Biomarkers: In Medicine, Drug Discovery, and Environmental Health

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BIOMARKERS


BIOMARKERS

In Medicine, Drug Discovery, and Environmental Health

Edited by

VishalS.Vaidya Joseph V. Bonventre Harvard Medical School Boston, Massachusetts

1WILEY A JOHN WILEY & SONS, INC., PUBLICATION

The cover art is called "Biofluid" and represents biological fluid with visible signs of biomarkers. Created by Dr. Ina Schuppe-Koistinen using watercolors, Dr. Schuppe-Koistinen is a senior principal scientist and molecular toxicologist at AstraZeneca, Sweden. Additional science watercolors by Dr. SchuppeKoistinen can be found at http://www.inasakvareller.se

Copyright © 2010 by John Wiley & Sons, Inc. All rights reserved. Published by John Wiley & Sons, Inc., Hoboken, New Jersey. Published simultaneously in Canada. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permission. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com. Library of Congress Cataloging-in-Publication Data: Biomarkers : in medicine, drug discovery, and environmental health / edited by Vishal S. Vaidya, Joseph V. Bonventre. p. ; cm. Includes bibliographical references and index. ISBN 978-0-470-45224-0 (hardback) 1. Biochemical markers. I. Vaidya, Vishal S. II. Bonventre, Joseph V. [DNLM: 1. Biological Markers. 2. Diagnostic Techniques and Procedures. 3. Drug Discovery. 4. Environmental Monitoring—methods. QW 541 B616 2010] QH438.4.B55B555 2010 616.07'5-dc22 2010013135 Printed in the United States of America. 10

9 8 7 6 5 4 3 2 1

To: My parents, Sudhakar and Suhasini; my wife, Alka; and my sons, Ariv and Rian. Vishal Vaidya

To: My wife, Kristie; my daughter, Joanna; my son, Andrew; my son-in-law, Brian; and my grandson, Daniel. Joseph Bonventre


CONTENTS

Preface

xxii

Contributors

xxv

Biomarkers: An Evolutionary Perspective Michael A. Ferguson and Vishal S. Vaidya References

1 4

SECTION I: TOOLS FOR BIOMARKER DISCOVERY

5

2

7

Genomics Weida Tong and Donna L. Mendrick Introduction Evaluation of the Technology Clinical Applications Bioinformatics Challenges Applications to Drug Toxicology, Medicine, and Environmental Health Improve Understanding of Basic Cellular Architecture and Function Mechanism of Toxicity and Disease Algorithmic Models to Predict Toxicity or Disease Strengths, Weaknesses, and the Road Forward Conclusion Summary Points Disclaimer References

3

7 8 10 12 14 15 16 17 18 20 20 20 20

Proteomics for Biomarker Discovery Timothy D. Veenstra

25

Introduction

25 vu

CONTENTS

Vlll

4

Tissue or Biofluid Technology Protein Identification Using Mass Spectrometry Sample Preparation Protein Quantitation Examples of Biomarker Discovery and Evaluation Challenges in Proteomic Biomarker Discovery The Road Forward: Targeted Verification and Validation Conclusion Summary Points Acknowledgments References

26 28 29 30 33 35 38 39 43 44 44 44

Metabolic Profiling for Biomarker Discovery

47

Hector C. Keun

5

Introduction: What is Metabolic Profiling? Analytical Strategies for Metabolic Profiling Data Pre-Processing, Analysis, and Pattern Recognition Preclinical Toxicology: Models for Pathological Biomarker Discovery Disease Biomarker Discovery Using Metabolic Profiling Inborn Errors of Metabolism Neuroscience Cancer Infectious Disease Metabolic Syndrome: Insulin Resistance, Cardiovascular Disease, and Hypertension Environmental Health and Metabolic Profiling Conclusion: Strengths, Weaknesses, and the Way Forward for Metabolic Profiling in Biomarker Discovery Summary Points References

47 50 54 56 58 58 59 59 61

The Bittersweet Promise of Glycobiology

75

61 62 63 64 64

Padmaparna Chaudhuri, Rania Harfouche, and Shiladitya Sengupta Introduction Glycosylation in Pathological States Congenital Disorders of Glycosylation (CDG) Glycomics of Immune Disorders Glycomics in Cancer Other Acquired Diseases Glycans in Therapeutics and as Therapeutic Targets Tools to Analyze the Glycome

75 75 75 76 77 79 79 80

CONTENTS

Analytical Chemical Microarray Molecular Strengths, Weaknesses, and the Road Forward Conclusion Summary Points References

IX

80 81 82 83 84 84 84 85

SECTION II: BIOMARKERS OF INJURY/DISEASE

89

6

91

Biomarkers of Alzheimer's and Parkinson's Disease Walter Maetzler and Daniela Berg

Definition and Prevalence of Alzheimer's and Parkinson's Disease 91 Alzheimer's Disease 91 Parkinson's Disease 92 Pathophysiology and Mechanisms 92 Alzheimer's Disease 92 Genetic Aspects 93 Pathology 93 Pathophysiological Mechanisms 94 Parkinson's Disease 95 Genetic Aspects 95 Pathology 96 Pathophysiological Mechanisms 96 Concluding Remarks to Pathological and Pathophysiological Aspects 97 Current Means for Diagnosis/Prognosis of the Diseases and Their Limitations 98 Alzheimer's Disease 98 Clinical Markers 98 Genetic Markers 98 In Vivo Markers from Pathology 98 Pathophysiological Mechanisms 100 Further Diagnostic Assessments 100 Parkinson's Disease 100 Clinical Markers 101 Genetic Markers 101 In Vivo Markers from Pathology 101 Pathophysiological Mechanisms 102 Further Diagnostic Assessments 102 Novel Biomarkers 102 Alzheimer's Disease 102

CONTENTS

X

Clinical Markers Genetic Markers In Vivo Markers from Pathology Pathophysiological Mechanisms Further Diagnostic Assessments Combination of Markers Parkinson's Disease Clinical Markers Genetic Markers In Vivo Markers from Pathology Pathophysiological Mechanisms Further Diagnostic Assessments Combination of Markers Methods to Quantify Biomarkers Conclusion Summary Points References

102 102 103 104 105 105 106 106 106 106 107 107 108 108 109 109 110

Biomarkers of Cardiac Injury

119

Anthony S. McLean and Stephen J. Huang Introduction Definition and Prevalence Pathophysiology and Mechanisms Diagnosis Biomarkers of Cardiac Injury Inflammatory Markers of Cardiac Disease C-Reactive Protein (CRP) Interleukins (IL) Tumor Necrosis Factor (TNF) and Fas CD40 Ligand Matrix Metalloproteinases (MMPs) Myeloperoxidase (MPO) Markers for Myocardial Cell Injury Creatine Kinase-Myocardial Band (CK-MB) Troponins (cTn) Heart-Type Fatty Acid Binding Protein (H-FABP) Markers for Cardiac Stress B-Type Natriuretic Peptide (BNP) and N-Terminal ProBNP (NT-ProBNP) Adrenomedullin (ADM) ST2 Multimarker Approach? Conclusion

119 119 123 125 126 127 127 129 129 130 130 131 131 131 132 134 134 134 137 138 138 139

CONTENTS

XI

Summary Points References

139 140

Lung Injury Biomarkers

157

Urmila P. Kodavanti Introduction Causes of Lung Injury Morphological and Cellular Targets of Lung Injury Airway and Mucosa Alveolar Macrophage The Surfactant Covering Alveolar Epithelial Cells Alveolar Epithelium, Interstitium, and Capillary Endothelium Pathobiologic Processes Involved in Lung Injuries and Diseases Airway Epithelial Damage, Mucus Hypersecretion, and Goblet Cell Hyperplasia Airway Inflammation in Asthma Airway Inflammation in Bronchitis and Chronic Obstructive Pulmonary Disease Airway Fibrosis, Bronchoconstriction, and Hyperresponsiveness Alveolar Epithelial, Capillary Endothelial, and Terminal Bronchiolar Injuries Pulmonary Edema Neutrophilic Inflammation, Alveolar Apoptosis, and Emphysema Pulmonary Fibrosis and Granuloma Alveolar Phospholipidosis Pulmonary Surfactant and Surfactant Protein Abnormalities Sampling Techniques for Biomarker Analysis Induced Sputum Bronchoscopy and Lung Biopsy Bronchoalveolar Lavage for Analysis of Biomarkers of Lung Injury Biomarker Assessments and Their Involvement in Lung Injury and Disease Lung Injury Biomarkers in Bronchoalveolar Lavage Fluid (BALF) and Sputum Total Protein and Albumin Lactate Dehydrogenase Activity 7-Glutamyl Transferase Activity N-acetyl Glucosaminidase Activity Cells in Bronchoalveolar Lavage Fluid as Biomarkers of Lung Inflammation Cytokines and Chemokines in Sputum and Bronchoalveolar Lavage Fluid Biomarkers of Oxidative Stress in Bronchoalveolar Lavage

157 158 159 159 161 161 162 162 162 164 164 165 166 166 167 167 169 169 170 170 171 171 172 174 174 175 175 175 176 177

Xll

CONTENTS

Fluid, Sputum, Lung, and Plasma Ascorbate Glutathione Extracellular Superoxide Dismutase Ferritin, Lectoferrin, Transferrin, and Iron-Binding Capacities 4-Hydroxynonenal F(2)-Isoprostanes Exhaled Nitric Oxide Heme Oxygenase-1 Asymmetric and Symmetric Dimethyl Arginine Surfactant Proteins in Bronchoalveolar Lavage Fluid and Plasma Matrix Metalloproteases as Biomarkers of Lung Injury Collagen and Elastin Fragments as Biomarkers of Lung Injury Circulating Lung-Cell-Specific Proteins as Biomarkers Blood Coagulation and Thrombosis Markers in Lung Injuries Novel Approaches for Biomarker Identification Acknowledgments Disclaimer References

177 178 179 179 180 180 180 181 181 182 182 183 184 184 185 185 187 187 187

Translational Biomarkers of Acute Drug-Induced Liver Injury: The Current State, Gaps, and Future Opportunities

203

JosefS. Ozer, William J. Reagan, Shelli Schomaker, Joe Palandra, Mike Baratta, and Shashi Ramaiah Intrinsic and Idiosyncratic Drug-Induced Liver Injury: Terminologies and Background Histological Manifestations of Liver Injury Hepatic Steatosis/Fatty Liver and Steatohepatitis Cholestatic Liver Injury Common Mechanisms of Acute Liver Injury Mechanistic Manifestations of DILI Processes of Hepatocyte Cell Death The Role of Immune Responses in Liver Injury Metabolic Idiosyncrasy in Liver Injury Underlying Inflammation Mechanisms with Liver Injury Mitochondrial Oxidant Stress and Dysfunction Inhibition of Tissue Repair Response Disruption of Calcium Homeostasis and Cell Membrane Damage in Liver Disruption of Cytoskeleton in Liver Injury Traditional Preclinical and Clinical Biomarkers of Drug-Induced Liver Injury Gaps in Traditional Hepatic Biomarkers Considerations to Predict Acute Liver Injury: Anatomy and Time-Course

203 204 204 204 205 205 205 206 206 207 207 208 208 208 209 209 210

CONTENTS

xiu

New and Emerging Serum Enzyme Biomarkers of Liver Injury Discovery and Application of Purine Nucleoside Phosphorylase (PNP), Paraxonase (PON-1), and Malate Dehydrogenase (MDH) as Hepatic Biomarkers PON1 Is a Functional Marker of Chronic Liver Injury Malate Dehydrogenase (MDH) Activity Is a Candidate Biomarker of DILI-1 Biomarker Qualification by the Predictive Safety Testing Consortium (PSTC) ALT Isozymes: ALT1 and ALT2 Historical Background of ALT Biology Gene Expression of ALT Isoforms The Localization of ALT Protein in Tissues ALT Protein Levels in Serum Current Knowledge on Biology of ALT Does Metabolic Syndrome Illicit a Conflicting ALT Signal for DILI? Anorexia Shows Metabolic Indicators of Liver Injury Including Subtle ALT Elevations Biomarkers of Biliary Injury Authors' Opinion on Future Biomarkers of Liver Injury, Novel Approaches, and Platforms Reactive Oxygen Species (ROS) as Potential Markers for Liver Injury Mechanisms Inflammation Markers as Potential Indicators of Liver Injury Novel Hepatocellular Leakage Enzymes as Early Biomarkers of Symptomatic Change Hepatic Regeneration Markers to Supplement Injury Biomarkers Unification of Diagnostic Metrics of Liver Fibrosis Analytical Biomarker Platforms to Assay Serum Biomarkers of Liver Injury Mass Spectrometry Technologies Can Fill Gaps to Detect Biomarkers When Antibody Approaches are Limited An Overview of Mass Spectrometry Technologies Mass Spectrometry Technologies to Potentially Detect Biomarkers and Rare Protein Antigens of Injury Improved Tagging Techniques for Mass Spectrometry Detection of Proteins High-Throughput Chromatography Enhances the Downstream Detection of Rare Serum Proteins by Mass Spectrometry Mass Spectrometry Approaches to Distinguish Novel Biomarkers of Renal and Liver Injury Mass Spectrometry Approaches to Detect Novel Serum Biomarkers of Liver Injury Conclusion

212

212 213 214 214 214 214 216 216 217 217 218 218 219 219 219 220 221 222 222 223 223 224 224 224 225 226 226 226

XIV

CONTENTS

Acknowledgments Summary Points References 10 Biomarkers of Acute Kidney Injury Frank Dieterle and Frank D. Sistare Introduction Definition and Prevalence of Acute Kidney Injury Pathophysiology and Mechanisms Current Standards for Diagnosing Acute Kidney Injury Novel Kidney Safety Biomarkers Kidney Function Biomarkers Serum Cystatin C Functional Biomarkers Urinary Total Protein Urinary Albumin Urinary p2-Microglobulin Urinary Cystatin C Leakage Markers Urinary GST-a and GST-u/ir Urinary NAG Expression Markers Urinary Kim-1 Urinary Clusterin Urinary NGAL Urinary Osteoactivin Urinary Osteopontin Urinary L-FABP Urinary Trefoil Factor 3 Immune Markers Urinary IL-18 Newest Technologies and Achievements Around Kidney Safety Biomarkers Assays and Technologies Consortia Achieving the First Regulatory Qualification of Kidney Safety Biomarkers Conclusion Summary Points References 11 In Search of Biomarkers for Drug-Induced Vascular Injury

227 227 227 237 237 237 238 242 244 245 245 246 246 247 248 249 250 250 251 251 251 253 253 255 255 256 257 257 257 258 258 260 261 263 263 281

James R. Turk History and Background of DIVI Overview

281 281

CONTENTS

Types of Compounds Implicated Descriptive Pathology of Drug-Induced Vascular Injury Vascular Anatomy Rat Dog Primate Spontaneous Lesions in Preclinical Species Comparison with Human Vasculitides Progress in Biomarker and Model Development for Drug-Induced Vascular Injury Alpha-1-Acid Glycoprotein Calprotectin (S100A9/A8) Caveolin-1 Circulating Endothelial Cells/Particles Complement Component 3 Connective Tissue Growth Factor (CTGF) C-Reactive Protein (CRP) Endothelin-1 Fibrinogen GRO/CINC-1 Haptoglobin Metallothionein-1 (MT-1) Monocyte Chemoattractant Protein-1 (MCP-1) Neutrophil Gelatinase-Associated Lipocalin (NGAL) Osteopontin (OPN) Smooth Muscle Actin Thrombospondin-1 (TSP-1) Tissue Inhibitor of Metalloporteinases-1 (TIMP-1) Tissue Plasminogen Activator (tPA) Vascular Cell Adhesion Molecule 1 (VCAM-1) Vascular Endothelial Growth Factor (VEGF) Von Willebrand Factor Conclusion References 12 Biomarkers of Immunotoxicity

xv 282 283 283 284 284 285 285 285 286 286 286 286 288 288 288 289 289 289 289 289 290 290 290 290 291 291 291 291 291 292 292 292 292 307

Rodney R. Dietert Introduction History of the Use of Biomarkers in Immunotoxicity Assessment Establishing the Testing Paradigm A "Challenging" Issue for Immune Biomarkers Targets of Immunotoxicity

307 308 308 308 309

XVI

CONTENTS

Diseases of Primary Concern Increased Susceptibility to Infections and Tumors Chronic Diseases and Conditions Based on Immune Dysfunction Developmental Immunotoxicity: Increased Vulnerability in Early Life Differential Exposure-Outcomes Between Genders A Disease-Based Approach to Immune Biomarker Selection Toxicogenomic and In Vitro Approaches Conclusion Summary Points Acknowledgments References 13 Biomarkers in Obstetric Medicine

310 310 310 313 314 314 315 316 316 317 317 323

Manish Maski, Sarosh Rana, and S. Ananth Karumanchi Aneuploidies-Trisomies 21, 18, and 13 Alpha Fetoprotein Human Chorionic Gonadotropin Pregnancy-Associated Plasma Protein-A Unconjugated Estriol InhibinA Detection of Trisomy 21 Detection of Trisomy 18 Detection of Trisomy 13 Amniocentesis and Chorionic Villi Sampling Other Novel Markers for Aneuploidy Screening Preeclampsia and Fetal Growth Restriction Vascular Endothelial Growth Factor Placental Growth Factor VEGF Receptors VEGFRl/Fltl (Fms-Like Tyrosine Kinase 1) Endoglin Role of Angiogenic Factors in the Pathogenesis of Preeclampsia The Ability of Angiogenic Proteins to Predict Preeclampsia Other Potential Biomarkers for the Prediction of Preeclampsia Angiogenic Factors and Intrauterine Growth Restriction Preterm Labor and Other Pregnancy Complications Preterm Labor Abruption Gestational Diabetes Summary Points References

323 323 324 324 325 325 325 327 327 334 334 335 335 336 337 337 338 338 339 340 341 342 342 343 343 344 344

CONTENTS

Biomarkers in Cancer

xvn 355

Roopali Roy, Christine M. Coticchia, Jiang Yang, and Marsha A. Moses Introduction Cancer Biomarker Discovery Strategies Cancer Biomarkers Breast Cancer Prostate Cancer Ovarian Cancer Pancreatic Cancer Conclusion Summary Points Acknowledgments References

355 356 357 357 362 364 367 369 370 370 370

Biomarkers of HIV

381

Lewis Kaufman and Michael J. Ross Introduction Novel Biomarkers Host Genetic Determinants of Susceptibility to HIV Infection Chemokines/Chemokine Receptors CCR5 Variants CCR2-64I Variant SDF1-3'A Variant Other Chemokine Polymorphisms Human Leukocyte Antigens HLA Heterozygosity Protects Against Progression to AIDS Protective HLA Alleles HLA Alleles Associated with Rapid Progression to AIDS Other Host Genetic Factors Associated with HIV-Related Outcomes Host Factors Associated with Non-Opportunistic HIV-Related Diseases HIV-Associated Nephropathy HIV-Associated Dementia Clinical Markers CD4+ T-Cell Depletion Plasma Viral Load Combination of Viral Load, CD4+ Count, and Proviral DNA Levels Generalized Immune Activation Conclusion Summary Points References

381 382 382 382 384 385 385 385 386 386 386 387 387 387 387 388 389 389 389 390 390 391 393 393

XVlll

CONTENTS

16 Biomarkers of In Vitro Drug-Induced Mitochondrial Dysfunction James A. Dykens and Yvonne Will Introduction Magnitude of the Problem Mitochondrial Physiology Drag-Induced Mitochondrial Dysfunction (DIMD) Has Been Overlooked Novel Methods to Detect Mitochondrial Dysfunction In Vitro An Emerging Model of Idiosyncratic Drug Toxicity Mitochondrial Diseases Potential Biomarkers of Mitochondrial Dysfunction Animal Models Summary Points References SECTION III: TECHNOLOGY FOR BIOMARKER DETECTION

401 401 402 403 406 407 409 411 413 416 417 417 423

17 Immunoassay-Based Technologies for the Measurement of Biological Materials Used for Biomarkers Discovery and Translational Research 425 Vincent Ricchiuti Introduction Immunoassay and Immunochemistry Background Basic Principles Radioimmunoassays Overview Principle of Radioimmunoassay Enzyme-Linked Immunosorbent Assay and Enzyme Immunoassay Overview Principle of Enzyme Immunoassay Fluorescent and Chemiluminescent Immunoassays Fluorescent Immunoassays Heterogeneous Fluorescent Immunoassays Homogenous Fluorescent Immunoassays Fluorescence Polarization Immunoassay (FPIA) Chemiluminescent Immunoassays Multiplexing Using Antibody Array and Bead Immunoassays Planar Protein Array Formats Suspension or Bead-Based Arrays Example of Multiplexing Technology Simultaneous Multi-Analyte Detection Introduction Multiple Bead Particle Technology

425 426 426 426 427 427 428 431 431 432 433 433 433 433 434 435 436 437 440 440 441 441 441

CONTENTS

xix

Applications The Future Electrochemiluminescence (ECL) Microarrays ECL Diagram Detection Multi Arrays Technologies Biochip Array Technology Biochip Manufacturing Applications Future of Immunoassays Summary Points Acknowledgments References

443 445 445 446 446 447 447 448 448 448 449 450 450

18 Nanoscale Techniques for Biomarker Quantification

457

Madhukar Varshney and Harold G. Craighead Introduction Nanoscale Sensing Techniques for Biomarker Quantification Optical Detection Bio-Barcode Assay-Based Sensors Quantum Dots-Based Sensors Dye-Doped Nanoparticles-Based Sensors Surface Enhanced Raman Spectroscopy-Based Sensors Dynamic Light Scattering Mechanical Detection Nanomechanical Cantilever-Based Sensors Electrical Detection Field Effect Transistor-Based Sensors Liposomes-Based Sensors Magnetic Detection Giant Magnetoresistance-Based Sensors Future Trends Conclusion Summary Points References

457 458 459 459 461 464 465 468 470 470 473 473 475 478 478 482 483 484 485

19 Immunodiagnostics with a Focus on Lateral Flow Point-of-Care Devices 495 Roy R. Mondesire, Glen M. Ford, Hannie F Ford, and Stephen C. Mefferd Introduction Antibodies in Immunoassays Structure and Function of Antibodies Kinetics of Antibody-Antigen Reactions

495 496 497 499

XX

CONTENTS

Polyclonal Antibodies Hybridoma Technology Rapid Manual and Rapid Automated Immunoassays Elements of Immunoassays: Soluble Labels and Detection Homogeneous Enzyme Immunoassays Signal Measurement Methods Colorimetry Fluorometry Time-Resolved Fluorescence Luminescence Principles of Binding Non-Speciflc Interactions in Immunoassays Colloidal and Particle Immunoassays Flow-Through Assays Particle Capture Fluorochrome-Dyed Microspheres Point-of-Care Lateral-Flow Assay Technology Introduction to Traditional Lateral Flow Tests Nucleic Acid Detection and Lateral Flow Principle of the Lateral-Flow Procedure for Nucleic Acid Detection Haptenized Primers Haptenized Detection Probes Molecular Detection of Chlamydia Trachomatis—A Major Agent of Sexually Transmitted Infections Pathogenic Bacteria Detection with Bacteriophage Sensitivity of Lateral-Flow Technology Summary Points Useful Information for Future Trends Emerging Technologies References

500 500 501 502 503 504 504 504 505 505 505 506 506 506 506 506 507 507 510 511 511 512 512 512 513 513 513 513 514

SECTION IV: HOT TOPICS IN BIOMARKER RESEARCH

517

20 Biomarkers for Environmental Exposure

519

Jane E. Gallagher, Elaine A. Cohen Hubal, and Stephen W. Edwards Introduction Need for Biomarkers to Support Environmental Risk Assessment Considerations for the Use of Biomarkers in Environmental Risk Assessment Applications Biomonitoring Studies Interpretation of Biomonitoring Data

519 520 522 524 524 527

CONTENTS

xxi

Cumulative Risk Assessment Molecular Epidemiology Emerging Issues Toxicity Pathway-Based Risk Assessment Systems Biology to Support Risk Assessment Summary Points Acknowledgments References

532 534 537 537 539 540 541 541

Clinical Study Design in Biomarker Research

549

Orfeas Liangos and Bertrand L. Jaber Overview of Clinical Study Design Case Study Case Series Cross-Sectional Study Case Control Study/Nested Case Control Study Cohort Study Experimental Studies Uncontrolled Trial Controlled Trial Blinded/Un-blinded Design Parallel Two-Arm/Multiple-Arm and Crossover Design Biomarkers in Observational Studies Biomarkers for Disease Detection and Diagnosis Biomarkers for Disease Monitoring Biomarkers for Disease Prognostication Biomarkers in Interventional Studies Biomarkers for Treatment Response Biomarkers for Monitoring Toxicity Conclusion Summary Points References

549 549 549 549 550 550 551 551 552 553 553 553 554 556 556 557 557 558 558 558 558

Statistical Issues in Biomarker Research

561

Daniel Holder and Matthew Schipper The Role of Statistics in Biomarker Discovery, Development, and Qualification Types of Biomarkers Stages of Development Kidney Project Background Statistical Methods/Metrics for Assessing Biomarker Performance Sensitivity, Specificity, and Receiver-Operator Characteristic Curves

561 562 563 564 566 566

XXII

CONTENTS

Assessing Whether a Marker Adds Value to Other Markers Errors in the Reference Standard Planning Human Clinical Trials Prognostic Biomarkers and Other Topics Biases in Biomarker Studies Discussion Summary Points Acknowledgments References

569 572 575 577 578 578 579 580 580

23 Regulatory Perspective for Biomarker Qualification from the U.S. FDA 581 Federico Goodsaid Overview Regulatory Paths in Biomarker Evaluation and Qualification Evidentiary Recommendations Harmonization Summary Points References 24 The European Medicines Agency Approach

581 583 586 586 587 587 589

Marisa Papaluca Amati and Spiros Vamvakas Introduction European Medicines Agency and Biomarkers: Briefing Meetings and Scientific Advice New Procedure for the Qualification of Novel Methodologies Current Status Index

589 590 592 594 595

PREFACE

A biomarker is defined as a characteristic that can be objectively measured and evaluated as an indicator of normal biologic or pathogenic processes of pharmacological responses to a therapeutic intervention.' Examples of biomarkers are proteins; lipids; genomic, metabolomic, or proteomic patterns; imaging patterns; electrical signals; and cells present on a urinalysis. In medicine, disease processes are heterogeneous in their pathophysiology and clinical presentation, making diagnosis and prognosis challenging. In drug development, biomarkers are critical at a variety of stages of the process, with the need for informative determination of efficacy and toxicity that spans the preclinical-clinical spectrum. In commenting on a major initiative of the FDA that focuses on biomarkers, Janet Woodcock, MD, deputy commissioner for operations and head of FDA's Critical Path Initiative, said, "Most researchers agree that a new generation of predictive biomarkers would dramatically improve the efficiency of product development, help identify safety problems before a product is on the market (and even before it is tested in humans), and facilitate the development of new types of clinical trials that will produce better data faster."2 The FDA has provided guidance that a biomarker can be considered "valid" if 1) it is measured in an analytical test system with well-established performance characteristics, and 2) there is an established scientific framework or body of evidence that elucidates the physiologic, pharmacologic, toxicologic, or clinical significance of the test result.3 We need better biomarkers to predict clinical efficacy and toxicity in preclinical studies, diagnose disease earlier, predict outcome in a patient with disease, and identify who will respond to an intervention and whether the intervention is working. In addition, better biomarkers will permit better stratification of patients for clinical trials and potentially lead to definition of new therapeutic targets. A good predictive biomarker will have a significant effect on evaluation of potential therapies because it will enable the identification of subgroups of patients who will have a high incidence of injury and hence reduce the number of patients needed to study in order to test potential therapeutic strategies. A clinically useful new biomarker will improve the sensitivity and specificity for the detection of and characterization of disease. It is also likely that some of these biomarkers will be useful to monitor severity and progression of disease. xxm

XXIV

PREFACE

Translational biomarkers that can be measured in blood or urine in both experimental animals and man are of particular interest. Biomarkers that have been well studied and characterized as very sensitive biomarkers of injury in animals, if they function similarly in man, may make it possible to monitor safety and efficacy in clinical trials when the ability to obtain kidney tissue is severely constrained and when the severity of the injury early on is insufficient to result in obvious alterations in clinical state. Given the importance to the clinical, pharmaceutical, and regulatory communities motivated by more specific and timely diagnoses, early intervention, and safer therapies, there has been a great deal of activity devoted to discovery and "fit for purpose" qualification of various potential biomarkers in a number of diseases that affect many different organs. In this book we have tried to capture the excitement and potential of biomarkers over a wide variety of applications spanning medical diagnostics to safety monitoring in therapeutic and environmental exposures. The early chapters are devoted to individual treatments of applicability of genomics, proteomics, glycomics, and metabolomics to this rapidly evolving field of biomarker discovery. The next set of chapters takes specific organs or disease processes and considers in depth the state of the biomarker art in this specific area. Individual chapters are devoted to Alzheimer's and Parkinson's disease, cardiac injury, lung injury, drug-induced liver injury, acute kidney injury, drug-induced vascular injury, immunotoxicity, and obstetric medicine. These are followed by chapters discussing biomarkers in cancer, HIV, and drug-induced mitochondrial dysfunction. The book then moves to a more technical perspective incorporating chapters on immunoassay-based technologies, nanoscale techniques, and lateral flow immunodiagnostics at point of care. Chapters on environmental exposure, clinical trial design, and statistical issues in biomarker analysis then follow. The last two chapters deal with the regulatory perspectives of the FDA and the European Medicines Agency. The chapters are written by leaders in their respective fields and we are very grateful to them for their comprehensive chapters. We hope that the readers will agree with us that the material in this book is timely and will go far to advance the field of biomarker research and facilitate the development of new drugs that are safe, add new biological targets to our therapeutic armamentarium, and ensure environmental safety. Joseph V. Bonventre, MD, PhD and Vishal S. Vaidya, PhD Brigham and Women's Hospital, Harvard Medical School References 1. Group BDW. Biomarkers and Surrogate Endpoints: Preferred Definitions and Conceptual Framework. Clin Pharmacol Ther. 2001;69:89-95. 2. FDA. FDA Unveils Critical Path Opportunities List Outlining Blueprint to Modernizing Medical Product Development by 2010. Biomarker Development and Clinical Trial Design Greatest Areas for Impact. FDA News. P06-39, March 16, 2006. 3. FDA. Center for Drug Evaluation and Reseach (CDER) CfBEaRC, and Center for Devices and Radiological Health (CDRH): Guidance for Industry. Pharmacogenomic data submissions. 2005;l-22.

CONTRIBUTORS

Marisa Papaluca Amati, The European Agency for the Evaluation of Medicinal Products, London, United Kingdom Mike Baratta, Pharmacokinetics, Dynamics, and Metabolism, Pfizer, Andover, Massachusetts Daniela Berg, Center of Neurology, Department of Neurodegeneration and Hertie Institute for Clinical Brain Research, University of Tubingen, Tubingen, Germany Joseph V. Bonventre, Renal Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Harvard Institutes of Medicine, Boston, Massachusetts Pamaparna Chaudhuri, Brigham & Women's Hospital, Harvard-MIT Division of Health Sciences & Technology, Cambridge, Massachusetts Christine Coticchia, Program in Vascular Biology and Department of Surgery, Karp Family Research Building, Children's Hospital Boston and Harvard Medical School, Boston, Massachusetts Harold G. Craighead, School of Applied and Engineering Physics, Cornell University, Ithaca, New York Frank Dieterle, Novartis Institutes of Biomedical Research, Translational Sciences, Basel, Switzerland Rodney R. Dietert, Department of Microbiology and Immunology, Cornell University, Ithaca, New York James A. Dykens, Pfizer, Drug Safety R&D, Sandwich, United Kingdom Stephen W. Edwards, National Health and Environmental Effects Research Laboratory, Immediate Office, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina Michael A. Ferguson, Division of Nephrology, Children's Hospital, Boston, Massachusetts XXV

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CONTRIBUTORS

Glen M. Ford, BioAssay Works, LLC, Ijamsville, Maryland Hannie F. Ford, BioAssay Works, LLC, Ijamsville, Maryland Jane E. Gallagher, Environmental Public Health Division, National Health and Environmental Effects Laboratory, U.S. Environmental Protection Agency, Office of Research and Development, Research Triangle Park, North Carolina Federico Goodsaid, Office of Clinical Pharmacology, Office of Translational Science, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland Rania Harfouche, Brigham & Women's Hospital, Harvard-MIT Division of Health Sciences & Technology, Cambridge, Massachusetts Daniel Holder, Merck Research Laboratories, West Point, Pennsylvania Stephen J. Huang, Department of Intensive Care Medicine, Nepean Hospital, University of Sydney, Sydney, New South Wales, Australia Elaine A. Cohen Hubal, National Center for Computational Toxicology, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina Bertrand L. Jaber, Division of Nephrology, Department of Medicine, St. Elizabeth's Medical Center, Boston, Massachusetts S. Ananth Karumanchi, Beth Israel Deaconess Medical Center, Boston, Massachusetts Hector C. Keun, Department of Biomolecular Medicine, Faculty of Medicine, Imperial College London, South Kensington, London, United Kingdom Urmila P. Kodavanti, Environmental Public Health Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina Orfeas Liangos, Division of Nephrology, Department of Medicine, St. Elizabeth's Medical Center, Boston, Massachusetts Walter Maetzler, Center of Neurology, Department of Neurodegeneration and Hertie Institute for Clinical Brain Research, University of Tubingen, Tubingen, Germany Manish Maski, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts Anthony S. McLean, Department of Intensive Care Medicine, Nepean Hospital, University of Sydney, Sydney, New South Wales, Australia Stephen C. Mefferd, BioAssay Works, LLC, Ijamsville, Maryland

CONTRIBUTORS

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Donna L. Mendrick, Division of Systems Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas Roy R. Mondesire, RoMonics, LLC, Boulder, Colorado Marsha A. Moses, Program in Vascular Biology and Department of Surgery, Karp Family Research Building, Children's Hospital Boston and Harvard Medical School, Boston Massachusetts Josef S. Ozer, Pharmacokinetics, Dynamics, and Metabolism, PGRD, Pfizer St. Louis Laboratories, Chesterfield, Missouri Joe Palandra, Pfizer Biotech, Pharmacokinetics, Dynamics, and Metabolism, Andover, Massachusetts Shashi Ramaiah, Pfizer Global Research and Development, Drug Safety Research and Development, St. Louis, Missouri Sarosh Rana, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts William J. Reagan, Pfizer Biotech, Drug Safety Research and Development, Andover, Massachusetts Vincent Ricchiuti, Division of Endocrinology, Diabetes and Hypertension, Brigham and Women's Hospital, Boston, Massachusetts Roopali Roy, Program in Vascular Biology and Department of Surgery, Karp Family Research Building, Children's Hospital Boston and Harvard Medical School, Boston, Massachusetts Michael J. Ross, Division of Nephrology, Mount Sinai School of Medicine, New York, New York Matthew Schipper, Innovative Analytics, Kalamazoo, Michigan Shelli Schomaker, Drug Safety Research and Development, Pfizer, Groton Pfizer Groton/New London Laboratories, Groton, Connecticut Shiladitya Sengupta, Brigham and Women's Hospital, Harvard-MIT Division of Health Sciences & Technology, Cambridge, Massachusetts Frank D. Sistare, Merck & Co, Inc., Laboratory Sciences and Investigative Toxicology, Westpoint, Pennsylvania Weida Tong, Division of Systems Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas James R. Turk, Amgen, Inc., Thousand Oaks, California

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CONTRIBUTORS

Vishal S. Vaidya, Laboratory of Kidney Toxicology and Regeneration, Renal Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Harvard Institutes of Medicine, Boston, Massachusetts Spiros Vamvakas, The European Agency for the Evaluation of Medicinal Products, London, United Kingdom Madhukar Varshney, School of Applied and Engineering Physics, Cornell University, Ithaca, New York Timothy D. Veenstra, Laboratory of Proteomics and Analytical Technologies, Advanced Technology Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland Yvonne Will, Pfizer, Compound Safety Prediction, Groton Connecticut

CHAPTER

BIOMARKERS: AN EVOLUTIONARY PERSPECTIVE Michael A. Ferguson and Vishal S. Vaidya

The official National Institutes of Health (NIH) definition of a biomarker (biologic marker) is "a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention." 1 This definition is broad and, though not explicitly stated, encompasses laboratory tests, radiologic studies, as well as physical exam findings. Although the term biomarker is a relatively new one that dates back to the late 1960s,2 biologic assessments and measurements in the evaluation of human disease were practiced in antiquity, evident in the writings of the ancient Egyptians. Not surprisingly, clinically useful biomarkers have evolved over time, reflecting the scientific and technologic progress made over the centuries (see Figure 1.1). As a result, an increasing number of clinically relevant tests and procedures are available to estimate organ injury and guide treatment. Recent discoveries in genetics and molecular biology have resulted in impressive advances in our understanding of the pathophysiologic processes of individual diseases and yielded an abundance of prospective therapies directed against novel targets. This has brought about an increased focus on biomarker identification, validation, and quantification, as well as the development of analytical technologies for biomarker measurement. It is anticipated that further characterization of novel biomarkers will enable improvements in diagnostic and prognostic strategies and facilitate accelerated development of new pharmacologic and non-pharmacologic therapies, ultimately resulting in improved patient outcomes. In their earliest incarnation, biomarkers were confined to objective physical findings and observations, such as heart rate and tactile temperature and general features of the patient's body. The diagnostic and prognostic utility of such physical biomarkers can be traced to the earliest known medical manu1

2

BIOMARKERS

FIGURE I. I The evolution of biomarkers in medicine.Through the centuries, biomarkers have taken on different forms. Physical indicators of health and disease were the earliest biologic markers, dating back to the seventeenth century B.C. Qualitative analysis of biologic fluids followed, with uroscopy representing the major diagnostic modality employed from the sixth century B.C. through the eighteenth century A.D. Clinical chemistry and microscopy altered the scope of laboratory specimen and allowed for the routine assessment of analytes in bodily fluids in twentieth century medicine.The twenty-first century has brought with it the emergence of "-omic" technologies, vastly increasing the number of biomarkers in medicine.

scripts, the medical papyruses of ancient Egypt (seventeenth century B.C.). The Edwin Smith Papyri, believed to be written around 1500 B.C. and based on the earlier work of Imhotep (twenty-seventh century B.C.), details 48 cases of trauma as well as therapeutic and prognostic considerations. Breasted's translation of this document reveals "an ancient Egyptian surgeon... as a man with the ability to observe, to draw conclusions from his observations... and maintain a scientific attitude of the mind."3 The examination of biologic specimens can also be traced to ancient times, with reference to the qualitative inspection of urine and stools referenced in the Assyrian Book of Prognoses (650 B.C.).4 Not surprisingly, the prognostic and diagnostic significance of findings around this time was based on a combination of observation and spiritual mysticism. The utility of biologic specimens in the diagnosis and prognosis of disease was further advanced by the ancient Greeks. Hippocrates (350 B.C.) advocated a systematic approach to the patient that included physical exam procedures as well as careful inspection of bodily fluids.5 The Hippocratic physician employed his five senses to study the patient's secretions and excretions to determine the prognosis of a disease and aid in treatment. Par-

BIOMARKERS: AN EVOLUTIONARY PERSPECTIVE

3

ticular emphasis was placed on the evaluation of urine, and Hippocrates is credited with relating specific urinary characteristics, including sediments and surface foam, to chronic illness.5 In the centuries that followed, the practice of uroscopy became paramount in patient evaluation, and by the Middle Ages the matula (urine flask) emerged as the most recognizable symbol of medical practitioners. The importance of urinary diagnosis became exaggerated in the seventeenth century when it often superseded direct evaluation of the patient, leading to isolation of the physician and patient.6 This resulted in a backlash against those who practiced uroscopy; however, macroscopic examination of the urine remained the primary biologic marker in clinical diagnosis until the Victorian era.7 The nineteenth century brought impressive advances that allowed for the generation of increasing amounts of clinical data. Instruments, such as the stethoscope, ophthalmoscope, laryngoscope, spirometer, electrocardiogram, and sphygmomanometer, allowed for improved physical exam assessment and physiologic measurement. In addition, the X-ray, microscope, as well as new laboratory-based chemical and microbiologic techniques, vastly expanded the physician's diagnostic capabilities. These developments allowed for an unparalleled degree of objectivity with respect to the assessment of biologic indicators of normal and abnormal biologic function. As a result, physicians were able to establish standards and evaluate deviations of human physiology.5 By the turn of the twentieth century, clinical laboratories were growing in favor and influence and clinico-pathological laboratories opened in an increasing number of hospitals. Systematic analysis of blood and urine samples established reference levels for a variety of analytes, correlated variations in disease states, and clarified metabolic pathways in health and disease.8 Advances in analytic techniques, including chromatographic separation and colorimetric quantification of analytes, facilitated clinical usefulness and resulted in the ability to assay a growing number of biologic markers to monitor the changing condition of the patient.8 Increasingly, the medical provider became dependent on the chemical analysis of bodily fluids in the monitoring of health, diagnosis, and prognosis of disease, and assessment of response to therapeutic interventions. With increased understanding of the pathophysiologic processes involved in specific disease processes, the quest to identify and characterize biologic markers with improved sensitivity and specificity for a variety of illnesses and associated outcomes has followed. Efforts at biomarker discovery and validation have intensified since the turn of the twenty-first century. Advanced genomic, proteomic, and metabolomic techniques now permit comparative analysis of specimens from healthy and diseased individuals, facilitating biomarker identification. As a result, biomarker initiatives have become ubiquitous in the scientific landscape, with considerable private and public resources now dedicated to clarifying the utility of biologic markers in virtually all aspects of health care. Novel measures of biologic function will prove critical in the research setting, serving as surrogate endpoints in clinical trials that promise to streamline pharmacologic and non-pharmacologic therapeutic development.9 In addition, it is anticipat-

4

BIOMARKERS ed that selected biomarkers and/or biomarker panels will revolutionize drug development, environmental health screening, and medicine, facilitating the movement toward personalized patient care. Ongoing and future biomarker studies are likely to enable individualized assessment of disease susceptibility, response to therapy, as well as disease progression/regression. An emphasis on concurrent development of technologies for rapid biomarker analysis, ideally point of care modalities, will help ensure rapid assimilation into clinical practice. The use of biologic measures in the assessment of health and disease is not new; however, the concept of what constitutes a useful biomarker has evolved considerably, closely paralleling technologic advances of the time. The current era of scientific discovery has brought seemingly limitless opportunities for improvements in medical care. Coordinated efforts at biomarker discovery and validation, as well as technologies for biomarker measurement, will help ensure that the ultimate goal of safer drugs, a cleaner environment, and improved patient outcomes is realized.

REFERENCES 1. Biomarkers and Surrogate Endpoints: Preferred Definitions and Conceptual Framework. Clin. Pharmacol. Ther. Mar 2001 ;69(3): 89-95. 2. DeCaprio, A. Introduction to Toxicologic Biomarkers. In DeCaprio AP, Ed., Toxicologic Biomarkers. New York, NY: Taylor and Francis Group;2006:l-15. 3. Breasted, J. The Edwin Smith Surgical Papyrus. Chicago, IL: The University of Chicago Press; 1930. 4. Keele, K. D. The Evolution of Clinical Methods in Medicine. London. Pitman Medical Publishing Co., Ltd;1963. 5. Berger, D. A Brief History of Medical Diagnosis and the Birth of the Clinical Laboratory. Part 1—Ancient Times Through the 19th Century. MLO Med. Lab Obs. July 1999;31(7):28-30, 32, 34-40. 6. Pardalidis, N. Kosmaoglou E., Diamantis A., and Sofikitis N. Uroscopy in Byzantium (330-1453 A.D.), J. Urol. April 2008;179(4):1271-1276. 7. Armstrong, J. A. Urinalysis in Western Culture: A Brief History, Kidney Int. March 2007;71(5):384-387. 8. Rosenfeld, L. Clinical Chemistry Since 1800: Growth and Development. Clin. Chem. January 2002;48(1): 186-197. 9. Varmus, H. Foreword, In Downing G. J., Ed., Biomarkers and Surrogate Endpoints: Clinical Research and Applications. New York, NY: Elsevier; 2000.

SECTION I TOOLS FOR BIOMARKER DISCOVERY


CHAPTER

GENOMICS Weida Tong and Donna L. Mendrick

INTRODUCTION The study of changes in gene expression levels in tissue and cells has shown tremendous promise in the identification of novel biomarkers and mechanisms for clinical application and risk assessment. However, some early technical concerns hampered the field, from which it has not fully recovered. In the earliest days of microarray technology, the optimal study design (i.e., number of biological replicates, time points of study, analytical approaches, etc.) was unknown and protocols used in laboratories were not standardized. These issues caused groups to report less than stellar comparability results when different labs performed experiments, although appropriate statistical analyses and standardization of protocols did improve the extrapolation.12 This chapter first summarizes the progress that has been made in the past five years, with emphasis on the consensus for use of microarray technology and statistical methods for determining the differential expression to understand underlying mechanisms of disease and toxicity and the prediction of adverse events. Next, an approach to develop microarray-based diagnostic and prognostic tests is discussed, with a description of the FDA-led community supportive consortium effort, to address the issues and challenges associated with this approach. Given the important role of bioinformatics in genomic research, we advocate an integrated approach of data management, analysis, and interpretation through, for example, the FDA genomic tool, Array Track™. Lastly, the application of the genomic technologies is further illustrated with a number of examples. The chapter concludes with the authors' views on the issues and challenges remaining in this field and the way of moving this discipline forward.

7

BIOMARKERS

EVALUATION OF THE TECHNOLOGY The use of cDNA or oligonucleotide microarrays as a molecular tool to measure transcript abundance has been prevalent for more than a decade.3 During this time the production of microarrays and the associated laboratory methods have improved and become more standardized. The technical improvements have been achieved in the manufacturing of microarrays (resulting in improved inter- as well as intra-platform reproducibility), and also in the laboratory procedures used to generate transcript abundance data using microarrays. These process improvements have resulted in decreased requirements for starting material, greater sensitivity, and dynamic range, while simultaneously resulting in more standardized methods, all of which have contributed to improved data reproducibility.' ■2-4-5 One major application of microarray technology is to identify genes differentially expressed between different states, for example, changes between treated and control groups, or between diseased patients and healthy individuals. These so-called DEGs (differentially expressed genes) should be biologically informative, and importantly, be reproducible across different laboratories and platforms. Many statistical methods have been applied for DEG selection, ranging from the simple T-test (ANOVA for multiple groups)6 and SAM (statistical analysis of microarrays)7 to the Benjamini-Hochberg8 method that controls false discovery rate (FDR)9 and the conservative Bonferroni correction approach10 (Figure 2.1). To a greater or lesser degree, all of these methods explicitly or implicitly assume that genes are expressed independent of one another, such that each gene's selection constitutes a null hypothesis test. In reality, mRNA is extracted from tissues of different phenotypes and most genes differentiating phenotypes are expected to act interdependently through a number of complex biological, signaling, and metabolic pathways. True phenotype differentiating genes would be expected to exhibit expression in cascades or constellations with temporal dependency. While much is known

FIGURE 2.1 A summary of the main statistical methods used for determining differentially expressed genes (DEGs) for two-class comparisons using microarrays.The simple t-test method normally produces high false positives while the Bonferroni criterion has low specificity. Given the fact that the correlations among genes being analyzed are unknown, the methods such as false discovery rate, permutation testing, and volcano plot attempt to balance the specificity and sensitivity for DEGs identification.

GENOMICS

9

about pathways and gene associations, the knowledge regrettably remains qualitative and incomplete, precluding direct accounting for such expression covariance. All of these difficulties have led to many proposed DEG selection methods that, unfortunately, produce disparate gene lists. Not long ago, a number of publications11-15 raised concerns about the microarray technology based on the lack of agreement in DEGs or predictive signatures obtained from different laboratories and array platforms for highly similar study designs and experiments. Other performance issues for the array technology were also discussed, including 1) array quality—what degree of experiment quality and individual array platform technical performance should be deemed achievable and adequate? 2) data analysis issues—what results can be anticipated from different algorithms and approaches, and its corollaryxan consensus be reached for a baseline approach to microarray data analysis? 3) cross-platform issues—what consistency can be expected among different microarray experimental platforms? and 4) reliability issues—whether is it still necessary and required for the microarray results to be verified by alternative and well-established gene expression platforms such as real time PCR? On February 11, 2005, the FDA formally launched the Micro Array Quality Control (MAQC) project (http://edkb.fda.gov/MAQC) to address these concerns as well as other performance, standards, quality, and data analysis issues. Phase I of the MAQC project (MAQC-I, from February 11, 2005 to September 8, 2006) focused on assessing technical reliability of microarray technology with participation of 137 scientists from 51 organizations. Gene expression data on four titration pools from two distinct, commercially available reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. The resulting rich reference data set consists of over 1300 microarray hybridizations, and additional measurements for over 1000 genes with alternative technologies such as qPCR. The MAQC-I project observed, when standard operating procedures (SOPs) were followed and the data analyzed properly, high intraplatform reproducibility across test sites, as well as interplatform concordance in terms of genes identified as differentially expressed. Platforms with divergent approaches to the assay generated comparable results in terms of differential gene expression. In other words, the differential gene expression patterns reflected the same biology despite differences in platform technology. Similar results were observed from a realistic rat toxicogenomics experiment, in support of the major findings of data generated from the reference RNA samples. The MAQC-I results were published in six research papers in the September 2006 issue of Nature Biotechnology }^2X The MAQC-I project suggested that the common practice of ranking genes solely by a statistical significance measure like p-value from the simple T-test, and selecting DEGs using a stringently significant p-value threshold was the cause of an apparent lack of reproducibility in microarray experiments and thus recommended selecting genes using fold-change ranking together with a non-stringent p-value cutoff filter to balance specificity/sensitivity and reproducibility of DEGs. Several studies verified this recommendation.22'23

10

BIOMARKERS

It is now generally understood that reproducibility is a complicated issue that is affected by many factors, including the performance metrics used for assessing reproducibility and how to balance reproducibility with statistical power of a study. In general, the more samples for a study, the higher the reproducibility. Reproducibility has an inverse relationship with statistical significance, however this relationship is a complex one (i.e., not a simple trade-off). It appears that reproducibility is dependent on the number of DEGs pre-selected for assessing the reproducibility and, furthermore, the dependency is a complicated one and less understood. But most importantly, irreproducible gene lists resulting from different analysis approaches for the same data set could be all biologically relevant (i.e., true discovery).

CLINICAL APPLICATIONS Clinical diagnostic and prognostic assessments rely on, for example, accurate histopathology, cytomorphology, or immunophenotyping. Unfortunately, some diseases remain hard to classify by these current clinical techniques. The maturation of microarray technology provided the necessary groundwork for the recent deployment of two different microarray-based diagnostic tests that measure transcript abundance related to cancer.24,25 These recent advances highlight the utility of transcript-based molecular signatures (or classifiers) measured by microarrays in clinical applications and suggest their potential application to other fields, such as drug development and risk/safety assessment. Molecular classification uses supervised learning techniques to first identify a molecular signature that separates subjects (known as a training set) into known categories, such as diseases. The derived signature is then verified by predicting new subjects with known diseases (a test set).26-28 Once biologically qualified and validated, the molecular signature could be used to improve early detection of diseases, provide better diagnostic capabilities, etc. Developing classifiers from microarray data is often problematic because: 1) the predictor variables (i.e., genes from a microarray experiment) normally far outnumber the samples (i.e., the number of subjects), increasing the likelihood of a random solution with little or no predictive value; 2) the sample classes are often skewed between, for example, disease and healthy subjects; 3) microarray data tends to have a low signal to noise ratio (i.e., considerable random variability); and 4) diseases that are difficult to diagnose with clinical techniques could result in false positives and false negatives within the sample. Although MAQC-I20 demonstrated the technical reliability of microarray technology in detecting differential gene expression, questions remained regarding reliability of the technology in clinical applications such as disease diagnostics or prognostics, and for tailored patient treatment based on gene expression profiles. Specifically, the reliability and utility of classification models for the prediction of patient outcomes has been questioned in recent literature.14-1529 To investigate the capabilities and limitations of microarray technology in such practical applications, MAQC-II was launched on September 21, 2006 to address technical and scientific issues involved in the

GENOMICS

11

development and validation of predictive models or classifiers. The project aimed to develop best practices through comprehensively evaluating different machine learning approaches and modeling parameters for the development and validation of predictive models or classifiers for clinical and preclinical (toxicogenomics) applications. Multiple existing data sets were selected and distributed to participating organizations for independent data analyses (Table 2.1). A three-step approach was implemented to determine the best practices for gene expression based on predictive signature development (Figure 2.2): 1) Step 1 or initial discovery: predictive models were developed based only on the training set of each of the six MAQC data sets (Table 2.1) and were "frozen" (i.e., cannot be altered further) before receiving the test sets; 2) Step 2 or independent validation: prediction models were challenged by data sets that were set aside and unseen by the analysis teams in the previous step to determine best practices; and 3) Step 3: in this final step of the project, new sets of data will be generated from different labs or platforms to further challenge the "best practices." At the time of this writing, over 10 manuscripts were submitted to Nature Biotechnology and The Pharmacogenomics Journal. The manuscripts can be grouped into three areas of focus: 1) assessing the impact of modeling factors; 2) the process of qualifying a classifier; and 3) generating consensus documents on developing a classifier.

FIGURE 2.2 A three-step approach was implemented in the MAQC-II to develop the best practices for molecular classifiers. Six MAQC data sets (Table I) were divided into the training and test sets. In Step I, the training sets were distributed to 36 analysis teams and the resulting classifiers, signature genes, and the data analysis plans (DAPs) were locked down. In Step 2, the test sets with the samples' classification blinded were released to the analysis teams to challenge the classifiers and the best practices were constructed. In Step 3, new data will be generated to challenge the best practice. At the time of this writing, the manuscripts summarizing the first two steps have been submitted while Step 3 is proceeding in parallel.

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TABLE 2.1

MAQC-1 I data sheets. Datasets*

Clinical data

Step 1

Step 2

Training set

Test set

(n)

(n)

Breast cancer

130

100

Multiple myeloma

340

214

Neuroblastoma

246

253

Lung tumor

70

88

Toxicogenomics

Non-genotoxic hepatocarcinogenicity

216

201

data

Liver injury (necrosis)

214

204

* (I) The breast cancer data set was provided by the MD Anderson Cancer Center; (2) The multiple myeloma data set was provided by University ofArkansas for Medical Sciences; (3) The neuroblastoma data set was provided by University of Cologne; (4) The lung tumor data set was provided by the Hamner Institutes for Health Sciences; (5) The non-genotoxic hepatocarcinogenicity data set was provided by Iconix Biosa'ences, Inc, (now part of Entelos, Inc); and (6) The liver injury data set was provided by the National Institute of Environmental Health Sciences.

BIOINFORMATICS CHALLENGES While large community efforts, such as the MAQC projects, start to address the challenges remaining in the microarray field for clinical application and risk/safety assessment, a robust bioinformatics capability is also widely acknowledged as central to realizing the promises of the microarray technology. Successful application of microarray approaches inextricably relies on a bioinformatics solution for appropriate data management, the ability to extract knowledge from massive amounts of data, and the availability of functional information for data interpretation. Data management—The database and associated software organizes and enables access to all data from a study along with the microarray experimental design information. A microarray experiment involves multiple steps and the data in each step need to be appropriately managed, annotated, and, most importantly, stored in an appropriate data structure for ready analysis and correlation with the study observations. This enables efficient and reliable access for subsequent data analysis normally done by a multidisciplinary group of scientists. Furthermore, re-analysis is likely as new or more accepted analytic methods evolve, a process much more easily carried out with a well-managed and annotated database. Data analysis—With the price for the microarrays and reagents, as well as microarray service, continually declining, larger scale studies using microarrays become feasible—allowing a systematic test of the hypothesis. Consequently, a single microarray study yields a large amount of data and a formidable data analysis and visualization undertaking. The immensity of data analysis scales directly with the complexity of the experiment, such as the number of technical and biological replicates, and temporal and dose response parameters. In addition, since a plethora of potential sources of vari-

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13

ability inevitably complicate, and possibly confound, interpretation, public data can be important for comparison. Thus, the ability to search, filter, and apply mathematical and statistical operations and graphically visualize data quickly with an intuitive user interface becomes essential to facilitate the laborious process. Data interpretation—This is a highly contextual process incorporating known and unknown functions of genes, proteins, and pathways. Efficient and effective interpretation demands that relevant knowledge residing in public sources such as gene annotations, protein functions, and pathways are readily available and integrated with the data analysis process. In addition, the periodic re-examination of the data in light of the continual evolution of gene annotation information and pathways in the public domain is desirable, which further enforces the need of data and analysis results to be properly managed in a structured database. Several commercial vendors and institutes have developed bioinformatics solutions for microarray studies, such as Rosetta Resolver System (http:// www.rosettabio.com/products/resolver) and NIEHS CEBS (Chemical Effects in Biological Systems, http://cebs.niehs.nih.gov/cebs-browser/cebsHome.do). In addition, the public data repositories such as GEO (Gene Expression Omnibus, http://www.ncbi.nlm.nih.gov/geo) and Array Express (http://www.ebi. ac.uk/microarray-as/ae) are available with the primary goal of ensuring that microarray results published in peer-reviewed scientific journals are available for independent evaluation. However, a recent attempt to reproduce the work of 18 published studies in well-recognized prestigious journals met with great difficulty due to the lack of available data and/or accurate description of analytical methods in the papers or referenced public repositories.30 Thus, a data standard (i.e., ontology) is urgently needed beyond the MIAME guideline (Minimum Information About a Microarray Experiment, http://www.mged.org/workgroups/MIAME/miame.html) to report accurately the study design, microarray array experiment, and analysis methods. The data models corresponding to the standards need to be established with robustness to accommodate the evolving nature of the technology and other emerging molecular technologies. One effort with such potential is the bioinformatics solution developed at the FDA's National Center for Toxicological Research (NCTR) described below. The NCTR/FDA is developing a public data management, analysis, and interpretation software called ArrayTrack™31,32 (http://www.fda.gov/ScienceResearch/BioinformaticsTools/Arraytrack/default.htm). The tool is primarily used in the FDA for reviewing genomic data submitted by sponsors through the Voluntary Genomics Data Submission (VGDS) program (http://www.fda. gov/OHRMS/DOCKETS/98fr/2003d-0497-gdl0002.pdf). ArrayTrack stores all data and information related to DNA microarrays and the clinical and nonclinical study, as well as the processed data derived from proteomics and metabonomics experiments. In addition, ArrayTrack provides a rich collection of functional information about genes, proteins, and pathways drawn from various public biological databases for facilitating data interpretation. Many data analysis and visualization tools are available within ArrayTrack for individual

14

BIOMARKERS

FIGURE 2.3 A typical workflow using ArrayTrack to identify DEGs distinguishing treatment and control groups, followed by pathway and Gene Ontology (GO) analyses. (A) DEGs are identified using the Volcano plot or other means in ArrayTrack. DEGs can also be identified using other commercial or public tools and uploaded into ArrayTrack (B) DEGs are summarized in a table format and can be readily linked to ArrayTrack library functions for biological interpretation; (C) Significantly altered KEGG pathways are identified based on DEGs; (D) DEGs are submitted to the Gene Ontology tool in ArrayTrack to identify GO terms associated with significantly altered gene expression.

platform data analysis, multiple omics data integration and integrated analysis of omics data with study data. Importantly, gene expression data, functional information, and analysis methods are fully integrated so that the data analysis and interpretation process is simplified and enhanced. Using ArrayTrack, users can select an analysis method from the ArrayTrack toolbox, apply the method to selected microarray data, and the analysis results can be directly linked to individual gene, pathway, and gene ontology analysis (Figure 2.3). ArrayTrack is publicly available online.

APPLICATIONS TO DRUG TOXICOLOGY, MEDICINE, AND ENVIRONMENTAL HEALTH Genomics has been applied to a) improve our understanding of basic biological processes as well as the diversity of these processes, b) delineate mechanisms of efficacy and toxicity of xenobiotic compounds (e.g., drug, dietary supplements, and environmental agents), c) understand disease processes, and d) generate predictive models or molecular classifiers to provide better predictive and diagnostic accuracy.

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15

Improve Understanding of Basic Cellular A r c h i t e c t u r e and Function Examination of gene expression levels in various cells and tissue types has expanded our understanding of basic cellular function and opened many doors to new hypotheses. For example, the Japanese Toxicogenomics Project evaluated gene expression in discrete areas of the kidney, a highly heterogeneous organ with distinct functional units that are more or less susceptible to individual xenobiotic compounds.33 Although this can serve as a valuable source to improve our understanding of specific units' functioning, it is impractical routinely to isolate each of the functional units for analysis. Although genomics has been utilized most often to investigate an adverse event or disease, it can be employed to study basic issues such as animal husbandry, species, aging, and gender differences. Examination of genes expressed in the liver of normal rats can discriminate genders, fasted from rats fed ad libitum, and can distinguish Wistar from Sprague-Dawley rats, two outbred strains of albino rats.34 The Japanese initiative mentioned above also examined acetaminophen hepatotoxicity as an effect of aging and these investigators reported differences in the time course of response that may suggest older rats are more susceptible.35 Age also seems to play a role in human reactions to idiosyncratic drugs, compounds that fail to induce signs of hepatotoxicity in the classical tests performed in nonclinical species such as rodents.36 The same study found that gender seems to play a role in the extent of liver failure seen in such patients. As noted above, expression of genes in the liver of normal rats can discriminate the genders and thus may lead to clues as to the differential severity of hepatic injury seen in females.34 Gene expression in disease states that affect one sex more than another is a source of much research in areas other than liver disease, such as inflammatory diseases that affect women more than men,37-38 and vice versa in the case of hepatocellular carcinoma.3940 Another use of genomics is to identify the similarities and differences between cells in situ and those used in vitro. To reduce the use of animals and provide higher throughput assays, investigators would prefer to use in vitro hepatocyte systems rather than treating the rats in vivo. However, no clarity exists on which in vitro system most closely replicates the normal liver under normal situations or upon toxicant exposure. Some have used genomics to try and answer those questions. For example, Boess, et al. reported that liver slices were more similar to intact liver than primary hepatocytes.41 In contrast, Jessen, et al. studied the expression of genes within the rat liver, liver slices and primary rat hepatocytes prior to and after treatment with hepatotoxicants. Better correlation exists in the untreated state between the two in vitro systems (R2 = 0.87) than between cultured hepatocytes and liver slices with in vivo (R2 = 0.80 for both).42 They reported that both in vitro systems exhibited fewer gene expression alterations than did animals treated in vivo, an understandable reaction as neither in vitro system reflects circulatory aspects of the liver and replicates the pharmacokinetic aspects of the drug, to name a few differences. These authors concluded that they could not identify the best in vitro system to

BIOMARKERS

be used for the study of hepatotoxicity, although most utilize cultured hepatocytes versus tissue slices. Some of the different conclusions reached as to the best in vitro system to be used (i.e., slices versus hepatocytes) could be due to culture conditions, as there are no generally accepted standards of media and substratum used for such cells.

echanism of T o x i c i t y and Disease Examination of the mechanisms whereby drugs, dietary supplements, environmental agents, or disease processes alter cellular function and cause injury can lead to the discovery of new biomarkers and thus preventive intervention. In the area of drug or chemical development, such biomarkers can be incorporated into a screening assay and used to avoid similar liabilities with other compounds.43 Researchers at the EPA used a toxicogenomics approach to understand the different types of hepatic effects caused by triazole fungicides and derivatives of perfluoroalkyl acid. They found that this approach could be used to categorize the chemicals, provide mechanistic insight and predict downstream pathological damage.44 They concluded that toxicogenomics can be useful in the assessment of environmental risk. Much is being learned in terms of disease processes and such knowledge can lead to the identification of biomarkers that can span divergent needs such as diagnoses and providing new drug targets.45 A recent review by Margulies, et al. discussed the insight learned from applying genomics technology to the study of heart failure in animals and humans.46 Likewise, genomics approaches are leading us closer to a systems biology understanding of renal diseases.47 To assist in these endeavors, the European Renal cDNA Bank was created in 1998 to store samples of kidney biopsies for transcriptomics study. Such collaborative studies, for example, revealed genomic fingerprints that potentially can stage diabetic nephropathy, revealing potential new therapeutic targets to halt the progression of renal disease.48 Drugs are known to have heterogeneous effects on individual patients and between species. Since many xenobiotics undergo metabolic activation, genomics has been applied to the study of drug-metabolizing genes to help identify patient-specific susceptibility biomarkers and population (e.g., species) differences. Mattes, et al. examined the expression levels of genes involved in the glutathione pathway, a major route of detoxification, and reported major differences in basal tissue levels among mouse, rat, and canine.49 Several other groups have examined species similarities and responses to drugs and environmental agents to help explain in-life observations (reviewed in 50). Genomics can be used to study the pharmacology of drugs. For example, actions of immunosuppressive drugs were studied in mice and revealed common genes and pathways that can be used for future compound screening.51 The ability of drugs to inhibit the liver X receptor that controls cholesterol efflux from peripheral tissues can be studied with a genomics approach using peripheral blood from rodents, non-human primates, and humans.52 In an effort to find a noninvasive way to study cholesterol metabolism, a transcriptomics approach was utilized on circulating mononuclear cells and liver tissue

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17

from guinea pigs. The results indicated that the former could accurately reflect changes induced by drug therapy.53 The use of circulating white blood cells has shown great potential in the differential diagnosis of many human diseases and is discussed below. Another area of research involves mitochondria function. Mitochondrial dysfunction has been shown to contribute to disease, metabolic abnormalities, and drug induced toxicity. Desai and collaborators generated a microarray containing genes involved in the structure and function of the mitochondria (MitoChip)54 and have used this to identify dysfunction due to an idiosyncratic drug,55 nucleoside reverse transcriptase inhibitors,56 and a dietary supplement.57 Thus, genomics identifies many potentially useful biomarkers of normal cellular function, differences between sexes, response to xenobiotics, and disease processes. It also can provide an understanding of the mechanism of drug actions, environmental agents, and diseases. However, identification of individual biomarkers with understandable roles in such processes requires specialized training, subjective evaluation, and may lead to the lack of sufficient power to provide needed accuracy.

Algorithmic Models to Predict Toxicity or Disease Biologically-meaningful (i.e., easily interpretable) changes in gene expression provide context and thus are most acceptable to investigators. However, in some cases one does not obtain the needed accuracy if only utilizing genes with an understandable role to play in the adverse event or disease processes. Additionally, some biomarkers contribute more to the phenotype than others, thus requiring a weighting scenario employing a mathematical model. Thus, many investigators are using a statistical approach to identify and predict adverse events or disease. (Such approaches have been extensively evaluated in the aforementioned MAQC-II project.) For example, in the field of environmental chemicals, Thomas and colleagues at The Hamner Institute utilized a toxicogenomics approach to find methods to shorten the exposure time in animal studies used to identify lung tumor carcinogens. They identified six genes that could discern, after 13 weeks of exposure, chemicals known to induce lung tumors after a two-year exposure, thus suggesting a faster system to identify carcinogens.58 This data set was also extensively investigated and analyzed by the MAQC teams. Since hepatotoxicity remains a major issue during drug development and upon approval of drugs, many investigators have focused on a toxicogenomics approach to improve detection of such compounds using in vivo exposure in the rat and/or treatment of primary rat hepatocytes in culture. Successful predictive toxicogenomic algorithmic models have been generated in many pharmaceutical companies (e.g., AstraZeneca, GlaxoSmithKline, and Millennium Pharmaceuticals) and in commercial companies (Gene Logic and Iconix) yet, in most situations, the biomarker panels identified have not been subjected to public qualification since there is no business incentive to do so. Some predictive models, based on algorithms using a large number of genes in the rat, have been found capable of identifying compounds that can cause

18

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phenotypically-obvious damage in rats and humans and those of a more idiosyncratic nature, i.e., drugs for which no liver adverse event is seen in rats.59"61 This suggests that a toxicogenomics approach in drug discovery might 1) provide more complete compound information at a relatively early stage of drug discovery to assist in decision-making, and 2) offer clues as to potential biomarkers that might be useful to follow or prevent adverse events. The use of a multiplex algorithmic model has shown promise in multiple clinical indications. For example, as noted above, MammaPrint® is an FDA-approved test that examines gene expression in breast cancer tissue and provides the physician and patient with a report detailing the risk of cancer recurrence. Another FDA-approved assay, AlloMap®, monitors changes in 20 genes present in circulating white blood cells to assess the risk of cardiac allograft rejection. Using gene expression alterations as biomarkers of tissue injury is not restricted to allograft rejection although, to date, the other biomarkers have not been qualified for use. These include the examination of changes in the gene expression in circulating white blood cells as a measure of disease activity and/or disease type. Examples include distinguishing patients with ulcerative colitis from those suffering from Crohn's disease,62 identifying various forms of neurological disease,63 and predicting the future of allograft rejection.64 A very promising study suggests the use of such an approach may be more accurate than classical endpoints at detecting toxic levels of drugs in animals and humans.65

S T R E N G T H S , W E A K N E S S E S , A N D T H E ROAD FORWARD Since microarrays monitor the expression levels of tens of thousands of genes at one time, genomics is an ideal discovery tool to identify biomarkers of interest in understanding mechanisms of drug actions, exposure to environmental agents, and disease processes. However, in some settings, such as clinical medicine, access to the tissue itself is not feasible. Therefore, the search tends to focus on biomarkers accessible in body fluids. As discussed above, one method is to employ a genomics approach to study the effects of exogenous compounds and disease on circulating white blood cells since they 1) may have the same biological process as the tissue of interest and thus reflect the exact biological change, and 2) serve as a sentinel system in the body and may reflect abnormalities within the body. Another approach is to mine the genomics data in search of genes that encode secreted or cell surface proteins, thus providing starting points to pursue a proteomics approach.66 However biomarkers are discovered, there are major hurdles to their acceptance and use. Those identified and tested side-by-side with a drug can be qualified and the testing platform validated during this process. Even then, different regulatory centers at the FDA evaluate drugs and testing devices. Yet more challenging is when biomarkers are identified outside the clinical trial paradigm. Who will determine if biomarkers meet biological and testing standards?

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Who will pay for the appropriate testing? Goodsaid and Frueh at the FDA were instrumental in establishing a path forward at that agency.67 Working together with the Critical Path Institute's Predictive Safety Testing Consortium and its member pharmaceutical companies, a series of seven urinary protein biomarkers in rats were qualified for regulatory use by the FDA and EMEA68 (http:// www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/2008/ucml 16911. htm). The establishment of a working method to obtain regulatory approval of biomarkers not tied to a specific drug and a consortia-led effort to obtain the necessary data to do so seems to be the best method to successfully qualify biomarkers. Multiple consortia have been established to investigate both nonclinical and clinical biomarkers within the Critical Path Institute. However, each effort will need to be decided on a case-by-case basis. This can be a difficult task, particularly for clinical qualification as the costs likely will be high. Assuming clinical biomarkers are successfully qualified, the next hurdle they face will be patient/physician acceptance and third party reimbursement. The Genetic Information Nondiscrimination Act (GINA) of 2008 was written to protect individuals against discrimination based on their genetic information. However, it does not cover all types of insurance (e.g., life insurance) and covers genotype testing only.69 These gaps may discourage individuals from being tested with new genomic and proteomics biomarkers particularly if they provide predictive information as to eventual disease or likelihood of an adverse event. Another impediment is to obtain third-party payment. Most insurance companies follow the lead of the Centers for Medicare and Medicaid Services (CMS), a United States federal agency. However, coverage for tests may be limited by the law stating that reimbursement for services must be "necessary for the diagnosis or treatment." Thus, tests used to screen individuals without current complaints requires new legislation by Congress as was necessary, for example, for breast cancer screening.70 CMS and insurance carriers are requiring clear evidence that new biomarkers improve patients' health, lower costs, etc. and this can be difficult to obtain. Warfarin is a useful example to illustrate the complexity of the issue. It is a widely used anticoagulant with a narrow therapeutic window and large patient-to-patient variability. Warfarin causes 15 percent of all severe adverse events in the U.S. and a number of studies have found that variants of two genes along with other factors such as age can account for 31 to 79 percent of the inter-patient variability.71 Thus, the FDA revised warfarin labeling to include pharmacogenetic information and has approved multiple genetic tests. A recent clinical trial found that this algorithm approach using pharmacogenetic and clinical factors was successful in selecting a starting dose closer to the stable maintenance dose needed for a patient.72 However, CMS recently refused to cover the cost of the pharmacogenetic test even for this widely-used drug known to cause many serious adverse events. They stated that there was insufficient evidence to demonstrate it improved health outcomes (https://www.cms. hhs.gov/mcd/viewdraftdecisionmemo.asp?from2-viewdraftdecisionmemo. asp&id=224&), but will cover the cost of testing when in concert with a clinical trial to study the effectiveness of this approach.

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It is not clear who will pay for expensive clinical trials using generic drugs such as warfarin, particularly since diagnostic companies generally do not make sufficient profit to cover research costs and there is little or no incentive for drug companies to limit their markets with such tests. Once again, a consortium-type effort may be the best avenue, although it can be expected that only a limited number of biomarkers can be tested under such costly circumstances.

CONCLUSION Acceptance of microarray technology has benefited from the work of the MAQC consortia as discussed above. The work of the first effort showed, for example, that data generated in one laboratory can be replicated in another, addressing a major criticism at the time. It also demonstrated that similar biological networks and pathways are identified when different platforms are used. Newer consortia efforts should help establish appropriate methods to develop algorithmic models as well as methods to test their accuracy. Although the field of genomics is more standardized than some of the other omic technologies (metabolomics and proteomics), improvement is still needed, particularly in the discussion of when data are of sufficient quality and robustness to be analyzed. For example, if the RNA shows some signs of degradation, when is it deemed of insufficient quality to continue? There are no universally accepted pass/fail criteria for measurements of RNA integrity and data quality originating from microarrays, so the field will need to set some standards. Even after such technical considerations are met, it is no surprise that, once again, technology is outpacing human acceptance. It will take time and the efforts of many to address privacy concerns and resolve third-party payment issues before the usefulness of genomics will be fully appreciated.

SUMMARY P O I N T S 1. 2. 3. 4.

Genomics is achieving widespread acceptance. Great strides have been made in the discovery of potential genomic biomarkers in tissues and in circulating white blood cells thus enabling clinic use. Issues still remain in areas such as mining the data and determining when data is of sufficiently high enough quality to merit review. Privacy issues and the lack of third-party payment is hampering clinical acceptance of new biomarkers.

DISCLAIMER The views presented in this article do not necessarily reflect those of the U.S. Food and Drug Administration.

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BIOMARKERS 55. 56. 57. 58. 59. 60. 61. 62. 63. 64.

65. 66. 67. 68. 69. 70. 71. 72.

Kashimshetty, R., Desai, V. G., and Kale, V. M., et al. Underlying Mitochondrial Dysfunction Triggers Flutamide-Induced Oxidative Liver Injury in a Mouse Model of Idiosyncratic Drug Toxicity. Toxicol. Appl. Pharmacol. 2009. Desai, V. G., Lee, T., and Delongchamp, R. R., et al. Nucleoside Reverse Transcriptase Inhibitors (NRTIs)-Induced Expression Profile of Mitochondria-Related Genes in the Mouse Liver. Mitochondrion. 2008;8:181-195. Joseph, A., Lee, T., and Moland, C. L., et al. Effect of (+)-Usnic Acid on Mitochondrial Functions as Measured by Mitochondria-Specific Oligonucleotide Microarray in Liver of B6C3F1 Mice. Mitochondrion. 2009;9:149-158. Thomas, R. S., Pluta, L., Yang, L., and Halsey, T. A. Application of Genomic Biomarkers to Predict Increased Lung Tumor Incidence in 2-Year Rodent Cancer Bioassays. Toxicol. Sci. 2007;97:55-64. Hultin-Rosenberg, L., Jagannathan, S., and Nilsson, K. C , et al. Predictive Models of Hepatotoxicity Using Gene Expression Data from Primary Rat Hepatocytes. Xenobiotica. 2006;36:1122-1139. Martin, R., Rose, D., Yu, K., and Barros, S. Toxicogenomics Strategies for Predicting Drug Toxicity. Pharmacogenomics. 2006;7:1003-1016. Kenne, K., Skanberg, I., and Glinghammar, B., et al. Prediction of Drug-Induced Liver Injury in Humans by Using In Vitro Methods: The Case of Ximelagatran. Toxicology in Vitro. 2008;22:730-746. Burczynski, M. E., Peterson, R. L., and Twine, N. C , et al. Molecular Classification of Crohn's Disease and Ulcerative Colitis Patients Using Transcriptional Profiles In Peripheral Blood Mononuclear Cells. J. Mol. Diagn. 2006;8:51-61. Sharp, F. R., Xu, H., and Lit, L., et al. The Future of Genomic Profiling of Neurological Diseases Using Blood. Arch. Neurol. 2006;63:1529-1536. Mehra, M. R., Kobashigawa, J. A., and Deng, M. C , et al. Clinical Implications and Longitudinal Alteration of Peripheral Blood Transcriptional Signals Indicative of Future Cardiac Allograft Rejection. J. Heart Lung Transplant. 2008;27:297-301. Bushel, P. R., Heinloth, A. N., and Li, J., et al. Blood Gene Expression Signatures Predict Exposure Levels. Proc. Natl. Acad. Sci. USA. 2007;104:18211-18216. Mendrick, D. L. and Daniels, K. K. From the Bench to the Clinic and Back Again: Translational Biomarker Discovery Using In Silico Mining of Pharmacogenomic Data. Biomarkers Med. 2007;1:319-333. Goodsaid, F. M. and Frueh, F. W. Biomarker Qualification Pilot Process at the U.S. Food and Drug Administration. AAPS J. 2007;9:E105-E108. Goodsaid, F. M., Frueh, F. W., and Mattes, W. Strategic Paths for Biomarker Qualification. Toxicology. 2008;245:219-223. Rothstein, M. A. Currents in Contemporary Ethics. GINA, the ADA, and Genetic Discrimination in Employment. J. Law Med. Ethics. 2008;36:837-840. Secretary's Advisory Committee on Genetics HaS. Realizing the Potential of Pharmacogenomics: Opportunities and Challenges. 2009. Kim, M. J., Huang, S. M., Meyer, U. A., Rahman, A., and Lesko, L. J. A Regulatory Science Perspective on Warfarin Therapy: A Pharmacogenetic Opportunity. J. Clin. Pharmacol. 2009;49:138-146. Anderson, J. L., Home, B. D., and Stevens, S. M., et al. Randomized Trial of Genotype-Guided versus Standard Warfarin Dosing in Patients Initiating Oral Anticoagulation. Circulation. 2007;116:2563-2570.

CHAPTER

PROTEOMICS FOR BIOMARKER DISCOVERY Timothy D. Veenstra

INTRODUCTION While it can be argued that genomics is the foundation of all "omics" that came subsequently, there is no denying the need for these other fields of study. Figure 3.1 shows that progression of complexity and regulatory events that occur as biological molecules are created from the DNA template. The human genome contains in the range of 25,000 genes. The sequence of bases within these genes dictates the protein that is ultimately translated and mutations can lead to errors that cause diseases such as cancer and neurological disorders. The transcription of these genes is regulated by outside agents such as transcription factors, but also by methylation and acetylation of individual bases within promoter regions. These genes are transcribed into mRNA transcripts that are post-transcriptionally regulated by microRNAs and events such as alternative splicing. Finally, these transcripts are translated into proteins that are further regulated by post-translational modifications such as phosphorylation, acetylation, glycosylation, etc. The net effect of all of these regulation events is 25,000 genes giving rise to potentially hundreds of thousands of proteins. The exact number is unknown and may never be known as the proteome complement of a cell is dynamic and sensitive to internal and external stimuli. While it is a daunting challenge, it is important to be able to piece together the puzzles of the proteome as these proteins play a major part in dictating the phenotype of the cell, tissue, or organism from which they are derived. This hope of being able to comprehensively scan complex mixtures has been the foundation of what drives the discovery of biomarkers for human diseases, such as neurological disorders and cancers. There are major tech25

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nologies that have enabled complex biological samples to be interrogated at the genomic, transcriptomic, proteomic, and metabolomics. For genomics, high-throughput sequencers (such as the 454 sequencer) are able to sequence 400-600 megabases of DNA per 10-hour run. This throughput allows whole genomes to be sequenced or sequences within large numbers of samples to be compared in genome-wide associated studies (GWAS) for the discovery of disease-specific mutations. For transcriptomics, DNA microarrays containing tens of thousands of probes enable comparative analysis of messenger RNA (mRNA) from various patients to discover differences in the abundances of transcripts within complex samples. For proteomics and metabolomics, mass spectrometry (MS) has been the driving technology as it possesses the capability of detecting thousands of proteins or metabolites in the time frame of hours. While genome sequencing has provided incredibly valuable insight into individual susceptibility to diseases such as cancer, and is now providing information directing the proper types of treatments, many scientists are looking to proteomics to provide biomarkers that indicate the early onset of disease or the response to a chosen therapy.

T I S S U E OR B I O F L U I D It is this ability to identify thousands of proteins within complex samples (such as blood, urine, tissue, etc.) that has spurred the hope of using MS to find

FIGURE 3.1

Flow of information from genes to proteins.

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novel biomarkers. The goal is to simply identify and quantitate differences in proteins between comparative samples (e.g., healthy versus diseased) and hope that any of these proteins can be directly associated with the disease of interest. Before initiating any biomarker discovery project it must first be determined what types of samples will be studied and what need will the biomarker fill.1 For example, let's assume the disease of interest is renal cell carcinoma (RCC). This carcinoma arises within the proximal renal tube and is the most most common type of kidney cancer in adults.2 Detection of RCC most often occurs when noninvasive imaging is being used to evaluate non-specific symptoms. Tumors detected in this manner are generally smaller, early stage tumors than if they are detected in RCC patients that are exhibiting symptoms related to paraneoplastic disorders (e.g., hypertension, anemia, abnormal liver function, etc.), pain or mass related to metastatic disease. Fortunately, approximately 60% of RCCs are diagnosed while the disease is still localized.3 Renal cell carcinoma is, however, notoriously chemo-resistant.2 Therefore, while an early stage diagnostic marker would benefit RCC patients, what would be even more beneficial is a biomarker that would predict therapy response, enabling correct selection of the most effective therapies for each individual. On the other hand, ovarian cancer has a different need. Ovarian cancer affects over 22,000 women in the U.S. annually.4 This cancer is treatable when detected at an early stage; as reflected by the statistic that greater than 90% of women diagnosed with ovarian cancer prior to its spreading beyond the ovary live at least five years after detection. Unfortunately, less than 20% of ovarian cancers are detected at this early stage. Therefore, the greatest impact on individuals with ovarian cancer would come in the form of a biomarker that diagnoses early stage disease. For conducting the proteomic comparative studies, obtaining case and control samples that are properly matched based on parameters such as gender, age, ethnicity, and lifestyle is critically important. Another major decision that needs to be considered is whether to study biofluids or tissues.1,5 A partial list of advantages and disadvantages in working with either type of sample is provided in Table 3.1. In a large number of biomarker discovery efforts, biofluids such as serum, plasma, or urine have been the sample of choice for a number of reasons. Biofluids are easier to obtain than tissue samples. Urine collection, for example, is almost completely noninvasive, and blood samples are generally drawn as part of a routine physical. Tissue collection requires invasive procedures that may include general or local anesthesia. If the purpose is to discover a protein biomarker for early-stage diagnosis, it is much easier to obtain a biofluid sample to measure. It is impractical to acquire a tissue sample on an annual basis for a disease that may affect only a small subpopulation of individuals. The analysis of biofluids also has a number of disadvantages if the aim is to discover disease-specific biomarkers. The main disadvantage is the chance of finding a biomarker that is highly specific for the disease being investigated. Every cell in the body is within four cell units of the circulatory system. Taking this fact and the role of the circulatory system in molecular transport, it can be concluded that the proteome of these biofluids is made up of com-

BIOMARKERS

28

TABLE 3.1 Comparison of advantages and disadvantages of analyzing tissues and biofluids for the discovery of disease-specific biomarkers.

Characteristic Easy to acquire

Tissue

Biofluid

No

Yes

Higher

Lower

Can be localized to specific cell environment

Yes

No

Can study function in cell

Yes

No

Can compare levels in surrounding tissue

Yes

No

Useful for early disease detection

No

Yes

Useful for therapeutic monitoring

Yes

Yes

Concentration of biomarker

ponents originating from cells throughout the entire body.6 Discovery of a cancer-specific biomarker in serum for example, requires the ability to analytically detect a protein that has been secreted by a tumor and is diluted within a complex matrix prior to retrieving the sample. In addition, circulating proteins are subject to a number of proteolytic modifications that can occur during its transport from the site of disease. These modifications may render the protein unrecognizable depending on the analytical methods chosen. While the odds of finding a biomarker in any biofluid using a completely discovery driven approach are small, discovering one would be an enormous benefit to public health. This fact is what keeps scientists working in this area. The use of tissue samples also has its own advantages and disadvantages. Most biomarker discovery studies address a disease that is localized to a specific area in the body (e.g., tumor, neurological disorder), therefore examining the source tissue provides the greatest chance of recognizing a verifiable disease-specific biomarker. Determining other proteins with which the biomarker may interact can be performed to understand its function within a cellular context. The most obvious disadvantage when designing a study to analyze tissues is their availability as their procurement is invasive and the amount of material obtained is small. Even if a tissue-related biomarker was discovered, its use would be limited to therapeutic monitoring as biopsies cannot be routinely performed during yearly physical checkups for use in early-stage diagnostic testing.

TECHNOLOGY Finding disease-specific protein biomarkers can be the ultimate discoverydriven study. These studies are totally unbiased and are approached with no a priori knowledge of what the biomarker will be or to what class of proteins it belongs. Sometimes these experiments are referred to by the unflattering term "blind fishing expeditions," since the scientists have no idea what type of "fish" they are going to catch. If we build upon this analogy, MS-based biomarker discovery fishes with a net with the goal to capture and identify as many fish (i.e., proteins) as possible.7 Unfortunately, the mass spectrometer


29

(net) is not very selective and you end up identifying (catching) whatever proteins (fish) end up being selected (trapped). The major development in proteomics that has allowed the field to consider biomarker discovery has been the exponential increase in the size of the net and the number of fish that can be caught in a single experiment. How is it then that investigators believe that this unbiased approach will result in the discovery of disease-specific biomarkers? Their faith is built on the development of MS technologies that can identify thousands of proteins in a matter of hours. A subtle point that needs to be clarified is that mass spectrometers are primarily designed to identify peptides not proteins. It is important to understand that a protein that is identified using MS is in almost all cases identified through its peptides that are correlated back to their protein of origin. Therefore, most complex proteome samples are digested into tryptic peptides prior to MS analysis. Trypsin is the proteolytic enzyme of choice because of its high degree of specificity, which enables a defined search constraint to be added when identifying the peptides (i.e., every peptide must end with an Arg or Lys residue).

Protein I d e n t i f i c a t i o n Using Mass S p e c t r o m e t r y An illustration on how MS is able to identify thousands of proteins in complex proteome samples is shown in Figure 3.2. The base-peak chromatogram of a complex proteome sample that has been analyzed using an ion-trap mass spectrometer is shown in panel A. The base-peak chromatogram is a recon-

FIGURE 3.2 Schematic of how mass spectrometers select and identify peptides and proteins within complex biologic samples.

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BIOMARKERS

struction of the peptide signals that are observed by the mass spectrometer. Examining a defined slice within the base-peak chromatogram (Panel B) reveals several peptide signals that were observed at this specific time point. To identify the peptides present, the mass spectrometer is instructed to sequentially isolate individual peptides and subject them to collisional induced dissocation (CID), popularly known as tandem MS (MS2). In most studies, peptides are sequentially selected based on their signal intensity (from highest to lowest). In the example provided in panel C, the peptide at m/z 1785 is isolated and subjected to MS2. The peptide at m/z 1821 is then isolated and subjected to MS2, followed by the peptide at m/z 1404, then 1912, etc. This sequence is conducted until a designated number of peptides (usually between 5 and 10) have been analyzed in this fashion. The mass spectrometer then records another mass spectrum to see if new peptide signals are detected before repeating the sequence of isolating peptides and recording their MS2 spectra. State-of-the-art mass spectrometers are able to repeat this sequence of peptide isolation followed by MS2 approximately 7000 times per hour. The entire set of recorded MS2 spectra is analyzed using software that converts the raw data into peptide identifications. While not every MS2 spectra provides a reliable identification, approximately 1000 peptides can be confidently identified in a single one-hour LC-MS2 experiment.

