 Patel P, 314, 332 Patel R, 314, 332 Patel RN, 436, 443 Patterson GA, 265, 278, 384, 390, 399 Patton LM, 352, 362 Pattulo S, 36, 55 Paul K, 326, 340, 404, 414 Paulson SE, 227, 230 Pavord ID, 322, 338 Pawliszyn J, 225, 230 Payen DM, 410, 418 Peach MJ, 7, 21, 41, 47, 60 Pearl JM, 32, 66 Pease-Fye ME, 108, 113 Pedersen S, 313, 332 Pederson TC, 160, 164, 177, 183 Pedoto A, 32, 33, 67 Pedroletti C, 36, 37, 55, 208, 215, 433, 442 Peersson MG, 237, 239, 240, 244 Pegelow DF, 34, 38, 39, 58, 107, 113 Pekins DJ, 435, 443 Pelech SL, 144, 155 Pelizzoni A, 31, 54 Pelligrino DA, 51, 65 Pellizzari ED, 223, 228, 230 Pendergast DR, 34, 36, 56, 58, 69, 107, 113, 202, 216 Pendino KJ, 160, 164, 177, 183, 186 Peng HB, 167, 189, 242, 245 Peng T, 123, 129 Peng Y, 369, 378 Penney DG, 134, 149 Pepper JR, 271, 279, 385, 399 Perez J, 163, 185, 327, 341 Perez Leiros C, 432, 441 Perillo IB, 34, 69, 107, 113, 267, 278 Perkins PT, 34, 69, 107, 113, 267, 278 Perler BA, 288, 290, 294, 296, 298, 301, 304 Pernerstorfer T, 35, 71 Peroni D, 309, 329 Peroni DJ, 76, 77, 87

Author Index Perrella MA, 118, 122, 128, 140, 141, 153 Perrella MS, 126, 131 Perretti M, 410, 418 Perrotta MG, 160, 164, 177, 183 Persichini T, 167, 189 Persson MG, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 47, 48, 48, 49, 50, 51, 51, 52, 53, 54, 56, 57, 58, 64, 65, 66, 67, 68, 71, 73, 83, 85, 89, 91, 95, 97, 102, 103, 106, 111, 112, 113, 200, 203, 207, 211, 213, 267, 268, 278, 308, 328 Pesci A, 31, 54, 325, 340 Peter ME, 168, 189, 190 Peterson C, 432, 442 Peterson JE, 207, 215 Peti-Peterdi J, 180, 194 Petkov V, 262, 277 Petrache I, 123, 130 Petreas M, 225, 230 Petri M, 425, 438 Petrova MA, 326, 341 Petrusz P, 77, 87 Petty TL, 345, 346, 361, 363, 376 Pfeifer A, 13, 25 Pfeiffer S, 168, 190 Pham SM, 260, 275 Phelan MW, 242, 245, 262, 277 Phelan P, 324, 339 Pheng LH, 410, 418 Philippe C, 163, 185, 327, 341 Phillips CR, 34, 35, 38, 58, 107, 113, 202, 212, 316, 335 Phillips M, 219, 220, 224, 227, 228, 229, 229, 230, 231, 368, 369, 371, 372, 377, 378, 379 Piacentini GL, 31, 54, 92, 111, 202, 212, 309, 329 Piantadosi CA, 39, 59, 118, 122, 128 Piasecka G, 321, 337 Pickering AC, 309, 329 Piedbouef B, 122, 129 Pieramiei SP, 6, 20 Pierre AF, 265, 278 Pietras T, 321, 337, 338

Author Index Pietropaoli AP, 34, 69, 107, 113, 267, 278 Piguet PF, 123, 129 Pillai R, 261, 276 Pilz RB, 144, 155, 167, 189 Pinard E, 134, 150 Pinsky DJ, 262, 263, 264, 277 Pinsky MR, 32, 67 Piper P, 43, 61 Piper S, 428, 440 Pipis S, 309, 329 Pippen AM, 424, 438 Pisa E, 432, 441 Pisetsky DS, 424, 435, 437, 438, 443 Pison U, 164, 187 Pitsillides AA, 41, 60 Pitt BR, 424, 437 Pittet JF, 171, 192, 345, 346, 361 Pitzele BS, 11, 24 Pizzichini E, 199, 211 Pizzichini MM, 199, 211 Platts LAM, 432, 441 Platts-Mills TA, 34, 70, 203, 213, 248, 253, 263, 277, 314, 333 Platts MT, 316, 335 Pleil JD, 223, 230 Plotkowski MC, 164, 186, 411, 419 Poderoso JJ, 167, 171, 188, 192 Pogliaghi S, 36, 56 Pohlman TH, 261, 276 Pokhaznikova MA, 327, 341 Pokreisz P, 250, 254 Polack JM, 9, 23 Polak J, 37, 56, 106, 113, 268, 278 Polak JM, 17, 27, 48, 64, 249, 253, 308, 315, 328, 334, 385, 390, 393, 399, 405, 413, 415, 432, 441 Polissar NL, 32, 67, 237, 238, 244 Polizzi S, 425, 426, 427, 438 Pollock JS, 6, 7, 11, 19, 21, 23, 74, 76, 85, 87, 164, 186, 249, 253, 314, 315, 333, 334 Pollock WH, 371, 379 Polunovsky VE, 122, 129 Pomerance A, 385, 390, 393, 399 Ponaman M, 325, 340

