Advances in Clinical Chemistry, Volume 47

ADVANCES IN CLINICAL CHEMISTRY VOLUME 47

Advances in CLINICAL CHEMISTRY Edited by GREGORY S. MAKOWSKI Clinical Laboratory Partners, LLC Newington, Connecticut USA

VOLUME 47

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

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Copyright ß 2009, Elsevier Inc. All rights reserved 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 or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://www.elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-374796-9 ISSN: 0065-2423 For information on all Academic Press publications visit our website at www.elsevierdirect.com Printed and bound in USA 09 10 11 12 10 9 8 7 6

5 4 3 2

1

CONTENTS CONTRIBUTORS

................................................................................

ix

PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xi

Amyloidosis KOSTANDINOS SIDERAS AND MORIE A. GERTZ 1. 2. 3. 4. 5. 6.

Abstract ... ................................................................................... Historical Perspective....................................................................... Pathogenesis ................................................................................. Diagnosis.. ................................................................................... Classification, Clinical Presentation, and Prognosis of Amyloidosis.................. Treatment . ................................................................................... References. ...................................................................................

2 2 3 14 20 28 36

Urinary Markers in Colorectal Cancer BO FENG, FEI YUE, AND MIN-HUA ZHENG 1. 2. 3. 4. 5.

Abstract ... ................................................................................... Introduction ................................................................................. Potential Urinary Markers for Colorectal Cancer ...................................... Analytical Techniques and Data Analysis ............................................... Conclusions .................................................................................. References. ...................................................................................

45 46 47 50 53 53

Effect of Hormone Replacement Therapy on Inflammatory Biomarkers PANAGIOTA GEORGIADOU AND EFTIHIA SBAROUNI 1. 2. 3. 4. 5. 6.

Abstract ... ................................................................................... Introduction ................................................................................. Inflammation and Vascular Disease ...................................................... Mechanisms of Action of HRT in Vascular Biology ................................... Effects of HRT on Inflammatory Markers .............................................. Conclusion ................................................................................... References. ...................................................................................

v

60 60 62 65 71 82 83

vi

CONTENTS

Personalized Clinical Laboratory Diagnostics KEWAL K. JAIN 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Abstract....................................................................................... Introduction.................................................................................. Basic Concepts of Personalized Medicine ................................................ Molecular Diagnostic Technologies for Personalized Medicine. ....................... Role of PCR in Development of Personalized Medicine.. .............................. Combined PCR–Enzyme-Linked Immunosorbent Assay (ELISA).................... Non-PCR Methods.......................................................................... Direct Molecular Analysis Without Amplification ...................................... SNP and Personalized Medicine ........................................................... Genetic Variations in the Human Genome Other Than SNPs ......................... Role of Biomarkers in Personalized Medicine ........................................... Application of Biochip Technology in Developing Personalized Medicine ...................................................................... Role of Nanobiotechnology-Based Diagnostics in Personalized Medicine ...................................................................... Role of Cytogenetics in Personalized Medicine .......................................... Integration of Molecular Diagnostics and Therapeutics ................................ Concluding Remarks and Future Prospects.. ............................................ References ....................................................................................

96 96 96 99 99 102 103 103 103 105 108 109 111 114 116 117 118

Verification of Method Performance for Clinical Laboratories JAMES H. NICHOLS 1. 2. 3. 4.

Abstract....................................................................................... Introduction.................................................................................. ISO Quality Management System: The Fundamentals of Quality..................... Laboratory Quality Standards in Regulations and Accreditation Guidelines ................................................................... 5. Comparison of Quality Requirements .................................................... 6. Performing Method Verification........................................................... 7. Summary ..................................................................................... References ....................................................................................

121 122 123 129 131 132 136 136

Interpreting the Proteome and Peptidome in Transplantation TARA K. SIGDEL, R. BRYAN KLASSEN, AND MINNIE M. SARWAL 1. 2. 3. 4. 5.

Abstract....................................................................................... Introduction.................................................................................. Application of Proteomics and Peptidomics in Transplantation....................... Important Issues ............................................................................. Conclusion ................................................................................... References ....................................................................................

140 140 155 160 162 163

CONTENTS

vii

Biomarkers in Long-Term Vegetarian Diets IRIS F.F. BENZIE AND SISSI WACHTEL-GALOR 1. Introduction ................................................................................. 2. Possible Nutritional Deficiencies in Association with Long-Term Vegetarian Diets. ............................................................................ 3. Biomarkers of Oxidant/Antioxidant Balance in Association with Vegetarian Diets....................................................................... 4. Biomarkers that Reflect Lower Risk of Disease in Long-Term Vegetarians ......... 5. Biomarkers to Differentiate the Vegetarian from the Nonvegetarian................. 6. Summary and Recommendations for Clinical Chemistry .............................. References. ...................................................................................

172 173 186 194 207 209 210

Effect of Caloric Restriction on Oxidative Markers JAN SˇKRHA 1. 2. 3. 4. 5. 6. 7. 8.

Abstract ... ................................................................................... Introduction ................................................................................. Foods and ROS Generation ............................................................... Mitochondria as a Source of Reactive Oxygen and Nitrogen Species ................ Caloric Restriction and Oxidative Stress ................................................. Oxidative Stress Markers by Caloric Restriction........................................ Data Interpretation ......................................................................... Conclusions .................................................................................. References. ...................................................................................

224 224 225 226 229 232 240 241 242

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . COLOR PLATE SECTION AT THE END OF THE BOOK

249

CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

IRIS F.F. BENZIE (171), Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Kowloon, Hong Kong BO FENG (45), Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China PANAGIOTA GEORGIADOU (59), 2nd Department of Cardiology, Onassis Cardiac Surgery Center, Athens, Greece MORIE A. GERTZ (1), Division of Hematology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA KEWAL K. JAIN (95), Jain PharmaBiotech, Basel, Switzerland R. BRYAN KLASSEN (139), Department of Pediatrics—Nephrology, Stanford University Medical School, Stanford University, Stanford, California 94305, USA JAMES H. NICHOLS (121), Professor of Pathology, Tufts University School of Medicine and Medical Director, Clinical Chemistry, Baystate Health, Springfield, Massachusetts 01199, USA MINNIE M. SARWAL (139), Department of Pediatrics—Nephrology, Stanford University Medical School, Stanford University, Stanford, California 94305, USA EFTIHIA SBAROUNI (59), 2nd Department of Cardiology, Onassis Cardiac Surgery Center, Athens, Greece

ix

x

CONTRIBUTORS

KOSTANDINOS SIDERAS (1), Division of Hematology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA TARA K. SIGDEL (139), Department of Pediatrics—Nephrology, Stanford University Medical School, Stanford University, Stanford, California 94305, USA JAN SˇKRHA (223), Laboratory for Endocrinology and Metabolism and 3rd Department of Internal Medicine, 1st Faculty of Medicine, Charles University in Prague, U Nemocnice 1, 128 08 Prague 2, Czech Republic SISSI WACHTEL-GALOR (171), Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Kowloon, Hong Kong FEI YUE (45), Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China MIN-HUA ZHENG (45), Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

PREFACE I am pleased to present volume forty‐seven of Advances in Clinical Chemistry series. In this first volume for 2009, an array of relevant clinical laboratory topics is presented. The biochemistry of amyloidosis is explored with respect to the microenvironment, mechanisms of organ dysfunction, and the role of toxic intermediates. The importance of low molecular weight urinary biomarkers associated with colorectal cancer, one of the most commonly diagnosed cancers worldwide, is reviewed using a metabolomic approach. The role of hormone replacement therapy is investigated with respect to inflammatory biomarkers and vascular disease in women. It is noteworthy that cardiovascular disease risk is typically underestimated in the female population. A wonderful review on personalized clinical laboratory diagnostics is presented by a leader in the field of pharmacogenomics. Verification of method performance is reviewed with respect to a number of international quality standards, accreditation agencies, and regional laws. Another topic, application of the proteome to impact on organ transplantation outcomes, is also presented. This volume is concluded by two reviews on diet. In the first paper, biomarkers associated with vegetarian diets are explored. The second review investigates the role of caloric restriction on lifespan as evidenced by impact on oxidative markers. I extend my appreciation to each contributor of volume forty‐seven and thank colleagues who contributed to the peer review process. I extend my thanks to my Elsevier editorial liaison, Gayathri Venkatasamy. I sincerely hope the first volume of 2009 will be enjoyed by our diverse readership. As always, I warmly invite comments and suggestions for future review articles for the Advances in Clinical Chemistry series. In keeping with the tradition of the series, I would like to dedicate volume forty‐seven to my father‐in‐law Dr Gale R. Ramsby. GREGORY S. MAKOWSKI

xi

ADVANCES IN CLINICAL CHEMISTRY, VOL. 47

AMYLOIDOSIS Kostandinos Sideras and Morie A. Gertz1 Division of Hematology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA

1. Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Historical Perspective. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Structure of the Amyloid Fibril . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Amyloid Aggregation Mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Interactions of the Amyloid Fibril with the Microenvironment . . . . . . . . . . . . . 3.4. Tropism of Amyloid Proteins for DiVerent Organs . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Mechanisms of Organ Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Suspecting the Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Screening for Amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Establishing the Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Typing of Amyloid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Imaging of Amyloid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Classification, Clinical Presentation, and Prognosis of Amyloidosis . . . . . . . . . . . . . . 5.1. Primary Amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Secondary Amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Familial Amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Senile Amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Localized Amyloidosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Strategies Aimed at Eradicating the Production of the Amyloid Precursors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Native Protein Structure Stabilizing Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Amyloid Fibril Destabilizing Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Immunologic Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. Supportive Care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6. Treatment of Localized Amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

2 2 3 3 6 9 12 13 14 14 15 16 17 18 20 20 21 23 25 26 28 28 32 33 34 35 35 36

Corresponding author: Morie A. Gertz, e‐mail: [email protected] 1

0065-2423/09 $35.00 DOI: 10.1016/S0065-2423(09)47001-X

Copyright 2009, Elsevier Inc. All rights reserved.

2

SIDERAS AND GERTZ

1. Abstract Amyloidosis is a heterogeneous group of diseases in which an otherwise normal protein, with or without proteolytic cleavage, forms insoluble amyloid fibrils. These, in turn, deposit in various organs and cause dysfunction. A wide range of diseases are associated with amyloidosis such as Alzheimer’s disease, multiple myeloma, other plasma cell disorders, and chronic inflammation, either as a cause, or result, of amyloid production. This heterogeneity in cause and presentation leads to an incomplete understanding of the pathophysiology of amyloid disease. As such, study of this complicated disease process presents significant challenges. The purpose of this review article is to introduce the biochemistry of amyloidosis including ultrastructure analysis, models of monomer aggregation, the importance of the amyloid microenvironment, and the mechanisms of organ dysfunction, including the role of ‘‘toxic intermediates.’’ Pathophysiologic analysis of amyloidosis will focus on diagnostic tools as well as the classification of the various forms of amyloidosis. Finally, treatment of amyloidosis will be reviewed including traditional and established modalities. We will also introduce new and novel treatment options as they relate to the basic pathophysiology of this complex and heterogeneous disorder.