Sample Preparation Sample preparation prior to MS analysis is critical to the success of any proteome biomarker discovery project. The type of sample selected for the project will dictate many of the strategies used to prepare the sample. If plasma or serum is to be analyzed, it is critical to deplete the high abundant proteins. Approximately 99% of the protein content of serum and plasma is made up of 22 proteins, with albumin itself representing about 50% of their protein content (Figure 3.3A).8 If albumin and some of these other high abundant proteins are not removed from the sample, the mass spectrometer will not select peptides from lower abundant proteins and the net result will be a long list of identified peptides that originate from proteins such as albumin, transferrin, immunoglobulins, etc. Albumin depletion has been accomplished through its binding to Cibacron blue,9 specific antibodies,10 and peptides." Others have relied on chromatography12,13 or ultrafiltration14 to remove albumin, however, because of their lack of specificity, these methods can result in the loss of other potentially important proteins. Over the past two to three years immunoaffinity systems that remove large numbers of highly abundant proteins from serum and plasma have been developed and are commercially available. One of the first was the multiple affinity removal system (MARS) from Agilent that immunodepletes six high abundant proteins (i.e., albumin, IgG, IgA, transferrin, haptoglobin, and alpha-1-antitrypsin).15 An example of the depletion capabilities of this column is shown in Figure 3.3B. Studies of the robustness and reproducibility of MARS columns showed high reproducibility (standard deviation between runs >7%) over 250 serum samples analyzed during a six week period.15 Other commercial vendors have built upon


31

FIGURE 3.3 A) Relative abundance of proteins within human serum. B) Depletion of high abundance proteins in serum using the immunoaffinity-based multiple affinity removal system (MARS). Lane 2: serum standard; Lane 3: raw plasma; Lanes 4 and 7: plasma proteins elute from MARS column during first washing step; Lanes 5 and 8: plasma proteins that elute from MARS column during second washing step; Lanes 6 and 9: high abundant proteins that are retained by the MARS immunodepletion column. (See color insert for a full color version of this figure.)

the success of MARS technology to create columns capable of depleting up to 20 of the highest abundant proteins from serum or plasma.16 The effectiveness and ease of use of these columns have made them a standard procedure in the processing of serum or plasma prior to biomarker discovery by MS. Immunodepletion is typically not used with urine, however, it is useful for measuring CSF as this fluid can contain substantial amounts of albumin depending on the circumstances surrounding sample collection. Tissue samples are not generally immunodepleted, however, these can also contain albumin due to vascularization. Even if a sample is immunodepleted it is still much too complex to be directly infused into the mass spectrometer with the anticipation of obtaining thousands of peptide identifications. The basic strategy is to "divide and

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BIOMARKERS

conquer." The peptides need to be effectively separated (i.e., divided) so that the mass spectrometer has the opportunity to identify (i.e., conquer) as many of them as possible. Probably the earliest separation technique used for biomarker discovery was two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).17 State-of-the-art 2D-PAGE gels that utilize pi range focusing strips can resolve upwards of 3000 protein spots. While 2D-PAGE is routinely criticized for being laborious and low-throughput, the fact that it visualizes changes in protein abundance at the gel level requires only certain spots be selected for further MS analysis. One of the issues raised in the past in using 2D-PAGE for comparing complex biofluid proteomes for discovering biomarkers is the difficulty in aligning gels. While software programs for proper gel alignment have been developed and work quite effectively, nothing can replace reproducibility of separation within the gel itself. The development of 2D differential gel electrophoresis (DIGE) in 1997. has helped to ease the problem of gel-to-gel irreproducibility.18 This method has become increasingly popular, as illustrated by the over 200 manuscripts listed on PubMed for the year 2008 that used this technique. Up to three different proteome samples can be compared on a single 2D-PAGE gel using the DIGE method. In a typical 2D-DIGE experiment, three different samples (e.g., healthy, diseased, and internal control) are covalently labeled using different fluorophores, l-(5-carboxypentyl)-l'-propylindocarbocyanine halide Af-hydroxysuccinimidyl ester (Cy3), l-(5-carboxypentyl)-l'-methylindodicarbocyanine halide iV-hydroxysuccinimidyl ester (Cy5), and 3-(4-carboxymethyl)phenylmethyl)-3' -ethyloxacarbocyanine halide N-hydroxysuccinimidyl ester (Cy2). Equal amounts of the labeled proteomes and control sample are combined and resolved on a single 2D-PAGE gel. The resolved gel is scanned using different wavelengths corresponding to the excitation of the Cy2, Cy3, and Cy5 dyes. The individual fluorescent images are then merged to detect differences between the abundance levels of proteins from the three different proteomes from the different dyes to be compared. The ability to co-separate samples on the same gel ensures accurate quantitation of the same spots, eliminating confusion related to gel mis-alignment. As with conventional 2D-PAGE, differentially abundant protein spots, as determined by differences in the fluorescent images, are cored from the gel, enzymatically digested, and identified using MS. The other major prefractionation strategy used in biomarker discovery is referred to as solution-based. These "non-gel" based strategies rely on liquid chromatography (LC) methods to fractionate the proteome prior to MS analysis. A number of solution-based prefractionation methods have been proposed, however, they universally end with a reversed-phase (RP) LC separation that is coupled directly to the mass spectrometer for direct elution of peptides into the instrument. Most solution-based fractionations rely on at least two-dimensions of LC, however, strategies employing up to four dimensions have been utilized. The most commonly used fractionation to be combined with RPLC is strong cation exchange (SCX). This combination, popularly known as MudPIT for multidimensional protein identification technology, was popularized


33

by Dr. John Yates III (Figure 3.4).19 This combination has been used to analyze a variety of clinically relevant proteome samples including plasma, serum, urine, cerebrospinal fluid, plasma filtrate, and blood ultrafiltrate, often resulting in the identification of hundreds or thousands of proteins.20

Protein Quantitation A challenge in conducting solution-based separations with MS for comparative proteomics for the discovery of biomarkers is how to quantify differences between the peptides that are observed. While isotope-labeling methods such as isotope-coded affinity tags (ICAT) and 0 16 /0 18 labeling can be used in a solution-based approach, these methods only allow two samples to be directly compared with each other. Biomarker discovery requires several samples to be directly compared. To enable multiple samples to all be compared with each other, investigators have relied on methods that contrast the number of peptides identified for a specific protein or the actual peak intensities provided by specific peptides in different spectra. In one method, the relative abundance of proteins is based on the number of peptides identified for that specific protein in different samples.2' The hypothesis behind the utility of this method is illustrated in the analysis of a single serum sample. If tryptically digested serum is analyzed directly by LCMS2, large numbers of albumin peptides will be identified since this protein is present in the range of 40-80 mg/mL in serum. The number of peptides identified from a protein in the abundance range of ng/mL range (such as a chemokine) will probably not exceed one, if any are identified. It is unlikely, however, that even one peptide from a low-abundance protein, such as a chemokine, will be identified. In a practical example, the identification of nine peptides for cancer antigen-125 (CA-125) in serum sample A and two peptides for this protein from sample B, is taken to conclude that CA-125 is 4-5 fold more abundant in sample A. This approach, known as subtractive proteomics or peptide count, is a very attractive method for biomarker discovery because it requires minimal sample preparation and most proteomic laboratories have the capability of identifying thousands of proteins within serum/plasma. In addition, this quantitative measurement allows an unlimited number of comparative samples. Unfortunately, like most proteomic applications, it is still relatively low-throughput. The method does not have great quantitative precision with the minimum difference that can be reasonably measured being on the order of three-fold. This minimal difference means that low abundance proteins, which are generally only identified through one or two peptides, may not be accurately quantifiable. Quantitative measurements from the same type of data set can be made by directly comparing the peak areas of individual peptides identified in different samples.22 Using this approach, selected ion chromatograms of the peptides of interest are generated so that the peak area of each peptide can be measured. Many proteins will be represented by multiple peptides, therefore the abundance ratios provided are calculated by averaging the peptide peak area ratios for the same protein.

34

BIOMARKERS

FIGURE 3.4 Multidimensional protein identification technology for comprehensive analysis of complex proteome samples.


35

E X A M P L E S OF BIOMARKER DISCOVERY A N D EVALUATION A recent study comparing quantitative results provided by subtractive proteomics and measuring peak areas was conducted by analyzing plasma samples obtained from a human subject prior to (untreated) and nine hours after treatment with lipopolysaccharide (LPS).23 This endotoxin is known to induce inflammatory reactions, such as cell migration, cytokine production, and production of acute-phase proteins. The untreated and LPS-treated plasma samples were tryptically digested and the resultant peptides fractionated into 50 aliquots using SCX chromatography. Each of these aliquots was analyzed by reversed-phase LC-MS2. Combining the results obtained from analysis of both samples (i.e., treated and untreated) resulted in the identification of 5176 unique peptides corresponding to 804 proteins. The group plotted the number of peptides identified for 74 specific proteins against their literature documented concentrations in plasma to determine if the number of peptides identified for a specific protein correlated with their relative abundances. The correlation was quite good, suggesting that the number of peptides identified per protein provides a semiquantitative assessment of a protein's relative abundance in a complex mixture. The peak areas for peptides that were identified in both samples were also compared to identify proteins that were differentially abundant in LPStreated plasma and this calculated ratio was evaluated against that obtained using the number of peptide identifications. Eight out of the nine proteins in which a protein abundance ratio was determined showed an increase in concentration following LPS administration by both the protein abundance ratios and the ratios of peptide hits. Unfortunately, the overall correlation was not extensive as many of the up-regulated proteins were identified in only one of the two methods (and not by both methods). However, cases in which proteins were identified using both quantitative methods can provide a quick confirmation of the results without having to completely reproduce the entire study. At present, most comparative studies of serum and plasma are conducted using either of these two computational approaches as it is important to be able to measure the relative quantitation of proteins in numerous samples. As described later, the low-throughput nature of MS-based analysis of complex biologic samples hampers the confidence levels that are attainable when perceived abundance differences in proteins are observed. So how do we move forward in finding biomarkers? One possible solution is to increase the number of samples analyzed. If a difference between a protein's abundance is seen across a large number of samples, the confidence in that difference increases as well. This increased confidence, however, comes at the expense of time. Another option is to use a variety of proteomic platforms to conduct comparative proteomic analyses. In a study aimed at finding biomarkers for Down Syndrome (DS), first- and second-trimester maternal serum samples of DS were compared to gestational age-matched controls using 2D-DIGE,

36

BIOMARKERS

two-dimensional liquid chromatography-chromatofocusing (2D-CF), MudPIT, and MALDI-TOF-MS peptide profiling [15].24 Twenty-eight and 26 proteins were differentially abundant in first- and second-trimester samples compared to matched controls, respectively. Nineteen and 16 were specific for the first and second trimesters, respectively. Ten were differentially abundant in serum samples obtained from patients in either trimester. Twenty of these potential markers were identified by at least two of the four methods (Table 3.2), while a-1-acid glycoprotein 1 was observed using all four. The greatest overlap of potential biomarkers was observed when the results using 2D-CF and 2D-DIGE were compared. Somewhat surprisingly, only four out of the 20 markers were observed in both the 2D-CF and MudPIT analyses. Many of the potential biomarkers are serum glycoproteins that may play a role in cellular differentiation of fetal growth. As with increasing the total number of samples that are analyzed, using multiple platforms increases the confidence in the observed differences; however, it also substantially increases the study time. A recent, exciting development in the search for biomarkers using MS, has been the demonstration of the ability to compare proteomes extracted from formalin-fixed paraffin-embedded (FFPE) tissues.2526 Owing to the covalent crosslinking used to preserve the morphology of these samples, FFPE tissues had been considered intractable to MS analysis. What needs to be remembered is that biomarker discovery using MS does not rely on identification of the intact protein, rather tryptic surrogates are measured as a reflection of its protein of origin. Therefore, the protein extraction methods used for FFPE tissue only need to extract unmodified peptides in order to successfully identify and quantitate proteins within these samples. In a recent study, FFPE tissue sections obtained from poorly (PD), moderately (MD), and well-differentiated (WD) head and neck squamous cell carcinoma (HNSCC) tumors were compared using an LC-MS2 approach.27 Lasercapture microdissection (LCM) was used to extract approximately 20,000 cells from normal squamous epithelial tissue as well as tumors that were classified as PD, MD, or WD. The relative abundance of each protein was determined by measuring the number of peptides identified per individual protein. A number of significant abundance differences were seen between the cells. For example, cytokeratin 4 was found to be more abundant in normal epithelial tissue compared to tumor cells, while cytokeratin 16, vimentin, and desmoplakin were found to be more abundant in the tumor cells. The WD tumor cells showed a striking increase in the amount of the protein desmoplakin. As with any good study, the potential biomarkers found within the analysis of a complex mixture requires validation using an orthogonal method. While the MS discovery phase is slow, once the potential markers are identified, the validation methods (e.g., immuno assays) used to test the potential biomarkers are much faster and a large number of samples can be analyzed concurrently. For validation, archived tissues consisting of nor-


37

mal epithelial and tumor HNSCC were processed and used for immunohistochemical (IHC) detection with antibodies against cytokeratin 4, vimentin, and desmoplakin (Figure 3.5) using a tissue microarray (TMA). Ten normal tissue sections were used for each antibody tested, while for the PD, MD, and WD tumors between 33 and 105 tissues were analyzed. Scoring of the tissues was based on tissue differentiation and the intensity of staining by each individual antibody. For cytokeratin 4, light gray represents >5% and 5% and 10,000 fold.52 Such enhancement has made in vivo 13C and 15N imaging practical5355 and could also be a route to exploiting putative biomarkers detected by conventional NMRbased metabolic profiling. Despite the fact that many metabolites produce very characteristic NMR spectra, and the recent arrival of metabolomic databases with substantial NMR content (e.g., the Madison Metabolomics Consortium Database (MMCD)56 and the Human Metabolome Database (HMDB)57), automated deconvolution of NMR-based metabolic profiles has not as yet proved a very successful strategy in biomarker applications. However "manual" or semiautomatic pattern matching and deconvolution can be routine in ID NMR data analysis and some commercial solutions are becoming more popular. NMR and MS are clearly different, varied, and complementary approaches. NMR is perhaps more limited in the scope of metabolism that can be covered but has many valuable features and a strong historical link to clinical

54

BIOMARKERS

applications. A mixture of multiple strategies is the best solution where possible. Both have off-the-shelf solutions but neither are trivial to implement. It is important to note that, as for other types of molecular profiling studies, great care must be taken to avoid introduction of sample bias58 during collection and analysis and indeed to assume that some bias is envitable and to characterize the effect of common problems such as freeze thaw cycles.50 Different biological matrices and platforms have different susceptibilities to bias, so such factors may need to be assessed separately in each study.

DATA P R E - P R O C E S S I N G , A N A L Y S I S , A N D PATTERN R E C O G N I T I O N Several aspects of pre-processing of raw spectral data are highly instrument dependent such as calibration, baseline correction, peak extraction, and alignment. The aim of all these steps is to produce a data table in which variations from instrumental artefacts are minimized. They are often intrinsic to the instrument software although many freeware options are available and popular in metabolic profiling (e.g., AMDIS or XCMS59). Metabolite identification and spectral annotation is often a distinct process which can occur either before or after statistical analysis, the latter approach, suitable for biomarker studies, providing the opportunity to reduce the task to just those metabolites that appear to be relevant to the study endpoint. However, other often necessary pre-processing steps such as variable transformation ("scaling") and normalization make a significant impact on the numerical analysis itself. Scaling is self-explanatory; the values for each variable in the analysis (a metabolite concentration or peak intensity) are mathematically transformed to make the distribution more normal or to reduce the dominance of highly abundant species over low concentration ones. Normalization in the context of metabolic profiling refers to a transformation that occurs across the values that constitute a profile, typically converting the metabolite levels into a relative value that has more biological relevance or removes some specific experimental artefact. A common example is the removal of urinary dilution or concentration by dividing the urine metabolic profile by the total metabolite intensity, the average fold-change or a more physiologically relevant quantity such as the osmolality or creatinine excretion. After these stages the data table is ready for whatever statistical analysis is deemed appropriate. From a statistical perspective there are no major reasons why metabolite data should be different from any other profile data. Within standard caveats, e.g., assumptions of normality should be tested, all standard methodologies are applicable. It is important to bear in mind however that as with all highly multivariate profiles, issues of multiple testing become very important60 and the risk of false positive associations is usually quite high and should be estimated, e.g., by the approach of Benjamini and Hochberg61 or resampling techniques. Pattern recognition (PR) techniques have and continue to be very important in the analysis of metabolic profile data (reviewed by Lindon, et al.62). The term PR, in the context of profiling analysis, refers to a range of statistical

METABOLIC PROFILING FOR BIOMARKER DISCOVERY

55

techniques that generate models that give a description of the statistical association (or more simply, similarity) between profiles or profile measurements (unsupervised PR) and/or between profiles and some target factor (supervised PR). One can then interrogate the model to find out those features of the profile that correlate to your factor of interest (potential biomarkers or collectively a "combination biomarker" or "biomarker signature") and one can use the model to predict information about new samples from the profiles alone, typically done to validate to the model. PR has played a particularly large role to date in applications where spectral data are analyzed directly prior to peak picking and annotation of metabolites (i.e., "fingerprinting"). The most widely used (and abused) techniques include bi-linear covariance modelling methods such as principal components analysis (PCA) and partial least squares regression (PLS) that are also synonymous with the terms "chemometrics" and "multivariate analysis" (reviewed by Trygg, et al.63). PCA and PLS produce parsimonious descriptions of multivariate data that allow quick visualization of clustering in the most important variables and can to some degree deal with interferences that coincide with signals of interest but vary in an uncorrelated (orthogonal) manner. Other PR techniques familiar across many scientific disciplines have been used in metabolic profiling including but not limited to: hierarchical clustering analysis and K-nearest neighbours;64 density estimation;24'65 self-organizing maps (Kohonen neural networks);66 and genetic algorithms.67'68 The main advantage of PR is that it can quickly extract patterns of response in metabolic profiles that are defined across many metabolites at once and are not straightforward to detect by visual inspection of the raw data. It can also be applied in an unbiased fashion, demonstrating a relationship between the profile as a whole and a factor of interest, rather than selecting just a few peaks that may have associations with the factor by chance. Finally, it is important to note that univariate analyses typically ignore correlations between predictors, whereas PR methods can reveal that many dozens of metabolites may respond to an interference in a common fashion while only one may appear to be significantly associated using standard methods of false discovery correction. Hence, even where we move to more well annotated datasets and away from fingerprinting, PR approaches remain popular and effective. PR approaches such as statistical total correlation spectroscopy (STOCSY) or statistical heterospectroscopy (SHY) can also assist metabolite annotation itself.69-72 These methods highlight the covariance in intensity between different spectral signals to facilitate structure determination. While the benefits of PR are well aligned with the goals of biomarker discovery and a top-down approach to systems biology, other often more basic research into metabolism and "bottom-up" approaches requires more detailed biochemical models that can provide information on flux through defined pathways and predict the behaviour of a living system from molecular parameters. Several groups are also currently seeking to extend established metabolic modelling methods to a genome-wide scale,73-76 sometimes including analytical approaches that can be referred to as "fluxomics."74'77 Metabolic fluxes can

56

BIOMARKERS

be inferred from the model or measured more directly via measurement of isotopomer dynamics after enrichment of specific pathways by an isotopicallylabeled substrate. While these approaches are not usually considered as biomarker discovery strategies, alterations in pathway flux can clearly act as biomarkers and are important considerations for metabolic biomarkers exploited by imaging of isotopologues (e.g., 18FDG-PET and 13C-pyruvate MRI). Identifying target pathways can also be achieved by more informatic approaches to data modelling and particular by the interrogation and statistical integration of multiple omics data. Techniques such as pathway over-representation and gene enrichment analysis can be applied irrespective of whether the entities measured are metabolites or genes. Genome-wide association or quantitative trait loci mapping which exploit simple models of genetic variation have been used to associate genotypes to global metabolic phenotypes in microbes,73,74 plants,78 and mammals,20'79including man. Such systems' biology approaches represent an important area for future development in metabolic profile data analysis.

P R E C L I N I C A L T O X I C O L O G Y : MODELS FOR P A T H O L O G I C A L BIOMARKER DISCOVERY There is a clear need for better translational biomarkers in toxicity studies during drug discovery and development, and metabolic profiling has the potential to address this "biomarker gap" (reviewed in 80, 81). Metabonomics can be readily incorporated from an early stage in preclinical investigation since, providing adequate care is taken with collection procedures, it can utilize samples that are typically generated during routine ADMET studies. It has been shown that metabonomics can reveal otherwise silent lesions, providing information for candidate selection.82 Beyond this, it can provide mechanistic clues that aid risk assessment.83 Much of the early development of metabonomics was in application of biofluid NMR spectroscopy to characterize the biochemical effects of toxicant exposure. Initially, work by Nicholson and co-workers showed that urinary NMR profiles were sensitive reporters of renal damage.9 Subsequent studies examined acute exposure to a number of model nephrotoxins and showed that proximal tubular damage produced aminoaciduria distinct from papilliary toxins that produced higher excretion of renal osmolytes such as betaine and methylamines.28'84 Furthermore, it was demonstrated that these profile differences could be classified by pattern recognition approaches, suggesting the possibility of toxicity screening by metabolic profiling approaches. Toxicity studies provided the opportunity to show that metabonomics is sensitive to a number of other pathologies, including but not limited to: liver steatosis;85 phospholipidosis;13,86 hepatobilliary damage;25 testicular necrosis,87-88 vasculitis;89 hepatic glutathione depletion;43 and pancreatic toxicity.90,91 A number of studies have provided evidence supporting the use of several specific metabolites as toxicity biomarkers. Increases in urinary taurine, observed by NMR-based profiling, has long been proposed as a marker of he-


57

patotoxicity.92,93 Taurine is thought to have a protective role against oxidative stress and could reflect alterations to cysteine and glutathione metabolism.94 In a series of related publications, urinary metabolite profiles were shown elevated urinary levels of N-methylnicotinamide and N-mefhyl-4-pyridone3-carboxamide, both end products of the tryptophan-nicotinamide adenine dinucleotide (NAD+) pathway, and shown to correlate to the degree of the peroxisome proliferation induced by a number of PPAR agonists.95 Soga, et al.,43 using a CE-MS platform, shows that serum levels of opthalmic acid, an analogue of glutathione, specifically reflected hepatic glutathione depletion. Bollard, et al. have shown substantial increases in the metabolite betaine, in particular in urine, during liver regeneration using a partial hepatectomy model; betaine is converted to dimethylglycine (DMG) by the enzyme betainehomocysteine methyltransferase, which is largely liver specific. The partial hepatectomy study is one example of the work conducted as part of the Consortium on Metabonomics in Toxicology (COMET) project, an ongoing collaboration between the pharmaceutical industry and Imperial College London (ICL).96 In Phase I of the project (2001-2004) six industrial partners and ICL sought to establish a resource of metabonomic data to help evaluate the technology as applied to preclinical toxicological studies. In total >30,000 samples were analyzed by 'H NMR spectroscopy from 147 studies of exposure to model toxicants and other physiological stressors. Using these data it was possible to demostrate the high level of analytical49 and physiological17 inter-laboratory reproducibility of metabonomic responses using NMR spectroscopy of urine. A number of classification strategies and data analysis tools were explored, culminating in the development of an "expert system" that could match treatments based on the similarity of metabonomic response ("toxin-likeness").24 The second phase of the COMET project aims to focus more on developing metabonomics as a tool in mechanistic toxicology and in particular to understand individual variability in drug response. One of the model compounds investigated was the hepatotoxin galactosamine (galN), known to produce a lesion highly variable in severity.25 Coen, et al. used 'H NMR spectroscopy to explore why glycine protects against toxicity.83 Treatment with glycine alone was found to significantly increase hepatic levels of uridine, UDP-glucose, and UDP-galactose, and in view of the known effects of galactosamine, this suggested that the protective role of glycine against galN toxicity might be mediated by changes in the uridine nucleotide pool rather than by preventing Kupffer cell activation as widely believed. An important extension of earlier work on individual variability in galactosamine toxicity was to consider if baseline metabolic profiles were sensitive to any factors that could explain and/or predict variation in response to drug exposure. This notion—pharmacometabonomics—inspired the study by Clayton, et al., which examined the consequences of acetaminophen (paracetamol) exposure in individual rats.97 Using an intermediate dose level, a range of toxicity was produced together with considerable variation in the metabolic fate of paracetamol. Using NMR spectroscopic data of pre-treatment urines, it could

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be shown that: a) the paracetamol sulphate-to-glucoronide ratio could be convincingly predicted; b) a correlation was present between the metabolic profile and severity of hepatoxicity. The metabolite signals that allowed the prediction of pharmacokinetics (PK) included some that were likely to derive from glucoronides of dietary species such as cresol, perhaps indicating that such molecules affect endogenous capacity for various metabolic transformations leading to excretion and detoxification. The level of toxicity was correlated to baseline urinary excretion of taurine, trimethlamine-N-oxide (TMAO) and betaine. TMAO is a gut microbial metabolite of choline, which is also converted to betaine. Changes in both these metabolites have been associated with liver toxicity and susceptibility to fatty liver.98 Not only did the study by Clayton, et al. show the viability of pharmacometabonomics, it also suggested that metabolic profiling was a valuable probe for exploring the largely unknown role of gut microbial involvement in modulating each individual's drug metabolism and susceptibility to toxicity. Several other metabolic profiling studies have shown explicitly the effects of gut microbes on mammalian endogenous metabolic profiles99-101 and how they may affect drug toxicity.102

DISEASE BIOMARKER DISCOVERY U S I N G METABOLIC PROFILING Inborn Errors of Metabolism Perhaps the most obvious application for metabolic profiling in the clinical environment is in the diagnosis of inborn errors in metabolism. Since the pioneering work of Robert Guthrie, which led to the introduction of infant screening for phenylketonuria in the 1960s,103 LC-MS/MS (tandem MS) methods were established that by profiling a defined set of organic acids and amino acids could recognize up to 20 metabolic disorders simultaneously in a high-throughput manner.104 More recently, using adapted techniques, it has become possible to measure enzyme activities by reaction monitoring, and thus include the possibility of screening for other conditions such as lysosomal storage disorders.105' 106 Many countries now operate infant screening programs based on LC-MS/MC platforms with a throughput of 4 million samples annually in the U.S. alone. While clearly metabolic profiling of a kind, these screening protocols represent a very targeted and restricted output, measuring only pre-defined biomarkers. The use of less targeted profiling and systematic analysis with a view to biomarker discovery and definition of novel inborn errors of metabolism is also an important application. In this respect, NMR spectroscopy (reviewed in 107) has proved valuable, identifying new conditions such as aminoacylase I deficiency108 and guanidinoacetate methyltransferase deficiency.109 Even though the genetic lesion is incurable and such conditions are rare, screening biomarkers could be very important. In many types of metabolic disease, as in the classic example of phenylketonuria, dietary intervention started within the first month of life can be effective in preventing neurological damage and mental retardation.


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Neuroscience While metabolic profiles are sensitive to metabolic defects that can cause neurological effects, metabolomics has a wider role in characterizing neurological disorders and brain function. Metabolic profiling studies of animal models can clarify the consequences of genetic mutations that lead to neurological disease. For example, Pears, et al.110 characterized the metabolic alterations in brain tissue from a Cln3 knock-out mouse model of Batten's disease one of the neuronal ceroid lipofuscinoses. NMR spectroscopy of tissue from Cln3 null mutant mice was characterized by an increased glutamate concentration and a decrease in gamma-amino butyric acid (GABA) concentration, implying a change in neurotransmitter cycling between glutamate/glutamine and the production of GABA. Tsang, et al.111 investigated the metabolic consequences of Huntington's disease in the R6/2 mouse model using NMR spectroscopy. Global increases in relative brain concentrations of osmolytes, creatine, glutamine, and lactate, and decreases in acetate and N-acetylaspartate were found together with striatal-specific lower concentrations of GABA and choline. Clear differentiation of R6/2 and wild-type mice was also obtained for urine and blood metabolite profiles. The analysis of post-mortem tissue offers a more direct opportunity to discover clinically relevant biomarkers. Lan, et al." 2 used NMR spectroscopy to identify molecular changes in post-mortem brain tissue of patients with a history of bipolar disorder and compared discriminating features to the effect of chronic treatment with the mood-stabilising drugs lithium and valproate. Glutamate levels were increased in the post-mortem bipolar brain, while the glutamate/glutamine ratio was decreased following valproate treatment, and gamma-aminobutyric acid levels were increased after lithium treatment, suggesting that the balance of excitatory/inhibitory neurotransmission is central to the disorder. The level of N-acetyl aspartate, a clinically important metabolic marker of neuronal viability, was found to be unchanged following treatment. These findings show how metabolic profiling can provide new insight into the pathophysiology of bipolar disorder. In terms of direct clinical application, several metabolites can be measured noninvasively by MRS/MRI techniques, and in addition to blood and urine, a more directly relevant biofluid available is cerebrospinal fluid (CSF). Holmes, et al.113 showed that drug-naive or minimally-treated patients with first onset schizophrenia have a different CSF NMR profile compared to health controls (n=152 in total). In the disease group normalization of this profile was observed with treatment, prior to clinical improvement, whereas patients treated after more than one episode did not exhibit normalization, consistent with reports that suggest the efficacy of treatment is improved with earlier intervention.

Cancer It has long been known that tumour cells exhibit common metabolic phenotypes that can be exploited in diagnosis and therapy. For example, one of the manifestations of the Warburg effect, high glucose uptake and glycolysis to lactate, is the basis of 18-fluorodeoxyglucose (FDG) PET imaging of tumours.

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BIOMARKERS

FDG uptake and accumulation detected by PET is a sensitive and specific noninvasive metabolic marker of cancer, and can be used for diagnosis, staging, prognosis and early detection of drug response."4 Warburg's discovery, dating from the beginning of the last century, has received renewed interest of late as there is growing evidence that the phenomenon, together with other aspects of the tumour metabolic phenotype, is regulated by well-established tumour suppressors and oncogenes such as p53 and hypoxia inducible factor (HIF) (reviewed by 115, 116). Other common manifestations of the tumor metabolic phenotype, such as alterations to choline metabolism, have been detected by NMR spectroscopy.117, " 8 Typically an increase in tissue phophocholine (PC) is observed, which is due in part to increased choline uptake and upregulation of choline kinase activity. A large number of studies have shown this phenomenon to occur across many tumor types, to be detectable noninvasively in vivo and to translate to animal models, although some exceptions have been reported.119 In cell models of tumourigenic progression, increases in PC and the PC:GPC (glycerophosphocholine) ratio have been frequently reported. Importantly, it has been shown that alterations to choline metabolism can preempt tumour formation in morphological normal tissue, discriminating tissue susceptible to tumor formation possibly by detecting "field effects."47 This is one possible route to improving early detection of cancer with diagnostic biomarkers. A key target for metabolic profiling in oncology is a plasma or urine biomarker that can be used for early detection of cancer. Odunsi and co-workers were able to achieve complete distinction between epithelial ovarian cancer patients and healthy controls using NMR spectroscopy.120 In breast cancer, mass spectrometry-based analysis of nucleosides in urine samples from breast cancer patients and healthy volunteers was able to identify patients with a sensitivity and specificity which improved upon current breast cancer biomarkers.121 However, there have been marked failures in the history of molecular profiling biomarkers for detection of cancer, including early metabolic studies. Fossel, et al. reported accurate detection of malignancies using proton NMR spectroscopy of blood plasma, results which were not supported by subsequent studies.123 Tissue-specific metabolic processess are also potentially a source of cancer biomarkers. In the prostate, zinc accumulates to inhibit aconitase and citrate oxidation via the TCA cycle,124 hence citrate accumulates in prostate glandular tissues to extremely high concentrations. Loss of this phenotype, which can be detected ex vivo in biopsy by MAS-NMR and in vivo by MRS, is associated with the presence of prostate cancer and can distinguish between benign and malignant disease.119,125,126 More recently in a high-profile study, Sreekumar, et al.127 identified the metabolite sarcosine as a potentially important metabolic mediator of prostate cancer cell invasion and aggressivity. Sarcosine was highly increased during prostate cancer progression to metastasis and could be detected noninvasively in urine. Reduction in sarcosine availability attenuated prostate cancer cell invasion while increasing availability induced an invasive phenotype in benign prostate epithelial cells.


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Infectious Disease Metabolomics has a role to play in understanding factors that influence pathogenicity and virulence, and to date metabolism is an under-explored facet of the host-pathogen interface. Olszewski, et al.128 highlight the uptake of extracellular arginine by the malarial parasite Plasmodium falciparum and its role proliferation. Bundy, et al.129 show that metabolic profiles were able to distinguish between avirulent strains of Bacillus cereus and clinical isolates from meningitis patients where screening of genomic DNA for the presence of genes encoding known toxins gave no candidate genes that were unambiguously able to distinguish between the two groups. A number of publications have characterized the metabolic consequences of infection in mouse models of human pathogenic parasitic disease including Schistosoma mansoni;130,131 Trypanosoma brucei brucei;132-133 Plasmodium berghei;128,134 and Echinostoma caproni.135 Marked alterations in plasma metabolic profiles have been observed including elevated plasma concentrations of lactate, branched chain amino acids, and acetylglycoprotein fragments.132 Urine from mice infected with P. berghei134 showed increased levels of pipecolic acid (unique to infection with this parasite), and decreased concentrations of TMAO, suggesting a disturbance to gut microbial populations. If these putative biomarkers translate to the clinical setting they may have important consequences for management of therapy. A clinical study by Coen, et al.136 showed that metabolic profiling of CSF could distinguish patients with bacterial or fungal meningitis from patients with viral meningitis and control subjects and clearly distinguished patients with postsurgical ventriculitis from postsurgical control subjects. Early diagnosis and selecting the appropriate treatment for patients with conditions such as meningitis or postsurgical ventriculitis using metabolic profiling could potentially save lives.

Metabolic Syndrome: Insulin Resistance, Cardiovascular Disease, and H y p e r t e n s i o n Perhaps the most active and ambitious area of metabolic profiling research is in the characterization of facets of the metabolic syndrome (MetS), in particular insulin resistance, cardiovascular disease, and hypertension. Diabetes and insulin resistance are obvious cases where we expect diagnositic and prognostic changes in a systemic metabolic profile and this has been extensively explored, not only in animal models,79'98 but also in human populations. For example, Korpela, et al. reported the prognosis of diabetic complications in sera of 613 patients by 'H NMR spectroscopy.66 Self-organising maps (Kohonen neural networks) were used to demonstrate that metabolic profiles correlated to patients with associated renal disease and as a result to patient mortality. Coronary disease is another condition with an expected metabolic phenotype. Brindle, et al.16 showed an NMR-based metabolic signature, in particular an altered lipid profile, associated with the presence and severity of atherosclerosis. GC-MS has also been used to define novel serum biomarkers of heart failure, e.g., pseudouridine.137

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While the diagnostic value of NMR profiles in coronary disease is still debated,138 a number of prolific studies support the prognostic value of lipid signatures. NMR spectroscopy gives a unique viewpoint into the lipid/lipoprotein particle distribution in blood serum or plasma139-141 that may provide better prognostic markers for coronary events than standard clinical chemistry. For example, in a prospective nested case-control study (n=1061) NMR measurements showed that gemfibrozil treatment significantly reduced LDL particle number and size relative to the placebo group, while conventional measurement of LDL-cholesterol (LDL-C) was not significantly altered.142 LDL-particle number was also significantly prognostic of a coronary event (fatal myocardial infarction or cardiac death) during follow-up in both treated and placebo groups, again where LDL-C was not. However, the most recent and largest study published reported that NMR-based lipoprotein measurements were comparable to, but not better than, standard lipid profile measurements for the prediction of cardiovascular disease in women (n=27,673 with 1015 incidents of cardiovascular events over an 11-year period).143 While disappointing, this study was only in apparently healthy women with a low overall risk; the possibility remains that NMR lipoprotein data could provide a diagnostic or prognostic advantage in other patient groups. As with the use of metabolic profiling for newborn screening, these large clinical studies are targeted to defined biomarkers in profile data analysis. Researchers are now attempting unbiased metabolic profiling for biomarker discovery in uncontrolled human population studies, as shown recently in a study by Holmes of 4630 individuals from 16 sites across four countries, part of the INTERMAP project.21 Two matched 24h urine samples were analyzed by 'H NMR spectroscopy and a range of pattern recognition analyses applied. This work showed that metabolic profiles could be identified that differentiated each population centre and different dietary habits. Importantly, the metabolites responsible for these discriminations, in particular formate, were associated with blood pressure (hypertension) across individuals, a key health indicator, a prognostic factor for coronary disease, and a component of the MetS phenotype.

ENVIRONMENTAL HEALTH AND METABOLIC PROFILING There is a substantial body of work applying metabolic profiling to environmental research (reviewed in detail by Bundy, et al.144), particularly in the area of ecotoxicogenomics. Several studies have shown that toxicant exposure can produce metabolic signatures in "sentinel organisms" (indicator species), both in laboratory experiments and in the field. For example, Bundy, et al.145 reported the effects of heavy metal contamination in earthworms (Lumbricus rubellus) on NMR-based metabolic profiles. Yet such studies have not translated into biomonitoring programs and metabolic profiling studies of human exposure are limited. There is currently a need to improve exposure assessment in order to get a clearer picture of how specific risk factors interact with genotype to produce effects on human health.146 Several studies exploring the potential of


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metabolic profiling for chemical exposure and risk assessment in human populations are underway (e.g., the FP7 Envirogenomarkers consortium) and this is likely to be one of the growth areas for metabolic profiling in years to come.

C O N C L U S I O N : STRENGTHS, WEAKNESSES, A N D T H E W A Y F O R W A R D FOR M E T A B O L I C P R O F I L I N G I N BIOMARKER DISCOVERY Metabolic profiling has several strengths that make it a good complementary tool to the many other approaches to biomarker discovery. Metabolites are easily measured in body fluids, which are more readily obtained than tissue, facilitate measurement of response dynamics, and can simultaneously report on effects to several organs. Metabolites are chemically unique across cell types, species, or physiological states and changes in metabolism frequently represent a functional endpoint that is the closest to phenotype. Metabolism is also often the interface by which an organism interacts with extra-genomic/environmental factors, giving a unique viewpoint into the health effects of diet, gut microbes, and xenobiotic exposure. However, many of these features are also at the root of some of the major challenges in application of metabolic profiling. The majority of metabolites in central metabolism are common across many cell types, making tissue-specific metabolites or biomarker signatures difficult to find and validate. Constant exogenous input from nutrition and gut microbial activity has a major influence on the metabolic profile (in systemic body fluids and most tissues) and thus it is very difficult to disentangle the primary effect of a particular stressor and inevitable secondary effects on dietary intake and gastrointestinal function.147 For many detectable small molecules the distinction between exogenous and endogenous is blurred.148 While genotype is essentially constant for an individual, the metabolome or metabotype is rapidly fluctuating and constantly evolving. Underlying stable features are detected but are not easy to define precisely without several measurements over time. Arguably, determining metabolic flux and not concentrations per se is the key to understanding genotype-phenotype relationships and the role of metabolism in the etiology of chronic disease. However, while rapid progress has been made in the development of genome-wide metabolic models for microbial organisms and single cells, there are no models at present that can adequately explain metabolic profiles at the whole-system level for multiorgan, multigenome communities such as mammals. Reactive flux is only one element of such a hypothetical model; metabolite transport and the integrity of physiological compartments are often not considered in detail but are vital for understanding the causes and consequences of tissue damage and organ failure. While the rapid evolution of metabolic profiling platforms and analytical techniques is paying dividends in improving the coverage of the metabolome, there has historically been little consensus as to common protocols, standards, and reporting structures, with a dearth of metabolic data in public repositories. After a decade of exponential growth, metabolic profiling has not yet yielded a clear biomarker success story with widespread use or defined regulatory approval.

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Yet the future looks bright for metabolomics and metabonomics. Initiatives to standardize many aspects of metabolic profiling studies are underway as part of the Metabolomic Standards Initiative (MSI)149 and data repositories are being established. The establishment of resources such as the Human Metabolome Database that can act as a portal integrating spectral libraries, data analysis tools, pathway data and literature information will accelerate the growth of a collective knowledge base for the entire community. Investment in systems biology is driving forward the development of more sophisticated models and algorithms for interrogating and integrating molecular profiles from "-omics" technologies. In particular, pathway-driven informatic approaches to identifying drug and biomarker targets will come to the forefront as more substantial public datasets become available. The completion and continued expansion of major population metabolome mapping projects, such as HUSERMET35 and other large cohort studies, will provide a wealth of information on metabolome composition, metabolite distributions, correlations and relationships to basic parameters such as age, gender, weight, diet, and smoking in normal human populations: vital reference data for understanding the potential of putative biomarkers from discovery projects. Moving beyond exploratory laboratory and clinical studies, metabolic profiling is well poised to make a significant impact on our understanding of how an organism's environment, in terms of nutritional, drug, toxicant and microbial exposures, interacts with genotype to modulate health and disease risk at an individual level.

SUMMARY POINTS 1. 2.

3. 4. 5.

Metabolic profiling (metabonomics/metabolomics) is the unbiased characterisation of metabolite content in biological samples. The principal technologies for generating metabolic profiles are NMR spectroscopy, GC- and LC-coupled mass spectrometry, and frequently multivariate pattern recognition techniques and metabolic models are used in data analysis and integration with other "-omics" data. Metabolite analysis is highly translatable from the laboratory to the clinic and metabolic biomarkers can be measured noninvasively in body fluids or by imaging. Metabolic profiling has been demonstrated as a viable approach in uncontrolled human populations on an epidemiological scale. The metabolic phenotype offers a unique viewpoint into how genotype, parasites, drugs, environmental exposures, and diet interact to determine pharmacology, health, and disease.

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BIOMARKERS 50. 51. 52.

53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66.

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resolution MAS H-l NMR Spectroscopy. Journal of Proteome Research. Mar 2006;5(3):483^192. Lan, M. J., McLoughlin, G. A., and Griffin, J. L., et al. Metabonomic Analysis Identifies Molecular Changes Associated with the Pathophysiology and Drug Treatment of Bipolar Disorder. Molecular Psychiatry. Mar 2009;14(3):269-279. Holmes, E., Tsang, T. M., and Huang, J. T. J., et al. Metabolic Profiling of CSF: Evidence that Early Intervention May Impact on Disease Progression and Outcome in Schizophrenia. Plos Medicine. Aug 2006;3(8):1420-+. Frank, R. and Hargreaves, R. Clinical Biomarkers in Drug Discovery and Development. Nature Reviews Drug Discovery. Jul 2003;2(7):566-580. Kroemer, G. and Pouyssegur, J. Tumor Cell Metabolism: Cancer's Achilles' Heel. Cancer Cell. 2008;13(6):472-482. Vizan, P., Mazurek, S., and Cascante, M. Robust Metabolic Adaptation Underlying Tumor Progression. Metabolomics. 2008;4(1):1-12. Glunde, K., Jacobs, M. A., and Bhujwalla, Z. M. Choline Metabolism in Cancer: Implications for Diagnosis and Therapy. Expert Review of Molecular Diagnostics. 2006;6(6):821-829. Nimmagadda, S., Glunde, K., Pomper, M. G., and Bhujwalla, Z. M. Pharmacodynamic Markers for Choline Kinase Down-regulation in Breast Cancer Cells. Neoplasia. 2009;11(5):477-^184. Teichert, E, Verschoyle, R. D., and Greaves, P., et al. Metabolic Profiling of Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) Tissue by H-l-NMR Analysis: Evidence for Unusual Phospholipid Metabolism. Prostate. Jul 1, 2008;68( 10): 1035-1047. Odunsi, K., Wollman, R. M., and Ambrosone, C. B., et al. Detection of Epithelial Ovarian Cancer Using H-1-NMR-based Metabonomics. International Journal of Cancer. Feb 20, 2005;113(5): 782-788. Frickenschmidt, A., Frohlich, H., and Bullinger, D., et al. Metabonomics in Cancer Diagnosis: Mass Spectrometry-based Profiling of Urinary Nucleosides from Breast Cancer Patients. Biomarkers. 2008;13(4):435^149. Fossel, E. T., Carr, J. M., and McDonagh, J. Detection of Malignant-Tumors— Water-Suppressed Proton Nuclear-Magnetic-Resonance Spectroscopy of Plasma. New England Journal of Medicine. 1986;315(22):1369-1376. Okunieff, P., Zietman, A., and Kahn, J., et al. Lack of Efficacy of WaterSuppressed Proton Nuclear-Magnetic-Resonance Spectroscopy of Plasma for the Detection of Malignant-Tumors. New England Journal of Medicine. 1990;322(14):953-958. Mycielska, M. E., Patel, A., and Rizaner, N., et al. Citrate Transport and Metabolism in Mammalian Cells: Prostate Epithelial Cells and Prostate Cancer. Bioessays. Jan 2009;31(l):10-20. Swanson, M. G.,Zektzer, A. S., and Tabatabai, Z. L., etal. Quantitative Analysis of Prostate Metabolites Using 'H HR-MAS Spectroscopy. Magn. Reson. Med. Jun 2006;55(6): 1257-1264. Swanson, M. G., Vigneron, D. B., and Tabatabai, Z. L., et al. Proton HR-MAS Spectroscopy and Quantitative Pathologic Analysis of MRI/3D-MRSI-targeted Postsurgical Prostate Tissues. Magn. Reson. Med. Nov 2003;50(5):944-954. Sreekumar, A., Poisson, L. M., and Rajendiran, T. M., et al. Metabolomic Profiles Delineate Potential Role for Sarcosine in Prostate Cancer Progression. Nature. Feb 12, 2009;457(7231):910-U176.


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128. Olszewski, K. L., Morrisey, J. M., and Wilinski, D., et al. Host-Parasite Interactions Revealed by Plasmodium falciparum Metabolomics. Cell Host & Microbe. Febl9,2009;5(2):191-199. 129. Bundy, J. G., Willey, T. L., Castell, R. S., Ellar, D. J., and Brindle, K. M. Discrimination of Pathogenic Clinical Isolates and Laboratory Strains of Bacillus Cereus by NMR-based Metabolomic Profiling. Ferns Microbiology Letters. Jan 1,2005;242(1):127-136. 130. Wang, Y. L., Holmes, E., and Nicholson, J. K., et al. Metabonomic Investigations in Mice Infected with Schistosoma Mansoni: An Approach for Biomarker Identification. Proceedings of the National Academy of Sciences of the United States of America. Aug 24, 2004;101(34):12676-12681. 131. Li, J. V., Holmes, E., and Saric, J., et al. Metabolic Profiling of a Schistosoma Mansoni Infection in Mouse Tissues Using Magic Angle Spinning-nuclear Magnetic Resonance Spectroscopy. International Journal for Parasitology. Apr 2009;39(5):547-558. 132. Wang, Y L., Utzinger, J., and Saric, J., et al., Global Metabolic Responses of Mice to Trypanosoma Brucei Brucei Infection. Proceedings of the National Academy of Sciences of the United States ofAmerica. Apr 22, 2008;105(16):6127-6132. 133. Li, J., Saric, J., Wang, Y. L., Utzinger, J., Balmer, O., and Holmes, E. Metabolic Profiling of Co-Infection of Trypanosoma Brucei Brucei Strains in Mice. American Journal of Tropical Medicine and Hygiene. Dec 2008;79(6):104. 134. Li, J. V., Wang, Y., and Saric, J., et al. Global Metabolic Responses of NMRI Mice to an Experimental Plasmodium Berghei Infection. Journal of Proteome Research. Sep 2008;7(9):3948-3956. 135. Saric, J., Li, J. V., and Wang, Y L., et al. Metabolic Profiling of an Echinostoma Caproni Infection in the Mouse for Biomarker Discovery. Plos Neglected Tropical Diseases. Jul 2008;2(7). 136. Coen, M., O'Sullivan, M., Bubb, W. A., Kuchel, P. W., and Sorrell, T. Proton Nuclear Magnetic Resonance-based Metabonomics for Rapid Diagnosis of Meningitis and Ventriculitis. Clinical Infectious Diseases. Dec 1, 2005;41(11): 1582-1590. 137. Dunn, W. B., Broadhurst, D. I., and Deepak, S. M., et al. Serum Metabolomics Reveals Many Novel Metabolic Markers of Heart Failure, Including Pseudouridine and 2-oxoglutarate. Metabolomics. Dec 2007;3(4):413^126. 138. Kirschenlohr, H. L., Griffin, J. L., and Clarke, S. C , et al. Proton NMR Analysis of Plasma is a Weak Predictor of Coronary Artery Disease. Nat. Med. Jun 2006;12(6):705-710. 139. Ala-Korpela, M., Makela, S. M., and Salminen, A., et al. 'H NMR Metabonomics of Serum to Identify and Classsify Lipoprotein Subclass Profiles. Atherosclerosis Supplements. Jun 2007;8(1):39. 140. Ala-Korpela, M., Lankinen, N., and Salminen, A., et al. The Inherent Accuracy of H-l NMR Spectroscopy to Quantify Plasma Lipoproteins is Subclass Dependent. Atherosclerosis. Feb 2007;190(2):352-358. 141. Otvos, J. D., Jeyarajah, E. J., Bennett, D. W., and Krauss, R. M. Development of a Proton Nuclear-Magnetic-Resonance Spectroscopic Method for Determining Plasma-Lipoprotein Concentrations and Subspecies Distributions from a Single, Rapid Measurement. Clinical Chemistry. Sep 1992;38(9): 1632-1638. 142. Otvos, J. D., Collins, D., and Freedman, D. S., et al. Low-density Lipoprotein and High-density Lipoprotein Particle Subclasses Predict Coronary Events and

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144. 145.

146. 147.

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are Favorably Changed by Gemfibrozil Therapy in the Veterans Affairs Highdensity Lipoprotein Intervention Trial. Circulation. Mar 28, 2006;113(12): 1556-1563. Mora, S., Otvos, J. D., Rifai, N., Rosenson, R. S., Buring, J. E., and Ridker, P. M. Lipoprotein Particle Profiles by Nuclear Magnetic Resonance Compared With Standard Lipids and Apolipoproteins in Predicting Incident Cardiovascular Disease in Women. Circulation. Feb 24, 2009;119(7):931-U944. Bundy, J. G., Davey, M. P., and Viant, M. R. Environmental Metabolomics: A Critical Review and Future Perspectives. Metabolomics. Mar 2009;5(1):3-21. Bundy, J. G., Keun, H. C , and Sidhu, J. K., et al. Metabolic Profile Biomarkers of Metal Contamination in a Sentinel Terrestrial Species are Applicable Across Multiple Sites. Environmental Science & Technology. Jun 15, 2007;41(12): 4458^1464. Wild, C. P. Environmental Exposure Measurement in Cancer Epidemiology. Mutagenesis. Mar 2009;24(2): 117-125. Connor, S. C , Wu, W., and Sweatman, B. C , et al. Effects of Feeding and Body Weight Loss on the H-1-NMR-based Urine Metabolic Profiles of Male Wistar Han Rats: Implications for Biomarker Discovery. Biomarkers. Mar 2004;9(2): 156-179. Nicholson, J. K., Holmes, E., Lindon, J. C , and Wilson, I. D. The Challenges of Modeling Mammalian Biocomplexity. Nature Biotechnology. Oct 2004;22(10):1268-1274. Fiehn, O., Robertson, D., and Griffin, J., et al. The Metabolomics Standards Initiative (MSI). Metabolomics. Sep 2007;3(3): 175-178.

CHAPTER

THE BITTERSWEET PROMISE OF GLYCOBIOLOGY Padmaparna Chaudhuri, Rania Harfouche, and Shiladitya Sengupta

INTRODUCTION Glycosylation is one of the most common post-translational modifications in eukaryotes, affecting more than half of all known proteins as well as many lipids (Figure 5.1).1,2 Glycosylation is involved in key developmental roles including cell differentiation and innate immunity and modulating signal transduction. Furthermore, several inherited and non-genetic diseases such as pathogenic infections, immunity, tumor invasion etc. arise as a result of aberrant glycosylation and alterations in their structures.3 As a result, glycomics (the study of sugars in an organism) has become a very active field of research that may lead to new approaches in the diagnosis and prognosis of human diseases. Glycans thus provide an alternative, though they are an under-explored class of cellular biomarkers for diseases, and are attractive targets for broad novel therapeutics. However, the glycome has proven extensively difficult to study, mainly due to its inherent structural (e.g., linear or branched) and chemical (e.g., N- or O-sulfation, epimerization, acetylation) complexities.4 For instance, there are 41 different types of glyco-modifications, comprising 13 different sugar building blocks.5

G L Y C O S Y L A T I O N I N P A T H O L O G I C A L STATES Congenital Disorders of Glycosylation (CDG) In the past decade over 30 genetic disorders have been identified that alter glycan synthesis and structure, ultimately affecting the function of most organs.6 The largest number of these disorders affects N-glycosylation and usu75

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FIGURE 5.1 The glycome represents the most abundant class of post-translational modifications, yet it is the hardest to decipher

ally leads to hypoglycosylated proteins, resulting in unoccupied glycosylation sites on proteins. Patients with this type of CDG exhibit developmental delays, particularly in the brain.7 Other aberrations include point mutations that create novel glycosylation sites on proteins, causing their misfolding and rapid degradation.6 For example, a mutation that creates an N-linkage site in fibrillin 1 causes Marian Syndrome, an incurable connective tissue disorder.8 Recently, patients with heightened susceptibility to mycobacterial infections were found to carry a pathological point mutation that creates a novel glycosylation site in the interferon receptor IFN7R2.9 Defective O-glycosylation has also been implicated in several CDGs, including congenital muscular dystrophy, Walker-Warburg syndrome being the most severe.6,10 Mutations that affect various stages in glycosaminoglycans (GAG) synthesis also cause several human disease phenotypes such as hereditary multiple exostosis, a benign bone tumor leading to deformity.6'"

Glycomics of Immune Disorders The development and function of the mammalian immune system are largely modulated by the glycome.12 For instance, the immune-cell glycome is altered during cell differentiation, activation, and apoptosis, and these alterations affect homeostatis, leading to various immune diseases.2 Studies that bridge immunology and glycobiology therefore continue to provide new insights into the diagnosis, prognosis, and therapeutic strategies for immune-related disorders. In auto-immune diseases like rheumatoid arthritis (RA), there is a marked increase in the percentage of serum IgG glycans lacking sialic acid and galactose residues. In RA, aggregated agalactosyl glycoforms of IgG (IgG-GO) are specifically recognized by mannose-binding lectin leading to inappropriate

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activation of the innate immune system. Abnormally high IgG-GO levels in the serum are also characteristic of other diseases, including Crohn's disease, juvenile onset chronic arthritis, systemic lupus eryfhematosus, and tuberculosis.13 Recent studies analyzing the glycosylation pattern of immunoglobulin Al (IgAl) in patients with IgA nephropathy (IgAN) provided new insights into the autoimmune nature of pathogenesis of this common renal disease. Specifically, aberrant glycosylation of O-linked glycans in IgAl resulted in galactose-deficient IgAl and their subsequent recognition by IgG or IgAl antibodies. The resulting immune complexes are not efficiently cleared by the liver and deposit in the renal mesangium, thus inducing glomerular injury.14 Aberrant glycosylation has also been implicated in other autoimmune diseases. For instance, deficiency in mannoside 3-1,6 N-acetylglucosaminyltransferase V (Mgat5), an enzyme in the N-glycosylation pathway which modifies T-cell receptors, lowers the T-cell activation thresholds by enhancing their clustering, resulting in kidney autoimmune disease, enhanced delayed-type hypersensitivity and increased susceptibility to autoimmune encephalomyelitis.15 The aberrant synthesis of endogenous glycans can result in the exposure of cryptic epitopes that are perceived by the immune system as non-self and induce chronic inflammation. For example deficiency of ct-mannosidase-II blocks N-glycan maturation and increases the expression of physiologically primitive hybrid N-glycans at the cell-surface, which are recognized by endogenous mannose-binding lectin receptors.16 Lastly, abnormal catabolism of glycans can affect the virulence of pathogens. For instance, hyaluronan is a glycan that consists of a disaccharide repeat that modulates innate immunity by interacting with Toll-like receptors (TLRs). Some pathogens express hyaluronidase that cleaves hyaluronan into fragments not recognized by TLR, thereby evading the immune response.17

Glycomics in Cancer The remodeling of cell surface receptors through the modification of their oligosaccharide structures is often associated with malignant cellular transformations and there are currently more than 100 tumor markers which are mainly glycoproteins and glycolipids (Figure 5.2).18,19 In the tumor environment, changes in glycosylation allow neoplastic cells to metastasize by modulating receptor activation, cell adhesion, and motility.19 The crucial importance of the glycome in cancer pathology becomes apparent as aberrant glycosylation occurs in essentially all types of human cancers, as is described in this section. Tumor cells tend to produce increased levels of glycoconjugates containing sialic acid, which is associated with the increased invasive potential of tumor cells and hence translates to poor prognosis. For example hypersialylation of pi integrins in colon and ovarian adenocarcinomas has been shown to lead to a more metastatic phenotype.20 Fucosylation is regarded as another important post-translational modification and is increased dramatically in tumors such as that of the liver, lung, and stomach. Interestingly, the best-known markers for hepatocellular carcinoma and metastatic lung cancer are fucosylated a-fetoprotein and core fucosylated E-cadherin, respectively.21'22

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FIGURE 5.2

Important glycans implicated in tumor progression.

Several N- and O-glycosylation alterations underlie many neoplastic changes and hence offer excellent potential as tumor markers and therapeutics. As such, one of the most common forms of glycosylation in human tumors is the upregualtion of (31,6-GlcNAc branched N-linked glycans, which lead to integrin and E-cadherin clustering, altered cell-cell and cell-matrix adhesion, and enhanced metastasis.2324 Cancer-related glycosylation changes that occur in prostrate specific antigen (PSA), interestingly, is the best current diagnostic marker for prostrate cancer.25'26 Aberrant O-glycosylation has also been frequently described as a tumor-associated alteration resulting in expression of novel carbohydrate epitopes. Altered mucosal glycosylation of O-linked oncofetal antigens such as Thomsen-Friedenreich (TF) disaccharide and sialyl-Tn have been implicated in epithelial cancers, including colon cancers.27 Underlying mechanisms include increased binding to adhesion receptors such as selectins, thus increasing tumor cell interactions with platelets, leukocytes, and endothelial cells, thus promoting tumor cell survival and metastasis.28 Interestingly, aberrant expression of glycans, especially heparan sulphate proteoglycans (HSPG), has also been implicated in tumor angiogenesis, shed-

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ding new light on the angiogenesis hypothesis of cancer.29,30 HSPG on tumor cells act as co-receptors to stabilize growth-factor receptor signaling complexes, thus promoting tumor proliferation and invasion in such diverse cancers as that of the pancreas, breast, ovaries, and liver. In a positive feedback loop, tumor cells upregulate genes that modulate sulfation of cell-surface HSPG, explaining why anti-angiogenic drugs eventually fail in the long run.31 Like proteins, lipid glycosylation is also involved in tumor pathology, playing a major role in tumor growth and metastasis.32 More specifically, colon cancer specimens showed three specific alterations in their glycosphingolipids compositions: increased ratio of acidic type-2 oligosaccharides, a2-3 and/or a2-6 sialylation, and a 1-2 fucosylation, opening the door for novel biomarkers.29

Other Acquired Diseases Altered protein glycosylation is also an attractive tool for noninvasive diagnosis of liver diseases such as cirrhosis. Modifications that continually appear in all liver diseases are hyperfucosylation, increased branching, abnormal sialylation, and a bisecting N-acetylglucosamine.33 Likewise, carbohydrate-deficient transferrin (CDT) is the most commonly used marker of alcoholic liver disease. In fatty liver diseases, aberrant glycosylation of apolipoprotein-B or fucosylation of N-glycans alter biochemical parameters of lipid metabolism in the liver.33

G L Y C A N S I N T H E R A P E U T I C S A N D AS T H E R A P E U T I C TARGETS As we have just described, glycans play a crucial role in differentiation, immunity, and signal transduction. However, owing to their structural complexity, their therapeutic potential has not been fully exploited, with a few notable exceptions. Glycans play an important role in two distinct but related areas of drug development.34 Firstly, as a component of therapeutic biotechnologically derived glycoproteins (e.g., antibodies and erythropoietin), glycans modulate their activity, stability, half-life, and immunogenicity.34 For example, modification of the glycan coat of a protein has led to the second-generation anaemia drug, Darbepoetin, which shows improved therapeutical benefits as compared to traditional therapeutics.35 Secondly, complex glycans that are isolated from natural sources or are chemically synthesized are themselves active pharmaceutical agents. For example, a synthetic version of a truncated heparin oligosaccharide (fondaparinux sodium) has successfully been used for the treatment of thrombosis.34 These advantages of glycan-based therapeutics are due to their more increased stability and ease of formulation as compared with protein-based drugs.34 Additionally, they are highly specific and less immunogenic than other protein or RNA-based therapies. Since the role of glycans in tumor proliferation, metastasis, and angiogenesis is well-known, concerted efforts have been put forward to develop

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therapeutics which target the following: (a) N-glycosylation; (b) sialylation pathways; (c) HSPG; and (d) chondroitin sulphate proteoglycans (CSPGs).18 For instance, Swainsonine, a plant-derived competitive inhibitor of N-glycan processing in the Golgi, is currently in Phase II trial, whereas heparin is widely used as an antigoagulant in clinics.336 Furthermore, CSPGs overexpression in tumors has been successfully targeted in mouse melanoma models using chondroitin-sulphate-binding cationic liposomes loaded with chemotherapeutics.37 Mucin overexpression in cancer induces rapid cellular growth and survival, making it another attractive target for tumor therapeutics.38 As such, MUC16 antibodies are in Phase II clinical trials, whereas a peptide-based vaccine therapy targeting MUC 1 is undergoing Phase III trials for ovarian and breast carcinomas, respectively.18 Finally, glycans are also potential targets for vaccines against many microbial pathogens such as Leishmania, HIV, and the influenza virus.3'39 Glycan-based vaccines are based on creating an immune response toward the surface glycan antigen of the pathogen.

TOOLS TO ANALYZE THE GLYCOME

Although there has been tremendous progress in elaborating tools to study the glycome in both experimental and clinical settings, this still presents a formidable challenge. This is due to the facts that sugars are present in much lower amounts than proteins in the cells, cannot be template-based amplified like nucleic acids, and have inherent structural and chemical heterogenicities which can yield millions of different modifications to proteins or lipids.40 Glycome analysis falls into four main categories: analytical, chemical, microarray, and molecular, as will be described in the following section.

Analytical Mass spectometry remains the main tool for glycome analysis, due to its high resolution and sensitivity in separating various glycoproteins from a complex mixture including body fluids and organs (Figure 5.3).2,29,41 This method, which enables precise primary structure determination to be achieved, requires first ionizing the test compounds in order to analyze the mass-to-charge ratio of gas phase ions. The two main types of ionizing methods, "soft" versus "hard," refer to the intensity of ionization generated by the spectrometer and is used to determine molecular weight or molecular composition, respectively. Hard ionization methods include fast electron impact (El) and chemical ionization (CI), whereas soft ionization comprises atom bombardment (FAB), electrospraty (ES) and matrix-assisted laser desorption ionization (MALDI), the latter being the most sensitive of all three.42 In order to improve resolution, soft and hard ionizing methods can be combined with each other or with chromatography and enzymatic techniques. A useful chromatography method includes using activated graphitized carbon and C18 chips in order to discriminate between hydrophilic and hydrophobic glycoproteins, respectively.43 This method also has the advantage of resolving glycoprotein

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FIGURE 5.3 Analytical techniques for oligosaccharide profiling to distinguish between normal and cancerous cells. (See color insert for a full color version of this figure.)

anomers and isomers. Another method, regularly used in our laboratory, involves analyzing sulfated glycans using reverse polarity capillary electrophoresis, which can reliably yield the sugar signature of cells and zebrafish (Figure 5.4)4445

Chemical In the past few years, various synthetic fluorescent labeling techniques have been developed for molecular imaging of the cellular glycome. This method relies on metabolic labeling of target glycans with an unnatural monosach-

FIGURE 5.4 Capillary electrophoresis quantification of glycosaminogiycans between wild-type (bottom) and heparan sulfate-modifying enzyme-deficient (top) zebrafish.

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FIGURE 5.5 (A) Bioorthogonal chemical reporting strategy for imaging glycans. (B) Bioorthogonal reactions used to visualize chemical reporters appended to unnatural sugars. (C) Application of the bioorthogonal chemical reporter strategy for in vivo imaging of glycans in zebrafish. (See color insert for a full color version of this figure.)

haride substrate bearing a biologically orthogonal reactive group (such as azide or alkyne), and their subsequent ligation to fluorescent probes (Figure 5.5).46 For instance, in an interesting study, the versatile labeling technique based on Cu(I) catalyzed "click" chemistry between a fluorescent probe and azidomodified fucose was used to visualize protein fucolysation, a postranslational modification involved in metastasis and immunity.47 A similar chemical ligation strategy between alkynyl-glycans and biotin azides was employed to label and capture glycans in order to enrich the glycoprotein fraction of samples, thus facilitating subsequent analyses via mass spectrometry.48 Likewise, glycoprotein enrichment can be achieved by trapping them from biological samples onto synthetic polymers via a transoximization reaction.49 In an elegant study Bertozzi, et al. demonstrated for the first time glycome imaging in a living animal, the zebrafish (Figure 5.5).46M The authors metabolically labelled the zebrafish glycome with unnatural monosaccharide substrate reporters which subsequently fluoresced in the presence of bioorthogonal imaging probes. The ligation strategy used in the study, namely strain-promoted cyclo-addition of monosaccharide azides with fluoro-conjugated cyclooctynes (Cu(I)-free click chemistry), offers the advantages of improved kinetics under physiological conditions and superior analysis of glycan trafficking dynamics in live cells.

Microarray Like nucleic acids and proteins before them, lectins or antibodies have been used to build microarray s capable of monitoring specific glycoprotein-receptor interactions or determining the type of glycoprotein expressed under specific

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conditions, respectively (Figure 5.6).2,51,52 Major limitations of this technique, however, are that lectins usually have low affinity for their ligands and glycodirected antibodies are scarce due to the lack of information about selective epitopes. To overcome these limitations, gold nanoparticles have been used to increase lectin binding signals, which can increase the avidity of the system.52 Some researchers are also developing synthetic oligosaccharide microarray chips made up of heparan sulfate or chondroitin sulfate oligosaccharides.53 An added advantage of this technique is that the shorter substrate used (e.g., oligosaccharides vs. lectins or antibodies) renders the arrays more selective, thus offering the most promise for clinical applications.

Molecular Antibodies have long been used to analyze the glycome of tissues by standard immunohistochemistry, although due to the limitation described above, this technique is severely limited.46 For this reason, a more sensitive approach is a modified ELISA (Biotin-NeutrAvidin adhesion assay, BNAA) where various biotinylated glycoproteins are adhered on the reaction plate and then incubated with a specific neutrAvidin-modified receptor in order to decipher the types of sugars this receptor binds.39 This technique shows important therapeutic

FIGURE 5.6

Schematic representation of the multiple modifications present in glycan microarrays.

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potential, as it helped establish the type of glycoproteins bound by HIV during infection and thus brought researchers a step closer to developing novel HIV therapeutics.39 Due to the glycome's uniquely high degree of complexity, most groups, including ours, lean toward using a combination of tools, rather than a single one, in order to get a more accurate representation of glycomic modifications.45' 54 The same approach should also be taken in clinical settings.

STRENGTHS,WEAKNESSES,ANDTHE FORWARD

ROAD

Due to the global role glycans play in all aspects of physiology, glycomicbased therapies are emerging as a crucial and novel concept to diagnose and target a plethora of diseases. The glycomic field has several advantages that make it well-suited and highly sensitive as a novel disease biomarker. Changes in glycosylation can be more distinct than changes in protein expression and affect many proteins functions. Specific glycans that are not present, or are present in low amounts, in normal state may be upregulated in diseased states. Also, the location of glycans on the cell surface and on the matrix gives them access to most proteins in the body.3 Since many signaling pathways are mutated in cancer and other diseases, targeting a single glycomic event would hamper the function of multiple proteins, translating to more potent and selective therapeutics. The major challenge of glycomic-based therapies lies in the lack of rapid and efficient analysis tools, which reflects the inherent structural and chemical complexities of the glycome, as compared with proteins, lipids, and nucleic acids.40 These limitations could be overcome by a better understanding of all possible glyco-modifications present in physiological and diseased states. To that end, glycoinformatic databases are being put forward to regroup all general knowledge deciphered thus far on the glycome.2,3

CONCLUSION Whereas glycosylation events were once merely thought of as superficial modifications, their crucial roles in all aspects of homeostasis are now clearly established. This has paved the way for using glycans as diagnostic tools and targeted therapeutics. Although the field of glycomics has come a long way, much progress needs to be made based on several criteria, mainly the need for better characterization, quantification, and isolation of glycans.

SUMMARY P O I N T S 1. 2.

Glycosylation of proteins and lipids is essential for their proper functioning. The glycome mediates all aspects of human development and homeostasis.

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For this reason, alterations in the glycome underlies a plethora of diseases. Identifying and targeting disease glyco-biomarkers, as opposed to simply proteins or lipids, offers more therapeutic potential.

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Turnbull, J. E. and Field, R. A. Emerging Glycomics Technologies. Nat. Chem. Biol. February 2007;3(2):741. Haslam, S. M., Julien, S., and Burchell, J. M., et al. Characterizing the Glycome of the Mammalian Immune System. Immunol. Cell Biol. October 2008; 86(7):564-573. Packer, N. H., Von der Lieth, C. W., and Oki-Kinoshita, K. R, et al. Frontiers in Glycomics: Bioinformatics and Biomarkers in Disease. An NIH white paper prepared from discussions by the focus groups at a workshop on the NIH campus, Bethesda, MD (September 11-13, 2006). Proteomics. January 2008;8(l):8-20. Prescher, J. A. and Bertozzi, C. R. Chemical Technologies for Probing Glycans. Cell. September 8, 2006;126(5):851-854. Honorat, M., Mesnier, A., and Di, P. A., et al. Dexamethasone Down-Regulates ABCG2 Expression Levels in Breast Cancer Cells. Biochem. Biophys. Res. Commun. October 24, 2008;375(3):308-314. Freeze, H. H., Genetic Defects in the Human Glycome. Nat. Rev. Genet. July 2006;7(7):537-551. Marquardt, T. and Denecke, J. Congenital Disorders of Glycosylation: Review of Their Molecular Bases, Clinical Presentations and Specific Therapies. Eur. J. Pediatr. June 2003;162(6):359-379. Lonnqvist, L., Karttunen, L., Rantamaki, T., Kielty, C , Raghunath, M., and Peltonen, L. A Point Mutation Creating an Extra N-glycosylation Site in Fibrillin-1 Results in Neonatal Marfan Syndrome. Genomics. September 15, 1996;36(3):468^175. Vogt, G., Chapgier, A., and Yang, K., et al. Gains of Glycosylation Comprise an Unexpectedly Large Group of Pathogenic Mutations. Nat. Genet. July 2005; 37(7):692-700. Van, R. J., Brunner, H. G., and Van, B. H. Glyc-o-Genetics of Walker-Warburg Syndrome. Clin. Genet. 2005 April; 67(4):281-289. Zak, B. M., Crawford, B. E., and Esko, J. D., Hereditary Multiple Exostoses and Heparan Sulfate Polymerization. Biochim. Biophys. Ada. December 19, 2002;1573(3):346-355. Rudd, P. M., Elliott, T., Cresswell, P., Wilson, I. A., and Dwek, R. A. Glycosylation and the Immune System. Science. March 23, 2001;291(5512):2370-2376. Axford, J. S. Glycosylation and Rheumatic Disease. Biochim. Biophys. Ada. October 8, 1999;1455(2-3):219-229. Novak, J., Julian, B. A., Tomana, M., and Mestecky, J. IgA Glycosylation and IgA Immune Complexes in the Pathogenesis of IgA Nephropathy. Semin. Nephrol. January 2008;28(l):78-87. Demetriou, M., Granovsky, M., Quaggin, S., and Dennis, J. W. Negative Regulation of T-Cell Activation and Autoimmunity by Mgat5 N-Glycosylation. Nature. February 8, 2001;409(6821):733-739. Marth, J. D. and Grewal, P. K. Mammalian Glycosylation in Immunity. Nat. Rev. Immunol. November 2008;8(ll):874-887.

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Shriver, Z., Raguram, S., and Sasisekharan, R. Glycomics: A Pathway to a Class of New and Improved Therapeutics. Nat. Rev. Drug Discov. October 2004; 3(10):863-873. Egrie, J. C , Dwyer, E., Browne, J. K., Hitz, A., and Lykos, M. A., Darbepoetin Alfa has a Longer Circulating Half-Life and Greater In Vivo Potency Than Recombinant Human Erythropoietin. Exp. Hematol. April 2003;31(4):290-299. Goss, P. E., Baptiste, J., Fernandes, B., Baker, M., and Dennis, J. W. A Phase I Study of Swainsonine in Patients with Advanced Malignancies. Cancer Res. March 15, 1994;54(6):1450-1457. Lee, C. M., Tanaka, T., and Murai, T., et al. Novel Chondroitin Sulfate-Binding Cationic Liposomes Loaded with Cisplatin Efficiently Suppress the Local Growth and Liver Metastasis of Tumor Cells in Vivo. Cancer Res. August 1, 2002;62(15):4282^1288. Hollingsworth, M. A. and Swanson, B. J., Mucins in Cancer: Protection and Control of the Cell Surface. Nat. Rev. Cancer. January 2004;4(l):45-60. McReynolds, K. D. and Gervay-Hague, J. Chemotherapeutic Interventions Targeting HIV Interactions with Host-Associated Carbohydrates. Chem. Rev. May 2007;107(5):1533-1552. Raman, R., Raguram, S., Venkataraman, G., Paulson, J. C , and Sasisekharan, R. Glycomics: An Integrated Systems Approach to Structure-Function Relationships of Glycans. Nat. Methods. November 2005;2(11):817-824. Dove, A. The Bittersweet Promise of Glycobiology. Nat. Biotechnol. October 2001;19(10):913-917. Dell, A. and Morris, H. R. Glycoprotein Structure Determination by Mass Spectrometry. Science. March 23, 2O01;291(5512):2351-2356. Alley, W. R., Jr., Mechref, Y., and Novotny, M. V. Use of Activated Graphitized Carbon Chips for Liquid Chromatography/Mass Spectrometric and Tandem Mass Spectrometric Analysis of Tryptic Glycopeptides. Rapid Commun. Mass Spectrom. February 2009;23(4):495-505. Johnson, N. A., Sengupta, S., and Saidi, S. A., et al. Endothelial Cells Preparing to Die by Apoptosis Initiate a Program of Transcriptome and Glycome Regulation. FASEBJ. January 2004;18(1):188-190. Harfouche, R., et al. Glycome and Transcriptome Regulation of Vasculogenesis. 2009;Circulation 120:1883-1892. Laughlin, S. T. and Bertozzi, C. R. Imaging the Glycome. Proc. Natl. Acad. Sci. USA. January 6, 2009;106(l):12-7. Sawa, M., Hsu, T. L., and Itoh, T., et al. Glycoproteomic Probes for Fluorescent Imaging of Fucosylated Glycans In Vivo. Proc. Natl. Acad. Sci. USA. August 15, 2006;103(33):12371-12376. Hanson, S. R., Hsu, T. L., and Weerapana, E., et al. Tailored Glycoproteomics and Glycan Site Mapping Using Saccharide-Selective Bioorthogonal Probes. J. Am. Chem. Soc. June 13, 2007;129(23):7266-7267. Shimaoka, H. and Nishimura, S. I. One-Pot Solid Phase Glycoblotting and Probing by Transoximization for High Throughput Glycomics and Glycoproteomics. Chemistry—A European Journal 13. 2007; 1665-1673. Laughlin, S. T, Baskin, J. M., Amacher, S. L., and Bertozzi, C. R. In Vivo Imaging of Membrane-Associated Glycans in Developing Zebrafish. Science. May 2, 2008;320(5876):664-667. Cummings, R. and Etzler, M. E. Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, 2nd Edition, Chapter 45. 2009.

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Gao, J., Liu, D., and Wang, Z. Microarray-Based Study of Carbohydrate-Protein Binding by Gold Nanoparticle Probes. Anal. Chem. November 15, 2008; 80(22):8822-8827. de Paz, J. L. and Seeberger, P. H. Deciphering the Glycosaminoglycan Code with the Help of Microarrays. Mol. Biosyst. July 2008;4(7):707-711. Ito, H. and Narimatsu, H. Strategy for Glycoproteomics: Identification If Glyco-Alteration Using Multiple Glycan Profiling Tools. Journal ofProteome Research. 2009;8[3]: 1358-1367.

SECTION II BIOMARKERS OF INJURY/DISEASE


CHAPTER

BIOMARKERS OF ALZHEIMER'S AND PARKINSON'S DISEASE Walter Maetzler and Daniela Berg

D E F I N I T I O N A N D PREVALENCE OF A L Z H E I M E R ' S A N D P A R K I N S O N ' S DISEASE During the last centuries, the world population has shown a continuous increase in the proportion of elderly subjects and consecutively neurodegenerative diseases occurring primarily with increasing age, such as Alzheimer's disease (AD) and Parkinson's disease (PD). These disorders are accompanied by a large personal, occupational, but also social burden, and the improvement of diagnostic measures to detect early and subtle symptoms to modify the disease course is one of the central challenges of clinicians, scientists, and governments. Interest has not only focused on the phase in which clinical symptoms have appeared (to measure disease progression) but also on the phase in which prevention efforts are expected to have their greatest impact: the preclinical phase, putatively lasting up to decades in both diseases, in which less neurons have degenerated compared to the time of diagnosis.

Alzheimer's Disease AD is the prototypical and, by far, the most common dementia. The diagnosis is based on the criteria of the National Institute of Neurologic and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA), and includes a persistent decline in cognitive function from a previously higher level.1 Memory loss is the principal cognitive deficit, and at least one other cognitive domain must also

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be affected, leading to aphasia (language disturbance), agnosia (failure to recognize people or objects in presence of intact sensory function), apraxia (inability to perform motor acts in presence of intact motor system), and/ or disturbance of executive function (plan, organize, sequence actions, or form abstractions).2 Mild cognitive impairment (MCI) is a preclinical phase of AD during which subjects have measurable cognitive deficits, which are not sufficient to fulfill criteria for any specific dementing disease. However, not all people diagnosed as having MCI will develop AD, pointing to the urgent need of state biomarkers, especially in preclinical and very early disease phases.3 Disease-specific neurodegeneration has been estimated to start about 20 years before the diagnosis can be made.4 At this stage, with a clinical dementia rating (CDR) score of 0.5 ("very mild dementia"), about 60 percent of neurons at specific brain sites (i.e., the layer II entorhinal cortex) are lost.5 Prevalence of AD doubles every five years, beginning with one percent at 60 years of age, and reaching over 30 percent at 85 years of age.6 Incidence rate also increases with age: for people with 65 years of age, the annual risk of developing AD is 0.6 percent, for those older than 85 years it is 8.4 percent.7 The economic burden of caring for patients with dementia exceeds that of more common illnesses like diabetes and arthritis.8

Parkinson's Disease Idiopathic Parkinson disease (PD) is basically a clinicopathologic diagnosis. Clinically, asymmetric manifestation of bradykinesia and at least one of the following symptoms: resting tremor, rigidity, and/or decrease of postural reflexes is observable. At the time when motor symptoms allow the clinical diagnosis, more than 50 percent of the dopaminergic neurons at the SN are lost,9 indicating not only the enormous compensatory capacities of the brain, but also the urgent need to detect subjects in this process before this large amount of neurons is lost. Besides essential tremor, PD is the most common movement disorder and affects up to 200,000 people in Germany, and approximately 500,000 people in the United States.10 Like AD, it is a disease of the elderly, about 1 percent of people older than 50 years suffer from this disorder. Lifetime risk tables report the risk of developing PD to be two percent for men and 1.3 percent for women."

PATHOPHYSIOLOGY AND MECHANISMS Alzheimer's Disease The cause for neurodegeneration in AD is not entirely clear and appears to be multifactorial, with several biochemical processes operating sequentially and/ or in parallel. It is hypothesized that similar or identical pathological lesions are the consequence of multiple environmental (toxic exposure, infection, inflammation) and genetic susceptibility factors.

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Genetic Aspects Monogenetic defects leading to AD are extremely rare, however, the affected proteins point to an involvement of the amyloid cascade in the AD pafhogenesis. Mutations of the Abeta precursor protein (APP) gene, or mutations in the presenilin 1 or 2 genes result in an increase of the amyloidogenic Abeta peptide (the 42 amino acid version) in the brain.12 There is some evidence that the abnormally phosphorylated tau protein (which is found in neurofibrillary tangles) is a secondary effect from the Abeta deposition. Also, the e4 allele of the apolipoprotein E (ApoE) gene is linked to frequency and onset of AD. ApoE is involved in cholesterol transport in the periphery but, maybe more important, also in central nutritive and pathogenic pathways. Isoform-specific effects on neurite outgrowth, neuronal plasticity, neurotoxicity, lipid peroxidation, oxidative injury, binding to cytoskeletal proteins, and interactions with Abeta including neuritic plaque formation, have been shown.13 Pathology On macroscopic examination, the AD brain appears atrophic with enlarged ventricles and sulci, and overall brain weight is reduced. The deep layers of the temporal cortex and the hippocampus are most severely affected. Most of the clinical features can readily be explained by a loss of cholinergic transmission in cortical brain regions innervated by neurons arising in the nucleus basalis of Meynert. The dopaminergic and serotoninergic neurotransmitter systems are also affected, and their dysfunction may explain, at least partly, many of the non-cognitive symptoms of AD, e.g., mood and motivation disturbances. Intrinsic classical neurotransmitters (e.g., gamma amino butyric acid and glutamate) and cortically localized neuropeptides (e.g., somatostatin and corticotropin releasing factor) are also altered.14 Neuritic plaques and neurofibrillary tangles are the distinguishing microscopic features used in the pathological diagnosis of AD (Figure 6.1).15,16 Neuritic plaques are located extracellularly and are composed of higher-order Abeta fibrils with diameters of up to 10 nm, mostly occurring in radiating, star-like assemblies. They are initially found in cortical areas, and may then distribute to deeper brain areas following a distinct hierarchical sequence.17 Neurofibrillary tangles are abnormal intracellular hyperphosphorylated filaments that form a distinctive paired helical structure (Figure 6.1). They are found throughout the neocortex and limbic nuclei, but are also strongly represented in the basal forebrain and in brainstem structures like the substantia nigra (SN), raphe nuclei, and locus ceruleus. The protein components of neurofibrillary tangles have been identified as microtubules, tau (microtubular associated protein), and ubiquitin. Plaques and tangles may result from destructive processes involving the disruption of microtubule assembly and synaptic loss, rather than its causes. They may contribute to further neuronal damage and disease progression: especially in animal models, the reversal or removal of this amyloid plaque and tangle pathology has been shown to be beneficial on clinical symptoms and may slow disease progression.18,19 The Abeta peptide forms soluble oligomers,

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FIGURE 6.1 The pathological hallmarks of Alzheimer's disease, i.e., neuritic plaques (Gallays stain, A) and neurofibrillary tangles (Gallays stain, B), and the pathological hallmarks of Parkinson's disease, i.e., alpha-synuclein-positive Lewy bodies (arrows) and Lewy neurites (arrowhead, C). Neuritic plaques are located extracellularly and are composed of Abeta fibrils. They are initially present in cortical areas. Neurofibrillary tangles are abnormal intracellular neuronal hyperphosphorylated filaments. They are found mainly in limbic areas. A major component is tau, a microtubulus-associated protein. Lewy bodies and Lewy neurites are eosinophilic proteinaceous neuronal inclusions which are located primarily in brainstem neurons. All these aggregates are thought to be crucially involved in the pathogenesis of the diseases, but rather not at early stages. By courtesy of Dr Jens Schittenhelm, Institute for Brain Research, University of Tuebingen. (A) and (C) x200, (B) x400. (See color insert for a full color version of this figure.)

which then aggregate into insoluble fibrils. Growing evidence suggests that the oligomeric forms are more toxic than the mature senile plaques and may be more related to the primary degenerating processes.20 Pathophysiological Mechanisms

Environmental exposures: Several epidemiologic studies have examined whether a link exists between aluminium exposure and AD, but the results are conflicting. There is some evidence that monomeric organic aluminium in drinking water may be associated with AD.21 An association was observed between an elevation of iron in the brain and AD, but no significant association was detectable for occupational exposure to lead or mercury, or for mercury from dental amalgams.22 Pesticides, especially organophosphates and carbamates, are known to cause neuronal damage. A significant association was observed between occupational exposure to pesticides in general, and for fumigants and defoliants in particular, and AD.22 There is clear evidence that oxidative stress contributes to AD pathogenesis. At the site of neuritic plaques, expression of inducible nitric oxide synthase (iNOS) is increased, as are oxidative markers.23 Reactive oxygen species (ROS) and reactive nitrogen species (RNS), along with many other substances with free radical character, can react with proteins, lipids, carbohydrates, DNA, and RNA, starting a "vicious cycle" which damages or destroys cells.24 Mitochondria are the main energy-generating organelles of human cells, and they are key players in oxidative stress phenomena as they generate more than 90 percent of the cell's endogenous oxidant species.25 Mitochondrial impairment has been suggested to contribute to AD, as energetic enzymes have been shown to be markedly impaired, and mitochondrial DNA shows


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abnormally elevated oxidation products in the temporal, parietal, and frontal lobes of the AD brain.26 A deficit of ATP production capacity inside the mitochondria seems to contribute to altered glucose metabolism and tolerance in AD patients.27 Disruption of calcium homeostasis and excitotoxicity—mediated via ionotropic glutamate receptors—are likely determinants of neuronal vulnerability in AD because neurons in brain regions with high Abeta load (entorhinal cortex, hippocampus, inferior parietal cortex) degenerate, whereas neurons in regions with little or no Abeta accumulation (cerebellum, striatum, motor cortex) typically do not.28 Neurons primarily affected in AD typically express high levels of NMDA receptors and have relatively low levels of calciumbinding proteins (like calbindin) compared to resistant neurons.29 Changes in the expression of glutamate receptors may also contribute to altered neuronal Calcium levels in AD, as a significant increase of the NR2A subunit of the NMDA receptor has been shown to occur in subjects with neurofibrillary tangle neuropathology.30 Insufficient Calcium homeostasis may lead to selective neuronal vulnerability in AD through perturbation of the energy metabolism, of antioxidant systems, and neurotrophic factor support.28 A striking feature of neuritic plaques is the presence of activated microglia, cytokines, and complement components, suggestive of a local inflammatory process in AD.31 There is evidence from epidemiological studies that regular consumption of non-steroidal anti-inflammatory drugs (NSAIDs) leads to a reduced risk of AD.32 In line with the observation that NSAID treatment studies with AD patients basically failed to show any benefit, it has been hypothesized that inflammation may be a key player rather at the very early/preclinical stages of AD.33 Diverse inflammation markers are increased in the brain and body fluids of AD, and inflammation inhibitors have been shown to inhibit fibril formation and to facilitate re-entering the cell cycle.33

Parkinson's Disease The cause of PD is unknown. Environmental factors seem to play a major role, as underlined by a large epidemiologic study which found that concordance rates for parkinsonism in monozygotic and dizygotic twins were indistinguishable.34 Genetic Aspects Five to ten percent of cases are directly linked to genetic defects. To date, 13 genetic loci, and nine genes are known to be capable of causing PD in the case of alterations.35 It seems obvious that the molecular pathways identified in these monogenic forms—primarily mitochondrial impairment, protein aggregation, and oxidative stress—may also be implicated in sporadic PD, as some of the proteins involved in monogenic Parkinsonism are also able to cause PD, i.e., solely by dosage effects (e.g., alpha-synuclein through duplication or triplication36).

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Pathology

PD motor symptoms are primarily related to the degeneration of dopaminergic cells of the pars compacta of the SN.37 However, other areas (e.g., pigmented brainstem nuclei, autonomic nuclei, pyramidal cells in the presupplementary cortex) are also affected. The degenerative process has been suggested to follow a distinct pattern starting in the deep brainstem and reaching high cortical areas within years or decades.38 The pathological hallmark of PD are Lewy bodies, i.e., eosinophilic cytoplasmic proteinaceous inclusions which are located primarily in brainstem neurons, but are also often found in the cortex (Figure 6.1). They consist of a dense core surrounded by a halo of 10 nm wide radiating fibrils. The primary structural component of these fibers is alphasynuclein, a protein physiologically located at the vicinity of pre-terminal synapses. Alpha-synuclein may cause impaired dopamine storage, influences dopamine transport activity, catalyzes the formation of hydrogen peroxide (a product known to damage neuronal membranes), and is very probably directly involved in the protein aggregation process which finally leads to Lewy bodies. This accumulation is associated with neuronal death in animal models.39 Pathophysiological Mechanisms

Environmental factors are heavily debated in PD. A substantial number of epidemiologic studies suggest an association between an exposure to pesticides and PD (see below, for a review see reference 22). This is underscored by pathological data which show increased levels of pesticides in the brains of PD cases versus controls.40 Many epidemiological studies also argue for an increased risk for PD when living in rural areas, working with wood or in other forms of construction, and use of well water. This may be associated with a runoff of pesticides or other environmental contaminants.41 There is evidence from epidemiological studies that the combination of an increased exposure to iron and copper, iron and lead, and iron and manganese (but not iron alone) leads to an increased risk for PD.22 Iron plays a crucial role in the pathogenesis of PD due to its increase in SN neurons and reactive microglia and its capacity to enhance production of reactive oxygen radicals.42 Considering epidemiological and pathological data, conflicting results are observable for the occurrence of other heavy metals, like mercury, copper, aluminium, and manganese, and the risk for PD.22 Oxidative stress has been suggested to play a major role in the pathogenesis of PD. Free radicals react with membrane lipids and lead to lipid peroxidation, membrane injury, and cell death. The dopaminergic system may be especially prone to oxidative stress as dopamine metabolized by oxidation reactions is capable of generating free radicals.43 Because the SN of PD patients contains high iron levels—which facilitates oxidation—and decreased glutathione levels—which protects against free radical formation—nigral cells may be selectively vulnerable to oxidative stress.42 The interaction of dopamine with metal ions may lead to further damage.


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Mitochondrial respiratory failure appears to be an important contributor to neuronal death in the SN of patients with PD. Complex I deficiency is basically related to this respiratory failure, but it is not clear whether this defect is primary and inherited or secondary to environmental influences. l-Methyl-4phenyl-pyridin (MPTP), a designer drug, causes a clinical syndrome closely resembling PD without relevant accompanying multifocal neurotoxicity. It was industrially developed as a potential herbicide with a structure resembling paraquat—a herbicide also associated with parkinsonism—but was never produced commercially. Rotenone is a further herbicide with a potent inhibitory effect on complex I mitochondrial activity which has the capability to provoke parkinsonism.44 Excitotoxicity is a pathological process with overactivation of NMDA-, AMPA- and/or metabotropic receptors leading to elevated calcium influx into the cell resulting in nerve cells damage by glutamate and similar substances. As mitochondria and the endoplasmic reticulum are the principal cellular calcium sinks, increased calcium influx is able to impair ATP synthesis, to induce free radical formation, and to lead to lipid peroxidation and finally to cell death.45 Excitotoxicity is discussed to be one of the major sources of neurodegeneration in PD,46 which may be underscored by the fact that midbrain dopamiergic neurons are selective autonomic pacemakers which are driven by calcium channels. These cells are especially prone to calcium overload.47 There is growing evidence from post-mortem and in-vivo studies that inflammation contributes to the pathophysiology of PD.48 Activation of glial cells has been consistently found in PD brains. Diverse cellular and molecular events are associated with neuroinflammation, and the final steps are mostly mediated by activated glial and peripheral immune cells. This cellular response to PD-associated neurodegeneration triggers, e.g., oxidative stress and cytokine-receptor-mediated apoptosis, which in turn might provoke disease progression.

C o n c l u d i n g Remarks t o Pathological and Pathophysiological Aspects It is remarkable that in both neurodegenerative diseases, AD and PD, similar pathophysiological processes lead to different clinical symptoms, which progress relentlessly. Only the pathological picture differs in the majority of cases: AD is defined by the occurrence of Abeta plaques and neurofibrillary tangles, whereas PD presents with Lewy bodies. However, about every fifth AD patient has Lewy bodies,49 and the same percentage of PD cases present with AD pathology.50 This may also point toward similarities of the diseases. One of the most strikingly similar aspects, however, is the long preclinical neurodegenerative phase of both disorders which may last at least one decade. This is the time frame when potent neuroprotective therapies may be most successful, and it should be a primary aim to define biomarkers for this phase.

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C U R R E N T MEANS FOR D I A G N O S I S / P R O G N O S I S OF T H E DISEASES A N D T H E I R LIMITATIONS The diagnostic accuracy of the presence (trait) and the severity (state), (Figure 6.2) of AD and PD is limited, and sensitive and reliable biomarkers that reflect the underlying disease process are urgently needed, as a prerequisite for a more certain diagnosis and the development of novel disease-modifying therapeutic strategies.