503 Porter JW, 370, 378 Pospischil M, 163, 186 Poss KD, 119, 123, 124, 126, 128, 128, 130, 131, 134, 142, 143, 150, 153, 154, 205, 214 Poss WB, 171, 192 Post M, 43, 61, 62 Postma DS, 31, 34, 54, 68, 70, 95, 112, 200, 211, 314, 333, 426, 438 Potena A, 432, 433, 441, 442 Potenza B, 321, 338 Potter J, 288, 290, 294, 296, 298, 299, 304 Potter S, 163, 186 Pou S, 74, 85 Pou WS, 74, 85 Pourzand C, 123, 129 Prabhakar NR, 6, 20, 134, 135, 150 Pratico D, 324, 339 Prato P, 364, 377 Prazma J, 77, 87 Preas HL, 35, 71, 314, 333 Prehn J, 427, 439 Premknmar D, 6, 20 Prentiss RA, 203, 213 Prescott RJ, 427, 439 Presta A, 238, 244 Preusser S, 259, 275 Preziosi P, 134, 150 Price PV, 322, 339, 372, 375, 379 Price R, 76, 78, 87 Primiano T, 125, 131 Prince A, 409, 417 Prince R, 172, 193 Pritchard K, 7, 21 Pritchard KA Jr, 242, 245 Propst SM, 406, 415 Pryor WA, 15, 26 Przyjazny A, 225, 230 Przyk E, 225, 230 Psathakis K, 326, 341 Puddey IB, 322, 339 Puiatti P, 248, 253, 429, 430, 440 Punjabi CJ, 160, 164, 177, 183, 186 Purves MJ, 44, 62 Puyana JC, 321, 338

504

Author Index Q

Qayumi AK, 260, 276 Quaaz F, 163, 185 Quaggiotto P, 324, 339 Quertermous T, 7, 21 Quick RA, 316, 335 Quinlan GJ, 170, 180, 191, 195, 271, 279, 371, 379 Quinones S, 169, 190 Qureshi S, 406, 417

R Raake JL, 32, 66 Raatgeep HC, 321, 337 Racke MK, 423, 437 Radi R, 167, 168, 171, 189, 190 Radice S, 32, 48, 64 Radomski A, 49, 64, 409, 417 Radomski MW, 9, 23, 49, 64, 409, 417 Radzioch D, 411, 419 Rafice AL, 327, 341 Rai P, 125, 130 Raij L, 49, 65 Raine CS, 423, 437 Raivio KO, 120, 128 Rajavashisth TB, 167, 189 Ralston SH, 434, 442 Ramdev R, 168, 189 Ramnarine SI, 48, 64 Ramsay AJ, 17, 27, 165, 188 Randall YK, 180, 194 Randell SH, 411, 419 Rannels DE, 43, 61 Rao S, 6, 20 Rappaport SM, 225, 230 Rapport RM, 13, 24 Rascher W, 404, 414 Rasmussen K, 316, 335 Ratan RR, 76, 86 Rathi S, 204, 214, 250, 254 Rathmell JC, 168, 189 Ratjen F, 35, 71, 317, 326, 335, 340, 404, 411, 412, 414, 419

Ratnatunga CP, 260, 275 Ratner AJ, 409, 417 Ratner L, 301, 304 Rattan S, 204, 214, 250, 254 Rau M, 252, 256, 324, 339 Raung SL, 145, 155 Raushel FM, 238, 245 Rawlinson SC, 41, 60 Ray A, 316, 335 Raychaudguri B, 409, 413, 418 Raychaudhuri B, 235, 244 Raymer JH, 228, 230 Raymond JR, 406, 416 Raza R, 314, 332 Read RC, 145, 155 Reader JA, 385, 399 Reardon JE, 371, 379 Reaziekhaligh MH, 31, 54 Rebich S, 47, 63 Redington A, 17, 27, 78, 88, 308, 328 Rediske J, 424, 426, 427, 438 Reed RR, 6, 19, 162, 163, 184 Reelfs O, 123, 129 Reep B, 167, 168, 188 Rees RS, 345, 361 Reeve HL, 242, 246 Regine JE, 364, 377 Reginster JY, 410, 418 Reichner JS, 423, 437 Reid D, 393, 400 Reid DM, 434, 442 Reilly J, 168, 189 Reilly JJ, 37, 57, 83, 89, 308, 328 Reilly P, 282, 284, 303 Reily CA, 343, 359 Reily CM, 6, 20 Reinert P, 205, 214, 251, 255 Reinhardt D, 404, 414 Reinhold P, 209, 216 Reining U, 311, 331 Reis A, 260, 276 Reiser PJ, 6, 20 Reiter R, 283, 303 Reitz BA, 261, 276 Rektorik J, 368, 369, 378