2. Historical Perspective Although the term ‘‘amyloid’’ was first used in botany as early as 1838 by Matthias Schleiden to describe plant starch, and subsequently in 1854 by Rudolf Virchow to describe abnormal macroscopic deposits, the disease of amyloidosis, though with diVerent names, was known at least since the mid‐ seventeenth century [1–3]. Anatomists and pathologists frequently described organs with a ‘‘lardaceous’’ or ‘‘waxy’’ appearance and a major debate in the mid‐nineteenth century consisted of whether the disease was caused by deposition of a lard‐like or a starch‐like substance. Professor Karl von Rokitansky from Vienna was the main proponent of the idea of ‘‘lardaceous change’’ (wax). On the other hand, the German (Berlin) professor Rudolf Virchow believed that a starch‐like substance was responsible for the abnormal spleens he examined. He had come to that conclusion after he stained the abnormal deposits with a combination of iodine and sulfuric acid, and found that, just like starch, the tissues stained pale blue, and thus must be carbohydrate, coining the term amyloid. Both Rokitansky and Virchow were wrong. George Budd found no lardaceous substance in the liver of a patient with amyloidosis and in 1859

AMYLOIDOSIS

3

Friedreich, Nicolau, and Kekule found no starch‐like substance in the spleens described by Virchow, suggesting, to their credit, that the ‘‘amyloid’’ substance was probably albuminoid in nature [4]. In 1920, Schmiedeberg described the similarity of amyloid to serum globulin, which strongly suggested its proteinaceous nature. Early reports of amyloidosis were invariably described in patients with chronic inflammatory conditions like tuberculosis, syphilis, leprosy, and rheumatoid arthritis. These were the early reports of secondary (AA) amyloidosis. However, occasional reports in patients without inflammatory conditions were also made. Sir Samuel Wilks is credited to be the first physician to describe such a patient, a 56‐year‐old with ‘‘lardaceous change.’’ This was probably the first report of a patient with primary (AL) amyloidosis. Soyka was the first to describe both cardiac amyloidosis in patients of advanced age and senile amyloidosis. The Congo red stain, which since 1884 was used in the textile industry to stain cotton, was used by Bennhold in 1922 to stain amyloid, for which it was found to have a strong aYnity [1]. However, it was Divry and Florkin who in 1927 found that Congo red stained amyloid exhibited green birefringence under polarized light. The first description of the amyloid fibril came from Cohen and Calkins in 1959 who noticed the fibrillar structure of amyloid when viewed under the electron microscope, thus being the first to definitely conclude that amyloid was not ‘‘amorphous’’ as suggested by its appearance under the light microscope [5]. Finally, in 1968, Eanes and Glenner discovered the b‐pleated sheet nature of the amyloid fibril which explained some of the resistance of the structure to the action of solvents [6].

3. Pathogenesis 3.1. STRUCTURE OF THE AMYLOID FIBRIL Since Cohen and Calkins described the ‘‘fibrillar’’ nature of amyloid, multiple investigators used electron microscopy to further characterize the structure of the amyloid fibril [2]. Amyloid from diVerent human and animal sources was found to be composed of similar, rigid, nonbranching fibrils of indeterminate, long length (anywhere from 100 to 1600 nm), with an average width of 7.0–12 nm [2]. Each amyloid fibril in turn is composed of a number of b‐pleated sheets (protofilaments), each 2.5–3.5 nm in diameter, which run along the longitudinal axis of the amyloid fibril and slowly twist [7] creating a helical repeat of b‐pleated sheets of about every 11.5 nm. The b‐pleated sheets run perpendicular to the long axis of the fibril. This structure has

4

SIDERAS AND GERTZ

been called the continuous b‐sheet helix [8] or the cross‐b spine, and is responsible for the characteristic cross‐b X‐ray diVraction pattern of amyloid (Fig. 1). Further examination of the ultrastructure of the cross‐b spine has shown it to be a cross‐double b‐sheet, with side chains protruding from the two sheets forming a dry, tightly self‐complementing steric zipper, bonding the sheets [9]. Every segment is bound to its two neighboring segments through stacks of both backbone and side‐chain hydrogen bonds (Fig. 2). Despite this similarity at the protofilament level, the amyloid precursor proteins do not share a common sequence homology, size, function, or tertiary structure. At least 25 diVerent human proteins have been described as precursors to amyloid fibrils and cause a variety of diVerent amyloid‐ related diseases [10] (Table 1). Examples include the monoclonal l or k immunoglobulin light chain which causes AL amyloidosis (related to plasma cell disorders), the serum amyloid A protein (SAA) related to systemic inflammatory conditions (secondary or AA amyloidosis), and the Val30Met and Gly47Val transthyretin (TTR) variants related to hereditary amyloidosis. In addition to these 25 proteins that have the ability to form amyloid fibrils in vivo, there are a number of synthetic amyloid fibrils in existence which are used for research purposes. Moreover, there are many other proteins that are capable of forming fibrillar deposits when taken out of physiologic conditions but do not have the ability to cause amyloidosis in the human body. Another organizational diVerence between diVerent types of amyloid is at the number of protofilaments that form the amyloid fibril. The amyloid fibril, visible in vivo, is composed of anywhere from three to six protofilaments

115 Å 24 b-strands

FIG. 1. Molecular model of the common core protofilament structure of amyloid fibrils. A number of b‐sheets (four illustrated here) make up the protofilament structure. These sheets run parallel to the axis of the protofilament, with their component b‐strands perpendicular to the fibril axis. With normal twisting of the b‐strands, the b‐sheets twist around a common helical axis ˚ containing 24 that coincides with the axis of the protofilament, giving a helical repeat of 115.5 A b‐strands (this repeat is indicated by the boxed region). Reprinted from Ref. [7], Copyright 1997, with permission from Elsevier.

5

AMYLOIDOSIS

A Gln5

B

Gly1

Asn3

Asn2

Tyr7

Gln4

Asn6

Asn3 Gln5

4.87 Å

a

b

Gln5 Asn3

c C

Gln5

Asn3

Asn2

Tyr7

Gln4

b c D

a

a c

c

Wet interface Dry interface

E

Asn6

Asn2

Gln4

b

3.0 2.8

2.9

2.9

2.9

3.0 2.9 2.8

Gly1 Gln5

Asn3 2.9

2.9

2.8 2.9 2.8 2.9

3.1

3.1

Tyr7

3.2 2.8

2.8 3.0 2.9 2.7 3.2 2.9

2.9

2.8

2.8 3.0 2.9 2.7 3.2 2.9

2.9

3.2

2.8

a FIG. 2. Structure of GNNQQNY, a seven‐residue peptide segment from the yeast protein Sup35. Unless otherwise noted, carbon atoms are colored in purple or grey/white, oxygen in red, and nitrogen in blue. [9] (A) The pair‐of‐sheets structure of the fibril‐forming peptide GNNQQNY. The dry interface is between the two sheets, with the wet interfaces on the outside

6

SIDERAS AND GERTZ

which are laterally associated with each other. For example, the amyloid fibril of Val30Met TTR, the most common type of familial amyloidosis, is composed of four protofilaments arranged in a square array around an electron‐lucent center, whereas the amyloid fibril of amyloid Ab (which causes Alzheimer’s disease) is composed of five or six protofilaments [11]. 3.2. AMYLOID AGGREGATION MECHANISMS Despite this knowledge, advances in understanding the amyloid fibril at the atomic level have been more diYcult. It is not clear, for example, if the specific amino acid sequence plays a role in determining the ability of a protein to form a cross‐b spine, to what extent it aids in the stabilization of the amyloid fibril, and how this sequence aVects interaction of the amyloid fibril with the microenvironment (i.e., amyloid P component, heparan sulfate proteoglycans (HSPG), apolipoprotein E, extracellular matrix, lipid bilayer). A number of models have been proposed in this regard [12] (Fig. 3). Partially folded intermediates are thought to play an important role in the pathogenesis of amyloidosis. It is thought that thermodynamic instability of the protein native structure leads to partially folded intermediates several of which have been shown to form amyloid fibrils readily [13, 14]. The same mechanism is thought to lead to amorphous deposits of immunoglobulin light chains and it is unclear why some proteins favor deposition as amyloid fibrils versus amorphous deposits. The process appears to be dependent on the specific partially folded structure as diVerent intermediates of the same protein have been shown to deposit either as amyloid fibrils or amorphous deposits [15]. At a diVerent organizational level, oligomers can act as intermediates in the process of fibrilogenesis as well. For example, in the case of b‐2‐microglobulin, the formation of the final amyloid fibril is proceeded by the

surfaces. (B) The steric zipper viewed edge on (down the a‐axis). (C) The GNNQQNY crystal viewed down the sheets (i.e., from the top of panel a, along the b‐axis). Six rows of b‐sheets run horizontally. Peptide molecules are shown in black and water molecules are represented by redþ. ˚ ) alternating with a wider Notice that the sheets are in pairs, with a closely spaced pair (8.5 A ˚ ) pair. The wide spaces between sheets (wet interfaces) are filled with water spaced (15 A molecules, whereas the closely spaced interfaces (dry interfaces) lack waters, other than those hydrating the caroboxylate ions at the C‐termini of peptides. The atoms in the lower left unit cell are shown as spheres representing van der Waals radii. (D) The steric zipper. This is a close up view showing the remarkable shape complementarity of the Asn and Gln side chains protruding into the dry interface. (E) Views of the b‐sheets from the side (down the c axis), showing three b‐strands with the interstrand hydrogen bonds. Side chain carbon atoms are highlighted in yellow. Backbone hydrogen bonds are shown by purple or black dots and side chain hydrogen bonds by yellow ˚ units. Reprinted from Ref. [9], Copyright dots. The length of each hydrogen bond is noted in A 2005, with permission from Macmillan Publishers Ltd.

7

AMYLOIDOSIS TABLE 1 SOME OF THE PROTEINS KNOWN TO CAUSE CLINICAL AMYLOID DISEASE IN HUMANS

Precursor protein

Human disease

Major causative association

Major clinical manifestation Renal, cardiac, GI, peripheral nervous system Much less frequent than AL amyloidosis Renal

Immunoglobulin light chain

Primary (AL) amyloidosis

Plasma cell disorders

Immunoglobulin heavy chain Serum amyloid A

Primary (AH) amyloidosis Secondary (AA) amyloidosis Familial

Plasma cell disorders

Transthyretin (TTR)

Inflammation Mutation of ATTR

Senile

Wild‐type ATTR, aging

Apolipoprotein AI

Familial

Mutation of Apo‐AI

Apolipoprotein AII

Familial

Lysozyme

Familial

Fibrinogen Aa‐chain

Familial

Gelsolin

Familial (Finish type)

Point mutation of stop codon leading to additional 20 amino acid residues Mutation of lysozyme Mutation of fibrinogen Aa‐chain Mutation of gelsolin

Cystatin C

Familial (Islandic type)

Mutation of cystatin

b‐2‐Microglobulin

Ab2M

Hemodialysis

Peripheral nervous system, heart Multiple organs, cardiac most clinically prominent Very slowly progressive disease Renal

Kidney, liver, lungs, and spleen Renal

Corneal dystrophy, cranial neuropathy, and cutis laxa Amyloid angiopathy and cerebral hemorrhage Joints

formation of dimeric, tetrameric, and hexameric intermediates in time scales much faster than the time scale of fibrilogenesis itself (minutes to hours versus weeks to months) [16]. In this model, the monomer (b‐2‐microglobulin) requires activation to an ‘‘activated state’’ (in the presence of copper) which then rapidly forms oligomers, by the sequential addition of dimmers, within minutes to hours. These oligomers form by the process known as domain swapping. Oligomers then form mature amyloid in timescales of weeks to months.