Alzheimer's Disease The diagnostic accuracy for AD reaches 80 percent sensitivity and 70 percent specificity, and even in specialized centres the current approach for diagnosing AD leads to misdiagnosis in up to 10 to 15 percent of cases.51 C l i n i c a l Markers

The current approach to the diagnosis is based on clinical aspects, with careful physical and neurologic examinations, and mental status testing to identify the characteristic memory, language, and visuospatial deficits.' In the medical history, potential risk factors like depression, hypertension, heart disease, diabetes, transient ischemic attacks, environmental exposure to toxins (particularly lead), low educational achievement, lack of intellectual and physical activity, and lack of social interaction should be assessed. G e n e t i c Markers

There is no genetic test or a simple blood or urine test that can detect AD. Mutations in the presenilins and the APP gene are able to cause early-onset AD and have strengthened the amyloid hypothesis. However, they are extremely rare and do not play a role in the clinically based diagnostic work-up. Fifty to 70 percent of AD patients have at least one ApoE4 allele, and only 15 to 25 percent of elderly controls. However, for routine diagnosis the accuracy of the ApoE genotype assessment is too low and cannot be recommended.52 In Vivo Markers f r o m Pathology

Definitive diagnosis of AD can only be made by autopsy with appropriate numbers of plaques and tangles determined from specific regions of the brain, in the presence of a clinical history consistent with dementia. Cerebrospinal fluid (CSF) assays for soluble Abeta, total tau, and phosphorylated tau are commercially available and are useful trait markers for the differential diagnosis between AD and non-AD causes of dementia. In AD, tau becomes abnormally phosphorylated, aggregates, and loses its ability to maintain the microtubule tracks. Abeta peptides result from enzymatic breakdown of amyloid precursor protein (APP) by presenilins and display the main part of amyloid deposits in


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FIGURE 6.2 Markers of Alzheimer's disease and Parkinson's disease. Note that both neurodegenerative diseases develop relatively similan especially in the way that both have a long preclinical phase in which large numbers of neurons are progressively lost, and discrete changes are already detectable. This time frame is the most promising phase in which neuroprotective therapies are supposed to be successful and it should be the primary aim to find state and trait markers for this period. Adapted from reference 98.

AD. According to a longitudinal study with pharmacologically untreated patients up to six years with repeated serial CSF measurements,53 CSF p-tau231 levels may decrease with disease duration, suggesting that phosphorylated tau CSF levels may have the potential to serve as a state marker. In addition, a high CSF total tau/Abeta ratio has been shown to be highly associated with increased risk of conversion from healthy to MCI54 and from MCI to AD.55 This is an example which may stand for future developments: the diagnostic accuracy of a combination of the three pathophysiologically widely independent CSF biomarkers (Abeta, total tau, and phospho tau) is superior to every "single CSF biomarker, by increasing both sensitivity and specificity.56 The best studied candidate biomarker in plasma so far is Abeta (40 and 42), but the findings are contradictory and thus no recommendation concerning the value of plasma Abeta as biomarker can actually be given. Acetyl- and butyrylcholinesterase are basically involved in the cholinergic neurotransmission by inactivating acetylcholine in the synaptic cleft and are suggestive biomarkers for cholinergic degeneration. CSF acetylcholine may be decreased in untreated AD patients, and showed a significant positive correlation with dementia scale scores.57 CSF acetylcholinesterase levels

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have been shown to be either decreased or unchanged compared to controls (reviewed in reference 58), and may therefore have limited potential to serve as a biomarker. Butyrylcholinesterase is detectable in diverse brain tissues and in the CSF. CSF butyrylcholinesterase levels have been shown to be decreased in AD59 and may correlate negatively with cognitive capacities.60 Pathophysiological Mechanisms

Markers associated with oxidative stress: Elevated homocysteine levels (associated with oxidative stress and excitotoxicity) and elevated low-density lipoprotein cholesterol levels are weakly associated with increased AD risk, but are not useful markers on an individual level (for a review see reference 61). Mitochondrial markers: ApoE is involved in mitochondrial impairment, lipid transport, and cholesterol homeostasis and is attracting a lot of attention in the context of AD, however, as there are conflicting results concerning CSF and serum levels in AD patients compared to controls, ApoE protein level seems not to be a promising marker for AD pathology. Markers associated with excitotoxicity: Tests focusing on excitotoxicity processes are not included in clinically-based diagnostic assessments. Markers associated with inflammation: Despite being increased in affected brain tissue, there is no convincing evidence that levels of molecules like tumor necrosis factor (TNF) or interleukin 1 and 6 change relevantly in body fluids during AD.61 F u r t h e r Diagnostic Assessments

Structural brain imaging, assessment for depression and laboratory testing with particular emphasis on thyroid function and vitamin B12 levels increase diagnostic accuracy.51 Structural MRI helps to exclude secondary dementia due to, e.g., vascular or metabolic pathology. Fluorodeoxyglucose (FDG) PET scanning on average achieves 90 percent sensitivity in identifying AD, although specificity in differentiating AD from other dementias is lower. Clusters of low patterns of cerebral glucose metabolism are typically observable in the posterior cingulate cortex and the precuneus, in the inferior parietal lobule, and the middle temporal gyrus of AD patients. This method may also provide specific and sensitive measure at very early disease stages: In MCI patients with increased risk for AD, reduced patterns of cerebral glucose metabolism were detectable in the posterior cingulate cortex.62,63 Automated analysis algorithms are already available, providing clinicians with z-score maps for metabolic deviation.64

Parkinson's Disease The thorough diagnostic work-up for PD enables correct characterization of about 80 to 90 percent of cases (high sensitivity but lower specificity) and requires clinical skills, but also some subjective clinical experience.


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C l i n i c a l Markers

A general neurologic examination focuses on the assessment of cardinal PD motor symptoms. Assessment of pyramidal and/or cerebellar function, eye movement, and orthostatic blood pressure determines whether further parkinsonian syndromes should be considered. Medication history will establish likely drug-induced disease, and for information about genetic causes the family history of first degree relatives is assessed. Amelioration of parkinsonism after an acute challenge dose of apomorphine, a short-acting dopamine agonist, or levodopa enhances the likelihood of a long-term diagnosis of PD.65 G e n e t i c Markers

Genetic tests for hereditary subtypes (i.e., PARK1-13) are the ideal biomarkers as they have been proven to correlate to pathopysiology, and to be highly sensitive and specific. However, the number of affected individuals is low. In V i v o Markers f r o m Pathology

Special attention has been placed on a breakdown product of central dopamine, i.e., homovanillic acid. Although CSF levels of homovanillic acid may not correlate with disease severity, the ratio of homovanillic acid/xanthine may significantly differ between patients with mild PD and controls.66,67 2-methyl6,7-dihydroxy-l,2,3,4-tetrahydroisoquinoline (2-MDTIQ), a dopamine derivate with striking similarities to MPTP, has been detected in PD but not in control CSF, and levels correlated negatively with disease duration.68 A decrease of dopamine receptors in lymphocytes has repeatedly been shown, and the decrease of the D3 mRNA expression correlated with the degree of clinical severity in PD patients.69 The development of radioligands permits the study of the dopaminergic system, which is basically affected in PD, these ligands provide powerful tools in the differential diagnostic of PD. Thanks especially to longitudinal studies on the dopamine transporters (e.g., [123I]beta-CIT, [uC]d-threo-methylphenidate-PET) and dopamine metabolism (e.g., [18F] Fluorodopa-PET) it is widely accepted that the maximal deterioration of this neurotransmitter system takes place at early or even premotor disease stages. These ligands are, in addition to clinical evaluation which is prone to bias and therefore of limited use as a "biomarker," the only established markers in PD which serve not only as trait but also as state markers. To separate PD from non-PD (including multiple system atrophy) at early stages, cardiac iodine-123 metaiodobenzylguanidine measures have been recommended.70 This tracer detects defects of the peripheral autonomic system. Lower CSF levels and higher plasma levels of alpha-synuclein have been observed in PD compared to controls, but the overlap was large and there is actually no evidence that CSF or plasma alpha-synuclein levels may have the potential to serve as biomarkers.71,72 Glutathione independent prostaglandin D synthase is very likely to play an important role in both maturation and maintenance of the central nervous system, and has been shown to be altered in CSF of PD patients.73

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Pathophysiological Mechanisms

There are, to our knowledge, so far no established markers associated with oxidative stress, mitochondrial dysfunction, excitotoxicity, and inflammation, which add any information to the current diagnosis of PD. F u r t h e r Diagnostic Assessments

Especially in younger patients, more extensive biochemical testing is indicated for exclusion of symptomatic parkinsonism, including Wilson disease. MRI is typically normal in PD patients, but should be initially assessed to exclude secondary forms of parkinsonism like normal pressure hydrocephalus and leucencephalopathy.

N O V E L BIOMARKERS The increasing prevalence of AD and PD, accompanied by the diagnostic uncertainties actually evident, motivate the drive to develop new biomarkers to reliably define the diseases and their course particularly at a very early, or even preclinical stages, and to identify the pathology associated with these disorders. A putative model of what a sufficient diagnostic procedure could look like in the future is presented in Figure 6.3. From a pathophysiological point of view, a further issue must be considered: neurodegeneration in AD and PD is very probably multifactorial, whereby several genetic and biochemical processes operate sequentially and/or in parallel, and similar pathological lesions can be the consequence of different causes and pathways. It is therefore inevitable to consider biomarkers which are capable of differentiating between the pathophysiology and/or localization of processes associated with the evolution of AD and PD. Biomarkers of high value must also act as surrogate endpoints for clinical outcomes, filling the gap for objective measurements to test new disease-modifying strategies. It is not always easy to distinguish between a "current" and a "novel" marker, and the classification may be to a certain degree subjective. We decided to include markers which may be most promising to serve as a part of future diagnostic panels.

Alzheimer's Disease C l i n i c a l Markers

The actual research focuses more on preclinical than on clinical symptoms of AD. There are hints that hyposmia, slight neuropsychological defects, and depression may be preclinical markers of AD (Figure 6.3).74-76 This is of particular interest as a combination of such markers may have the potential to serve as a relevant diagnostic markers on an individual level. This is matter of ongoing research. G e n e t i c Markers

In addition to ApoE, numerous putative genetic risk markers like monoamine oxidase A, myeloperoxidase, CYP46A1, ABCA1, alpha-1-antichymotrypsin,


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FIGURE 6.3 Neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's disease (PD) lead to a progressive loss of abilities and increase of symptoms severity. Actual clinical diagnosis is mainly restricted to delineate patients from healthy subjects (i.e., to define that "something is wrong"), and to check for relevant differential diagnoses. For this purpose, trait markers are used (e.g., clinical parameters in A D and PD, cerebrospinal fluid (CSF) Abeta and tau levels, and neuropsychology in AD). State markers define progress/velocity of the disease course (e.g., CSF phospho tau levels in AD). As both A D and PD have a preclinical stage of more than a decade duration in which neurodegeneration takes place but symptoms are notyetvisible.it is of utmost importance to define both trait (e.g., hyposmia and depression in A D and PD, hyperechogenicrty of the substantia nigra in PD) and state markers, not only for the early clinical phase but also for the preclinical phase.These markers can define people at risk for developing the disease.

and ubiquilin 1 have been proposed for AD, although all suffer inconsistent replication suggesting that modest effect sizes are likely to be the norm (for a review see reference 77). However, it can be assumed that multiple weak genetic factors, together with ApoE4, account for a relevant genetic contribution to late-onset AD risk, and it should be the aim of future research to determine a panel of certain genetic risk factors that, as a whole, has the potential to increase diagnostic accuracy. In Vivo Markers from Pathology Molecular approaches to imaging the Abeta peptide with agents like Pittsburgh Compound B are in particular promising and argue for a preclinical appearance of amyloid deposits,78 but are actually limited to research settings. Furthermore, it is not clear at present whether the diagnostic accuracy of this

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method might be better than that of the more matured FDG PET as about 20-30 percent of healthy elderly with an age of 70 years also show cortical amyloid deposition, and it is not yet clear whether all of them will develop cognitive deterioration.79 One fundamental advantage of this method is its marker of a pathophysiologically relevant mechanism, making it a promising marker for application in treatment studies to investigate amyloid-modifying strategies. Beta-site APP-cleaving enzyme 1 (BACE1) is another promising biomarker: this protein is a transmembrane aspartyl protease with all the known characteristics of APP beta-secretase. Elevated CSF BACE1 protein levels in MCI were associated with an increased risk to concert to AD, and MCI subjects showed increased levels of BACE1 activity compared to healthy controls and AD patients.80 Thus, BACE1 may be able to detect very early changes, and may therefore serve as a marker for early detection, prediction, and for progression of AD. There is accumulating evidence that plasma concentrations of 24S-hydroxycholesterol reflect a mass of metabolically active neuronal cells. This oxysterol has been used as a marker of brain atrophy in patients with AD. A small fraction is entering the CSF; and CSF levels may reflect the rate of neuronal degeneration.81 Pathophysiological Mechanisms

Markers associated with oxidative stress: CSF levels of the oxidative stress markers 8-hydroxy-2'-deoxyguanosine, a DNA oxidation product, have been shown to be dramatically increased in AD patients, with no overlap to controls.82 In addition, 3-nitrotyrosine and isoprostanes are elevated in the CSF of AD (reviewed in reference 61). Isoprostanes are peroxidation products of arachidonic acid and structural isomers of prostaglandins, and 3-nitrotyrosine formation is a central event in nitrosative stress. An increase of isoprostanes was also found in the CSF of MCI subjects compared with controls, and levels increased over time.83 Thus, CSF isoprostanes and 3-nitrotyrosine levels are likely candidates for trait markers of oxidative stress in AD, and may at least partially dispose the capability to serve as state markers. In AD, CSF levels of (E)-4-hydroxy-2-nonenal (HNE), a neurotoxic product of lipid peroxidation, may be increased and positively correlated to in CSF homocysteine levels.84 Further important antioxidants are the vitamins A, B, C, and E. Serum levels of these vitamins can be influenced by dietary habits, and a low vitamin status may give an indication of the susceptibility of a subject to oxidative damage. Decreased vitamin E levels in AD compared to controls have been shown in the CSF and serum,85 and AD patients may, as a mean, also present with decreased serum levels of vitamins A, B6, B12, and C.61 Mitochondrial markers: Abeta may not only damage cells by the generation of plaques but may also have an influence on mitochondrial function as it has been found inside the mitochondria of AD neurons.86 Vice versa, mitochondrial dysfunction enhances Abeta accumulation in the neuronal cytoplasm.87 These phenomena might contribute to a "vicious cycle" involving


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amyloid deposition, mitochondrial failure, energetic failure, functional neuronal impairment, and cell death, and CSF Abeta levels may indirectly give an estimate about mitochondrial function. Markers associated with excitotoxicity: Abeta is able to induce calcium influx by inserting into the plasma membrane and forming ion-conducting pores.88 Vice versa, increase of intracellular calcium can cause tau phosphorylation and intracellular Abeta accumulation in neurons.89 Thus, CSF Abeta and phospho tau levels may give indirect information about excititotoxic pathways as they occur in AD. Markers associated with inflammation: To date, serum alpha(l)-antichymotripsin (ACT) concentration is the most convincing marker for CNS inflammation, and higher ACT levels have been observed to correlate positively with cognitive function (reviewed in reference 61). Further Diagnostic Assessments In particular magnetic resonance imaging (MRI) is useful in the diagnosis of presymptomatic and very early clinical symptomatic stages of AD. Even in preclinical stages of AD, significant atrophy of the hippocampal formation can be demonstrated by MRI, and predicts later conversion to AD with about 80 percent accuracy.90 This method may also serve as a useable state marker as annual atrophy rates of 3 to 7 percent have been demonstrated, compared to about 1 percent in healthy elderly (reviewed in reference 91). Automated and rater-independent methods like determination of cortical thickness, deformation-based (DBM) and voxel-based morphometry (VBM) have been shown to be associated with (the development of) AD and seem to have the potential to overcome confounding effects like general brain atrophy that occurs as a part of normal aging.92 A further promising approach of structural MRI is diffusion tensor imaging which detects white matter fiber tract alterations, a process occurring early in the AD disease course.93 Functional MRI (fMRI) allows for the measurement of brain activation during cognitive tasks at a high level of resolution without radiation exposure. In AD, fMRI studies suggest that one of the first factors that might be altered is the integration across neural networks. The changes in functional connectivity have been shown to precede differences in brain activation between the MCI and healthy control group.94 Combination of Markers With a molecular test for blood plasma, 18 signaling proteins were determined which classify AD, and patients who had MCI that progressed to AD, with very high accuracy. These proteins are associated with systemic dysregulation of hematopoiesis, immune responses, apoptosis, and neuronal support.95 This is an example how effective the combination of biomarkers may be to diagnose AD and persons at risk for AD. A combination of not only a specific set of different neurochemical markers in one specific compartment (CSF, blood), but also the combination of parameters obtained from different compartments and methods seems to be

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the most promising strategy to achieve a more accurate early and differential diagnosis. First examples are given: in MCI studies it has been shown that the combined measurements of the CSF markers Abeta, total tau and phospho tau, and regional cerebral blood flow96 or mediotemporal lobe atrophy97 have higher predictive power than either diagnostic approach alone. Furthermore, such combinations enable a cross-evaluation of the markers.

Parkinson's Disease C l i n i c a l Markers

There is evidence that a number of clinical premotor markers and risk factors exist, e.g., depression, olfactory dysfunction (in more than 50 percent of subjects in the premotor phase), autonomic dysfunction (like constipation, bladder dysfunction, orthostatic hypotension), executive dysfunction, rapid eye movement (REM) sleep behavior disorder (RBD, in more than 30 percent of subjects in the premotor phase), and slight motor signs (like reduced arm swing).98 These markers can be partially assessed by medical history and clinical examination, but there is a lack of accepted recommendations on how to define and use these markers in clinical practise. Currently conducted studies define the value of combinations of these symptoms in the preclinical and early diagnosis of PD (see also Figure 6.2). G e n e t i c Markers

A genetic approach which would be usable for the majority of patients suffering from PD is the determination of disease-associated polymorphisms. Polymorphisms of alpha-synuclein, N-acetyltransferase-2, monoamine oxidase B, glutathione transferase, and the mitochondrial gene tRNAGlu have been suggested to serve as promising targets of PD diagnosis.99' 10° With the advent of high density microarrays, gene expression profiling of human SN pars compacta showed down-regulation of 68, and up-regulation of 69 genes.101 The down-regulated genes referred to pathways including signal transduction, protein degradation (e.g., ubiquitin-proteasome subunits), dopaminergic transmission/metabolism, ion transport, protein modification/phosphorylation, and energy pathways/glycolysis functional classes. The up-regulated genes referred mainly to biological processes involving cell adhesion/cytoskeleton, extracellular matrix components, cell cycle, protein modification/ phosphorylation, protein metabolism, transcription, and inflammation/stress. It is actually possible to screen in families with genetic parkinsonism for mutation carriers who have not yet developed the disease. These persons are ideal candidates to study preclinical aspects of the disease and to define preclinical markers. In Vivo Markers f r o m Pathology

Oligomeric forms of alpha-synuclein may be increased in body fluids and brain extracts.102,103 Early amyloid aggregates like oligomers are most likely


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the pathogenic aggregation components that drive neurodegeneration and neuronal cell death, rather than mature amyloid fibrils,104 and are detectable in the plasma and CSF of PD patients and controls.102 Although not yet sufficiently investigated, there is great hope that these species may serve as potent biomarkers. Pathophysiological Mechanisms Markers associated with oxidative stress: Two cross-sectional studies found increased homocysteine plasma levels in PD compared to controls, which correlated positively with disease duration.105'106 An increased level of serum uric acid, a natural antioxidant and free radical scavenger, is associated with reduced risk of PD as shown in two independent studies.107,108 In addition, serum uric acid levels were associated with disease progression, making it an attractive predictive trait marker for disease course.107 Another study showed that both CSF levels of oxidized glutathione (but not reduced gluthatione) and vitamin E were reduced in PD patients compared to controls,109 suggesting them to be, in combination with other markers, attractive variables for a comprehensive diagnostic panel. Mitochondrial markers: Complex I and IV activity may be lower in PD patients than in controls, and a negative correlation of complex I and IV activity in platelet mitochondria with disease duration has been shown at very early disease stages.110 Markers associated with excitotoxicity: Glutamate uptake in platelets from PD patients has been shown to be reduced compared to controls by about 50 percent.1" This points to the multisystem character of PD as not only the brain may suffer from excitotoxicity, giving the intriguing perspective to include different compartments and tissue components into a comprehensive diagnostic panel. Markers associated with inflammation: With the advent of 2D gel electrophoresis, different serum levels of nine complement factors were detected between PD and control samples.112 The complement system is part of the non-specific immune system. Osteopontin (OPN) is a molecule with diverse functions including modulation of inflammatory response of microglia. It is detectable in the CSF in much higher levels than in the serum, and higher CSF and serum levels have been detected in PD compared to controls.113 These data give further support to evidence suggesting that the immune system is basically involved in the pathogenesis of PD. Further Diagnostic Assessments Promising MRI protocols focus mainly on the measurement of the iron content in the SN and the basal ganglia. Using a three tesla MRI with a gradient echo sequence, a correlation between clinical (motor) symptoms and iron content of the corresponding SN has been described.114 In PD, functional imaging may be associated rather with cognitive than with motor impairment. Using a mask generated from hypometabolic areas displayed by FDG PET (this mask comprised temporo-parietal and occipital

108

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regions), only the cognitive assessment but not the motor score was significantly associated with reduced glucose utilisation.115 Cortical PIB uptake is also strongly associated with dementia in PD.116 Increased echogenicity of the SN, as determined by transcranial sonography (TCS), is characteristic of PD and can help to differentiate PD from atypical parkinsonian syndromes.1 "About 8 to 10 percent of healthy subjects also show this hyperechogenicity of the SN, and there is increasing evidence that these subjects are at increased risk for PD, thus SN hyperechogenicity may be a premotor, or a "vulnerability" marker of PD." 8 Combination of Markers With a proteomics-discovered multianalyte profile (MAP), Zhang and colleagues"9 investigated CSF from patients with probable AD and PD, and from control subjects. Using the best fitting eight proteins, MAP agreed with expert diagnosis for 95 percent of PD, 95 percent of control subjects, and 75 percent of AD. The MAP consisted of the following (in decreasing order of contribution): tau, brain-derived neurotrophic factor (BDNF), interleukin 8, Abeta42, beta2-microglobulin, vitamin D binding protein, ApoAII, and apoE. This result suggests that combinations of proteins are highly effective at identifying PD (and moderately effective at identifying AD), and points to new approaches which combine markers to reach higher predictive power than either diagnostic approach alone. Microarray studies investigating the SN of PD patients underscore the reasonability of such protocols.101

M E T H O D S T O Q U A N T I F Y BIOMARKERS A wide range of methods are in use for the detection of subtle early signs of the two prominent neurodegenerative diseases AD and PD, and to differentiate between AD/PD, and non-AD/non-PD, as well as for prediction and description of disease progression. This includes genetic, clinical, biochemical, and imaging methods. Genetic approaches include sequencing and microarray technique, and especially the analysis of gene expression profiles may have the potential to contribute relevantly to a more sophisticated diagnostic procedure in late-onset AD and PD, as the technique may soon be available broadly and at reasonable prices, and have the advantage to provide an overview about many pathophysiologically relevant processes.101 However, the donor structure of the mRNA has to be determined, but cannot be brain tissue. A promising target is skin tissue as this is relatively easy to obtain, shows histological features comparable to brain tissue,120 and is an ideal donor tissue for stem cell experiments ("induced pluripotent stem cells, IPS"). New methods arising in the biochemical field enable measurement of more than one marker at the same time, i.e., multiplex analyses based on flow cytometry. Here, up to 100 (protein) markers can be detected with a relatively small amount of body fluid. First results are promising." 9 ' m New insights into the pathophysiology of these neurodegenerative diseases have also been pro-


109

vided by results obtained with 2D gel electrophoresis, another method which enables analysis of a large number of proteins. However, methods like ELISA, western blot analysis, mass spectroscopy, high pressure liquid chromatography, electrochemical detection, and gas chromatography are still indispensable for the detection of new marker candidates as they are relatively broadly available, established, and comparably cheap. As a general remark, it must be stated that dealing with biological material has a main drawback compared to genetic (DNA), clinical, and imaging methods: assessment of samples is prone to bias and needs to be strictly standardized and optimized according to, e.g., time elapse from probe supply to freezing. Pre-analytical variability must be minimized with standardized procedures. Freezing period also influences some protein levels and is a relatively often neglected confounder in data analyses. Imaging methods may also have a great potential to refine diagnostic accuracy, especially as state markers, as they are noninvasive and can be repeatedly performed. MRI sequences enable, among others, detection of fiber tract pathologies (diffusion tensor imaging), composition of tissue (magnetic resonance imaging, gradient echo sequencing), and imaging of functional networks (functional MRI), but to date there is a lack of automated protocols which can be used in large cohorts. Radioligands and PET/SPECT provide, e.g., insights into metabolism (glucose utilisation) and pathophysiology (cholinergic system, amyloid deposition). The main drawback of these methods is that they are relatively cost-intensive and are not broadly available.

CONCLUSIONS Both neurodegenerative diseases, AD and PD, have a preclinical period of an undefined number of years, and a relentlessly progressive course. Both are not causatively treatable and suffer from a diagnostic procedure that is far from perfect. Efforts to identify biomarkers to assist with the early and differential diagnosis, as well as progression markers, are urgently needed, especially as neuroprotective therapies will hopefully soon be available. There exists a long list of promising candidate genes, proteins, and other biomaterials, and regions of interest, and there is broad acceptance that a single biomarker may not be adequate to image disease pathophysiology and progression. A panel of well-characterized biomarkers covering different pathophysiological aspects should be defined and validated in well-designed prospective studies, with the aim to serve for better diagnostic accuracy for all affected subjects and for subjects at risk.

SUMMARY POINTS 1.

Alzheimer's (AD) and Parkinson's disease (PD) are the two most common neurodegenerative diseases, and there is a strong correlation between age and the diseases' prevalence. Personal and societal burden is enormous.

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2.

3.

4.

5.

The pathophysiological mechanisms involved in AD and PD are widely comparable, and include genetic disposition (including monogenic forms), changes in neurotransmitter systems (mainly cholinergic in the former, dopaminergic in the latter disease), protein aggregation and dysfunction of elimination, oxidative stress, mitochondrial impairment (in particular in PD), and (chronic) inflammation processes. Current means for diagnosis of AD and PD under ideal conditions do only reach an accuracy of 80 to 90 percent and include an extensive medical history, neurological examination, exclusion of secondary forms of dementia/parkinsonism via imaging like MRI, and laboratory parameters (e.g., vitamin B12, folic acid, thyroid hormones). Up to now, most studies have emphasized discovery, characterization, and validation of several highly promising individual biomarkers, but their impact on different disease stages has hardly been extensively investigated. One of the primary goals of future studies on biomarkers of AD and PD should therefore be the evaluation and validation of given markers according to their impact on (a) diagnosis of subjects at risk (preclinical period), (b) differential diagnosis at early clinical stages, and (c) predication and description of disease course. Future research should also focus on the development and validation of cost-effective and broadly available high-throughput technologies for biomarker quantitation, as this seems the only way to come across the needs of highly accurate diagnosis and sufficient supervision of therapeutic strategies.

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97. 98. 99. 100. 101.

102. 103. 104. 105. 106.

107. 108.

Teipel, S. J., Stahl, R., and Dietrich, O., et al. Multivariate Network Analysis of Fiber Tract Integrity in Alzheimer's Disease. Neuroimage. Feb 1,2007;34(3): 985-995. Bokde, A. L., Lopez-Bayo, P., and Meindl, T., et al. Functional Connectivity of the Fusiform Gyrus During a Face-Matching Task in Subjects with Mild Cognitive Impairment. Brain. May 2006;129(Pt 5): 1113-1124. Ray, S., Britschgi, M., and Herbert, C , et al. Classification and Prediction of Clinical Alzheimer's Diagnosis Based on Plasma Signaling Proteins. Nat. Med. Nov2007;13(ll):1359-1362. Hansson, O., Buchhave, P., Zetterberg, H., Blennow, K., Minthon, L., and Warkentin, S. Combined Rcbf and CSF Biomarkers Predict Progression from Mild Cognitive Impairment to Alzheimer's Disease. Neurobiol. Aging. Feb 2009;30(2):165-173. Bouwman, F. H., Schoonenboom, S. N., and Van Der Flier, W. M., et al. CSF Biomarkers and Medial Temporal Lobe Atrophy Predict Dementia in Mild Cognitive Impairment. Neurobiol. Aging. Jul 2007;28(7): 1070-1074. Berg, D. Biomarkers for the Early Detection of Parkinson's and Alzheimer's Disease. Neurodegener. Dis. 2008;5(3-4): 133-136. Tan, E. K., Khajavi, M., Thornby, J. I., Nagamitsu, S., Jankovic, J., and Ashizawa, T. Variability and Validity of Polymorphism Association Studies in Parkinson's Disease. Neurology. Aug 22, 2000;55(4):533-538. Tan, E. K., Chai, A., and Teo, Y. Y., et al. Alpha-Synuclein Haplotypes Implicated in Risk of Parkinson's Disease. Neurology. Jan 13, 2004;62(1): 128-131. Grunblatt, E., Mandel, S., and Jacob-Hirsch, J., et al. Gene Expression Profiling of Parkinsonian Substantia Nigra Pars Compacta; Alterations in Ubiquitin-Proteasome, Heat Shock Protein, Iron and Oxidative Stress Regulated Proteins, Cell Adhesion/Cellular Matrix and Vesicle Trafficking Genes. J. Neural. Transm. Dec 2004;111(12):1543-1573. El-Agnaf, O. M., Salem, S. A., and Paleologou, K. E., et al. Detection of Oligomeric Forms of Alpha-Synuclein Protein in Human Plasma as a Potential Biomarker for Parkinson's Disease. Faseb. J. Mar 2006;20(3):419^25. Paleologou, K. E., Kragh, C. L., and Mann, D. M., et al. Detection of Elevated Levels of Soluble {Alpha }-Synuclein Oligomers in Post-Mortem Brain Extracts from Patients with Dementia with Lewy Bodies. Brain. Jan 20, 2009. Conway, K. A., Harper, J. D., and Lansbury, P. T., Jr. Fibrils Formed In Vitro from Alpha-Synuclein and Two Mutant Forms Linked to Parkinson's Disease are Typical Amyloid. Biochemistry. Mar 14, 2000;39(10):2552-2563. Dos Santos, E. E, Busanello, E. N., and Miglioranza, A., et al. Evidence That Folic Acid Deficiency Is a Major Determinant of Hyperhomocysteinemia in Parkinson's Disease. Metab. Brain Dis. Mar 18, 2009. Hassin-Baer, S., Cohen, O., and Vakil, E., et al. Plasma Homocysteine Levels and Parkinson Disease: Disease Progression, Carotid Intima-Media Thickness and Neuropsychiatric Complications. Clin. Neuropharmacol. Nov/Dec 2006; 29(6): 305-311. Schwarzschild, M. A., Schwid, S. R., and Marek, K., et al. Serum Urate as a Predictor of Clinical and Radiographic Progression in Parkinson's Disease. Arch. Neurol. Jun 2008;65(6):716-723. De Lau, L. M., Koudstaal, P. J., Hofman, A., and Breteler, M. M. Serum Uric Acid Levels and the Risk of Parkinson's Disease. Ann. Neurol. Nov 2005;58(5): 797-800.


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109. Tohgi, H., Abe, T., Saheki, M., Hamato, E, Sasaki, K., and Takahashi, S. Reduced and Oxidized Forms of Glutathione and Alpha-Tocopherol in the Cerebrospinal Fluid of Parkinsonian Patients: Comparison Between Before and After L-Dopa Treatment. Neurosci. Lett. Jan 16, 1995; 184(1):21-24. 110. Benecke, R., Strumper, P., and Weiss, H. Electron Transfer Complexes I and IV of Platelets are Abnormal in Parkinson's Disease but Normal in Parkinson-Plus Syndromes. Brain. Dec 1993;116 (Pt 6): 1451-1463. 111. Ferrarese, C, Tremolizzo, L., and Rigoldi, M., et al. Decreased Platelet Glutamate Uptake and Genetic Risk Factors in Patients with Parkinson's Disease. Neurol. Sci. Feb 2001;22(l):65-66. 112. Goldknopf, I. L., Sheta, E. A., and Bryson, J., et al. Complement C3c and Related Protein Biomarkers in Amyotrophic Lateral Sclerosis and Parkinson's Disease. Biochem. Biophys. Res. Commun. Apr 21, 2006;342(4): 1034-1039. 113. Maetzler, W., Berg, D., and Schalamberidze, N., et al. Osteopontin Is Elevated in Parkinson's Disease and Its Absence Leads to Reduced Neurodegeneration in the MPTP Model. Neurobiol. Dis. Mar 2007;25(3):473^182. 114. Kosta, P., Argyropoulou, M. I., Markoula, S., and Konitsiotis, S. MRI Evaluation of the Basal Ganglia Size and Iron Content in Patients with Parkinson's Disease. J. Neurol. Jan 2006;253(l):26-32. 115. Liepelt, I., Reimold, M., and Maetzler, W., et al. Cortical Hypometabolism Assessed by a Metabolic Ratio in Parkinson's Disease Primarily Reflects Cognitive Deterioration—[18F]FDG-PET. Mov. Disord. 2009;Accepted. 116. Maetzler, W., Liepelt, I., and Reimold, M., et al. Cortical PIB Binding In Lewy Body Disease Is Associated with Alzheimer-like Characteristics. Neurobiol. Dis. Apr 2009;34(1):107-112. 117. Gaenslen, A., Unmuth, B., and Godau, J., et al. The Specificity and Sensitivity of Transcranial Ultrasound in the Differential Diagnosis of Parkinson's Disease: A Prospective Blinded Study. Lancet Neurol. May 2008;7(5):417^t24. 118. Berg, D. Transcranial Sonography in the Early and Differential Diagnosis of Parkinson's Disease. J. Neural. Transm. Suppl. 2006;(70):249-254. 119. Zhang, J., Sokal, I., and Peskind, E. R., et al. CSF Multianalyte Profile Distinguishes Alzheimer's and Parkinson's Diseases. Am. J. Clin. Pathol. Apr 2008; 129(4):526-529. 120. Ikemura, M., Saito, Y, and Sengoku, R., et al. Lewy Body Pathology Involves Cutaneous Nerves. J. Neuropathol. Exp. Neurol. Oct 2008;67(10):945-953. 121. Lewczuk, P., Kornhuber, J., and Vanderstichele, H., et al. Multiplexed Quantification of Dementia Biomarkers in the CSF of Patients with Early Dementias and MCI: A Multicenter Study. Neurobiol. Aging. Jun 2008;29(6):812-818.


CHAPTER

BIOMARKERS OF CARDIAC INJURY Anthony S. McLean and Stephen J. Huang

INTRODUCTION D e f i n i t i o n and Prevalence Although the term "cardiac injury" is used widely in cardiology, it is not well defined clinically. The term is used broadly to denote some form of cardiac insult, which may encompass myocardial cell injury and/or cell death (autophagy, apoptosis or necrosis). The causes of cardiac injury are multifarious, including ischemia, direct trauma to the heart,' drug-induced myocardial toxicity,2 myocardial depression as a result of severe sepsis, viral myocarditis, end-stage renal failure,4 and increased wall stress from congestive heart failure and pulmonary embolism.5,6 However, amongst all these, myocardial ischemia is the most prevalent cause of cardiac injury and commonly the term "cardiac injury" is used interchangeably with "myocardial infarction" (MI) or "acute coronary syndrome" (ACS). Pathologically, myocardial infarction (MI) is defined as myocardial cell death due to prolonged ischemia. Clinically however, the definition of MI has been changing over time and is inseparable from the development of cardiac biomarkers. Before the era of cardiac biomarkers, the clinical definition of MI was inconsistent and relied solely on clinical signs, ECG, and symptoms, which have poor sensitivities and specificities. In 1979, the World Health Organization (WHO) made the first attempt to unify the definition of acute myocardial infarction (AMI) globally based on history (or symptoms), ECG, and serum cardiospecific enzymes, including creatine kinase (CK), creatine kinase-MB isozyme (CK-MB), lactate dehydrogenase, and aspartate aminotransferase (AST) (Table 7.1).7 Subsequently however, in view of the inconsistent criteria 119

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used for denning AMI in various studies, the WHO Multinational Monitoring of Trends and Determinants in Cardiovascular Diseases (MONICA) code was introduced in 1984 for epidemiological purposes.8 Although ECG and symptom of chest pain remained as criteria in the MONICA code, they provided no prognostic value whatsoever. On the other hand, CK-MB was the strongest predictor of one-year and five-year mortality.9 Further, using the enzyme profiles provided by the WHO definition or the WHO-MONICA code resulted in a higher false positive rate of definite MI when compared to other criteria."^12 The seed for a redefinition of MI was thus planted. The discovery of troponin and its subsequent proven correlation with histological myocardial necrosis in the 1990s accelerated the redefinition process.13,14 In 2000, the criteria for the diagnosis of MI were redefined by a consensus group of the European Society of Cardiology and the American College of Cardiology (ESC/ACC).15 The new criteria represented a paradigm shift in the thinking process: instead of the two out of three criteria used in the WHO definition, the new definition requires specifically the rise and fall of cardiac biomarkers, preferably troponin, plus symptoms or ECG changes (Table 7.1). Since then, the role of biomarkers has been lifted to a new high level, making their measurements mandatory in the diagnosis of MI. Not surprisingly, the new definition has identified more patients with MI compared with the WHO criteria. Studies comparing the identification of patients with MI using the WHO and the ESC/ACC 2000 criteria clearly demonstrated that the latter was more sensitive and identified more patients with MI.16," However, these studies also found the ESC/ACC criteria were not universally accepted for the diagnosis of MI. Several concerns were expressed about the new definition, including: excessive reliance on troponins, insufficient attention to ECG changes, failure to identify early fatal cases in the first few hours after the onset of MI before there is time for troponins to be released, as well as non-fatal cases where troponin tests were unavailable, and the inability to maintain the consistency and comparability of historical (trend) epidemiological data.18-19 In view of the above concerns, new case definitions were published as an American Heart Association Scientific Statement in 2003. Following that, a universal definition of acute MI was released by the Joint European Society of Cardiology/American College of Cardiology Foundation/American Heart Association/World Health Federation Task Force in 2007.20 There were several major advancements made in the new universal definition. First, the new universal definition made it clear that the term myocardial infarction should only be used in the setting of myocardial ischemia and not from any other cause. Secondly, it designated different classes of myocardial infarction (Table 7.2). Thirdly, it re-emphasizes the role of troponin in the diagnosis of MI, recommending a more stringent cut-off value (99th percentile of upper range limit). Fourthly, while cardiac imaging was mentioned in the 2000 definition, it is only in the 2007 universal definition that cardiac imaging is included as one of the inclusion criteria. The 2007 universal definition will most certainly increase the sensitivity and specificity of the diagnosis, and the new classification will help clinicians manage patients with different classes of MI.

BIOMARKERS OF CARDIAC INJURY

TABLE 7.1

Year

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The evolution of the definition of myocardial infarction since 1979.

Authority

Criteria

1979

World Health Organization (WHO)

Any two of the following three: 1. History of chest pain or any typical symptoms. 2. A typical ECG pattern with the development of Q waves. 3. Unequivocal change in serum enzymes (CK, CKMB, LD, or AST).

1984

WHO-MONICA code

1. Evolving diagnostic ECG; and/or 2. Diagnostic ECG and abnormal enzymes; and/or 3. Prolonged cardiac pain and abnormal enzymes.

2000

ESC/ACC

Either one of the following: 1. Typical rise and fall of troponin (or CK-MB, if troponin is not available) plus one of the following: • Ischemic symptoms; • Development of pathologic Q waves on the ECG; • ECG changes indicative of ischemia (ST segment changes); or • Coronary artery intervention. 2. Pathological findings of an acute Ml.

2007

ESC/ACCF/AHA/WHF

Any one of the following: 1. Typical rise and fall of troponin with at least one value above the 99th percentile of the URL, plus one of the following: • Ischemic symptoms; • Development of pathologic Q waves on the ECG; • ECG changes indicative of ischemia (ST segment changes or new LBBB); or • Imaging evidence. 2. Sudden, unexpected cardiac death before blood samples can be obtained or before biomarkers can appear in the blood, and accompanied by evidence of symptoms, ECG, coronary angiogram or autopsy. 3. For PCI patients with normal baseline troponin levels, troponin > 99th percentile of URL are indicative or peri-procedural myocardial necrosis. 4. For CABG patients with normal baseline troponin > 99th percentile of URL are indicative or peri-procedural myocardial necrosis. 5. Pathological findings of an acute Ml.

ACC: American College of Cardiology: ACCF: American College of Cardiology Foundation: AHA: American Heart Association; AST: aspartate aminotransferase; CK: Creatine kinase; CK-MB: creatine kinase-MB isozyme; ECG: electrocardiogram; ESC: European Society of Cardiology; LD: lactate dehydrogenase; LBBB: left bundle branch block; WHF: World Heart Federation.

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TABLE 7.2 Clinical classification of different types of myocardial infarction according to the 2007 universal definition.

Type 1

Spontaneous Ml related to ischemia due to a primary coronary event such as plaque erosion and/or rupture, Assuring, or dissection.

Type 2

Ml secondary to ischemia due to either increased oxygen demand or decreased supply, e.g., coronary artery spasm, coronary embolism, anaemia, arrhythmias, hypertension, or hypotension.

Type 3

Sudden unexpected cardiac death, including cardiac arrest, often with symptoms suggestive or Ml, accompanied by presumably new ST elevation, or new LBBB or evidence of fresh thrombus in a coronary artery by angiography and/ or at autopsy, but death occurring before blood samples could be obtained, or at a time before the appearance of cardiac biomarkers in the blood.

Type 4a

Ml associated with PCI.

Type 4b

Ml associated with stent thrombosis as documented by angiography or at autopsy.

Type 5

Ml infarction associated with CABG.

CABG: Coronary artery bypass graft sugery: LBBB: left bundle branch block; Ml: myocardial infarction: PCI: percutaneous coronary interventions.

Maintaining the consistency and comparability of historical data is a difficult task in epidemiological studies because of "diagnostic drift," which is largely driven by the availability of new cardiospecific biochemical markers. Examining a population-based MI registry data from 1997 to 2002, the 2003 definition, which included the use of troponins, identified 83% more definite Mis than the WHO-MONICA code.21 The Minnesota Heart Survey compared the incidence of MI for 1970 and 1980 with and without incorporating cardiospecific enzymes (CK or CK-MB) in the diagnostic criteria. When cardiospecific enzymes were not used, the incidences of definite MI were similar in 1970 and 1980 (0.174% vs 0.180%) regardless of whether or not autopsy findings were included in the algorithm. Inclusion of CK or CK-MB to the algorithm increased the rate of definite MI from 0.209% in 1970 to 0.277% in 1980.22 Examination of the Framingham Heart Study participants over four decades (1960 to 1999) revealed that the incidence rates of ECG-confirmed acute MI declined by approximately 50%, whereas the biomarkers-confirmed incidence increased by two-fold.23 While the decline in ECG-based diagnosis could be explained by improvements in primary prevention or intervention over that last few decades, as evident by a similar fall in MI case fatality, the increase in biomarker-based diagnosis confirmed the improved sensitivity of the diagnosis process. Despite the difficulties in defining MI, the Heart Disease and Stroke Statistics—2008 Update revealed that about one in three American adults have one or more types of cardiovascular disease. About 16 million Americans have coronary artery disease (CAD), of which 8.1 million had experienced an MI.


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The estimated annual incidence of MI is 600,000 new attacks and 320,000 recurrent attacks. In 2004, of the 451,326 deaths caused by CAD in the U.S., about one in five of all deaths, MI accounted for 156,816 deaths.24

Pathophysiology and Mechanisms The 2007 universal definition only confined its definition of MI to ischemic causes. In addition to coronary events, increases in myocardial oxygen demand or decreases in oxygen supply also constitute "ischemia" under the new definition. Hence, coronary artery spasm, coronary embolism, anaemia, arrhythmia, and hypoperfusion can all come under the ambit of "ischemia." The universal definition explicitly excludes other causes of MI, such as myocardial cell death associated with mechanical injury, renal failure, heart failure, cardioversion, sepsis, myocarditis, cardiac toxins, and infiltrative disease, although these can all lead to cardiac injury.20 CAD accounted for more than half of all cardiovascular events in Americans < 75 years of age and is the major cause of MI.24 CAD, which has previously been considered as a cholesterol disorder, is now recognized as an inflammatory disease. The cornerstone of CAD is the series of inflammatory responses triggered by various proinflammatory factors such as hypercholesterolemia, obesity, hyperglycemia, hypertension, and smoking. In hypercholesterolemia, the infiltration and retention of modified (oxidized) low-density lipoprotein (LDL) in the arterial intima leads to the release of phospholipids that can activate endothelial cells in large and medium-sized arteries.25 Segments of arteries exposed to hemodynamic strain are prone to develop atherosclerosis and have a higher endothelial cell expression of adhesion molecules and inflammatory genes.26 Adhesion of the platelets to the activated sites further enhances endothelial activation.27 The expression of leukocyte adhesion molecules is an important step in atherogenesis. In hypercholesterolemia, activated endothelial cells express vascular-cell adhesion molecule 1 (VCAM-1) which attracts monocytes and T lymphocytes to these sites.28 Once attached to the endothelium, these leukocytes transmigrate through the endothelial junction into the subendothelial layer in response to cytokines produced in this layer. The monocytes, now differentiated into macrophages under the effects of intima-released macrophage colony stimulating factor, will express scavenger receptor and start to internalize a wide range of molecules and particles, including bacterial endotoxin, apoptotic cell fragments, and oxidized LDL.29'30 The accumulation of cholesteryl esters inside the macrophages results in the formation of cytosolic lipid droplets and these macrophage acquire the foamylike appearance—and hence the name "foam cells." Also by upregulating the toll-like receptors, the macrophages will be activated by binding pathogen-like molecules, and produce inflammatory cytokines (tumor necrosis factor and interleukin 1), proteases, and cytotoxic oxygen radicals.31 Bacterial toxins, stress proteins, DNA motifs, heat-shock protein 60, and oxidized LDL can all activate the toll-like receptors.32 The T lymphocytes transform into T-helper 1 (Thl) effector cells after exposure to various antigens, including oxidized lipids, heat-shock protein 60, viral antigens, and lipid antigens.33 Activated

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Thl cells produce interferon-y that augments the production of inflammatory cytokines (tumor necrosis factor and interleukin-1) from macrophage.34 As the inflammatory process advances silently in the early stage, smooth muscles cells undergo a phenotypic modulation from a contractile, non-proliferative state to an activated proliferative and migratory state in response to growth factors.35 They make their way through to the intima from the tunica media. Acting in concert with endothelial cells and macrophages, the smooth muscle cells secrete matrix metalloproteinases (MMPs) in response to interleukin-18, a cytokine produced by macrophage and the endothelial cells.36 MMPs serve numerous functions in the vascular wall, including activation, migration, and cell death, as well as new vessel formation, remodelling, and destruction of extracellular matrix.37 As the inflammatory lesion progresses, calcification ensues.38 Cell death is common in established atherosclerotic lesions.39 The death of foam cells leads to accumulation of the extracellular lipid in the intima which coalesce to form the classic, lipid-rich necrotic core of the atherosclerotic plaque. The mature atherosclerotic plaque consists of a lipid-rich core separated from the vessel lumen by a fibrous cap composed of vascular smooth muscle cells, collagen, and extracellular matrix. Apoptosis of the smooth muscle cells leads to plaque destabilization and rupture, and the apoptotic process is promoted by various factors, including inflammatory cytokines (e.g., tumor necrosis factor) and proteases (MMPs and cysteine proteases) released by activated macrophage and oxidized LDL.40 Rupture of the fibrous cap exposes the underlying collagen, resulting in platelet activation, thrombosis, and potential occlusion leading to ischemia. While rupture is widely recognized as the main cause of MI, it only accounts for 40% to 65% of Mis.4' It is now believed that some of the episodes of MI could be due to massive endothelial cell apoptosis inducing thrombus formation.42 A number of pro-atherosclerotic factors can induce endothelial cell apoptosis, including elevated glucose concentration, oxidized LDL, and reactive oxygen radicals.43 MI is considered as necrotic myocardial cell death due to prolonged ischemia. Myocardial cell apoptosis is the first event in response to ischemia. It is characterized by cell shrinkage and aggregation of chromosomal DNA into small masses and preparation for exocytosis (seen as membrane blebbing under the microscope). Apoptotic bodies are rapidly removed by macrophages resulting in minimal inflammatory response.44 As apoptosis is an energy requiring process and, if energy (oxygen) supply fails to meet the demand, apoptosis will be stalled and followed by necrosis. Timely interventions aim to restore perfusion and significantly reduce infarct size and survival, possibly allowing apoptosis to complete.45 If necrosis prevails, the infarct areas are infiltrated by inflammatory neutrophils and macrophages. The release of inflammatory cytokines and proteases by these cells leads to inflammatory reactions as well as further cell loss and connective tissue disruption. In acute MI, the necrotic area is found predominantly at the center of the lesion and the apoptotic areas sandwiched between the necrotic and healthy tissues.46 MI is not the only form of cardiac injury (Figure 7.1). Cell death (apoptosis) is also a feature of cardiomyopathy, and contributes to the deteriorating


FIGURE 7.1

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Common causes of cardiac injury.

cardiac function observed.47"19 In the decompensated state of heart failure, volume overload is thought to be a causative factor of apoptosis.50 Other proapoptotic factors in heart failure include pressure overload, elevated angiotensin II, and catecholamine overproduction.51-53 Proinflammatory cytokines are also involved in heart failure. It is hypothesized that precipitating events, such as ischemic heart injury, trigger a series of inflammatory responses and the expressions of cytokines in the myocardium.54 These cytokines are associated with deleterious effects on the ventricular function and accelerate progression of heart failure.55 Heart failure aside, certain drugs and agents are also capable of inducing apoptosis and myocardial dysfunction, including doxorubicin,56 anthracyclin,57 arsenic trioxide,58 cyclophosphamide,59 alcohol,60 and bacterial toxins.61 Inhibition of the apoptotic mediator, caspase, reduces apoptosis and improves cardiac functions in various models.62

DIAGNOSIS The accuracy and reproducibility of a diagnosis depend on the clarity and validity of the definition of the disease. Prior to the introduction of cardiac biomarkers, the diagnosis of MI relied on clinical history, symptoms, and ECG changes. The most recent consensus for MI diagnosis is listed in Table 7.1.

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BIOMARKERS

Although ECG is more objective than clinical history and symptoms, its value in the diagnosis of MI is questionable in that it lacks sensitivity and specificjty 63-65 fjjg di a g nos tic accuracy of the ECG depends upon the extent of myocardial necrosis and its localization. For example, while it is most sensitive in patients with occlusion of the left anterior descending artery, its sensitivity is only about 50% in detecting left circumflex occlusion.66 Furthermore, ECG diagnosis of acute MI is difficult in situations where the electroconduction pattern is altered, e.g., left bundle branch block and ventricular pacing, and often leads to missed diagnosis.67-68 Over the past 50 years, since the discovery of the release of aspartate aminotransferance (AST) by necrotic myocardial cells,69 biomarkers have assumed a crucial role in the diagnosis of MI.70 Biomarkers have substantially increased the detection of acute MI cases due to an improvement in sensitivity. The first generation of biomarkers for MI, namely AST and lactate dehydrogenase, suffer from poor cardiospecificity.69-71 It is now known that both enzymes are present ubiquitously in the body, most notably in the liver and red blood cells.7273 This lack of specificity renders these enzymes of little diagnostic value for acute MI.74-75 Creatine kinase (CK), due to its rapid appearance in serum after acute MI and its specificity for MI, soon became the choice of cardiac biomarker for MI in the 1970s.76 Although CK has superior specificity to AST and lactate dehydrogenase, a high level of CK is found in striated muscle and the serum CK level is subject to muscle injury.77 The false positive rate remains high even when the more cardiospecific isoform of CK, CK-MB, is used.78 Cardiac troponins, since endorsement in 2000 by the MI diagnosis consensus document, remain the mainstay for the diagnosis of MI despite the troponin levels being increased in various noncardiac pathological states.15 A distinction should be made between MI and other cardiac injuries when applying cardiac biomarkers. While MI and cardiac injury may both result in the elevation of cardiac biomarkers (often the same biomarkers) the two conditions are not synonymous. Etiologically, MI is the result of ischemia and cardiac injury can be due to various causes. In terms of biomarkers, the outcomes are similar—myocardial cell injury or cell death resulting in the release of intracellular cardiospecific contents (biomarkers). Therefore, the meaning of "specificity" in cardiac biomarkers may bear two different meanings: tissue specificity (specific to myocardial tissues), and disease specificity (specific to the type of cardiac disease). Much effort in the past decades has been invested to address these two issues of biomarkers.

BIOMARKERS OF C A R D I A C INJURY The applications of biomarkers fall into four main categories: screening, diagnostic, monitoring disease progression and guiding therapy, and prognostication. Depending on the use, the requirements for the biomarkers can be different. For example, while sensitivity and specificity are important characteristics for screening and diagnosis, they are less important for monitoring purpose. The desirable properties for an ideal cardiac biomarker intending to be used


127

for diagnostic purpose are listed in Table 7.3.79 The etiological factors and the evolution of the disease are different for different cardiac disease, and hence to exhaustively list out all the cardiac biomarkers relating to different types of cardiac disease is impossible. The evolution of most cardiac diseases can be divided into three stages: Inflammation, myocardial cells injury or damage, and cardiac stress. While these stages are continuous, there is no clear demarcation for each stage and they may occur concomitantly. This chapter will discuss different cardiac biomarkers by grouping them into three categories: inflammatory markers, markers for cardiac injury, and for cardiac stress (Figure 7.2).

I n f l a m m a t o r y Markers of Cardiac Disease Inflammation plays a key role in CAD.80 All stages of plaque development and eventual rupture leading to ACS can be considered as inflammatory response.81 Inflammation also plays an important role in heart failure and myocarditis. 55,82,83 The detection of key inflammatory molecules or cytokines hence offers an attractive approach for detecting cardiac ischemia, heart failure, and predicting outcomes.84 C-Reactive Protein (CRP) CRP is produced mainly in the liver as an acute-phase reactant and is transcriptionally driven by interleukin-6. Recent data suggest that CRP is also produced locally in the atherosclerotic plaque especially by smooth muscle cells and macrophages, and is believed to have a direct role in the pathophysiology of atherosclerosis.85~87 CRP enhances macrophage uptake of low density lipoprotein and contributes to foam cell formation. It also causes plaque insta-

TABLE 7.3

Characteristics of an ideal cardiac biomarker

Sensitivity

High sensitivity to avoid missing diagnosis High concentration in myocardium Released rapidly for early diagnosis Reasonable half-life to permit adequate window for diagnosis

Specificity

Specific to myocardial tissue Specific to cardiac disease Specific to the type of cardiac disease

Assay

Accurate Reproducible Simple to perform and readily available Rapid turnaround time Favourable cost-benefit ratio Diagnostic cut-off well-defined

Other

Plasma or serum levels proportional to injury size

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BIOMARKERS

FIGURE 7.2 The evolution of most cardiac diseases involves three stages: I) inflammation, 2) acute injury to myocardial cells, and 3) cardiac stress where the myocardium is subjected to pressure and/or volume overload.The myocardium responds to each stage by releasing different biomarkers.While these stages can be identified by various means, co-existence of these stages is to be expected. By measuring the concentrations (or relative concentrations) of these biomarkers at the same time, it is possible to identify the stage in addition to the type of cardiac disease. CRP: C-reactive protein; IL: interleukins;TNF: tumor necrosis factor; Fas: a member of theTNF-receptor family; MMP: matrix metalloproteinase; MPO: myeloperoxidase; CK-MB: creatine kinase-MB isozyme; cTn: cardiac troponin; H-FABP: heart-type fatty acid binding protein; BNP: B-type natriuretic peptide; NT-proBNP: N-terminal proBNP; ST2: a member of the IL-I receptor family and binds IL-33.

bility, induces adhesion molecule expression, and is associated with endothelia dysfunction.88'89 CRP was elevated only in patients with unstable angina and not those with variant angina caused by vasospasm, indicating that CRP is associated with inflammation in coronary artery rather than in the ischemic myocardium.90 CRP was also increased in other inflammatory conditions such as acute injury, infection, and chronic renal failure.91,92 CRP was also elevated in patients with heart failure and was first detected in the 1950s.93 Higher levels of CRP were found in patients with more severe heart failure. High CRP concentrations are associated with increased incidents of cardiovascular disease, and have similar magnitude as other risk factors such as LDL cholesterol, systolic blood pressure, and smoking.94 Inclusion of CRP into risk classification procedures provides more accurate assessments of


129

cardiovascular disease risk.95'% High levels of CRP in unstable angina were associated with worsening outcome.97 Data from the JUPITER trial demonstrated that CRP can be used to target high risk patients who have typical LDL cholesterol and benefit from statin therapy. The study found that patients who achieved LDL cholesterol concentration of < 70 mg/dl (1.81 mmol/1) and CRP < 2 mg/L had the best long-term clinical outcomes.98 CRP is now a recognized independent marker of cardiovascular risk. The recommended cut-offs in clinical practice are < 1 mg/L for low-risk and > 3 mg/L for high-risk individuals.99 In heart failure, CRP is an independent predictor of adverse outcome in acute or chronic heart failure, and can predict the risk of future development of heart failure in asymptomatic older subjects.82,10° I n t e r l e u k i n s (IL)

IL-6, a pro-inflammatory cytokine produced by the macrophage in the atherosclerotic plaques, induces hepatic synthesis of all the acute phase proteins, including CRP.81,101 Elevated IL-6 (a 5 ng/ml) was associated with a 3.5-fold increase in one-year mortality in patients with ACS.9'102 IL-6 was also a predictor of mortality independent of troponin T and CRP. Healthy individuals with high IL-6 also had an increased risk for future myocardial infarction.103 IL-18 is also a pro-inflammatory cytokine that is highly expressed in atherosclerotic plaques (macrophages). Significantly higher levels of IL-18 mRNA were found in symptomatic (unstable) plaque than asymptomatic (stable) plaque, suggesting IL-18 destabilizes atherosclerotic plaque leading to ischemic syndromes.104,105 Adipocytes from obese patients secret a significant amount of IL-18, three-fold more than the non-obese counterparts, supporting the notion that adipocytes participate in innate immunity and that IL-18 contributes to the risks of development cardiovascular disease and type 2 diabetes.106 IL-18 was a strong predictor of death from cardiovascular causes in patients with CAD.107 Due to its high level in HF, IL-18 is not suitable for selectively diagnosing ischemic heart disease. IL-6 and IL-18 are also produced by nucleated cells in the heart and are significantly elevated in heart failure.108,109 Clinically, the peak IL-6 level correlates with the severity of decompensated heart failure. When compared to age-matched non-cardiac patients, IL-6 levels in acutely decompensated patients peaked at 12 hours and declined thereafter. The peak IL-6 levels significantly correlated with pulmonary artery wedge pressure on admission, and were higher in patients requiring mechanical ventilation.110 Treatment of acutely decompensated HF patients with levosimendan resulted in a decrease in IL-6 levels.1" IL-6 predicts one-year mortality and can identify patients at high risk for worsening of heart failure.112' " 3 T u m o r N e c r o s i s Factor ( T N F ) and Fas

TNF-ct levels are elevated in patients with HF.114TNF-a causes left ventricular dilatation, presumably via activation of matrix metalloproteinases (see be-

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low).115 High TNF-a levels were associated with higher incidence of HF in asymptomatic elderly subjects.116 Fas is a member of the TNF-a receptor family that is expressed on a variety of cell types, including cardiomyocytes. Fas is responsible for mediating apoptosis and plays a key role in the development of and progression of HF.117 On-going myocardial damage is related to activated TNF and the Fas system in patients with worsening heart failure.118 The soluble form (serum) of Fas is increased in patients with HF and the concentration is proportional to the severity of the disease."9 In congestive HF, soluble Fas levels were found to be a useful prognostic factor for 48-month mortality independent of neurohormonal factors.120 A recent genetic study found that a single nucleotide polymorphism (SNP) in the Fas promoter region (-670G/A) is associated with excess apoptosis of vascular smooth muscle cells in the atherosclerotic lesion and may be a risk factor for MI occurrence.121 CD40 Ligand Both CD40 and soluble CD40 ligand (sCD40L) are expressed by vascular cells and macrophages. Platelets also express CD40 ligands.122 CD40 promotes degradation of fibrous caps of atherosclerotic plaques and tissue factor production by stimulating macrophages.123 sCD40L is increased in patients with stable CAD documented by angiography.124 However, sCD40L does not identify subclinical atherosclerosis in the general population.125 Plasma sCD40L levels were higher among patients with evidence of intraplaque lipid in the carotid artery than among those without it.126 High plasma sCD40L levels are associated with increased cardiovascular risk in healthy women. When controlled for age and smoking history, patients with higher sCD40L concentrations have higher risks of MI, stroke, or cardiovascular death during a four-year follow-up.127 Examination of the data from the OPUS-IMI 16 trial demonstrated that patients with the highest levels of both sCD40L and cardiac troponin had a significantly higher cardiovascular risk (12:1) compared with patients with the lowest levels of both markers.128 Matrix Metalloproteinases (MMPs) MMPs are a family of proteinases that facilitate the degradation and reorganization of the extracellular matrix, and play a crucial role in the pathology of atherosclerosis and vascular disease.129 MMPs regulate various biological activities by activating mediators like TNF-a, growth factors and their receptors, plasminogen and its activators, and endothelin.129 The circulating concentration of one family member of the MMPs, MMP-9, is increased in patients with CAD.130 Plasma MMP-9 levels are transiently increased to two- to three-fold above normal during acute MI. The levels returned back to the control range within a week, suggesting an active role for MMP-9 in plaque rupture.131 Despite its roles in atherosclerosis, circulating MMP-9 concentrations are not associated with the severity of coronary stenosis as defined by angiography or with carotid atherosclerosis.130,132 However, MMP-9 correlates with cardiovascular risk as estimated by the Framingham


131

risk score.'33 Increased in MMP-9 in subjects with a 50% carotid stenosis is associated with a two-fold increased risk of stroke or cardiovascular death.'34 As MMPs are affected by various comorbidities and drug therapies, diagnostic application MMPs is limited.135 Myeloperoxidase (MPO) MPO is a pro-inflammatory enzyme involved in LDL cholesterol oxidation and nitric oxide scavenging. It is mainly released by activated neutrophils, and its activity is abundant within atherosclerotic plaques.136 Interestingly, MPO activity was found to be lower in circulating neutrophils, but higher in circulation, in patients with acute MI and unstable angina as compared with those with chronic stable angina.137 This is indicative of their active release by neutrophils under these conditions, possibly under the stimulation of CRP.138 Plasma MPO levels were significantly elevated within two hours after the onset of symptoms (chest pain) and return to baseline levels within one week, including those with MI.137,139 This has significant advantage over troponin T which takes three to six hours to rise to measurable circulating levels after MI, suggesting MPO may be useful in triage and as a marker of unstable angina preceding MI—a predictor of vulnerable plaque. As the circulating MPO levels did not correlate with CK-MB and troponin (markers of myocardial damage), MPO may assume a role as a marker of instability and not simply a marker of oxidative stress and damage.137 MPO can be used for risk stratification for CAD. Compared to normal controls, blood and leukocyte MPO activity were higher in patients with CAD and in those apparently healthy but who developed CAD during an eightyear follow-up study.140,141 In the CAPTURE trial involving 1090 patients with ACS, serum MPO concentrations correlated with rates of death and MI, even in patients with undetectable cardiac troponin T (< 0.01 |xg/ml).142 A cut-off of 350 (ig/L was associated with an adjusted hazard ratio of 2.25 (95% CI, 1.32-3.82). In the emergency setting, MPO predicted the risk of MI in patients with chest pain even in the absence of cardiac necrosis.139 Plasma MPO was also significantly elevated in HF patients and was associated with worsening conditions.143144 However, MPO was not predictive of acute decompensated heart failure in patients presented with dyspnea in the emergency department.145 It is apparent that increased MPO is unlikely to be specific for cardiac diseases. Any disorder associated with dyspnea is sufficient to stimulate MPO release, as can activation of neutrophils and macrophages in any infections or inflammatory processes.146

Markers f o r Myocardial Cell Injury Creatine Kinase-Myocardial Band (CK-MB) Myocardial injury leads to the release of specific cytosolic substances which can be used as a marker for the injury. CK-MB is an enzyme present primarily in cardiac muscles and its intracellular level may increase in response to pres-

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BIOMARKERS

sure overload or ischemia.147'I48 The enzyme is released rapidly (within four to six h) into the circulation after the onset of MI. It peaks at 24 h, and returns to normal by 36 to 72 h.149 While total CK is a sensitive marker of MI, it is clear from autopsy series and myocardial biopsy performed during coronary bypass surgery that CK-MB does not detect all myocardial necrosis in patients with suspected coronary ischemia.150'151 Further, increases in CK-MB do not occur in most patients with severe unstable angina.152 Since plasma CK-MB rise at about six hours after onset of symptoms, a single measurement on presentation has low sensitivity. Serial measurements over three hours in patients with nondiagnostic ECGs provides a better sensitivity (90%) and is more sensitive than serial ECG in patients with non-ST segment elevation MI (NSTEMI).153'154 Due to the transient change in concentrations, CK-MB cannot be used for late diagnosis of acute MI. However, a re-elevation of plasma levels may suggest infarct extension or re-infarction.155 Lack of specificity is also an issue for CK-MB. In patients with chest pain symptoms, about 10% had elevated CK-MB but normal troponin.156 Although CK-MB is present in small amounts (1-3% of total CK) in skeletal muscle, skeletal muscle injury can increase circulating CK-MB levels.157 In response to skeletal muscle damage, there is a re-expression of proteins that existed during ontogeny, resulting in excessive production of the CK-MB isozyme.158 Sufficient CK-MB can be released from damaged skeletal muscle to increase circulating levels, confusing the diagnosis of acute MI. Increased circulating CK-MB has been observed in patients who underwent surgery, with cardiac contusion after chest wall trauma and electrical injury.159'160 CK-MB is released for a longer period of time in response to skeletal muscle injury, and plasma levels decline more slowly than after an acute MI. Perhaps this prolonged appearance of plasma CK-MB can be used to differentiate injuries which are of myocardial or skeletal muscle origin. Serial CK-MB measurements have a reasonable sensitivity and specificity for diagnosing patients with ACS.156 However, CK-MB is less sensitive than troponin. It is not recommended as the first line of biomarkers for MI unless troponin is not available.20,161 In patients undergoing percutaneous coronary intervention, elevation in baseline CK-MB has no long-term prognostic value.162 Troponins (cTn) The troponins, troponin T and I, are part of the contractile apparatus of striated muscle, including cardiac myocytes. Cardiac troponins T (cTnT) and I (cTnl) are the most specific and sensitive markers of myocardial injury, and there is no clinical difference between cTnT and cTnl for diagnosing cardiac necrosis.163 The trigger for cTn release is necrosis, and cTn assays can detect as little as 1 g of myocardial necrosis.161 In myocardial injury where the cell membrane allows the escape of intracellular proteins, cTn begins to increase within two to four h after onset of symptoms, and remains elevated for 7 to 14 days depending on the extent of injury and reperfusion status. Early release in acute MI is attributable to the cytosolic pool, with subsequent release to the structural pool following degradation of the actin and myosin filaments


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in the area of damage. The latter degradation and release is responsible for the prolonged release. The transient increase is an important observation of acute injury insult. The lack of a "rise and fall" pattern often suggests false positives. There were concerns about the cardiospecificity of cTnT, especially in patients with renal failure and skeletal muscle disease.164 Subsequent studies on skeletal muscle from such patients established that isoforms of cTnT that are re-expressed in response to injury are not detected with the second- and third generations of cTnT assays.165 The cTnT are therefore highly specific for cardiac injury. When both skeletal muscle and cardiac injury are present, the improved specificity reduces the number of false-positives while maintaining high sensitivity. On the other hand, cTnl was not expressed in the skeletal muscles at any stage of neonatal development or during regenerative muscle disease processes like polymyositis and Duchenne muscular dystrophy.166 The ESC/ACC 2000 consensus recommends that cTn is the preferred cardiac marker for MI, and the upper limit be defined as the 99th percentile.167 The sensitivities and specificities for cTnl and cTnT measured at four to eight hours or beyond four hours are about 90%.168'169 As expected, the sensitivity for cTnT in early diagnosis (< six hours) is poor (about 55%) but the specificity remains high (> 90%).169' 17° Clinically, only the measurement of cTn is able to distinguish patients with unstable angina from those with NSTEMI.171 However, while increased cTn always indicates myocardial tissue damage, a positive test is unable to suggest the type of cardiac injury or the mechanism responsible for it. Studies in both symptomatic and asymptomatic patients have shown that renal failure is associated with chronic elevations of cTn.172 Sepsis or pulmonary embolism can also independently increase cTn.173 Other causes of cTn elevation include trauma, pericarditis, HF, hypertension, and inflammatory diseases (Table 7.4).174 Elevated cTn does not reflect the mechanism of myocardial damage and should not be used alone to diagnose myocardial infarction. Prognostically, elevation of serum cTnl or cTnT is associated with an increased risk of cardiac death or reinfarction at 30 days (OR 3.44,95% 2.944.03). Elevated cTn is also predictive of long-term (five months to three years) outcome in those with STEMI or non-STEMI.175 In the GUSTO-IIa trial, the 30-days mortality rate was 10% in those with a positive baseline cTnT test, 5% with a late positive test, and nil in those with a negative test.176 cTn is also useful in predicting outcomes in patients without or without ACS.177,178 cTn is also elevated in patients with heart failure (HF), even in the absence of overt ischemia.179-18° The percentage of HF patients with elevated cTn could be as high as 45%. The mechanism for this elevation is believed to be due to on-going myocyte injury and the progressive loss of cardiac myocytes, with on-going release of cTn into the circulation.182-183 cTn lacks both sensitivity and specificity for diagnosis of HF. As a prognostic tool, however, increased serum cTnl or cTnT in patients with HF has been demonstrated to be associated with increased risks of cardiac events, rehospitalization and mortality.179'181-184185

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BIOMARKERS TABLE 7.4

Conditions commonly associated with cTn elevations.

Arrhythmias Congestive heart failure Coronary artery disease Coronary vasospasm Critically ill patient Hypertension Myocarditis Pericarditis, acute Pulmonary embolism Pulmonary hypertension, severe Renal failure Sepsis/septic shock Sepsis-related myocardial dysfunction Systemic inflammatory diseases Takotsubo cardiomyopathy Trauma

Heart-Type Fatty Acid Binding Protein (H-FABP)

H-FABP is a small cytosolic protein found in cardiomyocytes responsible for fatty acids transportation.186 H-FABP is rapidly released into the circulation following myocardial injury, and is detectable within two to three h of onset of clinical symptoms.187 The diagnostic sensitivity of H-FABP for cardiac injury is 93.1%, which is higher than CK-MB and cTn.188 Compared to CK-MB andcTn, H-FABP is abetter candidate for early detection of myocardial infarct. In a study involving 108 patients admitted to a mobile intensive care unit, H-FABP showed a better sensitivity to identify myocardial infarction than cTnl, myoglobin, and CK-MB.189 It also offers better sensitivity than TnT for early detection of acute MI and for detecting ongoing myocardial damage in congestive HF.169,190 H-FABP is now available as a rapid bedside test, and a study is currently underway to assess its diagnostic value outside the hospital in the general practice setting.191 Elevated serum H-FABP is associated with an increased risk of death and major cardiac events in patient with ACS despite a negative serum cTn and BNP.192In patients with dilated cardiomyopathy, the incidence of acute deterioration was significantly higher in patients with higher values of H-FABP than in those with lower values of the markers.193

Markers f o r Cardiac Stress B-Type Natriuretic Peptide (BNP) and N-Terminal ProBNP (NT-ProBNP)

The pre-prohormone BNP is a 134 amino-acid peptide synthesized in the ventricular myocytes and cleaved into the 108 amino-acid prohormone BNP. The prohormone is released into the circulation during hemodynamic stress.194


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Once in the circulation, the prohormone BNP is cleaved by a circulating endoprotease, corin, into the inactive 76 amino-acid NT-proBNP and the biologically active 32 amino-acid BNP. BNP causes arterial vasodilation, diuresis and natriuresis, which collectively reduce both afterload and preload. BNP also reduces the activities of the renin-angiotensin-aldosterone system and the sympathetic system. It plays an important role in counteracting the deleterious responses in heart failure by maintaining circulatory homeostasis and preventing the cardiovascular system from volume overload (Figure 7.3). Circulating levels of BNP and NT-proBNP differ, partly due to the different half-lives. NT-proBNP has a slightly longer half-life than BNP (60-120 min vs. 20 min), and may account for its higher concentrations that are approximately 20 times greater than BNP. While BNP is mainly cleared via internalization by cells that express the BNP receptors, renal clearance is the main mechanism for NT-proBNP. The concentrations of NT-proBNP are higher in patients with renal dysfunction, probably due to the reduced clearance. A number of clinical and epidemiology studies have demonstrated the relationship between HF and BNP or NT-proBNP195197 BNP is now commonly used to assist the diagnosis of HF, and has been endorsed as a useful diagnostic marker for HF. 198, '" BNP has been used to differentiate cardiac causes of dyspnea from that of pulmonary causes in the emergency setting.200 In the BREATHING NOT PROPERLY study, a plasma BNP level greater than 100 pg/ml was demonstrated to predict congestive HF (sensitivity = 90% and specificity = 73%).201 Similar findings were reported for NT-proBNP in the PRIDE study, although with different cut-off.202 The cut-off for patients younger than 50 years old is 450 ng/1 and for 50 years and older is 900 ng/1. BNP, however, fails to correlate with the New York Heart Association (NYHA) class of dyspnea and does not predict the severity of HF.203 The cut-off values may differ in patients with acute versus chronic HF. For example, about 20% of the patients with chronic HF exhibited plasma BNP levels below 100 pg/mg, which is the cut-off suggested for diagnostic purposes.204 BNP and NT-proBNP have been reported to be increased in patients with coronary artery disease.205 These increases are believed to be associated with both isolated left ventricular diastolic dysfunction and systolic dysfunction and are independent of hemodynamic overload.206-207 A single NT-pro BNP value at 96 hours after onset of symptoms proved useful for estimation of left ventricular ejection fraction.208 BNP was found to be useful in predicting mortality in acute MI.209 However, a recent systematic review casts doubt about the prognostic utility of BNP in these patients. The main issue of using BNP or BT-proBNP in patients with coronary artery disease is the lack of welldefined cut-off.210-211 What should be done to these patients once when one exceeds those cut-offs is also unclear. BNP can also be elevated in a number of conditions and is not specific to heart failure (Table 7.5). In the intensive care setting, as BNP is increased in a variety of cardiac conditions, it offers little help in differential diagnosis.212 BNP levels are also significantly confounded by age, gender, and fluid loading.213-215 The variation according to age and gender may be mediated par-

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FIGURE 7.3 Pathophysiology of cardiac disease and the physiological actions of B-type natriuretic peptide (BNP). Heart failure or ischemic events result in reduction in cardiac function (both systolic and diastolic), which in turn leads to a reduction in cardiac output (CO). Hypotension, tissue hypoperfusion, and reduced oxygen deliver (D0 2 ) are the main manifestations. The body compensates by increasing both preload and afterload: increasing salt and water retention via the rennin-angiotension-aldosterone system (RAAS), and vasoconstriction via baroreflex. Overcompensations result in deleterious effects such as pulmonary edema and increased cardiac workload. In response to volume overload, the myocardium releases BNP which partly counteracts the deleterious effects of overcompensation by inducing vasodilation, inhibiting the RAAS, and exerting some lusitropic effects. However the ability for BNP to compensate is limited. (See color insert for a full color version of this figure.)


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tially by the development of hypertension, renal disease, and vascular disease. Therefore, interpretation of BNP and NT-proBNP require the knowledge of age, gender, and the co-morbidities of the patients. Nevertheless, due to its high sensitivity and negative predictive value, BNP can be used to rule out cardiac disease. Adrenomedullin (ADM) ADM, a peptide of 52 amino acids, is a member of the calcitonin gene-related peptide family, which was originally discovered in human adrenal medulla as a hypotensive factor produced by pheochromocytoma cells.216 The precursor, pre-proADM, is synthesized and present in the heart, adrenal medulla, lungs, and kidneys. ADM is produced by the ventricular myocytes and fibroblasts in response to pressure and volume overload.217'218 Plasma ADM levels are significantly elevated in congestive heart failure, MI, and hypertension.219-221 Tissue levels of ADM and mRNA levels are also increased in ischemia.222 In acute MI, levels of ADM expressed in patients correlate with the severity of illness and are also a prognostic indicator for mortality in acute MI.223'224 The biological activities of ADM are similar to BNP—exerting a protective mechanism against the deleterious overcompensated response to failing hearts. ADM is a potent vasodilator, and can induce diuresis and natriuresis.216 Administration of ADM into patients with congestive heart failure increased urine output and urinary sodium excretion. ADM also improved cardiac index, hemodynamics, renal function, and hormonal parameters in the same group of patients.225 Part of these are mediated by its positive inotropic effect.226 The lack of standardization of the assay and a well-defined cut-off render hinders its utility as a clinical tool at present.

TABLE 7.5

Conditions or factors associated with BNP or NT-proBNP elevations.

Age Arrhythmias Cardiomyopathy: hypertrophic, ischemic, or dilated Congestive heart failure Coronary artery disease Gender Hypertension Left ventricular diastolic dysfunction Pulmonary embolism Renal failure Right heart failure Right ventricular overloading: fluid, or pressure overloading Sepsis or septic shock Sepsis-related myocardial dysfunction

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BIOMARKERS

ST2 ST2 is a member of the IL-1 receptor family. Both the transmembrane isoform (ST2L) and the soluble form of ST2 (sST2) are expressed by cardiomyocytes in response to mechanical strain, and were found to be increased in patients day one after acute MI.227 Until the discovery of IL-33 in 2005, ST2 was believed to be a stray receptor without any significant physiological function.228 It is now clear that the IL-33/ST2 plays an important role in modulating various inflammatory processes. ST2 is associated with diseases such as asthma, pulmonary fibrosis, rheumatoid arthritis, and septic shock.229 IL-33 is known to possess some cardioprotective effect against pressure overload. In animal models, IL-33 blocks die effects of angiotensin II and reduces ventricular hypertrophy and fibrosis in the face of increased ventricular strain.230 IL-33 also possesses anti-atherosclerotic effects by reducing vascular inflammation of atherosclerosis.231 While the ST2L receptor mediates the effect of IL-33, the physiology of sST2 has yet to be determined.232 It is clear that sST2 binds to IL-33 and reduces the cardioprotective effects of IL-33—antagonizing the effects of IL-33 by acting as a "decoy receptor."233 The local tissue ratio of IL-33 and sST2 could regulate IL-33 mediated signalling. Serum sST2 levels were increased one day after MI. The levels correlated with creatine kinase and were inversely proportional to left ventricular ejection fraction.227 In patients with acute MI, baseline sST2 levels were associated with higher 30-day mortality and development of new congestive HE234 In an analysis of patients presenting to the emergency with acute dyspnea, sST2 concentrations were significantly higher in patients presenting with acute systolic heart failure than in patients presenting with other causes of dyspnea. A serum level above 0.23 ng/ml had an 11-fold increased risk of death at one year.235 In patients with acute HF, sST2 segregated with more severe New York Heart Association functional class symptoms, and correlated inversely with left ventricular ejection fraction. sST2 also strongly predicted morality from a few months after presentation to at least one year in this class of patients.236 A few aspects of ST2 need to be addressed before it can be proven to be clinically useful. First, the true pathophysiological meaning of serum sST2 levels in cardiac disease is not fully understood. Whether the ratio of IL-33 to sST2 would provide a better diagnostic and prognostic value than sST2 alone requires further investigation. Second, the specificity sST2 levels may be undermined as sST2 are also increased in various inflammatory diseases such as asthma, autoimmune disease, and sepsis. Third, the impact of treatmentinduced longitudinal changes in sST2 over time on the clinical outcome and long-term prognosis also requires investigation.

Multimarker Approach? The reliance on a single biomarker for diagnostic or prognostic purpose has in many cases proven to be unsatisfactory. For example, in the context of MI, ischemia can be due to a number of causes ranging from coronary flow obstructing thrombosis, supply-demand imbalance without thrombosis, to


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iatrogenic causes. To diagnose MI itself is already a difficult task, to determine the origin of the injury presents even more challenges. A number of studies have demonstrated the benefits of using multimarker approaches. For example, the prognostic value was better when CRP was used in conjunction with BNP or cTn than when used independently.237,238 Similarly, the use of several biomarkers of cardiovascular and renal dysfunction substantially improved the risk stratification for cardiovascular causes of death compared to the use of established risk factors.239 The combination of cTnT, ECG, and ischemia-modified albumin could identify 95% of patients whose chest pain was attributable to ischemic heart disease.240,241 However, a recent study has shown that the combined measurements of various biomarkers did not provide any additional clinically significant benefit than by using cTnl alone in the diagnosis of MI.242 The main reason for this observation is that the other additional biomarkers used, including NT-proBNP, CRP, MMP, MPO, as well as soluble CD40 ligand, have low specificities and that their levels are influenced by non-specific inflammatory and other stimuli in the absence of acute MI. Poor sensitivities of the other biomarkers are also a hindering factor. While the multimarkers approach seems to be attractive, further research is required to prove its value further. In particular, different combinations of markers are required for different types of cardiac dysfunction and clinical settings.

CONCLUSIONS The use and interpretation of cardiac biomarkers depends upon the specific clinical setting and what information is being sought. For example, biomarkers employed for diagnostic purposes have very different requirements than those used for prognostic purposes. The type, and stage of development, of the cardiac disease pose another level of requirement and difficulty for the development of biomarkers and their interpretations. Ideally, the biomarker should be specific for cardiac diseases, but this is both theoretically and practically impossible due to the sharing of common pathophysiological pathways in many diseases. To date, while a small number of biomarkers have proven clinical values, which are still limited, most are unfit to use clinically due to poor sensitivity and specificity. Despite the lack of sensitivity and specificity, this should not deter biomarkers from being used, provided they are used within the clinical context and the user is aware of their limitations. There is no doubt that knowledge about cardiac biomarkers will continue to evolve with technology, most notably proteomics, and with it, our understanding of their physiological roles. When properly used, biomarkers provide invaluable additional information in screening, diagnosis, monitoring, and prognosis of cardiac disease.

SUMMARY POINTS 1.

The term "cardiac injury" comprises miscellaneous cardiac insults that involve the processes of cell injury or cell death (autophagy, apoptosis, and necrosis). While myocardial infarction is a common cause of car-

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2.

3.

4.

diac injury, it is not the sole cause. Other causes include heart failure, myocarditis, sepsis-induced myocardial depression, trauma, and druginduced myocardial toxicity. It is estimated that about 16 million Americans have CAD and about half of these experienced an MI. The pathophysiological mechanisms for most cardiac diseases involve three stages: inflammation, myocardial cell injury (damage), and cardiac stress. While these stages are continuous, there is no clear demarcation for each stage and they may occur concomitantly. Traditional cardiac biomarkers mostly reflect the extent of myocardial cell damage. The most important biomarker in this category is cTn. A recent change of paradigm emphasizes the importance of inflammation and cardiac stress. Biomarkers for the latter two are now available in the clinical setting, and the more important ones are CRP and BNP. Biomarkers generally show poor sensitivity and specificity for diagnostic purpose. None of the biomarkers are ideal and multimarker approaches may provide better diagnostic and prognostic values. Further research are required in this area.

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230. Sanada, S., Hakuno, D., Higgins, L. J., Schreiter, E. R., and McKenzie, A. N., et al. IL-33 and ST2 Comprise a Critical Biomechanically Induced and Cardioprotective Signaling System. J. Clin. Invest. 117;2007:1538-1549. 231. Miller, A. M., Xu, D., Asquith, D. L., Denby, L., and Li, Y., et al. IL-33 Reduces the Development of Atherosclerosis. J. Exp. Med. 205;2008:339-346. 232. Chackerian, A. A., Oldham, E. R., Murphy, E. E., Schmitz, J., and and Pflanz, S., et al. IL-1 Receptor Accessory Protein and ST2 Comprise the IL-33 Receptor Complex. J. Immunol. 179;2007:2551-2555. 233. Chackerian, A. A., Oldham, E. R., Murphy, E. E., Schmitz, J., Pflanz, S., et al. IL-1 Receptor Accessory Protein and ST2 Comprise the IL-33 Receptor Complex. J. Immunol. 179;2007:2551-2555. 234. Shimpo, M., Morrow, D. A., Weinberg, E. O., Sabatine, M. S., and Murphy, S. A., et al. Serum Levels of the Interleukin-1 Receptor Family Member ST2 Predict Mortality and Clinical Outcome in Acute Myocardial Infarction. Circulation. 109;2004:2186-2190. 235. Januzzi, J. L., Jr., Peacock, W. F., Maisel, A. S., Chae, C. U., and Jesse, R. L., et al. Measurement of the Interleukin Family Member ST2 in Patients with Acute Dyspnea: Results From the PRIDE (Pro-Brain Natriuretic Peptide Investigation of Dyspnea in the Emergency Department) Study. J. Am. Coll. Cardiol. 50;2007:607-613. 236. Rehman, S. U., Mueller, T., and Januzzi, J. L., Jr. Characteristics of the Novel Interleukin Family Biomarker ST2 in Patients with Acute Heart Failure. 7. Am. Coll. Cardiol. 52;2008:1458-1465. 237. Stolker, J. M. and Rich, M. W. The Combination of B-Type Natriuretic Peptide and C-Reactive Protein Provides Incremental Prognostic Value Among Older Patients Referred for Cardiac Catheterization. Am. J. Geriatric Cardiol. 16;2007:229-235. 238. Foussas, S. G., Zairis, M. N., Makrygiannis, S. S., Manousakis, S. J., and Anastassiadis, F. A., et al. The Significance of Circulating Levels of Both Cardiac Troponin I and High-Sensitivity C Reactive Protein for the Prediction of Intravenous Thrombolysis Outcome in Patients with ST-Segment Elevation Myocardial Infarction. Heart. 93;2007:952-956. 239. Zethelius, B., Berglund, L., Sundstrom, J., Ingelsson, E., and Basu, S., et al. Use of Multiple Biomarkers to Improve the Prediction of Death from Cardiovascular Causes. N. Engl. J. Med. 358;2008:2107-2116. 240. Peacock, F, Morris, D. L., Anwaruddin, S., Christenson, R. H., and Collinson, P. O., et al. Meta-Analysis of Ischemia-Modified Albumin to Rule Out Acute Coronary Syndromes in the Emergency Department. Am. Heart J. 152;2006: 253-262. 241. Sinha, M. K., Roy, D., Gaze, D. C , Collinson, P. O., and Kaski, J. C. Role of "Ischemia Modified Albumin," a New Biochemical Marker of Myocardial Ischaemia, in the Early Diagnosis of Acute Coronary Syndromes. Emerg. Med. J. 21;2004:29-34. 242. Apple, F. S., Smith, S. W., Pearce, L. A., and Murakami, M. M. Assessment of the Multiple-Biomarker Approach for Diagnosis of Myocardial Infarction in Patients Presenting with Symptoms Suggestive of Acute Coronary Syndrome. Clin. Chem. 55;20O9:93-100.


CHAPTER

LUNG INJURY BIOMARKERS Urmila P. Kodavanti

INTRODUCTION Biomarkers indicate quantitative and qualitative changes in molecules that reflect alteration in normal functioning of the cell and the organ system in response to various stimuli. The determination of biomarkers is essential in identifying and understanding the types of injuries, the consequent disease, and therapeutic targets. More importantly, biomarker analysis allows diagnosis of the disease. Depending on the physicochemical nature of the injury-causing substances and the organs impacted, the types of biomarkers vary. However, in all types of injuries, there are a few commonalities in the biomarkers between different diseases and the organ systems being affected, for example, those that measure inflammation, oxidative stress, and antioxidant compensation.15 These biomarkers, unlike tissue- and injury-specific biomarkers, individually may not reveal full details about the mechanisms of pathogenic processes; however, with the knowledge of the injury-causing agent and the organ system encountered, diagnosis of the disease can be made. In this chapter, lung injury biomarkers are described. For detailed information, readers are directed to recent review papers on pulmonary disease biomarkers.6-13 The lung is a unique organ system with highly metabolically active oxygen exchange process within the delicate epithelial and capillary endothelial layers having direct encounter with the outside environment. To counter the pressure differences between the capillary and the inhaled air and to prevent collapse caused by these pressure differences, unique surface lining fluid is secreted by type II cells, whereas the oxygen exchange occurs primarily through type I lining cells within the alveolar unit of the lung.14 The inhaled injury-causing substances are carried to these alveolar sacs via the trachea and respiratory bronchioles. 157

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Lung injury is induced by (1) the inhalation of one or more toxicants or microbial organisms, (2) substances that circulate through pulmonary capillaries, (3) as a result of left ventricular diastolic dysfunction, or (4) use of ventilators. Among all, the most prevalent contributing factors for acute and chronic lung injuries and diseases are environmental exposures; cigarette smoke being the primary environmental cause of lung disease.15-17 Depending on the causative factor, site-specific injury is inflicted on the airways and the lung parenchyma. Lung injuries are measured by physiological, morphometric, pathological, biochemical, and molecular analyses of the sputum, bronchoalveolar lavage fluid (BALF), plasma, urine, and tissues from humans and animal models. In clinical settings, lung injuries are examined by pulmonary function maneuvers, chest radiography, and computerized tomography (CT) scans.18 More detailed analysis often is made by bronchoalveolar lavage (BAL) for determination of inflammation and lung vascular leakage in patients,19,20 representing complex pulmonary CT abnormalities. Biopsy specimens are evaluated for more severe chronic disease and lung carcinoma.21 There are a few circulating biomarkers that are specific to the type of lung injury;22 those when analyzed in conjunction with radiographic examination provide diagnostic values. I will describe the causes of lung injuries, the cellular targets, the pathobiological processes involved, and then elaborate on biochemical and molecular biomarkers used for assessing lung injury in clinical and experimental studies.