Author Index Rengasamy A, 39, 59 Rennard S, 34, 70 Rensen L, 206, 218 Rensing S, 52, 66 Renzi PM, 78, 88 Repine JE, 145, 155, 261, 276, 320, 337, 352, 362, 374, 380 Reynolds HY, 145, 155 Rhodes GC, 122, 129 Rhodes P, 16, 26 Ribas J, 35, 72, 313, 332 Ribeiro SP, 33, 67 Ricciardolo FL, 33, 48, 51, 64 Riccio M, 12, 25 Rice TW, 34, 48, 51, 55, 77, 87, 164, 186, 235, 236, 243, 262, 277, 406, 411, 413, 415 Rice-Evans CA, 139, 140, 152 Rich A, 126, 131 Rich GF, 33, 67 Richards MK, 177, 194 Richaud C, 118, 127 Richter K, 207, 215, 320, 337 Rico P, 32, 67 Riedel B, 35, 37, 56, 71, 96, 97, 105, 106, 112, 113, 268, 278 Riley D, 300, 304 Riley JM, 344, 346, 360 Riley L, 176, 193 Riley LW, 176, 193 Rimar S, 36, 37, 38, 56, 108, 113 Rinder J, 34, 35, 37, 47, 55, 56, 62, 69, 70, 236, 244 Ringqvist A, 35, 71 Rink TJ, 43, 61 Ripple R, 17, 26 Rippo MR, 163, 185 Risby T, 283, 290, 295, 298, 301, 303, 304 Risby TH, 283, 284, 286, 288, 290, 292, 294, 295, 296, 298, 299, 300, 301, 303, 304, 343, 359, 368, 371, 373, 378 Ritter JH, 265, 278 Ritter JM, 47, 63

505 Ritter T, 118, 128 Rivera JH, 322, 339 Riveros-Moreno V, 17, 27, 78, 88, 163, 185, 308, 328 Road J, 34, 38, 58 Robbins CG, 170, 176, 191, 194 Robbins RA, 18, 27, 34, 35, 48, 51, 64, 66, 69, 70, 71, 81, 88, 92, 111, 171, 191, 200, 203, 207, 211, 213, 271, 279, 308, 311, 312, 313, 314, 315, 327, 328, 331, 333, 334, 341, 375, 380, 405, 412, 415 Roberts AB, 406, 416 Roberts AM, 52, 66, 309, 329 Roberts JD, 36, 56 Roberts LJ, 252, 255, 322, 338 Roberts N, 428, 440 Roberts SJ, 47, 48, 63 Robertson HT, 32, 39, 43, 59 Robinson D, 311, 330 Robinson DS, 404, 413 Robinson JA, 344, 360 Robinson JK, 34, 55 Robinson LJ, 76, 86, 162, 184 Robson A, 309, 313, 329, 332 Robson SC, 142, 154 Roca J, 34, 35, 70, 72, 313, 332 Rochelle LG, 160, 164, 177, 183 Rochette F, 311, 331 Rochitte CE, 300, 304 Rocker G, 364, 377 Roczniak A, 408, 413, 417 Roddy MA, 39, 49, 59 Rodger IW, 321, 338 Rodgers P, 121, 129 Rodgers PA, 205, 215 Rodkey FL, 203, 213 Rodman DM, 240, 242, 245, 249, 254, 315, 334 Rodriguez D, 427, 439 Rodriguez I, 123, 129 Rodriguez M, 167, 189 Rodriguez-Roisin R, 34, 35, 70, 72 Roe P, 33, 68 Roerig DL, 160, 164, 177, 183

506 Rogers DF, 34, 48, 64, 70, 200, 203, 206, 207, 211, 212, 217, 320, 337 Roggeri A, 314, 333 Rohde CA, 284, 286, 290, 299, 304 Rojanasakul Y, 409, 413, 418 Roken BO, 52, 66 Rolfe VE, 409, 417 Rolik LV, 207, 215, 251, 255, 320, 337 Rolla G, 248, 253, 425, 426, 427, 429, 430, 431, 438, 440, 441 Rollag H, 382, 394, 398 Rollenhagen JE, 78, 87 Rollinghoff M, 163, 185 Roman LJ, 162, 163, 184, 186 Romling U, 404, 415 Ronco RJ, 29, 53 Ronnett GV, 126, 131, 140, 141, 153 Rosbe KW, 77, 87 Rose A, 77, 87 Rosen GM, 74, 85 Rosen H, 165, 188 Rosenberg ME, 118, 125, 127, 131, 139, 153 Ross BB, 51, 65 Ross D, 284, 304 Ross DJ, 394, 401 Rossaint R, 34, 39, 52, 58, 66, 68, 83, 89, 164, 187 Rossi GA, 309, 329 Rossier G, 123, 129 Rossitch E, 43, 61 Rossoni G, 32, 48, 64 Rothe M, 209, 216 Rottenberg ME, 171, 192 Rouot B, 411, 419 Rousseau DL, 238, 244 Roy CC, 229, 231, 368, 372, 377 Royall JA, 164, 186, 263, 277, 320, 321, 337, 359, 362 Royston D, 35, 71, 96, 97, 105, 106, 112, 260, 268, 275, 278 Ruan J, 176, 193 Rubbo H, 167, 171, 189, 263, 277, 278 Rubin BK, 404, 412, 414 Rubin LJ, 39, 42, 59, 61 Rubins J, 37, 57

Author Index Rubinstein I, 34, 48, 64, 68, 69, 171, 191, 203, 213, 314, 315, 327, 333, 334, 341, 372, 379 Rudehill A, 34, 70 Ruggieri MP, 314, 333 Ruiz P, 424, 438 Runer T, 202, 216 Ruskoaho H, 43, 62 Russell D, 163, 185 Russell MA, 206, 216 Russell SW, 163, 185, 406, 416 Rusznak C, 309, 330 Rutgers SR, 34, 70, 314, 333 Ruth P, 13, 25 Rutili G, 282, 301, 303 Ruzinski JT, 179, 180, 194 Ryan JL, 344, 360 Ryan US, 264, 268, 278