8

SIDERAS AND GERTZ

Model class

Native protein

Intermediate

Fibril

Refolding

Natively disordered

Gain-ofinteraction

FIG. 3. Cartoon depicting the three general types of models for the conversion of proteins from their native state to the amyloid‐like state. In refolding models, the protein unfolds and then refolds into a diVerent structure, which is stabilized largely by backbone hydrogen bonds. In natively disordered models, the cross‐b spine forms from protein segments that are poorly structured in the native state. In gain‐of‐interaction models, a change in the conformation of the protein frees a segment for interaction with segments from other molecules. An extensive portion of the native structure is maintained in the fibril. Reprinted from Ref. [12], Copyright 2006, with permission from Elsevier.

There are also kinetic intermediates of the amyloid fibril formation that are called protofibrils (not to be confused with the protofilaments we discussed earlier which are distinct substructural elements of the amyloid fibril) [17]. These protofibrils are in equilibrium with monomeric or dimeric forms of Ab molecules (in the case of Alzheimer’s disease), and tend to accumulate during fibrillogenesis on preformed fibrils and transition into mature fibrils themselves [16, 18]. In vitro, protofibrils have been shown to grow by both monomer elongation and by lateral association [19]. Recently, time‐resolved structure analysis of growing b‐amyloid fibrils, focusing on the first 2 h of fibrilogenesis, shows additional nonfibrillar intermediates present at these early stages in addition to the monomers and the protofibrils just described [20]. The propensity of diVerent proteins to form amyloid fibrils may be relatively common. However, what determines the ability of proteins to deposit in human tissues depends on the kinetics and thermodynamics of the specific ‘‘fibrillar state,’’ posttranslational modifications of the protein (i.e., proteolysis, as is the case with the immunoglobulin light chains and other proteins where only a fragment makes up the amyloid fibril), and interactions of the amyloidogenic protein with its microenvironment, namely the

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extracellular matrix, serum amyloid P (SAP) component, glycosaminoglycans, apolipoprotein E, and the lipid membrane bilayer itself [21]. In addition, the amyloidogenic protein is generally produced in suYcient amounts by an independent pathologic process (i.e., plasma cell proliferative disorders in the case of AL amyloidosis or inflammation in the case of AA amyloidosis) before a clinical amyloidosis syndrome can result from any given protein. This overproduction, however, does not always need to be the case since in certain situations, as in the case of some patients with senile amyloidosis, the plasma concentration of the amyloidogenic protein is in fact less than normal, but in suYcient quantity to result in the clinical syndrome [22]. Another mechanism for production of amyloidogenic proteins includes specific genetic mutations that make an otherwise normal protein carry the ability to interact with the microenvironment in a way that makes amyloid fibril formation and deposition possible as in the case of TTR. TTR also has the ability to form amyloid deposits in its wild form with advanced age and in this case it is unclear what triggers its deposition. Proteolytic cleavage is also an important part of amyloid formation for some of the amyloid‐forming proteins. For example, in AL amyloidosis it is usually only a variable region, a J‐segment and a variable part of the constant region that forms the amyloid fibril rather than the full length immunoglobulin light chain. SAA, which is responsible for amyloid formation in response to inflammation (secondary or AA amyloidosis), does so only after cleavage of the 76‐residue N‐terminal part of the protein. The requirement for proteolytic cleavage need not be exact and fragments of variable length can be found in amyloid deposits. In the case of Alzheimer’s disease, however, the proteolytic cleavage of the amyloidogenic protein APP needs to be at exact locations before the pathologic fragments Ab 1–40 and Ab 1–42 are formed. Figure 4 schematically depicts some of these mechanisms of amyloid production, from abnormalities at the genetic level, to requirements for posttranslational modification, to overproduction of the amyloidogenic protein.

3.3. INTERACTIONS OF THE AMYLOID FIBRIL WITH THE MICROENVIRONMENT Despite the knowledge of the proteinaceous nature of the amyloid fibril, as early as 1894 glycosaminoglycans were suspected to be part of amyloid deposits by the Italian surgeon and anatomist Ruggero Oddi. In fact, later immunohistochemical analysis of amyloid deposits, found other nonfibril‐

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X

Abnormal gene Amyloid fibril Post-translational events (s)

Expression

Degradation

Amyloid fibril

FIG. 4. Mechanisms for amyloid fibril formation are shown schematically. Open circles represent normal protein molecules, solid circles are amyloidogenic protein molecules, and overlapping circles are the fibrils made from these proteins. Reprinted from Ref. [154], reproduced with permission from Elsevier.

forming proteins in close association with the amyloid fibril. The glycoprotein ‘‘SAP component,’’ glycosaminoglycans (mainly heparan sulfate), and apoprotein E have been found in amyloid deposits of diVerent types of amyloidosis [2]. Amyloid P component is a pentameric (thus the P) globular protein that binds reversibly in a Ca2þ dependent fashion to amyloid fibrils [23] and it is a universal constituent of amyloid deposits. It constitutes anywhere from 12% to 20% of the dry weight of amyloid deposits [24, 25]. It is a normal plasma protein (SAP component) which, like C‐reactive protein, is a small pentraxin produced in the liver. It appears to function physiologically as a key component of innate immunity and inflammation [26]. Occasionally, it can form fibrillar deposits itself and causes a form of amyloidosis secondary to inflammation. However, its main interest lies with its nonfibrillar native form and the nature of its association with the amyloid fibril. When 123I‐labeled SAP component is injected, patients with amyloidosis clear the radiolabeled SAP from the plasma more rapidly, but retain it in tissues for significantly longer periods, indicating its rapid localization to amyloid deposits [27]. In fact, this property of amyloid P component has been used clinically

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for purposes of diagnosis by using scintigraphy to detect the accumulated amyloid P component [28] (Fig. 5). The diagnostic specificity in one study was 90% for AL and AA amyloidosis and only 48% for ATTR‐type amyloidosis with a sensitivity of 93%.

FIG. 5. Total body 123I‐SAP scan, anterior (A) and posterior (P) view, 24 h after injection. (A) Male control. Blood pool activity as well as minor nonspecific uptake can be seen in the (blocked) thyroid, nasopharynx, stomach, urine in bladder, and testicles. (B–F) Five patterns of organ uptake in AA amyloidosis: (B) kidney uptake only (and prostatism with urine retention in bladder); (C) intense splenic uptake only; (D) spleen and kidney uptake; (E) spleen, kidney, and adrenal gland; (F) spleen, kidney, and liver uptake (and nonspecific uptake in the stomach adjacent to the left liver lobe, providing the illusion of liver enlargement). (G–I) Three examples of organ uptake in AL amyloidosis: (G) joints only (8%); (H) spleen and liver (20%); (I) spleen, liver, and bone marrow (10%). Reprinted from Ref. [28], Copyright 2006, with permission from Elsevier.

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The role of SAP component is not understood but it appears to prevent the degradation of amyloid fibrils by proteolytic enzymes [25]. It is unclear how it is structurally related to the amyloid fibril and whether it plays a role in the formation of the amyloid fibril or simply stabilizes the fibril once it is formed. HSPG have also been found to be closely associated with amyloid fibrils. Glycosaminoglycans have been isolated from water extracted amyloid fibrils as well as directly from amyloidogenic tissues [29]. Glycosaminoglycans have been shown to promote the aggregation of amyloidogenic proteins [30]. Again the precise mechanism of interaction is unclear. One theory suggests that the chemical properties of glycosaminoglycans as polyanions have an important role in catalyzing protein aggregation and stabilization of amyloid fibrils. This is supported by the evidence that nucleic acid, another polyanion, has been isolated from the brains of patients with Alzheimer’s disease, a form of amyloid deposition disease [31]. In fact other polyanions, like ATP, DNA, and heparin are also able to promote aggregation of amyloidogenic proteins [30]. Although the precise role and interaction of these nonfibrillar components is not well understood, it is noteworthy that in vitro amyloid can be formed without the aid of glycoproteins and glycosaminoglycans adding confusion to their role in the pathogenesis of amyloid. 3.4. TROPISM OF AMYLOID PROTEINS FOR DIFFERENT ORGANS Very little is understood on why diVerent amyloidogenic proteins ‘‘prefer’’ to deposit in diVerent organs. Although multiple associations have been made, we have been unable to explain the reasons behind amyloid tropism. For example, familial TTR amyloidosis aVects primarily the peripheral nervous system while native TTR, which causes senile amyloidosis, aVects primarily the heart when present in suYcient quantities to cause disease. Mutations of Apolipoprotein AI lead to a rare form of familial amyloidosis. Patients with mutations at the amino‐terminal present with renal, hepatic, and occasional cardiac amyloid, while patients with mutations at the carboxy‐terminal present with cardiac, cutaneous, and laryngeal involvement [32]. No success in explaining this striking observation has yet been made. It is also well known that certain subtypes of l light chain amyloidosis carry a propensity to aVect diVerent organs [33]. In a retrospective study of 60 patients with AL amyloidosis patients with clones derived from the 6a V (lambda VI) germ line gene were more likely to present with dominant renal involvement, whereas those with clones derived from the 1c, 2a2, and 3r V (lambda) genes were more likely to present with dominant cardiac and multisystem disease [33]. Patients with V (kappa) clones were more likely to have dominant hepatic involvement and patients with multiple myeloma

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were more likely to present with dominant cardiac involvement independent of which germ line gene was responsible for the plasma cell clone producing the amyloid [33]. Again, despite this knowledge, no specific theory has explained these associations. 3.5. MECHANISMS OF ORGAN DYSFUNCTION One of the ways amyloid can cause organ dysfunction is through anatomic interference or destruction of the involved organ. In this case, the organ dysfunction is directly proportional to the amount of amyloid deposited. For example, in the heart, amyloid deposition can occur within the cardiac muscle interfering with the hearts ability to contract properly leading to heart failure. Deposition within the conducting system leads to fatal arrhythmias, a main cause of death for this population. Deposition of amyloid in the kidney interferes with the structure and function of the glomerulus causing heavy proteinuria, or interferes with the tubular system causing azotemia and nephritis. Deposition in the arterial vasculature causes friable vessels which can result in life threatening bleeding (another cause of bleeding diathesis involves the deposition of the procoagulant factor X into the amyloid deposits leading to systemic deficiency of factor X). However, the organ dysfunction is not always proportional to the amount of amyloid present. For example, even small amounts of light chain can cause organ dysfunction. In this case it is thought that the amyloid fibril is ‘‘toxic’’ to the organ involved. This is especially true in the case of AL amyloidosis and is part of the reason that a partial response after treatment of the disease may not lead to clinical improvement of the involved organs. Organ improvement is usually the case only after a relatively complete response and elimination of the production of amyloid. After disease relapse, the clinical manifestations of organ dysfunction return. In fact there is ample evidence to suggest that, at least in certain situations, it is the protofibril and oligomeric intermediates that are toxic to organs rather than the mature amyloid fibril itself. For example, transgenic mice that overexpressing the Ab‐peptide of Alzheimer’s disease have evidence of neural degeneration prior to the formation of amyloid plaque, suggesting the presence of toxic intermediates [34]. In fact, nonfibrillar oligomers and protofibrillar intermediates of the Ab‐peptide have been shown to directly induce neurotoxicity [35, 36]. These findings may explain why the plaque load in Alzheimer’s disease does not correlate well with the severity of disease. In another example when neuroblastoma cell lines have been treated with TTR, the protein that causes familiar amyloidosis, it is the immature amyloid which has not yet properly aggregated that causes cell death rather than the mature amyloid fibril [37].