CAUSES OF L U N G INJURY Inhaled reactive gases, respirable particulate matter, tobacco smoke, dusts, metals, silicates, other minerals, and fibers are the major contributors of lung injury and chronic pulmonary disease burden.23-26 Lung injury also occurs as a result of respiratory infections from bacteria.27,28 Viral infections to the upper respiratory tract often increase vulnerability to bacterial infection involving the lung parenchyma.29-31 In addition, lung edema can occur following left heart failure.32 Drug-induced pulmonary injury and phospholipidosis has been noted from long-term treatment with cationic amphiphilic, antipsychotic, and antiarrhythmatic drugs such as amiodarone.33-35 Clinical use of ventilators in infants also is associated with lung damage early in life that affects normal lung growth and increases subsequent vulnerability to adult lung diseases.36 In many cases of environmental lung diseases, the exposure occurs to mixtures of a variety of highly reactive inorganic or organic species, which may cause injury via different mechanisms.37 Therefore, the disease caused by such insults is complex and affects both airways and alveolar compartments with multiple pathologies and molecular mechanisms. The classical example of such complexity is exposure to cigarette smoke. Cigarette smoke contains thousands of chemical species, including gas components such as nicotine, carbon monoxide, benzene, formaldehyde, acetone, arsenic, ammonia, tar, cadmium, and a variety of poly cyclic aromatic hydrocarbons.37,38 Thus, the injuries that result from cigarette smoke exposures involve multiple biologi-

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cal signaling mechanisms and affect many cell types. The disease that results from cigarette smoke, thus, is actually a group of diseases, which is referred as COPD based on the functional outcome of obstruction of the airways.39,"° Some diseases occur following exposure to a single chemical, for example, berylliosis in beryllium-exposed individuals41'42 or fibrosis in bleomycin- or cadmium-exposed individuals.4344 Lung diseases from environmental exposures are modified by the contribution of heritable genetic and epigenetic abnormalities. One can inflict injury to the lung in a genetically homogenous population with no known genetic vulnerabilities and still find variation in response among individuals exposed, emphasizing how little is known about the genetic networks and biological mechanisms. It is possible that, if no known genetic or epigenetic factors regulating compensatory or adaptive processes are abnormal, the injury is repaired without producing chronic disease. In some instances, the contributing genetic polymorphisms or epigenetic deregulations have been associated with lung injuries, but the genetic bases for many lung diseases still remain largely unknown.45,46 One example that has been studied for decades is the presence of polymorphism in a-1 antitrypsin leading to increased emphysema in smokers.47 Similarly, the integrin (3-3 gene has been associated with asthma susceptibility.48 However, most of these are associations with only single genes. Many different gene targets are speculated to be different in chronic disease, emphasizing the complexities of lung pathogenesis resulting from environmental exposures. These very same genetic factors can serve as biomarkers of susceptibility variations in lung injury.

M O R P H O L O G I C A L A N D C E L L U L A R TARGETS OF L U N G INJURY The lung is composed of two major compartments: 1) airways for air passage and 2) parenchyma for gas exchange. Some chemical or microbial agents produce site-specific injury within pulmonary tissue, whereas other agents produce more widespread damage along the airways and the parenchyma. Standard histological and electron microscopy examination of experimental animal tissues or biopsies from human patients, in conjuction with immunohistochemistry and in situ hybridization approaches, allows one to identify the morphological targets within the lung. This information not only provides insights into where a given biomarker originates within the lung, and how it may be associated with impaired lung function, but also is critical in localized therapeutic interventions. Thus, a brief description of such lung structural components is warranted.

Airway and Mucosa The mucosal layer covers tracheal and bronchial epithelial cells along the airway surface. This mucosal layer and epithelial cells are the first to encounter inhaled toxicants that deposit on the airway surface. The depth of this layer is anatomically related to the diameter of the airway and the number of goblet cells

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contributing to secretion.4950 The composition of airway surface liquid may depend on secretion from airway glands, ion transport across the surface epithelium, transepithelial gradients in hydrostatic pressure, and surface tension. In addition to mucus-secreting goblet cells, the airway epithelium is comprised of the pseudostratified columnar ciliated cells. Intact and damaged airway epithelial structures are shown in Figure 8.1. The mucus layer functions by modulating innate immune response, detoxifying reactive inhaled substances, and removing particulates and pathogens via mucociliary clearance. Mucus is made up of large glycosylated proteins rich in serine and threonine to which large carbohydrate structures anchor." Some mucins remain associated with the cell membrane and function as receptors for pathogens and their components, whereas other mucins are secretory and layer airway surfaces. The composition of mucus can be modified by chemical exposures, which trigger mucus hypersecretion following airway injury and in a variety of diseases, such as asthma, bronchitis, COPD,

FIGURE 8.1 Airway structural alterations following inhalation of injury causing agents. A simplified sketch of major alterations is provided. Note that the physicochemical properties and the biological activities of inhaled substances will cause airway injuries by different mechanisms, and the end-results of pathologies will show a spectrum of alterations and temporality dependent on the nature of the inhaled single substance or mixtures. This airway damage shown in the figure is not representative of all injury processes; rather it illustrates major changes. Some of the figure components are copied from the slides obtained from Motifolio, Inc. (Ellicott City, MD).


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and cystic fibrosis.52-54 Mucin production is regulated by a number of genes and can be studied by the measurement of proteins and gene expression.52 The airway epithelium (Figure 8.1) is supported by the basement membrane, whereas smooth muscle layer surrounds the interstitial space supporting the basement membrane and the epithelium.55 Antigen-presenting dendritic cells project between these epithelial cells and capture antigens. On recognition of antigen (paniculate, microbial, or soluble), dendritic cells migrate to the draining lymph nodes and cause innate and humoral immune responses.56,57 The airway epithelium and also the deeper parenchyma are innervated by sensory C-fibers. These vagal C-fibers respond to inhaled noxious substances, such as capsaicin and, when stimulated, evoke classical defensive reflexes, such as bradycardia, systemic hypotension, increases in parasympathetic tone with bronchoconstriction, and cough.58

A l v e o l a r Macrophage Alveolar macrophages guard the air-blood interface by serving as the first line of defense against ingested particulates, bacteria, and other pathogens. Alveolar macrophages also clear degraded or excess surfactant material.59 They originate from blood monocytes and, when needed, migrate to the lung for host defense. In addition to killing, ingesting, and processing pathogens, alveolar macrophages play a significant role in innate immune response by synthesizing and secreting an array of cytokines and arachidonic acid metabolites.5960 These mediators are responsible for recruitment of neutrophils and other inflammatory cells into the airspaces and the subsequent inflammatory response. More recently it has been shown that macrophages play an equally important role in the resolution of inflammation through regulating the clearance of apoptotic neutrophils.61,62 These cells are recovered easily from the lung via bronchoalveolar lavage (BAL) in humans and laboratory animals, and serve as a major tool for identifying biomarkers that are responsible for a variety of lung injuries.

The Surfactant C o v e r i n g A l v e o l a r Epithelial Cells The surfactant is an essential component of the respiratory system. It is important for alveolar stability, prevention of collapse, and preserving patency. It also functions as the host defense system within the alveoli.63-66 The surfactant is made by the type II alveolar epithelial cells and is secreted at the apical surface in the form of lamellar bodies. Once secreted, it rapidly forms a thin layer over the alveolar surface. When alveoli are compressed during expiration, it reduces surface tension to prevent collapse.63-66 The surfactant material is composed of phospholipids (-80%) and other neutral lipids and proteins. While diplamitoyl phosphatidylcholine functions to reduce surface tension, the neutral lipids and proteins provide an appropriate backbone for its assembly and proper function. There are four surfactant proteins (SPs): SP-A, SP-B, SP-C, and SP-D. SP-A is the most abundant protein, followed by SP-D. SP-A and SP-D are large, water-soluble glycosylated proteins and play a role in

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host defense, whereas hydrophobic peptides SP-B and SP-C are important in the functioning of surfactant by enabling adsorption and spreading of the surfactant material along the alveolar lining.63-66 On injury to the airway lining, these proteins escape into the circulation and serve as important circulating biomarkers of lung injury, whereas their detection in the BALF provides more accurate information on the type of lung damage and alveolar defense.

A l v e o l a r Epithelium, I n t e r s t i t i u m , and Capillary Endothelium Respiratory bronchioles in humans terminate into small alveolar units called alveolar sacs, where the primary gas exchange occurs. Beneath a thin layer of surfactant, alveolar type I cells cover the most area of the alveolus, whereas type II cells, although the most abundant cell type of the alveoli, occupy hardly any space lining the alveolus (Figure 8.2). The pulmonary capillary network surrounds the alveolus, creating an extremely close distance between air and blood for diffusion of carbon dioxide from blood to air and oxygen from air to blood.67-69 Interstitial tissue supporting these structures is comprised of extracellular matrix, elastin, and pulmonary myofibroblasts. Abnormalities in these alveolar units are detected by a variety of biomarkers through BAL fluid analysis and histologically. Alveoli interlinked with connective tissue network and encapsulated by the pleural mesothelial layer, provide anatomical structure to the lung.

P A T H O B I O L O G I C PROCESSES I N V O L V E D IN L U N G INJURIES A N D DISEASES Pulmonary injuries can be classified based on where in the lung the injury occurs or what pathological processes may be involved in the development of the disease. One must be careful, however, because many injuries involve more than one structural or morphological units of the lung, and, thus, the specificity of the biomarkers selected to evaluate injury diminishes. However, as indicated previously, no single biomarker may be chosen for diagnosing a disease.

A i r w a y Epithelial Damage, Mucus H y p e r s e c r e t i o n , and G o b l e t Cell Hyperplasia There are several types of epithelial injuries that depend on the type of causative agent involved and the cell type primarily being injured. For example, ciliary damage by inhaled ozone or phosgene is associated with mucus hypersecretion and airway inflammation.70-72 In case of sulfur dioxide exposure, mucus hypersecretion is associated with airway inflammation.73,74 Inhaled reactive oxidant gases and respirable particles, including microbials, interact with the mucosal and ciliary components, chemically modifying proteins that render them nonfunctional. Cell epithelial membrane structures are injured, and ciliary beating is impaired. Often the entire layer of ciliary epithelia is sloughed off; rendering the basement membrane exposed to dam-


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FIGURE 8.2 Normal alveolus structure with capillary depicting interaction between pulmonary epithelial and endothelial cells. During injury, epithelial and endothelial cells are damaged, and macrophages are activated. Type II cell hyperplasia, protein leakage, neutrophilic inflammation, infiltration of macrophages, and secretion of inflammatory mediators are common features of acute injury. Depending on the nature of acute injury, macrophage accumulation, interstitial fibrosis, emphysema, granuloma, and other diseases including carcinoma are likely to occur Some of the figure components are copied from the slides obtained from Motifolio, Inc. (Ellicott City, MD). (See color insert for a full color version of this figure.)

aged cell debris and nonfunctional mucus components (Figure 8.1). This leads to the loss of epithelial integrity and basement membrane thickening.7577 Hyperplasia and mucus hypersecretion are triggered in goblet cells within the airway submucosal glands and airway epithelium.78-81 Mucus hypersecretion is a factor promoting increases in smooth muscle mass, airway vascularity, and airway fibrosis. The process of remodeling and repair may lead to functional impairment characterized by bronchoconstriction and airway hyperresponsiveness.82-84 Damaged airway epithelial and smooth muscle cells secrete cytokines and stimulate extravasation of inflammatory neutrophils, eosinophils, and monocytes at the damaged site.

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Impairment and chemical modification of mucus lining layers lead to a variety of pathogenic outcomes. Mucus secretion is a dynamic process and involves soluble N-ethyl-maleimide-sensitive factor attachment receptor (SNARE) proteins, myristoylated alanine-rich C kinase substrate (MARCKS), and Munc proteins for secretory granules processing and exocytosis.85 Excessive and defective mucus production has been associated with obstruction of small airways. The mechanism by which airway inflammation stimulates mucus production and transformation of Clara and ciliated cells into goblet cells involves epidermal growth factor receptor and IL-13 activation and coordinated involvement of FoxA2, TTF-1, SPDEF, and GABAAR.8687 These mediators are involved in transcriptional upregulation of mucin 5AC expression and increased mucin in goblet cell granules that fuse to the plasma membrane through actions of MARCKS, SNAREs, and Munc proteins.86,87 Thus, one or more of these proteins can be rate-limiting in the process and thus may serve as important biomarkers for diagnostic and therapeutic purposes.

A i r w a y Inflammation in Asthma Chronic inflammation and airway hyperresponsiveness are the central pathogenic processes involved in asthma.84-88~93 Asthma is a spectrum of abnormalities thought to be caused by interaction of environmental and genetic factors. Depending on the presence of an allergic or nonallergic component, the factors involved in the inflammatory process and the physiological and clinical outcomes vary.84-88-93 The primary contributors are the Th2 lymphocyte-mediated release of a set of cytokines, with recruitment and activation of mast cells, eosinophils, and macrophages. In the pathogenic process, neutrophil extravasation also is noted with involvement of CD4+ T lymphocytes and regulatory T cells.94-96 A variety of invasive and noninvasive biomarkers are available to diagnose the disease and to understand the pathogenic mechanisms of inflammation and bronchoconstriction in asthma. Inhaled particulates, including cellular components of gram-negative bacteria, and proteoglycans activate pattern-recognition receptors, including toll-like receptors that, through release of IL-7, stimulate dendritic cell receptors CD40, CD80, and OX40 to enhance Th2 polarization.97-99 Through production of Th2 cytokines IL-4, IL-5 and IL-9, IL-13, and cellular interactions between inflammatory cells and airway epithelium, fibroblasts, microvascular endothelial cells, smooth muscle cells, dendritic cells, and neuronal cells produce a chronic inflammatory phenotype associated with asthma.97-99 Mediators released in a temporal manner from the inflammatory cells have been, perhaps, the most extensively used biomarkers of inflammatory disorders.

A i r w a y Inflammation in Bronchitis and C h r o n i c O b s t r u c t i v e Pulmonary Disease Inflammation associated with chronic bronchitis resulting from cigarette smoking or inhalation of smoke from biomass burning presents with a different phenotypic expression than what is seen in asthma, however, with oc-


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casional overlap of both diseases.100,101 Mucus hypersecretion is moderate in asthma pathogenesis, whereas it is the predominant pathogenic factor in bronchitis.102-104 Through activation of nuclear factor (NFKB)-associated signaling, cigarette smoke components activate transcription of genes involved in production of inflammatory cytokines and subsequent infiltration of neutrophils and macrophages involved in innate immune response.102-104 The presence of adaptive CD8+ T lymphocytes has been central in inflammation associated with COPD.105 Alveolar macrophages play a central role in this inflammatory cascade. Lung epithelial cells secrete macrophage chemoattractant protein-1 and macrophage inflammatory protein-2, which attract and activate pulmonary macrophages and neutrophils. BAL of smokers with COPD contains more pulmonary macrophages than that of nonsmokers. These macrophages, however, are inefficient in phagocytosis and have longer half-lives. They express membrane glycoproteins essential for cell adhesion and phagocytosis and produce more oxygen radical species than do macrophages of nonsmokers. Macrophages of COPD patients have even greater elastolytic activity.106 CD8+ T cells also are increased in animal models of cigarette-smoke-induced inflammation and emphysema,107 suggesting that, in addition to modulating inflammatory response, these cells may play a role in alveolar destruction caused by imbalance between proteases and antiproteases. It is believed that CD8+ T cells secrete cytokines, such as interferon-7 and interferon-inducible protein-10, dominant features of the Thl phenotype.108 These mediators likely are involved in activation of proteases within macrophages, leading to destruction of the alveolar compartment. Because the pathobiologic and phenotypic presentation of COPD in different individuals is diverse, therapeutic approaches targeted to one particular phenotype have achieved limited success. Similarly, any one biomarker evaluation is less likely to be useful for diagnostic purpose. Numerous biomarkers have been analyzed in bronchial biopsies and sputum, including those involved in inflammation, mucus hypersecretion, and oxidative stress.

rway Fibrosis, B r o n c h o c o n s t r i c t i o n , d Hyperresponsiveness Bronchial smooth muscle hypertrophy and fibroblast proliferation likely occur as a consequence of chronic inflammation, mucus hypersecretion, and bronchoconstriction. More collagen is deposited along large and small airways affecting normal airway constriction. Because these multiple diseases coexist in different proportions in bronchitis, asthma, and COPD, it is not clear which initial stimuli perpetuate signaling events in causing multiple pathways to be stimulated. Subepithelial fibrosis occurs as a result of epithelial injury and mucus hypersecretion, leading to increased synthesis and deposition of extracellular matrix components, including collagens I, III, and V; fibronectin; laminin; tenascin; and biglacan. Collagen deposition is associated with smooth muscle cell migration, hypertrophy, and hyperplasia. A

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number of cytokines and co-stimulating factors specific to epithelial and smooth muscle cells might promote increased extracellular matrix production.82, 84,109' "° Myofibroblast migration has been shown to contribute to increased collagen deposition through transforming growth factor-(3 (TGF-P) signaling.111 It is believed that CD4+ T cells, along with other inflammatory cells, are central to airway remodeling and fibrosis.112 Cystic fibrosis is a classic example of an airway sodium-transport defect associated with a cascade of events leading to inflammation, fibrosis, mucus production, and airway dysfunction.113 Airway fibrosis associated with chronic bronchitis and asthma plays a significant role in producing bronchoconstriction.

A l v e o l a r Epithelial, Capillary Endothelial, and Terminal B r o n c h i o l a r Injuries There are a variety of environmental exposures, drugs, and idiopathic factors that inflict injuries to the deep lung with varying mechanisms, which are either persistent or reversible. Depending on the type of insult, the anatomical site, and the cell signaling pathway induction, the insult leads to specific pathological outcomes, such as inflammation, alveolar protein leakage, increased production of surfactants, defective surfactant production, stimulation of myofibroblasts through degradation of extracellular matrix (ECM) components, release of cytokines, stimulation of apoptosis, endothelial and type I cell injuries, and type II cell hyperplasia.62, n4-116 In a long-term situation, chronic fibrosis, emphysema, pulmonary hypertension, alveolar proteinosis, and often metastasis of the lung occur. However, there are marked variations in the degree of lung disease between individuals and laboratory animal species based primarily on multiple genetic differences.46'117,118 In subsequent sections, these processes will be discussed, along with focus on specific biomarkers. There are a limited number of circulating lung-specific biomarkers used clinically that provide insights into the type of pathology; these biomarkers in conjunction with patient exposure history, chest radiograms, and CT scans, make diagnoses possible in most cases, whereas in others, more invasive sampling of lung tissue is required via bronchoscopy.119_122BALF also is obtained and can be analyzed for a number of cytokines, inflammatory cells, and injury markers.

Pulmonary Edema Deep lung edema is a life-threatening complication that generally is associated with acute lung injury, infection leading to pneumonitis, and acute respiratory distress syndrome.123,124 Epithelial injury and disrupted function of sodium channels, along with increased endothelial damage, cause changes in alveolar and capillary permeability, which subsequently impairs gas exchange and causes secondary complications. Other than ventilation strategies, no standard treatment exists for permeability edema, making the search for novel regulators of endothelial and epithelial hyperpermeability and dysfunction important. A recent review provides an account of potential thera-


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peutic targets that attenuate oxidative stress, inflammation, epithelial barrier dysfunction, and hydrostatic and permeability edema. The understanding of these processes is critical in developing therapeutic strategies.125

N e u t r o p h i l i c I n f l a m m a t i o n , Alveolar A p o p t o s i s , and Emphysema The mechanism of airway and alveolar injuries involves receptor-mediated phosphorylation and cell signaling, leading to nuclear translocation of regulatory proteins, and transcription of genes that are responsible for inflammation and repair processes. Many proteins involved in these signaling events can serve as biomarkers of acute and chronic lung injuries. An example of how numerous pathological processes are regulated by NFKP-mediated signaling is provided in Figure 8.3. In the case of tobacco smoke, lung injury involves both airway and alveolar compartments, leading to bronchitis and emphysema. In the past decade or so, our understanding of the role of proteases, antiproteases, and vascular endothelial growth factor (VEGF) in cigarette-smoke-induced alveolar apoptosis has advanced significantly. The mechanisms that induce pulmonary apoptosis include growth factor deprivation, mitotic aberrations, extracellular matrix degradation, loss of cell-cell communication, activation of cell death receptors by soluble endogenous ligands, and epithelial and endothelial injuries.126,127 There are extrinsic and intrinsic pathways that trigger caspases (proteases) involved in the release of cytochrome C from mitochondria and ultimately formation of apoptotic bodies.62,128,129 These apoptotic bodies (Rho kinases) cause externalization of cell membrane phosphatidyl serine (normally associated with the inner side) and cell blabbing. Numerous antiproteases counteract the balance between proteases and antiproteases in maintaining structural integrity of the lung cell. Protease activated receptor-1, through inhibition of AKT phosphorylation, and other antiproteases such as tissue inhibitor of matrix metalloprotease-1, and inhibition of CD63-mediated ERK and AKT phosphorylation, inhibit apoptotic processes and protect cells.130 The antiprotease a-1 antitrypsin, implicated in protecting the lung from emphysema, directly can inactivate neutrophil elastase, which, through extrinsic and intrinsic mechanisms, cause apoptosis in smokers.131 The deficiency in a-1 antitrypsin has been associated with exacerbated emphysematous changes in smokers and in animal models of COPD and emphysema.131

Pulmonary Fibrosis and Granuloma A variety of environmental and occupational exposures, including ambient paniculate matter, reactive gases, asbestos, silica, and drug treatments induce fibrotic lung diseases and granulomas.132,133 Idiopathic pulmonary fibrosis, although rare, occurs in humans.134 In contrast to the destructive process of apoptosis induced in emphysema, the process of pulmonary fibrosis, especially bleomycin-induced, is associated with abnormal or aberrant tissue repair and dysregulated angiogenesis, likely involving similar pathways as in em-

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FIGURE 8.3 Major signaling events associated with nuclear factor-Kp} ( N F - K B ) activation and nuclear translocation in response to lung injury caused by inhaled substances. In addition to cytokines, growth factors, lipopolysaccharides (LPS), and lymphotoxins, oxidative stress infectious agents, inhaled particles, and reactive gaseous materials activate N F - K B family protein by dissociating inhibitor-KB via its phosphorylation through I-KB Kinase Complex. Upon activation, N F - K B homo- and heterodimers of Rel family including N F - K B (p50), N F - K B 2 (p52), RelA (p65), RelB, and c-Rel (Rel) translocate into the nucleus and induce gene expression. The phosphorylated I-KB is removed by proteosomal degradation. Note that some of the components of the figure and the basic pathways are extracted from the signaling pathways provided by SA Biosciences Inc. (http://www.sabiosciences.com/pathwaycentral.php) and Protein Lounge (San Diego, CA). VEGF, vascular endothelial growth factor;VEGFR.VEGF receptor; IL-1, interleukin-1; ILIR IL-1 receptor; LPS, lipopolysaccharide;TLRs,Toll-like receptors;Tumor necrosis factor,TNF;TNFR,TNF receptor;TCR,T-cell receptor; BCR, B-cell receptor; Lt-B, Lymphotoxin-S; Lt-B R, L t - p receptor; BAFFR, B-cell activating factor receptor;TRAFs,TNF receptor-associated factors; IKK-a, inhibitor kinase-a; IKK-a, inhibitor kinase-a; IKK-p, inhibitor kinase-p. (See color insert for a full color version of this figure.)

physema, but in an opposing manner.135,136 It is postulated that oxidative stress in bleomycin-induced lung injury activates PI3 kinase/AKT, which leads to increased transcriptional activation of collagen and fibroblast proliferation within the pulmonary interstitium.137 Activation of PI3 kinase/AKT also is known to activate HIF-1 and VEGF, which contribute to collagen- and fibroproliferative effects of bleomycin, unlike inhibition of AKT, and VEGF in emphysema.138 The role of TGF-(3 signaling through platelet-derived growth factor and its receptors in mediation of pulmonary fibrosis and aberrant tissue repair is well studied.139 A number of inflammatory cytokines, including tumor necrosis factor-a and interleukin-1, are involved in the signaling cascade that activates TGF-(3. Antioxidant interventions are known to reduce the propen-


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sity of fibrosis.140 Collagen deposition, viewed easily in histological sections of the lung, occurs in the interstitial space and at the terminal bronchiolar region often surrounding the area of proliferating myofibroblasts. In experimental studies, lung tissue hydroxyproline, a collagen component, is analyzed to determine in a quantitative manner the level of fibrosis.141 The evaluation of mediators, such as cytokines and growth factors such as platelet-derived growth factor and TGF-P, aids in determining fibrotic changes that might be occurring in the lung. Fibrosis and emphysema often coexist in the same lung at different locations, making targeted therapeutic interventions challenging. Depending on the injury-causing agent, the processes of aberrant tissue repair and morphology of collagen deposition differ; for example, in case of silica and asbestos exposure, granuloma surrounding toxic materials often is observed.

A l v e o l a r Phospholipidosis Dipalmitoylphosphatidyl choline and phosphatidyl ethanolamine are the primary surfactant phospholipids synthesized and assembled with surfactant proteins in lamellar bodies within type II alveolar epithelial cells and transported toward the apical surface of alveolar cells.65 Cationic amphiphilic drugs, such as amiodarone, gentamycin, and classes of other antiarrhythmic and antipsychotic drugs, when taken over a long period of time, cause pulmonary phospholipidosis characterized by increased accumulation of surfactant lipids in lamellar bodies within the alveoli and macrophages.34'35 These drugs are taken up selectively from circulation by lung cells in which they inhibit phospholipases, especially lysosomal phospholiases Al and A2, leading to reduced surfactant turnover and accumulation within the alveoli. Often, phospholipidosis is noted with silica exposure, in addition to granuloma development.142'143 Amiodarone is the most studied drug that, in addition to phospholipidosis, also induces fibrosis.144

Pulmonary Surfactant and Surfactant Protein A b n o r m a l i t i e s In an alveolus, the biochemical alterations are inevitable upon encounter of materials, such as proteins, hemoglobin, fatty acids, inflammatory cells, and exogenous substances that chemically modify surfactant and alter its function.145-147 Oxidation and nitration of surfactant molecules also can render it inactive. Surfactant deficiency during early lung growth predisposes newborn children to acute respiratory distress syndrome, which is treated by providing exogenous synthetic surfactant.145 Lysophospholipase from eosinophils, together with the enzyme phospholipase A2, catalyzes the hydrolysis of phosphatidylcholine, incapacitating the ability of the surfactant to maintain airway patency.148 Biochemical surfactant alterations and related functional abnormalities have been noted in a variety of obstructive lung diseases, cystic fibrosis, infections, pulmonary edema, and proteinosis. Surfactant abnormalities also accompany exposure to reactive gases and particulate matter.149-15°

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Given the chemical functionality of surfactant proteins and their interaction with alveolar macrophages and epithelial cells, it is possible that changes in surfactant proteins occurring with disease conditions may be detected in the serum, serving as biomarkers specific to lung cell injuries. Indeed, the levels of SP-A and SP-D in circulation are highly predictive of interstitial lung diseases and survival.151,152

S A M P L I N G T E C H N I Q U E S FOR BIOMARKER ANALYSIS Lung tissue sampling in humans may range from noninvasive collection and analysis of exhaled breath, sputum, blood, and urine to more invasive bronchoscopic techniques for BAL and biopsies. Site-specific fluid and biopsy samples of lung tissue are obtained using more advanced bronchoscopic methods assisted by endobronchial ultrasound. These sampling methods are selected based on the degree of complications and patient health. Induced Sputum Induced sputum is a noninvasive sampling technique that has been used to identify markers associated with injury in the airways.15'153,154 The use of sputum in identification of biomarkers of airways injury has improved our understanding of obstructive airway diseases, such as asthma and COPD. There are some differences in the consistency of biomarker identification between spontaneous and induced sputum. Induced sputum provides excellent consistency in identification of biomarkers associated with airway injury and disease.155 The determination of inflammatory cells in the induced sputum is highly accurate and reproducible and provides information about the severity of inflammation. Induced sputum also can detect bacterial infection in patients with tuberculosis156 or acquired immune deficiency syndrome.157 The protocol for collecting induced sputum includes administration of nebulized hypertonic saline at increasing volume, which provides consistent yields of inflammatory cells as opposed to those with normal saline.158 Generally, prior inhalation of albuterol is performed to produce bronchodilation, followed by inhalation of hypertonic or normal saline. Following administration of hypertonic saline, the patient is encouraged to expectorate sputum through voluntary coughing. In cases where coughing is not spontaneously elicited, the patient is asked to cough deeply. The specimen is collected in a sterile beaker and processed. The thick material contains mucus, cellular debris, and whole cells. The thick mucus is separated from saliva manually and analyzed for cellular markers following a quick staining protocol.159 Generally, patients with COPD and chronic bronchitis yield a sufficient quantity of sputum, whereas it is often difficult to collect sufficient material from healthy individuals. Inflammatory cells can be identified as biomarkers of underlying inflammation. Analysis of cytokine proteins can be accomplished using targeted antibodies for immunological techniques such as enzyme linked immunosorbant assay


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(ELISA) or western blotting. The sputum also can be examined in detail by microscopy and other techniques for the presence of inflammatory cells.160 Inhalation of hypertonic saline in some patients can induce a small degree of bronchoconstriction that is reversible. This potential bronchoconstriction can be reduced or prevented by prior inhalation of bronchodilators, such as albuterol.

Bronchoscopy and Lung Biopsy Airway epithelial cells are the first to encounter inhaled pathogens or toxicants. An endotracheal tube is used to sample upper airway cells without invasive bronchoscopy. However, most sophisticated cancer diagnosis is done by elaborate bronchoscopy-based technologies. Conventional white light bronchoscopy and forceps biopsy techniques have been modified to incorporate advanced computerized technologies. These are used in conjunction with the CT assessment and cryosampling techniques to provide better diagnostic field values and preserve sample integrity for molecular analysis.119'161,162 The bronchoscopic procedures are carried out with or without general anesthesia in patients. Different illumination modes, including blue-light filtering and auto-fluorescence mode, are used to get better views of the airways as deep as ninth-generation bronchioles. Medium- and large-airway wall dimensions have been measured reliably using CT. More recently, optical coherence tomography, a new micron-scale resolution imaging technique, has been employed that can image airways as little as 2 mm in diameter. This technique is more appropriate for understanding airway pathologies that are associated with changes in forced expiratory volume in individuals with obstructive airway disease.163 In the case of lung cancer, a solitary pulmonary nodule is sampled by means of transbronchial needle aspiration, brush, or transbronchial lung biopsy under fluoroscopy. Ultrasound technique allows more accurate localization and sampling of peripheral pulmonary, mediastinal, and hilar lesions. Generally, two-stage procedures are used to localize cancer within the lung and to obtain biopsy specimens. First, three-dimensional CT imaging is used, followed by interventional bronchoscopy.122 Because the specimens obtained from standard transbronchial lung biopsies lack sufficient quantity and quality due to crush artifact, flexible cryoprobes have been used in therapeutic bronchoscopy. These techniques are invasive and require special hospital procedures generally done under local or general anesthesia.

Bronchoalveolar Lavage f o r Analysis of Biomarkers of Lung Injury The BAL technique has been employed successfully in humans and in laboratory animals to sample lung lining fluid to determine a variety of injury and inflammation markers. In humans, BAL often is performed in conjunction with the use of a bronchoscope. After adequate sedation, a bronchoscope is advanced until wedged in a desired subsegmental bronchus at the desired loca-

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tion. 164 ' 165 Bronchial trauma needs to be avoided, especially for patients having hemorrhagic injury. At one time, generally 20 mL of sterile saline is infused with a syringe. The flow of saline is monitored through the bronchoscope tip. Then, the lavage fluid is withdrawn into a collection vessel by applying gentle suction (50 to 80 mmHg). This process is repeated with fresh saline infusion, followed by suction five or six times to collect a sufficient amount of fluid from a given lobular segment of the lung. Generally, 40% to 70% of the fluid is recovered. Ninety-five percent of sampled individuals experience no complications following lavage. However, occasionally cough, transient fever, or transient bronchospasm with a decrease in baseline partial pressure of oxygen occurs in nearly 5% of patients. Patients with persistent pneumonia, diffuse lung diseases, and alveolar hemorrhage are recommended for lavage. Lung lavage is also performed for clinical studies. This process can be diagnostic for infections, malignancies, and various types of inflammation. Generally, nearly 80% macrophages, 3% neutrophils, 1% to 2% eosinophils, and 4,16M (Figure 9.1).

GAPS IN T R A D I T I O N A L HEPATIC BIOMARKERS • Although ALT is considered the "gold" standard biomarker of liver injury, it has limitations as a specific predictor of acute liver injury. Serum ALT can increase in the absence of hepatocyte necrosis and in the presence of metabolic disease such as type 1 diabetes, NAFLD, or even skeletal muscle associated disorders. • No biomarker has been demonstrated to predict the potential of a compound to induce DILI-2 or determine the susceptibility of patients to develop DILI-2. • No biomarker differentiates between drugs that induce liver failure versus drugs that initiate transient liver injury despite continued drug treatment.

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FIGURE 9.1 Traditional and novel biomarkers of DILI. Widely employed historical predinical and clinical biomarkers include serum transaminases (ALT, AST) known to detect hepatocyte injury, while serum ALP andT bili are used to assess cholestatic or biliary injury. To address the gaps in the specificity of these traditional biomarkers, ongoing efforts are generating new biomarkers. Emerging enzyme biomarkers such as PON I, PNR MDH, and GLDH are being tested preclinically through a PSTC consortium and preliminary data shows promise. Furthermore, novel biomarkers continue to emerge based on exploratory studies reported in literature, although these analytes need additional testing. While efforts are ongoing to assess the usefulness of these emerging and new biomarkers, there is a need to test these analytes both in the predinical and clinical space to address translatability. In addition, the assay technologies and platforms employed to detect these biomarkers are also discussed in detail in this chapter

Nevertheless, ALT elevations alone still guide regulatory decisions in clinical trials to protect patient safety, even with some limited degree of uncertainty regarding what constitutes true injury. In this review, new hepatic biomarkers that can potentially translate to clinic to detect DILI-1 will be discussed. While ongoing biomarker efforts are geared toward understanding the specificity of current hepatic biomarkers, additional studies are needed to fill the existing gaps.

C O N S I D E R A T I O N S T O P R E D I C T A C U T E LIVER INJURY: A N A T O M Y A N D T I M E - C O U R S E Regarding the histologic designation of injury, biomarkers can differentiate between hepatocellular and biliary injury. Biomarkers to address regiospecific histologic change (e.g., centrilobular verses periportal) would certainly benefit compound developmental decisions, but these would not be considered nearly as critical as the biliary versus hepatocellular distinction. The time com-

TRANSLATIONAL BIOMARKERS OF ACUTE DRUG-INDUCED LIVER INJURY

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FIGURE 9.2 In spite of extensive studies to generate novel hepatic biomarkers, there appears to be a need for additional biomarkers. Although traditional and new hepatic biomarkers can detect hepatic injury, none of these address hepatic injury mechanisms. In addition, these markers do not assess hepatic regeneration, especially considering the role of hepatic regeneration/adaptability in the causation of DILI 2. The biomarkers listed in this figure are being assessed to address these gaps. While hepatic injury markers appear to be sensitive, based on the biology of these enzymes, additional information can also be obtained by refining the assays used to analyze these markers.

ponent for predictable liver injury is critical since developmental compounds and toxicants show effects that are both dose- and time-dependent and can not be readily ascertained prior to study execution. Biomarkers that show a relationship to the time course of liver injury would be extremely valuable for decision making when selecting compounds from a pool for further development. Truly predictive markers of liver injury have not yet been discovered and substantial evidence does not yet support or rule out their existence, although a consensus exists that these investigations would be resource intensive and difficult to perform. By predictive, we refer to biomarker changes that precede or are prodromal to anchored histopathology observations. For example, an algorithm of genomic markers of renal injury that are predictive at day five for injury manifested at day 28 have been reported, although the set of transcripts does not correlate to known disease mechanisms nor is their performance rigorously uniform across multiple platforms, which is a great limitation of such approaches.34 Soluble markers from serum and perhaps urine are translatable to the clinic, while genomic markers show mostly limited utility in this regard and will not be discussed further. In addition to biomarkers of degeneration and necrosis, biomarkers of reactive metabolites, inflammation, and recovery/liver regeneration are critical to obtain a comprehensive view of the relationship between dose and onset of hepatic injury, which will be discussed below.

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N E W A N D EMERGING SERUM E N Z Y M E B I O M A R K E R S OF LIVER INJURY To address the gaps in traditional hepatic biomarkers and to ensure efficient and effective drug discovery and development, it is critical that specific novel hepatic biomarkers are discovered and implemented. Both the pharmaceutical industry and regulatory agencies recognize the value in the development of novel biomarkers of drug toxicity and safety. The EMEA35 and the FDA36"38 have recently developed processes to guide the qualification of preclinical and clinical biomarkers for regulatory purposes. Pharmaceutical companies are working collaboratively with these regulatory agencies in consortia, like the Drug-induced Liver Injury Network (DILIN), the Innovative Medicines Initiative (Evil), and the Critical Path's Predictive Safety Testing Consortium (PSTC), with the common goal of identifying new biomarkers and qualifying both new and recognized biomarkers that are commonly employed in practice. These efforts will potentially benefit the development and subsequent acceptance of more reliable biomarkers of hepatic toxicity that can be utilized to monitor safety concerns in regulated preclinical and clinical studies. The utility and acceptance of these novel biomarkers will depend, to some extent, on their application across key model preclinical species and translation to humans, their presence in easily accessible tissues and/or biofluids such as blood or urine, and the ability for rapid analytical quantitation that sensitively and reproducibly correlates with well-defined preclinical histomorphologic changes. As mentioned previously, biomarkers of hepatic injury should be specific to liver, and outperform or add information to ALT and/or AST measurements.39-42

D i s c o v e r y and A p p l i c a t i o n of Purine Nucleoside Phosphorylase (PNP), Paraxonase ( P O N - I ) , and Malate Dehydrogenase ( M D H ) as Hepatic Biomarkers Utilizing proteomics as an approach to discover and develop novel biomarkers of hepatotoxicity is well documented in the literature.43-44^7 Three novel serum biomarkers, malate dehydrogenase (MDH), purine nucleoside phosphorylase (PNP), and paraoxonase (PON-1) were identified by proteomic methods as serum biomarkers associated with rat liver toxicity or hypertrophy48 (Figure 9.1). These authors used a series of archetypal hepatotoxicants to model specific modifications to the liver that are often encountered in safety evaluation studies and then searched for chemically induced alterations in the expression of highly specific gene products.48 Of particular interest in this study was the identification of biomarkers in the peripheral circulation that were quantitatively altered after exposure to liver toxicants. After dosing rats with either acetaminophen, a-naphthylisocyanate, Phenobarbital, or Wyeth (Wy)-14,632, four compounds that target the liver through different mechanisms, proteomic analysis of sera was completed and 19 possible biomarkers of altered liver function that correlated with actual hepatic effects were identified. After a critical evaluation of the 19 potential biomarkers, MDH, PNP, and PON1, were


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identified as proteins having the greatest potential to serve as early indicators of hepatotoxicity.48 Selection was based on a set of criteria which included a protein expression change at an early time point because of the potential for sensitivity, a known function or toxicity indication in published studies, origin primarily or exclusively in the liver, expression in human as well as rat, and the potential for adaptation of the analytical method to clinical chemistry autoanalyzers to allow rapid quantitation of enzymatic activities.48 Purine nucleoside phosphorlyase (PNP), a key enzyme in the purine salvage pathway, reversibly catalyzes the phosphorolysis of nucleosides to their respective bases and corresponding l-(deoxy)-ribose-phosphate. PNP is located mainly in the cytoplasm of endothelial cells, Kupffer cells, and hepatocytes and is released into hepatic sinusoids during necrosis. Serum PNP has been shown in the literature to be correlated with liver injury in the rat after treatment with galactosamine.49 Rat serum activities of PNP were also increased earlier than ALT following endotoxin treatment that resulted in cellular necrosis.50 Concurrent increases of PNP and ALT are indicative of hepatocyte damage following administration of several hepatotoxins.50 However, since ALT is found exclusively in hepatocytes and PNP is localized in hepatocytes, sinusoidal endothelial cells, and Kupffer cells, investigators proposed that PNP leakage may be a reliable marker of nonparenchymal cell injury in the liver when no concurrent ALT leakage is present.50 This premise was tested by measuring ALT and PNP concurrently after treatment with galactosamine and lipopolysaccharide. This study demonstrated that an elevation in PNP activity was an indicator of nonparenchymal cell damage in the absence of a change in ALT activity.49

P O N I Is a Functional Marker of C h r o n i c Liver Injury Paraoxonase-l (PON1) is a high density lipoprotein (HDL)-associated esterase secreted mainly by the liver that detoxifies organophosphates and protects low density lipoproteins from oxidative modifications. PON1 is released into normal circulation bound to HDL and it is a decrease in serum PON1 that is indicative of liver tissue damage. This is likely due to a reduction in PON1 synthesis and less secretion by the liver into blood.51 Decreases in serum PON1 have been reported after dosing male rats with phenobarbital52 and after endotoxin treatment in male hamsters.51 Decreases in PON1 have been linked clinically to chronic hepatic injury,53,54,55,56 but also to a number of other disease states including atherosclerosis57,58 and vasculitis.59 PON1 activity in humans displays a polymorphic distribution and can vary within a given population.60,61 Notwithstanding the variability in PON1 levels, a significant decrease (27%) in PON1 in patients with hepatosteatois was observed when compared to healthy controls,53 and PON1 was useful when testing patients for chronic hepatitis and cirrosis in conjunction with the following standard liver function tests: albumin, ALT, GGT, ALP, and bilirubin.54 PON1 has high diagnostic accuracy when distinguishing patients with liver disease from control subjects, increases the overall sensitivity without affecting specificity, and has a diagnostic accuracy equivalent to that of ALT in patients with chronic hepatitis.54

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Malate Dehydrogenase (MDH) Activity Is a Candidate Biomarker of DILI-1 Malate dehydrogenase (MDH) catalyzes the reversible conversion of malate into oxaloacetate utilizing NAD+ and is a constitutive enzyme in the citric acid cycle. MDH is a periportal enzyme that is released into the serum indicating tissue damage. While localized in two cellular compartments, the cytoplasm and the mitochondria, the enzyme found in the serum from leakage is primarily from cytoplasmic storage of the enzyme.41 Serum MDH has been reported as correlated with liver injury in the rat after treatment with acetaminophen,62 and thioacetamide.63 Clinically, this enzyme has been reported to be a useful measurement for estimating the severity of liver diseases64 and higher levels have been shown in cirrhotic patients when compared to noncirrhotic cases.65

Biomarker Qualification by the Predictive Safety Testing Consortium (PSTC) MDH, PNP, PON1, as well as glutamate dehydrogenase (GLDH) which has been considered for some time a sensitive measure of hepatotoxicity,66'67 are currently undergoing a regulatory qualification process being sponsored by the PSTC. These markers have been brought forward as putative safety biomarkers within the PSTC membership on the basis of evidence from peerreviewed literature and internal datasets. The sponsored qualification of these markers by the PSTC will include multi-site and company characterization and validation of technical attributes for each assay, an evaluation of the biological relevance, e.g., added value relative to aminotransferase activity, correlation in the preclinical setting to standardized histopathologic observations, and an extended evaluation in clinical settings with regulatory guidance. This qualification is anticipated to provide a substantial understanding of the performance characteristics for these biomarkers and determine how they might add value to currently employed markers in the detection and monitoring of hepatotoxicity in preclinical and clinical settings.

ALT I S O Z Y M E S : A L T I A N D ALT2 Historical Background of ALT Biology Alanine aminotransferase activity is a marker of hepatoxicity in humans, dogs, and rats based on high ALT levels in liver compared to other tissues.68 Although the liver contains greater levels of ALT, it is also present in skeletal muscle, heart, fat, intestines, and brain of rats, dogs, and humans.6871 The widespread distribution of ALT is probably related to the importance of this enzyme in carbohydrate and amino acid metabolism. Increases in liver ALT have been seen in conditions in rats that favor gluconeogenesis, such as in-


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creased protein intake, fasting, caloric restriction, diabetes, and treatment with corticosteroids.72'73 The exact origin of steady state ALT that is present in serum is not well understood, but is probably a result of normal cell senescence and associated release of enzyme. Historically, ALT increases in the circulation have been thought to be a markers of cellular necrosis, but there is some evidence that it can also be released from intact cells via a process referred to as membrane blebing during injury processes.74 These cytosolic packages of enzymes can then be released into the blood circulation with cellular injury and not reflecting necrosis per se.75 In drug development, significant increases in serum ALT without correlated histological evidence of hepatic necrosis or other organ injuries is observed infrequently. Thus, serum ALT may be increased without associated liver injury, which would indicate an adaptive response, which is not considered an adverse event.7677 Using classic biochemical and cellular fractionation techniques, it has been shown there are at least two different isoenzymes, and perhaps more, of ALT in rats.78 Historically, several investigators have shown that the major ALT isoenzyme is a soluble cytosolic form, but based on cellular fractionation, 4-20% of the ALT enzyme is mitochondrial in origin.78-80 Discrepancies in the amount of mitochondrial ALT form measured may be related to instability of this form of the enzyme. It has been shown that the ALT enzyme can be stabilized by glycerol or dimethylsulfoxide, which will ensure that activity is preserved.7881 Both ALT isoenzymes catalyze the reversible reaction of the transfer of the alpha amino group of alanine to the alpha keto group of ketoglutaric acid to form pyruvate and glutamate, and thus play an important role in amino acid metabolism and gluconeogenesis (Figure 9.3). The mitochondrial form favors the forward ALT reaction, thus favoring the pathway that can promote gluconeogenesis.78 It has also been shown that glucocorticoids, which stimulate gluconeogenesis, will increase the amount of the ALT mitochondrial form in the rat and mouse liver.78'82 The mitochondria form could also be purified from other tissues such as the pig heart.80 Until recently there have been limited tools for measuring and better understanding how these different forms of enzymes react to hepatocellular injury and adapt to different metabolic states in animals and humans.

FIGURE 9.3

ALT enzymatic pathway. Forward and reverse enzymatic reactions are shown.

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Gene Expression of ALT Isoforms The genes encoding different forms of ALT have been identified in the mouse,83 rats,71 dogs,84 and human.7085 In humans, the original ALT gene that was cloned was designated GPT which was the cytosolic form and encodes a 495 amino acid protein (54 Kd).70,85 More recently, cloning and expression of a homolog, designated GPT2 (hALT2), encodes for a protein 523 amino acid (58 Kd) in length.70 GPT2 (hALT2) shares 78% identity to GPT (hALTl) and shows ALT activity in recombinant systems. Expression of hALT2 occurs in the muscle, kidney, liver, brain and fat, and hALTl is expressed in the kidney, liver, heart and fat. Recently, two protein coding products from hALT2, designated as ALT2_1 and ALT2_2 were discovered (58 and 47 Kd, respectively).69 Comparison of the rat, human and murine sequences shows that rat ALT1 (rALTl) shares 97% and 88% homology to the murine and human sequences, respectively, with greater conservation present in rat ALT2 (rALT2), which is 98% and 94% identical to its murine and human sequences, respectively.71 The rALT2 gene encodes an N-terminal 28-amino acid extension which is a likely mitochondrial targeting sequence.86 How these different genes and other transaminases are regulated is starting to be more comprehensively understood.8789

The Localization of ALT Protein in Tissues Quantification of ALT protein in a panel of tissues by quantitative western blot analysis using antibodies generated against recombinant rat ALT isoenzymes showed very similar distribution patterns as seen with gene expression.71 Rat ALT1 and rALT2 are highly expressed in the liver and muscle. Rat ALT1 is highly produced in the small intestine and less in the colon. In contrast, rALT2 showed minimal production in intestinal tissue. Rat ALT 1 production is higher than rALT2 in heart and fat. Brown fat expresses more rALTl than the white fat tissue, but rALT2 was greater in white fat than brown fat. There were also some sex differences in rats where there was about 400% and 20% higher levels of rALT2 in liver and muscle, respectively in males than in females. Recently rALTl and rALT2 have been measured in normal rat livers with a kinetic based assay which uses D-cycloserine to differentially inhibit ALT1 versus ALT2 activity.81 Recombinant rALTl and rALT2 proteins are used as standards to measure the isoenzymes. Using this approach it has been determined that in 30 normal rat livers there is 134-381 and 26-118 mU/mg of rat ALT1 and ALT2 activity, respectively. It was shown by western blot that the majority of ALT in the liver is ALT1, which is present at an approximately seven-fold greater concentration than ALT2.71 Rat ALT2 is enriched 20-fold when rat liver mitochondria are isolated by subcellular fractionation. Distribution of human ALT proteins in tissue was characterized by western blot and immunohistochemistry using polyclonal antibodies against peptides of hALTl or hALT2.69In addition to the expression in human liver, hALTl was highly produced in skeletal muscle and kidney and lower levels in cardiac muscle and not in pancreas. In contrast, high levels of ALT2_2


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were found in cardiac and skeletal muscle, but not in kidney or liver, although recent unpublished data suggests that ALT2_1 is expressed in human liver (Reagan, WJ, unpublished observations). It has not been reported if ALT2_2 has enzyme activity. Immunohistochemistry results support data obtained by western blot.

ALT P R O T E I N LEVELS I N SERUM ALT isoenzymes in the serum of normal rats, mice, and humans has been detected by western blot,71 immunoprecipitation activity assays,69 and preliminary kinetic based assays. Using all three techniques it has been shown that the predominant form of ALT in the serum is ALT1. Via immunoprecipitation, 74-91% of normal ALT activity in human serum was depleted with the ALT1 specific antibodies.69 In contrast with the antibodies to hALT2, only 4-18% of the total ALT activity was depleted. Western blot analysis showed none or minimal amounts of ALT2 present in the blood of normal rats and mice.71

C U R R E N T K N O W L E D G E O N B I O L O G Y OF ALT Understanding the biology of ALT1 and ALT2 using new methodological tools is emerging. Experimental models of hepatoxicity in mice and rats with CCL4 or acetaminophen induced hepatotoxicity, respectively, have shown that ALT 1 and ALT2 are released into the blood.71 After a single dose of CCL4 given to mice, total serum ALT increased 66-fold as determined by a kinetic based assay with the ALT1 and ALT2 elevated 4.8-fold and 3.9-fold, respectively as determined by quantitative western blot.71 In rats treated with acetaminophen, total ALT activity increased 21-fold at 48 hours post dose, with ALT1 increasing four-fold and ALT2 increasing 16-fold. Total serum activity correlated better with protein levels of rALTl compared with rALT2. In contrast, there have been few in vitro and in vivo models tested to suggest the involvement of these isoforms in an adaptive response. Mice were treated with 25 to 75 mg/kg of dexamethasone for one to three days to induce a gluconeogenic state based on its ability to stimulate glycogen accumulation in the liver, but inhibit glycogen breakdown.82 Hepatic glycogen content peaked at 24 hr, with a 24-fold increase relative to controls and was elevated at 72 hr with a 19-fold increase. Total hepatic ALT levels were increased at 72 hr by 1.6-fold. Total serum ALT activity was increased twofold in the 75 mg/kg dose group at 24 hr. Elevations were due to ALT2 since hepatic ALT2 levels increased (up to three-fold) and ALT1 levels did not change from baseline as determined by quantitative western blot. There was no concurrent increase in serum GDLH, AST, or ALP at either time points nor any histological evidence of hepatic necrosis to suggest that the release of the ALT2 was associated with hepatocellular damage. Another study using methionine and a choline deficient diet as a model of non-alcoholic steatohepatitis given to mice was used to assess the changes in ALT isoenzymes.90 It was shown that after 12 wk of treatment there

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was a four-fold increase in serum ALT which correlated well to increases in ALT1 and ALT2 in the liver both at the protein and mRNA level with an approximate two times greater increase in ALT2 than ALT1 relative to control animals. The treatment induced hepatic steatosis with minimal inflammation, and no necrosis was found on liver histopathological examination. In this study there was no increase in hepatocyte apoptosis to explain the increase in the origin of the increase in serum ALT. Increases in ALT2 have also been noted in the livers of Ob/Ob deficient mice and increases in serum ALT in people have independently predicted type 2 diabetes.83,91 It is possible that this increase in human serum ALT is due mainly to ALT2. Clearly more studies need to be performed to understand the kinetics of ALT1 and ALT2 in different disease states in animals and man to determine how useful isoenzyme analysis may be to differentiate an adaptive versus cytotoxic state, but the evidence to date is intriguing that these types of assessments may be useful in an application to characterize drug-induced liver injury.

DOES M E T A B O L I C S Y N D R O M E I L L I C I T A C O N F L I C T I N G ALT S I G N A L FOR DILI? The incidence of metabolic syndrome associates with a variety of variables including: country of origin, ethnicity, and nutritional intake, and these patients are at increased risk for the development of cardiovascular disease (C VD) and type II diabetes. Plasma insulin and other components of metabolic syndrome show high correlation to simple waist measurement.92 Higher body mass index (BMI) associated with metabolic syndrome shows > 2 X ALT levels higher than upper limit of normal (ULN), while alcohol consumption also influences ALT elevations additively or even synergistically.93 Patients with metabolic syndrome showing ALT elevations might be falsely considered as individuals that show liver injury. Interestingly, obesity is reported to be associated with serum uric acid and ALT elevations.94 Based on these observations, serum uric acid associated with ALT elevations may be a potential signal that might differentiate metabolic syndrome from hepatotoxicity, although more investigations are recommended. No demonstrated relationship of uric acid elevations to liver injury has been reported in literature. Clearly, the high incidence of metabolic syndrome in the U.S. population underscores the complexity of new candidate drug evaluations in populations considered normal for first in human studies.

A N O R E X I A SHOWS METABOLIC INDICATORS OF LIVER INJURY I N C L U D I N G SUBTLE ALT ELEVATIONS Anorexic patients show a complex decrease in their glutathione homeostasis.95 Plasma glutathione levels are reduced and building blocks for its synthesis, homocysteine, glycine, and glutamine levels are elevated. Reduced capacity


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for detoxification is consistent with lower BMI and subtle ALT elevations in anorexic patients, revealing subtle liver injury.96 Drug-induced eating disorders have been documented,97 which can indirectly induce liver metabolic injury. Anorexia rarely induces acute liver insufficiency, where ALT > 50 X ULN in the absence of necrosis.98

BIOMARKERS OF BILIARY INJURY Histologic manifestations of biliary injury is usually accompanied by serum biochemical alterations including elevated T bili, alkaline phosphatase (ALP), gamma glutamyl transpeptidase/transferase (GGT) and 51 nucleotidase (5'-NT). In addition, total serum bile acids can also be elevated in cholestasis associated with biliary injuries in addition to hepatic function deficits. Other disorders, where elevations of serum bile acids is noted, include intrahepatic cholestasis of pregnancy (ICP),99 and gastro-intestinal disorders such as small intestinal bacterial overgrowth and pruritus.100 In addition to total serum bile acids (quantitative) used to assess biliary damage and hepatocellular function, there have been several reports on assessing individual bile acids as sensitive and specific markers of liver damage. In patients with steatohepatitis, levels of bile acids in the liver were elevated relative to control.101 Specifically, deoxycholic, chenodeoxycholic, and cholic acids were elevated in patients with steatohepatitis and liver damage. Elevation of serum bile acids can also be the result of xenobiotics inhibiting bile salt export pump (BSEP) transporter and subsequent liver damage as exemplified by nefazodone toxicity.102 Over 20 individual bile acids were detected in serum with CC14 and alpha-naphthylisothiocyanate (ANIT) treated rats using ultraperformance liquid chromatography-mass spectrometry.103 Untreated and CC14 and ANIT treated animals were discriminated by the unique bile acid profiles.103 Bile acids can be measured by liquid chromatography-electrospray tandem mass spectrophotometer or LC/MS/MS in a rapid one-step method that can be used for routine analysis.104

A U T H O R S ' O P I N I O N ON FUTURE BIOMARKERS OF LIVER INJURY, N O V E L A P P R O A C H E S , A N D PLATFORMS Reactive Oxygen Species (ROS) as Potential Markers for Liver Injury Mechanisms Oxygen free radicals are potent damaging agents to biomacromolecules and recent approaches are targeted toward measuring ROS in serum rather than within liver tissue. Total antioxidant response (TAR) can be measured in serum using a colorimetric clinical chemistry assay. Reduced uric acid, glutathione, and bilirubin in plasma suppresses color formation in a concentration dependent manner.105,106 For example, renal injury revealed reduced serum TAR levels,105' 106yet a broader evaluation taking into consideration other or-

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gan injuries has not been performed. TAR includes serum uric acid and its metabolites allantoin, 6-aminouracil, and triuret, which have been multiplexed in LC-MS/MS for urinary determination of purine metabolism endpoints and scavenging of singlet oxygen species107 (Figure 9.2). Measurement of serum uric acid and associated metabolites in a multiplex is recommended as a complement to TAR determinations. Also, metabonomic evaluation of urine samples by ultra-performance liquid chromatography/mass spectrometry UPLC/ MS and NMR reveal adenosylmethionine (SAMe) flux rate reductions and creatinine elevations in a panel of liver toxicity studies in rat.108 These urinary metabonomic data support the hapten carrier hypothesis stating that reactive metabolite formation and inability to detoxify are the initial steps of liver injury,108 which would be supported by more extensive investigations in blood. In addition to predicting early liver injury, potential mechanisms of DILI can also be potentially assessed by these approaches.

Inflammation Markers as Potential Indicators of Liver Injury Hepatic inflammation appears to be common to the pathogenesis and progression of both toxic and metabolic hepatic disease. Hepatic inflammation can manifest as a histologic change with inflammatory cell infiltration or increased proinflammatory cytokines in the hepatobiliary milieu. Although such hepatic inflammation exists, there may not be serum biochemical changes such as elevated transaminases or parameters that detect lack of hepatic function. Thus, there is a need to develop reliable biomarker(s) to predict/detect hepatic inflammation states. An alternatively spliced and soluble form of TNF-ctreceptor 2 (TNFR2DS) can be measured by ELISA and discriminated from the proteolyzed form of membrane bound TNFR2.109 Multiple regression analysis of Caucasian Spanish populations revealed that ALT and AST elevations were inversely correlated to TNFR2-DS levels, independent of sex, age, body mass index, adiponectin, and homeostasis model assessment of insulin resistance.1M Although the function of TNFR2-DS is not well understood, it is hypothesized to be anti-inflammatory in nature by sequestering soluble serum TNFa.110 TNFR2DS is a potential biomarker to add information regarding inflammation state to a subtle preclinical ALT elevation in the absence of histopathology data (Figure 9.2). A better understanding of the TNFR2-DS signal preclinically is considered a prerequisite to application in a clinical translation model. The cytokine macrophage migration factor (MIF) mediates innate and adaptive immunity and contributes to inflammatory pathogenesis including rheumatoid arthritis (RA).111, " 2 MIF is constitutively expressed by macrophages and released into the blood by TNF and to a lesser degree by LPS.113 In addition to its role in innate immunity, MIF is also essential for adaptive immune response and recruits leukocytes to the site of inflammation. MIF is regarded as a general marker of inflammatory response and a possible target of therapeutic intervention.111112


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MIF is constitutively expressed in liver and is released to serum in patients with hepatitis.114 Idiosyncratic drug-induced liver injury may depend upon the balance between pro- and anti-inflammatory mediators. Using a guinea pig model of liver injury induced by halothane exposure, the level of liver toxicity associated with an increase in serum MIF.115 MIF leakage from the liver into the sera preceded peak increases in toxicity following APAP administration and MIF null (-/-) mice were less susceptible to this toxicity at early times and knock-out mice showed improved survival compared to wild type115 (Figure 9.2). MIF was demonstrated as a pro-toxicant signal in drug-induced liver injury and further studies are recommended. Thus, MIF is a candidate biomarker to monitor the progression of liver inflammation, yet lacks specificity for the origin of the inflammation source. Acute phase proteins can also be potentially employed for predicting hepatic inflammation. Acute phase proteins are induced by proinflammatory cytokines such as TNF, interleukin-6 (IL-6), IL-11, and IL-17, and include both the positive (increase with inflammation such as C-reactive protein in human) and negative phase (decrease with inflammation such as albumin) proteins (Figure 9.2). Both the local and systemic inflammation can alter acute phase proteins with the response time being from a few hours to days. These acute phase proteins have a very rapid response with return to baseline and remain elevated for a sufficient time period with varied kinetics of responses between acute phase proteins. Importantly, striking species-specific differences in acute phase responses has been reported (e.g., alpha 2 macroglobulin specific for rat, serum amyloid A for rat, and CRP for humans) which makes clinical translation quite challenging.116 Although the alterations of these acute phase proteins, MIF, and TNFa-DS may not be specific to the liver, they can be applied to hepatic inflammation when the investigator has ruled out inflammatory component in other organs (Figure 9.2). The role of osteopontin (OPN) in hepatic inflammation processes has been reported during alcoholic liver disease (ALD).117-120 The native and thrombincleaved form of OPN was induced within hepatocytes in the rodent ALD model with a strong correlation between cleaved form of OPN and hepatic neutrophil infiltration.121 These studies implicate OPN as an important chemotactic factor in the pathogenesis of hepatic inflammation during ALD. In the same study, the authors showed that uncleaved and cleaved OPN upregulated CDllb/CD18, which correlates quantitatively with neutrophil infiltration (Apte, et al., 2004; Banerjee, et al., 2006). Furthermore, a time course study also showed OPN to be an important early biomarker that can predict hepatic inflammation.117'121

Novel H e p a t o c e l l u l a r Leakage Enzymes as Early Biomarkers of Symptomatic Change Argininosucccinate synthase (ASS) and estrogen sulfotransferase (EST-1) are leakage enzymatic markers that have been shown to be significantly elevated in serum within hours following ischemia122 (Figure 9.1). ASS and EST-1 were not detected during chronic liver injury, while ALT elevations were profound.

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BIOMARKERS

ASS was elevated with an acute toxicant six hours after insult, where ALT elevations were not yet detected. ASS is reduced in serum during cirrhosis of the liver.123 Although the data is intriguing, additional extensive evaluation of these markers is recommended with multiple hepatotoxicants and metabolic disease to assess their hepatic biomarker value. An early predictive set of markers within hours of injury, rather than days, would be a valuable tool for the lead optimization of developmental compounds.

Hepatic Regeneration Markers to Supplement Injury Biomarkers Analysis of traditional circulating biochemical markers of hepatic injury such as ALT and AST may not be adequate to assess the extent of hepatic damage. A decrease in the serum values for these leakage markers after drug-induced liver injury could be due to recovery from damage and associated increase in hepatic regeneration. A sustained decline in hepatic injury markers with a concurrent elevation in regenerative markers could potentially aid in the favorable outcome in clinical patients with acute liver injury. It is reported that analysis of hepatic growth factors using serum markers such as alpha fetoprotein (AFT*), retinol binding proteins, and des-gamma carboxy-prothrombin to be useful markers of hepatic regeneration in clinical situations such as with Amanita mushroom and acetaminophen toxicities, although additional studies are required to validate these biomarkers in hepatic regeneration models (Figure 9.2). Calcium metabolism and excretion is regulated in rodent bile duct systems by the Ca2+-binding protein regucalcin, which is expressed predominantly in kidney as well as liver.124 Over expression of hepatic regucalcin suppresses cell death and apoptosis making it a biomarker candidate of liver injury and regeneration.124 An acute D-galactosamine and lipopolysaccharide (GalN/ LPS) model of liver injury in mice revealed regucalcin serum elevations.125 CC14 treated rats showed two-fold ALT and AST elevations three-days posttreatment, whereas regucalcin serum elevations were observed 30-days posttoxicant treatment.126 Transient biomarkers of liver injury are a concern in clinical trials where sample collection may be as infrequent as monthly. Regucalcin elevations early after dosing are nearly 20-fold greater compared to one month later,126 nevertheless the detection of injury one month after dosing is a powerful signal to monitor reversible injury (Figure 9.2). Translation of regucalcin as a marker of liver injury has not yet been performed, but regucalcin is 93% conserved between rat and human. Although clinical threshold determinations for regucalcin elevations will be complex to determine and qualify, this marker would be a compelling addition to other available liver injury markers.

U N I F I C A T I O N OF D I A G N O S T I C METRICS OF LIVER FIBROSIS Liver fibrosis is a complex cascade of tissue remodeling following acute injury and repair that has been extensively investigated, although a unification of


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these observations has not yet been performed across pharmaceutical, medical, and academic centers of excellence. Consortia are initiating an exchange among many groups and there is great optimism for these new efforts. Serum soluble collagen fragments associated with liver fibrosis have been a focus of investigations for many years. The amino-terminal procollagen type III peptide is released into blood during collagen type III deposition in hepatic fibrosis, although this process is observed in other tissues that show similar disease progression. Elevations in PIIINP in rat serum were observed after just two weeks of CC14 treatment using RIA approaches, where hepatic tissue hydroxylproline was observed after six weeks of toxicant treatment.127 ELISA approaches detect PIIINP in bile duct ligation and dimethylnitrosamine models of liver fibrosis that develop one week after dosing.128'129 In chronic hepatitis C patients with biopsy examination, patients were screened with a large panel of biomarkers. PIIINP and MMP-1 combined showed diagnostic value in receiver operator curves (ROC) and logistic model analysis (Leroy score).130 Nearly 20 additional clinical numerical scores for liver fibrosis have been characterized using a diverse and broad collection of functional serum markers, clinical chemistry markers such as ALT, and clinical endpoints. Each score uses an independent algorithmic approach to assess clinical liver fibrosis injury.131 Notably, many of these scores contain overlapping biomarkers, although a comparison across the various scoring metrics has not yet been performed. A fibrosis multiplex containing PIIINP, hyaluronic acid, TIMP-1, a2-microglobulin, and MMP-1 would allow direct comparison of several tests including Leroy, Rosenberg, Patel, and Fibrometer.130,132-134 Early markers of liver fibrotic injury would be valuable in clinical trials that are lengthy with strict limits to the availability and frequency of patient biopsy. In CC14 treated rats, serum PIIINP elevation was observed after five to seven weeks, yet PIIINP mRNA was elevated 10-fold after one week.135 Whether a liver tissue RNA based clinical fibrosis model would perform better than the more traditional models is an approach to consider. PIIINP is an early predictor for joint destruction in RA, indicating that specificity is broader than for liver injury alone.136

A N A L Y T I C A L BIOMARKER PLATFORMS T O ASSAY SERUM BIOMARKERS OF LIVER INJURY Mass Spectrometry Technologies Can Fill Gaps to Detect Biomarkers When Antibody Approaches are Limited Immunoanalytical approaches to detect biomarkers, including MesoScale Discovery and Luminex (Millipore or Rules Based Medicine) are powerful approaches that allow multiplexing and parallel detection of analytes in a high throughput manner. These immune assays are also complex to analytically characterize and combine in a multiplex format since antibody interference or cross reactivity among assays might occur. Batch to batch variation in antibody reagent development can occur, making standardization approaches

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an extensive process when replenishing stocks from commercial sources. Immune reagents are often species specific and do not readily translate across preclinical species to human, although exceptions are noted. Analytical approaches, including HPLC coupled with tandem mass spectrometry (LC/MS/ MS), offer an attractive alternative to conventional antibody based approaches, at considerable equipment cost, maintenance, and required expertise.

An O v e r v i e w of Mass S p e c t r o m e t r y Technologies A tandem mass spectrometer has two mass analyzers. The mass analyzers are separated by a collision cell into which an inert gas (e.g., argon, nitrogen) is admitted to collide with the selected sample ions and bring about their fragmentation. The analyzers can be of the same or of different types, the most common combinations being quadrupole-quadrupole (or triple quadrupoles denoted as Qq-Q) and quadrupole-time of flight (denoted Qq-TOF or Q-TOF), where Q refers to a mass-resolving quadrupole, q refers to a collision cell, and TOF refers to a time-of-flight mass spectrometer. The selectivity of ion scans is superior on Q-TOF systems compared to triple quadrupoles because the high resolving power of the TOF mass analyzer permits high-accuracy fragment ion selection at no expense of sensitivity. This minimizes interferences from other peptide fragment ions (a-, b-, and y- type) of the same nominal mass but with sufficient differences in their exact masses. Triple quadrupoles, however, still remain the gold standard for targeted quantitative analysis (i.e., SRM analysis) due to their increased sensitivity and dynamic range compared to Q-TOF (Table 9.1).

Mass S p e c t r o m e t r y Technologies t o Potentially D e t e c t Biomarkers and Rare Protein Antigens of Injury Reports47 utilized Q-TOF technology for characterization of proteomic biomarkers of hepatotoxicity in rat liver, investigating the effect of carbon tetrachloride, acetaminophen, amiodorane, and tetracycline on protein elevations in the liver in vivo. Eight proteins were affected by the four toxicants including carbonic anhydrase III, 60 kD heat shock protein, glutamate dehydrogenase, adenylate kinase isoenzyme 4, NADP-dependent malic enzyme, 2-oxoisovalerate dehydrogenase, serotransferrin and N-Myc down regulated Gene 1, or NDRG1 related protein. A more common quantitative approach, utilized successfully for the quantitation of hidden allergenic peanut proteins, involves characterization and identification of proteins or peptides by Q-TOF and then subsequent targeted quantitation or single reaction monitoring (SRM) of select peptides using a triple quadrupole.137

Improved Tagging Techniques f o r Mass S p e c t r o m e t r y D e t e c t i o n of Proteins A new and novel mechanism helping the targeted SRM approach to quantitate peptides and proteins is the use of tagging reagents, such as the isotope coded


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TABLE 9.1 Comparison of mass spectrometry approaches. LC/MS/MS is the reference standard method for targeted analysis.

Quadrupole time of flight (Q-TOF)

Typically used for qualitative purposes (i.e., analyte scouting), taking advantage of the instrument's fast scanning speed and large dynamic mass range (for intact protein analysis)

Triple quadrupoles

Typically used for quantitative purposes (i.e., targeted quantitation) taking advantage of the instrument's sensitivity and quantitative dynamic range.

Ion trap hybrid (LTQ Orbitrap)

New technology recently introduced byThermo typically used for qualitative purposes. System has a lower dynamic mass range than Q-TOF, but generally a more powerful tool for smaller proteins or peptides < 4000 atomic mass units due to its high resolving power and accurate mass capabilities.

affinity taq (ICAT), isobaric tag peptide labeling (iTRAQ), and stable isotope labeled reagents (mTRAQ). Each labeling technique has its own distinct advantages, for instance ICAT is advantageous for cysteine containing peptides and proteins. Similar to ICAT, iTRAQ is based upon chemical tagging of N-terminus peptides generated from protein digests that have been isolated from cells in two different states. However iTRAQ suffers the same peptide overabundance problem and must be coupled with one or more dimensions of chromatographic or electrophoretic separation before MS analysis to limit the number of isobaric tagged peptides in the first MS dimension. mTRAQ is a non-isobaric variant of iTRAQ and is available in two labels. The ability to label the sample and reference peptides with either one of the two possible combinations is an inherent advantage of this method, as it provides a means for verification of the reported ratios. mTRAQ was used for absolute quantitation of the potential cancer marker pyruvate kinase M1/M2 in normal and diseased endometrial tissue.138

H i g h - t h r o u g h p u t C h r o m a t o g r a p h y Enhances the D o w n s t r e a m D e t e c t i o n of Rare Serum Proteins by Mass S p e c t r o m e t r y To characterize serum proteins, micro-chromatography approaches allow fractionation and removal of albumin and immunoglobulins from samples, allowing subsequent quantitation of trypic peptide analytes in liquid chromatography coupled to tandem mass spectrometry with electrospray ionization (LC/ ESI/MS/MS) for biomarkers that are in low ng/mL range.139 Use of disposable columns make this a high throughput approach to characterize analytes in the 1 ng/mL range, and a single antibody allows confirmation of the tryptic peptide rather than the two antibodies required for development of a sandwich as-

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say.139 Accurate measurement of low abundance peptides can also be detected by LC-ESI-Qq-TOF.139,14° Nearly all of the current biomarkers of liver injury discussed here have peptides that have been detected using this technological approach as part of serum proteome mapping approaches141'142 and personal communication, J. Marshall.

Mass S p e c t r o m e t r y Approaches t o Distinguish Novel Biomarkers of Renal and Liver Injury Cyclosporin A treatment blocks bile acid efflux resulting in concomitant cysteinyl leukotriene accumulation in kidney, thereby reducing glomerular filtration rate (GFR) and decreasing renal functional capacity.143 Bile acid accumulation in serum is an early marker for such injury and recent technology advances allow individual bile acid and conjugate measurements in addition to traditional total bile acid values from clinical chemistry methods. Ursodeoxycholic acid and conjugates are multiplexed in human plasma using a straightforward LC/ MS/MS methodology.144 A more comprehensive quantitative bile acid and conjugate profile in mouse plasma was demonstrated in LC/MS/MS indicating that detection of individual bile acids is routine enough to measure in drug development processes.145

Mass S p e c t r o m e t r y Approaches t o D e t e c t Novel Serum Biomarkers of Liver Injury Several candidate biomarkers of liver injury have been preliminarily characterized but have limited antibody reagent availability,41 making further analysis on these new platforms an attractive approach to the wider scientific community. Regucalcin, sorbitol dehydrogenase (SDH), hpd or 4-hydroxyphenylpyruvate dioxygenase (Serum Protein F), and ALT isoforms are all potential biomarkers of DILI-1 that are of high interest to investigators, yet a lack of available antibody reagents precludes further study. Since a large panel of serum protein are amenable to this approach using a standardized chromatography approach,141,142 mass spectrometry based proteomic approaches should be considered to develop multiplexes of proposed markers of DILI-1. Integration of multiple platforms for biomarker detection increases the rigor and confidence of both platform approaches. LC/MS/MS allows unprecedented quantitation of analytical standards for standard curve preparation, which cannot be as rigorously measured by antibody reagents alone. Having preliminary or validated antibody assays greatly reduces the cycle time for LC/MS/ MS assay development. Integration of technologies provides the most rapid and rigorous outcome for novel assay development.

CONCLUSIONS DILI is a complex injury process with multiple mechanisms and manifests either as intrinsic or idiosyncratic type. Idiosyncratic DILI is rare and is observed with marketed compounds clinically, while intrinsic DILI is observed


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throughout the entire drug development pipeline. Currently, ALT is considered the reference marker of predictable DILI, and in combination with T bili (in clinics), the reference marker for idiosyncratic DILI. While ALT is a sensitive biomarker of intrinsic DILI, additional biomarkers are sought to add value and enhance specificity. New serum enzyme biomarkers are being qualified to improve intrinsic DILI detection. Also, additional strategies under evaluation to add specificity to ALT include measuring ALT isoforms and individual bile acids. In addition to improving the value of ALT by the qualification of new hepatic biomarkers , there is also the need to understand precise mechanisms for DILI with the aid of undiscovered biomarkers. Parallel efforts in the area of new platform technologies will no doubt increase analytical rigor to assess DILI in conjunction with current technologies.

ACKNOWLEDGMENTS We sincerely thank PSTC Hepatic Working Group members and Pfizer Hepatic Biomarker Team for helpful discussions. We also thank Denise Robinson-Gravatt, Dale Morris, and Timothy Heath who provided constructive comments on the chapter; Rod Mathews, Jeff Prasakiewicz, and Jon Klover for helpful discussions on hepatic fibrosis; John Marshall for input on MS technologies and proteomics.


3.

4. 5.

Traditional hepatic biochemical parameters (serum transaminases; ALT, AST) and histology fail to discriminate between DILI-1 and -2. Significant biomarker gaps exist with the traditional preclinical and clinical biomarkers of DILI-1. These gaps are being addressed by emerging serum enzyme biomarkers which are evaluated in a qualification process with regulators to add value to serum aminotransferases. Isozymes ALT1 and ALT2 are being analyzed to increase the specificity of ALT in response to extrahepatic injury and gluconeogenesis. New biomarkers such as paraxonase 1 (PON1), purine nucleoside phosphorylase (PNP), malate dehydrogenase (MDH), and glutamate dehydrogenase (GLDH) are also being qualified to add value to serum aminotransferases. New biomarkers of hepatic oxidant stress, inflammation, injury mechanisms, and regeneration will add significant value to understanding of DILI and predicting ultimate outcome of hepatic injury. Mass spectrometry technologies can fill gaps to detect biomarkers when antibody approaches are limited and are proposed to detect novel serum biomarkers of liver injury.

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78. 79. 80. 81. 82. 83. 84.

85. 86. 87. 88. 89.

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TRANSLATIONAL BIOMARKERS OF ACUTE DRUG-INDUCED LIVER INJURY 233 90. 91. 92. 93.

94.

95. 96. 97. 98. 99. 100.

101. 102. 103.

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Liu, L., Zhong, S., and Yang, R., et al. Expression, Purification, and Initial Characterization of Human Alanine Aminotransferase (ALT) Isoenzyme 1 and 2 in High-Five Insect Cells. Protein Expr. Purif. Aug 2008;60(2):225-231. Hanley, A. J., Williams, K., and Festa, A., et al. Elevations in Markers of Liver Injury and Risk of Type 2 Diabetes: The Insulin Resistance Atherosclerosis Study. Diabetes. Oct 2004;53(10):2623-2632. Bauduceau, B., Vachey, E., and Mayaudon, H., et al. Should We Have More Definitions of Metabolic Syndrome or Simply Take Waist Measurement? Diabetes Metab. Nov 2007;33(5):333-339. Alatalo, P. I., Koivisto, H. M., Hietala, J. P., Puukka, K. S., Bloigu, R., and Niemela, O. J. Effect of Moderate Alcohol Consumption on Liver Enzymes Increases with Increasing Body Mass Index. Am. J. Clin. Nutr. Oct 2008;88(4): 1097-1103. Kang, Y. H., Min, H. G., Kim, I. J., Kim, Y. K., and Son, S. M. Comparison of Alanine Aminotransferase, White Blood Cell Count, and Uric Acid in Their Association with Metabolic Syndrome: A Study of Korean Adults. Endocr. J. Dec 2008;55(6): 1093-1102. Zenger, F., Russmann, S., Junker, E., Wuthrich, C , Bui, M. H., and Lauterburg, B. H. Decreased Glutathione in Patients with Anorexia Nervosa. Risk Factor for Toxic Liver Injury? Eur. J. Clin. Nutr. Feb 2004;58(2):238-243. Tsukamoto, M., Tanaka, A., and Arai, M., et al. Hepatocellular Injuries Observed in Patients with an Eating Disorder Prior to Nutritional Treatment. Intern. Med. 2008;47(16):1447-1450. Tanigawa, T, Higuchi, K., and Arakawa, T. Mechanism and Management of Drug-Induced Eating Disorder. Nippon. Rinsho. Mar 2001;59(3):521-527. Rautou, P. E., Cazals-Hatem, D., and Moreau, R., et al. Acute Liver Cell Damage in Patients with Anorexia Nervosa: A Possible Role of Starvation-Induced Hepatocyte Autophagy. Gastroenterology. Sep 2008;135(3):840-848, E841-843. Castano, G., Lucangioli, S., and Sookoian, S., et al. Bile Acid Profiles by Capillary Electrophoresis in Intrahepatic Cholestasis of Pregnancy. Clin. Sci. (Lond). Apr 2006; 110(4):459^165. Abrahamsson, H., Ostlund-Lindqvist, A. M., Nilsson, R., Simren, M., and Gillberg, P. G. Altered Bile Acid Metabolism in Patients with Constipation-Predominant Irritable Bowel Syndrome and Functional Constipation. Scand. J. Gastroenterol. 2008;43(12): 1483-1488. Aranhaa, M. M., Cortez, H., and Costad, A., et al. Bile Acid Levels Are Increased in the Liver of Patients with Steatohepatitis. European Journal of Gastroenterology & Hepatology. 2008;20(6):519-525. Kostrubsky, S. E., Strom, S. C , and Kalgutkar, A. S., et al. Inhibition of Hepatobiliary Transport as a Predictive Method for Clinical Hepatotoxicity of Nefazodone. Toxicol. Sci. Apr 2006;90(2):451^159. Yang, L., Xiong, A., and He, Y, et al. Bile Acids Metabonomic Study on the Ccl(4)- and Alpha-Naphthylisothiocyanate-Induced Animal Models: Quantitative Analysis of 22 Bile Acids by Ultraperformance Liquid ChromatographyMass Spectrometry. Chem. Res. Toxicol. Dec 15, 2008; 21(12):2280-2288. Tagliacozzi, D., Mozzi, A. F, and Casetta, B., et al. Quantitative Analysis of Bile Acids in Human Plasma by Liquid Chromatography-Electrospray Tandem Mass Spectrometry: A Simple and Rapid One-Step Method. Clin. Chem. Lab. Med. Dec 2003;41(12):1633-1641.

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BIOMARKERS 105. Erel, O. A Novel Automated Direct Measurement Method for Total Antioxidant Capacity Using a New Generation, More Stable ABTS Radical Cation. Clin. Biochem. Apr 2004;37(4):277-285. 106. Erel, O. A Novel Automated Method to Measure Total Antioxidant Response Against Potent Free Radical Reactions. Clin. Biochem. Feb 2004; 37(2): 112-119. 107. Kim, K. M., Henderson, G. N., and Frye, R. F., et al. Simultaneous Determination of Uric Acid Metabolites Allantoin, 6-Aminouracil, and Triuret in Human Urine Using Liquid Chromatography-Mass Spectrometry. J. Chromatogr. BAnalyt. Technol. Biomed. Life Sci. Jan 1, 2009;877(l-2):65-70. 108. Schnackenberg, L. K., Chen, M., and Sun, J., et al. Evaluations of the Trans-Sulfuration Pathway in Multiple Liver Toxicity Studies. Toxicol. Appl. Pharmacol. Feb 15, 2009;235(l):25-32. 109. Esteve, E., Botas, P., and Delgado, E., et al. Soluble TNF-Alpha Receptor 2 Produced by Alternative Splicing Is Paradoxically Associated with Markers of Liver Injury. Clin. Immunol. Apr 2007;123(l):89-94. 110. Baumel, M., Lechner, A., Hehlgans, T., and Mannel, D. N. Enhanced Susceptibility to Con A-Induced Liver Injury in Mice Transgenic for the Intracellular Isoform of Human TNF Receptor Type 2. / Leukoc. Biol. Jul 2008;84(1): 162-169. 111. Morand, E. F. New Therapeutic Target in Inflammatory Disease: Macrophage Migration Inhibitory Factor. Intern. Med. J. Jul 2005;35(7):419^26. 112. Morand, E. F. and Leech, M. Macrophage Migration Inhibitory Factor in Rheumatoid Arthritis. Front. Biosci. Jan 1, 2005;10:12-22. 113. Calandra, T, Bernhagen, J., Mitchell, R. A., and Bucala, R. The Macrophage Is an Important and Previously Unrecognized Source of Macrophage Migration Inhibitory Factor. J. Exp. Med. Jun 1, 1994;179(6):1895-1902. 114. Ohkawara, T., Nishihira, J., Takeda, H., Asaka, M., and Sugiyama, T. Pathophysiological Roles of Macrophage Migration Inhibitory Factor in Gastrointestinal, Hepatic, and Pancreatic Disorders. J. Gastroenterol. Feb 2005;40(2): 117-122. 115. Bourdi, M., Reilly, T. P., Elkahloun, A. G., George, J. W., and Pohl, L. R. Macrophage Migration Inhibitory Factor in Drug-Induced Liver Injury: A Role in Susceptibility and Stress Responsiveness. Biochem. Biophys. Res. Commun. Jun 7, 2002;294(2):225-230. 116. Schreiber, G., Tsykin, A., and Aldred, A. R., et al. The Acute Phase Response in the Rodent. Ann. NYAcad. Sci. 1989;557:61-85;Discussion 85-66. 117. Apte, U. M., Banerjee, A., McRee, R., Wellberg, E., and Ramaiah, S. K. Role of Osteopontin in Hepatic Neutrophil Infiltration During Alcoholic Steatohepatitis. Toxicol. Appl. Pharmacol. Aug 22, 2005;207(l):25-38. 118. Ramaiah, S., Rivera, C , andArteel, G. Early-Phase Alcoholic Liver Disease: An Update on Animal Models, Pathology, and Pathogenesis. Int. J. Toxicol. Jul/Aug 2004;23(4):217-231. 119. Ramaiah, S. K. A Toxicologist Guide to the Diagnostic Interpretation of Hepatic Biochemical Parameters. Food Chem. Toxicol. Sep 2007;45(9):1551-1557. 120. Ramaiah, S. K. and Rittling, S. Pathophysiological Role of Osteopontin in Hepatic Inflammation, Toxicity, and Cancer. Toxicol. Sci. May 2008;103(1):4—13. 121. Banerjee, A., Burghardt, R. C , Johnson, G. A., White, F. J., and Ramaiah, S. K. The Temporal Expression of Osteopontin (SPP-1) in the Rodent Model of Alcoholic Steatohepatitis: A Potential Biomarker. Toxicol. Pathol. 2006;34(4): 373-384.

TRANSLATIONAL BIOMARKERS OF ACUTE DRUG-INDUCED LIVER INJURY 235 122. Svetlov, S. I., Xiang, Y., and Oli, M. W., et al. Identification and Preliminary Validation of Novel Biomarkers of Acute Hepatic Ischaemia/Reperfusion Injury Using Dual-Platform Proteomic/Degradomic Approaches. Biomarkers. Jul/Aug 2006;ll(4):355-369. 123. Igarashi, M., Kawana, S., Iwasaki, H., and Namiki, A. Anesthetic Management for a Patient with Citrullinemia and Liver Cirrhosis. Masui. Jan 1995;44(1): 96-99. 124. Yamaguchi, M. Role of Regucalcin in Maintaining Cell Homeostasis and Function (Review). Int. J. Mol. Med. Mar2005;15(3):371-389. 125. Lv, S., Wei, L., Wang, J. H., Wang, J. Y, and Liu, F. Identification of Novel Molecular Candidates for Acute Liver Failure in Plasma of BALB/C Murine Model. J. Proteome. Res. Jul 2007;6(7):2746-2752. 126. Yamaguchi, M., Tsurusaki, Y, Misawa, H., Inagaki, S., Ma, Z. J., Takahashi, H. Potential Role of Regucalcin as a Specific Biochemical Marker of Chronic Liver Injury with Carbon Tetrachloride Administration in Rats. Mol. Cell. Biochem. Dec2002;241(l-2):61-67. 127. Schuppan, D., Dumont, J. M., Kim, K. Y, Hennings, G., and Hahn, E. G. Serum Concentration of the Aminoterminal Procollagen Type III Peptide in the Rat Reflects Early Formation of Connective Tissue in Experimental Liver Cirrhosis. J. Hepatol. 1986;3(l):27-37. 128. Cho, J. J. and Lee, Y S. Enzyme-Linked Immunosorbent Assay for Serum Procollagen Type III Peptide in Rats with Hepatic Fibrosis. J. Vet. Med. Sci. Nov 1998;60(11):1213-1220. 129. Gerling, B., Becker, M., Waldschmidt, J., Rehmann, M., and Schuppan, D. Elevated Serum Aminoterminal Procollagen Type-III-Peptide Parallels Collagen Accumulation in Rats with Secondary Biliary Fibrosis. J. Hepatol. Jul 1996; 25(l):79-84. 130. Leroy, V., Monier, F, and Bottari, S., et al. Circulating Matrix Metalloproteinases 1,2,9 and Their Inhibitors TIMP-1 and TIMP-2 as Serum Markers of Liver Fibrosis in Patients with Chronic Hepatitis C: Comparison with PIIINP and Hyaluronic Acid. Am. J. Gastroenterol. Feb 2004;99(2):271-279. 131. Gressner, O. A., Weiskirchen, R., and Gressner, A. M. Biomarkers of Liver Fibrosis: Clinical Translation of Molecular Pathogenesis or Based on Liver-Dependent Malfunction Tests. Clin. Chim. Ada. Jun 2007;381(2): 107-113. 132. Cales, P., Oberti, F, and Michalak, S., et al. A Novel Panel of Blood Markers to Assess the Degree of Liver Fibrosis. Hepatology. Dec 2005;42(6):1373-1381. 133. Patel, K., Gordon, S. C , and Jacobson, I., et al. Evaluation of a Panel of NonInvasive Serum Markers to Differentiate Mild From Moderate-to-Advanced Liver Fibrosis in Chronic Hepatitis C Patients. J. Hepatol. Dec 2004;41(6):935-942. 134. Rosenberg, W. M., Voelker, M., and Thiel, R., et al. Serum Markers Detect the Presence of Liver Fibrosis: A Cohort Study. Gastroenterology. Dec 2004; 127(6):1704-1713. 135. Kauschke, S. G., Knorr, A., and Heke, M., et al. Two Assays for Measuring Fibrosis: Reverse Transcriptase-Polymerase Chain Reaction of Collagen Alpha(l) (III) Mrna Is an Early Predictor of Subsequent Collagen Deposition While a Novel Serum N-Terminal Procollagen (III) Propeptide Assay Reflects Manifest Fibrosis in Carbon Tetrachloride-Treated Rats. Anal. Biochem. Nov 15, 1999;275(2): 131-140. 136. Tebib, J. G., Viguier, P., Noel, E., Colson, F, Barbier, Y, and Bouvier, M. Serum N Terminal Procollagen III Fragment: A Predictive Marker of Joint Destruction In Rheumatoid Arthritis? Clin. Rheumatol. Dec 1992;ll(4):502-507.

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BIOMARKERS 137. Careri, M., Costa, A., and Elviri, L., et al. Use of Specific Peptide Biomarkers for Quantitative Confirmation of Hidden Allergenic Peanut Proteins Ara H 2 and Ara H 3/4 for Food Control by Liquid Chromatography-Tandem Mass Spectrometry. Anal. Bioanal. Chem. Nov 2007;389(6):1901-1907. 138. Desouza, L. V., Romaschin, A. D., Colgan, T. J., and Siu, K. W. Absolute Quantification of Potential Cancer Markers in Clinical Tissue Homogenates Using Multiple Reaction Monitoring on a Hybrid Triple Quadrupole/Linear Ion Trap Tandem Mass Spectrometer. Anal. Chem. Mar 26, 2009. 139. Tucholska, M., Bowden, P., and Jacks, K., et al. Human Serum Proteins Fractionated by Preparative Partition Chromatography Prior to LC-ESI-MS/MS. J. Proteome. Res. Mar 6, 2009;8(3):1143-1155. 140. Tucholska, M., Scozzaro, S., and Williams, D., et al. Endogenous Peptides From Biophysical and Biochemical Fractionation of Serum Analyzed by MatrixAssisted Laser Desorption/Ionization and Electrospray Ionization Hybrid Quadrupole Time-of-Flight. Anal. Biochem. Nov 15, 2007;370(2):228-245. 141. Zhang, R., Barker, L., and Pinchev, D., et al. Mining Biomarkers in Human Sera Using Proteomic Tools. Proteomics. Jan 2004;4(l):244—256. 142. Marshall, J., Kupchak, P., and Zhu, W., et al. Processing of Serum Proteins Underlies the Mass Spectral Fingerprinting of Myocardial Infarction. J. Proteome. Res. Jul/Aug 2003;2(4):361-372. 143. Aleo, M. D., Doshna, C. M., and Fritz, C. A. An Underlying Role for Hepatobiliary Dysfunction in Cyclosporine a Nephrotoxicity. Toxicol. Appl. Pharmacol. Jul 1,2008;230(1):126-134. 144. Tessier, E., Neirinck, L., and Zhu, Z. High-Performance Liquid Chromatographic Mass Spectrometric Method for the Determination of Ursodeoxycholic Acid and Its Glycine and Taurine Conjugates in Human Plasma. J. Chromatogr. BAnalyt. Technol. Biomed. Life Sci. Dec 25, 2003;798(2):295-302. 145. Alnouti, Y., Csanaky, I. L., and Klaassen, C. D. Quantitative-Profiling of Bile Acids and Their Conjugates in Mouse Liver, Bile, Plasma, and Urine Using LC-MS/MS. / Chromatogr. BAnalyt. Technol. Biomed. Life Sci. Oct 1, 2008; 873(2):209-217.