S Saarelainen S, 34, 70 Saavedra JP, 6, 20 Sabas M, 228, 231, 371, 379 Sabatino G, 242, 245 Sachdev V, 76, 87 Saetta M, 314, 333, 433, 442 Sahgal N, 170, 191 Said SI, 165, 187 Saito N, 249, 254, 271, 279 Saito S, 34, 37, 57, 135, 151, 239, 245, 411, 419 Saito T, 322, 338 Saito Y, 35, 71, 249, 254 Saito Y, 202, 216 Saitoh M, 12, 25 Sakamaki T, 34, 35, 69, 71, 82, 89, 202, 216, 249, 254, 312, 331 Sakamoto H, 34, 69, 82, 89, 312, 331 Sakurai N, 404, 415 Sakurai T, 404, 415 Salama G, 12, 25 Salamonsen RF, 384, 398 Saleh D, 76, 84, 87, 89, 164, 186, 248, 249, 253, 308, 315, 316, 328, 334, 430, 440

Author Index Salkowski CA, 406, 416 Salloum J, 260, 275 Salome CM, 52, 66, 309, 329 Saloojee Y, 206, 216 Salter M, 81, 88 Salvemini D, 300, 304 Salzman AL, 81, 88, 141, 153 Sammut IA, 135, 150 Samouilov A, 263, 277 Sampson JB, 165, 188 Samuni A, 172, 193 Sanders L, 435, 443 Sanderson MJ, 43, 61, 62 Sandhaus RA, 261, 276 Sanghera JS, 144, 155 Sanna A, 17, 26 Sannomiya P, 51, 66 Santavirta N, 432, 441 Santiago E, 163, 184 Santillano DR, 76, 86 Santoni A, 163, 185 Sapa II, 208, 215 Sapienza MA, 33, 34, 35, 48, 51, 64, 70, 71, 202, 212, 308, 314, 328, 332 Sarathchandra P, 123, 130, 135, 139, 150, 153, 204, 214, 251, 255 Sarkharova S, 205, 214 Sarpel G, 136, 152 Sartori C, 33, 38, 49, 57, 65, 107, 113, 267, 278 Sasaki H, 135, 151, 206, 217, 317, 319, 335, 336, 337 Sasaki M, 76, 86 Sasidhar M, 124, 130 Sata H, 135, 150 Sato K, 34, 35, 69, 71, 82, 89, 123, 124, 130, 142, 143, 153, 154, 202, 216, 240, 242, 245, 249, 254, 312, 331 Sato M, 137, 152 Sato ST, 142, 143, 154 Satta MA, 134, 150 Sauls DL, 10, 23 Saumon G, 364, 377 Saura M, 316, 335, 406, 416 Sautebin L, 314, 332

507 Savill J, 123, 129 Savitskaia NV, 207, 215, 251, 255, 320, 337 Sawa T, 171, 192 Sawada Y, 34, 69, 428, 440 Sayan H, 34, 68, 202, 216 Saye J, 41, 47, 60 Scaffidi C, 168, 189, 190 Scappaticci E, 248, 253, 429, 430, 440 Scarim AL, 406, 416 Scavuzzo M, 383, 398 Schaberg T, 252, 256, 324, 339 Schafer S, 316, 335, 423, 437 Schaffer MR, 428, 439 Schaible TF, 145, 155 Schall TJ, 261, 276 Schanzenbacher P, 12, 25 Schapowal A, 424, 437 Scharte M, 135, 151, 206, 218, 320, 337 Schechter AN, 108, 113 Schedin U, 31, 35, 37, 38, 40, 49, 52, 54, 56, 57, 60, 66, 83, 89 Scheff PA, 34, 68, 372, 379 Scheffner M, 176, 193 Scheideler L, 208, 215, 327, 341 Schellekens SL, 321, 337 Scheppers LA, 314, 333 Scherer SW, 8, 21 Scherrer U, 38, 49, 57, 65 Schick R, 409, 417 Schiller H, 282, 284, 303 Schilling J, 34, 69 Schilling K, 6, 20 Schlensak C, 259, 275 Schmetterer L, 34, 35, 38, 39, 58, 59, 71 Schmid R, 117, 127, 137, 152 Schmidt H, 6, 19 Schmidt HH, 6, 19, 74, 76, 85, 87 Schmidt HHHW, 6, 7, 20, 21, 235, 236, 244 Schmidt JA, 11, 24 Schmidt K, 250, 255 Schmitt MP, 118, 127 Schmitz I, 168, 190

508 Schobersberger W, 49, 65 Schoeffield-Payne MS, 13, 25 Schoeller DA, 223, 230 Schoeniger LO, 295, 304 Scholte BJ, 409, 417 Schot R, 316, 335 Schroeder RA, 29, 53 Schubert JK, 365, 367, 368, 369, 372, 373, 374, 377, 378 Schueler S, 261, 276 Schult G, 13, 25 Schurch S, 170, 181, 191 Schussheim AE, 164, 187 Schutz W, 35, 71 Schwartz D, 369, 378 Schwartzman ML, 118, 128 Schwarz KB, 288, 290, 294, 296, 304 Schwiebert LM, 406, 415 Schwulera U, 208, 215, 327, 341 Sciorati C, 12, 13, 25, 25 Scmidt K, 204, 214 Scollo M, 311, 312, 331, 404, 414 Scott C, 33, 45, 62 Scott DE, 423, 437 Scott JA, 17, 27 Scott PG, 428, 439 Scott RPW, 288, 290, 294, 296, 304 Scriver CR, 403, 404, 412, 413 Sears S, 325, 340 Sebatiao N, 78, 88 Seeger W, 32, 37, 39, 40, 44, 56, 62, 239, 245 Segal BH, 162, 184 Segal I, 432, 441 Seguchi H, 160, 182 Sehajpal P, 167, 189 Sehnert S, 290, 298, 301, 304 Sehnert SS, 283, 284, 286, 288, 290, 292, 294, 295, 296, 298, 299, 303, 304 Sekizawa K, 135, 151, 206, 217, 317, 319, 335, 336, 337 Seldin MF, 424, 437, 438 Selve N, 433, 442 Selwyn AP, 40, 49, 60 Semenza GL, 126, 131, 134, 150 Sendo F, 354, 362