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4. Diagnosis 4.1. SUSPECTING THE DIAGNOSIS One of the greatest challenges in amyloidosis is suspecting the diagnosis. The majority of the signs and symptoms are not pathognomonic. On the contrary, signs and symptoms are nonspecific and they are shared by many other more common diseases. The symptoms are generally directly related to the organs involved. As a result renal involvement, which is the most commonly involved organ in amyloidosis, is expected to present with nephrotic range proteinuria resulting in edema, weight loss, and fatigue. Peripheral nerve involvement causes paresthesias, and cardiac involvement commonly causes dyspnea, edema, and weight gain. Gastrointestinal involvement can cause a variety of symptoms including malabsorption with resulting weight loss, diarrhea, or less commonly pseudo‐obstruction of the upper gastrointestinal tract. Senile amyloidosis commonly aVects the heart, and in the case of Alzheimer’s disease amyloid deposits in the brain lead to cognitive decline. In less common situations amyloidosis has been known to involve almost any organ, and in these cases the clinical manifestations are more atypical. Pathognomonic findings exist in amyloidosis but they are uncommon, and when they occur they are easily overlooked. For example, tongue enlargement, periorbital purpura, and periarticular amyloid infiltration (shoulder pad sign), although specific to amyloidosis, are only present in 15% of patients with AL amyloidosis. Thus, a high index of suspicion is needed by any clinician faced with these symptoms. In certain clinical scenarios, on the other hand, the diagnosis of amyloidosis should always be suspected. Nondiabetic patients who present with nephrotic range proteinuria should always be screened for amyloidosis since 10% of these patients are found to have the disease [38]. Patients with evidence of cardiomyopathy without symptoms of ischemia or evidence of atherosclerotic heart disease should also be screened for amyloidosis. Findings in the electrocardiogram such as low voltage and the pseudo‐infarction sign as well as specific findings in the echocardiogram such as wall thickening and poor filling in the absence of systemic hypertension, can lead clinicians to the diagnosis. Other clinical scenarios include peripheral neuropathy in a nondiabetic patient, unexplained hepatomegaly, patients with tongue enlargement, and patients with unexplained malabsorption, weight loss, or pseudo‐obstruction. Finally, patients with established multiple myeloma and symptoms that are not consistent with myeloma should also be screened always.

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4.2. SCREENING FOR AMYLOIDOSIS Tissue biopsy is the only method available for establishing the diagnosis of amyloidosis. However, tissue biopsy sometimes is diYcult to perform and can carry significant risks especially if organs like the heart, kidney, or liver are involved. In these cases screening for amyloidosis first is desirable. However, it needs to be kept in mind that noninvasive screening is available only for AL amyloidosis. This is due to the fact that in AL amyloidosis detection of the underlying plasma proliferative disorder and immunoglobulin light chains is possible. No such neoplastic underlying conditions exist for AA amyloidosis, hereditary amyloidosis, or localized amyloidosis, and as a result biopsy is the only means of detecting the disease. If screening is negative in a patient where there is a high index of suspicion for amyloidosis, as is the case of a patient with characteristic echocardiographic findings of cardiac amyloid but negative screening, a biopsy needs to be performed. The current standard for screening for AL amyloidosis is testing for the presence of immunoglobulin light chains by immunofixation of both the serum and urine and by a free immunoglobulin light chain assay. A commonly occurring error in clinical practice is screening for amyloidosis by serum or a urine protein electrophoresis without immunofixation. The reason is that although serum protein electrophoresis is a good test for screening for the circulating heavy chain produced by multiple myeloma cells, it generally does not detect light chains, because light chains are in small quantities in the serum and do not produce a spike on the electrophoresis. On the contrary, immunofixation allows for the identification of monoclonal proteins in the serum and/or the urine in 90% of cases of AL amyloidosis. Also, the reason for testing both the serum and the urine is that 25% of patients with AL amyloidosis that have a negative serum immunofixation will be found to have a positive urine immunofixation alone. However, the most sensitive test for screening for amyloidosis is an immunoglobulin free light chain assay [39]. This technique, which uses a nephelometric assay with antibodies that recognize only free light chains not bound to heavy chain, has the ability of detecting a quantitatively abnormal k or l free light chain population or an abnormal k to l ratio in 99% of patients with AL amyloidosis. In patients with a negative serum but positive urine immunofixation, the free light chain assay detects abnormalities in over 80% of patients. Even in patients with known amyloidosis, and both negative serum and urine immunofixation, the quantitative immunoglobulin light chain assay can detect abnormalities in 86% of patients with k and in 30% of patients with l light chain amyloidosis.

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4.3. ESTABLISHING THE DIAGNOSIS The diagnosis of amyloidosis can only be confirmed with tissue biopsy. The pathognomonic finding on biopsy is the presence of green birefringence on Congo red staining. The diagnosis requires pathologists experienced with the technique since it is not uncommon for patients to be given a diagnosis of amyloidosis and in further review of the biopsy to be found that the patient does not in fact have amyloidosis. Over fixation and trapping of the Congo red stain can result in false positives. Also staining of collagen and elastin in the skin and fat can be confused with amyloid tissue; however, in these cases there is no green birefringence. Congo red staining is a highly specific technique for diagnosing amyloidosis with a specificity of 100% in experienced hands. However, it is not as sensitive nor does it allow typing of the amyloid protein. The technique can be combined with immunohistochemistry and Congo red fluorescence to improve sensitivity. Congo red fluorescence can detect small amounts of amyloid deposits, does not interfere with immunohistochemical staining [40], and can be successfully applied in frozen sections. Typing of the amyloid (identifying the specific protein involved) cannot be done with the Congo red stain since all forms of amyloid deposits are identical at this level. Immunohistochemical staining of tissues with commercially available antisera is needed for such identification. Amyloid P component, which is present in all amyloid deposits irrespective of the specific protein involved, acts as a positive control. In contrast, apolipoprotein E does not appear to be uniformly present [41]. Immunohistochemical analysis has sometimes identified patients with amyloidosis of diVerent kinds that just happen to have an underlying plasma cell proliferative disorder. In one particular study as many as 10% of patients who were thought to have AL amyloidosis were found to have diVerent types of amyloidosis [42]. This included 5% of patients who had fibrinogen amyloid and 4% who had hereditary amyloidosis with mutations of TTR. Although biopsy of the organ involved is highly specific for the diagnosis of amyloidosis, this is not always desirable. Commonly involved organs include the kidney, heart, and liver and biopsy of these organs can lead to significant morbidity. Although fine needle aspirate can occasionally replace core needle biopsy in the diagnosis of amyloidosis thus reducing the risk of the procedure, biopsy of easily accessible sites is more desirable [43, 44]. For example, amyloidosis in patients with renal disease can be successfully diagnosed with duodenal biopsies, which are easier to perform [45]. Similarly, labial salivary gland biopsy has been used to diagnose amyloidosis in patients presenting with polyneuropathy [46]. Other sites that have been routinely used for the diagnosis of amyloidosis include the rectum and the skin.

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However, the most commonly used technique today for the diagnosis of amyloidosis is a subcutaneous fat aspirate [47]. Fat aspiration is an easy technique to perform, causes minimal discomfort to the patient, allows for diagnosis within 24 h and has an acceptable sensitivity ranging from 58% to 84% with a specificity of 99–100% [47–49]. Although weak nonspecific staining and collagen birefringence can lead to false positives [50], fat aspiration is the established diagnostic technique for suspected amyloidosis.

4.4. TYPING OF AMYLOID Because the treatment of the various forms of amyloidosis varies considerably, and multiple patients are known to have received the wrong therapy (i.e., chemotherapy for patients with secondary, hereditary, or localized amyloidosis who happen to also have an unrelated paraproteinemia) typing of the amyloid fibril is of the utmost importance in situations when a paraproteinemia is not found or the patient presents in an atypical location of disease. For example, involvement of the skin, larynx, tracheobronchial tree, vocal cords, bladder, urethra, ureter, macula, orbits, conjunctiva, atria of the heart, lung, pleura, and articular cartilage should raise the suspicion of localized amyloidosis. Renal amyloidosis could be due to immunoglobulin light chain deposition, AA amyloid deposition, as well as various hereditary mutant proteins making the need for proper typing important in situations when the diagnosis is not clear [51]. Because correct typing requires considerable amount of tissue, several microtechniques have been developed aiming to use smaller amounts of protein. Microextraction and micropurification techniques have successfully been used to extract and purify amyloid fibrils both from fresh tissue as well as from formalin fixed tissue [52, 53]. Several techniques have been used to identify the purified amyloid deposits. Although immunohistochemistry is the most commonly used of these techniques, there are several pitfalls that make its use controversial. The antibodies used for immunohistochemistry have poor sensitivity in detecting light chain amyloid fibrils although generally the sensitivity is much better for other types of amyloidosis. Although various synthetic peptides have been developed aiming at increasing the ability of immunohistochemical techniques to recognize light chain amyloid fibrils, in up to a third of the patient’s, immunohistochemistry can be negative or equivocal [54, 55]. This is partly due to the fact that the amyloid deposits are composed of the N‐terminal fragment of the light chain where most commercial antisera recognize the constant portion of the light chain. Inconsistent immunolabeling reactions and nonspecific background staining leads to some of these problems as well.

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Attempts at developing suitable antibodies against immunoglobulin light chains for use in immunohistochemistry are underway [56]. AA amyloidosis deposits are easily recognized by immunohistochemistry and thus the diagnosis is easily confirmed or excluded by this method. However, staining quality and observer experience always remain an issue and attempts to develop techniques with improved diagnostic accuracy are ongoing. One such technique involves quantification by ELISA of SAA using monoclonal antihuman serum amyloid A antibodies which was recently reported to have a sensitivity and specificity of 84% and 99%, respectively [57]. In another study ELISA has been used successfully in typing amyloid from subcutaneous fat biopsies correctly in 14 out of 15 patients studied [49]. Various other laboratory techniques exist for typing amyloid deposits [58]. This includes deposits in formalin fixed specimens [59]. Recent improvements in micromethods have enabled tandem mass spectrometry to precisely identify the protein nature of the pathologic deposits [60]. Mass spectroscopy, looking for TTR molecules with abnormal molecular weight, has been particularly helpful in classifying deposits in cardiac amyloidosis. Gene expression analysis can identify unique molecular profiles that can be used to discriminate AL amyloidosis from other subtypes [61]. Restriction fragment length polymorphism, various polymerase chain reaction techniques, single strand confirmation polymorpism, and nucleotide sequencing can recognize mutations in proteins that cause familial amyloidosis [58]. Ultrastructural studies of abdominal fat samples using immuno‐electron microscopy has shown significant specificity by correctly identifying amyloid deposits in 15 out of 15 patients in one study [62]. Western blot analysis combined with specific amyloid fibril protein antibodies has characterized successfully 32 out of 35 abdominal fat biopsies with amyloid deposits [63]. Proper typing, using one or a combination of the above techniques, is of the outmost importance in familial amyloidosis. This is true because family history is an inaccurate screening tool since half the patients have no family history and misdiagnosing patients with true familial amyloidosis as having AL amyloidosis is not uncommon when an unrelated monoclonal gammopathy is present [64, 65]. In the case of a previous unknown mutation a combination of the above techniques is necessary to accurately provide the diagnosis. 4.5. IMAGING OF AMYLOID Echocardiography, magnetic resonance imaging, radionuclide imaging, and radioiodinated amyloid P component scan are various imaging techniques used to detect amyloidosis in tissues. Echocardiography can diVerentiate amyloid heart disease from other types of cardiomyopathy. In general,