CHAPTER

BIOMARKERS OF ACUTE KIDNEY INJURY Frank Dieterle and Frank D. Sistare

INTRODUCTION D e f i n i t i o n and Prevalence of A c u t e Kidney Injury Kidney disease and renal injury are prevalent serious health problems significantly impacting patient short and long-term survival due to alterations or even complete loss of the renal detoxification capacity, deregulation of salt and water balance, and disturbance of kidney endocrine function. Although renal injury and impairment have a broad pathophysiological spectrum, a classification into acute kidney injury (AKI) and chronic kidney diseases (CKD) is typically performed according to the speed of progression. CKD is characterized by a low glomerular filtration rate with a slow steady loss of renal function over time. CKD can have various causes, such as hypertension, diabetes, chronic glomerulonephritis, polycystic kidney disease, or tubulointersitital fibrosis.12 In contrast, AKI refers to a spontaneous and sustained decrease in renal function. AKI, which has also often been referred to as acute renal failure (ARF), has been reported to complicate 1-7% of all hospital admissions3-6 and 1-25% of intensive care unit (ICU) admissions,7,8 and is associated with a high mortality rate of up to 80% in the ICU setting.9-13 In addition, AKI is a considerable risk factor for the development of non-renal complications contributing to mortality.14'15 Although mortality rates seem to have decreased in recent years, the incidence of AKI, or at least the identification of AKI, has increased over time,16 and the lack of widely accepted tools to detect AKI early, renders a timely intervention difficult. Extensive clinical and nonclinical research to understand, define, and develop new tools 237

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to detect AKI has been conducted in the last decades. Although significant progress has been made in understanding the biology and mechanism of AKI in animal models, translation of this knowledge into improved management and outcomes for patients has been limited.17 Only recently, great expectations for better and earlier tools to improve the early detection of AKI have been created with the development of new renal safety biomarkers, which are reviewed in this chapter.

Pathophysiology and Mechanisms AKI can result from a number of factors, such as decreased renal or intrarenal perfusion, toxic or obstructive insults to the renal tubules, tulolointerstitial inflammation and edema, or primary reduction in the filtering capacity of the glomeruli,18 whereby ischemia and nephrotoxicity account for the largest number of cases of AKI.19 In particular in critically ill patients, nephrotoxicity and ischemia often add to other risk factors such as sepsis, hematologic cancers, renal impairment, or acquired immunodeficiency syndrome. As a consequence, nephrotoxicity has been shown to contribute to 8-60% of all cases of AKI, depending on the patient population and definition of AKI. For example, in a recent biopsy study of 104 patients suffering from AKI or chronic kidney failure, approximately 35% seem to be drug related.20 Looking at the anatomy and function of the kidney, it is straightforward to understand that drug-induced nephrotoxicity is not an uncommon phenomenon. The kidney is basolaterally exposed to blood circulating chemicals and metabolites, and its function is to increase the concentration and excretion of these entities, resulting in high luminal exposure. For example, cisplatin accumulates in the S3 segment of tubules and the subsequent high local concentrations of this cytotoxic drug causes direct damage to the tubular epithelial cells. Furthermore, even drugs with a short half-life and low toxicity to other organs can be potential nephrotoxicants since renal blood flow accounts for more than one fourth of the cardiac output. A considerable number of known marketed drugs induce kidney injury. Often toxicity is considered a class effect, for example certain antibiotics, analgesics, or immunosuppressants. Various modes of drug-induced nephrotoxicity in human have been reported. Some drugs and drug candidates have very specific effects leading to nephrotoxicity, and are beyond the scope of this chapter. The most frequently observed mechanisms are briefly summarized below. Acute tubular necrosis is not an uncommon phenomenon for cytotoxic drugs excreted via the kidney, since the role of the tubule to concentrate and reabsorb the glomerular filtrate renders it vulnerable to direct injury. A number of aminoglycosides, such as neomycin, gentamicin, tobramicin, amikacin, and streptomycin are not metabolized but excreted by glomerular filtration and bind to the tubuloepithelial membrane in the proximal tubule due to their cationic properties and may subsequently interfere with normal lysosomal cellular function, protein synthesis, or mitochondrial function. The combination with other drugs, which show a similar direct toxicity on the tubular epithelial cells such as cisplatin (agent for chemotherapy), causes more nephrotoxic-

BIOMARKERS OF ACUTE KIDNEY INJURY

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ity than each agent alone. Also, a group of antiviral agents such as adefovir, cidofovir, and tenofovir are known to cause direct proximal tubular injury. They are actively reabsorbed by the human renal organic anion transporter-1, their structural similarity to naturally occurring nucleotides appearing to be an important factor for nephrotoxicity. A number of other drugs show similar direct tubular epithelial toxicity, such as the bisphosphonates ibandronate and zoledronate, contrast agents, amphotericin B, and many more. Direct tubular epithelial toxicity is one of the most frequent mechanisms observed in drug development leading to nephrotoxicity. Acute interstitial nephritis is an acute inflammatory condition that specifically affects the renal tubules and the interstitium caused by a cell-mediated hypersensitivity reaction to a drug. A number of drugs are associated with acute interstitial nephritis, such as vancomycin, penicillins, cephalosporins, diuretics, valproic acid, and many more. In most cases, acute interstitial nephritis is self-limited and reversible. Glomerular alterations and injury, also known as nephrotic syndrome or glomerulopathy, is marked by heavy proteinuria. Drugs such as gold therapy, doxorubicin, puromycin, penicillamine, and interferon can directly or indirectly affect mesangial cells or podocytes resulting in an altered permeability of the glomerular filtration wall. Glomerular injury is typically linked to subsequent tubular injury due to the protein overload resulting in poisoning of the tubules (see also Figure 10.1). Crystalline nephropathy, which is also known as obstructive uropathy, obstructive acute renal failure, or crystal nephropathy, can be traced back to the precipitation of crystals in the tubular lumens due to their relative insolubility in human urine. The risk of crystal deposition, which causes an obstruction of the tubular lumen, is increased by volume contraction caused by chronic diarrhea, anorexia with nausea/vomiting, adrenal insufficiency and renal salt wasting, but also pancreatitis, heart failure, and pleural effusions. Drugs associated with crystal deposition include antiretroviral agents such as indinavir, tenofovir, and acyclovir, but also the antibiotic sulfadiazine. Hemodynamic renal failure, which is also known as pre-renal nephropathy, is caused by modulation of the intra-renal blood flow. Intraglomerular pressure and consequently glomerular filtration rate are regulated by the vasomotor tone of the afferent and the efferent arterioles. In case of decreased renal blood flow, the intraglomerular pressure is maintained by vasoconstriction of the efferent arteriole and vasodilation of the afferent arteriole. Drugs that may interfere with the hemodynamic regulatory system can cause a critical further drop of the glomerular filtration rate and result in renal dysfunction. Angiotensin-convering enzyme (ACE) inhibitors and angiotensin receptor blockers inhibit the angiotensin II—mediated vasoconstriction of the efferent arteriole, whereas calcineurin inhibitors (cyclosporin A, tacrolimus) can cause a vasoconstriction, mainly of the afferent arteries. Nonsteroidal anti-inflammatory drugs (NSAIDS) and cyclooxygenase (COX) inhibitors can inhibit prostaglandin-induced vasodilation, affecting the afferent arteries. Contrast agents may impact renal perfusion in certain patients and precipitate nephropathy. Except

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FIGURE 10.1 Scheme of the glomerulus and tubules illustrating glomerular filtration and tubular reabsorption of low molecular weight (LMW) proteins and high molecular weight (HMW) proteins in a normal kidney, after glomerular injury, and after tubular injury. In the non-injured kidney, only low amounts of H M W proteins pass the glomerular filtration wall. LMW proteins pass freely the filtration wall and are reabsorbed to a great extent in the tubules with only a small fraction being excreted with the urine. In early stages of glomerular injury, H M W proteins pass the glomerular filtration wall and are reabsorbed in the tubules, competing with the reabsorption of the LMW proteins, which are subsequently excreted into urine to a large extent. With continued glomerular injury, the continuously reabsorbed H M W proteins "poison" the tubular reabsorption complex, and both LMW and H M W proteins are excreted into urine to a large extent.Thus, LMW proteins can be early and sensitive markers for glomerular injury or for a direct impairment of the tubular reabsorption complex. By contrast, with only tubular injury low amounts of H M W proteins appear in the urine while the LMW proteins continue to be reabsorbed and do not appear in the urine. (See color insert for a full color version of this figure.)

for the calcineurin inhibitors, which induce severe nephrotoxicity in a wide population limiting their clinical use, the other agents show their nephrotoxic potential mainly in individuals with reduced renal function, with other risk factors, and with co-medication of other potentially nephrotoxic drugs. The plurality of different modes of toxicity also has a consequence that nephrotoxicity is one of the major safety concerns in drug development and leads to the termination of a significant number of drugs from development in nonclinical and clinical stages. Further complications rendering the management of nephrotoxicity in drug development difficult are: Non-translatable nephrotoxicity: In some cases the nephrotoxicity observed in animal models does not exactly match the situation encountered in human. On the one hand, there are situations in which no nephrotoxicity in human is observed despite nephrotoxicity in one or several animal species at comparable doses. On the other hand, the nephrotoxicity observed in human may differ in terms of mechanisms, courses, and pathologies. An example is the acute toxicity observed with cyclosporine A. In rats, tubular degeneration, necrosis, and regeneration are observed with the distal tubules being more affected than the proximal tubules, whereas in human the same pathologies dominate more in the proximal tubules than in the distal tubules. Different modes of nephrotoxicity for the same drug: Various factors, such as dosing regimen (chronic or acute toxicity), individual renal performance, or metabolic activity can influence which mode of toxicity is domi-


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nant and which renal pathology will manifest. An example is the drug lithium, which is used in the treatment of bipolar disorders. Treatment can cause a socalled nephrogenic diabetes insipidus (NDI), which is linked to the lithiuminduced down-regulation of the vasopressin-regulated water channel aquaporin-2 expressed in the collecting ducts. Similarly, the acute toxicity primarily causes necrosis in the collecting ducts. On the other hand, chronic lithium toxicity induces chronic tubulointerstitial nephropathy visible as cysts in the distal tubules. A second example is chronic treatment with NSAIDS. Besides the hemodynamic renal failure in patients at risk described above, chronic use of analgesics can lead to necrosis of the loop of Henle, to chronic interstitial nephritis, to papillary necrosis and ultimately to chronic renal failure. In contrast to the hemodynamic renal failure, patients at risk are mainly middle-aged women taking combination analgesics for various disorders. The third example is the acute versus chronic toxicity of cyclosporine A. Whereas the acute toxicity is directly linked to the vasoconstriction and is blood-level dependent and can be managed by reducing the dose levels, the chronic nephropathy is largely irreversible and can occur independent of acute toxicity. Risk factors: Several types of drug-induced kidney injury in humans occur in combination with certain risk factors, which are human specific and which cannot be modeled preclinically. For example, in clinical situations with an impaired renal perfusion such as renal artery stenosis, true volume contraction (duretics, diarrhea, vomiting), or effective volume depletion (nephrosis, cirrhosis decompensated heart failure), ACE inhibitors, COX2 inhibitors and non-steroidal anti-inflammatory drugs (NSAIDs) show a significantly increased risk of renal hemodynamic failure. The background is that in these clinical situations the kidney tries to maintain the glomerular filtration rate by a vasodilatation of the afferent arteries and a vasoconstriction of the efferent arteries. ACE inhibitors reduce the vasoconstriction, whereas the COX-2 inhibitors and NSAIDs inhibit the vasodilatation. For most of the clinical risk factors and underlying diseases, animal models do not exist and in those cases where animal models are available, their use in routine toxicology assessment is not feasible. These aspects also highlight the requirements of new tools for monitoring kidney safety in drug development and in routine use of drugs: The new tools need to be available for preclinical and clinical use to allow the translation of drug candidates from animals to human to evaluate kidney safety in all species, and they need to be able to cover various pathologies, biochemical and physiologic processes, modes and mechanisms of toxicity, and compartments of the kidney. Although most of the mechanisms of kidney injury described above involve a proximal tubular injury also as secondary toxicity, a monitoring of different compartments of the kidney would allow in many cases more precise understanding, greater sensitivity, and may enable a differential diagnosis by identifying the primary injury. On a molecular and cellular level, injury to the tubular epithelium results in a rapid loss of the cytoskeletal integrity and depolarization of the cell, disordering adhesion and membrane molecules.21'22 Further consequences are

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apoptosis, shedding of the tubular brush border and necrosis. If a significant number of cells are no longer viable, the filtrate can leak through the exposed basement membrane and result in severe inflammation and vasoconstriction through the action of vasoactive mediators.19,23 Repair mechanisms of the kidney include epithelial cell spreading and migration to cover the denuded areas of the basement membrane, cell de-differentiation, proliferation, and differentiation to restore the integrity and functionality of the nephron.24 Recent evidence proposes that there is a thin line between successful tissue repair and failure thereof leading to progression of the injury. Thus, early detection of injury and timely intervention are crucial to prevent severe injury, chronic kidney disease and kidney failure.25

C u r r e n t Standards f o r Diagnosing A c u t e Kidney Injury Definitions and criteria for AKI are mainly based on rapid decrease of renal function, i.e., glomerular filtration rate, typically identified by monitoring serum creatinine in patients. Numerous definitions of AKI based on serum creatinine, with or without oliguria, lead to a plurality of different clinical endpoints, criteria for patient selection, and classification, rendering clinical and epidemiologic studies difficult to compare.26 To simplify and standardize the clinical definition of AKI, the Acute Dialysis Quality Initiative (ADQI) has recently published a consensus definition of AKI entitled the RIFLE criteria (Risk for renal dysfunction, Injury to the kidney, Failure of kidney function, Loss of kidney function, and Endstage renal disease)17,27 with the criteria for the classification shown in Table 10.1. This classification system accounts for the linear relationship between minor elevation of creatinine and progression to kidney failure manifested by the need for renal replacement therapy. Although the utility of this classification system in determining risk of renal replacement therapy and hospital morality has been well documented,28,29 its utility in

TABLE 10.1 Rifle classification criteria for acute renal failure: Risk of renal dysfunction, injury to the kidney, failure of kidney function, loss of kidney function, end-stage renal disease; SCr = serum creatinine, UO = urine output.The criteria that lead to the worst possible classification should be used.

Stage

GFR Criterion

Urine Output Criterion

Risk (1)

Increased SCr x 1.5 or GFR decrease > 25%

UO < 0.5ml/kg/h x 6h

Injury (2)

Increased SCr x 2 or GFR decrease > 50%

UO < 0.5ml/kg/h x 12h

Failure (3)

Increased SCr x 3 or GFR decrease > 75% or SCr a4mg/dl (acute rise a 0.5mg/dl)

UO < 0.3ml/kg/h x 12h or Anuria x 12h

Loss (4)

Persistent ARF = complete loss of kidney function > 4 weeks

ESKD (5)

End-stage kidney disease (> 3 months)


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treatment decisions and intervention might be limited due to the fundamental problem of using assessments of the glomerular filtration rate (GFR) for the identification and classification of kidney injury. The GFR is only indirectly linked to kidney injury and changes of the GFR reflect a late consequence in a chain of effects and changes associated with primary insult to the kidney. In addition, a large amount of the functioning renal mass can be lost without any significant changes of the GFR also often referred to as "renal reserve."3031 An illustrative example of the renal reserve is the situation of kidney donors. After donation of one kidney, which corresponds to the loss of 50% of the renal functioning mass, virtually no increases of serum creatinine as estimation of the GFR can be seen.32 34 The direct measurement of GFR by assessing the renal clearance of inulin is still considered the gold-standard.35-36 However, this approach is not suitable for routine clinical practice due to the need for continuous intravenous infusion, timed urine collection, and sometimes bladder catheterization. Also, other techniques based on iothalamate or other X-ray tracers37-39 allow measurement of GFR, but similarly to insulin are hampered by the need for urine collection and continuous intravenous infusion. An alternative method is GFR determination by plasma clearance of 51Cr-EDTA.40 Although it offers the advantage of not needing urine collection and continuous intravenous infusion, its use in clinical practice is very limited due to the special needs of handling radiolabled compounds. As a consequence, estimations of glomerular filtration rate by serum level measurement of endogenous constantly produced molecules, which are predominately cleared by glomerular filtration, have become the de facto standard in clinical practice, i.e., serum creatinine and blood urea nitrogen (BUN). Yet both methods have several drawbacks, complicating the timely and reliable detection of AKI. Serum creatinine is a small 113 Da molecule derived from the metabolism of creatine in skeletal muscle and from dietary meat intake. It is released into the plasma at a relative constant rate and is freely filtered by the glomerulus. Consequently, a decrease of GFR causes a rise of serum creatinine concentrations, showing an inverse relationship to GFR.41 Despite being the most widely accepted standard for assessing GFR, there are a number of limitations: 1) Production and release can be variable depending on sex, age, dietary intake muscle mass, and disease-related loss of muscle mass (e.g., rhabdomyolysis), resulting in significant variations of baseline serum creatinine. 2) In the case of normal kidney function, up to 25% of creatinine is secreted by the tubules into urine and is not filter by glomeruli, resulting in an overestimation of the GFR. If the GFR is decreased due to kidney impairment, the proportion of creatinine secretion is additionally increased, further contributing to an overestimation of GFR.42'43 Also, several drugs, such as cimetidine or trimethoprim, can competitively block the secretion of creatinine and consequently cause an increase of serum levels of creatinine in the absence of kidney injury, limiting its specificity.44 3) Rapid changes of GFR are not visible in real-time by changes of serum creatinine levels, but rather delayed, as creatinine needs time to accumulate to a new steady state, delaying the diagnosis of AKI by hours to days.45

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BUN: Serum urea is a low molecular weight waste product of protein metabolism and its serum levels are inversely correlated with GFR similarly to serum creatinine, but its use is problematic due to extra-renal factors: 1) The rate of urea production can be widely modified by protein intake, illness (chronic liver disease, sepsis, trauma) and drug intake, causing variable baseline BUN levels. 2) Approximately 40 to 50% of filtered urea is reabsorbed by proximal tubular cells. In the case of decreased circulating volume, more urea is reabsorbed, causing increased BUN levels under-representing GFR. Although urine output is routinely measured in crticially ill patients and can be helpful in assessing kidney function, its lack of specificity and sensitivity for AKI renders its use as a single criterion for detecting AKI doubtful. In summary, the deficiencies in current standards for detecting AKI, which have been used for decades, clearly show the urgent need for more sensitive and earlier tools for detecting AKI, and allowing for timely intervention and prevention of renal failure. In addition, more sensitive tools allow drug developers to assess better the potential of their compounds to induce kidney injury and to develop drugs that will show better overall renal safety.

N O V E L K I D N E Y SAFETY BIOMARKERS In recent years, kidney safety biomarkers have become promising tools to detect AKI in a much more sensitive and earlier way than the current standards of serum creatinine and BUN. Although the principle of measuring proteins in the urine or plasma to monitor kidney safety is not new, only recently has the availability of sensitive and reliable assays to measure many new biomarkers in systematic investigations involving preclinical studies, and clinical studies opened the door for these promising new tools and surfaced evidence of their utility. A number of proximal brush border enzymes, such as alanine aminopeptidase (AAP), alkaline phophatase (AP) or 7-glutamyltranspeptidase (GGT) have been measured in urine of patients for decades, but their instability and sometimes limited sensitivity have hindered their general breakthrough. In general, kidney safety biomarkers or a panel of biomarkers should have a number or properties to render them useful for different contexts: • The markers should be sensitive for detecting a diverse set of renal insults. A biomarker could be specific for a certain type of cellular injury; for a certain step in a pathologic process, physiologic function, or biochemical process; for only certain regional or anatomical compartments of the kidney; or could be a nonspecific marker of general kidney injury or dysfunction. Different complementary biomarkers might be combined on a kidney injury panel, offering the advantage that injury could be localized, more precisely characterized, and help to resolve ambiguities inherent in interpretations of more general traditional biomarkers. • The biomarkers should change early and sensitively allowing an early and accurate detection of AKI and thus a timely intervention. • The magnitude of change in the biomarker is expected to be proportional to the severity and extent of kidney injury or dysfunction.


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• Specificity to kidney-related injury prevents the generation of false positive AKI diagnoses in the context of other organ injuries or other interfering events, in particular in critically ill patients. • Accessibility in peripheral body fluids such as blood or urine is important as invasive procedures such as biopsies are not generally acceptable and feasible in routine clinical practice. In addition to analyte stability in the stored sample, the absence of particular sampling requirements are a big plus. Knowledge of the kinetics of the biomarker in the body is needed and should suffice to match the practical needs for patient monitoring. A very transient biomarker signal, for example, may have limited practical utility. • Availability of technologies to measure these markers at reasonable technical efforts and prices is a practical consideration. Many of the most promising kidney biomarkers are discussed below. The biomarkers are ordered and characterized by the mechanism through which their levels are modulated and by the compartment of the kidney for which these biomarkers are specific; such as general kidney function biomarkers, de-novo expression injury response biomarkers, leakage markers, functional biomarkers of glomerular filtration, tubular re-absorption, and inflammatory biomarkers.

Kidney Function Biomarkers Serum C y s t a t i n C

Serum cystatin C is a general renal function marker that is rapidly gaining increased use in different clinical settings. Cystatin C used to be called 7-trace and is a non-glycosylated low-molecular weight 13 kDa protein. It is continuously produced by all nucleated cells and functions as a housekeeping factor.46 Cystatin C is directly and freely filtered from blood into the glomerulus, and therefore is considered an improved estimator of GFR due to several positive attributes: 1. Compared to serum creatinine, a greatly reduced impact of age, sex, muscle mass, dehydration state, and circadian rhythm on cystatin C serum levels has been observed. 2. An unhindered straightforward filtration of cystatin C by glomeruli. 3. In contrast to serum creatinine, an absence of tubular secretion or extrarenal clearance.47 A limitation for certain clinical contexts might be the modulation of serum cystatin C levels seen with corticosteroids, e.g., in transplantation.48 It has been demonstrated in numerous clinical studies and meta-reviews that serum cystatin C outperforms or at least equally performs to SCr for the estimation of the GFR in various different contexts of AKI and CKD, with a better performance in particular for glomerular function impairment, for critically ill patients, and for mild changes of GFR.49-54 The FDA approval of an assay

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BIOMARKERS

to measure cystatin C also shows its increasing importance and value in clinical practice55 and it might be speculated that serum cystatin C may someday replace serum creatinine as a general renal function biomarker.

Functional Biomarkers U r i n a r y Total P r o t e i n

Total protein is the ensemble of all protein species measured together in urine. Abnormally high excretion of proteins is called proteinuria and has been highlighted in the literature as a clinical prognostic marker, as a preclinical and clinical diagnostic marker to detect AKI, and as a factor predicting progressive loss of renal function in the context of a variety of diseases.56"60 Alterations of the glomerular filtration barrier, such as damage to the glomerular podocytes, leads to leakage of plasma proteins into the ultrafiltrate.61The normal glomerular filtrate contains 10 mg protein/L, but only approximately 1% is normally present in the urine because of the strong re-absorption capacity of the proximal tubule. If this re-absorption reaches a saturation point due to excessive glomerular leakage with an often observed associated "poisoning" of the proximal tubules, or if the tubular protein reabsorption complex is directly damaged by toxic agents, proteinuria can be observed despite normal glomerular filtration rates.62 In progressive glomerular disease, dysfunctions of glomerular filtration and of tubular re-absorption are found together as tubuloglomerular proteinuria. Normal urinary proteins consist of low amounts of both high and low molecular weight proteins including albumin, other plasma proteins (beta 2-Microglobulin, alpha 1-microglobulin, retinol binding protein, haptoglobin, cystatin C) and Tamm-Horsfall glycoprotein secreted by tubular cells. As shown in Figure 10.1, glomerular injury and disease can result in a mixed picture of both high and low molecular weight proteins over the chronology of the injury; both types of protein contributing to the estimation of urinary "total protein." It has been postulated that the ratio between low and high molecular weight proteins in urine would allow a better prediction of the type and severity of damage than the quantity of proteinuria.63'M The clinical use of evaluating urinary total protein is many-fold, such as evaluating renal diseases including proteinuria, complicating diabetes mellitus, nephrotic syndromes (e.g., membranous proliferative glomerulopathies, lipoid nephrosis, systemic lupus erythematosus, amyloidosis, heavy metal poisoning by gold, lead, and cadmium), glomerulonephritis, Goodpasture's syndrome, Henoch-Schonlein purpura, kidney infection, polycystic kidney disease, multiple myeoloma, collagen diseases, drug-induced nephrotoxicity, renal tubular lesions, tubular proteinuria including Wilson disease and Fanconi syndrome, and hypertension (about 10-15% of patients with hypertension show proteinuria).65 Among these diseases, nephrotic syndromes cause the most severe proteinuria and nephrotic syndrome is defined usually by the extent of proteinuria. Proteinuria is diagnosed when total urinary protein excretion is greater than 300 mg/24h. For different laboratories, the reference ranges might vary slightly, but most commonly upper references ranges of 140-150mg/24h are


247

used for 24 hour urine collections, whereas an upper reference concentration of 45mg/mmol Creatinine is applied for a random spot urine collection specimen (Laboratory Corporation of America 2007). In the last decades, proteinuria has usually been diagnosed in a screening setting by simple dipstick tests, despite the possibility of the test being falsely negative even with nephrotic range proteinuria. False negatives can occur if the urine is diluted or if the proteinuria consists of mainly other proteins than albumin, since the reagent on the test strips, bromphenol blue, is highly specific for albumin. These qualitative assessments have significantly contributed to a mixed reputation of total protein as a marker of kidney injury as well as the fact that there has never been a formal assessment or qualification of total urinary protein as a biomarker for different pathologies, such as the glomerular injury, in a large-scale clinical context. The formal regulatory qualification of quantitative total urinary protein as a biomarker to monitor glomerular injury by the PSTC and its acceptance for specific preclinical use on a case-by-case translational contexts by the EMEA and FDA is expected to change the culture of using quantitative urinary protein in a more formal way in drug development and clinical settings (see "Consortia achieving the first regulatory qualification of kidney safety biomarkers"). A number of publications now highlight the diagnostic power of total protein for AKI induced by nephrotoxicants, such as cisplatin, thalidomide, pamidronate, aminoglycosides, ifosfamide, doxorubicin, or nonsteroidal anti-inflammatory drugs.66"71 Urinary Albumin

For more than four decades urinary albumin has been known to be a biomarker of kidney injury.7273 Albumin is a major high molecular weight serum protein larger than the pores of the glomerular filter, so albuminuria is best known as a biomarker of glomerulopathy, its appearance in urine presumed to represent a compromise to the integrity of the glomerular basement membrane.74 However a small but biologically significant fraction of albumin isfiltered,75*77and normally very efficiently absorbed by proximal tubule epithelium, degraded, and reutilized or excreted fragmented into the urine.78 The experimental data support therefore an interpretation that large quantities of intact albumin in urine in excess of 200 mg/g creatinine and referred to as macroalbuminuria exceed the absorptive capacity of normally functioning tubular epithelium and reflect dysfunction of the glomerular filter. The smaller quantities of intact albumin between 20 and 200 mg/g creatinine, and often referred to as microalbuminuria, may represent the significant fraction of albumin filtered by a normally functioning glomerulus that cannot be reabsorbed by tubular epithelium rendered dysfunctional by disease or chemical toxicants (see Figure 10.1). Concurrent dysfunction to both segments cannot be differentiated from albuminuria measurements alone. Furthermore, the traditional urinary dipstick protein detection method is insensitive to the lower critical ranges of urinary albumin useful for diagnosing renal tubular dysfunction. Clinically, urinary albumin has gained favor as a biomarker for monitoring chronic kidney disease progression and to monitor delay of progression with

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BIOMARKERS

treatment intervention, especially for managing nephropathy, cardiovascular disease, and renal hypertension in diabetics.79-83 In human studies albuminuria has clearly been demonstrated to be a relevant functional biomarker of acute chemically induced injury to the tubular epithelium following, for example, treatment with gentamicin,84 carboplatin,85'86 ifosfamide,87 or cisplatin.88 Recently numerous studies were conducted in rats administered renal toxicants, including gentamicin and cisplatin, or non-toxicants and comparing the relative performance of albuminuria to several urinary biomarkers and to serum creatinine. Albuminuria was shown to outperform serum creatinine for detection of histopathologically confirmed drug-induced renal tubular injury,217 with albuminuria appearing in rats presenting with tubular histology at doses and times when serum creatinine and blood urea nitrogen were unchanged. Decades of data, together with recent studies and greater understanding of renal handling of this protein, point to the underutilization of this important and versatile renal function biomarker. Since albumin is such an abundant protein appearing in serum and animal feed, important practical considerations must be made regarding interpretations of potentially spurious findings in animal studies as a result of an occasional toe nail bleed or from food droppings contaminating overnight urine collections when fasting may not always be possible. U r i n a r y (32-Microglobulin

2-microglobulin is a low molecular weight (11.8 kDa) protein component of the MHC Class 1 molecule. In healthy subjects 150-200 mg are synthesized daily and eliminated via the kidney. It is readily filtered by the glomeruli, and almost completely reabsorbed and metabolized by the tubules. Only 0.1% of the (32-microglobulin filtered by the glomeruli is normally excreted into urine. It has been demonstrated that an impairment of the tubular uptake causes increased excreted urinary p2-microglobulin levels, up to several hundred folds. So far, there are two identified mechanisms proven to be responsible for this impairment. At first, glomerular alterations, damage, and/or diseases allow higher molecular weight proteins to pass through the filtration membrane causing a high protein load in the tubule. As a consequence, higher molecular weight proteins like albumin compete for common transport mechanisms decreasing the tubular uptake of (32-microglobulin and increasing the excretion into urine (see also Figure 10.1).89 Secondly, the tubular re-absorption complex is directly impacted by treatment with drugs or by different tubular diseases. For spot urine collections, the concentration of (3 -2-microglobulin in healthy subjects is typically s 160 ug/L or £ 300 |ag/L/ g creatinine. (3 -2-microglobulin may be unstable in urine of pH < 5.5 or in urine with enzymes present which may be proteolytic (e.g., urinary tract infection), therefore the urine should be alkalinized and frozen at -80 C within minutes of collection.90 Increases of urinary |3 -2-microglobulin with renal injury have been described in a number of settings including cardiac surgery,9192 renal transplantation,93 and in particular nephrotoxicity in more than 200 peer reviewed publications, such as HIV patients treated with tenofovir, disoproxil fumarate,


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and other antiretroviral agents,94 patients with aminoglycoside treatments,95 cisplatin treatment regimens,9697 and gold treatment (sodium aurothiomalate) for rheumatoid arthritis98 to name only a few examples. In a review of 14 studies on urinary biomarkers in septic acute kidney injury, the authors noted that urine (3 -2-microglobulin was associated with a change in serum creatinine, can help to distinguish pre-renal azotemia from acute tubular necrosis, and can detect sub-clinical or predict acute kidney injury.99 However, they observed that the prognostic value of urine (3 -2-microglobulin in sepsis was unclear. Similarly, Herget-Rosenthal, et al. studied 73 patients with non-oliguric renal failure and found that while increased urine (3 -2-microglobulin was associated with the risk of requiring renal replacement therapy, it was not as predictive as markers such as NAG, RBP or alpha 1-microglobulin.100 Thus, despite its use for decades with a better performance than BUN and serum creatinine, there is a need to evaluate systematically the utility of |3 -2-microglobulin in different clinical contexts similar to the preclinical regulatory qualification of |32-Microglobulin by the PSTC, in a side-by-side comparison with urinary cystatin C, which shows better stability. Urinary Cystatin C

An impairment of re-absorption in proximal tubules by the same mechanisms as described for urinary (3 2-microglobulin can lead to several hundred-fold increases of urinary levels in humans and rats.101,102 Reported reference ranges and average control values of cystatin C are very consistent and indicate a normal urinary cystatin C concentration below 0.3mg/L including studies with nearly 2000 healthy subjects in total. For example, in a study with 1670 healthy subjects the average urinary cystatin C concentration was 5lug/1 ±25.2ugfl (Uchida, K. and Gotoh, A. 2002). Until now changes in urinary cystatin C levels have been characterized mainly in the context of different kidney diseases affecting glomerular integrity and proximal tubular re-absorption in humans.103'104 In a recent publication, urinary cystatin C levels were investigated in 50 patients with glomerular diseases and 22 patients with tubulointerstitial diseases, which were all proven by biopsy.101 Urinary cystatin/urinary creatinine ratios > 11.3 mg/mmol was highly associated with tubular proteinuria, biopsy-proven tubulointerstitial disease, and heavy proteinuria. The study identified both functional impairment due to protein overload in the case of heavy proteinuria (glomerular disease), as well as structural impairment due to tubulointerstitial disease as factors associated with increased urinary levels of cystatin C as also reported elsewhere. For instance, Uchida, et al identified increased urinary cystatin C concentrations for a number of subjects with proteinuria indicating tubular damage as a consequence of protein overload. In addition, highly increased urinary cystatin C levels were found in 56 patients with chronic renal failure.105 Tkaczyk, et al. showed that urinary cystatin C levels in 12 children with idiopathic nephrotic syndrome (INS) where higher than in children in clinical remission in the eighth week of INS treatment and higher than in healthy

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BIOMARKERS

children.106 Also, in critically ill patients with AKI, urinary cystatin C showed a better performance than a number of other urinary biomarkers to predict the subsequent need for acute renal replacement therapy.100 Although urinary (32-microglobulin has a much longer track record of clinical and nonclinical use, cystatin C shows higher stability in urine and consequently might be the preferred marker for future use, in particular as both markers monitor the same pathologies and as assays for cystatin C have now become available.

Leakage Markers U r i n a r y GST-a and G S T - U / T T

Glutathione-S-transferases are soluble cytosolic enzymes with two subtypes of equal size which are located in different segments of the tubules, such as the asubtype in the proximal tubules and the p/Tr-subtypes j n the distal tubules (ufor rodents and IT for humans). GSTs play a role in detoxification processes in the kidney. In the case of tubular cell necrosis, the content of the cells leaks into urine. Thus, urinary GST-a is a leakage marker specific to proximal tubular cells and GST-JJ/TT protein levels are leakage markers indicating distal tubular cell injury. In rat studies, increased levels of GST-a and increased levels of GST-p were associated with proximal and distal tubular injury after treatment with cisplatin, gentamicin and N-phenylanthranilic acid (NPAA).107,108 In animals and in humans, increased GST-a levels were reported after treatment with compound A, a nephrotoxic degradation product of sevofluorane.109, "° Increases of both GST-a and GST-TT were associated with treatment with amphotericin B in male patients, but not in female patients.1" In a small study with 26 critically ill patients admitted to the intensive-care unit, of whom four developed AKI, both GST-a and GST-TT showed an AUC of the ROC of 0.9.117In another small study in kidney-transplanted patients, increased levels of GST-a were associated with cyclosporine A nephrotoxicity, whereas increased levels of GST-TT were associated with acute allograft rejection.112 Urinary GST-a has been reported to rise faster than serum creatinine and to be a very sensitive marker when the kidney is exposed to heavy metals."3 In diabetic patients GST-TT but not GST-a levels were correlated with the degree of albuminuria,114 whereas increased levels of GST-a, but only to a small extent increased levels of GST-TT were reported for obese patients with normal serum creatinine."5 In another study with 76 patients undergoing cardiac surgery, increased levels of GST-a and GST-TT were reported in 36 patients developing AKI as defined by the AKIN criteria.116 In conclusion, it can be said that both GST-a and GST-p AIT are promising markers in different clinical contexts. Yet more evidence about this clinical utility, in particular if only one of the different isoforms is evaluated, is needed. In particular the preclinical evaluation with a histopathology anchor is more limited than for other markers. In addition, GSTs are not stable in urine under certain conditions. Whether the recommendation to add stabilizing buffer to urine sample corrections turns out to be a clinical "show-stopper" needs to be seen, in particular when more clinical evidence of the utility of the GSTs and other "concurring" biomarkers may become obvious.


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Urinary NAG Urinary NAG (N-Acetyl-ir-D-glucosaminidase) is a lysosomal brush-border enzyme of 140 kDa with two isoforms (A and B) and is mainly expressed in proximal tubules where its function is the breakdown of glycoproteins. Due to its size, plasma levels of NAG are normally not filtered by the glomeruli and its excretion into urine correlates with increased tubular lysosomal activity, tubular cell injury (leakage), and indirectly with increased proteinuria. NAG has been used for decades as it is stable in urine in constrast to many other urinary enzymes, and due to its specific localization in the proximal tubules. In the context of renal diseases (diabetic and hypertensive nephropathy, focal segmental glomerulosclerosis), AKI, and treatment with nephrotoxic compounds, increased urinary NAG levels have been observed typically before increases of serum creatinine and BUN.117-121 In hospitalized patients, increased NAG levels were associated with an adverse outcome (dialysis or death).122 Yet in the framework of exposure to metals and other nephrotoxicants and increased urinary urea, the activity of the enzyme is inhibited in urine by these molecules, therefore compromising its use.123-125 In addition, increased NAG levels have been reported in a variety of conditions without clinically significant renal injury, such as rheumatoid arthritis and hyperthyroidism.126,127 Finally, NAG has been criticized to be oversensitive in the absence of clinically relevant renal injury.118'128 This means that a broad utility of NAG, despite its high sensitivity under certain conditions, might be limited to a combination with other markers in a panel to compensate for its over-sensitivity, lack of specificity, and observed interferences.

Expression Markers Urinary Kim-1 Kidney injury molecule-1 (Kim-1 in rodents, KIM-1 in humans), which is also referred to as T-cell immunoglobulin mucin - 1 (TIM-1) and hepatitis A virus cellular receptor-1 (HAVCR-1), is expressed in very low levels in the body except on proximal tubular epithelial cells and lymphocytes. Kim-1 is a glycosylated type I cell membrane glycoprotein with a six-cystein immunoglobulin-like domain and a mucin domain in its extracellular region. An attractive characteristic of Kim-1 as a kidney safety biomarker is the fact that modulations of expression levels of Kim-1 in the body have only been reported upon proximal tubular injury (up to several folds induction of mRNA and protein levels) and to a much lower extent in the cochlea after cisplatin treatment.129 Kim-1 mRNA, and subsequently the protein, is expressed during dedifferentiation of proximal tubular epithelial cells. The protein is cleaved and the ectodomain is shed into the urine,130 and has been shown to be stable at room temperature for several hours. In numerous animal studies, the utility of Kim-1 as a biomarker to detect kidney injury has been demonstrated such as protein-overload nephropathy and aging-induced nephropathy,131 ischemiainduced renal injury,132 a model of polycystic kidney disease,133 and kidney injury induced by various nephrotoxicants such as cisplatin, folic acid, TFEC,134

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cadmium,135,136 contrast agents,137 gentamicin, mercury, chromium,138 ochratoxin A,139 cyclosporine,140 tacrolimus, lithium, furosemide, vancomycin, puromycin, and doxorubicin.240 The utility of Kim-1 as a biomarker to diagnose AKI and CKD in humans —and thus its utility as a translational marker—has been shown in different clinical contexts. In a study of 40 children undergoing cardiac surgery, for which originally NGAL levels were determined (see section about NGAL below), urinary KIM-1 levels could diagnose AKI 12 hours after surgery with an AUC of the ROC of 0.81, whereas rises of serum creatinine were observed between 24 and 72 hours only.141 The diagnostic performance of KIM-1 levels was lower than the diagnostic performance of NGAL, but repeated freezethaw cycles, a long-term storage of the samples between these measurements, and the small sample size may question the relevance of this difference. The same limitations in terms of sample size might apply to another study evaluating urinary KIM-1, NGAL, NAG, cystatin C, IL-18, and a 1-microglobulin in 103 patients undergoing cardiac surgery, with 13% of the patients developing AKI. KIM-1 showed the highest diagnostic performance (AUC 0.78) and was the only marker independently associated with AKI after adjusting for preoperative AKI score. The variance of reported results for the different markers in the context of cardiac surgery followed by AKI demonstrates that there is a pressing need to compile more evidence in different populations and assessing all markers together in these cohorts to obtain comparable evidence of their utility in different clinical contexts. A cross-section study is reported in which urinary KIM-1, MMP-9, and NAG levels of 29 patients with AKI (due to sepsis and hypoperfusion, nephrotoxins, and contrast-induced nephropathy) versus 45 control patients (healthy volunteers, CKD patients and patients with urinary tract infection, UTI) were determined.H1 The AUCs of the ROC were 0.74 for MMP-9, 0.90 for KIM-1, 0.97 for NAG, and 1.0 for all three biomarkers combined. In a study with 201 patients with clinically established AKI, urinary KIM-1 levels and NAG levels correlated with the clinical composite endpoint of death or dialysis requirement, also after adjustment for disease severity and comorbidity.142In a study with non-diabetic renal disease, urinary KIM-1 levels were increased in patients with proteinuria and decreased in those patients who were treated with a renin-angiotensin-aldosterone system inhibitor, sodium restriction, or diuretic therapies. In those patients, KIM-1 correlated with proteinuria decrease, rendering it a potential alternative clinical endpoint.143 The utility of KIM-1 was not only demonstrated as a peripheral marker, but also as a tool to assist the pathologist in evaluating kidney biopsies. For example, increased KIM-1 staining in kidney biopsies of patients with a pathology diagnosis of AKI was reported.144 Also increased KIM-1 staining in biopsies from 102 patients with a variety of kidney diseases was associated with tubulo-interstitial inflammation and fibrosis.145 In biopsies of transplanted kidneys, increased Kim-1 staining was detected in 100% of patients with deterioration of kidney function and pathological changes indicating tubular injury, in 92% of patients with acute cellular rejection, and in 28% of pa-


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tients with normal biopsy readouts, which might indicate a higher sensitivity of KIM-1 staining compared to current standards of histopathology assessment.146 Also, in kidney transplantation, increased tertiles of urinary KIM-1 excretion were prognostic of graft loss.147 In conclusion, Kim-1 is proving to be one of the most promising biomarkers to monitor AKI impacting proximal tubular epithelial cells due to its unique specificity, its sensitivity to detect various forms of tubular injury earlier than current diagnostic standards, its stability, its translatability between different species, and finally thanks to rapidly increasing evidence of its preclinical and clinical utility in numerous contexts. Thus, Kim-1 might have the potential to become the "troponin of the kidney." Urinary Clusterin

Clusterin has a secreted and a nuclear isoform. Only the secreted isoform, which is a 76-80 kDa glycosylated protein with extensive post-translational modifications, is considered relevant in the context of kidney injury. During early stages of renal development it is highly expressed, but later only in the case of injury to proximal and distal tubules. Secreted clusterin has been suggested to play an anti-apoptotic role, to be involved in cell protection, lipid recycling, cell aggregation, and cell attachment.148 Clusterin gene over-expression was induced by different types of kidney injury in glomeruli, tubules, and papilla of rats and dogs as a result of drug nephrotoxicity,149-151 surgery and ischemia,152-155 and in animal models of different renal diseases.156 Changes of protein levels of clusterin have been observed in kidneys and in the urine of some of these animal studies.151,152,155'156 In human there are very limited and non-conclusive data available.157,158 In the PSTC regulatory qualification of biomarkers, urinary clusterin has proven to be a powerful diagnostic biomarker to monitor proximal tubular injury and regeneration with a performance nearly as good as urinary Kim-1 in rat studies (see section "Consortia achieving the first regulatory qualification of kidney safety biomarkers"). Urinary NGAL

Neutropil gelatinase-associated lipocalin (NGAL), which is also known as human neutrophil lipocalin, lipocalin-2, siderocalin, 24p3, or LCN2, is a 25kDa protein initially identified bound to gelatinase in specific granules of the neutrophil. It is expressed in various tissues at low levels, but induced in epithelial cells upon inflammation or other types of injury.159-16C In mouse models, strongly increased mRNA levels of NGAL and increased protein levels in the kidney and urine were seen shortly after cisplatin administration and renal ischemia.161' 162NGAL has been associated with an antiapoptotic and antioxidative role. In particular for ischemia-related AKI, NGAL could be translated into a sensitive human peripheral biomarker with increased levels upon injury measurable in blood and in urine. One of the most-cited studies of NGAL is a cardiopulmonary bypass study in children in which AKI occurred in 28% of the patients defined by a serum creatinine rise one to three days

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after operation. Urinary and serum NGAL level elevations measured only a few hours after surgery predicted AKI with a sensitivity and specificity close to 100%.163 In a second similar study in adults, increased urinary NGAL levels were observed though at lower specificity, which might be attributed to different co-factors.164 For trauma patients, urinary NGAL could differentiate between patients developing AKI and patients not developing AKI with high sensitivity and specificity.165 Urinary and plasma NGAL have also predicted contrast-induced AKI with a high diagnostic power two hours after contrast administration166-167,168 and similar to IL-18, increased urine levels collected the day after kidney transplantation could predict recipients with subsequent delayed graft function and who needed dialysis.226 Modulations of NGAL have also been reported for a number of kidney diseases. For example, increased serum and plasma NGAL levels were reported in patients with polycystic kidney disease. In addition, patients with higher cystic growth had higher urinary and plasma levels than patients with lower cystic development.169 In CKD, there is less coherence between urinary and blood levels of NGAL than in AKI settings, which might be explained by the mechanism of NGAL processing in the kidney. For example, in patients affected by IgA nephropathy, higher grade patients showed increased urinary NGAL levels but normal blood levels.170 Also, patients with idiopathic glomerulonephritis had increased urinary NGAL levels, which were correlated with the extent of proteinuria and urinary levels predicting worsening of renal function in patients for one year follow-up.171' 172In a recent study, patients with diabetic nephropathy showed increased serum and urinary NGAL levels even before other clinical signs such as albuminura, and the levels correlated with severity of disease.173 The role of NGAL in proteinuria-related CKD may be explained by the mechanism of how NGAL is processed in the kidney, and which also might raise some concerns in terms of specificity. NGAL is filtered by the glomeruli and under physiological conditions nearly completely reabsorbed in the promixal tubule by binding to the protein transporter complex cubilin-megalin. In the case of proteinuria, the non-specific binding of urinary proteins to this complex can lead to a saturation of the re-absorption capacity leading to increased urinary levels of NGAL. In addition, increased protein loads could lead to increased expression and release of NGAL as defensive mechanisms, as also shown for other "tubular stress" proteins such as Kim-1.174 As a consequence, conditions which lead to a saturation or impairment of the re-absorption complex or which lead to highly increased plasma levels due to non-renal expression of NGAL, could cause increases of urinary NGAL levels. Since NGAL can be expressed in different tissues and organs upon injury, such as muscle injury and liver injury, further mechanistic studies are proposed to investigate potential limitations of specificity of NGAL. Despite the excellent diagnostic performance of NGAL for ischemia-related AKI, the effect of drug-induced injury on the de novo expression of NGAL in kidney and on the impairment of tubular NGAL re-absorption might prove of high practical utility for use of NGAL useful as a kidney biomarker in the context of drug-development.


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Urinary Osteoactivin Following discovery of osteoactivin (OA) in a rat model of osteopetrosis, OA has been shown recently to exist in transmembrane and glycosylated secreted isoforms acting as an anabolic factor regulating osteoblast differentiation and function.175 OA was initially identified as a transmembrane type I glycoprotein non-melanoma b in low metastatic human melanoma cell lines and is also referred to as gpnmb.176 OA was also identified in murine dendritic cells, termed cell heparan sulfate proteoglycan integrin dependant ligand (DC-HIL) and in human hematopoietic cells, termed growth factor inducible neurokinin (HGFIN). Additional studies suggest roles for OA as a receptor, ligand, or enzyme in regulating fibroblast differentiation and cancer metastasis, and attenuating degeneration induced in muscle, liver, and kidney.177 OA mRNA and protein expression has been shown to be robustly upregulated in ischemic rat kidney injury, localized to tubular epithelium and interstitial fibroblasts, suggesting a role for OA as an early trigger for renal interstitial fibrosis.178 OA has also been shown to be upregulated in the livers of a rat hepatic cirrhosis model, further suggesting a role of the protein in the pathogenesis of fibrosis.179 While at an early stage of evaluation, this promising biomarker's appearance in urine may prove to be a sensitive and insightful indicator of yet another unique aspect of the dynamic and complex renal injury response to acute kidney injury. Urinary OA may prove specific and useful for monitoring initial injury and continued kidney progression toward fibrosis if further studies conducted in animals and humans can demonstrate that acute damage to the liver and muscle do not yield OA secretions that will spill over from the blood compartment into the urine. Urinary Osteopontin Osteopontin is also known as secreted phophoprotein I (SPP1), 44kD bone phophoprotein, sialoprotein I, uropontin, and early T-lymphocyte activation-2 (Eta-1). It is synthesized at the highest levels in bone and epithelial tissues, but also expressed in macrophages, activated T cells, smooth muscle cells, and endothelial cells and it is widely distributed in normal adult human tissues, such as bone matrix, kidney, epithelial cells of gastrointestinal tract, gall bladder, pancreas, urinary and reproductive tracts, lungs, breasts, salivary glands, brain, arteries, urine, and milk.180 It plays a role in the regulation of osteoclast function during bone formation, tuomorigenesis, accumulation of marcrophages, and in the kidney in the protection versus NO, oxidative stress, and ischemia, and is also involved in regeneration processes. Osteopontin has also been associated with kidney stones, but it is unclear if it promotes or inhibits the formation of kidney stones.180'181 In the normal mouse, rat, and human kidneys, osteopontin is expressed in the thick ascending limbs of the loop of Henle and the distal convoluted tubules.182 Increased levels of osteopontin mRNA expression and protein expressions in the kidney have been observed in a number of diseases such as human progressive idiopathic membranous nephropathy, IgA neprhitis, lupus

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nephritis, cresentric glomerulonephritis, but also renal cell carcinoma. Similarly, increased mRNA levels and protein levels in kidneys of different animal models of renal injury have been reported, such as gentamicin, cisplatin, mercury chloride, cyclosporine, sevoflurane, angiotensin II, puromycin, bacitracin, ochratoxin, vancomycin, para-aminophenol, anti-thy-1 nephritis, unilateral urethral obstruction, and remnant kidneys in 5/6 nephrectomy-induced kidney injury.183-195 Although osteopontin has proven to be a very sensitive indicator of different forms of renal injury on a gene expression and a localized protein expression level, its value as a peripheral measurable urinary protein biomarker needs to be proven. The quantification of urinary osteopontin is complicated by the fact that in the kidney phosporylated and non-phosphorylated forms of osteopontin are secreted and that different fragments in urine are found, which are differently modulated by diseases, such as IgA nephritis.196 As recently commercially available protein assays for different platforms have been made available for the quantitation of osteopontin in the urine of rats, mice, and humans, it is expected that more evidence of the utility of osteopontin as a peripheral marker for detecting AKI will be generated soon. Urinary L-FABP Liver-type fatty acid-binding protein (L-FABP) is a 14-kDa protein normally expressed in the proximal tubules in the kidney.197 Cytoplasmic L-FABP in the proximal tubules bind free fatty acids, which are then transported to mitochondria or peroxisomes and metabolized there.198 Increased urinary levels of L-FABP have been associated with different types of kidney injury, in particular ischemia-related injuries, chronic kidney diseases, and some types of drug-induced acute kidney injury. Publications about the utility of L-FABP to detect drug-induced AKI include cisplatin, where urinary L-FABP was increased within 24 hours in contrast to serum creatinine (no increases until 72 hours) in mice and contrast-induced AKI, where increased urinary L-FABP levels also preceded increased serum creatinine levels, and after administration of cephaloridine urinary L-FABP levels indicated drug-induced renal injury seen in the absecene of rises in serum creatinine and BUN.199-201 In septic shock patients, only urinary L-FABP among a number of parameters showed a correlation with survival.202 Ischemia-related kidney injuries with subsequent increases of urinary L-FABP have been reported in the context of cardiac surgery where increases of L-FABP preceded increases of serum creatinine by a few days, and in kidney transplantation where increased urinary L-FABP levels were correlated with ischemia time and with peritubular capillary blood flow of the transplanted organ.203-204 In the context of chronic kidney diseases, increased urinary L-FABP levels have been identified in a number of studies such as non-diabetic chronic kidney disease, polycystic kidney disease, early diabetic nephropathy, and idiopathic focal glomerulosclerosis.205-209 Since L-FABP is predominantly expressed in the liver, urinary levels of L-FABP might not be specific to kidney injury and kidney diseases, but might be influenced also by changes of serum levels of L-FABP, if the proximal tubular re-absorption of the freely filtered L-FABP is saturated. Yet, until now no


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non-renal injury or condition has been reported, which caused highly increased levels as described for the renal injuries (several fold). Some studies investigating urinary L-FABP levels for CKD patients with impaired renal reabsortion capabilities show only a low contribution of serum L-FABP levels to urinary levels.210-211 With the availability of commercial assays for measuring L-FABP in different species, it is expected that further data about the utility of L-FABP for detecting AKI, as well as possible limitations in terms of specificity, will become available soon. Urinary Trefoil Factor 3 Trefoil factors are small proteins secreted by mucus-producing epithelial cells. They are believed to play a role in mucosal surface homeostasis and protection against injury, possibly by inhibiting apoptosis and promoting epithelial cell differentiation and mucin secretion.212-215 Trefoil Factor 3 (TFF3) is reported to be widely distributed to human pancreas, brain, respiratory tract, gall-bladder, bile ducts, salivary glands, and gastrointestinal tract (ref 1). Rat kidney is also a major site of TFF3 expression.216 Recently217 in situ hybrization results in rat kidney have localized TFF3 mRNA to abundant tubules of the outer stripe of the outer medulla, a site enriched for proximal straight tubules, while histochemical localization data has detected TFF3 binding sites in the collecting ducts of the rat kidney,2'8 suggesting an important role of TFF3 for communication between cells of the proximal tubule and the downstream collecting duct. The discovery of a profound decrease in tissue and urinary TFF3 mRNA and protein levels, respectively in response to numerous kidney tubular toxicants has been made only recently in rats217 and its relevance to humans remains to be evaluated. The specific stimulus that regulates TFF3, in the context of acute renal tubular injury, is unknown. Results from studies in gastrointestinal models indicate that TFF3 is regulated by inflammatory cytokines,219' 22° suggesting that acute reduction of TFF3 may be mediated by inflammatory cytokines produced during the course of renal tubular injury. Further work is required to investigate the potential utility of TFF3 as a translational biomarker beyond the rat into other test species and its relative value in humans for reflecting events associated with cytokine signaling in monitoring drug induced and other causes of acute renal injury.

Immune Markers Urinary IL-18 Interleukin 18 (IL-18) is a proinflammatory cytokine that is converted from its pro-form to the active form by the intracellular cystein protease capsase-1.221 It is involved in inflammation, ischemic tissue injury, and T-cell mediated immunity and plays an important role in the activation of macrophages and natural killer cells.222223 In the kidney it is induced and cleaved mainly in the proximal tubules and released into the urine under different conditions of kidney injury: In mechanistic studies of mice models, it was been shown that IL-18 is a mediator of ischemic AKI in mice and with increased kidney and urinary protein

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levels as biomarker ischemia-related AKI.222,224 In patients, the utility of IL-18 to detect ischemia-related AKI has been shown in children undergoing cardiopulmonary bypass. Hereby increased IL-18 levels raised at four to eight hours after operation and remained increased for the next 40 hours, whereas serum creatinine detected AKI only 48 to 72 hours after cardiopulmonary bypass operation.225 Also in the context of kidney transplantation, increased IL-18 levels in the first 24 hours post-transplant were highly predictive of delayed graft function and of the need for dialysis.226 Also, increased mRNA levels in kidney biopsies of patients with acute kidney allograft rejection were reported.227 Two studies demonstrate a significant value of IL-18 as a biomarker to detect AKI: In a cross-sectional study, urinary IL-18 levels were significantly increased in patients with established AKI, but not in patients with urinary tract infection, CKD, nephritic syndrome, or prerenal failure, making it an ideal marker on a biomarker panel to differentiate different conditions, which can lead to increases of serum creatinine and other kidney biomarkers.228 In a study investigating the potential of IL-18 as an early marker of AKI in acute respiratory distress syndrome patients, IL-18 predicted AKI 24 hours before serum creatinine and was also predictive in mortality independent of severity scores of the illness, serum creatinine, and urine output. Yet, evidence of the utility of IL-18 as a biomarker to predict or diagnose AKI in other conditions, such as drug-induced kidney injury, has not been generated. More seriously, the utility of IL-18 has been challenged in several studies under conditions when increased IL-18 levels were expected, such as AKI in cardiac surgery in adults or contrast-induced nephropathy, despite other positive reports in these settings.229-232 As the underlying factors and reasons for these contradictions are currently unknown, interpretations of IL-18 should be made with caution and it is recommended to collect as much information about the studies, co-factors, and technical details together with the generated biomarker data to compile enough evidence about the utility and limitations of this promising kidney biomarker.

NEWEST TECHNOLOGIES AND A C H I E V E M E N T S A R O U N D K I D N E Y SAFETY BIOMARKERS Assays and Technologies The recent advances in the discovery and evaluation of kidney safety biomarkers have also stimulated the development of new assays as well as new technologies for the measurement of these biomarkers. The traditionally used tubular urinary enzymes (AP, AAP, G-GT, NAG) are typically spectrophotometrically measured enzyme-substrate-based colorimetric activity assays. With the new urinary and plasma protein biomarkers, ELISA assays have became the new workhorse for accurate quantitation. ELISA assays are based on the detection of an analyte with one or two epitopically distinct antibodies (depending on the assay format such as direct, indirect, sandwich, or com-


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petitive assays) whereby one antibody is linked to an enzyme to generate a chromogenic, fluorogenic, or electrochemical detectable signal. A number of ELISA assays for the measurement of kidney biomarkers for human and several animal species have become commercially available, such as GST-a, clusterin, or NGAL.233"235 Elisa assays also have the advantage that no exotic hardware and training are required rendering them broadly usable, e.g., in hospital labs. Yet, ELISA assays also have certain drawbacks, in particular with respect to throughput and multiple-biomarker measurements. In particular a result is available only after several hours, a direct multiplexing of measurements is not supported (measurement of several biomarkers in the same sample/well), and the volume of sample needed can be limiting in nonclinical studies (typically 0.1-0.3 ml per biomarker). To overcome these limitations, kidney biomarker assays have been developed for two automated, user-friendly, high-throughput technologies, such as the Luminex® and the MesoScale Discovery® platforms. The principle of Luminex® xMAP® technology lies on the capture of antibodies conjugated to the surface of color-coded microspheres (beads), which react with specific antigens present in the sample similarly to sandwich assays. Then, detecting antibodies labeled with a fluorescent reporter molecule bind in proportion to the captured antigen. The quantification is performed by passing the suspended microspheres (beads) through the detection chamber of a flow-cytometer. A green laser detects the amount of analytes bound to the beads and a red laser identifies the color-coded beads (the nature of the target). Up to 100 colorcodes exist, which allow a theoretical multiplexing of up to 100 simultaneous assays, whereby only 10 jul sample volume is needed. For the Luminex technology, rules based medicine markets most of the kidney biomarkers mentioned in this chapter (rat and human) as well as kits (rat) for home use.236 The electrochemiluminescence-based MesoScale Discovery® assays also rely on the sandwich immunoassay format. Here, the antibodies are immobilized on planar arrays in microplate format and the readout is the light signal emitted by an electrochemiluminescence reaction. Each well is equipped with a working electrode and a counter electrode generating an electrical circuit, which initiates the electrochemical stimulation of the ruthenium-labeled detection antibodies, resulting in the emission of light. Different formats of plates are available. Multi-spotting up to 10 spots in 96-well format and up to 100 spots in 24-well format allows multiplexing.237 MesoScale Discovery® also offers a number of kidney safety biomarkers multiplexed on different plates for rat and for human. For both technologies, it is also possible for a customer to develop new ELISA assays and to transfer these and other existing ELISA assays onto these platforms. Whereas the MesScale Discovery® and Luminex® assays need specific platforms, which are not necessarily available in every clinical laboratory of medium-size and smaller hospitals, the transfer of biomarker assays on standardized clinical platforms ensures a broad availability of the assays in clinical units as recently reported for the urinary NGAL assay (ARCHITECT® analyzer from Abbott Diagnostics).238 Alternatively, the development of point-

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of-care devices will facilitate the distribution of the kidney biomarkers into smaller hospital units and to doctors' offices. An early example is the transfer of the serum NGAL assay onto the Triage® NGAL Device, from Biosite Incorporated. The device is deployable directly to the point of patient care and a measurement requires only micro liter quantities of whole blood or plasma and delivers results in approximately 15 minutes.238 Finally, the recent work by Vaidya and coworkers to develop a diagnostic dipstick for an easy Kim-1 quantitation (rat and human) is an exciting development in the direction of expanding the convenience of using the new kidney biomarkers for rapid diagnostic purposes. The dipstick test allows a visual readout within a few minutes and can be used directly by the patient. Current limitations are the absolute quantitation of Kim-1 by normalizing to urinary creatinine. At an attractive price, this dipstick test might not only revolutionize screening for renal injury in patients, but also animal studies in drug development due to its simple application without the need for complicated dedicated laboratory equipment.

C o n s o r t i a Achieving the First Regulatory Q u a l i f i c a t i o n of Kidney Safety Biomarkers The stagnation of medical product development has been pointed out in a widely recognized report from the U.S. Food and Drug Administration (FDA) 2004 entitled "Challenge and Opportunity on the Critical Path to New Medical Products."239 This signaled the start of the FDA Critical Path Initiative, aimed at increasing the awareness of the need for collaboration in particular for opportunities that imply considerable resources non-achievable for single entities (regulatory authorities, single companies, universities, or other government agencies) such as organ safety biomarker qualifications. As one of the first projects of the Critical Path Initiative, the C-Path Institute was founded in 2005 as neutral ground and a catalyst for programs. The initial project hosted by the C-Path Institute, the Predictive Safety Testing Consortium (PSTC), brought 16 pharma and companies, one patient organization, and advisors from academic institutions, the FDA, and the European Medicines Agency (EMA) to exchange data and methodologies with the goal to qualify organ safety biomarkers for regulatory decision making in preclinical, translational, and clinical contexts. Until recently, there had been no clear path forward on how organ safety biomarkers, such as the kidney safety biomarkers, could be qualified for regulatory decision making in drug development, such as adapting dosing regimens or stratifying patients for treatment. All new biomarkers, such as the kidney safety biomarkers, were considered as exploratory biomarkers for internal decision making only, despite evidence of superiority compared to the current BUN and serum creatinine standards, which had never been formally qualified but were the only accepted measures for kidney safety. The companies recognized the opportunity of collaboration under the PSTC and collected preclinical and clinical evidence for the utility of seven kidney safety biomarkers (urinary cystatin C, (32-microglobulin, Kim-1, clusterin, TFF3, albumin, and total protein). These data and claims about their performance relative to


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BUN and serum creatinine, together with claims about the intended use of these biomarkers, were submitted to the FDA and EMA for formal regulatory qualification and approval for specific contexts in June 2007. In May 2008, the decision by the EMA and FDA for the acceptance of these biomarkers for use in specific preclinical contexts and for translational contexts on a case-by-case basis was published. Following the FDA and EMA first ever approval of new renal safety biomarkers, the first level of usefulness of these new markers in drug development is expected to be translational between toxicology studies and the first safety trials in healthy subjects and patients.240241 With the formal qualification of these seven biomarkers and with the availability of validated assays, it is anticipated that their preclinical and clinical use in drug development programs will increase further supplementing the nonclinical and clinical evidence of their utility, but also further highlighting their limitations. Future clinical use of these biomarkers will include the early identification of adverse renal effects of potentially nephrotoxic drugs, patient and medication selection, as well as punctual intervention for an optimized therapy on an individual basis, often also referred to as personalized medicine. Also, routine clinical care beyond drug development will profit from this expected exponentially growing use of these renal safety biomarkers, such as diagnosing and staging diseases as well as AKI. In May 2008, the HESI Development and Application of Biomarkers of Toxicity Technical Committee also submitted preclinical data for the urinary biomarkers RPA-1, GST-a and GST-(JL, and clusterin to the FDA and EMA as part of the biomarker qualification review process, demonstrating evidence of superior diagnostic performance of site-specific injury of some of these novel markers (e.g., GST-a and RPA-1) relative to reference markers in the nonclinical setting.242 An acceptance of the preclinical use of some of these biomarkers by the health authorities will further increase the toolbox of biomarkers accepted for preclinical contexts. Also, in Europe a public-private consortium was formed under the Innovative Medicine Initiative (IMI) in 2009 with the goal of accumulating clinical evidence, developing assays, and obtaining regulatory acceptance for biomarkers to monitor kidney, liver, and vascular safety in translational and clinical contexts. This consortium, called SAFE-T (Safer and Faster Evidence-based Translation), consists of 11 pharmaceutical companies, four small-medium enterprises, five academic institutions, and the EMA as founding members, and has a research budget of 34 million Euro for five years. SAFE-T and PSTC will jointly accumulate enough critical evidence for the utility and limitations of organ safety biomarkers, and in particular for kidney safety biomarkers, to obtain regulatory acceptance as well as broad scientific consensus for their utility in clinical contexts.

CONCLUSION AKI is not an uncommon event in routine clinical care and in drug development and becomes a devastating situation due to the fact that current diagnos-

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tic standards are late, insensitive, and cannot localize the injury in the kidney. Recently, a considerable number of promising new biomarker candidates have been proposed, which are not only more sensitive than the current diagnostic standards in different preclinical and clinical contexts, but sometimes are also site-specific and injury type-specific. Most of these biomarkers are not only useful in humans but also in different preclinical test species, which is of crucial importance when translating potentially nephrotoxic compounds into first in man trials in pharmaceutical drug development. Until recently the use of the new biomarkers in preclinical and clinical studies in drug development has been hindered by the lack of regulatory acceptance of these new biomarkers by the health authorities. In 2008, the first regulatory qualification of seven kidney safety biomarkers for preclinical use and for translational use on a case-by-case basis by EMA and FDA opened the door of safety biomarkers to drug development and will help to obtain more preclinical and translational evidence of the utility and limitations of the kidney biomarkers. For most of the kidney biomarkers, significantly more clinical evidence about their utility and limitations in different clinical contexts is needed before a regulatory qualification for wider clinical contexts will be achieved to enable their broad use in clinical trials. The increasing evidence of their utility will also promote their use outside of drug development, such as standard clinical care and diagnosis of kidney diseases. Differences of performances of single biomarkers, or differences of relative performances between biomarkers reported for different studies but in the same clinical context, clearly indicate a pressing need of large studies with assessments of not only one but of a whole panel of kidney biomarkers implemented. Such studies will assess the comparable utility of different biomarkers for certain clinical contexts, demonstrating the opportunities and limitations of each biomarker. This will enable understanding of which biomarkers should be combined on a panel for diagnosing kidney injury and disease in specific clinical contexts, and ultimately for developing decision algorithms for a panel of biomarkers. A first step in this direction was recently published for the diagnostic evaluation of nine urinary kidney biomarkers (Kim-1, NGAL, IL-18, HGF, cystatin C, NAG, VEGF, CXCL10, and total protein) for the cross-sectional comparison of 204 patients with and without AKI.243 The study revealed that different biomarkers showed different sensitivity for AKI but also different specificity versus the different control groups. Ultimately, the best diagnostic performance for the diagnosis of AKI was obtained by a logistic regression model which included NGAL, HGF, total protein and Kim-1 levels. Other studies including several promising biomarker candidates for preclinical and clinical contexts have been published for AKI after cardiopulmonary bypass244 and for different nephrotoxicants in preclinical studies.135,240 These studies will help to connect publications of small studies of single biomarkers by generating broader evidence of utility and limitations of these biomarkers, obtaining regulatory buy-in, supporting the development of biomarker technologies, and promoting their use in drag development and in daily clinical care. Compared to other organs, new biomarkers to monitor kidney safety are more advanced and their preclinical and


263

clinical application will expand exponentially in the coming years, completely changing the management of renal safety in drug development and clinical care.


3.

4.

5.

Acute kidney injury is a common event in intensive care and can be a limiting factor in drug development. Current peripheral standards to detect kidney injury, blood urea nitrogen, and serum creatinine assess renal function and not kidney injury. Therefore these markers are late and insensitive and detect kidney injury only, when up to two thirds of the functional nephron mass has been lost. Therefore, AKI remains a clinical situation with a high mortality, despite advances in clinical care. A number of promising new kidney biomarker candidates have been proposed that monitor the function, integrity of different compartments of the kidney, and various molecular and biological processes induced by renal injury. Numerous publications demonstrate their utility (e.g., detecting AKI earlier than serum creatinine) and limitations in different preclinical and clinical contexts of detecting and prognosing AKI. Future research needs to be directed in compiling more coherent and comparative clinical evidence about the utility and limitations of new kidney biomarkers evaluated together in the same large studies for specific clinical contexts. In addition, technologies to measure several biomarkers in small laboratories and hospital units and simple bedside devices need to be developed. Already, assays for measuring the new biomarkers and the acceptance of seven biomarkers for preclinical and translational drug development contexts by EMA and FDA offer a unique toolset to manage kidney safety in drug development and in routine clinical care.

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179. Onaga, M , Ido, A., Hasuike, S., Uto, H., Moriuchi, A., Nagata, K., Hori, T., Hayash, K., and Tsubouchi, H. Osteoactivin Expressed During Cirrhosis Development in Rats Fed a Choline-Deficient, L-Amino Acid-Defined Diet, Accelerates Motility of Hepatoma Cells. J. Hepatol. 2O03;39(5):779-785. 180. Xie, Y., Sakatsume, M., Nishi, S., Narita, I., Arakawa, M., and Gejyo, F. Expression, Roles, Receptors, and Regulation of Osteopontin in the Kidney. Kidney Int. 2001 ;60(5): 1645-1657. 181. Giachelli, C. M., Lombardi, D., Johnson, R. J., Murry, C. E., and Almeida, M. Evidence for a Role of Osteopontin in Macrophage Infiltration in Response to Pathological Stimuli In Vivo. Am. J. Pathol. Feb 1998;152(2):353-358. 182. Hudkins, K. L., Giachelli, C. M., Cui, Y, Couser, W. G., Johnson, R. J., and Alpers, C. E. Osteopontin Expression in Fetal and Mature Human Kidney. J. Am. Soc. Nephrol. 1999;10(3):444-57. 183. Wang, E. J., Snyder, R. D., Fielden, M. R., Smith, R. J., and Gu, Y Z. Validation of Putative Genomic Biomarkers of Nephrotoxicity in Rats. Toxicology. 2008;246(2-3):91-100. 184. Rached, E., Hoffmann, D., Blumbach, K., Weber, K., Dekant, W., and Mally, A. Evaluation of Putative Biomarkers of Nephrotoxicity After Exposure to Ochratoxin A In Vivo and In Vitro. Toxicol. Sci. Jun 2008;103(2):371-381. 185. Xie, Y, Nishi, S., Iguchi, S., Imai, N., Sakatsume, M., Saito, A., Ikegame, M., lino, N., Shimada, H., Ueno, M., Kawashima, H., Arakawa, M., and Gejyo, F. Expression of Osteopontin in Gentamicin-Induced Acute Tubular Necrosis and Its Recovery Process. Kidney Int. Mar 2001;59(3):959-974. 186. Hudkins, K. L., Giachelli, C. M., Eitner, F., Couser, W. G., Johnson, R. J., and Alpers, C. E. Osteopontin Expression in Human Crescentic Glomerulonephritis. Kidney Int. Jan 2000;57(1):105-116. 187. Hudkins, K. L., Le, Q. C , Segerer, S., Johnson, R. J., Davis, C. L., Giachelli, C. M., and Alpers, C. E. Osteopontin Expression in Human Cyclosporine Toxicity. Kidney Int. Aug 2001;60(2):635-640. 188. Thomas, S. E., Lombardi, D., Giachelli, C , Bohle, A., and Johnson, R. J. Osteopontin Expression, Tubulointerstitial Disease, and Essential Hypertension. Am. J. Hypertens. 1998;11(8 Pt 1):954-961. 189. Ramankulov, A., Lein, M., Kristiansen, G., Meyer, H. A., Loening, S. A., and Jung, K. Elevated Plasma Osteopontin As Marker for Distant Metastases and Poor Survival in Patients With Renal Cell Carcinoma. Cancer Res. Clin. Oncol. Sep2007;133(9):643-652. 190. Yang, A., Trajkovic, D., Illanes, O., and Ramiro-Ibanez, F. Clinicopathological and Tissue Indicators of Para-Aminophenol Nephrotoxicity in Sprague-Dawley Rats. Toxicol. Pathol. 2007;35(4):521-532. 191. Mezzano, S. A., Droguett, M. A., Burgos, M. E., Ardiles, L. G., Aros, C. A., Caorsi, I., and Egido, J. Overexpression of Chemokines, Fibrogenic Cytokines, and Myofibroblasts in Human Membranous Nephropathy. Kidney Int. 2000;57(1):147-158. 192. Okada, H., Moriwaki, K., Konishi, K., Kobayashi, T., Sugahara, S., Nakamoto, H., Saruta, T., and Suzuki, H. Tubular Osteopontin Expression in Human Glomerulonephritis and Renal Vasculitis. Am. J. Kidney Dis. 2000;36(3):498-506. 193. Verstrepen, W. A., Persy, V. P., Verhulst, A., Dauwe, S., and De Broe, M. E. Renal Osteopontin Protein and Mrna Upregulation During Acute Nephrotoxicity in the Rat. Nephrol. Dial. Transplant. 2001;16(4):712-724.

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BIOMARKERS 194. Davis, J. W., II, Goodsaid, F. M , Bral, C. M., Obert, L. A., Mandakas, G., Garner, C. E., II, Collins, N. D., Smith, R. J., and Rosenblum, I. Y. Quantitative Gene Expression Analysis in a Nonhuman Primate Model of Antibiotic-Induced Nephrotoxicity. Toxicol. Appl. Pharmacol. 2004;200(1): 16-26. 195. Kharasch, E. D., Schroeder, J. L., Bammler, T., Beyer, R., and Srinouanprachanh, S. Gene Expression Profiling of Nephrotoxicity From the Sevoflurane Degradation Product Fluoromethyl-2,2-Difluoro-l-(Trifiuoromethyl)Vinyl Ether ("Compound A") in Rats. Toxicol. Sci. Apr 2006;90(2):419-431. 196. Gang, X., Ueki, K., Kon, S., Maeda, M., Naruse, T., and Nojima, Y. Reduced Urinary Excretion of Intact Osteopontin in Patients with Iga Nephropathy. Am. J. Kidney Dis. 2001;37(2):374-379. 197. Maatman, R. G., Van De Westerlo, E. M., Van Kuppevelt, T. H., and Veerkamp, J. H. Molecular Identification of the Liver and the Heart-Type Fatty Acid-Binding Proteins in Human and Rat Kidney. Use of the Reverse Transcriptase Polymerase Chain Reaction. Biochem. J. 1992;288(Pt l):285-290. 198. Sweetser, D. A., Heuckeroth, R. O., and Gordon, J. I. The Metabolic Significance of Mammalian Fatty-Acid-Binding Proteins: Abundant Proteins in Search of a Function. Annu. Rev. Nutr. 1987;7:337-359. 199. Negishi, K., Noiri, E., Sugaya, T., Li, S., Megyesi, J., Nagothu, K., and Portilla, D. A Role of Liver Fatty Acid-Binding Protein in Cisplatin-Induced Acute Renal Failure. Kidney Int. 2007;72(3):348-358. 200. Nakamura, T., Sugaya, T., Node, K., Ueda, Y, and Koide, H. Urinary Excretion of Liver-Type Fatty Acid-Binding Protein in Contrast Medium-Induced Nephropathy. Am. J. Kidney Dis. 2006;47(3):439^44. 201. Nakamura, K., Ito, K., Kato, Y, Sugaya, T., Kubo, Y, andTsuji, A. L-Type Fatty Acid Binding Protein Transgenic Mouse as a Novel Tool to Explore Cytotoxicity to Renal Proximal Tubules. Drug Metab. Pharmacokinet. 2008;23(4):271-278. 202. Noiri, E., Doi, K., Negishi, K., Tanaka, T., Hamasaki, Y, Fujita, T., Portilla, D., and Sugaya, T. Urinary Fatty Acid-Binding Protein 1: An Early Predictive Biomarker of Kidney Injury. Am. J. Physiol. Renal Physiol. 2009;296(4):F669-679. 203. Portilla, D., Dent, C , Sugaya, T., Nagothu, K. K., Kundi, I., Moore, P., Noiri, E., and Devarajan, P. Liver Fatty Acid-Binding Protein as a Biomarker of Acute Kidney Injury After Cardiac Surgery. Kidney Int. Feb 2008;73(4):465^72. 204. Yamamoto, T., Noiri, E., Ono, Y, Doi, K., Negishi, K., Kamijo, A., Kimura, K., Fujita, T, Kinukawa, T., Taniguchi, H., Nakamura, K., Goto, M., Shinozaki, N., Ohshima, S., and Sugaya, T. Renal L-Type Fatty Acid—Binding Protein in Acute Ischemic Injury. J. Am. Soc. Nephrol. 2007; 18(11):2894-2902. 205. Kamijo, A.,Sugaya, T.,Hikawa,A.,Yamanouchi,M.,Hirata,Y,Ishimitsu,T.,Numabe,A.,Takagi,M.,Hayakawa,H.,Tabei,F.,Sugimoto,T.,Mise,N.,andKimura,K. ClinicalEvaluationofUrinaryExcretionofLiver-Type Fatty Acid-Binding Protein As a Marker for the Monitoring of Chronic Kidney Disease: A Multicenter Trial. J. Lab. Clin. Med. 2005;145(3):125-133. 206. Nakamura, T., Sugaya, T., Kawagoe, Y, Ueda, Y, Osada, S., and Koide, H. Candesartan Reduces Urinary Fatty Acid-Binding Protein Excretion in Patients with Autosomal Dominant Polycystic Kidney Disease. Am. J. Med. Sci. 2005; 330(4):161-165. 207. Nakamura, T., Sugaya, T., Kawagoe, Y, Ueda, Y, Osada, S., and Koide, H. Effect of Pitavastatin on Urinary Liver-Type Fatty Acid-Binding Protein Levels in Patients with Early Diabetic Nephropathy. Diabetes Care. 2005;28(11): 2728-2732.


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208. Nakamura, T., Sugaya, T., Kawagoe, Y., Ueda, Y, Osada, S., and Koide, H. Urinary Liver-Type Fatty Acid-Binding Protein Levels for Differential Diagnosis of Idiopathic Focal Glomerulosclerosis and Minor Glomerular Abnormalities and Effect of Low-Density Lipoprotein Apheresis. Clin. Nephrol. 2006;65(l):l-6. 209. Hofstra, J. M., Deegens, J. K., Steenbergen, E. J., and Wetzels, J. F. Urinary Excretion of Fatty Acid-Binding Proteins in Idiopathic Membranous Nephropathy. Nephrol. Dial. Transplant. 2008;23(10): 3160-3165. 210. Oyama, Y., Takeda, T., Hama, H., Tanuma, A., lino, N., Sato, K., Kaseda, R., Ma, M., Yamamoto, T, Fujii, H., Kazama, J. J., Odani, S., Terada, Y, Mizuta, K., Gejyo, F, and Saito, A. Evidence for Megalin-Mediated Proximal Tubular Uptake of L-FABP, a Carrier of Potentially Nephrotoxic Molecules. Lab. Invest. 2005;85(4):522-531. 211. Kamijo, A., Sugaya, T., Hikawa, A., Yamanouchi, M., Hirata, Y, Ishimitsu, T., Numabe, A., Takagi, M., Hayakawa, H., Tabei, E, Sugimoto, T., Mise, N., Omata, M., and Kimura, K. Urinary Liver-Type Fatty Acid Binding Protein as a Useful Biomarker in Chronic Kidney Disease. Mol. Cell. Biochem. Mar 2006; 284(1-2): 175-182. 212. Taupin, D. R., Kinoshita, K., and Podolsky, D. K. Intestinal Trefoil Factor Confers Colonic Epithelial Resistance to Apoptosis. Proc. Natl. Acad. Sci. USA. 2000;97(2):799-804. 213. Kinoshita, K., Taupin, D. R., Itoh, H., and Podolsky, D. K. Distinct Pathways of Cell Migration and Antiapoptotic Response to Epithelial Injury: StructureFunction Analysis of Human Intestinal Trefoil Factor. Mol. Cell. Biol. 2000 (13):4680-4690. 214. Lesimple, P., Van Seuningen, I., Buisine, M. P., Copin, M. C , Hinz, M., Hoffmann, W., Hajj, R., Brody, S. L., Coraux, C , and Puchelle, E. Trefoil Factor Family 3 Peptide Promotes Human Airway Epithelial Ciliated Cell Differentiation. Am. J. Respir. Cell. Mol. Biol. 2007;36(3):296-303. 215. Hoffmann, W. TFF (Trefoil Factor Family) Peptides and Their Potential Roles for Differentiation Processes During Airway Remodeling. Curr. Med. Chem. 2007;14(25):2716-2719. 216. Suemori, S., Lynchdevaney, K., and Podolsky, D. K. Identification and Characterization of Rat Intestinal Trefoil Factor: Tissue- and Cell-Specific Member of the Trefoil Protein Family. Proc. Natl. Acad. Sci. USA. 1991;88(24): 11017-11021. 217. Yu, Yan; Jin, Hong; Holder, Daniel; Ozer, Josef S.; Villarreal, Stephanie; Shughrue, Paul; Shi, Shu; Figueroa, David J.; Clouse, Holly; Su, Ming; Muniappa, Nagaraja; Troth, Sean P.; Bailey, Wendy; Seng, John; Aslamkhan, Amy G.; Thudium, Douglas; Sistare, Frank D.; and Gerhold, David L. Biomarkers of Kidney Tubule Injury: Urinary Trefoil Factor 3 and Albumin. Submitted (2009). 218. Chinery, R., Poulsom, R., Elia, G., Hanby, A. M., and Wright, N. A. Expression and Purification of a Trefoil Peptide Motif in a Beta-Galactosidase Fusion Protein and Its Use to Search for Trefoil-Binding Sites. Eur. J. Biochem. 1993;212(2):557-563. 219. Baus-Loncar, M., Al-Azzeh, E. D., Romanska, H., Lalani, El-N., Stamp, G. W., Blin, N., and Kayademir, T. Transcriptional Control of TFF3 (Intestinal Trefoil Factor) via Promoter Binding Sites for the Nuclear Factor Kappab and C/Ebpbeta. Peptides. 2004;25(5):849-854.

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BIOMARKERS 220. Dossinger, V., Kayademir, T., Blin, N., and Gott, P. Down-Regulation of TFF Expression in Gastrointestinal Cell Lines by Cytokines and Nuclear Factors. Cell. Physiol. Biochem. 2002; 12(4): 197-206. 221. Melnikov, V. Y., Ecder, T., Fantuzzi, G., Siegmund, B., Lucia, M. S., Dinarello, C. A., Schrier, R. W., and Edelstein, C. L. Impaired IL-18 Processing Protects Caspase-1-Deficient Mice From Ischemic Acute Renal Failure. J. Clin. Invest. 2001 ;107(9): 1145-1152. 222. Lochner, M. and Forster, I. Anti-Interleukin-18 Therapy in Murine Models of Inflammatory Bowel Disease. Pathobiology. 2002/2003;70(3): 164-169. 223. Edelstein, C. L. Biomarkers of Acute Kidney Injury. Adv. Chronic Kidney Dis. 2008;15(3):222-234. 224. Melnikov, V. Y., Faubel, S., Siegmund, B., Lucia, M. S., Ljubanovic, D., and Edelstein, C. L. Neutrophil-Independent Mechanisms of Caspase-1- and IL-18-Mediated Ischemic Acute Tubular Necrosis in Mice. J. Clin. Invest. 2002;110(8):1083-1091. 225. Parikh, C. R., Mishra, J., Thiessen-Philbrook, H., Dursun, B., Ma, Q., Kelly, C , Dent, C , Devarajan, P., and Edelstein, C. L. Urinary IL-18 Is an Early Predictive Biomarker of Acute Kidney Injury After Cardiac Surgery. Kidney Int. 2006;70(1): 199-203. 226. Parikh, C. R., Jani, A., Mishra, J., Ma, Q., Kelly, C , Barasch, J., Edelstein, C. L., and Devarajan, P. Urine NGAL and IL-18 are Predictive Biomarkers for Delayed Graft Function Following Kidney Transplantation. Am. J. Transplant. 2006;6(7):1639-1645. 227. Simon, T., Opelz, G., Wiesel, M., Pelzl, S., Ott, R. C , and Siisal, C. Serial Peripheral Blood Interleukin-18 and Perform Gene Expression Measurements for Prediction of Acute Kidney Graft Rejection. Transplantation. 2004;77(10): 1589-1595. 228. Parikh, C. R., Jani, A., Melnikov, V. Y, Faubel, S., and Edelstein, C. L. Urinary Interleukin-18 Is a Marker of Human Acute Tubular Necrosis. Am. J. Kidney Dis. Mar 2004;43(3):405^114. 229. Bulent Gul, C. B., Gullulu, M., Oral, B., Aydinlar, A., Oz, O., Budak, E, Yilmaz, Y, and Yurtkuran, M. Urinary IL-18: A Marker of Contrast-Induced Nephropathy Following Percutaneous Coronary Intervention? Clin. Biochem. 2008;41 (7-8):544-547. 230. Ling, W., Zhaohui, N., Ben, H., Leyi, G., Jianping, L., Huili D, and Jiaqi Q. Urinary IL-18 and NGAL As Early Predictive Biomarkers in Contrast-Induced Nephropathy After Coronary Angiography. Nephron. Clin. Pract. 2008;108(3): C176-181. 231. Haase, M., Bellomo, R., Story, D., Davenport, P., and Haase-Fielitz, A. Urinary Interleukin-18 Does Not Predict Acute Kidney Injury After Adult Cardiac Surgery: A Prospective Observational Cohort Study. Crit. Care. 2008;12(4):R96. 232. Xin, C , Yulong, X., Yu, C , Changchun, C , Feng, Z., and Xinwei, M. Urine Neutrophil Gelatinase-Associated Lipocalin and Interleukin-18 Predict Acute Kidney Injury After Cardiac Surgery. Ren. Fail. 2008;30(9):904-913. 233. Argutusmedical Ltd Homepage, http://www.argutusmed.com. 234. R&D Systems Homepage, www.rndsystems.com. 235. Biovendor Homepage, http://www.biovendor.com. 236. Rules Based Medicine Website, http://www.rulesbasedmedicine.com. 237. Mesoscale Discovery Technology Website, http://www.mesoscale.com/catalogsystemweb/webroot/technology.htm.


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238. Devarajan, P. Neutrophil Gelatinase-Associated Lipocalin (NGAL): A New Marker of Kidney Disease. Scand. J. Clin. Lab. Invest. Suppl. 2008;241:89-94. 239. Http://www.fda.gov/oc/initiatives/criticalpath/whitepaper.html. 240. EMEA CHMP, Final Conclusions on the Pilot Joint EMEA/FDA VXDS Experience on Qualification of Nephrotoxicity Biomarkers. 2009, http://www.emea. europa.eu/pdfs/human/sciadvice/67971908en.pdf. 241. Http://www.fda.govtobs/topics/NEWS/2008/NEW01850.html. 242. HESI 2008 Annual Report, Page 10, http://www.hesiglobal.org/files/public/annual%20reports/HESI2008AnnualReportFinal.pdf. 243. Vaidya, V. S., Waikar, S. S., Ferguson, M. A., Collings, F. B., Sunderland, K., Gioules, C , Bradwin, G., Matsouaka, R., Betensky, R. A., Curhan, G. C, and Bonventre, J. V. Urinary Biomarkers for Sensitive and Specific Detection of Acute Kidney Injury in Humans. Clin. Transl. Sci. 2008; 1(3):200-208. 244. Liangos, O., Tighiouart, H., Perianayagam, M. C , Kolyada, A., Han, W. K., Wald, R., Bonventre, J. V., and Jaber, B. L. Comparative Analysis of Urinary Biomarkers for Early Detection of Acute Kidney Injury Following Cardiopulmonary Bypass. Biomarkers. 2009 In Press.


CHAPTER

IN SEARCH OF BIOMARKERS FOR DRUG-INDUCED VASCULAR INJURY James R. Turk

H I S T O R Y A N D B A C K G R O U N D OF D I V I Overview Rising attrition rates, especially in late phase drug development, are driving an unsustainable increase in expenditures required for research and development (R&D).1 The high cost of drug failure during clinical development, plus the requirement to minimize or prevent severe adverse events (AE) that occur after a drug has reached the market, emphasize an unmet need to develop translatable animal models and biomarkers that are predictive of toxicity in humans.2 There has been an increase in the incidence of an AE in preclinical toxicology studies that has been designated "drug-induced vascular injury" (DIVI). This AE is detected only by histology in animals that have exhibited no clinical signs or alterations of routine clinical pathology parameters at the time of sacrifice for standard toxicology studies. The morphological and pharmacological reversibility of DIVI is poorly understood. Noninvasive detection of DIVI is not currently possible due to the lack of specific and sensitive biomarkers of vascular cell injury.3,4-5 This lack of monitorable endpoints drives regulatory concern about the potential for DIVI to go undetected in early clinical trials in normal human volunteers while potentially promoting late phase or postmarketing progression of coronary and peripheral artery disease in human atherosclerosis, for which a number of circulating and functional biomarkers of endofhelial or vascular dysfunction have been proposed.6^16

281

282

BIOMARKERS

Types of Compounds Implicated Vascular injury in experimental animals has been associated with exposure to environmental toxins such as dioxin17 and compounds such as streptozotocin that is used to induce pancreatic beta cell death in animal models of diabetes.18 However, the focus of this chapter is DIVI that may halt the development of drugs to treat or prevent human disease. The observation that a compound as mundane as caffeine produces arteritis in rats19 illustrates species differences in cardiovascular pathophysiology20'21 and susceptibility to toxins, thereby challenging the translatability of preclinical DIVI to humans. Fenoldopam is a prototype compound that induces DIVI in rat mesenteric and canine coronary arteries22'23 for which there is no evidence of human AE. Indeed, fenoldopam is efficacious for indications that include increasing blood flow for cardio- and nephroprotection24-27 and the induction of controlled hypotension for oromaxillofacial and other surgery.28 During the 1980s and mid-'90s changes in mean arterial pressure (MAP) and heart rate (HR) were considered surrogate markers for DIVI in dogs.3'4i 29'30 If therapeutic doses of candidate drugs failed to induce hypotension and reflex tachycardia in humans, these drugs were considered to be safe. Using this guidance, many potent vasodilators that produced preclinical DIVI were developed due to monitorable hypotension with or without reflex tachycardia (Table ll.l). 5 ' 2 2 ' 2 3 ' 3 1 ^ 4 This paradigm shifted when endothelin receptor antagonists were shown to induce DIVI in the coronary arteries of dogs and non-human primates in the absence of significant alteration of HR or MAP, suggesting that these monitorable endpoints were insufficient criteria to exclude vascular injury in humans exposed to comparable doses.3'4'45"49

TABLE I I. I Pharmacological class, prototype drugs, and cardiovascular effects in preclinical druginduced vascular injury.