Author Index Senthilmohan R, 326, 340 Serrick C, 260, 276 Sessa WC, 7, 21, 41, 47, 60 Setoguchi K, 165, 187 Settergren G, 39, 52, 58, 66 Severin A, 17, 27 Severn A, 322, 437 Sevigny J, 142, 154 Seyedi N, 7, 21 Seylaz J, 134, 150 Shah M, 161, 183 Shah PL, 124, 130, 135, 151, 206, 217, 218, 318, 336, 404, 414 Shah SK, 171, 192 Shami PJ, 10, 23 Shamley PF, 352, 362 Shanley PF, 354, 362 Shapiro RA, 10, 16, 23, 26 Shapiro SD, 364, 377 Shariati B, 321, 337 Sharma HS, 6, 20, 118, 128 Sharma VS, 144, 155, 171, 192 Shasby DM, 145, 155 Shaskan EG, 224, 230 Shaul PW, 6, 20, 34, 35, 55, 68, 76, 86, 162, 184, 202, 212, 406, 413, 415 Shaw YH, 13, 25 Shearer BG, 11, 24 Sheel AW, 34, 38, 58 Shegog ML, 126, 131, 205, 214 Sheller JR, 323, 339 Shen XD, 118, 128 Shen YJ, 428, 439 Shenep JL, 163, 185 Sheng H, 6, 19, 74, 85 Shennib H, 84, 89, 260, 276, 385, 390, 393, 399, 400 Sherman PA, 11, 24, 162, 184 Sherman TS, 34, 68, 202, 212, 406, 413, 415 Sheta EA, 162, 184 Sheu FS, 248, 253, 327, 341 Shi X, 409, 413, 418 Shi XM, 8, 21 Shibahara S, 118, 128 Shichiri M, 409, 413, 418 Shiganaga MK, 359, 362

Author Index Shigenaga M, 284, 304 Shigenaga MK, 325, 340 Shiloh MU, 163, 176, 186, 193 Shimada SG, 33, 67, 346, 347, 348, 349, 362 Shimizu T, 44, 62 Shimokawa H, 406, 416 Shimouchi A, 7, 21 Shin WS, 167, 189 Shinano H, 34, 37, 57, 239, 245 Shinebourne EA, 18, 27, 34, 37, 51, 57, 66, 68, 92, 111, 200, 202, 207, 211, 212, 308, 311, 328, 375, 380 Shinoda Y, 135, 151 Shinomiya H, 411, 419 Shinozaki M, 427, 439 Shiraishi T, 384, 392, 399 Shirato K, 326, 340 Shirato M, 33, 68 Shires GT, 345, 360 Shiriati B, 321, 337 Shizuta Y, 409, 413, 418 Shoji S, 34, 69 Shokoohi V, 121, 129 Shulagin IuA, 204, 214 Shuler RL, 164, 186 Shultz PJ, 37, 57 Shuman H, 176, 194 Sibillle Y, 145, 155 Sica A, 242, 245 Siddiqui MT, 391, 400 Siddiqui RA, 404, 415 Sidorenko GI, 207, 215, 251, 255, 320, 337 Silkoff P, 388, 400 Silkoff PE, 37, 38, 47, 49, 57, 63, 84, 89, 96, 112, 200, 202, 207, 211, 212, 271, 278, 309, 310, 312, 313, 328, 330, 332, 392, 393, 394, 400 Silver GM, 370, 379 Silver IA, 242, 245 Silverman EK, 308, 328 Silvestri M, 309, 329 Sim JE, 121, 129 Simecka JW, 172, 192, 193 Simmons RL, 10, 16, 23, 26, 167, 172, 188, 193

509 Simon DI, 248, 253, 434, 442 Simon HU, 424, 437 Simon RH, 321, 338 Simpson A, 309, 329 Simpson CS, 409, 417 Simpson KL, 405, 412, 415 Simpson R, 345, 361 Singel DJ, 36, 56, 168, 189, 248, 253, 434, 442 Singer EA, 35, 71 Singer HA, 41, 47, 60 Singh I, 406, 416 Singh S, 29, 53 Siow RC, 135, 150 Siow Y, 136, 151 Sippel JM, 35, 47, 62, 202, 212, 318, 336 Sisson JH, 34, 48, 64, 69, 171, 191, 203, 213, 314, 315, 333, 334 Sitrin NF, 10, 23 Sittipunt C, 179, 180, 194 Sjoquist PO, 6, 20, 384, 390, 399 Skamene E, 411, 419 Skimming JW, 170, 181, 191 Skinner KA, 162, 184 Skinner SJ, 43, 61 Skoogh BE, 78, 87 Skopouli FN, 432, 441 Skosey J, 369, 378 Skosey JL, 368, 378 Skovron ML, 424, 426, 427, 438 Skulberg A, 346, 362 Skurpskii VA, 204, 214 Slama K, 164, 187 Slater T, 283, 303 Sleiman C, 260, 276 Slutsky AS, 33, 37, 38, 47, 49, 57, 63, 67, 84, 89, 96, 112, 200, 202, 207, 211, 212, 271, 278, 310, 312, 330, 364, 377, 388, 400 Slutsky S, 383, 398 Sly WS, 403, 404, 412, 413 Small T, 35, 71, 249, 254, 385, 386, 388, 392, 394, 399, 400, 401 Smart EJ, 76, 86, 162, 184 Smirnova EA, 208, 215 Smith A, 17, 27, 35, 72, 430, 440