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thickening of the left ventricular wall with reduced left ventricular compliance (diastolic dysfunction) is the most common finding. This is clinically followed by restrictive cardiomyopathy and systolic dysfunction. Amyloid fibrils depositing into the myocardium can cause local ischemia and disruption of the conducting system which eventually leads to arrhythmias, a common cause of death in these patients. Specific echocardiographic findings associated with cardiac amyloidosis include thickened interventricular septal wall and ventricular wall, reduced left ventricular systolic and diastolic diameters, restrictive physiology, and a characteristic granular appearance of the myocardium. When increased interventricular septum wall thickness is combined with the electrocardiographic findings of low voltage the pattern is highly suggestive of amyloid infiltrative cardiomyopathy [66]. When conventional echocardiography is combined with newer techniques, such as tissue Doppler and myocardial strain rate imaging, earlier stages of cardiac pathology can be detected [67, 68]. Similarly, with cardiac MRI, characteristic patterns of gadolinium kinetics and subendocardial late enhancement are specific to amyloid cardiomyopathy and thus cardiac MRI has found a clinical role in the diagnosis of this disease [69–71]. MRI can also aid in the identification of amyloid in bone thus helping to diagnose amyloid arthropathy [72]. Various radionuclide imaging techniques have been successful in detecting amyloid deposition with impressive results. Mean washout rates of Thallium‐ 201 from the heart can correlate with the severity of the disease [73]. Single proton emission computed tomography (SPECT) using Gallium‐67 and Thalium‐201 can diVerentiate active from inactive amyloid deposits in patients with dialysis‐associated amyloidosis [74]. Technetium labeled N2S2 conjugates of chrysamine G appear to have specificity for renal amyloidosis [75]. Technetium aprotinin has been shown to diVerentiate TTR‐related amyloidosis from AL amyloidosis of the heart [76]. Radioiodinated SAP component (I‐123 SAP) localizes in amyloid infiltrated tissues in proportion to the amount of disease. Patients with amyloidosis clear I‐123 SAP from the plasma quicker and show increased extravascular retention and interstitial exchange rate of I‐123 SAP. This characteristic has been used for evaluating the distribution of amyloidosis for prognostication and identifying possible biopsy sites as well as for monitoring the disease in a safe and noninvasive way [27, 77, 78]. Recent advantages in molecular imaging have been made through the use of small molecules that bind amyloid fibrils in a specific manner. Four of these small molecules (18F‐FDDNP, 11C‐PIB, 11C‐SB13, and 11C‐BF‐227), called positron emission tomography (PET) ligands, have found their way into imaging of amyloid tissue in patients with Alzheimer’s disease [79]. Specifically, 11C‐PIB, otherwise called Pittsburg compound B, has been

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shown in several studies to be highly retained in frontal, temporal, parietal, and occipital cortices and the striatum of patients with early Alzheimer’s disease compared to healthy controls [80]. It is noteworthy that as years go by the retention of the Pittsburg compound B in the brains of Alzheimer’s patients does not increase despite clinical worsening of the disease [81]. Thus, since the amyloid load in any given patient does not seem to directly correlate with clinical disease status, neural damage seems to be caused very early in the disease process. Also, as we discussed previously in Section 3.5, these findings could argue for the presence of toxic intermediates that do the damage instead of the mature amyloid fibril.

5. Classification, Clinical Presentation, and Prognosis of Amyloidosis 5.1. PRIMARY AMYLOIDOSIS Patients with AL amyloidosis have a detectable (by immunofixation) monoclonal light chain in the serum or urine and/or a detectable circulating free light chain level in addition to a clonal plasma cell disorder in the bone marrow. The bone marrow clonal plasma cell proliferative disorder, responsible for the generation of the amyloid paraprotein need not be of large quantity, as in the case of multiple myeloma, and in fact it is typically small. Patients with amyloidosis do not go on to develop multiple myeloma if it is not present at the time of diagnosis indicating the separate pathogenesis of these diseases. Three quarters of patients with amyloidosis present with l light chain restricted disease in contrast to MGUS where two‐thirds of patients are k light chain restricted, indicating an intrinsic amyloidogenic potential of l light chain immunoglobulin fragments. This appears to be due to the increased amyloidogenic potential of certain l light chain genes. Specifically, the genes 3r and 6a, belonging to the lIII and lVI families, encode 42% of amyloid variable l regions and can potentially account for the overrepresentation of the l restricted disease seen in AL amyloidosis [82]. Two‐thirds of patients with AL amyloidosis are male, and the median age of presentation at the Mayo Clinic is 67 years [83]. The most common organs involved clinically include the heart in 37% (with about half presenting with symptoms of heart failure), the kidney in 27% (with nephrotic range proteinuria being the most common manifestation), and the peripheral nervous system in 15% of the patients (presenting as paresthesias, carpal tunnel syndrome, and pain). The peripheral neuropathy is frequently axonal and demyelinating [84]. Hepatomegaly is seen in 17% of the patients but it is

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clinically dominant only in about 5%. Finally, gastrointestinal involvement in the form of pseudo‐obstruction, bleeding, and diarrhea is seen in 7% of patients. Other rarer presentations include involvement of the tongue, joints, and soft tissues. Of all the known prognostic factors cardiac involvement is the most important. Median survival of patients with cardiac amyloidosis is measured in months, where if there is no significant cardiac involvement the median survival is measured in years [85]. A prognostic model using biomarkers of cardiac injury (Troponin T or I and N‐terminal probrain natriuretic peptide) has been developed in patients with AL amyloidosis [86]. Specifically, patients are separated into three stages depending whether none (Stage I), one (Stage II), or both (Stage III) of these cardiac biomarkers are elevated with a median survival of 26.4, 10.5, and 3.5 months for each stage, respectively [87]. This model holds true for patients with amyloidosis undergoing bone marrow transplantation as well [86]. In bone marrow transplantation specifically the absolute value of the immunoglobulin free light chain is another important prognostic indicator [88]. Another important prognostic marker includes b‐2‐microglobulin despite the fact that in amyloidosis, unlike multiple myeloma, the overall disease burden is low indicating that b‐2‐microglobulin is not simply a reflection of neoplastic burden. 5.2. SECONDARY AMYLOIDOSIS Secondary amyloidosis is caused by the deposition of SAA fragments during a sustained inflammatory state. Serum amyloid A is an acute phase reactant whose levels can increase 1000‐fold during an acute inflammatory reaction [89]. Serum amyloid A is a 104 amino acid long apolipoprotein that binds to high‐density lipoprotein (HDL) particles during inflammation and replaces apolipoprotein A‐I which normally is the major apolipoprotein bound to HDL [90]. The reason for this binding is unclear but the SAA‐rich HDL may be involved in manipulating cholesterol metabolism and macrophage chemoattraction during inflammation [91, 92]. The amyloidogenic N‐terminal fragment of serum amyloid A can be produced by macrophage‐ induced proteolytic cleavage of SAA at position 76 in vitro [93]. Thus macrophages appear to have a central role in the pathogenesis of AA amyloidosis. SAA is actually a family of proteins with SAA1 and SAA2 being the two loci at the genetic level thought to be involved with amyloidogenesis due to their function as acute phase reactants. AA amyloidosis is seen during longstanding chronic inflammatory conditions such as rheumatoid arthritis, juvenile idiopathic arthritis, and ankylosing spondylitis, during chronic infections such as tuberculosis, osteomyelitis,

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syphilis, bronchiectasis, leprosy, and chronic infections associated with the paraplegic state (pressure sores, urinary tract infections), during periodic fever syndromes such as familial Mediterranean fever, TNF‐receptor‐ associated periodic fever syndrome, hyperimmunoglobulinemia D and periodic fever syndrome, cryopyrin‐associated periodic syndrome, and during other inflammatory conditions such as Crohn’s disease, cancer, Castleman’s disease, vasculitis, etc. In the western world AA is more strongly associated with rheumatoid arthritis in contrast to the rest of the world where infections are the most common cause. Amongst the periodic fever syndromes familial Mediterranean fever is the most frequent entity [94]. The most frequent organ involved is the kidney, in over 80% of patients and renal dysfunction has been described in up to 97% of patients [95]. The classical presentation of AA amyloidosis is a patient with rheumatoid arthritis who presents with nephrotic range proteinuria, although proteinuria is not the only renal manifestation of AA amyloidosis seen. Cardiac involvement can occur, although less commonly than in patients with AL amyloidosis. Hepatic involvement is clinically apparent in 9% of patients although SAP scintigraphy shows evidence of disease in up to 23% of patients [95]. Neuropathy is considered rare. Although the prognosis of AA amyloidosis is generally considered much better than AL amyloidosis, selected patients can have a rapidly deteriorating clinical course and poor prognosis. The predictors of poor outcomes are likely related to the ability to adequately control the underlining inflammatory condition. Cardiac involvement, as in the case of AL amyloidosis, again appears to predict for worse outcome with a 5‐year survival of 31% versus 63% for patients without cardiac involvement in a retrospective series of 42 patients from Japan [96]. In addition, the degree of renal involvement is important, with patients who have elevated creatinine levels doing worse compared to patients with a normal creatinine. The pattern of renal involvement is also important. Specifically, glomerular involvement with amyloid and fibrosis appear to have a clinical course characterized by deteriorating renal function compared to patients with other types of renal involvement [97, 98]. Generally, however, the median survival is over 5 years. In the largest prospective series of 334 patients from the United Kingdom (which excluded patients with cardiac involvement) elevated absolute annual median SAA concentration was strongly associated with poor outcome [95] (Table 2). End stage renal disease (RR 2.97) and older age (RR 1.53) were also associated with poor outcome where evidence of radiographic improvement by SAP scintigraphy (RR 0.13) and underlying periodic fever syndrome (RR 0.36) were associated with better outcome.

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AMYLOIDOSIS TABLE 2 UNADJUSTED RELATIVE RISK OF DEATH ASSOCIATED WITH THE MOST RECENT MEDIAN ANNUAL SAA CONCENTRATION [95] SAA (mg/l)

Relative risk (95% CI)

P value

<4 4 to <9 9 to <16.7 16.7 to <28 28 to <45.6 45.6 to <87 87 to <155 155

1.0 3.9 (1.5‐10.4) 5.1 (2.7‐9.4) 7.0 (3.7‐13.4) 9.1 (4.8‐17.2) 12.1 (6.9‐21.4) 17.0 (8.6‐33.8) 17.7 (8.7‐36.0)

0.007 0.003 0.07 0.008 <0.001 <0.001 <0.001

5.3. FAMILIAL AMYLOIDOSIS Familial amyloidosis is caused when inherited mutations (usually point mutations) lead to the production of proteins with amyloidogenic potential. The proteins known to cause amyloidosis by this mechanism are TTR, fibrinogen Aa‐chain, apolipoprotein AI, apolipoprotein AII, lysozyme, gelsolin, and cystatin C. TTR amyloidosis is the most common cause of familial amyloidosis in the world. FA syndromes can be divided into two main types based on their ability to cause peripheral neuropathy or not. TTR familial amyloidosis typically presents with peripheral neuropathy as the dominant symptom. This condition is often referred to as familial amyloidotic polyneuropathy (FAP). The only other protein that presents with peripheral neuropathy is the Gly26Arg mutation of Apolipoprotein AI. Of the 12 known Apolipoprotein AI mutations only the Gly26Arg mutation is known to cause peripheral neuropathy. On the other hand, the rest of the proteins do not cause peripheral neuropathy and are occasionally referred to as ‘‘Ostertag type’’ after Dr. Benno Ostertag’s description of a family in 1950. Proteins that cause familial amyloidosis are synthesized by diVerent organs and treatments vary significantly depending on the specific protein involved. 5.3.1. Transthyretin Over 100 diVerent mutations are known to be associated with TTR amyloidosis [99]. TTR was previously known as prealbumin due to its position in front of albumin during protein electrophoresis but it is now known to be a hepatically synthesized protein which functions as a carrier of thyroxine and retinol. The Val30Met mutation is the most common and frequently presents in large foci around the world [99] (Fig. 6). In northern Sweden it is reported that in a population of 500,000, 7500 people carry the mutation although the penetrance of the disease is only 2% [100]. The clinical phenotype varies even