Pharmacological class

Prototype drug

Cardiovascular effect

A l receptor agonist

Adenosine

BP/reflex tachycardia

(3-adrenergic agonist

Isoproterenol

BP

Dopamine agonist

Fenoldopam

No change

Endothelin antagonist

Bosentan

No change

K-channel openers

Minoxidil


PDEIII inhibitor

Milrinone/theophylline


PDEIV inhibitor

Cilomilast/CI-1044

No change

PDEV inhibitor

Taladafil

BP

Vasoconstrictor

Angiotensin II

BP

Vasodilator

Hydralazine


IN SEARCH OF BIOMARKERS FOR DRUG-INDUCED VASCULAR INJURY

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D E S C R I P T I V E P A T H O L O G Y OF D R U G I N D U C E D V A S C U L A R INJURY Vascular Anatomy The microscopic anatomy of the vasculature is relatively simple, consisting of three tunicae: (1) intima, composed of endothelial cells; (2) media, composed of smooth muscle cells and bounded by porous internal and external elastic laminae; and (3) adventitia, composed primarily of fibroblasts, that in epicardial conduit and other larger vessels are surrounded by and interdigitate with vasa vasorum and adipose tissue that contains not only adipocytes, but also capillaries, lymphocytes, macrophages, mast cell/basophils, and nerves.50-57 Additional cells from the circulating blood including erythrocytes, neutrophils, monocytes, eosinophils, and others influence vascular health and disease (Figure 11.1).

FIGURE I I. I Cells of the vasculature that may be involved in vascular injury. (See color insert for a full color version of this figure.)

BIOMARKERS

284

Rat In the rat DIVI primarily affects muscular resistance arteries of the mesentery with an external diameter of 100-800 microns. Necrosis and hemorrhage occur within the tunica media within the first 24 hours (Figure 11.2B). These lesions diminish over the next 3-14 days as leukocytes (macrophages, T-cells and B-cells) accumulate and fibroplasia occurs in the tunica adventitia (Figure 11.2C, D).22,23,32,35'37'u'52,58_67 The lesions are typically random and segmental. Inflammation may occur in the adjacent mesentery.63 DIVI also may occur in arteries of the heart, testis, and the pampiniform plexus.36-65

Dog In the dog DIVI typically affects 200-500 micron epicardial segments of the left and right coronary arteries, although smaller intramyocardial branches may be involved. The histologic changes and time frame are similar to those in the rat mesentery, consisting initially of necrosis of the tunica media with accumulation of hyaline droplets and pockets containing proteinaceous material and/or erythrocytes. In advanced lesions, the hemorrhage may bridge all vascular tunicae with pyknotic and karyorrhectic debris and a few leukocytes in the tunica media.The endothelium of affected arteries may become enlarged and overlain by adherent leukocytes. Hemorrhage, accumulation of mononu-

FIGURE I 1.2 A: Normal rat mesenteric artery; B: Necrosis and hemorrhage occur within the tunica media at 24 hours after fenoldopam; C and D: Accumulation of leukocytes and fibroplasia at seven days after fenoldopam. (See color insert for a full color version of this figure.)


285

clear and polymorphonuclear leukocytes, and proliferation of fibroblasts also occurs in the tunica adventitia.29'30,33,34,39Jl3,49,68~74

Primate In non-human primates the lesions of DIVI are similar to those in the dog, consisting of medial necrosis with cellular debris and mixed inflammatory cell response in the intima, media, and adventitia. Arterial lesions are sometimes present in extracoronary sites including the small and large intestines and testis.75'76

Spontaneous Lesions in Preclinical Species Spontaneous necrotzing arteritis develops with age in hypertensive rats and occurs more frequently in males than females. Lesions are common in mesenteric and testicular arteries, with only the lungs, brain, and aorta spared in males. In females the lesions may be restricted to the tongue, parametrium, and mesentery.77,78 Spontaneous rupture of the pancreatoduodenal artery and hemoabdomen has been reported in ACI/SegHsd rats.79 Necrotizing arteritis of the extramural coronary arteries is a poorly understood spontaneous lesion in dogs. 29,33,34,80 This background lesion has been reported in nearly a quarter of studies performed during drug development.81, 82 Much like the rat, this lesion occurs more frequently in male than female dogs.83 Polyarteritis affecting the testis, epididymis, thymus, and other vascular beds is also observed in young dogs exhibiting febrile response (104-106°F), pain, and neutrophilia.84-86 Segmental necrotizing arteritis of variable severity with transmural mixed inflammatory cell infiltrate, fibrinoid necrosis of the tunica media, and loss of the internal elastic lamina affecting vessels of the kidney, small intestine, colon, heart, spleen, mesentery, urinary bladder, and pancreas have been reported in a cynomolgus monkey. Immunohistochemical staining showed that many of the infiltrating cells were T lymphocytes and histiocytes, suggesting a cell-mediated component to the pathogenesis.87 Spontaneous or background lesions that occur in vehicle control and dosed animals may confound the identification of DIVI as a toxicity that limits drug development. The clinical signs as well as the nature and distribution of lesions may aid in differentiation of background arteritis from DIVI. DIVI tends to be restricted to the mesenteric arteries of rats, and the coronary arteries of dogs and primates; whereas spontaneous arteritides involve medium to small arteries of many organs in addition to the mesenteric or coronary arteries, for which the term polyarteritiis nodosa may be applied. Hemorrhage in the tunicae media and adventitia is absent in spontaneous arteritides, but is consistently associated with DIVI. Animals with DIVI show no clinical signs.

C o m p a r i s o n w i t h Human Vasculitides Vascular toxicity associated with antineoplastic agents is clinically heterogeneous, ranging from asymptomatic arterial lesions to a fatal thrombotic

286

BIOMARKERS

microangiopathic syndrome.87" The term "systemic vasculitis" describes a heterogeneous group of rare diseases characterized by inflammation and fibrinoid necrosis of blood vessel walls. Vasculitis may be primary (with no identifiable cause) or it may be secondary to infection, malignancy, or autoimmune disease. Evidence suggests that accelerated atherosclerosis is a complicating feature of most, if not all, autoimmune diseases including rheumatoid arthritis (RA), scleroderma, sarcoidosis, and systemic lupus erythematosus.88-91 Polyarteritis nodosa is an idiopathic necrotizing vasculitis affecting smallto medium-sized arteries in multiple vascular beds in humans91,92 and may resemble spontaneous background lesions in preclinical species. As discussed above, DIVI is typically restricted to the coronary and mesenteric arteries. Rash is a common manifestation of human drug-induced cutaneous vasculitis in which eosinophils may be prominent in biopsies.93'94 Neither rash nor eosinophils are typical of DIVI.

PROGRESS I N B I O M A R K E R A N D M O D E L D E V E L O P M E N T FOR D R U G - I N D U C E D V A S C U L A R INJURY Noninvasive detection of acute DIVI is not currently possible due to the lack of specific and sensitive biomarkers; however, Table 11.2 contains a nonexhaustive shortlist of some of the most promising candidates that have been nominated in recent years.3"5-32- «• 46-58-59- M- «•69'95

Alpha-1-Acid Glycoprotein Alpha-1-acid glycoprotein, also known as orosomucoid 1, is an acute-phase serum protein produced primarily by hepatocytes, but also by vascular endothelial cells and adipocytes.96-97 Alpha-1-acid glycoprotein inhibits the effects of histamine on endothelial cells.98 Alpha-1-acid glycoprotein has been reported to increase in a dose-dependent fashion within 24 to 72 hours in the serum of rats with mesenteric DIVI.64 Alpha-1-acid glycoprotein can be detected by ELISA in rat and human,64,99 radioimmunoassay in dog100 and by proteomics with mass spectrometry in humans.101

Calprotectin (SI00A9/A8) Calprotectin, also known as S100A9/A8, is a serum biomarker of human vascular inflammation that is expressed by neutrophils, monocytes, and vascular smooth muscle cells.101-106 Calprotectin has been shown by immunohistochemistry to be expressed at day four in canine coronary DIVI.74 Calprotectin can be assayed by RIA in dog106 and ELISA in human.101-106

Caveolin-1 Caveolin 1 is expressed by multiple cell types associated with the vasculature including endothelial cells, smooth muscle cells, fibroblasts, and mac-


TABLE I 1.2

287

Candidate biomarkers for drug-induced vascular injury.

Biomarker

Vascular expression

Detection

Species

Alpha-1 -acid glycoprotein (orosomucoid)

EC,Mac,Adp

ELISA, RIA, proteomics

Rat, dog, human

Calprotectin (S100A8/9)

Mon, Neu

ELISA, IHC, RIA,

Dog, human

Caveolin-1

EC,SMC,Mac,Adpa

IHC RT-PCR, western of PBMC

Brott, 2006; Dalmas 2008; Louden, 2006

Circulating endothelial cells/particles

EC

Ultracentrifugation and flow cytometry

Human

Complement 3

Mon/Mac

ELISA, microarray, proteomics

Rat, dog, human

Connective tissue growth factor

EC, SMC, uptake by Mac, pre-Adp

ELISA

Rat, human

C-Reactive protein

Hepatocyes, SMC

ELISA

Rat, dog, human

Endothelin-I

EC, SMC, Mac, Mst

ELISA

Rat, dog, human

Fibrinogen

Hepatocytes

ELISA

Rat, dog, human

ELISA

Rat

GRO/CINCI Haptoglobin

Hepatocytes.Adp

ELISA, proteomics

Rat, dog, human

Metallothionein-I

EC,Fbl,Mon,Adp

ELISA

Rat, dog, human

Monocyte chemoattractant protein-1

ECSMCMacAdp

ELISA

Rat, dog, human

NGAL

Neu

ELISA

Rat, dog, human

Osteopontin

EC, SMC, Mac, Fbl, Adp

ELISA

Rat, dog, human

Smooth muscle actin

SMC (EC)

Microarray

Rat, human

Thrombospondin-1

EC,SMC,Mac,Adp

ELISA

Rat, dog, human

Tissue inhibitor of metalloproteinases-1

EC,SMC,Mac,Adp

ELISA

Rat, dog, human

Tissue plasminogen activator

ECSMC, Fbl, Mac, Neu

ELISA

Rat, dog, human

Vascular cell adhesion molecule-1

EC, SMC, Mac

ELISA

Rat, dog, human

Vascular endothelial growth factor

ECSMCMacAdp

ELISA

Rat, dog, human

vonWillebrand factor

EC

ELISA

Rat, dog, human

288

BIOMARKERS

rophages.108-114 Decreased immunoreactivity for caveolin-1 has been shown at the onset of canine coronary and rat mesenteric DIVI.4 Caveolin-1 negatively regulates the activity of endothelial nitric oxide synthase (eNOS) and its absence is associated with excess basal release of NO and excessive vascular relaxation.110115 The observation that caveolin-1 induces smooth muscle cell apoptosis116 suggests that it may be a potential marker of DIVI-induced lesions in the tunica media as corroborated by its upregulation in smooth muscle cell-enriched fractions obtained by laser capture microdissection of rat mesenteric DIVI within one to four hours of induction.32 Noninvasive assay for caveolin-1 in humans has been performed by RTPCR, immunohistochemistry or western blotting of isolated peripheral blood mononuclear cells (PBMC),"7 or circulating microparticles.118 Antibodies against rat, dog, and human caveolin-1 are commercially available.

C i r c u l a t i n g Endothelial Cells/Particles Elevation of circulating endothelial cells (CECs) and microparticles has been proposed as a marker of human vasculitis.119'120 CECs and microparticles can be assayed by ultracentrifugation and flow cytometery.121 CEC in human vascular disease correlate with tissue factor and von Willebrand factor6'10,122 that are additional potential markers of DIVI as described by increased expression in endothelial cell-enriched samples of rat mesenteric DIVI discussed below.32 This technique has not yet been fully validated in the rat or dog.

Complement Component 3 Plasma complement component 3a (C3a) correlates with CEC in human autoimmune vasculitis.119 Complement component 3 also has been detected in human serum by proteomics with mass spectrometry.101 Elevated C3a has been assayed by microdialysis catheters in human thermal vascular injury.123 Complement component 3a receptor 1 (C3AR1) has been reported to increase by RNA microarray analysis of PBMC in human vascular thrombosis.124 Complement 3 is expressed by, and induces the conversion of, vascular smooth muscle cells from the contractile to synthetic phenotype associated with expression of osteopontin in rats.125 Complement component 3 expression detected by RNA microarray analysis is upregulated in canine coronary and rat mesenteric DIVI.32,58,59 69 In addition to assays for mRNA in tissues and PBMC, plasma C3a can be assayed by ELISA.118 Commercial ELISA kits are available for rat, dog, and human C3a.

C o n n e c t i v e Tissue G r o w t h Factor (CTGF) Connective tissue growth factor (CTGF) is expressed by multiple cell types associated with the vasculature including endothelial cells, smooth muscle, fibroblasts, and pre-adipocytes.126-131 Non-uniform shear induces CTGF expression in human endothelial cells129 and carotid atherosclerosis correlates


289

with increased plasma CTGF as detected by ELISA.132Upregulation of CTGF has been detected by RNA microarray analysis of vascular smooth muscle cell-enriched samples of rat mesenteric DIVI, suggesting that further study of its potential as a plasma biomarker of DIVI is warranted.32 ELISA kits for rat and human CTGF are commercially available; however these are not reported to be cross reactive for canine CTGF, for which there are no commercially available antibodies.

C-Reactive Protein (CRP) C-reactive protein (CRP) is an acute phase protein produced primarily by the liver, but also by vascular smooth muscle cells.134 CRP has been proposed as a leading candidate biomarker for inflammation and human cardiovascular disease;134,135 however species differences may hamper translational use of CRP since it is a poor marker of acute inflammation in the rat.136 Plasma CRP has been shown by ELISA to be elevated in rat DIVI64 and in dogs with arteritis.137 Commercial ELISA kits are available for rat, dog, and human CRP.

Endothelin-1 Endothelin-1 (ET-1) is expressed by multiple cell types associated with the vasculature including endothelial cells, smooth muscle cells, fibroblasts, macrophages, and mast cells.138-141 Plasma ET-1 detected by ELISA is increased in human vascular disease.142 The induction of DIVI by endothelin receptor antagonists47-49,73,74 and the observations that the expression of endothelin and endothelin receptors are upregulated in rat mesenteric DIVI32-58> 59 suggest that further study ET-1 as a plasma biomarker of DIVI is warranted. Commercial ELISA kits are available for rat and human ET-1, for which cross-reactivity with canine ET-1 is likely.143'144

Fibrinogen Fibrinogen is a plasma glycoprotein synthesized in the liver. Plasma fibrinogen is increased in human peripheral arterial disease15 and increases in canine coronary and rat mesenteric DIVI.58,59, m Fibrinogen mRNA expression is upregulated in rat mesenteric DIVI.32'58'59 Commercial ELISA kits are available for rat, dog, and human fibrinogen.

GRO/CINC-I Rat CINC is a homolog of human GRO and interleukin-8 homolog that has recently been shown to increase as early as four hours post DIVI in the rat mesentery.64'145

Haptoglobin Haptoglobin is expressed primarily by hepatocytes; however ischemia upregulates haptoglobin mRNA expression in adipocytes.146 Haptoglobin is stored in neutrophil granules and is released on activation to exert anti-inflammatory

290

BIOMARKERS

effects.147 Plasma haptoglobin has been shown by ELISA to be elevated in rat DIVI.64 Plasma haptoglobin has been identified by proteomics and mass spectroscopy as a biomarker of vascular disease in humans101-148 and rats.149 Commercial ELISA kits are available for rat, dog, and human haptoglobin.

Metallothionein-I

(MT-I)

Metallothionein-1 (MT-1) binds heavy metals and exerts antioxidant activity. It is expressed in endothelial cells, macrophages, fibroblasts, and adipocytes, and in human vascular lesions.150-153 MT-1 expression is decreased in humans with low plasma HDL-cholseterol.154 MT-1 mRNA is upregulated in rat mesenteric DIVI.58,59 MT-1 expression in endothelial cells, smooth muscle cells, and fibroblasts was demonstrated by in situ hybridization as was upregulation of mRNA in endothelial cell-enriched samples obtained by laser capture microdissection of rat mesenteric DIVI.32 Commercial ELISA kits are available for rat, dog, and human metallothionein.

M o n o c y t e C h e m o a t t r a c t a n t Protein-1 ( M C P - I ) Monocyte chemoattractant protein-1 (MCP-1) is expressed by multiple cell types associated with the vasculature including endothelial cells, smooth muscle cells, fibroblasts, macrophages, mast cells, and adipocytes.54- "3-155-159 MCP-1 gene polymorphisms correlate with serum MCP-1 and prognosis in human vascular disease.160-161 MCP-1 mRNA is upregulated in canine coronary DIVI.69 Commercial ELISA kits are available for rat, dog, and human MCP-1.

N e u t r o p h i l Gelatinase-Associated Lipocalin ( N G A L ) Neutrophil gelatinase-associated lipocalin (NGAL) is expressed in endothelial cells, smooth muscle cells, and macrophages in human vascular disease.162 Vascular injury upregulates the expression of neutrophil gelatinase-associated lipocalin (NGAL) mRNA and protein in an NF-kappaB-dependent manner in rat and human vascular smooth muscle cells.163 Serum neutrophil gelatinaseassociated lipocalin (NGAL) correlates with relapse of vasculitis in humans.164 Commercial ELISA kits are available for NGAL in rat, dog, and human.

Osteopontin (OPN) Osteopontin (OPN) is expressed by endothelial cells, smooth muscle cells, macrophages, adventitial fibroblasts, and adipocytes.165-171 As described above, complement component 3 is expressed by and induces the expression of osteopontin in smooth muscle cells of rats.125 Plasma OPN is associated with the presence and extent of human vascular disease.160-172,173 Osteopontin (OPN) mRNA expression is upregulated in rat mesenteric DIVI.32-58-59 Commercial ELISA kits are available for rat, dog, and human OPN.


291

Smooth Muscle A c t i n Alpha-smooth muscle actin is expressed primarily in SMC; however, it also may be expressed by microvascular endothelial cells and adventitial fibroblasts.174' "5 Increased numbers of large spindle cells expressing alpha-smooth muscle actin have been isolated from human peripheral blood mononuclear cells in human coronary artery disease.176 Smooth muscle actin is upregulated in smooth muscle cell-enriched fractions obtained by laser capture microdissection of rat mesenteric DIVI one to four hours after induction.32 At present there are no validated assays to assess smooth muscle actin in plasma or circulating PBMN for the rat or dog.

T h r o m b o s p o n d i n - 1 (TSP-I) Thrombospondin-1 (TSP-1) is expressed by multiple cell types of the vasculature including endothelial cells, smooth muscle cells, fibroblasts, macrophages, mast cells, and adiopcytes.177-180 Endothelial cell injury correlates with TSP-1.181 Thrombospondin-1 precursor mRNA expression is upregulated in canine coronary DIVI69 and in endothelial cell-enriched samples in rat mesenteric DIVI at the one-hour and/or four-hour time points.32 Commercial ELISA kits are available for rat, dog, and human TSP-1.

Tissue I n h i b i t o r of Metalloporteinases-1 ( T I M P - I ) Tissue inhibitor of metalloproteinases-1 (TIMP-1) is expressed by endothelial cells, smooth muscle cells, macrophages, fibroblasts, and adipocytes.182'184 TIMP-1 expression is greater in aortic smooth muscle cells from male than female rats.185 Plasma levels of TIMP-1 increase in human vascular disease.186- 187 TIMP-1 mRNA is upregulated in canine coronary69 and rat mesenteric DIVI.5859 Commercial ELISA kits are available for rat, dog, and human TIMP-1.

Tissue Plasminogen A c t i v a t o r (tPA) Tissue plasminogen activator (tPA) is expressed by endothelial cells, smooth muscle cells, fibroblasts, macrophages, and neutrophils.188-190 Serum tPA is increased in Henoch-Schonlein purpura, a childhood vasculitis.191 tPA mRNA is upregulated in rat mesenteric DIVI.32'58,59 Commercial ELISA kits are available for rat, dog, and human tPA.

Vascular Cell Adhesion Molecule I ( V C A M - I ) VCAM-1 is expressed by endothelial cells, smooth muscle cells, and macrophages.192, 193 Serum VCAM-1 increases in human vasculitis.194 VCAM-1 mRNA expression is upregulated in endothelial cell-enriched samples from rat mesenteric DIVI.32 Commercial ELISA kits are available for rat, dog, and human VCAM-1.

292

BIOMARKERS

Vascular Endothelial Growth Factor (VEGF) Vascular endothelial growth factor (VEGF) is expressed in endothelial cells, smooth muscle cells, fibroblasts, macrophages, mast cells, and adipocytes.195-198 Serum VEGF is an early marker of vascular damage that increases in acute human vasculitis and falls in remission.199-200 Serum VEGF increasd in rat mesenteric DIVI64 and VEGF mRNA was upregulated in endothelial cell-enriched samples microdissected from rat mesenteric DIVI.32 Commercial ELISA kits are available for rat, dog, and human VEGF.

Von Willebrand Factor Von Willebrand factor (VWF) is a plasma glycoprotein involved in platelet adhesion at the site of vascular damage, which acts as a bridge between the injured subendothelium and the platelet receptors.201 vWF in CEC correlates with human vascular disease.6202 In patients with preexisting vascular disease, VWF rises during acute coronary syndromes, and the extent of this VWF release is an independent predictor of adverse clinical outcome.203 vWF mRNA is upregulated in endothelial cell-enriched samples microdissection from rat mesenteri DIVI.32 Commercial ELISA kits are available for rat, dog, and human vWF.

CONCLUSIONS Drug-induced vascular injury in pre-clinical species currently halts the development of many drugs that have the potential to treat or prevent human disease. The noninvasive detection of DIVI is not currently possible due to the lack of specific and sensitive biomarkers. Table 11.2 contains a number of promising candidates that require further validation to facilitate their use for translation of preclinical findings to risk assessment of drugs for vascular injury in humans.2

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IN SEARCH OF BIOMARKERS FOR DRUG-INDUCED VASCULAR INJURY 301 136. Giffen, P. S., Turton, J., Andrews, C. M., Barrett, P., and Clarke, C. J., et al. Markers of Experimental Acute Inflammation in the Wistar Han Rat with Particular Reference to Haptoglobin and C-Reactive Protein. Arch. Toxicol. 2003;77:392-402. 137. Bathen-Noethen, A., Carlson, R., Menzel, D., Mischke, R., and Tipold, A. Concentrations of Acute-Phase Proteins in Dogs with Steroid Responsive Meningitis- Arteritis. J .Vet. Intern. Med. 2008;22:1149-1156. 138. An, S. J., Boyd, R., Zhu, M., Chapman, A., Pimentel, D. R., and Wang, H. D. NADPH Oxidase Mediates Angiotensin Il-Induced Endothelin-1 Expression in Vascular Adventitial Fibroblasts. Cardiovasc Res. Sep 1, 2007;75(4):702-709. 139. Lepailleur-Enouf, D., Valdenaire, O., Philippe, M., Jandrot-Perrus, M., and Michel, J. B. Thrombin Induces Endothelin Expression in Arterial Smooth Muscle Cells. Am. J. Physiol. Heart Circ. Physiol. 2000;278:H1606-1612. 140. Li, Z., Niwa, Y., Rokutan, K., and Nakaya, Y. Expression of Endothelin-1 in Macrophages and Mast Cells in Hyperplastic Human Tonsils. FEBS Lett. 1999;457:381-384. 141. Turk, J. R. Leading Edge of Medicine. Physiologic and Pathophysiologic Effects of Endothelin: Implications in Cardiopulmonary Disease. J. Am. Vet. Med. Assoc. 1998;212:265-270. 142. Videm, V., Wiseth, R., Gunnes, S., Madsen, H. O., and Garred, P. Multiple Inflammatory Markers in Patients with Significant Coronary Artery Disease. Int. J. Cardiol. 2007;118:81-87. 143. Biondo, A. W., Wiedmeyer, C. E., Sisson, D. D., and Solter, P. F. Comparative Sequences of Canine and Feline Endothelin-1. Vet. Clin. Pathol. 2003;32: 188-194. 144. Prosek, R., Sisson, D. D., Oyama, M. A., Biondo, A. W., and Solter, P. F. Plasma Endothelin-1 Immunoreactivity in Normal Dogs and Dogs with Acquired Heart Disease. J. Vet. Intern. Med. 2004;18:840-844. 145. Zagorski, J. and Delarco, J. E. Rat CINC (Cytokine-Induced Neutro-phil Chemoattractant) Is the Homolog of the Human GRO Proteins but Is Encoded by a Single Gene. Bichem. Biophys. Res. Commun. 1993;190:104-110. 146. Wang, B., Wood, I. S., and Trayhurn, P. Dysregulation of the Expression and Secretion of Inflammation-Related Adipokines by Hypoxia in Human Adipocytes. Pflugers Arch. 2007;455:479^92. 147. Theilgaard-Monch, K., Jacobsen, L. C , Nielsen, M. J., Rasmussen, T, and Udby, L., et al. Haptoglobin Is Synthesized During Granulocyte Differentiation, Stored in Specific Granules, and Released by Neutrophils in Response to Activation. Blood. 2006;108:353-361. 148. Pavon, E. J., Munoz, P., Lario, A., Longobardo, V, and Carrascal, M., et al. Proteomic Analysis of Plasma from Patients with Systemic Lupus Erythematosus: Increased Presence of Haptoglobin Alpha2 Polypeptide Chains Over the Alpha 1 Isoforms. Proteomics. 2006;6 Suppl l:S282-292. 149. Kiga, C , Sakurai, H., Goto, H., Hayashi, K., and Shimada, Y, et al. Proteomic Identification of Haptoglobin as a Stroke Plasma Biomarker in Spontaneously Hypertensive Stroke-Prone Rats. Life Sci. 2008;83:625-631. 150. Daskalopoulou, S. S., Daskalopoulos, M. E., Theocharis, S., Kavantzas, N., and Perrea, D., et al. Metallothionein Expression in the High-Risk Carotid Atherosclerotic Plaque. Curr. Med. Res. Opin. 2007;23:659-670. 151. Miyashita, H. and Sato, Y Metallothionein 1 Is a Downstream Target of Vascular Endothelial Zinc Finger 1 (VEZF1) in Endothelial Cells and Participates in the Regulation of Angiogenesis. Endothelium. 2005;12:163-170.

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BIOMARKERS 152. Pauwels, M., Van Weyenbergh, J., Soumillion, A., Proost, P., and De Ley, M. Induction by Zinc of Specific Metallothionein Isoforms in Human Monocytes. Eur. J. Biochem. 1994;220:105-110. 153. Trayhurn, P., Duncan, J. S., Wood, A. M., and Beattie, J. H. Regulation of Metallothionein Gene Expression and Secretion in Rat Adipocytes Differentiated from Preadipocytes in Primary Culture. Horm. Metab. Res. 2000;32:542-547. 154. Sarov-Blat, L., Kiss, R. S., Haidar, B., Kavaslar, N., and Jaye, M , et al. Predominance of a Proinflammatory Phenotype in Monocyte-Derived Macrophages from Subjects with Low Plasma HDL-Cholesterol. Arterioscler. Thromb. Vase. Biol. 2007;27:1115-1122. 155. Demicheva, E., Hecker, M., and Korff, T. Stretch-Induced Activation of the Transcription Factor Activator Protein-1 Controls Monocyte Chemoattractant Protein-1 Expression During Arteriogenesis. Circ. Res. 2008;103:477-484. 156. Nakayama, T., Mutsuga, N., Yao, L., Tosato. Prostaglandin E2 Promotes Degranulation-Independent Release of MCP-1 from Mast Cells. J. Leukoc. Biol. 2006;79:95-104. 157. Simionescu, M. Implications of Early Structural-Functional Changes in the Endothelium for Vascular Disease. Arterioscler. Thromb. Vase. Biol. 2007;27: 266-274. 158. Takahashi, M., Suzuki, E., Takeda, R., Oba, S., and Nishimatsu, H., et al. Angiotensin II and Tumor Necrosis Factor-Alpha Synergistically Promote Monocyte Chemoattractant Protein-1 Expression: Roles of NF-Kappab, P38, and Reactive Oxygen Species. Am. J. Physiol. Heart Circ. Physiol. 2008;294:H2879-2888. 159. Tsuchiya, K., Yoshimoto, T, Hirono, Y, Tateno, T., and Sugiyama, T, et al. Angiotensin II Induces Monocyte Chemoattractant Protein-1 Expression via a Nuclear Factor-Kappab-Dependent Pathway in Rat Preadipocytes. Am. J. Physiol. Endocrinol. Metab. 2006;291:E771-778. 160. Brenner, D., Labreuche, J., Touboul, P. J., Schmidt-Petersen, K., and Poirier, O., et al. Cytokine Polymorphisms Associated with Carotid Intima-Media Thickness in Stroke Patients. Stroke. 2006;37:1691-1696. 161. De Lemos, J. A., Morrow, D. A., Blazing, M. A., Jarolim, P., and Wiviott, S. D., et al. Serial Measurement of Monocyte Chemoattractant Protein-1 After Acute Coronary Syndromes: Results from the A to Z Trial. J. Am. Coll. Cardiol. 2007;50:2117-2124. 162. Hemdahl, A. L., Gabrielsen, A., Zhu, C , Eriksson, P., and Hedin, U., et al. Expression of Neutrophil Gelatinase-Associated Lipocalin in Atherosclerosis and Myocardial Infarction. Arterioscler. Thromb. Vase. Biol. 2006;26: 136-142. 163. Bu, D. X., Hemdahl, A. L., Gabrielsen, A., Fuxe, J., and Zhu, C , et al. Induction of Neutrophil Gelatinase-Associated Lipocalin in Vascular Injury via Activation of Nuclear Factor-Kappab. Am. J. Pathol. 2006;169:2245-2253. 164. Chen, M., Wang, F, and Zhao, M. H. Circulating Neutrophil Gelatinase-Associated Lipocalin: A Useful Biomarker for Assessing Disease Activity of ANCAAssociated Vasculitis. Rheumatology (Oxford). 2009;48:355-358. 165. Campos, A. H., Zhao, Y, Pollman, M. J., and Gibbons, G. H. DNA Microarray Profiling to Identify Angiotensin-Responsive Genes in Vascular Smooth Muscle Cells: Potential Mediators of Vascular Disease. Circ. Res. 2003;92:111-118. 166. Dorheim, M. A., Sullivan, M., Dandapani, V., Wu, X., and Hudson, J., et al. Osteoblastic Gene Expression During Adipogenesis in Hematopoietic Supporting Murine Bone Marrow Stromal Cells. J. Cell Physiol. 1993;154:317-328.

IN SEARCH OF BIOMARKERS FOR DRUG-INDUCED VASCULAR INJURY 303 167. Li, G., Oparil, S., Kelpke, S. S., Chen, Y. F., and Thompson, J. A. Fibroblast Growth Factor Receptor-1 Signaling Induces Osteopontin Expression and Vascular Smooth Muscle Cell-Dependent Adventitial Fibroblast Migration In Vitro. Circulation. 2002;106:854-859. 168. Oki, Y., Watanabe, S., Endo, T., and Kano, K. Mature Adipocyte-Derived Dedifferentiated Fat Cells Can Trans-Differentiate Into Osteoblasts in Vitro and In Vivo Only by All-Trans Retinoic Acid. Cell Struct. Fund. 2008;33:211-222. 169. Partridge, C. R., Williams, E. S., Barhoumi, R., Tadesse, M. G., and Johnson, C. D., et al. Novel Genomic Targets in Oxidant-Induced Vascular Injury. J. Mol. Cell. Cardiol. 2005;38:983-996. 170. Seipelt, R. G., Backer, C. L., Mavroudis, C , Stellmach, V., and Cornwell, M., et al. Osteopontin Expression and Adventitial Angiogenesis Induced by Local Vascular Endothelial Growth Factor 165 Reduces Experimental Aortic Calcification. 7. Thorac. Cardiovasc. Surg. 2005;129:773-781. 171. Sugiyama, T., Yoshimoto, T., Hirono, Y, Suzuki, N., and Sakurada, M., et al. Aldosterone Increases Osteopontin Gene Expression in Rat Endothelial Cells. Biochem. Biophys. Res. Commun. 2005;336:163-167. 172. Minoretti, R, Falcone, C , Calcagnino, M., Emanuele, E., and Buzzi, M. R, et al. Prognostic Significance of Plasma Osteopontin Levels in Patients with Chronic Stable Angina. Eur. Heart J. 2006;27:802-807. 173. Ohmori, R., Momiyama, Y, Taniguchi, H., Takahashi, R., and Kusuhara, M., et al. Plasma Osteopontin Levels Are Associated with the Presence and Extent of Coronary Artery Disease. Atherosclerosis. 2003; 170:333-337. 174. Ando, H., Kubin, T., Schaper, W., and Schaper, J. Cardiac Microvascular Endothelial Cells Express Alpha-Smooth Muscle Actin and Show Low NOS III Activity. Am. J. Physiol. 1999;276:H1755-1768. 175. Stenmark, K. R., Gerasimovskaya, E., Nemenoff, R. A., and Das, M. Hypoxic Activation of Adventitial Fibroblasts: Role in Vascular Remodeling. Chest. 2002;122(6 Suppl):326S-334S. 176. Sugiyama, S., Kugiyama, K., Nakamura, S., Kataoka, K., and Aikawa, M., et al. Characterization of Smooth Muscle-Like Cells in Circulating Human Peripheral Blood. Atherosclerosis. 2006;187:351-362. 177. McLaughlin, J. N., Mazzoni, M. R., Cleator, J. H., Earls, L., and Perdigoto, A. L., et al. Thrombin Modulates the Expression of a Set of Genes Including Thrombospondin-1 in Human Microvascular Endothelial Cells. J. Biol. Chem. 2005;280:22172-22180. 178. Scott-Burden, T., Resink, T. J., Hahn, A. W, and Biihler, F. R. Induction of Thrombospondin Expression in Vascular Smooth Muscle Cells by Angiotensin II. J. Cardiovasc. Pharmacol. 1990;16 Suppl 7:S17-20. 179. Stenina, O. I., Krukovets, I., Wang, K., Zhou, Z., and Forudi, E, et al. Increased Expression of Thrombospondin-1 in Vessel Wall of Diabetic Zucker Rat. Circulation. 2003;107:3209-3215. 180. Varma, V, Yao-Borengasser, A., Bodies, A. M., Rasouli, N., and Phanavanh, B., et al. Thrombospondin-1 Is an Adipokine Associated with Obesity, Adipose Inflammation, and Insulin Resistance. Diabetes. 2008;57:432-439. 181. Reed, M. J., Iruela-Arispe, L., O'Brien, E. R., Truong, T., and Labell, T, et al. Expression of Thrombospondins by Endothelial Cells. Injury Is Correlated with TSP-1. Am. J. Pathol. 1995;147:1068-1080. 182. Castoldi, G., Di Gioia, C. R., Pieruzzi, F., D'Orlando, C , and Van De Greef, W. M., et al. ANGII Increases TIMP-1 Expression in Rat Aortic Smooth Muscle Cells In Vivo. Am. J. Physiol. Heart. Circ. Physiol. 2003;284:H635-643.

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BIOMARKERS 183. Maury, E., Ehala-Aleksejev, K., Guiot, Y., Detry, R., and Vandenhooft, A. Adlpokines Oversecreted by Omental Adipose Tissue in Human Obesity. Am. J. Physiol. Endocrinol. Metab. 2007;293:E656-665. 184. Vaalamo, M., Leivo, T., and Saarialho-Kere, U. Differential Expression of Tissue Inhibitors of Metalloproteinases (TIMP-1, -2, -3, and -4) in Normal and Aberrant Wound Healing. Hum. Pathol. 1999;30:795-802. 185. Woodrum, D. T, Ford, J. W., Ailawadi, G., Pearce, C. G., and Sinha, I. Gender Differences in Rat Aortic Smooth Muscle Cell Matrix Metalloproteinase-9. J.Am. Coll. Surg. 2005;201:398^M)4. 186. De Leeuw, K., Freire, B., Smit, A. J., Bootsma, H., and Kallenberg, C. G., et al. Traditional and Non-Traditional Risk Factors Contribute to the Development of Accelerated Atherosclerosis in Patients with Systemic Lupus Erythematosus. Lupus. 2006;15:675-682. 187. West, M. J., Nestel, P. J., Kirby, A. C , Schnabel, R., and Sullivan, D., et al. The Value of N-Terminal Fragment of Brain Natriuretic Peptide and Tissue Inhibitor of Metalloproteinase-1 Levels as Predictors of Cardiovascular Outcome in the LIPID Study. Eur. Heart J. 2008;29:923-931. 188. Diamond, S. L., Eskin, S. G., and Mclntire, L. V. Fluid Flow Stimulates Tissue Plasminogen Activator Secretion by Cultured Human Endothelial Cells. Science. 1989;243:1483-1485. 189. Helenius, G., Hagvall, S. H., Esguerra, M., Fink, H., and Soderberg, R., et al. Effect of Shear Stress on the Expression of Coagulation and Fibrinolytic Factors in Both Smooth Muscle and Endothelial Cells in a Co-culture Model. Eur. Surg. Res. 2008;40:325-332. 190. Takamiya, M., Saigusa, K., Kumagai, R., Nakayashiki, N., and Aoki, Y. Studies on Mrna Expression of Tissue-Type Plasminogen Activator in Bruises for Wound Age Estimation. Int. J. Legal Med. 2005;119:16-21. 191. Besbas, N., Erbay, A., Saatci, U., Ozdemir, S., and Bakkaloglu, A., et al. Thrombomodulin, Tissue Plasminogen Activator and Plasminogen Activator Inhibitor-1 in Henoch-Schonlein Purpura. Clin. Exp. Rheumatol. 1998;16:95-98. 192. Hastings, N. E., Feaver, R. E., Lee, M. Y, Wamhoff, B. R., and Blackman, B. R. Human IL-8 Regulates Smooth Muscle Cell VCAM-1 Expression in Response to Endothelial Cells Exposed to Atheroprone Flow. Arterioscler. Thromb. Vase. Biol. 2009;29:725-731. 193. Trogan, E., Choudhury, R. P., Dansky, H. M., Rong, J. X., and Breslow, J. L., et al. Laser Capture Microdissection Analysis of Gene Expression in Macrophages from Atherosclerotic Lesions of Apolipoprotein E-Deficient Mice. Proc. Nat. Acad. Sci. USA. 2002;99:2234-2239. 194. Lee, A. B., Godfrey, T., Rowley, K. G., Karschimkus, C. S., and Dragicevic, G., et al. Traditional Risk Factor Assessment Does Not Capture the Extent of Cardiovascular Risk in Systemic Lupus Erythematosus. Intern. Med. J. 2006; 36:237-243. 195. Boesiger, J., Tsai, M., Maurer, M., Yamaguchi, M., and Brown, L. R, et al. Mast Cells Can Secrete Vascular Permeability Factor/Vascular Endothelial Cell Growth Factor and Exhibit Enhanced Release After Immunoglobulin E Dependent Upregulation of Fc Epsilon Receptor I Expression. J. Exp. Med. 1998; 188:1135-1145. 196. Inoue, M., Itoh, H., Ueda, M., Naruko, T., and Kojima, A., et al. Vascular Endothelial Growth Factor (VEGF) Expression in Human Coronary Atherosclerotic Lesions: Possible Pathophysiological Significance of VEGF in Progression of Atherosclerosis. Circulation. 1998;98:2108-2116.

IN SEARCH OF BIOMARKERS FOR DRUG-INDUCED VASCULAR INJURY 305 197. Jin, X., Ge, X., Zhu, D. L., Yan, C, and Chu, Y. E, et al. Expression and Function of Vascular Endothelial Growth Factor Receptors (Flt-1 and Flk-1) in Vascular Adventitial Fibroblasts. J. Mol. Cell. Cardiol. 2007;43:292-300. 198. Strande, J. L. and Phillips, S. A. Thrombin Increases Inflammatory Cytokine and Angiogenic Growth Factor Secretion in Human Adipose Cells In Vitro. J. lnflamm. (Lond). 2009;6:4. 199. Tsai, W. C , Li, Y H., Huang, Y. Y, Lin, C. C., and Chao, T. H., et al. Plasma Vascular Endothelial Growth Factor as a Marker For Early Vascular Damage in Hypertension. Clin. Sci. (Lond) 2005;109:39-43. 200. Wikman, A., Lundahl, J., and Jacobson, S. H. Sustained Monocyte Activation in Clinical Remission of Systemic Vasculitis. Inflammation. 2008;31:384-390. 201. Perutelli, P. and Molinari, A. C. Von Willebrand Factor, Von Willebrand FactorCleaving Protease, and Shear Stress. Cardiovasc. Hematol. Agents Med. Chem. 2007;5:305-310. 202. Blann, A. D. and McCollum, C. N. Von Willebrand Factor and Soluble Thrombomodulin as Predictors of Adverse Events Among Subjects with Peripheral or Coronary Atherosclerosis. Blood Coagul. Fibrinolysis. 1999;10:375-380. 203. Spiel, A. O., Gilbert, J. C , and Jilma, B. Von Willebrand Factor in Cardiovascular Disease: Focus on Acute Coronary Syndromes. Circulation. 2008;117: 1449-1459.


CHAPTER

BIOMARKERS OF IMMUNOTOXICITY Rodney R. Dietert

INTRODUCTION The immune system is universally recognized as playing a central role in the health and well-being of humans, wildlife, and domesticated animals. For this reason, identification of environmental health risks and application of the information for effective risk reduction can provide significant health benefits and should be a core of any safety screening strategy. But while protection of the immune system is an obvious and worthwhile goal, successful immunotoxicologic assessment is not necessarily a simple pursuit. The immune system represents one of the more daunting targets for toxicologic assessment. In part, this is directly related to its duality of function in which immune regulation of cell and tissue integrity and organ homeostasis is juxtaposed against a robust defense against external assault. The novel aspects of immunotoxicity evaluation are based on four fundamental features of the immune system: 1) the immune system is dispersed with representation in virtually every organ and tissue; 2) immune system components residing in different tissues may have their own range of toxicologic sensitivities; 3) the immune system is integrally wired to several other physiological systems (e.g., neurological, endocrine) so that both direct and indirect toxicologic effects can occur; and 4) the immune system is multifunctional, and significant alteration of any single function presents its own set of health risks. Immunotoxicology as the scientific basis for immune safety assessment has existed since as least the 1970s.1"1 During that interval, specific strategies have evolved for the identification and application of biomarkers to detect en307

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vironmental insult of the immune system. This chapter will consider both the background of existing biomarkers for immunotoxicity and more recent approaches to providing cost-effective safety assessment for the immune system.

HISTORY OF T H E USE OF BIOMARKERS I N I M M U N O T O X I C I T Y ASSESSMENT Establishing the Testing Paradigm Because of the complexity and multi-functional nature of the immune system, the challenge in immunotoxicity testing has been to determine: 1) what could be measured; 2) what should be measured and is sufficient for effective immune safety testing; and 3) what constitutes clinically- and/or biologically-significant alterations among the immune biomarkers measured? While not all of the questions have single definitive answers with universal agreement among scientists and regulators, there has been significant progress in defining the features of effective immunotoxicity testing strategies. Since the early 1980s, immune biomarkers have been used to replace what were then more specialized, cumbersome, and costly host resistance (HR) assays.5' 6 The HR assays (viral, bacterial, parasitic, and tumor challenges) were considered the gold standard. Sets of immune biomarkers were examined for their potential effectiveness in replacing HR assays. Additionally, a tieredapproach to testing was instituted whereby information on general immune status was obtained first. If these data collected caused concern or raised additional questions, more specific information using other immune parameters and assays could be collected in second or third tiers of assessment. In the early 1990s Luster and colleagues published a series of papers concerning the effectiveness of immune biomarkers for identifying immunotoxicants (also identified via HR evaluation).7'8 These studies utilized the National Toxicology Program database to enable a comparison of numerous xenobiotics as well as immune biomarkers. The fundamental concepts that developed from this analysis provided an important basis for immunotoxicity testing and continue to drive immunotoxicity testing strategies today. The concepts are: 1) no single immune parameter is sufficient as a screening tool to identify immunotoxicants and 2) functional tests (preferably multiple functional tests) are required to package biomarkers for achieving immunotoxic predictive success. For accurate identification of immunotoxicants, Luster, et al.7 found that eight different combinations, each employing three immune biomarkers, provided 100% predictive success (although the database was modest for some three-way combinations). Among those successful combinations, none were without immune functional measures. Five combinations included cell-mediated immune measures and three included humoral immune measures.

A "Challenging" Issue for Immune Biomarkers Remarkably, despite the observations of Luster, et al.7 that functionally-linked biomarkers were important for successful identification of immunotoxicants,

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regulatory agencies have not been uniform in requiring challenge, vaccination, or immunization of the immune system before initial immune safety screening data are collected. In part, the argument for not challenging the immune system has been that immune-relevant safety data should be collected using test animals where developmental, reproductive, and/or neurological data will also be collected. Because immunization, challenge, or vaccination might change the physiological status of the animals, there has been concern it might alter reproductive or neurological baselines. But not challenging the immune system presents severe limitation in the opportunity to detect toxicant-induced immune dysfunction. It is akin to assuming that you can evaluate how well a car drives without ever starting it.

TARGETS OF IMMUNOTOXICITY One of the reasons that immunotoxicity testing and the use of biomarkers can be a significant challenge is that virtually every immune cell population and every immune maturational event represents a potential toxicologic target. The routes to potential pathology and diseases are numerous. This has been discussed in several reviews for the developing immune system where toxicants are known to disrupt critical immune maturation events during specific windows of prenatal or neonatal development.9-" Additionally, the existence of numerous pathways of xenobiotically induced immune damage-alteration is also reflected in the mode of action of therapeutic agents designed to correct immune dysfunction. Numerous cell signaling and receptor mediated pathways have been utilized to treat allergic and autoimmune disease alone. Xenobioticmediated immune damage is as diverse as the therapeutic pathways pursued in drug discovery. Examples of toxicant-induced specific immune disruption are: 1) the capacity of alcohol to impair macrophage maturation and function by depleting glutathione;12-13 2) the ability of lead to skew dendritic cells for promotion of T helper (Th) 2-biased immune responses;14 3) the ability of 2,3,7,8-tetrachlorodibenzo-jo-dioxin (TCDD) to cause exaggerated inflammation;15-16 4) the action of cyclosporine A to suppress natural T regulatory (Treg) cell population expansion;17 and 5) the capacity of tributyltin, an organotin, to induce apoptosis in thymocytes producing thymus atrophy.18 Additionally, a single toxicant is capable of disrupting multiple immune targets depending upon the exposure concentration, the timing of exposure, and the route of exposure. For example, TCDD is a potent immunotoxicant. However, it cannot be characterized simply as an immunosuppressor. While TCDD and similar chemicals can impair acquired immune responses,19-20 it also causes improper inflammatory responses15-21 and an elevated the risk of later life autoimmune responses.22-23 For this reason, it is most useful to think of immunotoxicants not as simply immunosuppressive compounds or alternatively, as immune enhancers, but rather as inducers of dysfunction. In reality, it seems likely that a majority of immunotoxicants are capable of causing more than one adverse immune outcome.

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DISEASES OF PRIMARY

CONCERN

Increased Susceptibility t o Infections and Tumors Immunotoxicology emerged as an interdisciplinary area of research and testing during a time when safety testing concerns were focused on immunosuppression (HIV- and chemically-induced) and the identification of occupationally-related sensitizing chemicals and drugs. Not surprisingly, application of biomarkers to identify immunotoxicants focused on these health concerns, and these health concerns are still important concerns today. The driving force was protection against infectious diseases and tumors. The resulting biomarkers used in routine screening (e.g., primary IgM production against sheep erythrocytes and histopathology of an unchallenged immune system) have utility for the detection of overt immunosuppression. This is good news relative to those goals established in prior decades. But there is a potential downside to the continued reliance on the application of historic biomarkers of immunotoxicity for safety testing. For example, Luebke et al. 24 pointed out that the spectrum of diseases associated with mild to moderate immunosuppression are quite distinct from those associated with more pronounced immunosuppression. Additionally, the prevalence of several chronic diseases has increased since the 1980s, and these required additional attention in terms of use of biomarkers in immunotoxicity safety testing. The disconnection that has emerged between prior versus future biomarkers involves the realization that a much wider spectrum of health risks needs to be identified during front-line immunotoxicity testing. A comparison of the derivation and application of original HR-defined biomarkers vs. a more relevant range of disease-based-defined biomarkers is illustrated in Figure 12.1. The problem that has arisen is that application of the flow chart shown on the left in Figure 12.1 utilizes only HR-derived biomarkers (with a possible addition of chemical sensitizer tests). These do not account for protection against the full range of immune-dysfunction based diseases known to have environmental risk factors (shown included in the flow chart on the right of Figure 12.1). The flow strategy based only on HR is effective for the detection of toxicant-induced overt immunosuppression. But this is not the concern with the environmental risk of asthma and other allergic diseases, type 1 diabetes, rheumatoid arthritis, autoimmune thyroiditis, lupus, celiac disease, multiple sclerosis, inflammatory bowel disease, and atherosclerosis. As discussed in the following subsection, it is becoming imperative that we protect against this broader group of immune-dysfunction based diseases.

C h r o n i c Diseases and C o n d i t i o n s Based on Immune Dysfunction Table 12.1 lists several different categories of immune-dysfunction based diseases that could be reduced via use of disease-based biomarkers for immunotoxicity testing. Many of the chronic diseases have known environmental risk factors and have increased in prevalence in the past two to three decades.


311

FIGURE 12.1 The flow chart illustrates a comparison of traditional immune biomarkers designed to represent surrogates of host resistance and their use (shown on the left) versus biomarkers derived from the spectrum of diseases linked to immune dysfunction and their application (shown on the right).

This suggests that additional environmental risk factors exist and need to be identified.25'26 Dietert and Zelikoff26 recently surveyed the prevalence of pediatric-onset chronic diseases having an immune dysfunction basis and environmental risk factors. These impact at least a quarter of the pediatric populations in some developed countries. Among those of concern are allergic diseases including asthma.27-32 Table 12.1 illustrates the problem in early safety detection for a potential elevated risk of allergic disease. The biomarker IgM antibody in a primary rodent antibody response against xenogeneic cell-immunogens like sheep erythocytes was never intended as a biomarker to detect an elevated risk of allergic disease. Therefore, it is not surprising that it is unlikely to perform well in that specific role. Instead, evaluation using relevant biomarkers such as IgE and IgG subclass levels, IL-4 levels (following appropriate challenge), and eosinophil activation for detection is precisely what is needed in current safety testing based on prevalence of human disease. Autoimmune diseases represent a second category of immune-based diseases that have increased in prevalence in recent decades.33 These diseases have the potential for multifactorial mechanisms of pathogenesis.34 The need for better prevention of these diseases has led to a search for predictive biomarkers35 and has become a significant concern within immunotoxicity testing.36Table 12.1 illustrates biomarkers such as quantitation of Treg and Thl7 populations and autoantibody measures that would be useful as indicators of

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BIOMARKERS

TABLE 12.1

Predictive versus current biomakers o f immune-based health risks.

Predominant Health Concerns with Immunotoxicity

Potentially Predictive Biomarkers

Closest RoutinelyCollected Parameters*

Childhood or occupational asthma and other allergic diseases**

IgE responses, IgG subclasses, Eosinophil measures,Th2dependent function, inflammatory mediators (cytokines, metabolites)

IgM response

Autoimmune diseases (e.g., type 1 diabetes, inflammatory bowel disease, autoimmune thyroiditis, rheumatoid arthritis, lupus, multiple sclerosis, celiac disease)

IgG subclass and IgA responses, autoantibody screening.T regulatory (Treg) cell andThl7 analysis, B cell analysis.Tcell receptor usage

IgM response, CD4 and CD8T cell quantitation

Otitis media and other recurrent infections

IgG subclass and IgA responses, Inflammatory responses to host challenge.Thl vs.Th2 functional comparisons, marginal zone B cell function

IgM response, Natural Killer (NK) cell activity

Ineffective vaccine responses and unanticipated immune reactions after vaccination

Host responses to vaccine challenge and/or infectious agents including, antibody (multiple isotypes) and CMI/ CTL responses, and inflammatory profiles, autoantibody production

IgM response, NK activity, lymphocyte cell surface analysis; In humans: antibody titers to the vaccine agent are occasionally measured

Childhood and adult cancers

Th 1 ,Treg and Th 17-dependent NK cell activity, lymphocyte cell surface analysis functional profiles, CTL and NK activity in response to host challenge, regulation of inflammatory cell activity

Inflammatory-associated diseases (e.g., athlerosclerosis, schizophrenia, myalgic encephalomyelitis)

Inflammatory responses and proinflammatory cytokine profiles in resting and challenge states;Th and macrophage functional balance

NK cell activity

*8osed on 2008 USEPA and FDA routinely expected immunotoxicity data. **Note that the local lymph node assay (LIMA) is used to test the sensitizing potential of chemicals.

potential risk of autoimmunity. Examples of autoimmune diseases important as targets for improved safety evaluation include: type 1 diabetes,37,38 rheumatoid arthritis,39,40 autoimmune thyroiditis,41 celiac disease,42 multiple sclerosis,43 and lupus.44


313

Among other diseases and conditions associated with immunotoxicity are increased risk of infections,45 vaccine failures,46 unanticipated responses to vaccinations,47 and otitis media.45'48 Cancer is also a potential outcome of immunotoxicity and biomarkers should reflect this disease risk. Recently, it has been suggested that some forms of childhood leukemia involve immune dysfunctional responses to common pediatric infections.49 Using a rodent model, Ng, et al.50 showed that maternal smoking during pregnancy reduced the cellmediated immune cytotoxic lymphocyte (CTL) response and also produced increased tumor growth in the offspring. Several other diseases are linked with environmental factors, immune dysfunction and misregulated inflammation (Table 12.1). Calderon-Garciduenas, et al.51 recently found that children exposed to high levels of air pollutants exhibited misregulated inflammatory responses compared with urban children with lower air pollution exposures. Because misregulated inflammation can produce pathologies that can occur in virtually any tissue or organ, this category of immune-based diseases provides important biomarkers for use in safety testing. For this reason measurement of inflammatory cell function, including production of cytokines as well as oxygen radicals and nitric oxide, are useful for detecting potential inflammation-linked disease risk. Among the diseases of concern are atherosclerosis,52'53 schizophrenia,54-56 and myalgic encephalomyelitis.57-59 Other diseases/conditions such as autism and autism spectrum disorders are also candidates for possible environment-immune involvement."•60~62

DEVELOPMENTAL IMMUNOTOXICITY: I N C R E A S E D V U L N E R A B I L I T Y I N EARLY LIFE Developmental immunotoxicity (DIT) concerns exposure of the prenatal, neonatal, juvenile, and adolescent immune system to environmental factors that produce adverse health outcomes in later life. DIT warrants a special consideration for the selection and use of biomarkers in immunotoxicity evaluation. The developing immune system has been shown to be more sensitive to environmental insult than that of the adult.63-M Additionally, the early-life insult may take different forms and be more persistent compared with that seen in the adult.10 Because of the very nature of immune development and the occurrence of one-time maturational events,965 adult-derived safety data has limited relevance to the non-adult.1066 Prenatal-neonatal exposure to even low-levels of specific chemicals and drugs has been linked to an elevated risk of specific later-life diseases.25-26-28-67 Given the increased vulnerability of fetuses and neonates to environmentallyinflicted immune insult and the long-term implication of subsequent chronic disease, DIT testing may be more cost effective than adult-exposure immunotoxicity testing. Several different reviews have discussed possible strategies for collecting relevant DIT data in animal models68-73 and children.74 While suggested approaches and application of biomarkers may differ, there is widespread agreement on the utility of having DIT biomarker data for health protection.

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BIOMARKERS

DIFFERENTIAL EXPOSURE-OUTCOMES B E T W E E N GENDERS Among recent findings in immunotoxicology for specific disease risk is the observation that major differences can exist between genders in exposure outcome. The gender-based differences in immunotoxic outcomes can be qualitative. In this case, one gender may experience a health risk that is not observed in the other gender following exposure to the same toxicant at the same dose during the same time frame. Alternatively, the comparison between genders may be more quantitative in nature. In this case, the genders differ in the toxicant dose range that produces a given immunotoxic outcome. The end result is that specific exposures to drugs, chemicals, or other environmental factors can present different health risks and/or impact the likelihood of different diseases for women versus men. This has implications for immunotoxicity testing in the need to examine both genders for exposure-assessment and for disease risk management. For example, a majority of autoimmune diseases occur predominately in females.75-77 Surprisingly, gender differences in immunotoxicity are particularly evident following early-life exposure to xenobiotics.73 These are life stages when one might have expected gender effects to be less prominent than those seen in the adult. In some cases, these differences can be explained by the potential endocrine disrupting nature of toxicants.78-80 But not all xenobiotics showing sex-based differences in immunotoxic outcomes are known endocrine disrupters.81,82

A DISEASE-BASED A P P R O A C H T O I M M U N E BIOMARKER S E L E C T I O N One of the concerns is that several categories of immune-based diseases (e.g., asthma and allergy, autoimmunity, inflammation-driven conditions) have risen in prevalence in recent decades despite ongoing immunological safety testing of chemicals and drugs. For some select diseases, a portion of this increased prevalence could be related to improved disease diagnosis (e.g., myalgic encephalomyelitis and some autoimmune diseases). Another part of the increase may reflect a less than adequate protection of populations to previously-identified immunotoxicants (e.g., heavy metals). However, currently identified immunotoxicants cannot account for the full extent of disease increases that have been observed. For this reason, it has become obvious that there are additional immunotoxic risk factors remaining to be identified. Because these diseases have increased in the face of ongoing immunotoxicity testing, a reduction in immune-based chronic diseases is likely to require a new approach to the use of biomarkers in immunotoxicity testing. As previously discussed, immunotoxicity testing has been based on biomarkers intended for the detection of overt immunosuppression (Figure 12.1, left half). But as shown in Figure 12.1, this is only one of several health risks that need to be detected. For this reason, a reverse engineered approach to


315

the selection of biomarkers for use in developmental immunotoxicity testing has been proposed.72 However, a similar approach would be useful whether the testing is performed following adult-exposure or for DIT. Such an approach is shown on the right side of Figure 12.1. This approach begins with the diseases of greatest concern and would drive testing using the biomarkers that are altered in association with those diseases. By including diseases from many different categories (allergy, autoimmunity, inflammation, cancer, infectious diseases), the subsequent immunotoxicity testing would move beyond immunosuppression to include problematic immune enhancement and misregulation. But to move beyond detection of simple immunosuppression, it may be necessary to modify traditional testing approaches. Different challenge methodologies are needed that would facilitate evaluation of a broader spectrum of immune and inflammatory parameters than is usually found in traditional tier one testing. For example, given that risk of allergic disease is a major concern, then measurement of antibodies involved in mast cell degranulation (e.g., IgE) as well as their promoting cytokines (e.g., IL-4) would seem to be a minimum testing expectation. Additionally, appropriate immune homeostasis is a major issue for inflammatory-related diseases of concern. Hence, there needs to be a better evaluation strategy for detecting the risk of misregulated inflammation. Presumably this would include measurement of such biomarkers as production of proinflammatory cytokines, their receptors, and tissue-damaging inflammatory mediators such as reactive oxygen species (ROS) and nitric oxide production. These should be measured early in immunotoxicity testing priorities and not relegated to second or third tiers of evaluation. To do this requires appropriate challenge models (e.g., influenza virus infection and airway evaluation). Additionally, if risk of autoimmune disease is to be adequately considered, then it is useful to screen for autoantibodies and to determine the status of regulatory cell populations such as the Tregs and Thl7 cells. In summary, while there are several options for ways to optimize these disease-associated biomarkers into a unified immunotoxicity testing protocol, protocols that are effective in identifying the needed spectrum of immunotoxicity-associated health risks are likely to have certain features: 1) the spectrum of parameters measured will be broader than those used in traditional testing emphasizing immunosuppression; 2) the immune system must be challenged in such a way as to elicit a broad range of acquired, innate, and inflammatory responses for evaluation; and 3) immunotoxicity safety data need to be relevant and predictive for the age group and gender under consideration.

TOXICOGENOMIC AND IN VITRO APPROACHES Most immunotoxicity evaluations to date have employed in vivo and in vivo/ex vivo strategies using biomarkers. However, there have been efforts to examine the potential for alternatives to animal testing or direct human evaluation. Two general alternative strategies have been examined: gene expression/toxicog-

316

BIOMARKERS

enomic systems83-85 and in vitro immune assays.86,87 At present, these alternative methodologies are in their infancy as potential substitutes. However, the best opportunity for which they may gain wider use is for initial screening of direct-acting (those not requiring metabolism) immunotoxicants.

CONCLUSIONS Biomarkers have been a pivotal component of immunotoxicity assessment since the origins of the interdisciplinary area. Originally, they were established as surrogates of host resistance to infectious agents and tumors. Additionally, identification of biomarkers of chemical sensitization has been an early goal within immunotoxicology. These have proved useful for the detection of xenobiotics that produce overt immunosuppression or sensitization. However, it is now recognized that health risks involve a wide range of diseases with underlying immune dysfunction rather than merely profound immunosuppression. Among these are allergic and autoimmune disease as well as leukocytic cancers and inflammatory conditions. These diseases impact more than a quarter of the population in some developed countries and detection of environmental risk factors for these diseases requires use of a broader set of biomarkers than is needed to detect overt immunosuppression. As a result, new immunotoxicity testing protocols capable of detecting any form of immune dysfunction/misregulation are being examined. Most utilize a challenged immune system to be able to detect any significant deviation from the expected response (suppression, enhancement, or misdirected responses induced by exposure to an environmental factor). Additionally, recent research has investigated the potential to apply toxicogenomic biomarkers and/or in vitro measures of immunotoxicity as substitutes for more complex in vivo/ex vivo assays.

SUMMARY POINTS 1. 2. 3. 4. 5. 6.

Immunotoxicity assessment using biomarkers is a core component of drug and chemical safety testing. Immune biomarkers were initially developed as substitutes for more complex measures of host resistance. Traditional immunotoxicity testing had a primary goal of detecting immunosuppression and utilized biomarkers designed to meet this goal. Under recent safety testing regulations, prevalence of environmentallyinfluenced immune-associated diseases (e.g., asthma, allergy, and type 1 diabetes) has risen. To reduce the prevalence of these diseases, immune biomarkers used in safety testing need to be more directly connected with those diseases of concern. To better address the full spectrum of health concerns including allergic and autoimmune diseases, more effective host challenge strategies and application of immune biomarkers are needed.

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ACKNOWLEDGMENTS The author thanks Janice Dietert for her editorial assistance and Burleson Research Technologies, Inc. (Morrisville, NC) for their continued support of immunotoxicity research and testing.

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80. 81. 82. 83. 84.

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Selgrade, M. K. Immunotoxicity: The Risk Is Real. Toxicol. Sci. 2007;100: 328-332. Luster, M. I., Dean, J. H., and Germolec, D. R. Consensus Workshop on Methods to Evaluate Developmental Immunotoxicity. Environ. Health Perspect. 2003;111:579-583. Ladies, G. S., Chapin, R. E., and Hastings, K. L., et al. Developmental Toxicology Evaluations—Issues with Including Neurotoxicology and Immunotoxicology Assessments in Reproductive Toxicology Studies. Toxicol. Sci. 2005;88:24-29. Dietert, R. R. and Holsapple, M. P. Methodologies for Developmental Immunotoxicity (DIT) Testing. Methods. 2000;41:123-131. Burns-Naas, L. A., Hastings, K. L., Ladies, G. S., Makris, S. L., Parker, G. A., and Holsapple, M. P. What's So Special About the Developing Immune System? Int. J. Toxicol. 2008;27:223-254. Dietert, R. R. Developmental Immunotoxicity (DIT) in Drug Safety Testing: Matching DIT Testing to Adverse Outcomes and Childhood Disease Risk. Curr. Drug Saf. 2008;3:216-226. Dietert, R. R. Developmental Immunotoxicology (DIT): Windows of Vulnerability, Immune Dysfunction and Safety Assessment. J. Immunotoxicol. 2008; 5:401^112. Luster, M. I., Johnson, V. J., Yucesoy, B., and Simeonova, P. P. Biomarkers to Assess Potential Developmental Immunotoxicity in Children. Toxicol. Appl. Pharmacol. 2005;206:229-236. Zandman-Goddard, G., Peeva, E., and Shoenfeld, Y Gender and Autoimmunity. Autoimmun. Rev. 2007;6:366-372. Lleo, A., Battezzati, P. M., Selmi, C, Gershwin, M. E., and Podda, M. Is Autoimmunity a Matter of Sex? Autoimmun. Rev. 2008;7:626-630. Maul, R. W. and Gearhart, P. J. Women, Autoimmunity, and Cancer: A Dangerous Liaison Between Estrogen and Activation-Induced Deaminase? J. Exp. Med. 2009;206:11-13. Rooney, A. A., Matulka, R. A., and Luebke, R. W. Developmental Atrazine Exposure Suppresses Immune Function in Male, but Not Female Sprague-Dawley Rats. Toxicol. Sci. 2003;76:366-375. Karrow, N. A., Guo, T. L., and Delclos, K. B., et al. Nonylphenol Alters the Activity of Splenic NK Cells and the Numbers of Leukocyte Subpopulations in Sprague-Dawley Rats: A Two-Generation Feeding Study. Toxicology. 2004;196:237-245. Guo, T. L., Chi, R. P., Germolec, D. R., and White, K. L. Jr. Stimulation of the Immune Response in B6C3F1 Mice by Genistein Is Affected by Exposure Duration, Gender, and Litter Order. J. Nutr. 2005;135:2449-2456. Bunn, T. L., Parsons, P. J., Kao, E., and Dietert, R. R. Gender-Based Profiles of Developmental Immunotoxicity to Lead in the Rat: Assessment in Juveniles and Adults. J. Toxicol. Environ. Health A. 2001;64:223-240. Peden-Adams, M. M., Keller, J. M., Eudaly, J. G., Berger, J., Gilkeson, G. S., and Keil, D. E. Suppression of Humoral Immunity in Mice Following Exposure to Perfluorooctane Sulfonate. Toxicol. Sci. 2008;104:144-154. Luebke, R. W, Holsapple, M. P., and Ladies, G. S., et al. Immunotoxicogenomics: The Potential of Genomics Technology in the Immunotoxicity Risk Assessment Process. Toxicol. Sci. 2006;94:22-27. Baken, K. A., Vandebriel, R. J., Pennings, J. L., Kleinjans, J. C , and Van Loveren, H. Toxicogenomics in the Assessment of Immunotoxicity. Methods. 2007;41:132-141.

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Baken, K. A., Pennings, J. L., and Jonker, M. J., et al. Overlapping Gene Expression Profiles of Model Compounds Provide Opportunities for Immunotoxicity Screening. Toxicol. Appl. Pharmacol. 2008;226:46-59. Gennari, A., Ban, M., Braun, A., and Casati, S., et al. The Use of In Vitro Systems for Evaluating Immunotoxicity: The Report and Recommendations of an ECVAM Workshop. J. Immunotoxicol. 2005;2:61-83. Carfi', M., Gennari, A., and Malerba, I., et al. In Vitro Tests to Evaluate Immunotoxicity: A Preliminary Study. Toxicology. 2007;229:11-22.

CHAPTER

BIOMARKERS IN OBSTETRIC MEDICINE Manish Maski, Sarosh Rana, and S. Ananth Karumanchi

A N E U P L O I D I E S - T R I S O M I E S 2 1 , 18, A N D 13 Maternal age is an inadequate screening criterion alone for the detection of autosomal trisomies. The utility of measurement of maternal serum biomarkers for the prenatal detection of autosomal trisomies was initially suggested in 1984 by the observation that levels of alpha fetoprotein were lower in mothers who subsequently delivered trisomic babies.1 The characterization of several other serum biomarkers over the next several years has lead to the routine use of various screening protocols for the prenatal detection of trisomies 21 (i.e., Down syndrome), 18 (i.e., Edward syndrome), and 13 (i.e., Patau syndrome.) Five maternal biomarkers are currently in routine clinical use, usually in combination: alpha fetoprotein (AFP); human chorionic gonadotropin (hCG), either in its total form or as its beta subunit (total hCG or free beta-hCG, respectively); pregnancy-associated plasma protein-A (PAPP-A); unconjugated estriol (uE3); and inhibin A (inh A). Each of these biomarkers will be discussed in turn, followed by a discussion of their use in combination, as well as in combination with ultrasonography, for the detection of Trisomies 21, 18, and 13.

Alpha Fetoprotein Alpha fetoprotein (AFP) is a 69-kDa glycoprotein synthesized in the fetal liver and yolk sac. AFP is a member of the Albuminoid superfamily and is located on chromosome 4. AFP binds and transports many ligands, including bilirubin, fatty acids, retinoids, steroids (including estrogens), and various

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drugs, but it has also been proposed to serve as a potential circulating reservoir of biologically-active peptides that can be produced via cleavage of the native protein. Furthermore, AFP contains several amino acid sequences that are homologous to cellular adhesion sequences found in other proteins.2 Investigation into the biologic activities of AFP (and its subfragments) is ongoing, but it has been shown to promote growth in a variety of cell and animal models, and, in some instances, has also been shown to inhibit proliferation.3,4 In addition to its use as a biomarker in the second trimester for the prediction of Trisomies 18 and 21, as will be discussed below, AFP is the test of choice in screening for neural tube defects.

Human C h o r i o n i c G o n a d o t r o p i n Human chorionic gonadotropin (hCG) is a glycoprotein hormone consisting of two subunits, alpha (92 amino acids) and beta (145 amino acids), joined noncovalently. It exists in three active forms: regular hCG, hyperglycosylated hCG, and the free beta-subunit of hyperglycosylated hCG. Regular hCG is made by fused villous syncytiotrophoblast cells of the placenta. The classically-recognized function of regular hCG is the promotion of corpus luteal progesterone production from gestation weeks three to six. However, more recent research suggests that regular hCG maintains angiogenesis in the myometrial spiral arteries throughout the length of pregnancy, and it has also been shown to promote the fusion of villous cytotrophoblast cells to form syncytiotrophoblast. Both of these more recently described functions are critical to effective placentation and represent the more logical prime functions of regular hCG over the length of gestation.5 Hyperglycosylated hCG is made by extravillous invasive cytotrophoblast cells and serves as an autocrine factor on these cells to initiate and control invasion, as occurs at implantation of pregnancy, and the establishment of hemochorial placentation, as occurs in malignancy, such as invasive hydatiform mole and choriocarcinoma.6 Hyperglycosylated hCG has been shown to inhibit apoptosis in extravillous invasive cytotrophoblast cells,7 thereby promoting cell invasion and growth. Screening tests for Down syndrome measure hyperglycosylated hCG or its free beta-subunit.

Pregnancy-Associated Plasma P r o t e i n - A Pregnancy-associated plasma protein-A (PAPP-A) is an 820-kDa homotetrameric glycoprotein (each monomer composed of 1547 amino acids), produced mainly by placental syncytiotrophoblast. A remote member of the alpha-mac roglobulin plasma protein family, PAPP-A can consistently be detected in maternal circulation four to six weeks after conception. PAPP-A is an inhibitor of bovine trypsin and human plasmin, and has been shown to bind a variety of cytokines. It specifically cleaves insulin-like growth factor binding protein-4 (IGFBP-4), which can increase IGF bioavailability and lead to stimulatory effects in a variety of systems.8 For example, PAPP-A knock-out mice demonstrate skeletal insufficiency in density, ar-

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chitecture, and strength, likely via loss of cleavage of inhibitory insulin-like biding proteins that, in turn, decreases local IGF-1 bioavailability in bone.9 Moreover, PAPP-A is felt to be important in allowing maternal immunological tolerance of the fetus.10 The lymphocyte proliferation response to alloantigens and lectin, as well as expression of HLA-DR molecules on monocytes, is predominantly suppressed in vitro by PAPP-A.8

Unconjugated Estriol The biosynthesis of estrogens during pregnancy involves a coordinated effort between the fetal adrenal glands and the placenta. The fetal adrenal cortex provides the immediate androgen precursors to the placenta for conversion to estrogens. Estrogens have been postulated to have a number of important functions in maintaining pregnancy and preparing the reproductive tract for parturition, such as increasing blood flow in the vascular beds of the myometrium and endometrium, inducing neovascularization within the placenta, and inducing myometrial gap junction formation that coordinates contractions late in gestation. Because unconjugated estriol (uE3) is almost exclusively a fetal product that is secreted into the maternal circulation, maternal concentrations of uE3 may reflect abnormalities in fetal and placental development. For example, fetal death during the second or third trimester results in a significant drop in maternal uE3 concentrations within a few hours.11

Inhibin A Inhibin A (inh A) is a heterodimeric glycoprotein that consists of an alpha subunit (18 kDa) and a beta subunit (14 kDa) linked by disulfide bridges.12 It is a distant member of the transforming growth factor-beta (TGF-beta) superfamily. There also exists a molecule known as inhibin B (inh B) that consists of the identical alpha chain of inh A linked to a unique beta chain (termed inhibin beta B); inh B shares approximately 64% homology to inhibin A,13 but its regulation and temporal pattern in maternal serum is distinct from that of inh A.14 Inh A is produced by the granulosa cells of the developing follicle in response to FSH and LH, and its secretion tracks with gonadotropin-mediated dominant follicle growth and demise. Maternal serum levels of inh A begin to rise in the late follicular phase, reaching peaks in the midcycle and, subsequently, in the midluteal phase. The main function of inh A is in the negative regulation of FSH synthesis and secretion, and it plays a critical role in follicular development.14

D e t e c t i o n o f T r i s o m y 21 Down syndrome is the most common chromosomal abnormality among newborns, occurring in approximately one in 800 to 1000 live births.15'16 It is the result of three copies of chromosome 21, most often secondary to meiotic nondysjunction, which occurs at increasing frequency with advancing maternal age. Down syndrome results in a variety of dysmorphic features, con-

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genital malformations, and other health problems, such as brachycephaly with short stature, mental retardation, and congenital heart disease. Screening for trisomy 21, in one form or another, has been a well-established part of routine prenatal care in many countries for over 20 years. While previously performed most often in the early second trimester, since the early 1990s, screening for trisomy 21 has become routine in the late first trimester. The option for earlier screening is preferable to many women, as it allows more privacy in decision-making, more timely reassurance, or the option of earlier and safer termination of pregnancy. Several studies have evaluated the use of the aforementioned maternal serum analytes in the prenatal detection of trisomy 21 in singleton pregnancies, and the most important of them are summarized in Table 13.1. In the first trimester, two serum biomarkers stand out: free beta-hCG and PAPPA.17 These two biomarkers are usually combined with maternal age and fetal nuchal translucency to generate an overall risk of trisomy 21. Nuchal translucency is the lucent (i.e., hypoechoic) zone of fluid between the skin and soft tissues at the posterior fetal neck that is observed sonographically, and it is increased in fetuses affected by Down syndrome. Nuchal translucency is also often used in the determination of risk for trisomies 18 and 13,18 as well as other chromosomal abnormalities (e.g., Turner syndrome). It should be noted that in order to incorporate nuchal translucency into estimates of risk for aneuploidy, the sonographer must be stringently trained in the technique and gain sufficient experience, and external quality control is required.19 Furthermore, even despite adequate technical skill on the part of the sonographer, a small percentage of fetuses will be unable to be visualized secondary to fetal position or maternal body habitus. In the second trimester, inh A, AFP, uE3, and total hCG are commonly measured as the so-called "quadruple test." It is generally thought that free beta-hCG is the preferred form of hCG for screening in first trimester, while total hCG improves in performance in the second trimester. For example, Evans, et al.. performed a Monte Carlo simulation trial based on a literature review of the PUBMED database from 1966 to 2005.20 This group found that detection of Down syndrome increased by 4, 5, 6, and 7 percentage points when free beta-hCG was added to PAPP-A and nuchal translucency, as compared with 0, 0, 2, and 4 percentage points for total hCG, at 9-12 weeks gestation, respectively. Furthermore, these investigators found that free beta-hCG was associated with a greater reduction in false positive screening results at each week (9-12), as compared to total hCG. Each maternal biomarker is measured in units (usually ng/mL or IU/ mL) and then converted to a gestational age-specific multiple of the median value (MoM), based on a representative population of women with unaffected singleton pregnancies. Compared to unaffected pregnancies, levels of first trimester free beta-hCG and PAPP-A in Down syndrome are about 1.8 MoM and 0.4 MoM, respectively, and levels of second-trimester AFP and uE3 are, on average, 0.70-0.75 MoM. Second trimester levels of total beta-hCG and inh A are about 2.0 MoM in pregnancies affected by Down syndrome.

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Detection ofTrisomy 18 Trisomy 18 is the second most common autosomal trisomy in newborns, after Down syndrome, occurring in approximately 1 in 8000 live births. In the second trimester, the prevalence ofTrisomy 18 is approximately one in 2400, but there is a third trimester fetal loss rate of about 70% that results in the reduced prevalence of liveborn fetuses.21 As with Down syndrome, the majority of these cases are the result of meiotic nondysjunction. Trisomy 18 can produce abnormalities in any organ system, but congenital heart disease (most commonly ventricular septal defects and patent ductus arteriosus), gastrointestinal abnormalities (usually Meckel's diverticulum and malrotation), craniofacial abnormalities, and mental retardation occur in the majority of affected fetuses. Trisomy 18 is a largely lethal condition, with only 5 to 10 percent of newborns surviving past the first year. Several studies utilizing various combinations of the aforementioned biomarkers (minus inh A) have been performed to find an efficient screen for trisomy 18. The most important of these are summarized in Table 13.2. The most striking difference between a positive screen for Down syndrome and that for trisomy 18 lies in the levels of hCG and free beta-hCG, which are elevated in Down syndrome but reduced in trisomy 18. In affected pregnancies, the median free beta-hCG during the first trimester is 0.20 MoM, and the median total hCG during the second trimester is about 0.30 MoM.

Detection ofTrisomy 13 Trisomy 13 is a rare aneuploidy, with an observed incidence between one in 10,000 to 17,000 live births.2223 Trisomy 13 is almost uniformly lethal in the post-natal period, with median survival under three days and only 5% of affected infants surviving beyond six months. Again, meiotic nondysjunction related to increasing maternal age is the most common etiology, but Robertsonian translocation and mosaicism do occur. Trisomy 13 is usually phenotypically characterized by severe neurological defects, including holoprosencephaly, mental retardation, and, often, neural tube defects. Other common clinical features include severe eye defects, facial clefting, omphalocele, and congenital heart defects, such as patent ductus arteriosis, ventricular and atrial septal defects, and dextrocardia. Prenatal diagnosis of affected fetuses can often be suggested via ultrasonographic demonstration of the associated structural abnormalities. However, efforts have been made to determine a pattern of maternal serum biomarkers that reliably predicts trisomy 13. Sailer, et al. examined second-trimester maternal serum levels of AFP, uE3, and total hCG in 28 cases of fetal trisomy 13, each case matched with five unaffected pregnancies.24 They found that only uE3 levels (median MoM=0.71) were statistically different (p47,000 pregnancies; Measurements performed between 9 and 20 weeks gestation

Biomarkers Examined AFP, total hCG, free beta- hCG, uE3, PAPP-A, inhA, nuchal translucency

% Detection Rate (DR) for Trisomy 21 at % False Positives (FP) FP% for DR=85% (all combinations include maternal age):

Comb. Risk Cutoff for Positive Screen Not given

(1) free beta-hCG + PAPP-A at 10 weeks gestation + nuchal translucency=6.1 % (2) [AFP + uE3 + free beta-hCG + inh A] at 14-20 weeks gestation ("quadruple test")=6.2% (3) nuchal translucency + PAPPA at 10 weeks gestation + [AFP + uE3 + free beta-hCG + inh A] at 14-20 weeks gestation ("Integrated test")= 1.2% (4) "Serum integrated test" [same as (3) but without nuchal translucency]=2.7%

Malone, et al, 2005 (FASTER Research Consortium) (135)

Case-Control; Multinational; Over 38,000 women screened between gestation weeks 10 to 18; Fetuses found to have cystic hygroma were excluded

Free beta-hCG, 1:150 for (1) Maternal age-related risk PAPP-A, and + free beta-hCG + PAPP-A + 1st nuchal nuchal translucency ("combined trimester translucency, screening"): screening measured at 10 - D R at FP=5%: weeks + 3 days to 87% at 1 1 weeks gestation; 13 weeks+ 6 days 85% at 12 weeks; gestation 82% at 13 weeks. AND AFR total hCG, uE3, and inh A, at 15 through 18 weeks gestation (all calculations included maternal age-related risk)

(2) AFP+total hCG + uE3 + inh A at 15 to 18 weeks gestation ("second-trimester quadruple screen"): DR=8l%atFP=5%. (3) "Stepwise sequential screen" [risk results after each of (I) and (2) above]: DR=95% at FP=5%. (4) "Fully integrated screen" [single estimation of risk after (l)and(2)]: DR=96% at FP=5%, with first trimester measurements at 1 1 weeks gestation. (5)"Serum integrated screen" [same as (4) but without nuchal translucency]: DR=88% at FP=5%.

1:300 for 2nd trimester screening

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TABLE I 3.1

331

Studies evaluating the prenatal detection of Trisomy 21, (continued)

Investigators

Design

Biomarkers Examined

Canick, et al, 2006 (for the FASTER Research Consortium) (136)

Case-Control: 79 cases of Down syndrome each matched to five controls, from 1 1 through 13 weeks gestation from the FASTER specimen bank (see previous)

Free beta-hCG, total hCG, inhibin A, PAPP-A (measured previously), and nuchal translucency (measured previously)

% Detection Rate (DR) for Trisomy 21 at % False Positives (FP) Maternal age-related risk + nuchal translucency + PAPP-A at 12 weeks gestation:

Comb. Risk Cutoff for Positive Screen Not given

-plus free beta-hCG: DR=84% at FP=5%; -plus total hCG: DR=83% at FP=5%; -plus inh A: DR=85% at FP=5%. (no statistically significant difference in screening performance among the three biomarkers)

Kagan, et al, 2008 (27)

Case-Control: >56,000 controls and 395 cases; Measurements performed between gestation weeks 1 1 and 13 + 6 days

Free beta-hCG, PAPP-A, nuchal translucency, and fetal heart rate

Maternal age + combined seNot given rum biomarkers + nuchal translucency + fetal heart rate: DR=90% at FP=3%

et al.,26 among the 15 cases of trisomy 13 identified among the more than 36,000 pregnancies screened, the following was observed: six were identified with cystic hygroma on ultrasound, and of the remaining nine cases, only four were identified using first-trimester nuchal translucency, maternal age-related risk, free beta-hCG, and PAPP-A, and only three of the seven cases still viable in the second trimester were identified using AFP, uE3, total hCG, inh A, and maternal age-related risk. More recently, in a study by Kagan, et al.,27 61 cases of trisomy 13 were identified out of nearly 57,000 pregnancies screened in the first trimester and were analyzed via a trisomy 13-specific algorithm. This algorithm incorporated maternal age-related risk, fetal nuchal translucency, fetal heart rate, free betahCG, and PAPP-A, and it identified 87% of affected pregnancies with a false positive rate of 0.2%, at a combined risk cutoff according to the distribution of pregnancies in England and Wales from 2000 to 2002. There are a number of approaches available to patients to assess their risk of aneuploidy. The various approaches include the first trimester combined tests (serum biomarkers and ultrasound), as well as integrated testing, which includes full integrated, serum integrated, step-wise sequential screening, and contingent sequential screening. The management of individual patients to

332 TABLE 13.2

Investigators

BIOMARKERS Studies evaluating the prenatal detection of Trisomy 18.

Design

Biomarkers Examined

Palomaki, et al, Case Series of 89 AFR uE3, total pregnancies; Multi- hCG 1995(137) national; Measurements performed between 13 and 22 weeks gestation (89% between 15-20 weeks)

% Detection Rate (DR) for Trisomy 18 at % False Positives (FP) Combined biomarkers + maternal age-related risk DR=53%at5% and 10 years old, and the ranking varies from year to year, they highlight the fact that additional mechanistic insight into potential drug toxicities and DDIs is needed. One previously-overlooked potential source of drug toxicity may be offtarget mitochondrial impairment.^23 Mitochondrial toxicity is typically not assessed in drug-development programs, but even when it was evaluated, it was not detected because in most cases cell viability is not dependent on mitochondrial function (see below). Moreover, several new technologies and changes in cell culture protocols now reveal potential mitochondrial toxicity. Such toxicity may be due to inhibition of respiration (the electron transport system that generates mitochondrial membrane potential), and/or coupling to ATP production, (oxidative phosphorylation; OXPHOS). The detailed physiology of mitochondrial electron transport and coupling of membrane potential

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to ATP production exceed the scope of this review, and interested readers are directed to the comprehensive books by Nicholls and Ferguson24 and Scheffler.25 Nevertheless, the basic process warrants review, if only to underscore the physiological interdependence between the respiratory complexes and the integrity of the inner mitochondrial membrane.

MITOCHONDRIAL PHYSIOLOGY Biological energy is captured by removing electrons with high potential energy, and then allowing these electrons to cascade sequentially down a redox gradient via protein complexes in the inner mitochondrial membrane. Electrons enter the electron transport system (ETS) at complexes I and II, and are passed to the lipophilic carrier ubiquinone, which shuttles the electrons to complex III (Figure 16.1). From here, they pass to cytochrome c and thence to complex IV which accumulates four electrons to tetravalently reduce molecular oxygen to water. Together, ETS and oxygen consumption to yield water constitute respiration. At complexes I, III, and IV, the magnitude of the redox reaction is sufficient to translocate protons from the matrix across the inner membrane, which generates a membrane potential of approx. 220mV (inside negative). This potential energy is harnessed by complex V (aka, ATP synthase) where protons flow down their gradient coupled to the phosphorylation of ADP to ATP. Thus, OXPHOS depends not only on the integrity of coupled redox reaction centers of the ETS and function of complex V, but also on the impermeability of the inner membrane to protons. Should the inner membrane become porous to protons, the membrane potential dissipates, and ATP is not generated despite robust respiration; ETS is said to be "uncoupled" from phosphorylation.24'25 As an aside, generations of students learned this process as the electron transport chain, which connotes a one-to-one linkage between the various components. However, the molar ratios between the various components are not equal; for every mole of complex I, there are three moles of complex III, seven of complex IV, nine of cytochrome c, and 50 of ubiquinone.26 Only when the lateral diffusion coefficients are included in the analysis does the equation approach unity for rates of electron transport, which underscores the importance of integrity of the inner membrane for proper mitochondrial function. In this light, this process is more akin to an electron transport system (ETS) rather than a serial chain. Moreover, recent evidence indicates that the respiratory complexes aggregate into "super complexes" that facilitate the requisite redox reactions.27 Regardless of ETS architecture, mitochondria produce >90% of the energy required for viability of most aerobically poised cells. In so doing, they also produce the vast majority of the reactive oxygen- and nitrogen-centered free radicals that, because of high and indiscriminate reactivity, can damage and even kill cells. Moreover, mitochondria integrate a host of physiological pathways, so that when mitochondrial function declines, cell viability is imperilled. More catastrophically, when mitochondria die, the cell dies. When such failure

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is acute and profound, the cell dies via necrosis. When mitochondrial failure is less profound or less widespread, several pro-apoptotic proteins are released from the organelles, and the cell dies via apoptosis. These are the extremes of a continuum, but the key point is that mitochondrial viability is a proximate determinant of cell viability. Moreover, mitochondria are complex organelles, both anatomically and physiologically. The name comes from fusion of the Greek words mitos, "a thread," plus chondros, "a cereal grain," which accu-

FIGURE 16.1 Mitochondrial function can fail in a variety of ways. Many drugs directly inhibit one or more of the four respiratory complexes of the Electron Transport System, or complex V, a.k.a. ATP Synthase (upper left panel).6"23,30 Several sites are capable of univalently reducing molecular oxygen to superoxide, notably complex I, ubiquinone, and complex III. 2 " 7 Many antivirals and antibacterials also impede mtDNA synthesis or gene expression occurring in the matrix, resulting in erosion of mitochondrial capacity. Xenobiotics that undermine integrity of the inner membrane, or that serve as proton shuttles within it, uncouple the ETS from phosphorylation by ATP Synthase, and some inhibit mitochondrial pathways that fuel ETS, such as b-oxidation, Kreb's Cycle, or the transmembrane adenine nucleotide translocator (ANT).24-27 Most of the above deleterious effects precipitate the irreversible formation of the "permeability transition pore" (PT) that collapses membrane potential and permits release of cytochrome c and other pro-apoptotic factors into the cytosol.24'27 (See color insert for a full color version of this figure.)


405

rately describes the constant fission and fusion they undergo to form "bean" or circular shaped individual organelles, which join to form thread-like organelles. These individual organelles can fuse to form a reticulum, which is typically simultaneously budding off organelles (Figure 16.2). Mitochondria contain their own DNA (with a genetic code different from that in the nucleus), all the components required for DNA replication and protein expression, for organellar replication, for the ability to fuse and then undergo fission, plus the capacity for OXPHOS. Mitochondria can fail via a variety of mechanisms, including repression of DNA replication and expression, loss of inner membrane impermeability, and inhibition of ETS and supporting metabolism (Figure 16.1). Mitochondrial ultrastructural anatomy underscores this notion that failure can arise from anatomical perturbations, such as loss of inner membrane stability, especially given the invaginations of it into cristae that serve to increase surface area (Figure 16.3). From a drug-safety perspective, mitochondria are a "targetrich" environment, which is also reflected by the large number of known inhibitors of respiration and OXPHOS, including rotenone, antimycin, oligomycin, and cyanide. Indeed, there are 60 classes of compounds that inhibit complex I alone.28 In this context, it should not be surprising to learn that many ethical pharmaceuticals also may have, to varying degrees, important effects on mitochondrial function.6-23' 26_3°

FIGURE 16.2 Cos cell stained with a potentiometric dye that enters the mitochondria as a function of mitochondrial membrane potential (tetramethylrhodamine) and nuclear stain (Hoechst). Note the individual mitochondria shaped like beans and threads, and the fused reticulum.21"27 Image by Sandra Wiley. (See color insert for a full color version of this figure.)