510 Smith AW, 406, 411, 416, 419 Smith C, 165, 188 Smith CD, 165, 188 Smith D, 167, 188 Smith JJ, 411, 418 Smith JR, 413, 419 Smith JW, 428, 440 Smith L, 36, 55, 83, 84, 89 Smith LM, 202, 212 Smith M, 283, 303 Smith NC, 33, 45, 62 Smith PL, 260, 275 Smith RN, 142, 154 Smith TW, 164, 187 Smolen JS, 145, 155, 435, 443 Smyrnios N, 321, 338 Snapper JR, 176, 194 Snashal DC, 309, 329 Snelder JB, 31, 34, 54, 309, 330 Snell G, 386, 393, 400 Snell GI, 384, 385, 387, 398, 399, 406, 413, 415 Snider MT, 368, 369, 378 Snider S, 271, 279 Snover D, 122, 129 Snyder A, 248, 253, 263, 277, 314, 333 Snyder SH, 6, 16, 20, 26, 51, 66, 74, 85, 86, 125, 131, 140, 141, 153, 162, 163, 184 Soares MP, 123, 124, 130, 142, 143, 153, 154 Sobol BJ, 242, 246 Sobotka PA, 368, 369, 371, 373, 377, 378, 379 Soda D, 38, 58, 107, 113 Sofia M, 35, 72 Sogos V, 167, 189 Soifer SJ, 49, 65, 249, 253, 315, 334 Sokabe M, 43, 61 Sokol K, 163, 186 Soler G, 16, 26 Soler P, 364, 377 Somervell CE, 43, 61 Soodaeva E, 205, 214 Sooranna SR, 34, 68 Soreson E, 346, 362

Author Index Sorrentino R, 9, 23 Sorsa T, 432, 441 Southan GJ, 81, 88 Souza JM, 167, 169, 189, 190 Spain DA, 136, 151 Spallarossa D, 309, 329 Sparrow J, 406, 416 Speckin P, 207, 215, 320, 337 Spector EB, 16, 26 Speiser W, 35, 38, 58 Spencer MJ, 41, 47, 60 Spicer M, 200, 211 Spiesser S, 411, 419 Spittler K-H, 367, 377 Spitz DR, 121, 129, 205, 214 Spitzer SA, 432, 441 Spock A, 411, 418 Sporn MB, 406, 416 Sprague RS, 49, 50, 65, 110, 113 Spriestersbach R, 32, 37, 39, 40, 44, 56, 62, 239, 245 Springall DR, 9, 17, 23, 27, 48, 64, 78, 88, 249, 253, 308, 315, 328, 334, 385, 390, 393, 399, 405, 413, 415 Sptiz DR, 126, 131 Squadrito GL, 15, 26 Sridhar K, 118, 121, 128, 129, 205, 214, 251, 255 Srimal S, 406, 416 Srinivasan A, 168, 189 St Clair EW, 435, 443 St Croix CM, 34, 38, 39, 58, 107, 113 St John G, 176, 193 Stadler J, 167, 188 Stadtman ER, 168, 170, 190, 191 Stalenheim G, 432, 441 Stamler JS, 6, 19, 34, 35, 36, 39, 48, 48, 49, 55, 56, 59, 63, 81, 84, 88, 89, 108, 113, 164, 168, 186, 187, 189, 200, 207, 211, 235, 236, 242, 243, 248, 253, 253, 256, 309, 311, 312, 322, 325, 330, 331, 340, 344, 345, 346, 360, 362, 424, 434, 436, 437, 442 Standiford TJ, 51, 66, 411, 419 Stanescu D, 17, 26

Author Index Stansberry L, 123, 130 Stansbury L, 144, 155 Stanton BA, 12, 25 Star RA, 6, 13, 20, 25, 35, 55 Stark GR, 406, 417 Staub NC, 176, 194 Steagall WK, 405, 415 Steer PJ, 34, 68 Steerenberg PA, 31, 34, 54, 203, 212, 309, 330 Stefanovic-Racic M, 433, 436, 442, 443 Steimle CN, 262, 277 Stein RA, 370, 371, 378, 379 Steinberg AD, 427, 439 Steinberg KP, 122, 129, 143, 154, 179, 180, 194 Steiner JP, 74, 86 Steinhauser ML, 423, 437 Steinhoff G, 385, 392, 399 Steinhorn RH, 249, 253, 315, 334 Steinmuller C, 385, 392, 399 Stelts D, 78, 88 Steltzer H, 374, 380 Stelzner TJ, 249, 254, 315, 334 Stepanov E, 205, 214 Stepanov VE, 204, 214 Stephan H, 252, 256, 324, 339 Sterin-Borda L, 432, 441 Sterk PJ, 310, 312, 316, 330, 335 Stetkiewicz PT, 343, 359, 368, 371, 373, 378 Steuhr DJ, 167, 189 Steupfel M, 144, 155 Stevens K, 163, 186 Stevenson DK, 203, 205, 206, 213, 215, 217 Stewart RD, 207, 215 Stewart RJ, 8, 21 Stewart TE, 33, 67 Stichtenoth DO, 423, 433, 434, 437, 442 Stick S, 34, 70, 202, 216 Stirling RG, 206, 217, 311, 318, 330, 336, 404, 413 Stitt JT, 33, 67, 346, 347, 348, 349, 362