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Sweden

Portugal

Japan

FIG. 6. Distribution of FAP in the world. Locations of foci of patients with FAP amyloidogenic mutated TTR Val30Met described in previous reports and obtained from personal communications are presented. The size of the circles is related to the number of patients at each location. Reprinted from Ref. [99], Copyright 2005, with permission from the American Medical Association.

amongst patients with the same type of mutation. Peripheral neuropathy is the most common presentation clinically manifesting in over half the known mutations followed closely by cardiac involvement. Cardiac involvement appears to be more dominant in late‐onset cases [101]. Peripheral neuropathy and cardiac dysfunction are often seen in the same patient. Other presentations include leptomeningeal and central nervous system involvement (cerebral hemorrhage, stroke, hydrocephalus, ataxia, paralysis, seizures, dementia), ocular involvement, autonomic involvement (sexual dysfunction, diarrhea, constipation, orthostatic hypotension, urinary incontinence), and carpal tunnel syndrome. The kidney, liver, and the skin are less frequently involved. The peripheral neuropathy usually begins with sensory symptoms of the lower extremities (especially changes in pain and temperature) followed by motor symptoms and muscle wasting. Amyloid deposits are present in the nerve trunks, plexuses, and ganglia, and fiber degeneration is axonal, beginning in the unmyelinated and small myelinated fibers [102]. Although patients with TTR amyloidosis live longer than patients with AL amyloidosis (median survival over 5 years) involvement of the heart, and the presence of significant peripheral neuropathy, are powerful predictors of survival, with the majority of patients dying of cardiac disease (heart failure or arrhythmias) or complications of progressive peripheral and autonomic neuropathy [65].

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5.3.2. Other Types of Familial Amyloidosis Mutations of Apolipoprotein AI can be at the amino‐ or carboxy‐terminal of the protein. It is generally a slowly progressive disease. Fibrinogen Aa‐chain amyloidosis is very common in Europe and presents with glomerular involvement of the kidney. Patients have hypertension and heavy proteinuria [32]. Glomerular involvement appears to also be the presentation with Apolipoprotein AII amyloidosis. Of interest is the fact that the point mutations in this type of amyloidosis lead to deletion of the stop codon of the protein and to the subsequent addition of 20 residues. The new protein with the additional 20 residues is responsible for the generation of the amyloidogenic peptide. The clinical presentation is one of slowly progressive glomerular and interstitial renal disease. Finally, mutations of lysozyme, a bacteriolytic enzyme produced by polymorphonuclear leukocytes and macrophages, leads to amyloid deposits in the kidney, liver, lungs, and spleen of patients [32]. Gelsolin amyloidosis is associated with a characteristic triad of corneal dystrophy, cranial neuropathy, and cutis laxa and it is known as the Finish type. Finally, cystatin C amyloidosis presents with amyloid angiopathy and cerebral hemorrhage and it is referred to as the Icelandic type.

5.4. SENILE AMYLOIDOSIS Senile amyloidosis is caused by deposition of normal, ‘‘wild’’ TTR in tissues, especially the heart and lung, in patients with advanced age. It is probably due to the amyloidogenicity of native TTR that patients with familial amyloidosis treated with liver transplantation can progress due to the deposition of wild‐type TTR in the heart [103]. Wild‐type TTR amyloid is an extremely common finding at autopsy of elderly patients, found in up to 25–28% of cases [104]. The atria, ventricles, aorta, the lung, and the gastrointestinal tract are most commonly involved and the involvement is generally asymptomatic. Renal involvement is unusual both at autopsy as well as clinically. The deposits are typically weakly stained by Congo red and organ involvement tends to be patchy making the diagnosis easy to miss [105, 106]. Massive wild‐type TTR deposition can occur during a patient’s lifetime causing symptomatic disease. The patients are almost invariably males over the age of 75. When this happens the most frequent organ involved is the heart. Peripheral neuropathy is rare despite the high prevalence of this presentation in patients with mutant TTR amyloidosis (familial amyloidosis). Also despite the fact that autopsy series find a very high prevalence of pulmonary alveolar and small blood vessel involvement, symptomatic disease from these anatomic locations is rare.

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Prognostically, patients with cardiac involvement do much better than their AL counterparts, despite indistinguishable pathologic and echocardiographic findings and advanced age (median survival over 5 years compared to only a few months in AL amyloidosis with cardiac involvement [105, 107]). Although various theories have tried to explain this paradox it is likely that the immunoglobulin light chain in AL amyloidosis may possess toxic eVects in the tissue independent of the structural damage caused by the amyloid deposition. 5.5. LOCALIZED AMYLOIDOSIS Localized amyloidosis is defined as amyloid deposition in one organ or tissue, without evidence of systemic disease, and after exclusion of a plasma cell proliferative disorder. The etiology for this presentation is unclear but the presence of a local population of plasma cells is hypothesized based on two observations. First, the amyloid deposits in these cases are almost invariably immunoglobulin light chain in nature (although SAP and TTR have also been found). Second, clonal plasma cells have been detected in the vicinity of amyloid deposits in some patients, and the amyloid has been linked to the plasma cell clones by DNA sequencing data [108–110]. However, in other situations the amyloid deposition has been found to have a heterogeneous sequence confusing the issue [111]. Although almost any anatomic location and tissue in the human body has been reported to present with amyloid deposition, in case reports presentation of localized amyloidosis is most common in the urinary bladder, tracheobronchial tree, the larynx, and the skin. It is the rule that patients with localized amyloidosis almost never present with subsequent systemic amyloidosis and after exclusion of systemic amyloidosis at diagnosis further follow up for the development of systemic amyloidosis is generally not needed (although follow up for recurrent localized amyloidosis is necessary). Patients with localized amyloidosis do not require systemic treatment and their prognosis is generally excellent. 5.5.1. Urinary Bladder Localized amyloidosis in the urinary bladder most frequently presents with hematuria but can also present with dysuria. Massive bleeding can occur on occasion. It can involve a single spot or it can be diVuse. Findings on cystoscopy are frequently misinterpreted as bladder carcinoma and the correct diagnosis is made only after immunohistochemical typing of biopsy specimens. In a series of 31 patients from the Mayo Clinic 24 out of 27 patients tested had immunoglobulin light chain disease and 3 had TTR [112]. Although localized symptoms from interventions and recurrence can be a problem for patients, many patients do not experience recurrence after treatment and patients typically die from other causes. About half the

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patients present with multiple recurrences, about one‐quarter have stable lesions and one‐quarter have no recurrence after intervention. 5.5.2. Tracheobronchial Tree and Larynx The number of patients in the literature with isolated tracheobronchial amyloidosis is just over 100 indicating the rarity of the disease. Presenting symptoms are cough, hoarseness, dyspnea, and hemoptysis. Patients are commonly treated for pneumonia, asthma, and bronchitis prior to diagnosis. It can involve the proximal, mid, or distal trachea and under bronchoscopy amyloid deposits can look like diVuse plaques or single nodules (in which case diVerentiation from a neoplasm may be diYcult). The prognosis is frequently one of progressive or recurrent disease, and death from respiratory failure may occur [113–116]. The lung parenchyma can be involved as well, either in isolation or concurrently, but symptomatic disease is less frequent and prognosis is better. Laryngeal involvement presents typically with hoarseness. Although recurrences are frequent, and death from the disease possible, most patients do very well and live many years [117]. 5.5.3. Skin Cutaneous amyloidosis, in the absence of systemic disease, most commonly presents with macular or lichen‐like lesions, in which the amyloid protein in the superficial dermis is composed of altered keratin filaments derived from degenerated keratinocytes [118]. Less commonly, it presents with nodules in what is known as nodular‐ or tumefactive‐type, or otherwise nodular primary localized cutaneous amyloidosis (NPLCA [119–121]). This is the most aggressive form and the pathogenesis appears to be always localized to clonal plasma cells producing amyloidogenic light chain [110]. The presence and clonality of plasma cells have not been consistently documented, however, and in some cases the immunoglobulin has been found to be of polyclonal origin. This type of nodular amyloidosis clinically presents with ovoid, shiny, pink‐yellow nodules, or plaques, and most frequently presents in the extremities. The clinical course can be one of frequent local relapses. The prognosis depends on whether there is systemic amyloidosis present at the time of diagnosis or not. There has been intense debate on whether localized cutaneous amyloidosis can progress to systemic amyloidosis or not, with anywhere between 7% and 50% of patients reported to progress to systemic illness. However, it appears that all patients progressing have either been suspected or demonstrated to have dysproteinemias at the time of diagnosis. To date there has not been a clear documented case of pure localized cutaneous amyloidosis progressing to systemic amyloidosis reported.

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5.5.4. Medin Associated Aortic Medial Amyloid Special attention should be payed to a type of localized amyloidosis that is present in virtually all individuals above the age of 60 and presents as amyloid deposition in the aortic media [122]. While its pathophysiologic significant is somewhat unclear, the amyloid monomer has been identified as medin, an integral fragment of lactadherin produced by aortic smooth muscle cells. Medin amyloid has been implicated in the pathogenesis of giant cell arteritis since it has been found present in 14 of 21 temporal artery biopsies in patients with this inflammatory disease in one study [123]. More recently, when tissue of patients with aortic aneurysm and aortic dissection was studied, medin amyloid was surprisingly found to be in significant lower concentrations in the patients with the above diseases compared to normal controls [124]. However, medin, when not associated with amyloid, was found in larger quantities in the diseased patients compared to controls. This potentially argues again for the role of toxic intermediates in the pathogenesis of amyloidosis and in fact aggregated medin induces apoptosis of aortic smooth muscle cells in vitro [124].

6. Treatment 6.1. STRATEGIES AIMED AT ERADICATING THE PRODUCTION OF THE AMYLOID PRECURSORS Treatment of amyloidosis depends on the specific type of the amyloid deposits, the organs involved, and the severity of the disease. To date there are many diVerent general strategies attempting to treat amyloidosis. By far the most common treatment strategy is aimed at eradicating the production of the amyloidogenic protein. In AL amyloidosis this means treatment with chemotherapy agents aiming at eradicating the clonal plasma cell population that produce the immunoglobulin light chain in the bone marrow. Although these strategies are successful in some individuals, given the otherwise relatively benign nature and small volume of the clonal plasma cells that cause amyloidosis (in contrast to the generally high volume aggressive nature of plasma cells that cause multiple myeloma) the majority of patients succumb to their disease due to eventual resistance to treatment. In AA amyloidosis, eradication of the amyloid fibril generation is generally easier since it usually implies simply treating the underlining chronic infection or inflammatory condition responsible for the amyloid production. In familial amyloidosis transplantation of the liver, when the source of the abnormal mutated protein, as in the case of TTR, fibrinogen Aa‐chain and apolipoprotein AI, can significantly improve or even cure (in the case of fibrinogen Aa‐chain) the disease.