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FIGURE 16.3 Electron tomogram of an individual mitochondrion.The extensive invagination of the inner membrane to form cristae is evident in the left panel. The irregularities on the surface are the respiratory complexes of the Electron Transport System and complex V. The right image highlights four cristae to indicate anatomical diversity and show how they form multiple junctions with the inner boundry membrane.80

DRUG-INDUCED MITOCHONDRIAL DYSFUNCTION (DIMD) HAS BEEN OVERLOOKED Potential mitochondrial impairment has not been recognized as an important source of drug toxicity, in large measure, as a consequence of circumstances in cell culture methods.30 Heretofore, to avoid having to change media daily, most cells have traditionally been cultured in high glucose media containing 25 mM glucose, which is five times physiological. Eighty years ago, two principles of metabolic physiology were reported independently by Crabtree and Warburg.31,32 The Crabtree Effect describes repression of respiration in the presence of elevated glucose, while the Warburg Effect notes that aerobic glycolysis yields lactate despite competent mitochondria. As a result of these two effects, transformed cells in contemporary culture generate almost all of their ATP from glycolysis, not from OXPHOS. Such cells typically have low rates of respiration, and are correspondingly resistant to mitochondrial toxins. For example, cells grown in high glucose are not killed by rotenone, antimycin, oligomycin, or cyanide.33 Potential in vivo toxicity of most drugs in devel-


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opment has typically been evaluated in cells under these culture conditions where mitochondrial liabilities are least likely to be detected, especially using viability assays; that the nascent drug is not toxic to these cells has often lead erroneously to the conclusion that it lacks mitochondrial toxicity. However, retrospective surveys of drugs having organ toxicities continue to indicate that many xenobiotics undermine function of isolated mitochondria, and under some conditions in intact cells as well.6"23,2930 To render cells susceptible to mitochondrial toxicity, glucose in the media can be replaced by galactose. The net ATP yield from glycolysis using glucose as substrate is 2 ATP, whereas it is closer to 0 when galactose is substrate (an investment of 2 ATP equivalents is required for galactose to enter glycolysis).33 Using galactose, cells must use OXPHOS to survive, and respiration accelerates. Now that the cells are dependent on OXPHOS, they become susceptible to mitochondrial impairment For example, cells grown in galactose are completely killed by a concentration of oligomycin at which more than 80% of cells grown in glucose remain viable.33 In our laboratories, potential drug toxicity is now routinely evaluated in cells grown in either glucose or galactose, and increased susceptibility in the latter is considered prima facia evidence of mitochondrial liabilities that can be further defined using additional assays described below.

NOVEL METHODS TO DETECT MITOCHONDRIAL DYSFUNCTION IN VITRO Another reason drug-induced mitochondrial dysfunction has been overlooked is because the polarographic Clark electrode experiments required to detect it have been the purview of specialists able to isolate functioning organelles. As noted above, in the absence of cell toxicity, there has been no motivation to examine potential effects on isolated mitochondria. Moreover, monitoring respiration by isolated mitochondria usually takes 15-30 min per sample, hardly conducive to high throughput assessments needed in the drug development arena (Figure 16.4). To circumvent this bottleneck, new assays of mitochondrial respiration in 96-well formats have been developed based on quenching of Pt-based fluorescent probes by molecular oxygen; as respiration depletes 0 2 in the well, the signal increases34,35 Well over 650 drugs, ranging from "nontoxic" to those with known organ toxicities, have been evaluated in this type of assay, and approximately 33% of them have demonstrated some level of direct and acute effects on mitochondria, either inhibiting respiration, or uncoupling it from phosphorylation, with many drugs affecting both.36'37 In several important drug classes, the potency of mitochondrial toxicity reflects clinical disposition. For example, of the thiazolidinediones, three (ciglitazone, troglitazone, dargitazone) most potently uncouple electron transport from phosphorylation, and all three, plus muraglitizar, most potently inhibit respiration.38 All four of these compounds have either been withdrawn from the market because of hepatotoxicity, or were dropped in development for organ toxicity.38 Rosiglitazone and pioglitazone also significantly, but less potently, uncouple and inhibit respiration;38 both have received Black Box

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FIGURE 16.4 Polarographic Clark electrode to monitor mitochondrial oxygen consumption. Respiration by mitochondria is slow in the absence of substrate, and when basal respiration is added (state 2) proceeds until ADP is added, at which point respiration accelerates to the maximum rate (state 3) until all ADP is phosphorylated and respiration returns to state 2. Drug induced inhibition is detected by repressed respiration in either state, and uncoupling by accelerated respiration, or failure to return to basal after ADP is phoshorylated. A typical assay takes between 16-30 minutes, which prompted development of a higher-throughput assay.3"8

Warnings for congestive heart failure, and both are associated with hepatotoxicity. Mitochondrial dysfunction is a proximate determinant of the latter pathology, and bioenergetic crisis and remodelling also figure prominently in congestive heart failure.39 Although a justified inference, it remains to be determined whether the severity of congestive heart failure correlates with the magnitude of mitochondrial impairment, or whether such impairment presages onset. Classical biochemical methods can demonstrate which of the electron transport complexes is being inhibited. With new technologies, the individual respiratory complexes can now be immunocaptured intact and their activity determined in 96-well plates.40 In this format, respiratory inhibition can be localized to an individual complex, and structure-activity studies can be conducted. For example, rosiglitazone inhibits complex I, while muraglitizar inhibits complex V.38 However, darglitazone inhibits complex IV profoundly, but also complexes II/III and V, while both troglitazone and ciglitazone inhibit all the complexes.38 Given the diversity of the respiratory complexes, it seems


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unlikely that these drugs are interacting with a motif shared by them all. It remains to be determined how these pleotrophic inhibitors are interacting with each of the individual complexes, but once elucidated, this could generate more specific assays to support SAR studies and drug discovery. As the mitochondrial capacity is diminished by drug-induced impairment, the cell compensates by accelerating glycolyticflux.41"*3Since oxidation of the resulting pyruvate is increasingly impeded, lactate effluxes into the blood. As a result, lactic acidosis is a classical, but not a pathognomonic, marker for mitochondrial impairment both in vivo and in vitro. For example, extracts of the plants in the lily family have long been used to treat diabetes, and they do lower blood glucose. These biguanides have been associated with lactic acidosis, and the first two to reach the market, phenformin and buformin, were later withdrawn because of fatal lactic acidosis, while metformin remains on the market despite rare instances of lactic acidosis, likely attributable to its lower potency. 4142 For example, Wang, et al.42 determined the EC50 for lactic acidosis in rat for phenformin, buformin, and metformin to be ~ 5, 120, and 735 uM, respectively. Traditionally, lactate efflux can be assayed in culture media via enzyme-linked assays, and oxygen consumption can be determined polarographically. With newer technologies, both indices can now be monitored simultaneously via fluorescent probes encapsulated at the end of a light pipe that is inserted into the sample,43 and with this technology, biguanide-induced media acidification can be shown to increase just as oxygen consumption declines with the same rank order of potencies reported by Wang, et al.42 However, although suggestive, elevated blood lactate cannot be pathognomonic for DIMD because it is also affected by exercise and other physiological variables. Nevertheless, lactate can be interrogated via noninvasive imaging techniques, so it may well become more useful as DIMD becomes more widely studied in the clinic. As an aside, the biguanide studies illuminate the role of bioaccumulation in drug toxicity, and reciprocally, efficacy. Greater than lOOuM concentrations of the drugs were needed to accelerate media acidification and repress 0 2 consumption in HepG2 cells.43 Similarly, inhibition of immunocaptured respiratory complexes by phenformin, buformin, and metformin required concentrations orders of magnitude greater than those that yielded lactic acidosis in vivo.43 Both observations suggest that bioaccumulation is required to detect toxicity, and indeed, detection of effects on isolated mitochondria required 40 min preincubation, where the same rank order of potency was found.4M5 Note also that the increased glycolytic flux to compensate for loss of OXPHOS yields the desired clinical outcome, viz. decreased plasma glucose, albeit by an unanticipated mechanism of mitochondrial impairment.

AN EMERGING MODEL OF IDIOSYNCRATIC DRUG TOXICITY The question remains, why, given such potentially potent and acute deleterious effects on mitochondrial function, certain drugs are not universally toxic,

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BIOMARKERS

i.e., why is frank organ toxicity idiosyncratic. Data like those discussed here support a novel model of idiosyncratic drug toxicity based on two concepts; threshold effects, plus heterogeneity of organ history and genetics. The threshold at which pathology emerges is relatively fixed and varies among different cell types. For example, an unstressed hepatocyte needs a set amount of ATP to conduct normal business such as albumin secretion, xenobiotic detoxification, glucose homeostasis, and a host of other basal processes. More aerobically demanding cells such as myocardiocytes and neurons have higher ATP turnover, but like hepatocytes and all other cells, they also require a minimum amount of energy for homeostasis. So although the basal energetic demand varies among different cell types, it is relatively constant across the population, and therefore is unlikely to underlie the idiosyncratic nature of many toxic drug responses. Rather, the bioenergetic reserve capacity above the threshold needed to maintain basal function varies more widely among individuals. For example, the mitochondrial capacity in a hepatocyte from an alcoholic is substantially less than that found in a drug-naive person.46 Cells with less reserve capacity (physiological scope) are closer to the bioenergetic threshold below which viability is compromised. As a result, such cells are more susceptible to drug-induced erosion of mitochondrial capacity, and hepatotoxicity will be apparent in some individuals under conditions that may be well tolerated by an individual with greater physiological scope. In this way, organ history, i.e., bioenergetic scope, plays a key role in the etiology of idiosyncratic drug toxicity. In an elegant review, Ulrich47 identifies a series of risk factors that converge in an individual to yield an idiosyncratic response. As several of these risk factors accumulate, the probability of drug-induced organ toxicity increases. The single factor that contributes the largest risk is increasing age,4748 although age in itself does not necessarily predict outcome. However, Ulrich also identified other risk factors including: inhibition of a key cellular function, extent of physical activity, genetics and inherited metabolic defects, plus concurrent drug exposures, heterogeneity in drug metabolism and bioactivation, presence of underlying disease, nutritional state, innate immune response, and gender (females more likely). Compellingly, there are parallel counterparts of all of these risk factors from the perspective of mitochondrial impairment. For example, mitochondrial capacity declines as we age, reducing the physiological scope and lowering the bioenergetic threshold for cell endangerment.48 As noted above, mitochondrial capacity is certainly a key cellular function, and its erosion compromises the cell's ability to respond to stressors or even to maintain basal function. Mitochondrial capacity increases with physical exercise, and conversely declines toward the minimal amount required for basal function with inactivity. Compared to a sedentary individual, an athlete has greater mitochondrial reserves, and hence can tolerate more drug-associated mitochondrial impairment before pathology emerges. This is particularly true for tissues where the bioenergetic capacity is subject to conditioning, such as skeletal muscle and heart, but less so for organs like liver where bioenergetic capacity reflects organ history more than conditioning.49 In terms of genetics and inherited metabolic defects, mu-


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tations or deletions in mitochondrial DNA cause a host of pathologies, and exposure to drugs with potential mitochondrial impairment can reveal previously silent mtDNA disorders,50 or to exacerbate already frank pathology.51 Concurrent exposures to several drugs with mitochondrial liabilities are at least additive,38 and surely, heterogeneity in drug metabolism will contribute to idiosyncratic responses, regardless of whether the toxicant is the parental or derivative molecule. Similarly, genetic variability in abundance and activity of plasma membrane transporters will also influence idiosyncratic responses to a given drug, with increased bioaccumulation predisposing toward toxicity. The risk factor of "underlying disease," in addition to mitochondrial disorders, clearly has bioenergetic sequelae, and "nutrition" also has direct influences on metabolic capacity and scope. Of the risk factors Ulrich identified, only "innate immune system" lacks a clear mitochondrial counterpart. But this risk factor is crucial to hapten-induced hypersensitivity reactions, not to cytotoxicity per se where the xenobiotic is a primary determinant of toxicity rather than the source of an inflammatory response. Given the relative high-throughput of the assays described here, especially in light of the pleotrophic effects of many drugs that are still poorly predicted by SAR, we propose that mitochondrial assessments be performed well before lead selection in the drug development process.36,37 At this stage, there is typically sufficient chemical diversity in the portfolio so that any observed mitochondrial impairment can be circumvented or minimized. It bears reiteration that assays with isolated organelles will likely yield more false positives than cell or intact animal models, and that the latter are required to characterize fully the nature of drug responses. However, in the latter models, the effects of bioaccumulation are in force, so that compounds with modest mitochondrial impairment that are substrates for transporters could be quite toxic at high localized concentrations, whereas in the absence of bioaccumulation, potent mitochondrial toxicants might never obtain sufficient concentrations in vivo to yield frank toxicity.

MITOCHONDRIAL DISEASES The fact that severe mitochondrial insufficiency translates into organ and systemic pathology is apparent on first principles by considering inherited mitochondrial syndromes and diseases. There are at least 75 such diseases, many of which are due to deletions and mutations in mtDNA, but also many are due to defects in nDNA encoded proteins destined for import in the mitochondria.50-52 An example of the latter is Freidreich's Ataxia (FA), where a triplet repeat in the gene for frataxin impairs mitochondrial iron homeostasis and hence Fe-S cluster assembly.53 FA typically has a pediatric onset, with progressive debilitating neuromuscular and CNS impairment, and is usually lethal via cardiomyopathy with median age of death at 35,52-53 Not all mitochondrial diseases are as relentless or severe, and such diseases range from symptomatically undetectable, to mild exercise intolerance, to sensory disturbances, to fatal ataxias. As might be expected, highly aerobi-

412

BIOMARKERS

cally poised and energy demanding tissues like CNS, sensory axis, and heart are frequently involved, but so also are metabolic pathologies like diabetes. For example, diabetes can be found in patients with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke), but typically not with MEERF (mycologist epilepsy and ragged-red fiber disease).52 Multi-organ system involvement is a hallmark of mitochondrial diseases.54 Using what we know about tissues affected by mitochondrial insufficiencies, we can extrapolate to DIMI and determine whether these are the same tissues first at risk, and in many instances they are. For example, hearing loss is associated with many mitochondrial insufficiencies and syndromes, and is among the first symptoms of aminoglycoside toxicity, plus mutations in mtDNA exacerbate the response.5155 Depending on which cells of the body are affected, symptoms might include: poor growth; loss of muscle coordination and muscle weakness; visual and/or hearing problems; developmental delays with learning disabilities or mental retardation; heart, liver, or kidney disease; gastrointestinal disorders such as severe constipation; respiratory disorders; diabetes; increased risk of infection; neurological problems including seizures; thyroid dysfunction; and dementia.52-56 However, in patients with mitochondriopafhies, distribution of defective copies of the causative genes is the result of several confounding processes, such as developmental segregation, and heteroplasmy, which is diversity of mtDNA within a cell and tissue. Plus, each mitochondrion contains multiple copies of mtDNA, which can also be heterogeneous. As a result, the same mutation can yield different symptoms in different patients, so called genocopies.50~54 Unfortunately, in the context of biomarkers there are no pathognomonic characteristics for mitochondrial diseases, and diagnosis is usually via family history, biopsy, histopathology, and detailed genetic analysis. However, this underscores the notion that bioenergetic capacity, fixed by genetics, mitochondrial disease, or organ history, is a prime determinate of idiosyncratic toxicity. For example, the mitochondrial capacity in a hepatocyte from an alcoholic is substantially less than that found in a drug-naive person.57 Therefore, the impaired hepatocyte will tolerate loss of mitochondrial capacity via drug exposure less than the cell with robust reserve capacity, and so will show organ toxicity sooner, and at a dose that does not yield pathology in a healthy person, i.e., idiosyncratically. We turn over our body weight in ATP everyday at rest.58 However, humans have aerobic scope and can increase metabolism between 10-20 fold over rest. This aerobic scope suggests that existing mitochondrial capacity would have to be eroded by any drug by more than 90% before resting metabolism in the cell would be imperilled. But note that the 20-fold range of aerobic scope largely reflects adaptations to training, where persistent increases in ATP turnover result in increased mitochondrial biomass. As such, the athlete has a higher bioenergetic threshold, and hence greater resistance to drug-induced mitochondrial impairment, but as noted above, only in those tissues subject to training, such as skeletal and cardiac muscle. However, it is in organs not capable of being trained, including liver (which is exposed to higher concentrations),


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kidney (which is enriched in transporters), peripheral and central nervous systems (assuming the drug can pass the blood-brain barrier), and sensory organs (e.g., aminoglycoside deafness), among others, where mitochondrial capacity is more static and the scope between the maximum ATP production and what is required to maintain viability is more constrained, that drug-induced toxicity is more typically observed. This is also influenced by the ability of mitochondria to replicate in response to reduced ATP availability so that drug-induced mitochondrial impairment would need to exceed the rate of replacement that is compensating for it. Interestingly, many drugs without organ toxicity often elicit transient increases in plasma liver enzymes ALT & AST, reflecting hepatocyte death. This "adaptation" seems a likely reflection of loss of cells with reduced aerobic capacity, and the lack of more severe organ toxicity a reflection of persistent viability of cells capable of replacing lost mitochondrial potential. In this way, if the ability to replace mitochondria exceeds the potency of the toxin, the cell will remain viable, although such replacement is expensive, i.e., requires energy and material that could have been used for other purposes, so that cell function and reserve capacity are correspondingly diminished.

POTENTIAL BIOMARKERS OF MITOCHONDRIAL DYSFUNCTION Chapters in this text provide information on biomarkers for various types of organ toxicity, such as muscle where myalgia, elevated serum muscle markers such as creatine kinase or myoglobin, and various troponins may signal rhabdomyolysis. However, any such biomarker reflects the final pathology, the organ toxicity, not mitochondrial dysfunction per se. Gradual mitochondrial erosion due to chronic repression of gene expression and/or mitochondrial replication can be monitored by assessing relative protein amounts. For example, monitoring the ratio of a nuclear-encoded protein and one encoded by mtDNA will reveal depletion of the mitochondrial capacity. This has been done using molecular biology techniques and peripheral blood samples.5960 A simple dipstick technology is available that can generate such information, and this technology works well for preclinical cell culture assessments. For example, repression of protein expression by several oxazolidinones and macrolide antibacterials, and nucleoside analogue antivirals can be detected after one cell population doubling.61 However, acute DIMD is difficult to detect in vivo. For example, uncouples dissipate the potential energy inherent in the mitochondrial membrane potential as heat, and can be detected as such in cells and under some circumstances in small animals.62 As noted above, this is the basis for many weight-loss dietary supplements. However, in intact animals, homeostatic mechanisms, such as increased sweating, compensate for extra heat production, so that core temperature is defended and is not a reliable biomarker of mitochondrial uncoupling. Moreover, depending on the extent of bioaccumulation and the tissue in question, mitochondrial uncoupling in one organ is unlikely to increase whole body temperature.

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Absent surrogate biomarkers, direct interrogation of mitochondrial function can be done by monitoring 0 2 consumption, C0 2 production, and heat production using direct and indirect technologies.63 Such metabolic assessments are well-established, and typically entail confining the patient in an airtight metabolic chamber where air input/output is finely controlled. Oxygen and C0 2 , and sometimes heat production, can be monitored in real time over extended periods. Portable devices, essentially a closed helmet, suffice for resting metabolism over shorter time periods, and the treadmill test where 0 2 consumption is monitored during exercise is well-known. Any of these techniques is likely capable of revealing DIMD, especially if a paired comparison of before-after drug exposure could be used, although to our knowledge none have been used in this capacity. However, without the benefit of paired-comparison experimental design that facilitates use of such technologies in the lab, intra-individual variability will substantially undermine utility for clinical evaluations of DIMD. Nevertheless, one could envision a pathological threshold for resting V0 2 , a surrogate below which the risk of organ toxicity from ETS inhibitors increases. Conversely, there could be a corresponding threshold of increased V0 2 for OXPHOS uncouplers above which the risk of frank lesion correspondingly increases. Similar indices could also be envisioned for C0 2 efflux and heat production. Another index of mitochondrial functional status is NADH/NAD reduction state, and for many years NADH fluorescence has served as a reliable indicator of cellular energetic and mitochondrial status. This technology is comprehensively reviewed by Mayevsky and Rogatsky,64 who discuss the historical development, and also state of the art in this area. For example, the advent of flexible light pipes and short-wavelength diodes has allowed development of compact fluorometers suitable for monitoring organ NADH fluorescence in situ.64 Although it is difficult to calibrate such systems in absolute terms, this issue can be circumvented by use of paired-comparison designs. The use of stable isotopes, i.e., non-radioactive atoms with the same number of protons, but different neutrons, for metabolic assessments has been the "gold standard" for years, being first described in 1949.65 The patient drinks water containing 2H2180, and using periodic blood tests over days to weeks, the clearance rates of the two isotopes is quantified via mass spec.66 The 0 2 equilibrates with C0 2 via carbonic anhydrase, and with water, which is lost as urine, sweat, etc. However, 2H2 depletion accounts for water loss, so that C0 2 production serves as a surrogate index of 0 2 consumption. Note, however, that it really is a surrogate for mitochondrial Krebs Cycle function, not OXPHOS, which under normal circumstances is a valid assumption.66 But during oxidative stress, C0 2 is also produced by the hexosemonophosphate shunt (HMS), branching off from glycolysis, that generates NADPH required as a cofactor by many enzymes, such as glutathione reductase. As such, HMS flux can serve as a surrogate index of glutathione turnover, which accelerates during oxidative stress, and correspondingly confounds C0 2 as a surrogate for OXPHOS. This confounding effect would be exacerbated by inflammation, where transmembrane NADPH oxidases generate free radicals necessary for


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antibacterial activity. However, the long duration of the test and the repeated examinations serve to moderate this variable. Currently, elegant, noninvasive exhalation breath techniques for monitoring mitochondrial function based on stable isotope analysis are under clinical evaluation.6768 Substrate selection and pattern of 13C labelling allow this technology to selectively interrogate several cellular processes in different tissues. For example, 13C-methionionine is preferentially metabolized via a transmethylation pathway in the liver that is not found, or has very low activity, in other tissues. When 1-carbon labelled methionine is used, the activity of a-ketobutyrate decarboxylase is interrogated, since this liver mitochondrial enzyme is a limiting step of substrate oxidation to CO r 68 Using 3- or 4-carbon labelled methionine it is possible to assess the trans-sulphuration pathway via release of the carbon as a-ketobutyrate, further metabolized into C0 2 via the tricarboxylic acid cycle. In addition, methyl-13C-labelled methionine can also be used to interrogate hepatic mitochondrial status. The latter is metabolized to sarcosine, which is oxidized by sarcosine-dehydrogenase to produce a one carbon fragment that can be converted into CO r 6 9 In rat liver, the sarcosine oxidase system is present exclusively in the mitochondria.69 Stable isotope C0 2 exhalation for mitochondrial assessment is also done by monitoring decarboxylation of ct-ketoisocaproate, and although in patients with primary biliary cirrhosis the signal is the same as that in normal patients, it is significantly lower in patients with alcoholic liver disease.70 Another stable isotope 31P can also be used to assess mitochondrial function using NMR. For example, using a surface coil to monitor NMR signals from the three phosphates of ATP, phosphocreatine and inorganic phosphate, one can follow bioenergetic status of a muscle noninvasively. These signals decline when the muscle is paced or rendered hypoxic, and the rate of adenylate recovery after exercise ceases, or the muscle is reperfused, reflects mitochondrial capacity (OXPHOS remains at maximum rates until all available ADP is phosphorylated).71,72 Recent advances in visible-wavelength spectroscopy permit noninvasive determination of haemoglobin and myoglobin oxygenation states, and when combined with 31P monitoring, provide noninvasive 0 2 consumption and hence determination of coupling efficiencies.73 Such a protocol should be able to detect DIMD, but to our knowledge has not been applied in this manner. However, as noted above, not having the advantage of a paired-comparison design, determining the adenylate recovery rate of a patient with suspected mitochondrial impairment forces reliance on population thresholds. Another potential biomarker of DIMD could be an index of free radical production. For example, deoxyguanosine in DNA is preferentially oxidized by hydroxyl radicals to 8-OH-deoxyguanosine (8-OH-dGUA), which is excised and excreted intact in the urine.74 As such, 8-OH-dGUA can serve as a noninvasive biomarker for endogenous radical production in patients undergoing radiation therapies, and also for radicals generated during an inflammatory response.74 Whether it, or some other marker of oxidative stress, can be developed as a biomarker for DIMD will depend on its variability and poten-

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tial thresholds, but also on whether the drug in question increases radical production, a result of how the drug is undermining mitochondrial function. For example, inhibitors of complexes III and IV, which serve to more fully reduce the "up stream" components of ETS and correspondingly increase the probability of their autoxidation to yield superoxide, will yield more robust signals from an oxidative biomarker than inhibitors of complex II, or some other component of mitochondrial metabolism, such as a Kreb's Cycle enzyme.24-27

A N I M A L MODELS As noted above, all the technologies discussed have been used in animal models, but such preclinical models offer additional benefits, including the possibilities of genetic manipulation. For example, Ong, et al.75 have described use of a heterozygous mouse where the manganese form of superoxide dismutase found in mitochondria (SOD2) is knocked down by 50%. In these Sod2(+'") animals, the previously silent hepatotoxicity of troglitazone is detected as increased serum alanine aminotransferase activity and co-occurrence of midzonal areas of hepatic necrosis.75 In hepatocytes isolated from Sod2(+/>, but not wild-type mice, troglitazone caused a concentration-dependent increase in superoxide production detected using a mitochondrial-targeting fluorescent probe. It is instructive in this context to reconsider the convergence of risk factors discussed previously; potential DIMI needs to be evaluated in animal models most likely to reveal it. Typically, potential in vivo toxicity of nascent drugs is evaluated in young, drug-naive animals with full mitochondrial capacity, precisely the circumstances under which it is least likely to be detected. Rather, old animals, preferably with prior chronic alcohol or hepatotoxic drug exposure to decrease hepatic mitochondrial capacity, would be a more physiologically—and pathologically—realistic model. Finally, genetic variability can be manipulated in preclinical animal models to increase, or decrease, susceptibility to various agents. Although the initial assumption is that all in-bred strains of rats or mice should be comparable physiologically or behaviourally responsive to xenobiotic exposure, this is not the case.7677 For example, mice from 14 standard inbred strains were evaluated for sensitivity to pentobarbital (PB) by monitoring low-dose stimulation and highdose depression of locomotor activity, reduced rearing, hypothermia, and ataxia assessed via rotarod. The strains significantly differed in all responses, with a > 4-fold range in the amount of PB present in the brain when failing the rotarod test, and a > 5-fold range in latency of response.77 Such rodent strains, thought to derive from a mixture of four subspecies, have been inbred for over 100 years, which has also fostered the tacit assumption that variations between mtDNA should be inconsequential. But this is also not the case. Recently, the complete sequence of mtDNA from 16 strains indicates that they all descend from the same wild type Mus musculus domesticus female ancestor.78 Moreover, the rate of accumulation of replacement substitutions in mtDNA is faster in the inbred strains of both mice and rats than in the wild types.78-79 As a result, there is more diversity in mtDNA from inbred strains, and hence larger range of possible phenotype in the in-

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bred animals compared to their outbred counterparts. More recently, these data have been extended and corroborated; 50 of the 52 inbred mice strains directly descended from the same initial female ancestor, with mtDNA mutations in 26 strains.79 These researchers then generated conplastic strains on the C57BL/6J background for 12 mtDNA variants with one to three functional mtDNA mutations, plus comparable strains with the four M. musculus subspecies, to yield a panel of 16 mtDNA variants. Phenotypic analysis of these conplastic strains revealed that mtDNA variations alter susceptibility to experimental autoimmune encephalomyelitis and anxious behavior. In this case, contrary to expectations, mtDNA apparently affected complex traits. In this light, it might be informative to evaluate these conplastic strains for differential susceptibility to known hepatotoxicants and to drugs associated with idiosyncratic hepatoxicity, or other organ toxicity.

SUMMARY POINTS 1.

2. 3. 4.

5.

The evidence is rapidly accumulating that many marketed drugs have direct "off target" mitochondrial liabilities, either inhibiting ETS and/or uncoupling OXPHOS, which are increasingly implicated in the development of idiosyncratic drug toxicities. Organ toxicity is a function of potency, but also of bioaccumulation, with aerobically poised organs typically at highest risk, as is the case of inherited mitochondrial diseases. The availability of animal strains with diversity in mtDNA provides potentially useful tools to help illuminate how phenotypic diversity might contribute to idiosyncratic responses. The current absence of biomarkers for drug-associated mitochondrial dysfunction can be circumvented by determining mitochondrial capacity directly. Several techniques are available, although they have not yet been used in this capacity. This situation will undoubtedly change as the importance of xenobiotic mitochondrial impairment gains wider appreciation, and such data along with thorough preclinical mitochondrial assessments early in the drug discovery and development process will help improve the safety profile for future drugs.

REFERENCES 1. 2. 3. 4.

http://www.fda.gov/cder/drug/drugreactions/default.htm#adrs: %20prevalence%20and%20incidence. http://www.fda.gov/cder/livertox/default.htm. Committee on Quality of Health Care in America: Institute of Medicine. To EnIs Human: Building a Safer Health System. Washington, D.C. National Academy Press, 2000. Lazarou, J., Pomeranz, B., and Corey, P. N. Incidence of Adverse Drug Reactions in Hospitalized Patients: A Meta-Analysis of Prospective Studies. JAMA. 1998;279:1200-1205.

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20. 21. 22.

Gurwitz, J. H., Field, T. S., Avorn, J., Mccormick, D., Jain, S., and Eckler, M., et al. Incidence and Preventability of Adverse Drug Events in Nursing Homes. Am. J. Med. 2000;109:87-94. Boelsterli, U. A. and Lim, P. L. Mitochondrial Abnormalities—A Link to Idiosyncratic Drug Hepatotoxicity? Toxicol. Appl. Pharmacol. 2007;220:92-107. Brunmair, B., Staniek, K., and Gras, F, et al., Thiazolidinediones, Like Metformin, Inhibit Respiratory Complex I: A Common Mechanism Contributing to Their Antidiabetic Actions? Diabetes. 2004;53:1052-1059. Cardoso, C. M., Custodio, J. B., Almeida, L. M., and Moreno, A. J. Mechanisms of the Deleterious Effects of Tamoxifen on Mitochondrial Respiration Rate and Phosphorylation Efficiency. Toxicol. Appl. Pharmacol. 2001;176:145-152. Dykens, J. A., Jamieson, J. D., and Marroquin, L. D., et al. In Vitro Assessment of Mitochondrial Dysfunction and Cytotoxicity of Nefazodone, Trazodone, and Buspirone. Toxicol. Sci. 2008;103:335-345. Fau, D., Eugene, D., and Berson, A., et al. Toxicity of the Antiandrogen Flutamide in Isolated Rat Hepatocytes. J. Pharmacol. Exp. Ther. 1994;269:954-962. Haasio, K., Koponen, A., Penttila, K. E., and Nissinen, E. Effects of Entacapone and Tolcapone on Mitochondrial Membrane Potential. Eur. J. Pharmacol. 2002; 453:21-6. Keller, B. J., Yamanaka, H., and Thurman, R. G. Inhibition of Mitochondrial Respiration and Oxygen-Dependent Hepatotoxicity by Six Structurally Dissimilar Peroxisomal Proliferating Agents. Toxicology. 1992;71:49-61. Krause, M. M., Brand, M. D., and Krauss, S., et al. Nonsteroidal Anti-Inflammatory Drugs and a Selective Cyclooxygenase 2 Inhibitor Uncouple Mitochondria in Intact Cells. Arthritis Rheum. 2003;48:1438-1444. Masubuchi, Y., Yamada, S., and Horie, T. Diphenylamine as an Important Structure of Nonsteroidal Anti-Inflammatory Drugs to Uncouple Mitochondrial Oxidative Phosphorylation. Biochem. Pharmacol. 1999;58:861-865. Maurer, I. and Moller, H. J. Inhibition of Complex I by Neuroleptics in Normal Human Brain Cortex Parallels the Extrapyramidal Toxicity of Neuroleptics. Mol. Cell. Biochem. 1997;174:255-259. Nulton-Persson, A. C , Szweda, L. I., and Sadek, H. A. Inhibition of Cardiac Mitochondrial Respiration by Salicylic Acid and Acetylsalicylate. J. Cardiovasc. Pharmacol. 2004;44:591^t95. Pessayre, D., Mansouri, A., Haouzi, D., and Fromenty, B. Hepatotoxicity Due to Mitochondrial Dysfunction. Cell. Biol. Toxicol. 1999;15:367-373. Souid, A. K., Tacka, K. A., Galvan, K. A., and Penefsky, H. S. Immediate Effects of Anticancer Drugs on Mitochondrial Oxygen Consumption. Biochem. Pharmacol. 2003;66:977-987. Tay, V. K., Wang, A. S., Leow, K. Y, Ong, M. M., Wong, K. P., and Boelsterli, U. A. Mitochondrial Permeability Transition as a Source of Superoxide Anion Induced by the Nitroaromatic Drug Nimesulide In Vitro. Free Radic. Biol. Med. 2005;39:949-959. Wallace, K. B. and Starkov, A. Mitochondrial Targets of Drug Toxicity. Ann. Rev. Pharmacol. Toxicol. 2000;40:353-388. Zhou, S. and Wallace, K. B. The Effect of Peroxisome Proliferators on Mitochondrial Bioenergetics. Toxicol. Sci. 1999;48:82-89. Benbrik, E., Chariot, P., and Bonavaud, S., et al. Cellular and Mitochondrial Toxicity of Zidovudine (AZT), Didanosine (Ddi) and Zalcitabine (Ddc) on Cultured Human Muscle Cells. /. Neurol. Sci. 1997;149:19-25.

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34. 35. 36. 37. 38.

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Riesbeck, K., Bredberg, A., and Forsgren, A. Ciprofloxacin Does Not Inhibit Mitochondrial Functions but Other Antibiotics Do. Antimicrob. Agents Chemother. 1990;34:167-169. Nicholls, D. G. and Ferguson, S. J. Bioenergetics 3. p. 317. 2002, Academic Press; London. Scheffler, I. Mitochondria, 1st Ed., p. 367. 1999;John Wiley, New York. Chazotte, B. and Hackenbrock, C. R. The Multicollisional, Obstructed, LongRange Diffusional Nature of Mitochondrial Electron Transport. J. Biol. Chem. 1988;263:14359-14367. Dykens, J. A. Redox Targets: Enzyme Systems and Drug Development Strategies for Mitochondrial Dysfunction, p. 1053-1087 In Comprehensive Medicinal Chemistry II (Triggle, D. J., Taylor, J. B., Eds.), 2007, Elsevier, Oxford. Chan, K., Truong, D., Shangari, N., and O'Brien, R J. Drug-Induced Mitochondrial Toxicity. Expert Opin. Drug Metab. Toxicol. 2005;1:655-669. Mokhova, E. N. and Khailova, L. S. Involvement of Mitochondrial Inner Membrane Anion Carriers in the Uncoupling Effect of Fatty Acids. Biochemistry Mosc. 2005;70:159-163. Drug-Induced Mitochondrial Dysfunction. (Dykens, J. A., Will, Y, Eds.) 2008; p. 616:Wiley, New York, NY. Rodriguez-Enriquez, S., Juarez, O., Rodriguez-Zavala, J. S., and Moreno-Sanchez, R. Multisite Control of the Crabtree Effect in Ascites Hepatoma Cells. Eur. J. Biochem. 2001;268:2512-2519. Warburg, O., Geissler, A. W., and Lorenz, S. On Growth of Cancer Cells in Media in Which Glucose Is Replaced by Galactose. Hoppe Seylers Z. Physiol. Chem. 1967;348:1686-1687. Marroquin, L. D., Hynes, A., Dykens, J. A., Jamieson, J. D., and Will, Y Circumventing the Crabtree Effect: Replacing Media Glucose with Galactose Increases Susceptibility of Hepg2 Cells to Mitochondrial Toxins. Toxicol. Sci. 2007;97:539-547. Hynes, J., Marroquin, L. D., and Ogurtsov, V. I., et al. Investigation of DrugInduced Mitochondrial Toxicity Using Fluorescence-Based Oxygen-Sensitive Probes. Toxicol. Sci. 2006;92:186-200. Will, Y, Hynes, J., Ogurtsov, V. I., and Papkovsky, D. B. Analysis of Mitochondrial Function Using Phosphorescent Oxygen-Sensitive Probes. Nat. Protoc. 2006;1:2563-2572. Dykens, J. A. and Will, Y. The Significance of Mitochondrial Toxicity Testing in Drug Development. Drug Dis. Today. 2007;12:777-785. Dykens, J. A., Marroquin, L. D., and Will, Y Strategies to Reduce Late Stage NCE Attrition Due to Mitochondrial Toxicity: Development of a High Throughput Respiration Screen. Exp. Opin. Mol. Diag. 2007;7:161-175. Nadanaciva, S., Dykens, J. A., Bernal, A., Capaldi, R. A., and Will, Y Mitochondrial Impairment by PPAR Agonists and Statins Identified via Immunocaptured OXPHOS Complex Activities and Respiration. Toxicol. Appl. Pharmacol. 2007;223:277-287. Ingwall, J. S. Energy Metabolism in Heart Failure and Remodelling. Cardiovasc. Res. 2009;81:412^tl9. Nadanaciva, S., Bernal, A., Aggeler, R., Capaldi, R., and Will, Y Target Identification of Drug Induced Mitochondrial Toxicity Using Immunocapture Based OXPHOS Activity Assays. Toxicol. In Vitro. 2007;21:902-911.

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Chan, N. N., Brain, H. P., and Feher, M. D. Metformin-Associated Lactic Acidosis: A Rare or Very Rare Clinical Entity? Diabet. Med. 1999;16:273-281. Wang, D. S., Kusuhara, H., Kato, Y, Jonker, J. W., Schinkel, A. H., and Sugiyama Y. Involvement of Organic Cation Transporter 1 in the Lactic Acidosis Caused by Metformin. Mol. Pharmacol. 2003;63:844-848. Dykens, J. A., Jamieson, J., Marroquin, L., Nadanaciva, S., Billis, P. A., and Will, Y. Biguanide-Induced Mitochondrial Dysfunction Yields Increased Lactate Production and Cytotoxicity of Aerobically-Poised Hepg2 Cells snd Human Hepatocytes In Vitro. Toxicol. Appl. Pharmacol. 2008;233:203-210. El Mir, M. Y, Nogueira, V, Fontaine, E., Averet, N., Rigoulet, M., and Leverve, X. Dimethylbiguanide Inhibits Cell Respiration via an Indirect Effect Targeted on the Respiratory Chain Complex I. J. Biol. Chem. 2000;275:223-228. Owen, M. R., Doran, E., and Halestrap, A. P. Evidence That Metformin Exerts Its Anti-Diabetic Effects Through Inhibition of Complex 1 of the Mitochondrial Respiratory Chain. Biochem. J. 2000;348:607-614. Sastre, J., Serviddio, G., and Pereda, J., et al. Mitochondrial Function in Liver Disease. Front Biosci. 2007;12:1200-1209. Ulrich, R. G. Idiosyncratic Toxicity: A Convergence of Risk Factors. Annu. Rev. Med. 2007;58:17-34. Cortopassi, G. A. and Wong, A. Mitochondria in Organismal Aging and Degeneration. Biochim. Biophys.Acta., 1999;1410:183-93. Fromenty, B. and Pessayre, D. Impaired Mitochondrial Function in Microvesicular Steatosis. Effects of Drugs, Ethanol, Hormones and Cytokines. J. Hepatol. 1997;26:43-53. Dimauro, S. Mitochondrial Diseases. Biochim. Biophys. Ada. 2004;1658:80-88. Schon, E. A., Hirano, M., and Dimauro, S. Drug Effects in Patients with Mitochondrial Diseases, p. 311-324. In Drug-Induced Mitochondrial Dysfunction (Dykens, J. A. and Will, Y, Eds.), 2008;Wiley, New York, NY. Finsterer, J. Mitochondriopathies. Eur. J. Neurol. 2004;11:163-186. Delatycki, M. B. Evaluating the Progression of Friedreich Ataxia and Its Treatment. J. Neurol. 2009;256 Suppl 1:36-41. Naviaux, R. K. Mitochondrial DNA Disorders. Eur. J. Pediatr. 2000;3: S219-226. Bindu, L. H. and Reddy, P. P. Genetics of Aminoglycoside-Induced and Prelingual Non-Syndromic Mitochondrial Hearing Impairment: A Review. Int. J. Audiol. Nov 2008;47(11):702-707. Schapira, A. H. V. Mitochondrial Disease. The Lancet. 2006;368:70-82. Sastre, J., Serviddio, G., Pereda, J., Minana, J. B., Arduini, A., Vendemiale, G., Poli, G., Pallardo, F. V, and Vina, J. Mitochondrial Function in Liver Disease. Front. Biosci. 2007;12:1200-1209. Dykens, J. A. and Will, Y Preface, pp. xiii-xvii. In Drug-Induced Mitochondrial Dysfunction (Dykens, J. A. and Will, Y, Eds), 2008; Wiley, New York, NY, 616. Saitoh, A., Fenton, T., Alvero, C, Fletcher, C. V, and Spector, S. A. Impact of Nucleoside Reverse Transcriptase Inhibitors on Mitochondria in Human Immunodeficiency Virus Type 1 -Infected Children Receiving Highly Active Antiretroviral Therapy. Antimicrob. Agents Chemother. 2007;51:4236-4242. Shikuma, C. M., Gerschenson, M., and Chow, D., et al. Mitochondrial Oxidative Phosphorylation Protein Levels in Peripheral Blood Mononuclear Cells Correlate with Levels in Subcutaneous Adipose Tissue Within Samples Differ-


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Goios, A., Pereira, L., Bogue, M., Macaulay, V., and Amorim, A. Mtdna Phytogeny and Evolution of Laboratory Mouse Strains. Genome Res. 2007;17:293-298. Yu, X., Gimsa, U. and Wester-Rosenlof, L., et al. Dissecting the Effects of mtDNA Variations on Complex Traits Using Mouse Conplastic Strains. Genome Res. 2009;9:159-165. Frey, T. G., Renken, C. W., and Perkins, G. A. Insight Into Mitochondrial Structure and Function From Electron Tomography. Biochim. Biophys. Ada. 2002;1555:196-203.

SECTION III TECHNOLOGY FOR BIOMARKER DETECTION


CHAPTER

IMMUNOASSAY-BASED TECHNOLOGIES FOR THE MEASUREMENT OF BIOLOGICAL MATERIALS USED FOR BIOMARKERS DISCOVERY AND TRANSLATIONAL RESEARCH Vincent Ricchiuti

INTRODUCTION This chapter summarizes various methods employed to characterize and quantify biological materials from human and animal sources. The measurement of biological compounds in body fluids and tissues is a critical component of clinical diagnostics, clinical research and translational, and represents an objective endpoint for many clinical trials, especially those involving therapeutic interventions and biomarkers of toxicity. Over the past decade, there have been significant technological advances made to characterize and quantify biological compounds from in vivo sources and many of these can be exploited in translational research and biomarkers of toxicity. The purpose of this chapter is to provide an overview of select methods that are available to the clinical researcher to assess biological compounds from human or animal material. The six areas of technologies that will be discussed in this chapter are: 1. Immunoassay and immunochemistry 2. Radioimmunoassays 3. Enzyme-linked immunoabsorbent assay 4. Chemiluminescence and fluorescence immunoassays 425

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5. Multiplex microarrays and beads immunoassays 6. Future of immunoassays Alternative technologies used to characterize and quantify biological materials such as chromatographic methods (including high pressure liquid chromatography and gas chromatography), mass spectrometry used for genomics (gene expression and genotyping), proteomics, and metabolomics are discussed in separate chapters of this book (see Chapters 1, 2, and 3). Immunoassays, chromatography, and mass spectrometry are significantly more established technologies than are genomics, proteomics, and metabolomics. Additionally, immunoassays, chromatography, and mass spectrometry methodologies provide quantitative results as opposed to the three latter methods that are more semi-quantitative or qualitative.

IMMUNOASSAY AND IMMUNOCHEMISTRY Background Immunoassay methodologies represent, perhaps, the most frequently used approach to measure biological compounds in translational, clinical research and biomarkers discoveries. Immunoassays are either approved by the Food and Drug Administration (FDA-approved) or for "research use only" (non FDAapproved). Either way, assays must be fully validated by laboratory prior to being utilized for precision, accuracy, reproducibility, analytical sensitivity, and linearity verification of dynamic range of assay. When validated, immunoassays can be used for the detection of small and large molecules such as hormones and lipids, as well as larger peptides and proteins that are present in human body fluids and tissues.1 In addition, a number of synthesized molecules such as therapeutic agents can be measured by immunoassays. Immunoassays can measure antigens and antibodies as well. Many immunoassays are extremely sensitive and can detect as little as 0.1 pg of compound/ml of body fluid.2 Basic Principles Regardless of the method used, all immunoassays rely upon the interaction of an antigen with an antibody.1 The extent to which this interaction occurs (the amount of antigen that is bound to antibody versus free) allows one to measure, either qualitatively or quantitatively, the amount of that particular antigen that is present in a biological fluid or tissue. Detection methods for particular assays vary and depend on the approach used to detect the antigenantibody complex. Antigens are defined as any substance that possesses antigenic sites (epitopes) that produce corresponding antibodies.1 Antigens can be small molecules such as peptides or steroids hormones, etc. or they can be very large compounds such as glycolipids and proteins. Antibodies that are generated in response to antigens (haptens) can be one of five types and include IgG, IgM, IgA, IgE, and IgD. Antibodies consist of heavy chain and either

T H E MEASUREMENT OF BIOLOGICAL MATERIALS

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K or X light chains and possess constant and variable regions. The hypervariable region can be assembled to recognize a wide variety of epitopes (Figure 17.1).2 Although antibodies can serve as antigens, for purposes of immunoassays, they are reactants used to detect antigens. Different types of antibodies can be obtained from several sources. Polyclonal antibodies are generated by immunizing an animal with an antigen. In this case, multiple antibodies are generated which recognize different epitopes. As a consequence, the affinity of polyclonal antibodies for a complex antigen is usually stronger than is that of a monoclonal antibody.3 Monoclonal antibodies are generated using somatic cell fusion and hybridoma selection.4 The resulting established cell line generates a homogeneous antibody population that represents a single epitope.2 While specific for a certain epitope, monoclonal antibodies may cross-react with different antigens that possess the same epitope. Nonetheless, the development of monoclonal antibodies has revolutionized immunoassay methodologies because monoclonal antibodies are well defined and specific reagents and their production can yield a nearly limitless supply of antibody.5 Further, monoclonal antibodies can be prepared through immunization of a non-purified antigen. A more recent approach to the development of antibodies for use in immunoassays is phage display in which antibody fragments of predetermined binding specificity are encoded in phage and expressed in bacteria.6 Figure 17.2 shows the classification of the various immunoassays available and their characteristics. Each of these methods (except chromatography) is discussed in this chapter.

RADIOIMMUNOASSAYS Overview Radioimmunoassay (RIA) was first described in 1960 for measurement of endogenous plasma insulin by Solomon Berson and Rosalyn Yalow of the Veterans Administration Hospital in New York.7 Yalow would later be awarded the 1977 Nobel Prize for Medicine for the development of the RIA for peptide hormones,8 but because of his untimely death in 1972, Berson could not share the award. Also in 1960, Dr. Roger Ekins of Middlesex Hospital in London published his findings on saturation analysis used to estimate thyroxine in human plasma.9 The immunoassay technique with a radioactive label immediately caught the imagination of many researchers and clinicians, and in the ensuing decade RIA for new analy tes were published at a rapid pace and variants of the method were rapidly developed. In 1968, Miles and Hales published their first results of an immunoradiometric technique with radioactive labeled antibodies rather than labeled antigen for measuring insulin in human plasma.10 In many laboratories around the world, special facilities were built in which investigators could safely work with the amounts of radioactivity required for the labeling of antigens or antibodies, but concern persisted with regard to the safety of laboratory personnel, the radioactive waste problem, the requirements of building special laboratory facilities, and the procurement of expensive counting equipment. It should be recalled that in the original studies,7-9 iodine-131 (131I) (6 and

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FIGURE 17.1 Antibodies consist of heavy chain and/either/or light chains and possess constant and variable regions.The hypervariable region can be assembled to recognize a wide variety of epitopes.

7 radiation) was used for the labeling because no alternatives were available at that time. The potential health problems related to the use of radioactive materials were greatly diminished when manufacturers such as Amersham began marketing iodine-125 (l25I) (weak radiation) preparations of sufficiently high specific activity and purity.

Principle of Radioimmunoassay RIAs are heterogeneous assays, meaning they require a washing step to separate antibody bound and free radiolabel. Typical radioisotopes used in RIAs include 125 131 3 1, 1, H, 14C, and 32P, although the majority of assays utilize 125I because of its ready ability to conjugate antigens without altering their biological activity. RIAs can be either competitive or non-competitive. Competitive assays are very common and utilize conditions of antigen excess as opposed to non-competitive assays that employ an excess of antibody.1-2 To some extent radioimmunoassays have been replaced by the enzyme immunoassay (EIA) method, which we discuss later in this chapter.


429

FIGURE 17.2 Classification of various assays from the most sensitive (chemiluminescence) to the lesser sensitive (chromatography). RIA: radioimmunoassay; IRMA: immunoradiometric assay; ELISA: enzyme-linked immunosorbent assay; EIA: enzyme immunoassay.

Various competitive RIA methods have been developed to measure a plethora of different biological compounds. Figure 17.3A shows one of the general methods that is routinely employed.2 Briefly, a known amount of labeled antigen and antigen from a biological specimen are combined and reacted with a known amount of antibody that is usually coated on a solid phase such as sepharose beads or on the inner wall of plastic tubes. After the mixture equilibrates, it is washed to remove unreacted antigens and the immune complex containing both labeled and unlabeled antigen is trapped in the solid phase. The washing step is referred to as bound versus free (B/F) separation.2 Radioactivity can be detected by scintillation counting and is expressed as counts per minute (CPM). Applying the concept of competition between labeled and unlabeled antigen, the antigen-bound percentage of total radioactivity against logarithmic concentration of the antigen can be compared to a standard curve as shown in Figure 17.3B. The CPM plot on the standard curve gives the concentration of antigen. To prepare a standard curve, known amounts of both labeled and unlabeled antigen are reacted as above. Various other competitive RIA methods exist in which a second antibody is utilized to capture antigen-antibody complexes in the solid phase. In addition, non-competitive assays are available

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FIGURE 17.3 A) Antigen and labeled antigen were added to antibodies that are bound to a solid phase. Antigen-antibody went through a reaction where both antigen and labeled antigen were bound to antibodies. Both bound and free antigens were washed and reaction was measured by counter B) The CPM plot on the standard curve gives the concentration of antigen. (Modified from Ashihara, et al. 2001, reference 2).

that employ conditions of antibody excess. These include techniques that are termed immunoradiometric assays (IRMAs) or sandwich type assays. These latter approaches can increase greatly the level of sensitivity of detection of compounds that are present in biological samples at very low concentrations." In summary, radioimmunoassays offer a number of advantages over other immunoassays in that they are highly sensitive and precise. Radioimmunoassays are the most precise method to assess steroids and endocrine hormone levels over more recent technologies such as chemiluminescence methods. The disparity in the hormonal values obtained from different assay methods warrants clinicians to be aware of their clinical interpretation. Using the same reference range for different assay methods is not appropriate. A comparative study between the new and standard assays is essential.12 In addition, radiolabeled compounds are easily prepared. Disadvantages include the fact that radioisotopes must be utilized within a few weeks (125I labeled), which may have a short half-life of 4-6 weeks, laboratory must have a radiation permit in good standing and an adequate facility to ensure the safety of laboratorians and proper disposal of wastes. Radioimmunoassays are heterogeneous assays and therefore cannot be easily fully automated.


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E N Z Y M E - L I N K E D I M M U N O S O R B E N T ASSAY A N D ENZYME IMMUNOASSAY Overview Enzyme-linked immunosorbent assay (ELISA) and enzyme immunoassay (ElA) were developed independently and simultaneously. Between 1966 and 1969, the group in Villejuif (Paris, France) reported their successful results of coupling antigens or antibodies with enzymes such as alkaline phosphatase (EC 3.1.3.1), glucose oxidase (EC 1.1.3.4), and others.13-14 Avrameas and colleagues13-14 described the optimal coupling of these molecules by means of glutaraldehyde. Their purpose was to use the enzyme-labeled antigens and antibodies to detect antibodies or antigens by immunofluorescence, and they applied their tools to histopathology. In Los Angeles, Pierce and colleagues15 had successfully developed the same line of research, also for histochemical purposes. The Uppsala group had developed a so-called (radio)immunosorbent technique in which antibodies were insolubilized by coupling them to cellulose or Sephadex beads. Engvall and Perlmann published their first paper on ELISA in 197116 and demonstrated quantitative measurement of IgG in rabbit serum with alkaline phosphatase as the reporter label. In the same year, van Weemen and Schuurs17 published their innovative work on enzyme immunoassay (EIA) and reported that it was possible to quantify human chorionic gonadotropin concentrations in urine. They used the enzyme horseradish peroxidase (EC 1.11.17), coupled by means of glutaraldehyde, as the reporter label.18,19 Perlmann's further research included cytotoxicity of human lymphocytes20 and immunogen selection and epitope mapping for malaria vaccine development.21 Engvall's group applied the ELISA measurement tool to parasitology, (e.g., malaria22 and trichinosis23), microbiology,24 and oncology.25-27 Engvall then focused her scientific interests on the biochemistry of tissues, e.g., fibronectin, laminin, integrins, and muscular dystrophies. Engvall's laboratory is currently investigating the use of differentiation factors for muscle regeneration and myogenic cells from non-muscle tissues for muscle cell replacement.28 During the late 1960s and early 1970s, many RIA test systems were essentially "home-brew" methods developed by individual researchers who could not keep pace (particularly financially) with the possibilities and facilities of commercial manufacturers such as Boehringer-Mannheim (Germany), Abbott (United States), and Organon Teknika (The Netherlands), to name only a few. Commercialization of EIA/ELISA test kits had started. Solid-phase techniques29'30 were used in the development of microtiter plates (96 wells) in which either an antigen or an antibody is noncovalently bound to a solidphase support. Technical advances led to automated pipetting devices (Micromedics; Hamilton), multichannel pipettes (Lab Systems), and microtiter plate readers and washers. In the 1980s fully automated test instruments were manufactured by Boehringer-Mannheim and Abbott, among others. Such automated systems have come to stay in medical laboratories. The spectacular invention of EIA/ELISA generated a whole series of test formats, from the

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immunoenzymometric (already mentioned in ref. 13) to the many variants of "sandwich" test procedures. For a comprehensive review of the possibilities the reader is referred to reference 31. The Dutch group at Organon/Organon Teknika successfully developed EIA systems in the field of reproductive endocrinology, including assays for human chorionic gonadotropin,32'33 total estrogens, and human placental lactogen34 in plasma. However, the new tests did not become commercially successful until the late 1970s and early 1980s, when they matched the exquisite sensitivity of existing RIA systems for the same analytes. In the early 1970s, blood-bank screening for virologic diseases such as hepatitis B antigen was done either by (semi) automated RIA or nonradioactive, but rather cumbersome, hemagglutination tests. In 1976, Organon Teknika developed and marketed a highly successful EIA system for the hepatitis B surface antigen (HbsAg),35 featuring a 96-well microtiter plate format. This test became the first commercially available EIA. Other microbiological and virologic diagnostic tests soon followed, e.g., for hepatitis B "e" (HBe) antigens,36 rubella antibodies, toxoplasma antibodies, and in the 1980s, an EIA system for detection of human immunodeficiency virus antibodies.

Principle of Enzyme Immunoassay In terms of methodologies, heterogeneous EIAs are similar to RIAs, although detection of antigen-antibody interactions is afforded by cleavage of substrates by enzymes linked to antibodies. Heterogeneous EIAs are at least as sensitive as RIAs, and in some cases is more sensitive (Figure 17.2). Various enzymes can be utilized in EIAs. The most common are alkaline phosphatase, x-galactosidase, glucose oxidase, urease, and catalase. The development of substrates cleaved by enzymes initially employed colorimetric and fluorometric detection and later chemiluminescent methods. EIAs are readily amendable to adaptation to fully automated techniques. An important advantage of EIAs over RIAs is that the former can be developed as homogeneous assays in which the tedious washing step to remove free antigen is eliminated, although homogeneous EIAs are frequently less sensitive than RIAs or heterogeneous EIAs. The first homogeneous EIA developed was enzyme multiplied immunoassay technique (EMIT).37 In summary, the number of analytical and clinical investigations relying on EIA/ELISA measurement procedures worldwide is exceedingly large. Thus, the numbers of measurements and determinations using immunoassay for routine patient care are astronomical. The clinical impact of EIA/ELISA as nonradioactive variants of immunoassays is indeed overwhelming. Perlmann, Schuurs, Engvall, and van Weemen were honored for their inventions when they received the German scientific award of the "Biochemische Analytik" in 1976 in Germany, five years after they had published their first papers. Given the impact that their inventions have had on clinical diagnosis and healthcare in general, as well as on the development of a well-established in vitro diagnostic industry, these inventors deserve to be honored again.


FLUORESCENT AND IMMUNOASSAYS

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CHEMILUMINESCENT

Fluorescent Immunoassays Several decades ago, it was demonstrated that antibodies could be labeled with molecules that fluoresce.38Jl0 These fluorescent compounds are called fluorophores or fluorochromes. They have the ability to absorb energy from an incident light and convert that energy into light of a longer wavelength and lower energy as the excited electrons return to the ground state. Fluorophores are typically organic molecules with a ring structure, and each has a characteristic optimum absorption range.39 The time interval between absorption of energy and emission of fluorescence is very short and can be measured in nanoseconds. Ideally, a fluorescent molecule should exhibit high intensity, which can be distinguished easily from background fluorescence. It should also be stable and have a high molar extinction coefficient (a measurement of how strongly a chemical species absorbs light at a given wavelength). The two compounds most often used are fluorescein and rhodamine, because these can be readily coupled with antigen or antibody. Fluorescein absorbs maximally at 490 to 495 nm and emits a green color at 517 ran.41 It has a high intensity, good photostability, and a high quantum yield. Tetramethylrhodamine absorbs at 550 nm and emits red light at 580 to 585 nm. Because their absorbance and emission patterns differ, fluorescein and rhodamine can be used together. Newer compounds that are beginning to be used are phycobiliproteins derived from algae, porphyrins, and chlorophylls, all of which exhibit red fluorescence at over 600 nm.42 H e t e r o g e n e o u s Fluorescent Immunoassays

Heterogeneous Fluorescent Immunoassays (FIAs), which require a separation step, include the following: indirect, competitive, and sandwich assays. These are based on the same principles as those of EIAs, but in this case the label is fluorescent. Such a label can be applied to either analyte or antibody. Use of solid phase is the typical means of separation in heterogeneous assays. Microbeads made of polysaccharides and polyacrylamides have been used by a number of manufacturers. Either analyte or antibody can be attached to the beads and reacted with analyte and a fluorescent labeled analyte. Then the reaction mixture is centrifuged, the supernatant is discarded, and the beads are analyzed for fluorescence. More recently, the use of magnetic particles allows separation by applying a magnetic field.43 H o m o g e n o u s Fluorescent Immunoassays

Homogenous FIAs require no separation of procedure, so they are rapid and simple to perform.44'45There is only one incubation step and no wash step, and usually competitive binding is involved. The basis for this technique is the change that occurs in the fluorescent label on analyte when it binds to a specific antibody. Such changes can be related to wavelength emission or polarity.

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There is a direct relationship between the amount of fluorescence measured and the amount of analyte in the patient sample. As binding of patient analyte increases, binding of the fluorescent analyte decreases and hence more fluorescence is observed. Typically, homogenous assays including enzyme assays have suffered from low sensitivity. Hence, most research has aimed at increasing sensitivity and newer procedures have been developed that include florescence polarization immunoassay (FPIA),46 florescence excitation transfer immunoassay, and time-resolved fluorescence immunoassay.44'45 All of these require specific instrumentation. Fluorescence P o l a r i z a t i o n Immunoassay (FPIA)

FPIA is based on the change of polarization of fluorescent light emitted from a labeled molecule when it is bound by antibody.4M1 Incident light directed at the specimen is polarized with a lens or prism so the waves are aligned in one plane. If a molecule is small and rotates quickly enough, when it is excited by polarized light, the emitted light is unpolarized. If, however, the labeled molecule is bound to antibody, the molecule is unable to tumble as rapidly, and it emits an increased amount of polarized light. Thus, the degree of polarized light reflects the amount of labeled analyte that is bound. In FPIA, labeled analytes compete with unlabeled analyte in the patient sample for a limited number of antibody binding sites (Figure 17.4). The more analyte that is present in the sample, the less the fluorescence labeled analyte is bound and the less the polarization that will be detected (Figure 17.4). With competitive binding, analyte from the specimen and analyte-fluorescein (AF) labeled reagent competes for binding sites on the antibody. As a homogeneous immunoassay, the reaction is carried out in a single reaction solution, and the bound Ab-AF complex does not require a wash step to separate it from "free" labeled AF. FPIA is utilized to provide an accurate and sensitive measurement of small toxicology analytes such as therapeutic drugs, and drugs of abuse, toxicology, and some hormones. FPIA utilizes three key concepts to measure specific analytes in a homogeneous format: fluorescence, rotation of molecules in solution, and polarized light. Fluorescence: Fluorescein is a fluorescent label. It absorbs light energy at 490 nm and releases this energy at a higher wavelength (520 nm) as fluorescent light. Rotation of molecules in solution: Larger molecules rotate more slowly

FIGURE 17.4 Competitive fluorescence polarization immunoassay. Competitive binding, analyte from the specimen and analyte-fluorescein (AF) labeled reagent compete for binding sites on the antibody.


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in solution than do smaller molecules. This principle can be used to distinguish between the smaller analyte-fluorescein molecule, AF, which rotates rapidly, and the larger Ab-AF complexes, which rotate slowly in solution. Polarized Light: Fluorescence polarization technology distinguishes antigen-fluorescein (AF) label from antibody bound-analyte-fluorescein (Ab-AF) by their different fluorescence polarization properties when exposed to polarized light (Figure 17.5). Polarized light describes light waves that are only present in a single plane of space. When polarized light is absorbed by the smaller AF molecule the AF has the ability to rotate its position in solution rapidly before the light is emitted as fluorescence. The emitted light will be released in a different plane of space from that in which it was absorbed and is therefore called unpolarized light. With the larger sized Ab-AF complex, the same absorbed polarized light is released as polarized fluorescence because the much larger Ab-AF complex does not rotate as rapidly in solution. The light is released in the same plane of space as the absorbed light energy, and the detector can measure it (Figures 17.5 and 17.6A). Measurement of large complexes using fluorescence, rotation, and polarized light in FPIA is shown in Figure 17.6A. FPIA results in an inverse dose response curve such that lower levels of patient analyte result in a higher signal (in this case, the signal is polarized light) (Figure 17.6B). High signal at low patient analyte levels results in a highly sensitive assay. Table 17.1 shows the advantages and disadvantages of florescence assays.

Chemiluminescent Immunoassays Several recently developed immunoassays use the principle of chemiluminescence to follow analyte antibody combination.52 Chemiluminescence is the emission of light caused by a chemical reaction producing an excited molecule that decays back to its original ground state. A large number of molecules are capable of chemiluminescence, but some of the most common

FIGURE 17.5 Polarized Light. Fluorescence polarization technology distinguishes analyte-fluorescein (AF) label from antibody bound-AF (Ab-AF) by their different fluorescence polarization properties when exposed to polarized light.

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FIGURE 17.6 A) The light is released in the same plane of space as the absorbed light energy, and the detector can measure it. Measurement of large complexes (AF) is performed using fluorescence, rotation, and polarized light in fluorescence polarization immunoassay (FPIA) 490nm. B) Measurement of large complexes using fluorescence, rotation, and polarized light in FPIA. FPIA results in an inverse dose response curve such that lower levels of patient analyte result in a higher signal (in this case, the signal is polarized light).

substances used are luminol,53 acridium esters,54 peroxyoxalates55,56 ruthenium derivative, and dioxetanes57 (Figure 17.7A). When these substances are oxidized, typically using hydrogen peroxide and an enzyme for a catalyte, intermediates are produced that are of a higher energy state. These intermediates spontaneously return to their original state, giving off energy in the form of light (Figure 17.7B). Light emissions range from a rapid flash of light to a more continuous glow that can last for hours. Different types of instrumentation are necessary for each kind of emission.58Table 17.1 shows advantages and disadvantages of chemiluminescence assays.

M U L T I P L E X I N G U S I N G A N T I B O D Y ARRAY A N D BEAD I M M U N O A S S A Y S Innovation in immobilization surfaces and detection strategies has increased the number of planar arrays and bead-based technologies. Planar antibody arrays are the most common type of protein arrays. This section describes the main formats of planar arrays and the differences between planar arrays and bead-based assays (Figure 17.8).


TABLE 17.1

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Advantages and disadvantages of chemiluminescence versus florescence assays.

Method

Florescence

Chemiluminescence

Advantages

• Sensitivity is higher than those of radiolabels and enzyme reactions.

• An excellent sensitivity comparable to EIA and RIA.

• The methodology is simple and there is no need to deal with and dispose of hazardous substances

• Reagents are stable and relatively nontoxic. • The sensitivity of some assays has been reported to be in the range of attamoles (10-18) to zeptomoles (10-21). • Because very little reagent is used, they are inexpensive to perform. • Detection systems basically consist of photomultiplier tubes which are simple and relatively inexpensive.

Disadvantages

• The main problem is the separation of the signal on the label from background fluorescence because of different organic substances normally present in serum. • Nonspecific binding to substances in serum can cause diminishing of the signal and change the amount of fluorescence generated.

• False results may be obtained if there is lack of precision in injection of the hydrogen peroxide. • Some biological materials such as urine or plasma may cause diminishing of the light emission.

• Any bilirubin or hemoglobin present can absorb either the excitation or emission energy. • It requires expensive dedicated instrumentation, which may limit its use in smaller laboratories.

Planar Protein Array Formats The main planar label-based assays are 1-antibody assays, which use one antibody to capture the target molecule, and sandwich assays, which use two antibodies to capture the target protein.59-61 One-antibody and sandwich assays both have advantages and pitfalls. In one-antibody label-based assays, the targeted proteins are captured by an immobilized antibody and detected through labeling with a tag (Figure 17.8A). In direct labeling, proteins are labeled with a fluorophore such as cyanine (Cy3 or Cy5). In indirect labeling, proteins are labeled with a tag that is later detected by a labeled antibody. One-antibody label-based assays allow the incubation of two different samples, each labeled

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FIGURE 17.7 A) Shows luminol as an example of chemiluminescence where the emission of light caused by a chemical reaction produces an excited molecule that decays back to its original ground state. B) Illustrates when these chemiluminescence molecules (such as luminol) are oxidized, typically using hydrogen peroxide and an enzyme for a catalyst, intermediate products are produced that are of a higher energy state.These intermediates spontaneously return to their original state, giving off energy in the form of light.

with a different tag on the arrays. These types of assays, therefore, allow the use of a reference sample that is co-incubated with a test sample and facilitates normalization.59-61 Another advantage of these types of assays is that they are competitive as the analytes in the test and reference solutions compete for binding at the antibodies,59-61 leading to improvement in linearity of response and dynamic range compared with noncompetitive assays.61 The main disadvantage of these types of assays is the disruption of the analyte-antibody interaction by the label, which may limit detection as well as sensitivity and specificity. In the sandwich label-based format, immobilized antibodies capture unlabeled proteins, which are detected by another antibody, with the signal for detection generated by several methods (Figure 17.8B). The use of two antibodies targeting each analyte confers greater specificity than label-based assays. The reduced background of these assays also increases the detection limit. The sandwich format allows only non-competitive assays, because only one sample can be incubated on each array.59-61 Noncompetitive assays have sigmoidal binding responses, which are linear in competitive formats, and require standard curves of known concentrations of analytes to achieve accurate calibration of concentrations.61 Sandwich assays are more difficult to develop in a multiplexed manner than label-based assays because matched pairs of antibodies and purified analytes may not be available for each target,


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FIGURE 17.8 A) In one-antibody (Ab) label-based assays, the targeted proteins are captured by an immobilized antibody and detected through labeling with a tag. B) In the sandwich label-based format, immobilized antibodies capture unlabeled proteins, which are detected by another antibody, with the signal for detection generated by several methods. C) Other antibody and protein array approaches are modifications of one-antibody and sandwich label-based arrays. These alternative strategies of protein arrays allow detection of proteins on whole cells without protein isolation. D) A growing area of cancer research that uses protein arrays on serum specimens entails the development and design of tumorassociated antigen (TAA) arrays to enhance detection of autoantibodies againstTAAs for cancer diagnosis. E) Complex protein extracts can also be spotted onto membranes and probed with antibodies targeting specific proteins on the so-called reverse-phase arrays. F) Proteins in suspension can also be detected by use of bead arrays. (See color insert for a full color version of this figure.)

and the potential cross-reactivity among detection antibodies increases with additional analytes.60'6I The size of multiplexed sandwich assays is limited to 30 to 50 different targets,59-61 in contrast to one-antibody assays, for which

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only the availability of antibodies and the space on the substrate limit the number of targets analyzed. Other antibody and protein array approaches are modifications of one-antibody and sandwich label-based arrays. These alternative strategies of protein arrays allow detection of proteins on whole cells without protein isolation (Figure 17.8C).60,62 A growing area of cancer research that uses protein arrays on serum specimens entails the development and design of tumor-associated antigen (TAA) arrays to enhance detection of autoantibodies against TAAs for cancer diagnosis (Figure 17.8D). The rationale is related to the presence of antibodies in the cancer sera that react with a unique group of autologous cellular antigens or TAAs.63-M Complex protein extracts can also be spotted onto membranes and probed with antibodies targeting specific proteins on the so-called reverse-phase arrays65,66 (Figure 17.8E). Proteins in suspension can also be detected by use of bead arrays (Figure 17.8F)62,67-70

Suspension or Bead-Based Arrays Suspension or bead-based arrays use different fluorescent beads. Each bead is coated with a different antibody, and all beads are spectrally resolvable from each other.62,67-70 The beads are incubated with a sample to allow protein binding to capture the antibodies, and the mixture is incubated with a cocktail of detection antibodies, each corresponding to one of the capture antibodies. The detection antibodies are tagged to allow fluorescent detection. The beads are passed through a flow cytometer system, and each bead is probed by two lasers, one to read the color or identity of the bead, and another one to read the amount of detection antibody on the bead.62,67~70 Multiplexed bead-based flow-cytometry assays represent an active area of development. Differentially identifiable beads coated with proteins, autoantigens, or antibodies use a cytometer system to identify a variety of bound antibodies or proteins.62,67_7° Advances in instrumentation and bead chemistries will probably make this approach valuable for the detection of circulating diseases markers and cancer cells in clinical practice. In another version of this method, suspensions of cells are incubated on antibody arrays, and the number of cells that bind each antibody is quantified by dark-field microscopy. These arrays enable characterization of multiple membrane proteins in specific cell populations or changes in cell surfaces induced by drug therapies.

Example of M u l t i p l e x i n g Technology The use of protein array technology over conventional assay methods has advantages that include simultaneous detection of multiple analytes, reduction in sample and reagent volumes, and high output of test results. The susceptibility of ligands to denaturation, however, has impeded production of a stable, reproducible biochip platform, limiting most array assays to manual or, at most, semi-automated processing techniques. Such limitations may be overcome by novel biochip fabrication procedures.


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Simultaneous Multi-Analyte Detection Introduction The world of Healthcare and Drug Discovery encompasses many arenas including understanding the nature of the problem—discovery research; targeting the potential candidates for regulation and amendment—target profiling & lead optimization; testing dose response and effects—ADME; and finally clinical testing to prove efficacy—clinical trials & diagnostics. In each phase, several biomarkers may need to be screened. Cytokines, chemokines, and cell signaling targets are a common area of biomarker analysis in many fields like cardiovascular disease, diabetes research and treatment, immunological disorders, or even the cosmetic industry. One may also target more specific analytes like osteocalcin for bone metabolism or adiponectin for obesity research. Conventional assays like radioimmunoassays and ELISAs are restricted in sample volume and number of tests that can be conducted to get the adequate information. Most assays may not address the complete dynamic range to measure both basal and elevated concentrations, requiring repetition. This requires a large amount of time and cost involvement, and may also challenge the integrity of the sample, and hence data generated. The ideal testing method would be a convenient, simple tool that enables measurement of all appropriate analytes, in a small sample volume, to provide a relevant conclusion. It would need to be comparable and compatible with the conventional assays to enable effortless adoption. It should be flexible to incorporate new tests and be easily transferred for reproducibility. In the following section of the chapter we will present three commercially available platforms for multiplexing: bead particles, arrays electro-chemiluminescence, and biochip array technology. M u l t i p l e Bead Particle Technology

The xMAP technology used in the Luminex (Luminex Corporation, Austin, TX) and equivalent instruments meets the requirements described above. Luminex's xMAP® (Multi Analyte Profiling, "x" being the variable depicting the type of assay) technology is built on proven, existing methods of flow cytometry, microsphere-based assays, and traditional chemistry, combined in a unique way71 (Figure 17.9). xMAP technology can be configured to perform a wide variety of bioassays quickly and could be cost-effective when used as multiple (save operator time) (Figure 17.10). However, the sensitity of assays may vary. The technology is based on uniformly sized 5.6 micron carboxylated polystyrene hydrophobic beads. Each bead contains a mixture of two fluorescent dyes that provide a "unique signature" which can be identified by the Luminex instrument. Thus, when a specific antibody is attached to a specific bead, the instrument can define and quantify the analyte being measured (Figure 17.11). These beads are dyed by adding them to an organic solvent containing the two fluorescent dyes in a defined ratio. The dyes are absorbed into the beads.

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FIGURE 17.9 The multiplex Luminex® assay format differs from conventional ELISA in one significant way—the multiplex capture antibody is attached to a polystyrene bead, whereas the ELISA capture antibody is attached to the microplate well. The use of the suspension bead-based technology enables the multiplexing capabilities of the Luminex® assays.The xMAP® technology uses 5.6 micron polystyrene microspheres (Luminex beads), which are internally dyed with red and infrared fluorophores of differing intensities. Each bead is given a unique number or bead region, allowing differentiation of one bead from another Beads covalently bound to different antibodies (capture antibodies) can be mixed in the same assay, utilizing a 96-well microplate format. At the completion of the sandwich immunoassay, beads can be read, using the Luminex® detection system, in single-file by dual lasers (633 nm and 532 nm wavelength) for classification and quantification of each analyte. (Kindly provided by Millipore.)

The beads are then dropped into an aqueous solution whereby the dyes are now trapped inside each bead set due to their hydrophobic nature. The basic and most commonly used instrument can detect a hundred different bead sets, where each has a designated unique number. Theoretically, this enables assaying up to 100 different analytes from the same sample; however, so far the highest number of tests multiplexed is closer to 31 analytes. Here is an example of a multiplexed immunoassay format. A specific antibody is attached to a specific bead set. Thus, to measure five analytes, the specific capture antibody for each analyte would be covalently conjugated tofivedifferent carboxylated bead sets. These beads are mixed together and added to the standard or sample containing the analytes in a 96-well plate format. This is followed by incubating with the biotinylated detection antibody mixture which binds to the bead immobilized analyte, followed by the reporter fluorescent dye phycoerythrin, conjugated to streptavidin forming an analyte-antibody sandwich. The Luminex instrument (or equivalent) collects the reaction mixture from each well and, following the fluidics principle of flow cytometry, transports these beads into the pathway of two different lasers in a flow cell. One laser identifies the specific bead, which in turn identifies the analyte being assayed; while the other laser measures the intensity of the fluorescent signal from the reporter dye. The data is analyzed and reported in real time by dedicated analysis software.


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FIGURE 17.10 Example of multiplex of several human cytokines/chemokines in one reaction vial as shown by the standard curves in serum matrix. (Kindly provided from Millipore.) (See color insert for a full color version of this figure.)

Applications

Since its inception in 1999, the Luminex instrument has played a significant role in both human and veterinary drug discovery research, genetic analysis, pharmacogenomics, clinical diagnostics, and the general healthcare industry.72-77 These liquid microarrays have been used in neonatal blood screening. A drop of blood from a toe prick can be used directly in the assay to measure multiple analytes, thus ensuring the health of newborns.73 Alternately, a blood spot may be dried onto a filter and sent to any global location. This can be eluted in a small volume of buffer and used for screening. This has proven to be a useful, safe, and effective method for population studies across many demographics. It has proven to be a valuable tool in clinical trials for the Pharmaceutical Industry. A complete array of biomarkers may be screened when selecting a potential drug target. From this, a select few targets and diagnostic biomarkers may be chosen to continue testing, in vitro and in vivo, in small

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BIOMARKERS

FIGURE 17.1 I The reader uses a 532 nm green laser ("assay" laser) to excite the phycoerythrin (PE) dye of the assay (streptavidin-PE).The 635 nm solid state laser (red "classify" laser) is used to excite the dyes inside the beads to determine their "color" or"region" and is also used for doublet discrimination by light scatter. The reader has four detectors, one for each of the optical paths shown in the figure. Detectors are used to measure the fluorescence of the assay, to make bead determination (I -100), and lastly to discriminate between single and aggregate beads. (Kindly provided by Millipore.) (See color insert for a full color version of this figure.)

animal models. The biomarker arrays may be expanded to include the upstream or downstream regulators to understand their mechanism of action and ADME. As drug development progresses, toxicity panels may be included to assess negative effects. Once the target drug is selected in its appropriate formulation for administration, this moves into clinical trials where hundreds of subjects may need to be tested before the drug can enter the marketplace. The xMAP technology helps to accomplish this in a very short time, as opposed to the many years and resources it would take using conventional methods. The flexibility of the Luminex technology has enabled multi-analyte testing in sample matrices that would have been impractical to test in the past, e.g.,


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tears,78,79 breath condensates,80,81 gingival crevicular fluids,72 and cerebral fluid, among others.73 The Future While the Luminex instruments have been ideal for high-content screening, they were not considered high-throughput. They were not easily adaptable to liquid handling systems for automation. A maximum of 6-8 plates could be run in a typical day. It was also susceptible to sample matrix effects as insoluble paniculate matter could not be washed away. However, these limitations have been overcome by the introduction of two innovations in this technology. The use of magnetic beads in place of the standard polystyrene beads now enables use of automated liquid handlers and reduces matrix-related interferences in the assay. FlexMAP 3D—an upscale Luminex model that can detect up to 500 bead sets—can analyze 384 well plates and takes less than 20 minutes to read a plate. It also provides higher sensitivity and dynamic range. The software is compatible with automation and liquid handling instrumentation. With these improvements, one may significantly reduce the level of technical errors in sample handling and assay setup, which may be a significant application in studies requiring large numbers of test samples. The Luminex xMAP technology and instrumentation is highly adaptable and flexible. It has been effectively used in small labs and academic settings, as well as in the biopharmaceutical industry. Its applications range from in-depth cell-based studies to clinical diagnostics. One can visualize this becoming an integral part of global healthcare.

Electrochemiluminescence (ECL) Microarrays Electrochemiluminescence (ECL) methods use electric current to generate light-emitting reactions.82 Upon application of a voltage, the ruthenium label on the detection molecule and the co-reactant tripropylamine are oxidized. A high-energy electron transfer from the tripropylamine radical to ruthenium puts ruthenium in an excited state. Relaxation of the excited state ruthenium to the ground state generates chemiluminescence emission at 620 nm.82,83 Like other chemiluminescent assay methods,84 ECL methods have a high signal and low background, since no external light source is used to generate signals. In addition, this ECL technology requires that the ruthenium be in close proximity to the electrodes, thus further reducing background produced by unbound detection molecules. An ECL methodology using magnetic beads and a fluidics system (BioVeris, Gaithersburg, MD) has been used for immunoassays.82,83,85 However, it is not amenable to intact cell-based assays. Recently, an ECL methodology using microwell plates with carbon electrodes built into the bottom became available. A charge-coupled device camera within the reader records ECL signals (Meso Scale Discovery, Gaithersburg, MD). The carbon surface electrodes, originally used to coat soluble capture molecules for immunoassays, were later found to also bind cells tightly.86 Consequently, suspension cells can be immobilized on the carbon surface plates and the plates

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BIOMARKERS

can be washed on a microplate washer. This eliminates the tedious centrifugation steps used in the live suspension cell-based ELISAs. MSD's assays are based on MULTI-ARRAY® technology, a proprietary combination of ECL detection and patterned arrays. ECL detection offers a unique combination of sensitivity, dynamic range, and convenience. Arrays bring speed and high density of information to discovery through miniaturization, organization, and parallel processing of biological assays. ECL Diagram Electrochemiluminescence detection uses labels that emit light when electrochemically stimulated. Background signals are minimal because the stimulation mechanism (electricity) is decoupled from the signal (light). Labels are stable, non-radioactive, and offer a choice of convenient coupling chemistries. They emit light at -620 nm, eliminating problems with color quenching. Few compounds interfere with electrochemiluminescent labels so you can use large, diverse libraries with confidence. Multiple excitation cycles of each label amplify the signal to enhance light levels and improve sensitivity (Figure 17.12). Detection MSD's instruments use custom-designed optics and photodetectors to collect and quantitatively measure light emitted from the microplates. Proprietary electronics and signal processing algorithms convert the measured signal into useful data.

FIGURE 17.12 Multiple excitation cycles of each label amplify the signal to enhance light levels and improve sensitivity. (Kindly provided by Meso Scale Discovery.)

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Multi Arrays Technologies MULTI-ARRAY® microplate footprints are integrated electrodes and arrays form conveniences and performance. The technology offers different formats of arrays: the multi-array or multi-spot plates. The multi-array plates feature flexibility to coat on surface in either 96 or 384 wells format. This is very useful for high throughput screening. The multi-spot offers the flexibility of high throughput multiplexing of biomarkers in a 96-well single spot or 4-, 7-, and 10-spot array (Figure 17.13).

Biochip A r r a y Technology The biochip array technology (Figure 17.14) may be novel, but the methodology is familiar, featuring competitive and sandwich immunoassays.87 The capture agents can be antigens or antibodies. Antibodies are covalently bound in the correct orientation to the test regions on the surface of the biochip. Analytes in the patient sample are bound by their complementary antibodies on the biochip. The enzyme-labeled detection agent also binds to the analytes. The signal reagents produce a chemiluminescent reaction that is used to quantify the amount of each analyte present. The biochip is the foundation of biochip array technology. A single 9 mm biochip acts as the reaction vessel, replacing multiple cuvettes. Randox biochips (Randox Laboratories Ltd, United Kingdom) currently hold up to 25 tests but are capable of carrying over 100 different assays. Evidence uses one biochip per patient sample to produce a panel of test results: the patient profile.

FIGURE 17.1 3 The multi-spot offers the flexibility of high thoughput multiplexing of biomarkers in a 96- and 384-well single spot (A) or 4-, 7-, and 10-spot array (B). (Kindly provided by Meso Scale Discovery.)

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FIGURE 17.14 The biochip is the foundation of biochip array technology. A single 9 mm biochip acts as the reaction vessel, replacing multiple cuvettes. Randox biochips currently hold up to 25 tests but are capable of carrying over 100 different assays. (Kindly provided by Randox.)

Biochip M a n u f a c t u r i n g

Biochip manufacturing requires a multidisciplinary approach and strict quality control procedures. The surface of the ceramic biochip is activated using silanation and a polymer method technique to create a uniform hydrophobic surface. Spots are precisely created using piezoelectric nano-dispensing of 330 pL droplets sequentially to achieve a total volume of 10 nL of ligand solution. The hydrophobic surface of the biochip prevents the fluid from spreading out, containing it in a uniform drop with a diameter of 0.3 nm. Dispense-head cameras monitor production to automatically detect deviations in size, shape, and position.87 The charge-coupled device (CCD) camera is used to quantify the chemiluminescence generated. The image and numerical data are archived along with QC and calibration data (Figure 17.15). Applications

Evidence assays are arranged into disease-orientated test panels (e.g., fertility) or related groups of tests (e.g., drugs of abuse, renal function profile). The tests have been carefully selected to offer established, emerging, and novel assays of clinical significance arrays in the fields of cytokines,88'89, n-93 adhesion molecules,90,91and cardiac markers.94,95 Customized arrays can be manufactured to customer requirements.

F U T U R E OF I M M U N O A S S A Y S The first immunoassay was described by Berson and Yalow in 1959. Their work resulted in their receipt of the Nobel Prize in Medicine in 1977. Since this introduction, immunoassays have evolved considerably. There have been several milestones that have led to the proliferation of modern immunoassays.


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FIGURE 17.15 The charge-coupled device camera is used to quantify the chemiluminescence generated.The image and numerical data are archived along with QC and calibration data. (Kindly provided by Randox.)

The development of monoclonal antibodies from mouse hydridoma cells by Millstein and Kohler (Nobel Prize in 1984) enabled the production of high quantities of antibodies with well characterized epitope specificity. The first homogenous immunoassay (no separation step required) was the EMIT, which enabled adaptation of this assay onto automated chemistry platforms. EMIT was also one of the first immunoassays that made use of non-isotopic labels. Other non-isotopic labels became available, such as chemiluminescence, to improve the analytical sensitivity of immunoassays. The advantages of highsensitivity immunoassays have created expanded diagnostic roles for some existing assays such as thyroid stimulating hormone for hyperthyroidism, C-reactive protein for cardiovascular risk assessment, and other applications. The development of instrumentation capable of automated heterogeneous immunoassays (separation step to improve sensitivity) has enabled movement of this technology from the "special chemistry" sections of a clinical laboratory into the "core" laboratory with other high-volume testing. Today, immunoassays play a prominent role in the analysis of many clinical laboratory analytes such as proteins, hormones, drugs, and nucleic acids. The future involves development of assays with higher sensitivities which will enable the discovery of new biofnarkers for disease diagnosis, and technology that will enable simultaneous multimarker analysis of tests whose needs are naturally grouped together (e.g., cytokines and allergens).


Immunoassay methodologies represent the most frequently used approach to measure biological compounds in translational, clinical research and biomarkers discoveries. The new generation of non-isotopic immunoassays using chemiluminescence and fluorescence labeling molecules are safer than isotopic immunoassays.

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4.

5.

Sensitivity is higher than those of radiolabels and enzyme reactions. The methodology is simple and there is no need to deal with and dispose of hazardous substances. Microarray, bead-based multiplex technologies and instrumentation are highly adaptable and flexible. They have been effectively used in small labs and academic settings; as well as in the biopharmaceutical industry. The future of immunoassay-based technology involves development of assays with higher sensitivities which will enable the discovery of new biomarkers for disease diagnosis, and technology that will enable simultaneous multimarker analysis of tests whose needs are naturally grouped together (multiplex panels).

ACKNOWLEDGMENTS I would like to thank Sonali Nayak (Millipore Corporation), Karma Carrier (Meso Scale Discovery), and Rajnesh Mathur (Randox) for providing material for the figures. Also, a particular thanks to Loc Tran (Brigham and Women's Hospital) for the tremendous help in the preparation of this chapter.

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CHAPTER

NANOSCALE TECHNIQUES FOR BIOMARKER QUANTIFICATION Madhukar Varshney and Harold G. Craighead

INTRODUCTION Miniaturized analytical devices and "labs-on-a-chip" are developing based on microfabrication technology. These systems are typically based on existing methods for biochemical analysis made more compact and rapid by the use of small fluid volumes and microfluidic systems for performing chemical reactions. Nanostructure science and engineering utilizing structures and devices with dimensions typically measuring in micrometers and nanometers, enables access to new physical length scales, and enables new approaches for molecular detection and analysis. The use of nanostructures can enable identification, detection, enumeration and isolation of small numbers of analyte molecules in complex mixtures. The small size of the materials and structures is comparable to the size of most biological materials, such as proteins, nucleic acids, cells, viruses, etc. (Figure 18.1). Chemical recognition may be combined with optical, electrical, mechanical, or magnetic signal transduction for very sensitive detection of biomarkers or other chemical compounds. These nanoscale techniques are being explored for a range of new analytical approaches rather than simply miniaturizing existing chemical analysis methods. These approaches are still the topic of research and are rapidly evolving as the technical capabilities and understanding of the nanoscale processes are developing. In this chapter we will provide a review of some of the methods and research directions in these areas. The specificity of the overall detection system is based on attaching biorecognition elements (antibodies, nucleic acid probes, aptamers, enzymes, and proteins) to nanomaterials or nanostructures, which can bind with target 457

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FIGURE 18.1 Size comparison between biomolecules and several nanomaterials. (Image reproduced with permission from reference 60.)

analytes in a complex medium consisting of non-targeted analytes along with other interfering chemical moieties. The existing surface modification and protein modification techniques are ready to apply to nanoscale techniques. Some detection techniques are only sensitive at nanometer distance. These techniques are complemented by the small size of nanomaterials. For example, giant magneto resistance (GMR) based detection techniques and surface enhanced Raman scattering (SERS) are most sensitive for detecting material at nanometer distance from the device surface. When nanomagnetic particles are specifically attached to the surface of the GMR sensor, it has shown that even a single magnetic nanoparticle can be detected. Shrinking of sample or interrogation volume has enabled the study of an individual molecule, otherwise obscured by ensemble averaging. Reduction in volume is achieved by using nanostructures or by confining samples in the nanofluidic channels. Observing a single molecule provides an opportunity to measure the distribution of behavior as opposed to examining only the average behavior. These single molecule detection techniques not only make it possible to detect extremely low number of molecules, but also significantly enhance signal to noise ratio.

NANOSCALE SENSING TECHNIQUES FOR BIOMARKER QUANTIFICATION This chapter includes a survey of nanoscale sensing techniques used for the quantification of biomarkers. Other techniques used specifically for imaging and studying biological systems are beyond the scope of this chapter. Conventionally, nanotechnologies refer to techniques that employ nanomaterials with at least one critical dimension in the range of 1-100 nm. In this chap-

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ter we have categorized nanoscale sensing techniques based on the detection technique (optical, electrical, mechanical, and magnetic) used for the system. In some cases there is more than one detection technique that can be used for a particular assay, in which case we have categorized that assay based on the currently prevalent detection technique used for that assay or the detection technique that is used for the most sensitive detection.