511 Stocker R, 119, 125, 128, 128, 139, 140, 148, 152, 156 Stoclet JC, 345, 361 Stohlawetz P, 35, 71 Stoltz RA, 118, 128 Stolzenberg ED, 411, 418 Stone BG, 368, 370, 371, 378 Stone RT, 203, 206, 213 Stopper VS, 6, 20 Storm V, 311, 331 Stoyanovsky D, 12, 25 Straathof KC, 310, 312, 330 Strang LB, 51, 65 Strenn K, 34, 39, 59 Stresemann E, 322, 338 Stretton CD, 17, 26, 33, 48, 64 Strieter RM, 51, 66, 261, 276, 411, 419, 423, 437 Stromberg S, 31, 32, 33, 37, 38, 41, 43, 54, 57, 95, 103, 112 Stuehr DJ, 9, 23, 34, 48, 51, 55, 76, 77, 86, 87, 162, 163, 164, 167, 184, 186, 188, 235, 236, 237, 238, 239, 240, 242, 243, 244, 245, 262, 277, 406, 411, 413, 415, 416 StuehrDJ, 97, 109, 112 Stuepfel M, 142, 154 Stull JT, 41, 47, 60 Sturrock RD, 435, 436, 443 Stutts MJ, 411, 418 Su Y, 314, 333 Suberville S, 163, 185, 327, 341 Sucher NJ, 168, 189 Sud A, 425, 438 Sue-Chu M, 309, 329 Suematsu M, 135, 150, 151 Suffrendini AF, 35, 71, 314, 333 Suga M, 165, 187 Sugai N, 44, 62 Sugarbaker D, 6, 19, 34, 55, 76, 87, 164, 186, 235, 236, 243, 346, 362 Sugarbaker DJ, 168, 189 Sugiura H, 326, 340 Sugiyama T, 432, 441 Suhasini M, 167, 189 Sullivan K, 260, 275

512

Author Index

Sullivan P, 124, 130, 135, 151, 206, 217, 314, 318, 333, 336, 404, 414 Sullivan PM, 384, 390, 399 Sullivan SK, 409, 417 Sumino H, 34, 35, 69, 71, 82, 89, 202, 216, 249, 254, 312, 331 Summers RJ, 47, 48, 63 Sumpio BE, 41, 47, 60 Sundler F, 48, 64 Sutter TR, 125, 131 Suttner DM, 118, 128, 205, 214, 251, 255 Suzuki H, 34, 37, 38, 57, 58, 107, 113, 167, 189 Suzuki K, 44, 62, 160, 164, 177, 183 Suzuki M, 171, 191, 436, 443 Suzuki N, 135, 151, 319, 337 Suzuki T, 135, 151 Suzuki Y, 202, 212, 309, 329 Svanholm C, 171, 192 Svenungsson E, 425, 438 Sviland L, 383, 398 Swedler I, 372, 379 Swedler WI, 34, 68, 369, 378 Swenson ER, 32, 39, 43, 59, 67, 237, 238, 244, 250, 254 Swiderek KM, 9, 23, 76, 86, 163, 185 Swierkosz TA, 14, 26 Sylvester SL, 118, 120, 121, 122, 128, 129, 143, 154 Synder S, 9, 23 Synder SH, 6, 19 Szabo C, 16, 26, 81, 88, 141, 153, 165, 188, 410, 418 Szalai JP, 37, 38, 57, 200, 211 Szefler SJ, 311, 331 Szidon JP, 229, 231 Szodoray P, 432, 441 Szondy Z, 432, 441

T Tabibi S, 409, 417 Tadjikarimi S, 48, 64, 406, 415 Taguchi Y, 298, 299, 304 Taha R, 321, 338

Taintor RR, 16, 26 Taira E, 76, 86 Taira M, 326, 341 Tajigami K, 142, 143, 154 Takahashi T, 35, 71, 125, 131, 202, 216, 249, 254 Takahashi Y, 313, 332 Takamiya R, 135, 150 Takao M, 32, 38, 39, 56, 82, 89, 107, 113, 267, 278 Takasaka T, 135, 151, 206, 217, 319, 337 Takasaki Y, 427, 439 Takasawa S, 406, 416 Takehara K, 430, 440 Takehara Y, 39, 59, 202, 216 Takemura H, 47, 63 Takemura K, 36, 55 Takeshita A, 406, 416 Takeyama K, 124, 130 Takigami E, 123, 124, 130 Takigami K, 143, 153 Talanian RV, 424, 437 Talbot JA, 317, 335 Tamaoki J, 40, 47, 60, 63, 326, 341 Tamatani T, 135, 150 Tamayo L, 135, 150 Tamioka H, 432, 441 Tamura M, 242, 245 Tan S, 162, 184 Tanabe E, 432, 441 Tanaka H, 322, 338 Tanaka M, 126, 131 Tanaka N, 313, 332 Tanaka S, 9, 23, 180, 195 Tanaka Y, 427, 439 Tang N, 125, 130 Tang W, 316, 335 Tangermann A, 371, 379 Taniguchi N, 374, 380 Taniguchi T, 406, 416 Tannenbaum SR, 345, 361 Tannous Z, 34, 69 Tanooka H, 284, 304 Tanoury GJ, 11, 24 Tanswell AK, 43, 61, 62