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6.1.1. Melphalan and Steroids The oldest treatment for AL amyloidosis consists of the combination of melphalan and prednisone. Melphalan and prednisone doubles the survival of patients with amyloidosis when compared to colchicine from a median of 6–8 months to a median of 12–17 months. The regimen is well tolerated by patients of all age groups and was considered standard therapy when no stem cell transplant is contemplated [125, 126]. Melphalan in combination with another steroid, dexamethasone has been shown to also be well tolerated, and to cause improvement in organ dysfunction in about half of the patients [127]. Pulse dose, single agent dexamethasone can cause a complete hematologic remission in 25% of treated patients and improvement of organ dysfunction in 45% of patients with a median time to respond of 4 months [128]. 6.1.2. The Immunomodulating Agents Thalidomide and Lenalidomide Due to the eYcacy of these agents in patients with multiple myeloma, thalidomide and lenalidomide have found their way in treatment for patients with amyloidosis. However, high doses of these drugs are toxic and are not tolerated by these patients. Thalidomide, in combination with intermediate dose dexamethasone, can produce a hematologic response in 48% of patients when used as second line treatment with 19% of patients experiencing a complete response [129]. However, significant toxicity included progressive edema, cognitive diYculties, constipation, dyspnea, dizziness, and rash occurring in 50–75% of patients [130]. Deep venous thrombosis is another serious consideration with thalidomide. Lenalidomide, a structural ‘‘cousin’’ of thalidomide, appears to be better tolerated at the reduced dose of 15 mg per day although the risk of thromboembolic events is still high at approximately 10%. In a phase II clinical trial of patients, most of who were previously treated, the overall response rate was 67% with a complete hematologic response of 29% [131]. The combination of chemotherapy, immunomodulating agents, and steroids in the form of cyclophosphamide, thalidomide, and dexamethasone has been tried in 75 patients with AL amyloidosis, with an overall response rate of over 60% and a complete response rate of 21% which is very promising [132]. 6.1.3. Tumor Necrosis Factor Alpha (Etanercept) Etanercept was tested in 16 patients with AL amyloidosis who had failed all previous therapies and 50% of patients were found to have an objective response (88% had a subjective response) [133]. Another seven Japanese patients with rheumatoid arthritis and AA amyloidosis (carrying the SAA1.3 allele) were treated with Etanercept and were found to have significant improvement in

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SAA levels, creatinine clearance, and proteinuria in addition to other inflammatory markers and rheumatoid arthritis specific symptoms [134]. 6.1.4. Stem Cell Transplantation for AL Amyloidosis Again, because of the survival advantage of high dose chemotherapy followed by stem cell transplantation in patients with multiple myeloma this treatment approach has made its way to the treatment of patients of amyloidosis. Major diVerences, however, exist, the most significant of all being that the mortality rate of transplantation is anywhere between 11% and 40% compared to less than 5% in patients with multiple myeloma. This is due to the intolerance of patients with amyloidosis to fluid shifts and decreased tolerance to physical stress from widespread organ dysfunction, which is not the case in multiple myeloma (with the occasional exception of the kidney). After chemotherapy followed by stem cell transplantation complete hematologic responses are seen in 16–50% of patients and organ responses from 34% to 64% [135]. Despite its ‘‘standard’’ use in the treatment of well selected patient’s direct evidence for this approach comes only from case controlled studies [86]. One randomized trial of 50 patients showed no survival advantage of high dose melphalan followed by stem cell transplantation when compared to standard oral melphalan and high dose dexamethasone; however, the transplant related mortality was 24% which may be biasing the results of this trial [136]. Although most single institution experiences have shown lower mortality rates, this trial is not the only one to report such high mortality. It becomes apparent that patient selection is key, and currently only 25% of patients with amyloidosis at the Mayo Clinic are eventually treated with transplantation [137]. Selection criteria have been suggested and it appears that patients with advanced amyloid cardiomyopathy or more than two major viscera involved with disease are poor candidates for SCT [138]. When age and renal function are factored in stratification of patients regarding mortality risk from autologous stem cell transplant is possible. Tandem transplant has been successfully attempted but the impact of such an approach remains unknown [139]. 6.1.5. Transplantation for Familial Amyloidosis In the case of TTR familial amyloidosis, liver transplantation results in removal of the main source of the amyloidogenic protein. However, treatment‐related mortality of both the patient and the donor, the need for long‐term immunosuppressive therapy, shortage of donors, and the fact that on occasion the native, TTR can continue to deposit into amyloid in the heart, are strong considerations. Results from a 10‐year registry in 16 countries of patients treated with liver transplantation found 539 patients with a median 5‐year survival after

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transplantation of 77% [140]. Seventeen of the patients underwent combined liver and kidney transplantation and six patients underwent combined liver and heart transplantation. After transplantation, amongst 149 evaluated patients improvement in neurological, musculoskeletal, and gastrointestinal involvement was reported in approximately 30–50% of patients. However, only 20% of patients with cardiac symptoms reported an improvement. This may be due to the fact that native, normal, TTR continues to deposit in the heart after transplantation. Over the last decade livers explanted from patients with familial amyloidosis during transplantation have in turn been used as grafts to patients with other diseases, for example, in patients with cancer. Since the livers of patients with familial amyloidosis (and other metabolic diseases) are otherwise normal, except for the production of the amyloidogenic protein, these livers can safely be used to save the lives of other patients in immediate need. However, the risks of development of amyloidosis in the recipients and the time frame involved are generally unknown. In a recent study, biopsy of the gastroduodenal mucosa in recipient patients (patients who do not have amyloidosis but received a liver from a patient with familial amyloidosis) found that in two out of the five patients biopsied, amyloid was deposited 47 months following the transplant [141]. This finding raises certain concerns regarding the development of systemic amyloidosis in recipient patients much sooner than previously thought. Regarding other types of familial amyloidosis transplantation depends on the organs involved as well as the organs producing the abnormal protein. Apolipoprotein AI is produced 50% by the liver and 50% by the gastrointestinal tract. Even though liver transplantation only removes part of the source, patients clinically improve after transplantation due to the generally slowly progressive course of the disease. Combined kidney and liver transplantations as well as cardiac transplantations have also been performed with good results [32]. On the other hand, fibrinogen Aa‐chain is produced exclusively in the liver and thus hepatic transplantation appears to be curative. This is in contrast to TTR amyloidosis, which, despite also being produced exclusively in the liver, deposition of the native protein in amyloid deposits after transplantation is responsible for disease progression. In fibrinogen Aa‐chain amyloidosis renal transplantation alone results in amyloid deposition and dysfunction of the transplanted kidney within 1–10 years. On the other hand, combined hepatic and renal transplantation does not cause this problem since the amyloidosis is cured. Lysozyme is produced by polymorphonuclear leukocytes and macrophages and as a result no disease modifying strategies exist as of yet. Renal transplantation has had satisfactory results overall. Little evidence exists regarding treatment of patients with Apolipoprotein AII amyloidosis although kidney transplantation has been successful.

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6.1.6. Specific Treatments for AA Amyloidosis Control of the underlining inflammatory disease is of the utmost importance as has been clearly demonstrated by the relationship of SAA levels to survival [95, 142]. In the case of familial Mediterranean fever, colchicine is of known benefit with significant reduction of the clinical symptoms of the disease as well as stabilization and improvement of the kidney disease [143]. It is unclear whether colchicine is beneficial in other inflammatory conditions. It is also unclear how specific anti‐inflammatory treatments, such as anticytokine therapy or cyclophosphamide, impact AA amyloidosis directly since it is diYcult to link the benefits patients receive from such therapies to specific improvements of the coexisting amyloidosis versus improvement of the underlying inflammatory condition.

6.2. NATIVE PROTEIN STRUCTURE STABILIZING AGENTS A diVerent strategy in treating amyloidosis involves the attempted stabilization of the native structure of the amyloidogenic protein in order to prevent the conformational change that leads to the b‐pleated sheet conformation and the subsequent formation of the amyloid fibril. One such agent, diflunisal, has been recently investigated in the setting of familial amyloidosis [144]. Diflunisal is a nonsteroidal anti‐inflammatory agent, which has the propensity to bind to T4 binding sites of the TTR and kinetically stabilize the TTR tetramers. This is in direct response to the hypothesis that kinetic destabilization and disassociation of the tetramer structure of mutant TTR leads to partial monomer unfolding and generation of amyloid fibrils [145, 146]. Serum from 37 patients with familial amyloidosis was treated with diflunisal under acidic conditions and tetramer stability measured by a combination of acid denaturation, cross‐linking, SDS‐PAGE, immunoblotting, and densitometry. TTR from diflunisal treated serum was significantly more kinetically stable than TTR from untreated serum. Moreover, diflunisal treated TTR tetramers was more stable than the TTR from 26 healthy control subjects suggesting that diflunisal can stabilize TTR beyond the level of normal controls. Four of these patients were orally administered diflunisal and the same observations were made in vivo. The results were less impressive when flufenamic acid was used, another nonsteroidal anti‐inflammatory agent. Although multiple compounds are in existence that are known to have the ability to kinetically stabilize TTR most are delayed in entering clinical trials due to unknown toxicity profiles and pharmacokinetics (something which is not the case with the above tested nonsteroidal anti‐inflammatories which are FDA approved for other indications).

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6.3. AMYLOID FIBRIL DESTABILIZING AGENTS Another novel strategy in treating amyloidosis involves attacking the nonfibrillar components of the amyloid fibril, namely the serum amyloid P protein (SAP) and the glycosaminoglycans. A palindromic compound named R‐1‐[6‐[R‐2‐carboxy‐pyrrolidin‐1‐yl]‐6‐oxo‐hexanoyl]pyrrolidine‐2‐ carboxylic acid, abbreviated to CPHPC, has the ability to bind the ligand binding sites of pentameric SAP as well as crosslink pairs of SAP molecules [147] (Fig. 7). CPHPC was identified by screening the Roche compound library for inhibitors of SAP binding to amyloid fibrils and then chemically modifying the resulting compounds to enhance the avidity of the protein‐ligand interaction. After encouraging results in the laboratory and in mice, CPHPC was tested by a 48‐h intravenous infusion in seven patients with systemic amyloidosis and it resulted in a rapid and consistent depletion of SAP by the end of the infusion. The resultant SAP depletion persisted for several days in patients with a high load of amyloid deposition, but returned to normal soon after the infusion in patients with a low load of amyloid deposition. Quantitative whole body scintigraphy using 123I‐labeled SAP hours after the infusion confirmed the successful depletion of SAP from all tissues except the liver, where there was significant accumulation, indicating that the liver is

FIG. 7. Two SAP pentamers crosslinked by means of their B faces by five molecules of CPHPC (blue), viewed perpendicular to the fivefold axis. A face helices are shown in red. The two calcium ions bound to each SAP subunit are yellow. Reprinted from Ref. [147], Copyright 2002, with permission from Macmillan Publishers Ltd.