OPTICAL

DETECTION

Bio-Barcode Assay-Based Sensors A combination of large amplification and high specificity of a typical immunoassay or DNA hybridization lends bio-barcode assays significant importance. The working principle of bio-barcode involves using two types of particles— one is used for the separation of target analyte, while the other is primarily used for amplification and specific binding with target analyte. Generally, a micro-sized magnetic particle conjugated with the biorecognition elements is used for separation. In the case of nucleic acid, the biorecognition element is an oligonulceotide, complementary to the statistically unique region of the target,' while in the case of individual protein or cell surface protein, the recognition element is an antibody (polyclonal or monoclonal), peptide, or aptamer. The particle used for amplification is a nanoparticle coated with another biorecogniton element to sandwich target analyte with the microparticles. In addition to biorecognition elements, the surface of nanoparticles is coated with hundreds of oligonucleotides (used for amplification) referred to as bio-barcodes. These bio-barcodes are used for the amplification of signal and can also be used for multiplex detection of analytes. Bio-barcodes typically comprise 15-20 mer oligonucleotides, allowing the user to pair a unique barcode with every conceivable recognition element, since for a 20-mer there are 420 unique combinations. After choosing the appropriate combination, these micro and nanoparticles are added to the sample concurrently. Following incubation, target analytes along with nanoparticles and magnetic particles are separated by applying magnetic force and unattached nanoparticles and analytes are washed off. The barcodes are released in buffer chemically (e.g., by dithiothreitol, DTT)2 or by heating the solution,2'* and are detected using microarray via scanometric using nanoparticle probes (Figure 18.2).7 If biomarkers carry fluorescent tags, then in situ fluorescence-based approaches are applied. However, in principle, any appropriate readout mechanism can be integrated with the system. Until now, the scanometric method has provided the lowest detection limit for both nucleic acid (high zeptomolar, 1021 M)1 and protein targets (low attomolar, 10~18 M).8 The potential of bio-barcode assay for multiplex detection is exploited by detecting four types of DNA using synthetic oligonucleotide sequences of 30-33 base long associated with (a) hepatitis B virus surface antigen gene, (b) variola virus, (c) ebola virus, and (d) human immunodeficiency virus at concentrations as low as 5 pmol/L in 40 min using capillary DNA Analyzer.9

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FIGURE 18.2 Schematic representation of a bio-barcode assay.The target analyte is sandwiched between magnetic microparticles conjugated with the biorecognition elements and nanoparticles coated with biorecognition elements and bio-barcodes.The bar-code oligonucleotides are released, and detected using the scanometric method. (Image reproduced with permission from reference 10.)

The bio-barcode assay is up to 106 times more sensitive than ELISA-based technology and is comparable to PCR in terms of its sensitivity.10 However, bio-barcode assay is much simpler in use and thus is viewed as an efficient alternative to PCR that will soon be available for widespread use in research and clinical applications. Due to the extremely low detection limit of these assays, they are also suitable for pre-mortem tests, where most non-PCR methods are not applied due to the extremely small amount of analytes present in the body fluid. In one such application, the bio-barcode method is used to detect amyloid-derived-diffusible ligands (ADDL, responsible for Alzheimer's disease through study of the brain)3 in the cerebral spinal fluid of subjects afflicted with the disease. This is a pre-mortem method and can be used to monitor the progress of Alzheimer's disease. Sometimes the use of sophisticated tools such as microarrays and chipimaging limits the portability of scanometric based bio-barcode assays. This

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issue can be overcome by a simple electrical readout from the oxidative release of metals from nanoparticles. One such electrochemical bio-barcode assay is used to detect human-fetoprotein (AFP), a tumor marker related to heptocellular cancer, yolk sac cancer, and liver metastasis from gastric cancer, testicular cancer, and nasopharyngeal cancer. Here, oligonucleotide bio-barcodes used in optical methods were replaced by CdS nanoparticles. This electrochemical biobarcode method was used to detect a minimum of 9.6 pg/ml of AFP.11 Bio-barcode assays offer several advantages that make them suitable for biomarker detection. They provide necessary specificity, sensitivity, multiplex detections and low detection limit required to detect extremely small amounts of biomarkers. This is beneficial not only in providing better diagnosis, but also in monitoring various stages of disease, which is imperative for early detection and cure. Due to these advantages, the use of these assays will extend and play a critical role in diagnosing a wide variety of fatal diseases such as cancer, HIV, and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. A comparison between bio-barcode assay and conventional techniques is shown in Figure 18.3 in the area of medical diagnostics.

Q u a n t u m Dots-Based Sensors The use of organic fluorophores is well established. During the past decade, advances in synthesis and biofunctionalization of colloidal semiconductor nanocrystals called quantum dots (QDs) have replaced organic fluorophores

FIGURE 18.3 The comparison of bio-barcode assay with other conventional detection technologies for medical diagnostic applications, (Image reproduced with permission from reference 10.)

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in some cell biology applications. These nanometer sized crystalline particles are composed of elements from periodic groups II-IV (e.g., CdSe) or III-V (e.g., InP) and are extremely bright and resistant to photobleaching that is a limitation of organic fluorphores. Advantages of QDs, including their intense brightness, makes them suitable for single molecule detection,12 and their size turnable emission wavelengths and single excited wavelength for multiple emission QDs make them suitable for the design of a simple and compact system to simultaneously detect multiple biomolecules (Figure 18.4).13 Additionally, owing to their robust optical properties in complex biochemical media, QDs are extensively used in the area of fluorescence immunolabeling for probing structures and locating signal transduction-related molecules. Ness and coworkers developed an immunohistochemical (IHC) protocol that combines conventional enzymatic signal amplification and QD labeling to detect intracellular antigens in rat and mouse brain tissue sections. Their study showed that QD IHC labeling resulted in greater sensitivity as compared to similar IHC approaches using conventional dyes.14Wu and coworkers developed reliable and specific QD probes to localize breast cancer cell surface marker Her2, cytoskeleton fibers, and nuclear antigens in fixed cells, live cells, and tissue sections, with a substantial increase in brightness and photostability as compared to organic dyes.15

FIGURE 18.4 A. Fluorescence emitted from quantum dots. Blue fluorescence can be emitted from small particles of approximately 2 nm in diameter, green from ~3 nm particles, yellow from ~4 nm particles, and red from large particles of ~5 nm. The wavelength of the excitation light is 365 nm. B. Fluorescence emission spectra depending on the size of quantum dots. (Image reproduced from http:// www.aist.go.jp/aist_e/aist_today/2006_2l/hot_line/hot_line_22.html with permission from National Institute of Advanced Science and Technology.) (See color insert for a full color version of this figure.)


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Although QDs are used in numerous applications for the quantification of bacterial cells and nucleic acids,16-18 a limited amount of work has been done in the use of QDs for quantifying biomarkers in medical diagnostics. Most QD based assays employ optical detection techniques, but few researchers focus on developing QD-based electrochemical sensors. Ho and coworkers developed sandwich immunoassay to capture carcinoembryonic antigen (CEA) between anti-CEA antibodies and alpha-CEA antibodies-CdS quantum dots. Anti-CEA antibodies were functionalized on carbon nanoparticle/ poly(ethylene imine)—modified screen printed graphite electrodes. The immobilized QDs were released by acid from the sandwich complex and square wave anodic stripping voltammetry was used to amplify the signal response obtained from the dissolved CdS QDs. The detection limit of the sensor was 32 pg/ml with a linear detection range of 0.032-10 ng/ml of CEA.19 In place of solid surface for the capture of analyte, magnetic particles were used to immobilize organophosphorylated acetylcholinesterase (OP-AChE) along with anti-phosposerine conjugated QDs (CdS ZnS).20 Following incubation, excess QDs were removed by washing and then square wave voltammetry was used to quantify the amount of Cd released from QDs. The detailed schematic of the methodology is shown in Figure 18.5. This magnetic immunoassay electrochemical assay was able to detect OP-AChE over a broad concentration range of 0.3-300 ng/ml in human plasma with a detection limit of 0.15 ng/ml.

FIGURE 18.5 Schematic illustration of magnetic electrochemical immunoassays of OP-AChE:A. Plasma samples, B. Magnetic capture of OP-AChE using amorphous MP-Ab I conjugates, C. Selective recognition of bound OP-AChE using QD-Ab2 labels, D. Electrochemical SWV analysis of cadmium released by acid from the captured QDs, and E. Representative SWV signal output. (Image reproduced with permission from reference 20.)

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Finally, more efforts are required to apply quantum based optical detection for the quantification of biomarkers in medical diagnostics. Additionally, in order to broaden the use of quantum dots, it can be combined with other assays, such as liposomes, and doped nanoparticles.

D y e - D o p e d Nanoparticles-Based Sensors Numerous types of luminescent probes are used in cell biology. These include quantum dots (QDs), fluorescent polymer particles and dye-doped nanoparticles (also called FloDots). While QDs have advantages compared to organic dyes in imaging and detection of biomolecules because of brightness, resistance to photobleaching and fluorescent linewidth, they display poor solubility, agglutination, blinking, and low quantum yield. Similarly, fluorescent particles, such as dye containing polystyrene particles and fluorescent polymethacrylic nanoparticles, used in biological applications2122 also exhibit some shortcomings. Owing to limited agglomeration, swelling and dye leaking, these polymer particles are not highly suitable for bioanalysis. FloDots as coined by the researchers are superior to some of the contemporary luminescent probes and have been studied extensively by Tan group at the University of Florida. These particles are dye-doped silica nanoparticles, which consist of a large number of luminescent organic or inorganic dye molecules dispersed inside the silica matrix. It has been shown that at optimal excitation and emission wavelengths, the luminescence intensity of a single 70-nm Rubpy FloDot is equivalent to that of many quantum dots or thousands of dye molecules. They are highly photostable because of the shielding effect of silica protecting dye-doped molecules from environmental oxygen. Additionally, silica is a desirable matrix for its role in the dispersion of particles in water and can be functionalized with a variety of functional groups suitable to conjugate FloDots with biotin, antibodies, nucleic acid, and enzymes. Wu and coworkers23 reported a tris (2,2'-bipyridyl) ruthenium (II) chloride hexahydrate (Rubpy) dye encapsulated silica nanoparticles based immunoassays for the detection of inflammatory biomarker IL-6. In this microarray approach primary anti-IL-6 antibodies were printed on an amino-functionalized slide followed by incubation with IL-6 and dye-doped silica particles functionalized with secondary anti-IL-6 antibodies. Following washing, quantitative analysis of the fluorescent images was performed by Scan Array Express HT microarray scanner. The detection limit was linear over a range of 0.1 to 10 ng/ml of IL-6 with a detection limit of 0.1 ng/ml.23 The schematic of the methodology and the measurement curves are shown in Figure 18.6. The Wiesner group24,25at Cornell University is also making monodisperse fluorescent core-shell silica nanoparticles (C dots) with enhanced brightness and photostability as compared to parent free dye for the development of molecularly targeted probes that exhibit low toxicity, high biostability, biocompatibility, and efficient clearance profiles through biological barriers in the body. They have been used for the in-vivo specific target of tumor and treatment,24-25 but have great potential to develop some quantitative detection of biomarkers similar to FloDots.


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FIGURE 18.6 I -A. Schematic illustration of the procedure forthe preparation of antibody conjugated Rubpy doped silica (RuDS) nanoparticles. l-B.The scheme of the RuDS label-based fluorescent immunoassay of IL-6 on a protein microarray format, (a) Capture anti-IL-6 antibody was printed on the slide, (b) Antigen, IL-6, was attached to the slide via antibody/antigen recognition, (c) Anti-IL-6 antibody-RuDS conjugates were coated on the slide to form a sandwich immunocomplex with RuDS as tags. 2-A. Fluorescence images of protein microarray with different concentrations of antigen, IL-6 (control, 0.1, 1, 10, 30, 60, 100 ng mL-1). 2-B. Calibration curve of fluorescence intensity versus IL-6 concentration. (Image reproduced with permission from reference 23.) (See color insert for a full color version of this figure.)

Surface Enhanced Raman Spectroscopy-Based Sensors Surface enhanced Raman spectroscopy (SERS) is an enhancement of Raman scattering by molecules absorbed on rough metal surfaces. The collective resonant excitation (surface plasmon excitation) of free electrons in metal nanostructures can enhance electromagnetic fields near the particle surface by many orders of magnitude.26 In short range plasmonic interactions, the field

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may be enhanced by a factor of 1014-1015 and is useful in identifying or detecting single molecule under ambient conditions.27-29 In a typical format of a SERS-based sensor, an oligonucleotide probe with a Raman dye is conjugated on gold nanoparticles while another probe is conjugated on the chip. The target oligonucleotide sequence is sandwiched between the chip and nanoparticles. Raman spectroscopic finger print reading is taken after impregnating gold with silver metal. Silver impregnation is known to enhance the signal30 however this is not always required. The number of commercially available Raman dyes makes it possible to identify or detect multiple oligonucleotide probes in a single test. Cao and coworkers, reported the detection of six nucleotide sequences for hepatitis A virus vall7 polyprotein gene, hepatitis B virus surface antigen gene, human immunodeficiency virus, ebola virus, variola virus (smallpox), and bacillus anthracis protective antigen gene using six Raman dyes (Cy3, TAMRA, Texas-red, Cy3.5, rhodamine 6G, and Cy5). In this work, nanoparticles functionalized with oligonucleotides and Raman labels were used to perform multiplex detection of oligonucleotide targets (Figure 18.7a). The unoptimized detection limit of the system was 20 fM.30 In short, six types of Raman dye-labeled and oligonucleotide-modified gold nanoparticles (diameter 13 nm) were prepared with sequences that were complementary to 30-36 bases oligonucleotide sequence for the target analyte. Initially, a chip spotted with 15 mer oligonuleotide sequence probes was used to hybridize the target sequence. Following this, excess unhybridized target sequences were washed off and the overhang of the target sequences were hybridized with Raman active oligonucleotide modified gold particles. Silver enhancement around gold particles was used before measuring Raman spectrum. The Raman spectra of six dye-labeled nanoparticle probes after silver enhancing on a chip is shown in Figure 18.7b. Although SERS is commonly used for short range plasmonic coupling interactions, it is also used for long-range plasmonic coupling as it allows the detection of proteins, clustered receptors on cell membranes, and intact viruses based on the coupling of adjacent metallic NPs in a no-wash/single step format. Qian and coworkers used gold nanoparticles modified with malachite green and thiolated nucleotide probes to prepare SERS NP beacons.26 These beacons were turned on and off by biomolecular binding and dissociation events. Figure 18.8 shows the design and preparation of SERS NP beacons by using monodispersed colloidal Au in two sizes (40 and 60 nm) and their operating principles. The NPs were first encoded with a reporter molecule such as malachite green (with distinct Raman signatures) and then functionalized with thiolated DNA probes. Long range plasmonic coupling was formed between two or more gold particles conjugated with complementary nucleotide sequence (direct sandwich assay) or a target nucleotide sequence sandwiched between two or more gold nanoparticles conjugated with the complementary oligonucleotide sequence for the target nucleotide (indirect sandwich assay). The resulting NP aggregates caused long-range plasmonic coupling interactions and enhanced the Raman signals. The direct sandwich formed between 60 nm SERS beacons and NP aggregates caused a SERS contrast ratio of 40-50 (calculated by


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FIGURE 18.7 A. Schematic illustration of nanopartides functionalized with oligonucleotides and Raman labels, coupled with surface-enhanced Raman scattering (SERS) spectroscopy to perform multiplexed detection of oligonucleotide targets, B.The Raman spectra of six dye-labeled nanopartides probes after Ag enhancing on a chip (after background subtraction). Each dye correlates with a different color in our labeling scheme (see rectangular boxes).TAMRA,tetramethyl rhodamine, and C. Six D N A sandwich assays with corresponding target analysis systems. AlO is an oligonucleotide tether with 10 adenosine units. (Image reproduced with permission from reference 30.)

using the areas of Raman peaks before and after hybridization). For indirect sandwich assay, the target sequence from cDNA of a cancer biomarker CD97 was sandwiched between two probes on different NPs. The SERS NP beacons aggregates showed excellent sequence specificity and were able to discriminate single-base mismatches with an improved on/off intensity ratio of 200-300. Nanoparticles-based SERS detection offers several advantages when compared to fluorescence based chip detection. The Raman signal can be extracted by excitation with single-laser excitation (also common with quan-

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FIGURE 18.8 Schematic diagrams showing the design and operating principles ofSERS NP beacons. A. Colloidal Au nanocrystals are encoded with a Raman reporter molecule, functionalized with thiolated D N A probes, and are stabilized and protected with low MWPEGs. B. Long-range plasmonic coupling induced by direct binding between two D N A sequences. C. Long-range plasmonic coupling induced by one target molecule binding to two NPs. (Image reproduced with permission from reference 26.)

turn dots). Secondly, the number of available Raman dyes are much more than available fluorescent dyes.31 Newer Raman dyes can be easily designed by chemically modifying two similar dyes.32 Therefore, nanoparticle-based SERS method offers potentially greater flexibility, a larger pool of available and non-overlapping labels, and higher multiplexing capabilities than conventional fluorescence-based detection approaches.30

Dynamic Light Scattering Gold and silver nanoparticles, including spherical particles, nanorods, and nanoshells within the size range of tens of nanometers to hundreds of nanometers are known to have a large absorption and scattering cross section in the surface plasmon resonance wavelength regions.33"35 The magnitude of light scattering from gold particles can be higher than light emission from fluorescent dyes.36 This unique property of metal nanoparticles has enabled a wide range of applications in the biomedical field. These include molecular


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and cell imaging, biosensing, and photothermal therapy.37-39 Dynamic light scattering (DLS), also known as photon correlation spectroscopy or quasielastic light scattering, is a technique conventionally used to determine the size distribution profile of small particles in a solution. Because of the established protocols of gold conjugation chemistry and biomolecular interactiondirected nanoparticle assemblies, the DLS technique can be directly applied to quantitative immunoassays. The capabilities of DLS to differentiate between free nanoparticles vs. nanoparticle dimers and clusters (due to nanoparticleanalyte conjugation) based on size difference make them a potential analytical tool for quantitative immunoassay. Liu and coworkers employed nanosized gold nanoparticles (diameter 37 nm) and nanorods (10 nm x 40 nm) for the quantitative detection of free prostate specific antigen (PSA).36 A pair of anti-PSA antibodies (capture and detection antibodies) was conjugated on nanoparticles, and mixed with f-PSA in the solution. The binding of f-PSA caused nanoparticles to form dimers, oligomers, or aggregates depending on the concentration of the antigen. Through DLS anlaysis, the relative ratio of nanoparticles dimers, oligomers, or aggregates vs. individual nanoparticles was measured. In principle, this ratio should increase with an increase in the concentration of antigen and such correlation would measure the concentration of f-PSA in the solution. In this study, the results showed the correlation between the scattering light intensity of gold nanoparticles and nanorods and the concentration of f-PSA in the picomolar range. A detection limit of 0.02 pM for gold particles and 0.4 pM for gold nanorods was established. Figure 18.9 shows the schematic of the immunoassay involving gold nanoparticles and nanorods and detection curve for the PSA. Scattering properties of gold nanoparticles have often been used in microscopic imaging for qualitative evaluation, but not as frequently in quantitative analysis. Other areas where DLS has been successfully applied include analyzing size and size distribution polymers, proteins, and nanoparticles. In some cases DLS has shown to be useful in monitoring the concentration of gold nanoshell in blood samples after intravenously injecting nanoparticles in a murine tumor model (Xie, et al., 2007).

FIGURE 18.9 A. Schematic of the formation of aggregates of gold nanoparticles, nanorods in an immunoassay. B.TEM micrographs of (a) gold nanoparticles (scale bar: 50 nm), (b) gold nanorods (scale bar: 60 nm),and (c) their dynamic light scattering intensities and linear regression curves for the detection of PSA. (Image reproduced with permission from reference 36.)

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MECHANICAL

DETECTION

Nanomechanical Cantilever-Based Sensors Detection of extremely small forces using micro- and nanoelectromechanical systems (MEMS and NEMS) is well established. The adsorption of molecules on the surface resulting in nanomechanical forces offers an exciting opportunity for the development of highly sensitive and miniaturized label-free biological sensors.41-42 In the past MEMS and NEMS have been developed as sensitive chemical and biological sensors capable of detecting small amounts of analytes,43 as light as 7 zeptogram (1 zg = 1021 g) in vacuum.44 In general, sensors built with this technology are operated in either static or dynamic sensing mode. In static sensing mode, a micro-sized cantilever undergoes bending due to surface stresses created by molecular adsorption confined to one side of the cantilever. The surface stress change can be read in the form of nanometer displacement. Dynamic mode sensors are excited at natural resonant frequencies, and shifts in resonant frequency as a result of analyte binding signify detection. The adsorption-induced bending and frequency variations can be measured by using several techniques such as variations in optical beam deflections, piezoresistivity, piezoelectricity, capacitance, and using optically interferometric techniques. Static deflection-based sensors are suitable for in-situ detection of analytes, while in most cases, dynamic sensors require measurements performed in air or vacuum to improve sensitivity, which is strongly limited by viscous damping effects in fluids. However, there has been some concern that the drying process and transport to and from solution may result in increased noise and non-specific binding. Recently, Burg and co-workers demonstrated a novel approach for operating dynamic resonators in the solution while maintaining the advantage of high quality factor by working in vacuum.45 They designed a suspended cantilever with a built-in microfluidic channel used to flow solutions. The measurement was done in the vacuum while the solution was flowing through the microchannels. The effects of stiffness and thickness on resonant frequency have been experimentally demonstrated and analyzed in recent works .45~48 The location of the bound analyte on the sensor can also determine to what extent changes in mass or stiffness affect the resonant frequency. Tamayo and co-workers have shown that bacteria adsorbed at the free end of a cantilever, where the motion is maximum, results in negative frequency shifts due to mass related effects while adsorption near the clamped end, where the motion is minimum, gives way to positive frequency shifts due to increased stiffness.46 Resonant sensors for mercury vapors have also shown positive and negative shifts, depending on how the mercury is adsorbed on the sensor.49 For cantilevers entirely coated in gold, the frequency increases, however, if gold is coated only at the tip of the resonator, the same mercury vapor will decrease the frequency. With proper experimental design, one or more of these effects can be neglected, thus facilitating data analysis and interpretation of results.

N A N O S C A L E T E C H N I Q U E S FOR BIOMARKER Q U A N T I F I C A T I O N

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Although cantilever-based sensors are extremely sensitive, they offer no intrinsic selectivity for biomolecules. Selectivity is achieved by affinity binding of biomolecules on the surface of cantilevers functionalized with selfassembled monolayers, nucleic acids, antibodies, or peptides. While the absolute mass sensitivity is an advantage point for resonant sensors, sensitivity to a particular concentration of analyte in a biologically relevant medium can be more important for early detection of disease or trace constituent analysis. For low analyte concentrations, static deflection-based sensors are limited by the need for a minimum surface coverage required to bend the cantilever by a measurable distance.50 On the other hand, dynamic sensors are limited by the total amount of mass bound to the devices.51 One method which may overcome some limitations of resonant sensors is secondary mass labeling for signal amplification. If additional mass is added to only those devices with target analytes, then the frequency shift will be enhanced and the detection limit improved.52-53 Gerber group demonstrated the first nanomechanical cantilever arraybased sensor for multiple differential gene expression without the use of sample amplifications or labels. The cantilever was able to detect specific transcripts without employing amplification steps in total RNA derived from human or rat cell lines. The markers that are upregulated to a high expression level upon drug exposure were detected and the cantilever array sensors were used as a tool for the fast detection of expression of significant marker genes in the field of personalized medical diagnostics. Specific target hybridization events were monitored by cantilever bending as a result of changes in surface stress. Figure 18.10 shows the working principle of a cantilever-based detection system. The detection limit of the device is -10 pM and is directly comparable with conventional gene-chip technologies where the detection limit is 1-6 pM, applying fluorescent probes.54 Other label-free techniques such as SPR55 are not capable of detecting as small as 12 mers nucleic acid sequences. Nanowire sensors56 were shown to be very sensitive with detection limits of fM range, however detection was performed in a non-competitive environment.

FIGURE 18.10 A. Schematic of the working principle of the static mode of cantilever B. Biofunctionalized cantilever array with different types of thiol-functionalized ssDNA using microcapillaries. (Image reproduced with permission from reference 54.)

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Craighead group at Cornell University used arrays of cantilever for the detection of prostate specific antigen (PSA), a protein biomarker associated with prostate cancer.57 Cantilevers in the form of trampolines (diameter 6 urn and thickness 90 nm) supported with four arms were used for the capture of PSA alone with anti-PSA antibodies on the device, or sandwich capture of the PSA between anti-PSA capture antibodies in the device and anti-PSA antibodies for the capture of nanoparticles as mass labels. PSA alone was detected with a detection limit of 50 ng/ml, while with mass amplification the detection limit was improved by six orders of magnitude to 50 fg/ml. A different shape of the cantilever, paddle lever, was used for the detection of prion protein, responsible for mad cow disease. The working principle and assay format was the same as discussed above. With mass amplification the detection limit was 2 ng/ml of prion protein in pure buffer and 200 pg/ml in blood serum.53, "Different shapes of the cantilevers were used in order to maximize the frequency change for the small change in mass on the cantilever. A comparative study of experimental results and modelling has been presented for different shapes and sizes of cantilever for the point mass detection.59 Several challenges must be overcome before cantilever array sensors can be widely used. Advances in developing superior bio-affinity molecules and

FIGURE 18.1 I SEM of A.An array oftrampolines, B.Trampoline resonator with nanoparticles mass tag for the detection of 50 ng/ml of PSA, C.Trampoline resonator for the control sample with no PSA, and D. Calibration curve for the detection of PSA. Scale bars in SEM are 6 pm.


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improved functionalization techniques are required in order to enhance analyte binding, thereby improving detection limits. Advances in nanofabrication and nanoscale motion detection techniques and miniaturization of optical detection systems are crucial for the viability of nanomechanical detection platforms. While technology for designing electronic chips has progressed, the integration of electronic, mechanical, and fluidic design still requires much work before integrated devices can be used as a viable sensing platform.

ELECTRICAL

DETECTION

Field Effect Transistor-Based Sensors A diversity of sensor architectures such as cantilevers, quantum dots-based fluorescent detectors, giant magneto-resistance, and others have been designed and fabricated at nanoscale dimensions. Most of them require integration of optics, and/or some labels to translate the surface-binding event into a readable signal. In contrast, sensors designed to operate likefieldeffect transistors (FET) are label-free detection techniques as they do not require any labels and are capable of translating analyte-binding directly into a readable signal, without using elaborate optical components. These devices utilize the electronic properties of the sensing element, such as conductance, to produce signal output.60 The use of ID nanomaterials such as nanowires (NWs) and nanotube(NTs) into electrical devices offers substantial advantages for biological detection. The diameter of NWs and NTs is comparable to the size of biological entities, and they are several micrometers long, providing a size compatibility between electrical components and biological analyte, as well as convenient interface with micrometer scale device components. The presence of surface charge on the surface of biological analyte or the charge transfer during a biological process could be directly detected by electronic nanocircuits based on NWs and NTs.61 Therefore, electronic devices based on NWs and NTs can serve as one of the most efficient strategies for the integration of biology and electronics into a common platform in biological sensing and detection.62 A typical structure of an FET sensor is illustrated in Figure 18.12. All FET sensors have gate, drain, and source terminals and there is a slight variation in the working of FET based on NWs and NTs depending on their physical and material properties. In the case of single walled carbon NTs, every atom is on the surface and exposed to the environment, and thus even a small change in the environment can cause drastic changes to the electrical properties of NTs. NWs are equally sensitive due to high surface to volume ratio as all electrical current flows through the nanometer-scale cross section. In principle, the devices based on NTs and NWs should have a detection limit at a single particle level. Lieber group demonstrated the use of silicon NWs for the detection of virus and was able to improve the sensitivity to a single virus.63 The signal transduction in NWs is caused by the depletion or accumulation of charge carriers as a result of charged biomolecules that are bound at the surface. There are two types of charge carriers in the NWs- holes for a p-type

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FIGURE 18.12 Structure of an FET nanobiosensor (a) Cross-sectional view: source and drain electrodes bridge the semiconductor channel. The gate electrode can be used to modulate the conductivity of the semiconductor channel. A receptor molecule attached to the surface of the semiconductor material can specifically recognize and capture a target molecule from a buffer solution. (b)Top view: SEM image of a typical nano-FET In these structures, the channel length is the S-D distance and the channel width is the S or D electrode width. Examples of nano-FET fabricated using either (c) carbon nanotubes or (d) indium oxide NWs as semiconductor materials. (Image reproduced with permission from reference 60.) (See color insert for a full color version of this figure.)

semiconductors or electrons for n-type semiconductor. The carrier density in the NWs is proportional to the conductance of the wire, which can be determined from the source-drain current of the device. Depending on the charge of the anlayte molecules the charge carriers will accumulate or deplete, causing a respective increase or decrease in the conductivity of the device. When a negatively (such as DNA) or positively charged molecule (such as protein below its isoelectric point) binds to the p-type NWs, it causes an increase or decrease in the conductivity of the NWs, respectively.61 The mechanism leading to signal transduction in single wall nanotube (SWNT) biosensors has, until recently, been poorly understood. For example, some researchers have reported that SWNT will show an increase in conductivity for every protein tested, independent of the overall charge on the protein.64 This contradicts the observation made in the case of NWs65,66 and cannot be explained as an electrostatic gating effect caused by the charged analyte perturbing the charge carries in the NTs. In order to resolve this anomaly, it was suggested that the dominant sensing mechanism is a modulation of the Schottky barrier at the electrodes-nanotube


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interface caused by binding of analyte and receptor.64 However, Heller and coworkers believed that electrostatic gating can also play an important role depending on the nature of the analyte.67 Lieber group demonstrated the capabilities of silicon nanowires-based FET sensors for multiplex detection of PSA, carcinoembryonic antigen (CEA), and Mucin-1. They used a bottom-up fabrication technique to fabricate p- and n-type doped silicon NWs. A linker molecule, aldehyde propyltrimethoxysilane (APTMS), was used to functionalize anti-PSA, anti-CEA, and anti-Mucin-1 antibodies. The detection limit for PSA, CEA, and Mucin-1 was 2 fM, 0.55 fM, and 0.49 fM, respectively.68 Some other work performed by Zhou group demonstrated the use of n-type ln 2 0 3 NWs and p-type CNTs in detecting PSA. They claimed to have developed a novel approach to link anti-PSA antibodies to ln 2 0 3 NWs via onsite synthesis of a succinimidyl linking molecule. The system detected a minimum of 5 ng/ml of PSA under unoptimized conditions.69 Both NTs and NWs are extensively used to detect protein,66' 70~72 nucleic acids,73-75 and cancer biomarkers.68

Liposomes-Based Sensors Liposomes are versatile structures used for labeling, drug delivery, and other therapeutic applications. Structurally liposomes are the vesicles whose membranes are made of phospholipids with hydrophobic chains forming the bilayer. The polar head groups of the lipids are oriented toward the extravesicular solution and inner cavity (Figure 18.13). Liposomes offer large surface area, large entrapment volume, and lipid bilayer can be conjugated with a variety of biorecognition elements. A wide variety of hydrophilic molecules can be encapsulated within the inner cavity of liposomes, including enzymes, DNA, vaccines, fluorescent dyes, electrochemical and chemiluminescent markers, and pharmaceutical compounds.76 Liposomes provide wider use in encapsulating drug molecules inside vesicles and also control the release of the drugs to minimize toxic effects of drugs and maximize their therapeutic

FIGURE 18.13 Structure of a liposome modified with biorecognition elements. Lipids form a bilayer entrapping an aqueous core entrapping highly water-soluble marker molecules. (Image reproduced with permission from reference 76.)

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index. This chapter will limit its discussion to the applications of Hposomes in numerous detection techniques. There are a number of different lipids that can be used in the lipid bilayer and can be easily modified to attach numerous biorecognition elements. Typically, modified lipids include phosphatidylethanolamine (PE)77 (amine modified), N-glutaryl-PE (carboxyl modified),78-80 maleimidomethyl cyclohexane-carboxamide (MCC)-PE or maleimidophenyl butyramide (MPB)-PE (maleimide modified),81"83 pyridyldithio propionate (PDP)-PE (disulfide modified),84 and cholesterol,85 or polyethylene glycol86 (hydroxyl modified). The modified lipid membrane is conjugated to biorecognition elements covalently using commonly used heterobifunctional and homobifunctional cross linkers, or with non-covalent interactions such as those provided by the biotin-streptavidin interaction, or protein A/G mediated protein association. Because Hposomes can entrap various hydrophilic molecules, they can be suitably modified as a signal enhancer for optical and electrochemical detection techniques. The detection based on lipososomes is performed by either keeping Hposomes intact during signal generation or lysing them before signal readout. The intact form of liposome is generally used in optical detection while the lysed form is used for the optical as well as electrochemical detection. Electrochemical detection systems based on Hposomes are increasingly getting attention from the researchers due to their high sensitivities, which at times are better than the sensitivities of the compared optical detection systems. Therefore, we have decided to discuss Hposomes based sensors under this section although they are equally used with optical detection systems. Baeumner group developed an electrochemical biosensor based on a PMMA substrate with interdigitated electrodes and microfluidic channels.87 The hsp70 mRNA was isolated from Cryptosporidium parvum and amplified using nucleic acid sequence-based amplification (NASBA). The amplified target sequences were detected by a sandwich hybridization between capture probes on superparamagnetic beads and reporter probes on tagged Hposomes. The electrochemical marker potassium ferro/ferrihexacyanide present inside the liposome was lysed for the amperometric quantification of the target DNA sequence. Amplified mRNA from only 1 oocytes was detectable with the electrochemical biosensors based on tagged Hposomes. Figure 18.14 shows the SEM of the gold interdigitated electrodes, micrograph of an embossed channel on PMMA with gold electrodes, and the microfluidic chip with channels and interconnects. An optical biosensor was developed using a membrane-based lateral flow system for the detection of 10 viable Mycobaterium avium subsp. paratuberculosis (MAP).88 The assay was based on the extraction of RNA from MAP and amplified using reverse transcriptase PCR. A nucleic acid hybridization sandwich format was used for the capture of target DNA sequence between capture DNA probes on the membrane surface and reporter DNA probes on the Hposomes encapsulating sulforhodamine B dye. The presence of dye inside Hposomes was quantified using a hand-held reflectometer or a fluorescence reader. This format has been used for the detection of astrovirus,89 Bacillus anthracis,90 Dengue virus,9192 and E. coli.93


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FIGURE 18.14 A. SEM of gold interdigitated microelectrodes formed on a PMMA substrate. B. A PMMA sheet containing a hot embossed channel was then bonded to the PMMA containing the electrodes. The finished device contained a 500pm channel positioned along the electrodes. C. The chip with channels and interconnects. (Image reproduced with permission from reference 87.)

In the past, liposomes have been encapsulated with several electrochemical markers such as potassium ferrocyanide used for the assessment of poreforming toxin,94 horseradish peroxidase used for the detection of theophylline,95 and ascorbic acid used for the detection of atrazine.96 The use of liposomes as signal enhancing labels in detection techniques has proven to improve the sensitivity,77,97 detection limit,98 and in most cases their use has reduced the total detection time99 as compared to conventional biosensing techniques. Researchers have found one to three orders of magnitude improvement in the sensitivity and detection limit when tagged liposomes are compared with antibody tagged fluorophores,100 analyte tagged fluorophores,10' HRP tagged

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BIOMARKERS

antibodies,97 and biotin tagged enzymes.77 However, in most cases the enhancement was not proportional to the number of molecules entrapped inside liposomes. This has been attributed to the steric hinderance and multivalency of liposomes. The relatively large size of liposomes prevents binding of multiple liposomes with the adjacent antigens.76-98,102 Moreover, liposomes have multiple biorecognition elements on their surface, which prevents them from binding with one to one molar ratio of the antigen, possible with the smaller labels.7698 Although liposomes have been used for a variety of applications due to their flexibility in encapsulating numerous labels, the search for newer labels and using them with other formats, such as bio-barcode assays, FET, magnetic, and others will further broaden their use.

MAGNETIC

DETECTION

Giant Magnetoresistance-Based Sensors Giant magnetoresistive (GMR) is a spin-dependent transport effect observed in thin film structures composed of alternating ferromagnetic and non-magnetic layers. The term "giant" refers to the large change in electrical resistance in a magnetic field. In the absence of an external magnetic field, the direction of magnetization of adjacent ferromagnetic layers is antiparallel due to weak anti-ferromagnetic coupling between layers. Current is sent through the device and the first magnetic layer spin polarizes the current. Since the layers are antiparallel there will be a high degree of spin scattering of the electron current resulting in high resistance. On the contrary, when an external magnetic field is applied, the magnetization of the adjacent ferromagnetic layers is parallel resulting in lower spin scattering and resistance. More recently, the GMR effect, which is widely used in the read heads of modern hard disk drives, is used in numerous biodetection techniques based on the labeling of protein and nucleic acids with magnetic tags. GMR was discovered in 1988 by a research team led by Peter Griinberg of the Jiilich Research Centre. It was also simultaneously but independently discovered by the Albert Fert group at the University of Paris-Sud (FR). For this discovery, Griinberg and Fert won numerous prestigious awards including the 2007 Nobel Prize in Physics. Two variations of GMR, namely, spin valve (SV) and magnetic tunnel junction (MTJ), have been used for various readout mechanisms and sensing arrays. In SV configuration, a conductor is used between magnetic layers while an insulator layer is used in MTJ. Neither of them has shown an absolute superiority over the other.103 However, SV biosensors are easy to fabricate and they are in an advanced stage of development, while MTJs have a high magnetoresistance ratio (MR) resulting in a potentially high single to noise ratio. The signal generation on GMR sensors is due to the attachment of magnetic tags to the protein or nucleic acids in close proximity to the sensor surface. The size of the magnetic tag ranges from as small as tens of nm to as large as 3 um. This includes paramagnetic polystyrene beads and similar sized paramagnetic particles. The larger tags mismatch in size with nucleic acids or


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protein targets in biological assays, thus prejudicing the quantitative capabilities of the system. The use of magnetic nanoparticle tags (also called nanotags) in the diameter of 100-1000 A, which are comparable to the size of the target biomolecules to be assayed, are expected to enhance the performance in real biological assays. Typically, this type of measurement is performed in the form of an array of devices on a microchip and is also categorized as magnetic microarray. The basic methodology of such a magnetic microarray in the case of nucleic acid detection is shown in Figure 18.15. In short, (a) SV and MTJ sensors are bound with known DNA probes which are complementary to the target DNA fragments, (b) Unknown DNA fragments are labeled with magnetic nanotags using binding mechanisms as biotin and streptavidin, and

FIGURE 18.15 Principle of magnetic DNA microarray with direct labeling of target nucleic acid with the magnetic nanotags I. Schematic illustrations of a. the top view of a MagArray SV sensor and b. its cross section. A single magnetic nanoparticle label is shown bound to the sensor through hybridized probe and target DNAs in the biologically active area. The aluminum leads define the electrically active area where an electrical sense current passes in the SV. (Image reproduced with permission from reference 103.)

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(c) Tagged DNA fragments are selectively captured by binding to the surface of the sensor by DNA hybridization mechanism.103 Eventually, the presence of magnetic nanotags is detected by measuring the change in the resistance before and after attaching magnetic nanotags, and by applying voltage or current across the device. Osterfeld and coworkers designed a magnetic nanotag-based protein assay chip consisting of 64 sensors in an 8 x 8 array. Each sensor has an active area of 90 x 90 (am2 consisting of 32 linear GMR segments, each 1.5 um wide connected in series.104 This SV stack of materials Ta 3/Seed layer 4/PtMn 15/ CoFe 2/Ru 0.85/CoFe 2/Cu 2.3/CoFe 2/Cu 1/Ta 4 (all thicknesses in nm) were deposited on an Si/SiO2 substrate (Figure 18.16). The chip was manually as well as robotically spotted with capture antibodies while the control experiment was performed using spotted BSA. This was followed by incubation of samples with single or multiple analytes. Following the capture of target analyte by respective capture antibodies, biotinylated antibodies specific to target analyte were incubated and used to attach magnetic nanotags. Seven analytes (TNF-a, IL-la, G-CSF, lactoferrin, CEA, eotaxin, and IFN-7) present in the same sample were simultaneously detected at 1 pg/ml level. In order to improve the detection limit, the signal was amplified by adding more nanotags. By doing so, the detection limit was as small as 57 fM, 56 fM, 53 fM, 13 fM, 5 fM, 119 fM, 59 fM of TNF-a, IL-la, G-CSF, lactoferrin, CEA, eotaxin,

FIGURE 18.16 Magnetic nanotag-based protein assay chip. The chip has a 200 pi reaction well and is supported by an 84-pin ceramic base a. Embedded in the bottom of the reaction well are 64 sensors in an 8 x 8 array, b. Each sensor has an active area of roughly 90 x 90 um2 and consists of 32 linear giant magnetoresistive (GMR) segments, each 1.5 um wide, which are connected in series. c.The edge of one such sensor segment and bound nanotags are imaged with a scanning electron microscope (d). (Image reproduced with permission from reference 104.) (See color insert for a full color version of this figure.)


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FIGURE 18.17 GM- based multiplex protein assay with nanotag amplification. Seven different capture antibodies were used to functionalize different regions of a chip to detect seven different protein analytes. A mixed sample containing each of the seven analytes at a concentration of I pg/ml in PBS was incubated on the chip followed by linker incubation and two rounds of nanotag amplifications. The graph shows signal response after second round of nanotag amplification. The numbers above the bars indicate the average signals for each set of sensors. (Image reproduced with permission from reference 106.)

and IFN-7, respectively (Figure 18.17). When compared to other magnetic detection, use of 50 nm diameter nanotag combined with 30 nm passivation layer on SV valve was credited to have improved the detection limit of the current work.104 In another study conducted by Martin and coworkers,105 it was observed that magnetically assisted hybridization improved the detection limit of GMR sensors by three orders of magnitude as compared to diffusion controlled hybridization. In addition, when compared to diffusion kinetics, the packing density of the particles was also improved by field assisted kinetics. The magnetic field assisted separation and concentration of magnetically labeled biomolecules have shown promising results with a detection time of less than 30 min.106,107The ssDNA from the conservative region of 16S rDNA from Escherichia coli was biotinylated at 5' end and was tagged with 250 nm streptavidin coated magnetic nanotag. Measurements were made using an in-plane transverse external excitation field of 1.1 (kA/m)rms (211 Hz) in combination with a DC bias field of 2.4 kA/m to magnetically attract nanotag tagged target ssDNA to the sensor surface functionalized with complementary probe. The presence of magnetically assisted hybridization gave a 25% higher saturation signal and an improved detection limit by three orders of magnitude (from pM to fM) as compared to diffusion assisted hybridization. The capability of GMR sensors in terms of high sensitivity, multiplex detection, ease of use, scalability, and system integration make them ideal for use in portable instruments in medical diagnostics, and point-of-care applicaitons. In the near future, capture agents with higher affinity to the analyte, and analyte-sized nanotags with higher magnetic moment are expected to enhance the analytical sensitivity of nanotag-based GMR sensors.104

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FUTURE TRENDS This chapter was intended to elucidate a variety of ways the nanomaterials, structures, and devices are being utilized for the quantification of biomarkers. Some of the approaches are being used in diagnostic devices or in research while others are still being studied for their full potential. It is likely that continuing technology development and possibilities for further integration of techniques will result in the emergence of new approaches. For example, total internal reflection fluorescence (TIFR) microscopy has been extensively used for understanding the action of enzymes or inhibitory molecules in the progression of disease or biological systems;108-110 visualization of molecular dynamics;111-113 transportation of proteins;"4'115 and binding of molecules to the membrane surface in a complex matrix.116,117 Surprisingly, TIRF has not been used for quantification of biomarkers except very few reports.118, " 9 Single molecule techniques are very sensitive and can be applied for the quantification of biomarkers, but not all of them are suitable for high throughput analysis. For example, trapping of molecules (optically and magnetically), dynamic force spectroscopy, and fluorescence correlation microscopy (FCS) are appropriate for single molecule interrogation but they are not yet high throughput techniques. However, they could become part of future analytical concepts. Additionally, the sorting of molecules could potentially be used for the quantification of biomarkers. In our group, we have extensively studied the analysis of nucleic acids in nanofluidic channels, and these techniques can be used for the quantitative measurement of nucleic acid-based biomarkers.120-123 In this technique, DNA molecules are driven electrophoretically through nanofabricated cavities to confine and dynamically elongate them beyond their equilibrium length for repeated detection via laser-induced fluorescence spectroscopy. This technique would be a significant improvement over measurement techniques in bulk by exposing all biomarkers along the length of nucleic acid which would otherwise not be detected in coiled or aggregated form when measured in the bulk. In a variant of this technique, nanofluidic channels consisting of narrow constrictions and wider regions are used to create size dependent trapping of DNA and as a result separate different sizes of DNA into bands.124-126 Single molecule confocal microscopy-based techniques are used to visualize DNA strands in both of the above mentioned techniques. By confining molecules in the nanoliter volumes, more uniformly illuminated probe volume is created and the molecular resolution is permitted beyond the capabilities of a diffraction limited system.123 Single molecule approaches are also used for the sorting, counting, and color co-incidence measurement of labeled biomolecules as they pass through the optically excitation volume.127131 The exact concentrations of different species can be obtained by counting the number of biomolecules in real-time. The signal-to-noise ratio is improved by reducing the excitation volume in order to reduce the background noise from scattering or intrinsic fluorescence of unlabeled biomolecules in the excitation volume. The optical excitation reduced below the width of the channel makes it possible to detect only one


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molecule at a time. Nanofluidics channels with the width of a few hundred nanometers are fabricated and comparable size of laser excited volume is used for the single molecule detection of labeled biomolecules. The multiple channels in a single chip and use of spectrally distinguishable fluorescent labels could be used for multiplex detection and high throughput real-time quantitative measurement of biomarkers. In place of physically confining sample to a small volume in nanofluidic channels, the optical confinements using zero-mode waveguides are also used for the optically excited and interrogated studies of bound and unbound biomolecules. In this case, subwavelength metallic holes are used for the attenuation of the incident light. With the diameter of holes in few tens of nanometers, this attenuation length becomes less than the film thickness.132-133 The net effect of this is an optical excitation volume which is of the order of zeptoliters (1021), significantly smaller than could be obtained, for example, by total internal reflection illumination, which can confine light to the evanescent field in one direction only. The FCS can be used to determine the concentration of entities by measuring the diffusion constant of freely diffusing fluorescently labeled biomolecules as they pass through nanoholes. A million holes can be fabricated on a single coverslip and that could be used for the highly parallel and high throughput quantitative analytical tool. Not only the flow of molecules on the surface is used for the single molecule analysis, the flow through the nanoscale pores is also pursued to study molecular structures.134-139 In this case, as the molecule passes through the pore, the ion current through the pore is modulated and it can lead to the knowledge of the structures of molecules, such as single strand, double strand DNA, or any other combination. Although, nanopores are simple but powerful biophysical probes of molecular confirmation, some separation and purification steps may be required to make this technique applied for the quantification of biomarkers.

CONCLUSION Nanoscale techniques are providing new opportunities for sensitive bio-chemical detection. Their utility will likely increase as they are incorporated in more integrated and miniaturized systems. The scope of the nanoscale technologies for the biomarker quantification can be improved either by developing highly sensitive detection techniques or making systems with massive parallelization. Nanomaterials are being used for the sensitive and also multiplex detection to some extent, but fabrication techniques will be highly appropriate for the multiplexing or parallelization. In general, fairly simple structures are fabricated to improve the sensitivity of detection techniques for handling small volumes or for the high throughput parallel detection. Microfabrication techniques scaled down to make nano-fabricated structures can be used for the massive parallel replication of identical units with 105,106, or more individual reactions performed on a single unit. It is clear that the scale and levels of integration are compatible with active optical and electronic devices in today's

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integrated electronic and optoelectronic systems, and carrying out millions of parallel optical or electronic measurements is a realistic possibility. In this respect, optical systems are best suited as they do not require direct contact with the analyte necessitating the use of interconnects and wires complicating the integration of a massive number of nanostructures with the external electrical measurement system. Other potential detection systems based on magnetic field either require specially designed structures, such as GMR or electo-magnets, and lend themselves to the same level of complexity as with electrical systems or require a huge mismatch in the size of permanent magnets, rendering them unfit for the highly parallel systems on a small chip. In order to greatly improve disease diagnostics, the advances in detection technologies are as important as the advances in the field of genomics and proteomics. The availability of multiple biomarkers is necessary for the diagnosis of highly complex diseases like cancer, where the heterogeneity of the disease makes a single test inadequate. Detection of multiple biomarkers might provide not only the information required for the robust diagnosis of the disease, but also about the stages of the disease that could facilitate early detection and cure of the disease.

SUMMARY P O I N T S 1.

2.

3.

4.

5.

6.

Nanostructure science and engineering utilizing structures and devices with dimensions typically measured in micrometers and nanometers enables access to new physical length scales and enables new approaches for molecular detection and analysis. Conventionally, nanotechnologies refer to techniques that employ nanomaterials with at least one critical dimension in the range of 1-100 nm. In this chapter we have categorized nanoscale sensing techniques based on the detection technique (optical, electrical, mechanical, and magnetic) used for the system. Shrinking of sample or interrogation volume has enabled the study of an individual molecule, otherwise obscured by ensemble averaging. Reduction in volume is achieved by using nanostructures or by confining samples in the nanofluidic channels. The specificity of the overall detection system is based on attaching bio-recognition elements (antibodies, nucleic acid probes, aptamers, enzymes, and proteins) to nanomaterials or nanostructures, which can bind with target analytes in a complex medium consisting of non-targeted analytes along with other interfering chemical moieties. While technology for designing electronic chips has progressed, the integration of electronic, mechanical, and fluidic design still requires much work before integrated devices can be used as a viable sensing platform. It is likely that continuing technology development and possibilities for further integration of techniques will result in the emergence of new approaches.

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8.

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Single molecule techniques are very sensitive and can be applied for the quantification of biomarkers, but not all of them are suitable for high throughput analysis. However, they could become part of future analytical concepts. In the future, nanofluidics and nanostructure-based single molecule analysis such as stretch of DNA, sorting of molecules, color coincidence measurement, diffusion through nanoholes, and zero mode waveguides would become part of the unltrasensitive and high throughput analytical tools applied for the quantification of biomarkers.

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Stern, E., Klemic, J. E, Routenberg, D. A., Wyrembak, P. N., Turner-Evans, D. B., Hamilton, A. D., Lavan, D. A., Fahmy, T. M , and Reed, M. A. LabelFree Immunodetection with CMOS-Compatible Semiconducting Nanowires. Nature. 2007;445:519-522. Cui, Y., Wei, Q. Q., Park, H. K., and Lieber, C. M. Nanowire Nanosensors for Highly Sensitive and Selective Detection of Biological and Chemical Species. Science. 2001;293:1289-1292. Heller, I., Janssens, A. M., Mannik, J., Minot, E. D., Lemay, S. G., and Dekker, C. Identifying the Mechanism of Biosensing with Carbon Nanotube Transistors. NanoLett. 2008;8:591-595. Zheng, G. R, Patolsky, F, Cui, Y, Wang, W. U., and Lieber, C. M. Multiplexed Electrical Detection of Cancer Markers with Nanowire Sensor Arrays. Nature Biotechnol. 2005;23:1294-1301. Li, C , Curreli, M., Lin, H., Lei, B., Ishikawa, F. N., Datar, R., Cote, R. J., Thompson, M. E., and Zhou, C. W. Complementary Detection of Prostate Specific Antigen Using In(2)0(3) Nanowires and Carbon Nanotubes. J. Am. Chem. Soc. 2005;127:12484-12485. Balavoine, F., Schultz, P., Richard, C , Mallouh, V., Ebbesen, T. W., and Mioskowski, C. Helical Crystallization of Proteins on Carbon Nanotubes: a First Step Towards the Development of New Biosensors. Angew. Chem. Int. Ed. 1999; 38:1912-1915. Bradley, K., Briman, M., Star, A., and Griiner, G. Charge Transfer from Adsorbed Proteins. Nano. Lett. 2004;4:253-256. Boussaad, S., Tao, N. J., Zhang, R., Hopson, T, and Nagahara, L. A. In Situ Detection of Cytochrome C Adsorption with Single Walled Carbon Nanotube Device. Chem. Commun. 2003;1502-1503. Hahm, J. and Lieber, C. M. Direct Ultrasensitive Electrical Detection of DNA and DNA Sequence Variations Using Nanowire Nanosensors. Nano Lett. 2004; 4:51-54. Li, J., Ng, H. T., Cassell, A., Fan, W, Chen, H., Ye, Q., Koehne, J., Han, J., and Meyyappan, M. Carbon Nanotube Nanoelectrode Array for Ultrasensitive DNA Detection. Nano Lett. 2003;3:597-602. Nguyen, C. V., Delzeit, L., Cassell, A. M., Li, J., Han, J., and Meyyappan, M. Preparation of Nucleic Acid Functionalized Carbon Nanotube Arrays. Nano Lett. 2002;2:1079-1081. Edwards, K. A. and Baeumner, A. J. Liposomes in Analyses. Talanta. 2006; 68:1421-1431. Jones, M., Kilpatrick, P., and Carbonell, R. Preparation and Characterization of Bifunctional Unilamellar Vesicles for Enhanced Immunosorbent Assays. Biotechnol. Prog. 1993;9:242-258. Weissig, V, Lasch, J., Klibanov, A., and Torchilin, V. A New Hydrophobic Anchor for the Attachment of Proteins to Liposomal Membranes. FEBS Lett. 1986;202:86-90. Kung, V. and Redemann, C. Synthesis of Carboxyacyl Derivatives of Phosphatidylethanolamine and Use as an Efficient Method for Conjugation of Protein to Liposomes. Biochim. Biophys. Ada. 1986;862:435^139. Maruyama, K., Kennel, S., and Huang, L. Lipid Composition Is Important for Highly Efficient Target Binding and Retention of Immunoliposomes. Proc. Natl. Acad. Sci. USA. 1990;87:5744-5748.

490

BIOMARKERS 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98.

Rule, G., Montagna, R., and Durst, R. Characteristics of DNA-Tagged Liposomes Allowing Their Use in Capillary-Migration, Sandwich-Hybridization Assays. Anal. Biochem. 1997;244:260-269. Bredehorst, R., Ligler, R, Kusterbeck, A., Chang, E., Gaber, B., and Vogelt, C. Effect of Covalent Attachment of Immunoglobulin Fragments on Liposomal Integrity. Biochemistry. 1986;25:5693-5698. Katoh, S., Kishimura, M., and Tomioka, K. Immune Lysis Assay of Antibodies by Use of Antigen-Coupled Liposomes. Colloids Surf. A. 1996;109:195-200. Hansen, C , Yao, G., Moase, E., Zalipsky, S., and Allen, T. Attachment of Antibodies to Sterically Stabilized Liposomes: Evaluation, Comparison and Optimization of Coupling Procedures. Biochim. Biophys. Ada. 1995;1239:133-144. Carroll, T., Davison, A., and Jones, A. Functional Cholesteryl Binding Agents: Synthesis, Characterization, and Evaluation of Antibody Binding to Modified Phospholipid Vesicles. J. Med. Chem. 1986;29:1821-1826. Zalipsky, S., In: Lasic, D., Martin, F. (Eds.). Stealth Liposomes. CRC Press. 1995:93-102. Nugan, S. R., Asiello, P. J., Connelly, J. T., and Baeumner, A. PMMA Biosensors for Nucleic Acids with Integrated Mixer and Electrochemical Detection. Biosens. Bioelectron. 2009;24:2428-2433. Kumanan, V., Nugen, S. R., Baeumner, A., and Chang, Y. A Biosensor Assay for the Detection of Mycobacterium Avium Subsp. Paratuberculosis in Fecal Samples. J. Vet. Sci. 2009;10:35^12. Tai, J., Ewert, M., Belliot, G., Glass, R., and Monroe, S. Development of a Rapid Method Using Nucleic Acid Sequence-Based Amplification for the Detection of Astrovirus. J. Virol. Method. 2003; 110:119-127. Hartley, H. and Baeumner, A. Biosensor for the Specific Detection of a Single Viable B. Anthracis Spore. Anal. Bioanal. Chem. 2003;376:319-327. Baeumner, A., Schlesinger, N., Slutzki, N., Romano, J., Lee, E., and Montagna, R. Biosensor for Dengue Virus Detection: Sensitive, Rapid, and Serotype Specific. Anal. Chem. 2002;74:1442-1448. Zaytseva, N., Montagna, R., Lee, E., and Baeumner, A. Multi-Analyte SingleMembrane Biosensor for the Serotype-Specific Detection of Dengue Virus. Anal. Bioanal. Chem. 2004;380:46-53. Baeumner, A., Cohen, R., Miksic, V, and Min, J. RNA Biosensor for the Rapid Detection of Viable Escherichia Coli in Drinking Water. Biosens. Bioelectron. 2003;18:405-413. Xu, D. and Cheng, Q. Surface-Bound Lipid Vesicles Encapsulating Redox Species for Amperometric Biosensing of Pore-Forming Bacterial Toxins. JACS Commun. 2002;124:14314-14315. Haga, M., Sugawara, S., and Itagaki, H. Drug Sensor: Liposome Immunosensor for Theophylline. Anal. Biochem. 1981;118:286-293. Baeumner, A. and Schmid, R. Development of a New Immunosensor for Pesticide Detection: A Disposable System with Liposome-Enhancement and Amperometric Detection. Biosens. Bioelectron. 1998;13:519-529. Suita, T. and Kamidate, T. Preparation of Antibody-Coupled Liposomes Containing Horseradish Peroxidase as a Marker Molecule. Anal. Sci. 1999;15:349-352. Singh, A., Kilpatrick, P., and Carbonell, R. Application of Antibody Fluorophore-Derivatized Liposomes to Heterogeneous Immunoassays for D-Dimer. Biotechnol. Prog. 1996;12:272-280.

NANOSCALE TECHNIQUES FOR BIOMARKER QUANTIFICATION 99. 100. 101. 102. 103. 104.

105.

106. 107.

108. 109. 110.

111. 112. 113. 114.

491

Jones, M., Kilpatnck, P., and Carbonell, R. Competitive Immunosorbent Assays for Biotin Using Bifunctional Unilamellar Vesicles. Biotechnol. Prog. 1994;10:174-186. Lee, M., Durst, R., and Wong, R. Comparison of Liposome Amplification and Fluorophor Detection in Flow-Injection Immunoanalyses. Anal. Chim. Acta. 1997;354:23-28. Choquette, S., Locascio-Brown, L., and Durst, R. Planar Waveguide Immunosensor with Fluorescent Liposome Amplification. Anal. Chem. 1992;64:55-60. Singh, A., Kilpatrick, P., and Carbonell, R. Noncompetitive Immunoassays Using Bifunmctional Unilamellar Vesicles or Liposomes. Biotechnol. Prog. 1995;11:333-341. Wang, S. X. and Li, G. Advances in Giant Magnetoresistance Biosensors with Magnetic Nanoparticle Tags: Review and Outlook. IEEE Trans. Magnet. 2008;44:1687-1702. Osterfeld, S. J., Yu, H., Gaster, R. S., Caramuta, S., Xu, L., Han, S., Hall, D. A., Wilson, R. J., Sun, S., White, R. L., Davis, R. W., Pourmand, N., and Wang, S. X. Multiple Protein Assays Based on Real-Time Magnetic Nanotag Sensing. Proc. Natl.Aca. Sci. USA. 2008;105:20637-20640. Martin, V. C , Cardoso, F. A., Germano, J., Cardoso, S., Sousa, L., Piedada, M., Feitas, P. P., and Fonseca, L. P. Femtomolar Limit of Detection with a Magnetoresistive Biochip. Biosen. Bioelectron. 2009;Doi:10.1016. J. Bios. 2009.01.040. Ferreira, H. A., Feliciano, N., Graham, D. L., Clarke, L. A., Amaral, M. D., and Freitas, P. P. Rapid DNA Hybridization Based on AC Field Focusing of Magnetically Labeled Target DNA. Appl. Phys. Lett. 2005;87:013901-013903. Graham, D. L., Ferreira, H. A., Feliciano, N., Freitas, P. P., Clarke, L. A., and Amaral, M. D. Magnetic Field-Assisted DNA Hybridisation and Simultaneous Detection Using Micron-Sized Spin-Valve Sensors and Magnetic Nanoparticles. Sens. Actuators B. 2005;107:936-944. Hodgkinson, G. N., Tresco, P. A., and Hlady, V. The Influence of Sub-Micron Inhibitory Clusters on Growth Cone Substratum Attachments and CD44 Expression. Biomaterials. 2008;29:4227^1235. Schmidt, P. M., Lehmann, C , Matthes, E., and Bier, F. F. Detection of Activity of Telomerase in Tumor Cells Using Fiber Optical Biosensors. Biosen. Bioelectron. 2002;17:1081-1087. Morin, N. A., Oakes, P. W, Hyun, Y. M., Lee, D., Chin, Y E., King, M. R., Springer, T. A., Shimaoka, M., Tang, J. X., Reichner, J. S., and Kim, M. Nonmuscle Myosin Heavy Chain IIA Mediates Integrin LFA-1 De-Adhesion During T Lymphocyte Migration. J. Exp. Med. 2008;205:195-205. Fu, G., Wang, C , Liu, L., Wang, G. Y, Chen, Y. Z., and Xu, Z. Z. Heterodimerization of Integrin Mac-1 Subunits Studied by Single Molecule Imaging. Biochem. Biophys. Res. Commun. 2008;368:882-886. Sohn, H. W., Pierce, S. K., and Tzeng, S. J. Live Cell Imaging Reveals That the Inhibitory Fcgammariib Destabilizes B Cell Receptor Membrane-Lipid Interactions and Blocks Immune Synapse Formation. J. Immunol. 2008;180:793-799. Keller, P., Toomre, D., Diaz, E., White, J., and Simons, K. Multicolour Imaging of Post-Golgi Sorting and Trafficking in Live Cells. Nat. Cell Biol. 2001 ;3: 140-149. Varadi, A., Tsuboi, T, and Rutter, G. A. Myosin Va Transports Dense Core Secretory Vesicles in Pancreatic MIN6 Beta-Cells. Mol. Biol. Cell. 2005;16: 2670-2680.

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BIOMARKERS 115. Kolin, D. L., Ronis, D., and Wiseman, P. W. K-Space Image Correlation Spectroscopy: A Method for Accurate Transport Measurements Independent of Fluorophore Photophysics. Biophys. J. 2006; 91:3061-3075. 116. Gesty-Palmer, D. and Thompson, N. L. Binding of the Soluble, Truncated Form of an FC Receptor (Mouse FC Gamma RII) to Membrane-Bound Igg as Measured by Total Internal Reflection Fluorescence Microscopy. J. Mol. Recognit. 1997;10:63-72. 117. Poglitsch, C. L. and Thompson, N. L. Interaction of Antibodies with FC Receptors in Substrate-Supported Planar Membranes Measured by Total Internal Reflection Fluorescence Microscopy. Biochemistry. 1990;29:248-254. 118. Kapoor, R., Kaur, N., Nishanth, E. T, Halvorsen, S. W., Bergey, E. J., and Prasad, P. N. Detection of Trophic Factor Activated Signaling Molecules in Cells by a Compact Fiber-Optic Sensor. Biosens. Bioelectron. 2004;20:345-349. 119. Conboy, J. C , McReynolds, K. D., Gervay-Hague, J., and Saavedra, S. S. Quantitative Measurements of Recombinant HIV Surface Glycoprotein 120 Binding to Several Glycosphingolipids Expressed in Planar Supported Lipid Bilayers. J. Am. Chem. Soc. 2002;124:968-977. 120. Levy, S., Mannion, J., Cheng, J., Reccius, C , and Craighead, H. G. Entropic Unfolding of DNA Molecules in Nanofiuidic Channels. Nano. Lett. 2008;8: 3839-3844. 121. Reccius, C. H., Mannion, J. T., Cross, J. D., and Craighead, H. G. Compression and Free Expansion of Single DNA Molecules in Nanochannels. Phy. Rev. Lett. 2005;95:268101-268104. 122. Reccius, C. H., Stavis, S. M., Mannion, J. T, Walker, L. P., and Craighead, H. G. Conformation, Length snd Speed Measurements of Electrodynamically Stretched DNA in Nanochannels. Biophys. J. 2008;95:273-286. 123. Mannion, J. T, Reccius, C. H., Cross, J. D., and Craighead, H. G. Conformational Analysis of Single DNA Molecules Undergoing Entropically Induced Motion in Nanochannels. Biophys. J. 2006; 90:4538-4545. 124. Han, J. and Craighead, H. G. Entropic Trapping and Sieving of Long DNA Molecules in a Nanofiuidic Channel. /. Vac. Sci. Technol. A. 1999;17:2142-2147. 125. Han, J. and Craighead, H. G. Separation of Long DNA Molecules in a Microfabricated Entropic Trap Array. Science. 2000;288:1026-1029. 126. Han, J. and Craighead, H. G. From Microfluidics to Nanofiuidics: DNA Separation Using Nanofiuidic Entropic Trap Array Device. Proc SPIE. Microfluidic Devices and Systems III, Santa Clara, CA. 2000:42-48. 127. Stavis, S. M. Single Molecule Studies of Quantum Dot Conjugates in a Submicrometer Fluidic Channel. Lab Chip. 2005a;5:337-343. 128. Stavis, S. M. Detection and Identification of Nucleic Acid Engineered Fluorescent Labels in Submicrometre Fluidic Channels. Nanotechnology. 2005b; 16: S314-S323. 129. Stavis, S. M. Single-Molecule Mobility and Spectral Measurements in Submicrometer Fluidic Channels. J. Appl. Phys. 2005c;98:044903-1-044903-5. 130. Foquet, M. DNA Fragment Sizing by Single-Molecule Detection in Submicrometer-Sized Closed Fluidic Channels. Anal. Chem. 2002;74:1415-1422. 131. Tegenfeldt, J., Prinz, C , Cao, H., Huang, R., Austin, R., Chou, S., Cox, E., and Sturm, J. Micro- and Nanofluidics for DNA Analysis. Anal. Bioanal. Chem. 2004;378:1678-1692. 132. Levine, M. J., Korlach, J., Turner, S. W., Foquet, M., Craighead, H. G., and Webb, W. W. Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations. Science. 2003;299:682-686.


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133. Moran-Mirabal, J. M. and Craighead, H. G. Zero-Mode Waveguides: SubWavelength Nanostructures for Single Molecule Studies at High Concentrations. Methods. 2008;46:11-17. 134. Lemay, S. G. Nanopore-Based Biosensors: The Interface Between Ionics and Electronics. ACS Nano. 2009;3:775-779. 135. Kasianowicz, J. J., Brandin, E., Branton, D., and Deamer, D. W. Characterization of Individual Polynucleotide Molecules Using a Membrane Channel. Proc. NatLAcad. Sci. USA. 1996;93:13770-13773. 136. Storm, A. J., Storm, C , Chen, J., Zandbergen, H., Joanny, J. E, and Dekker, C. Fast DNA Translocation Through a Solid-State Nanopore. Nano Lett. 2005; 5:1193-1197. 137. Heng, J. B., Ho, C , Kim, T., Timp, R., Aksimentiev, A., Grinkova, Y. V., Sligar, S., Schulten, K., and Timp, G, Sizing DNA Using a Nanometer-Diameter Pore. Biophys. J. 2004;87:2905-2911. 138. Li, J., Gershow, M., Stein, D., Brandin, E., and Golovchenko, J. A. DNA Molecules and Configurations in a Solid State Nanopore Microscope. Nature Mater. 2003;2:611-615. 139. Mathe, J., Visram, H., Viasnoff, V., Rabin, Y., and Meller, A. Nanopore Unzipping of Individual DNA Hairpin Molecules. Biophys. J. 2004;87:3205-3212.


CHAPTER

IMMUNODIAGNOSTICS WITH A FOCUS ON LATERAL FLOW POINT-OF-CARE DEVICES Roy R. Mondesire, Glen M. Ford, Hannie F. Ford, and Stephen C. Mefferd

INTRODUCTION Before the advent of immunodiagnostics, the diagnosis of infectious diseases required the demonstration of the presence of an organism by conventional methods. In many instances, these procedures were not useful because of technical difficulty and/or time required to achieve results. Immunodiagnostics provided a new and important set of tools for infectious disease diagnosis indirectly by detection of antibodies or directly by detection of antigen associated with the presence of the organism. Many immunodiagnostic tests are accurate, affordable, and user-friendly with a fairly rapid turnaround time. Infection by foreign antigen or organism results in antibody responses to antigens recognized by and appropriately presented to the body's immune system. Detection of antibody responses is accomplished by measurement of blood or serum reactivity to a known antigen reagent system. This involves production of polyclonal or monoclonal antibodies to the antigen of interest and this is used as the reagent detection system. The detection of either antibody or antigen in patient blood or serum is the basis for immunodiagnostic tests. These types of immunodiagnostic technologies have been employed for many years and are still the most important diagnostic tools for infectious disease. A limitation of the technology is that detection of antibody does not necessarily equate to active infection. This interpretation of an antibody response depends on the biology of the specific disease or host-agent interaction. There are many infectious diseases in which an antibody response clears the body of 495

496

BIOMARKERS

infection. In these instances, the interpretation of positive antibody tests is that the animal has been infected by the organism, but the status of active infection is not certain. For infectious agents that are not effectively eliminated by antibody responses, the detection of antibody responses may correlate better with an interpretation of active infection. Detection of antigen is a more direct determination of the presence of active infection. Rosalyn Yalow and Solomon Berson1 were the first to describe the principles of immunoassay technology. By the 1970s this technology evolved from research and development into incorporation in many large central and local hospital laboratories. Immunoassays have been applied to both qualitative and quantitative analyses and have been applied to most biomedical research. The application of impressive scientific and technological innovations into the in vitro medical device industry resulted in a dramatic increase in the use of immunodiagnostic products.

ANTIBODIES IN IMMUNOASSAYS Antibodies are secreted by B lymphocytes and comprise the primary arm of the humoral immune response. B lymphocyte development occurs in specific inductive microenvironments and includes both antigen-independent and dependent processes. Antigen specific naive B lymphocytes are retained in secondary lymphoid organs upon recognition of receptor-specific antigen. Secondary lymphoid organs contain B lymphocyte-rich follicles in which these naive antigen-specific B lymphocytes undergo clonal expansion which results in the generation of memory B lymphocytes or antibody-secreting plasma cells. Vertebrate immune systems have evolved a variety of strategies to achieve diversification of antigen receptor molecules. DNA recombination events result in mature antigen receptor genes from separate gene segments. Three gene segments are involved in this event. These genes have been designated variable (V), diversity (D), and joining (J). As the B cell matures, it rearranges or shuffles these gene segments and selects among hundreds of DNA segments. Specific sequences of DNA are cut and then selected pieces spliced together (Figure 19.1). For antibody, the V gene segment encodes the majority of the variable domain including complementarity-determining regions 1 and 2 (CDR1 and CDR2). The D segments and the DJ junctions encode CDR3. In the mouse, an extensive Ig repertoire is achieved by a large group of genes encoding the variable regions of the heavy (H) and light (L) chains (VH and VL). In humans however, the fewer VH and VL are compensated for by the relatively longer CDR3. For the heavy chain of antibody, the variable regions are derived from gene rearrangement and recombinations of the VH, DH, and JH. In the case of the light chain the variable regions are derived from the VL and JL gene segments. In humans there are approximately 105 V(D)J germline exons and 103 VL exons. Thus there are 108 different possibilities in the germline. Maturation and selection of B -cell clones in the germinal center results in the production of high affinity antibodies. During this time, antigen-specific B-cell clones

497

IMMUNODIAGNOSTICS

Germ-line DNA

5'—]

v

|

H8enes

1 D H ssgments

C H genes

JH segments

1

r-3'

11 Gene rearrangement

1

B-cell DNA

V J t Transcription and RNA splicing

1

B-cell mRNA

Heavy chain

FIGURE 19.1 chain.

NH 2 _|

ID

v

|| CDRl

1!

vH

CDR2

j

j

1

rn

1

J \ Translation

II

|

CH

| — COOH

CDR3

General pattern of rearrangement of genes in the production of an antibody heavy

undergo isotype switches and somatic hypermutation. In the T-cell-dependent regions of the peripheral lymphoid organs, B cells react with specific antigen and proliferate with the assistance of T cells and accessory cells.

S t r u c t u r e and Function of A n t i b o d i e s Antibodies are glycoproteins belonging to the immunoglobulin (Ig) supergene family. Five major isotypes occur within this family and are present in the majority of higher mammals. These have been designated IgG, IgM, IgA, IgD, and IgE. Size, charge, amino acid composition, and carbohydrate content distinguish the isotypes. IgG, usually depicted as the basic immunoglobulin molecule (H2L2), is a monomeric glycoprotein composed of two identical heavy chains (H) and two identical light chains (L). It is the predominant antibody in normal serum and accounts for 70-75% of the total Ig content. Human IgG has a sedimentation coefficient of 7S and a molecular mass (MM) of approximately 160 kDa. Four subclasses are recognized for human IgG. These have been designated IgGl, IgG2, IgG3, and IgG4. Figure 19.2 illustrates the general structure of IgG. IgM is the first immunoglobulin isotype detected early in a primary immune response and accounts for about 10% of the total antibody pool. It is pentameric, each unit consisting of a MM of 180 kDa. The relative MM is thus approximately 900 kDa. Monomers of IgM are linked by disulfide bonds in a circular array. A cysteine-rich "J chain" joins two of the monomers to complete the circle (Figure 19.3). IgA is a carbohydrate-rich immunoglobulin. In humans it is the second most abundant immunoglobulin. In most other mammals, IgA forms a relatively small part of the plasma pool. Dimeric IgA predominates in mammals and consists of two IgA molecules in association with a secretory component (SC). A J chain links the dimeric form via the Fc. IgA is the predominant immunoglobulin at the mucosae and plays an important role in protection at these surfaces. IgD (MM =170 kDa) accounts for a small fraction of circulating Ig.

498

BIOMARKERS

Antibody Combining Site (Paratope)

1 vL Q Hinge Region

1

v„

Fab —

1 s

11 s

1 s -S-S-

H^

1

-s-s-s-s,,

CH2 Fc CH3

1

s

1i CH1

J

1

s

1 1

r

1

s

*\.

Interchain disulfide bonds Intrachain disulfide bonds

s

1 FIGURE 19.2 Schematic of an antibody molecule showing the domains which make up the heavy (H) and light (L) chains.The variable region of the molecule is designated V. Note theVL andVH regions together form the antibody combining site. In IgG, there are three constant (CH) domains designated C I, C 2, and C 3,The domains C 2 and C 3 constitute the Fc region of the molecule.There are intra and inter-chain disulfide bonds (-s-s-). The hinge region, which imparts segmental flexibility to the molecule, is shaded.

FIGURE 19.3 General schematic of IgM showing its pentameric configuration, inter-chain disulfide bonds, and J-chain.

IMMUNODIAGNOSTICS

499

The structure of IgD confers a high susceptibility to proteolysis and heat. It is present primarily on the surface of circulating B lymphocytes in association with IgM and although its function has not been established, it may play a role in B cell differentiation. IgE (MM = 200 kDa) is primarily found associated with the high affinity IgE receptor (FccRI) on the membranes of mast cells and basophils. It mediates Type I hypersensitivity reactions and may play a role in immunity to some parasitic diseases. Two distinct functions, antigen binding and effector functions, are associated with antibodies. The binding site on an antibody is located on the hypervariable domain of the Fab region of the Ig. This binding site or "paratope" is located in the N-terminal portion of the molecule. This paratope is capable of recognizing and binding to an epitope on a corresponding antigen. The effector functions reside in the C-terminal portion of the molecule in the Fc constant domain region. Examples of effector functions are complement fixation and cell surface binding by means of Fc receptors expressed on phagocytic or other effector cells. The binding of antigenic epitopes to antibody paratopes involves multiple non-covalent bonds. The attractive forces involved in binding include hydrogen bond, electrostatic, van der Waals, and hydrophobic interactions. Hydrophobic interactions account for approximately 50% of the total strength of bonding and are primarily due to the interaction of non-polar hydrophobic residues where contact with water molecules is greatly reduced. These non-covalent interactions are very dependent on the distance between the interacting residues.

Kinetics of A n t i b o d y - A n t i g e n Reactions Immunoassays are based on the reversible binding reaction of an antibody molecule with a corresponding antigen where there is a significant amount of binding energy. These antigen-antibody reactions can be described by the law of mass action. The kinetics of this reversible reaction can be represented thus: [Ag]+[Ab]

-

K

[AgAb]

(19.1)

Where: [Ag] = free antigen (Ag) concentration [Ab] = free antibody (Ab) concentration [AgAb] = Ag complexed with Ab kj = the association rate k2 = the dissociation rate Equation 1 assumes a single antigenic epitope binding to a single Fab on an antibody. The rate of formation of the AgAb complex and law of mass action can be represented thus: d[AgAb] dt

=k 1 [Ag][Ab]-k 2 [AgAb]

(19.2)

500

BIOMARKERS

From equation 1, at equilibrium (i.e., when the net rate is zero): k,

[AgAb]

k2

[Ag] [Ab]

Where Keq is the equilibrium constant in liters mole-1 (LM-1). For analytes which exist at sub-nanomolar concentrations, for example 10-11 M levels, it is important to have antibody at greater than 100-fold excess (> 10-9 M), in order to provide a reasonably high frequency of interaction between the antigen and antibody. Typically a Keq of > 109 LM-1 is required for immunoassays. Antibody affinity, a measure of the strength of the bond between a single antigen-combining site and an antigenic determinant, is traditionally determined with the Scatchard equation: r/[Ag] = -Kr + nK

(19.4)

Where r is the number of occupied sites on the antibody, [Ag] is the concentration of free antigen, and n is the antibody valency. In the case of polyclonal antibodies, the affinity measurement is a reflection of the average affinities of the different antibodies present in the sample.

Polyclonal Antibodies A specific antibody reacts only with a small region (epitope) contained on the molecular structure of the antigen. Thus, the physical size and complexity of the antigen influences the number of different specific antibodies produced. Larger and more complex immunogens contain many different epitopes, each producing and reacting with its own specific antibody. The technology used to develop polyclonal antibodies involves injecting animals (rabbits, sheep, goats, donkeys) with the antigen of interest and collecting the serum portion of the blood. Of the total serum immunoglobulins from an immunized animal only 0.1-10% contain specific antibodies reacting with the injected material. Various protein purification techniques are used to isolate specific antibody from nonspecific antibody and other serum proteins. In affinity purification, the serum is applied to a solid phase column containing a chromatographic resin with immobilized antigen to which the antibody was raised. This results in absorption of the antibody from the liquid to solidphase. By various chemical treatments, the specific antibody is eluted and separated from nonspecific serum proteins.

Hybridoma Technology In 1976, immunology was revolutionized by Kohler and Milstein 2 when they showed that individual antibody producing cells could be immortalized when fused with a myeloma cell line, making it possible to produce a virtually unlimited supply of antibodies with the same specificity. These are called monoclonal antibodies (MAbs). Large quantities of specific antibodies can be produced, purified, and adapted as tools for in vitro diagnostics, in vivo diag-

IMMUNODIAGNOSTICS

501

nostics, or therapeutics. They are generally derived by immunizing mice with an antigen, isolating antibody-producing cells from the spleen or lymph nodes and fusing them with an immortalized plasmacytoma cell to obtain a hybridoma cell that can be cultured in vitro or grown in mice. As the hybridoma cells replicate, continuous production of large quantities of monoclonal antibody occurs. The ability of monoclonal antibodies to react with a single epitope allows development of immunoassays that have high specificity. Monoclonal antibodies have been used for cancer immunotherapy, where a radioactive or cytotoxic compound is attached to the antibody and injected into a patient. Thus, the toxic material concentrates primarily at the cancer site, leading to death of the cancerous cells. Anti-CD3 monoclonal antibodies have been used for allograft rejection treatment.

RAPID M A N U A L A N D RAPID A U T O M A T E D IMMUNOASSAYS With the desire of health practitioners to obtain clinical results in minutes rather than hours, a major milestone occurred in the past two decades with the development and optimization of rapid immunoassays. These can be manual, automated, or machine run. While the rest of the clinical diagnostic market expands at rates below 10%, the use of rapid immunoassays is growing at an annual rate of greater than 15% for the past few years, with revenues in the billions by biotechnology companies in 2008. Rapid manual immunoassays are used in point-of-care testing, where the need to have a result is of critical importance. Two types of rapid point-of-care assays have been developed. These are lateral-flow and flow-through assays. Many rapid automated-machine run tests are performed using membrane or nanoparticle solid surfaces. Because these have significantly greater surface areas than the wells, tubes or macroparticles used in conventional enzyme-linked immunosorbent assay (ELISA), more capture antibody or antigen can be immobilized. Combined with the inherent property of membranes to channel analytes into close proximity with the coated solid-phase, reaction rates occur significantly faster. Nanoparticles have the advantage of a mobile colloidal liquid-phase that also brings the reactants into close proximity, thereby increasing the reaction rate. Since the reaction of analyte with the solid-phase is usually complete after 10 to 30 minutes, high degrees of precision and reproducibility are realized. Nanoparticles used in rapid automated immunoassays include magnetic particles or latex particles coated with antigen or antibody. A robotic arm removes a sample from a primary collection tube, dispenses a precise amount into a reaction well containing the nanoparticles, the reaction mixture is incubated, and the particles washed automatically. Magnetic particles are easier to wash since a magnet is used to pull the particles to the side of the reaction tube or well during aspiration and washing. Latex nanoparticles are trapped on glass or cellulose fibers and washed. After the washing step, the particles are exposed to conjugate, washed again, substrate added, and the extent of reaction measured optically.

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BIOMARKERS

The sensitivity of rapid automated tests for their corresponding analyte is in the sub-picogram range with linear dose-responses over a four-log range.

E L E M E N T S OF I M M U N O A S S A Y S : SOLUBLE LABELS A N D D E T E C T I O N The ELISA is a heterogeneous enzyme immunoassay (EIA). Heterogeneous enzyme immunoassays are those that have at least one separation step to distinguish reacted from unreacted reagents. Figures 19.4 and 19.5 illustrate some of the principles of heterogeneous enzyme immunoassays. This technique, first described by Engvall and Perlmann,3 applies to all immunoassays in which one or more of the reactants is immobilized onto a solid phase. This solid phase is typically used to immobilize specific antibody or antigen depending on the assay configuration. Other components of immunoassays are the enzyme-labeled antibody or antigen. These conjugated reagents are used to probe any molecules that have reacted with the surface-bound antibody or antigen. Verification of the reaction sequence, in the case of a colorimetric assay, is achieved with a chromogenic substrate. Enzymes are more widely used than any other label in immunoassays. They generate colored, fluorescent, or luminescent compounds from neutral substrates. The various enzymes used in ELISA include horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, c-galactosidase, glucoamylase, carbonic anhydrase, and acetylcholinesterase. Several covalent conjugation methods are available for the coupling of enzymes to antigens or antibodies. The enzymes HRP (44 kDa) and AP (140 kDa) are the most commonly used in heterogeneous immunoassays. HRP catalyzes the conversion of the substrate H 2 0 2 to H20 and 0 2 . It then oxidizes another substrate resulting in a colored, fluorescent, or luminescent derivative, depending on the nature of the substrate. The enzyme AP catalyzes

Sandwich ELISA for the detection of specific IgG

+Substrate

1

2

3

Wash

4

5

Wash

FIGURE 19.4 Typical sandwich ELISA for the detection of specific IgG. In this configuration, species specific antibody (I) is bound to a solid phase. A sample containing IgG, (2) is added.The antibody (Y shaped molecule) in the sample is captured by the bound antibody (3). Following a wash step.anti species IgG enzyme (E)-conjugate is added (4).The conjugate binds to the captured IgG, forming a "sandwich"(5). Following a wash step, the addition of the appropriate chromogenic substrate will result in a color change, thus revealing binding of the specific antibody of interest.

IMMUNODIAGNOSTICS

503

FIGURE 19.5 Typical sandwich ELISA for the detection of antigen. In this configuration specific antibody (I) is bound to a solid phase. A sample containing antigen (2) is added.The antigen in the sample is captured by the bound antibody (3). Following a wash step, an antibody enzyme conjugate is added (4). The conjugate binds to a site on the captured antigen, forming a 'sandwich' (5). Following another wash, the addition of the appropriate chromogenic substrate will result in a color change, thus revealing binding of specific analyte of interest.

the hydrolysis of phosphate esters of primary alcohols, phenols and amines. Another commonly used approach employs biotin-avidin binding reactions with one of the components complexed with a chromogenic enzyme. Avidin (MM = 67 kDa) can be isolated from purified egg white. This molecule has a very high affinity (association constant = 1015 LM-1) for the small water soluble vitamin biotin (MM = 0.244 kDa). Four biotin molecules can bind to one avidin molecule. In a typical ELISA, biotinylated antibody and avidin-labeled enzyme are used instead of the enzyme-labeled antibody. This combination offers a significant enhancement in signal.

HOMOGENEOUS ENZYME IMMUNOASSAYS In homogeneous EIAs, the immunological reactions and the detection of changes in enzymatic activity are carried out in the same solution. There is no need for the separation of bound and free labels. As for the heterogeneous EIAs, the substrates used can be chromogenic, fluorogenic or chemiluminescent. Enzymes used for homogeneous EIAs include c-D-galactosidase, glucose6-phosphate dehydrogenase (G6PDH), hexokinase and glucose oxidase. The substrates for these labels are the fluorogenic substrate 4-methylumbelliferyle-D-galactopyranoside, D-glucose 6-phosphate + NAD+, D-hexose + ATP and glucose, respectively. Homogeneous EIAs can be competitive or noncompetitive binding assays. Competitive assays are based on the modulation of enzyme activity due to the competitive reaction of antibody with labeled and free antigen. Here the enzyme activity is either activated or inhibited as a result of immune complex formation. Noncompetitive assays utilize enzyme-labeled antibody conjugates.

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BIOMARKERS

One approach involves a proximal linkage assay based on substrate channeling due to the close proximity of the one enzyme with a corresponding coupling enzyme. In another method changes in enzyme activity take place due to the binding of antigen to the corresponding antibody enzyme conjugate.

SIGNAL MEASUREMENT METHODS Colorimetry The most common and simplest detection system is colorimetry. This can be determined visually or with the aid of a spectrophotometer. A common chromogenic substrate for peroxidase is 3,3',5,5'-tetramethylbenzidine (TMB), described earlier.

Fluorometry Fluorescence begins with the absorption of photons by fluorophores. At the appropriate wavelength, the electrons are energized from a ground energy state to an excited singlet state. As the molecule returns to the ground state, it emits a photon of light at a lower energy (i.e. longer wavelength). The Stokes' shift is the difference between the excitation maximum and the emission maximum (Figure 19.6). The standard label, fluorescein isothiocyanate (FITC) has an absorptionemission time interval of only one nanosecond (ns). Many other compounds, however, exhibit delayed fluorescence with much higher time intervals. The use of fluorescence techniques in immunoassays has been reviewed.4

Relative fluorescence

Emission Excitation

Wavelength (nm) FIGURE 19.6 Principle of fluorescent measurement. The Stokes' shift represents the difference between the maximum excitation and the maximum emission.

IMMUNODIAGNOSTICS

505

Fluorometric enzyme immunoassays (FIAs) are more sensitive than the colorimetric immunoassays and therefore may be used to detect or measure small concentrations of analyte. A disadvantage is increased complexity of the procedure and the need for instrumentation. A common substrate for AP in fluorometric El As is 4-methylumbelliferyl phosphate (4-MUP). AP dephosphorylates 4-MUP to the form the fluorophore 4-methylumbelliferone (4-MU).

Time-Resolved Fluorescence Background interference by light scattering and intrinsic fluorescence of sample components are some of the limitations of the traditional fluorescent compounds. Time-resolved fluorescence is based on the principle that some lanthanides, such as europium (Eu3+), form fluorescent chelates with certain organic ligands. These fluorophores have very large Stokes' shifts and decay times (200 nm and >500 nanoseconds respectively). Time-resolved fluorescence takes advantage of these long decay times and large Stokes' shifts. Thus any short-lived fluorescence background signals or scattered excitation radiation are eliminated. Signals are thus measured under conditions of virtually no background. The use of time-resolved fluorescence techniques in immunoassays has been reviewed.5 Several homogeneous and heterogeneous FIAs for the detection and quantitation of various diseases markers have been developed and many of them have been automated. Luminescence In chemiluminescent immunoassays, luminescent compounds emit light during the course of a chemical reaction. Luminol derivatives or acridinium esters have been used as labels. The kinetics are very fast and light is emitted within seconds of substrate oxidation. These assays are generally very sensitive with high dynamic ranges. In an electrochemiluminescence (ECL) technique,6 a ruthenium metal chelate and tripropylamine are utilized. Both of these molecules become oxidized at the surface of an electrode where they react to form an excited state of ruthenium that decays, releasing a photon at 620 nm. This technology is easily adapted to immunoassays and molecular diagnostics.

P R I N C I P L E S OF B I N D I N G Biological macromolecules can bind to plastic surfaces in various ways. The adsorption of molecules to surfaces is primarily due to intermolecular attraction forces. These forces are of two types—alternating polarities, known as hydrophobic interaction, and stationary polarities. Alternating polarities occur when molecules in close proximity create disturbances in their electron clouds. Molecules which possess stationary polarities can bind to each other through dipoles. Hydrogen bonding can result between two dipoles. Chemical groups which mediate H bonding include -OH, =0, -NH2, =NH, and gN. Prior to the immobilization of a molecule to a surface, it is useful to know the properties of both the solid phase and the molecule to be bound. For example, some plastics

506

BIOMARKERS

are available in either hydrophobic or hydrophilic forms, and others have both properties. The geometry of a globular molecule will dictate the maximum number of molecules that can be packed in the densest monolayer on a surface. The packing density is a function of the orientation of the molecule after binding. For an elongated molecule such as IgG (approximately 150 A x 3 A), it can be shown that vertical packing will result in more molecules bound per unit area when compared to a horizontal packing of the molecules.

NON-SPECIFIC INTERACTIONS IN IMMUNOASSAYS Non-specific interactions can have adverse effects on immunoassays. This effect is manifested primarily in reduced specificity. Many factors can contribute to this effect. The elimination of non-specificity is central to the performance of a reliable test. Several rational and empirical approaches are used to reduce or eliminate non-specific interactions. One approach requires the chemical modification of the immunoglobulin. In order to circumvent this effect, F(ab)

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