Author Index Tantry U, 428, 439 Tarakhan LI, 207, 215, 251, 255, 320, 337

513

Lung Biology in Health & Disease Volume 170 Disease Markers in Exhaled Breath

Lung Biology in Health & Disease Volume 186 Pleural Disease

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Lung Biology in Health & Disease Volume 157 Respiratory-Circulatory Interactions in Health & Disease

Lung Biology in Health & Disease Volume 195 Asthma Prevention

Lung Biology in Health & Disease Volume 180 Venous Thromboembolism

Lung Biology in Health & Disease Volume 210 Sarcoidosis

Lung Biology in Health & Disease Volume 135 Control of Breathing in Health and Disease

Lung Biology in Health & Disease Volume 176 Non-Neoplastic Advanced Lung Disease

Lung Biology in Health & Disease Volume 151 Fetal Origins of Cardiovascular and Lung Disease

Lung Biology in Health & Disease Volume 211 Tropical Lung Disease 2nd Edition

Lung Biology in Health & Disease Volume 176 Non-Neoplastic Advanced Lung Disease

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Lung Biology in Health & Disease Volume 191 Long-Term Intervention in Chronic Obstructive Pulmonary Disease

Lung Biology in Health & Disease Volume 182 Pharmacotherapy in Chronic Obstructive Pulmonary Disease

Monitoring Asthma (Lung Biology in Health and Disease, Volume 207)

Severe Pneumonia (Lung Biology in Health and Disease, Volume 206)

Lung Biology in Health and Disease Volume 153 Environmental Asthma

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Lung Development and Regeneration (Lung Biology in Health and Disease)

Lung Biology in Health & Disease Volume 184 Lung Volume Reduction Surgery for Emphysema

Lung Biology in Health & Disease Volume 181 Upper and Lower Respiratory Disease

Lung Biology in Health & Disease Volume 183 Acute Exacerbations of Chronic Obstructive Pulmonary Disease

Lung Biology in Health & Disease Volume 156 Genetic Models in Cardiorespiratory Biology

Lung Biology in Health & Disease Volume 201 Lung Surfactant Function and Disorder

Lung Biology in Health & Disease Volume 200 Functional Lung Imaging

Lung Biology in Health & Disease Volume 215 Ventilator-Induced Lung Injury

Lung Biology in Health & Disease Volume 160 Gravity and the Lung: Lessons from Microgravity

Lung Biology in Health & Disease Volume 162 Drug Delivery to the Lung

Lung Biology in Health & Disease Volume 162 Drug Delivery to the Lung

Lung Biology in Health & Disease Volume 170 Disease Markers in Exhaled Breath

Lung Biology in Health & Disease Volume 186 Pleural Disease

Proteoglycans in Lung Disease (Lung Biology in Health and Disease)

Lung Biology in Health & Disease Volume 157 Respiratory-Circulatory Interactions in Health & Disease

Lung Biology in Health & Disease Volume 195 Asthma Prevention

Lung Biology in Health & Disease Volume 180 Venous Thromboembolism

Lung Biology in Health & Disease Volume 210 Sarcoidosis

Lung Biology in Health & Disease Volume 135 Control of Breathing in Health and Disease

Lung Biology in Health & Disease Volume 176 Non-Neoplastic Advanced Lung Disease

Lung Biology in Health & Disease Volume 151 Fetal Origins of Cardiovascular and Lung Disease

Lung Biology in Health & Disease Volume 211 Tropical Lung Disease 2nd Edition

Lung Biology in Health & Disease Volume 176 Non-Neoplastic Advanced Lung Disease

Pleural Disease, Second Edition (Lung Biology in Health and Disease)

Lung Biology in Health & Disease Volume 191 Long-Term Intervention in Chronic Obstructive Pulmonary Disease

Lung Biology in Health & Disease Volume 182 Pharmacotherapy in Chronic Obstructive Pulmonary Disease

Monitoring Asthma (Lung Biology in Health and Disease, Volume 207)

Severe Pneumonia (Lung Biology in Health and Disease, Volume 206)

Lung Biology in Health and Disease Volume 153 Environmental Asthma

Bronchopulmonary Dysplasia, Volume 240 (Lung Biology in Health and Disease)

Lung Development and Regeneration (Lung Biology in Health and Disease)

Lung Biology in Health & Disease Volume 184 Lung Volume Reduction Surgery for Emphysema

Lung Biology in Health & Disease Volume 181 Upper and Lower Respiratory Disease

Lung Biology in Health & Disease Volume 183 Acute Exacerbations of Chronic Obstructive Pulmonary Disease

Lung Biology in Health & Disease Volume 156 Genetic Models in Cardiorespiratory Biology

Lung Biology in Health & Disease Volume 201 Lung Surfactant Function and Disorder

Lung Biology in Health & Disease Volume 200 Functional Lung Imaging

Lung Biology in Health & Disease Volume 215 Ventilator-Induced Lung Injury

Lung Biology in Health & Disease Volume 160 Gravity and the Lung: Lessons from Microgravity

Lung Biology in Health & Disease Volume 162 Drug Delivery to the Lung

Lung Biology in Health & Disease Volume 162 Drug Delivery to the Lung

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