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the site of action of CPHPC. In a subsequent study, 19 patients with end stage systemic amyloidosis were treated with CPHPC for a period of 1.2– 9.5 months with the majority of the patients experiencing stability of disease. At autopsy, although the involved organs still contained significant amounts of amyloid they only contained 15% of the amount of SAP typically seen in such patients. No studies have been reported yet on the clinical eYcacy of the compound in patients with earlier stages of amyloidosis. A number of low molecular weight anionic compounds have been found to mimic the glycosaminoglycan molecules that bind to amyloid fibrils [148]. As a result, binding of these small molecules to amyloid fibrils have the ability to destabilize them. Eprodisate (Kiacta, Neurochem) is such a negatively charged, sulfonated molecule structurally similar to heparan sulfate and has been tested in a recent prospective clinical trial of 187 patients with AA amyloidosis [149]. The patients were randomized to receive eprodisate or placebo for 24 months. Although there was a trend towards slower decline in renal function in patients receiving eprodisate there was no diVerence observed in the progression to end stage renal disease or death. Despite this apparent lack of survival benefit, this and other similar agents deserve further attention to discover their possible role treating patients with amyloidosis of various types and various stages, and investigate potential benefit in combining them with other standard treatment approaches. 6.4. IMMUNOLOGIC THERAPY 6.4.1. Monoclonal Antibodies Against Immunoglobulin Light Chain Monoclonal antibodies have been developed against human immunoglobulin light chains, and specifically epitopes of the b‐pleated sheet structure of these proteins, in hopes of stimulating passive immunity and resolution of the amyloid deposits. This hypothesis has been tested in mice that were subsequently exposed to the monoclonal antibody and there was neutrophil infiltration of the amyloid deposits and opsonization of the amyloid fibrils. Although this hypothesis has been only tested in mice thus far, there are planned phase I and phase II clinical trials in patients with AL amyloidosis [150]. 6.4.2. Vaccines There have also been multiple vaccination attempts to treat amyloidosis. These approaches generally begin with apheresis of the patient’s mononuclear cells and their subsequent diVerentiation into dendritic cells. These dendritic cells are subsequently exposed to various antigens and are reinfused into patients in hopes of mounting an immune response against the target antigen. Alternatively, synthetic target peptides can be infused in patients in

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similar hopes of mounting an immune response against amyloid fibrils. Although there are occasional responses observed with such therapy, vaccines have not yet found a foothold in clinical practice in general, and major breakthroughs are still to come. In addition, testing of vaccine strategies in patients with Alzheimer’s disease, by exposure of patients to the pathogenic Ab fibril of this disease, has led to significant side eVects in the form of aseptic meningoencephalitis and concerns regarding cerebral microhemorrhages [151]. Despite this, eVorts in this direction are ongoing. 6.5. SUPPORTIVE CARE Frequently diuretics are used in both cardiac and renal amyloidosis to prevent fluid accumulation. Ramipril has also been able to reduce proteinuria in patients with nephrotic syndrome [152]. Hemodialysis is commonly performed in patients with end stage renal disease from amyloidosis. Both the use of diuretics and hemodialysis is complicated in patients with cardiac concomitant disease due to the presence of orthostatic hypotension and sensitivity to fluid shifts. Pacemaker implantation can sometimes help patients with recurrent syncopal episodes [153]. Calcium channel blockers have been reported to worsen heart failure related to amyloidosis. 6.6. TREATMENT OF LOCALIZED AMYLOIDOSIS Treatment of localized amyloidosis does not involve systemic therapy. Localized treatment modalities (i.e., surgery, endoscopic procedures) are successful in controlling the disease and it is uncommon for patients to die from the amyloidosis. Localized amyloidosis of the tracheobronchial tree carries a higher risk of life threatening complications and death due to the potential for obstructing the airway. Although cystectomy is curative for localized amyloidosis of the bladder it should be avoided as much as possible in favor of attempting localized treatments first, such as fulguration therapy and laser treatment [112]. Transurethral resection, partial or total, is reserved for more diVuse or persistent symptomatic disease. Patients with diVuse bladder involvement have the option of using intravesical dimethyl sulfoxide (DMSO) therapy to control the disease. Four out of six patients treated at the Mayo Clinic with intravesical DMSO had disease stabilization for 2–6 years, and two patients had primary failure of the technique requiring further intervention. Tracheobronchial and laryngeal localized amyloidosis can be observed or treated with intermittent bronchoscopic resection or surgical resection. Laser therapy is being used more frequently recently and good results have been seen with carbon dioxide laser ablation as well as Nd:YAG (neodymium: yttrium‐aluminium‐garnet) laser ablation [116].

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A high rate of local recurrence is common with the nodular‐type presentation of cutaneous amyloidosis and various treatment options have been recommended including surgical excision, CO2 laser treatment, and cryotherapy. REFERENCES [1] J.D. Sipe, Amyloid Proteins. The Beta Sheet Conformation and Disease, Wiley‐VCH, Weinheim, 2005. [2] J.D. Sipe, A.S. Cohen, Review: history of the amyloid fibril, J. Struct. Biol. 130 (2–3) (2000) 88–98. [3] R.L.K. Virchow, Cellular Pathology, [Translated from the 2nd ed of the original by F Chance ed], DeWitt RM, New York, 1860. [4] N. Friedreich, A. Kekule, zur amyloidfrage, Virchows Arch. Pathol. Anat. 16 (1859) 50–65. [5] A.S. Cohen, E. Calkins, Electron microscopic observations on a fibrous component in amyloid of diverse origins, Nature 183 (1959) 1202–1203. [6] E.D. Eanes, G.G. Glenner, X‐ray diffraction studies on amyloid filaments, J. Histochem. Cytochem. 16 (11) (1968) 673–677. [7] M. Sunde, L.C. Serpell, M. Bartlam, P.E. Fraser, M.B. Pepys, C.C. Blake, Common core structure of amyloid fibrils by synchrotron X‐ray diffraction, J. Mol. Biol. 273 (3) (1997) 729–739. [8] C. Blake, L. Serpell, Synchrotron X‐ray studies suggest that the core of the transthyretin amyloid fibril is a continuous beta‐sheet helix, Structure 4 (8) (1996) 989–998. [9] R. Nelson, M.R. Sawaya, M. Balbirnie, A.O. Madsen, C. Riekel, R. Grothe, et al., Structure of the cross‐beta spine of amyloid‐like fibrils, Nature 435 (7043) (2005) 773–778. [10] P. Westermark, M.D. Benson, J.N. Buxbaum, A.S. Cohen, B. Frangione, S. Ikeda, et al., Amyloid: toward terminology clarification. Report from the Nomenclature Committee of the International Society of Amyloidosis, Amyloid 12 (1) (2005) 1–4. [11] L.C. Serpell, M. Sunde, M.D. Benson, G.A. Tennent, M.B. Pepys, P.E. Fraser, The protofilament substructure of amyloid fibrils, J. Mol. Biol. 300 (5) (2000) 1033–1039. [12] R. Nelson, D. Eisenberg, Recent atomic models of amyloid fibril structure, Curr. Opin. Struct. Biol. 16 (2) (2006) 260–265. [13] R. Khurana, J.R. Gillespie, A. Talapatra, L.J. Minert, C. Ionescu‐Zanetti, I. Millett, et al., Partially folded intermediates as critical precursors of light chain amyloid fibrils and amorphous aggregates, Biochemistry 40 (12) (2001) 3525–3535. [14] R. Khurana, P.O. Souillac, A.C. Coats, L. Minert, C. Ionescu‐Zanetti, S.A. Carter, et al., A model for amyloid fibril formation in immunoglobulin light chains based on comparison of amyloidogenic and benign proteins and specific antibody binding, Amyloid 10 (2) (2003) 97–109. [15] Z. Qin, D. Hu, M. Zhu, A.L. Fink, Structural characterization of the partially folded intermediates of an immunoglobulin light chain leading to amyloid fibrillation and amorphous aggregation, Biochemistry 46 (11) (2007) 3521–3531. [16] C.M. Eakin, F.J. Attenello, C.J. Morgan, A.D. Miranker, Oligomeric assembly of native‐ like precursors precedes amyloid formation by beta‐2 microglobulin, Biochemistry 43 (24) (2004) 7808–7815. [17] D.M. Walsh, A. Lomakin, G.B. Benedek, M.M. Condron, D.B. Teplow, Amyloid beta‐ protein fibrillogenesis. Detection of a protofibrillar intermediate, J. Biol. Chem. 272 (35) (1997) 22364–22372.

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[126] M. Skinner, J. Anderson, R. Simms, R. Falk, M. Wang, C. Libbey, et al., Treatment of 100 patients with primary amyloidosis: a randomized trial of melphalan, prednisone, and colchicine versus colchicine only, Am. J. Med. 100 (3) (1996) 290–298. [127] G. Palladini, V. Perfetti, L. Obici, R. Caccialanza, A. Semino, F. Adami, et al., Association of melphalan and high‐dose dexamethasone is effective and well tolerated in patients with AL (primary) amyloidosis who are ineligible for stem cell transplantation, Blood 103 (8) (2004) 2936–2938. [128] M.V. Dhodapkar, M.A. Hussein, E. Rasmussen, A. Solomon, R.A. Larson, J.J. Crowley, et al., Clinical efficacy of high‐dose dexamethasone with maintenance dexamethasone/ alpha interferon in patients with primary systemic amyloidosis: results of United States Intergroup Trial Southwest Oncology Group (SWOG) S9628, Blood 104 (12) (2004) 3520–3526. [129] G. Palladini, V. Perfetti, S. Perlini, L. Obici, F. Lavatelli, R. Caccialanza, et al., The combination of thalidomide and intermediate‐dose dexamethasone is an effective but toxic treatment for patients with primary amyloidosis (AL), Blood 105 (7) (2005) 2949–2951. [130] A. Dispenzieri, M.Q. Lacy, S.V. Rajkumar, S.M. Geyer, T.E. Witzig, R. Fonseca, et al., Poor tolerance to high doses of thalidomide in patients with primary systemic amyloidosis, Amyloid 10 (4) (2003) 257–261. [131] V. Sanchorawala, D.G. Wright, M. Rosenzweig, K.T. Finn, S. Fennessey, J.B. Zeldis, et al., Lenalidomide and dexamethasone in the treatment of AL amyloidosis: results of a phase 2 trial, Blood 109 (2) (2007) 492–496. [132] A.D. Wechalekar, H.J. Goodman, H.J. Lachmann, M. Offer, P.N. Hawkins, J.D. Gillmore, Safety and efficacy of risk‐adapted cyclophosphamide, thalidomide, and dexamethasone in systemic AL amyloidosis, Blood 109 (2) (2007) 457–464. [133] M.A. Hussein, J.V. Juturi, L. Rybicki, S. Lutton, B.R. Murphy, M.A. Karam, Etanercept therapy in patients with advanced primary amyloidosis, Med. Oncol. 20 (3) (2003) 283–290. [134] T. Nakamura, S. Higashi, K. Tomoda, M. Tsukano, S. Baba, Efficacy of etanercept in patients with AA amyloidosis secondary to rheumatoid arthritis, Clin. Exp. Rheumatol. 25 (4) (2007) 518–522. [135] N. Leung, A. Dispenzieri, F.C. Fervenza, M.Q. Lacy, R. Villicana, J.L. Cavalcante, et al., Renal response after high‐dose melphalan and stem cell transplantation is a favorable marker in patients with primary systemic amyloidosis, Am. J. Kidney Dis. 46 (2) (2005) 270–277. [136] A. Jaccard, P. Moreau, V. Leblond, X. Leleu, L. Benboubker, O. Hermine, et al., High‐ dose melphalan versus melphalan plus dexamethasone for AL amyloidosis, N. Engl. J. Med. 357 (11) (2007) 1083–1093. [137] M.A. Gertz, M.Q. Lacy, A. Dispenzieri, S.R. Hayman, S. Kumar, Transplantation for amyloidosis, Curr. Opin. Oncol. 19 (2) (2007) 136–141. [138] R.L. Comenzo, M.A. Gertz, Autologous stem cell transplantation for primary systemic amyloidosis, Blood 99 (12) (2002) 4276–4282. [139] V. Sanchorawala, D.G. Wright, K. Quillen, K.T. Finn, L.M. Dember, J.L. Berk, et al., Tandem cycles of high‐dose melphalan and autologous stem cell transplantation increases the response rate in AL amyloidosis, Bone Marrow Transplant. 40 (6) (2007) 607. [140] G. Herlenius, H.E. Wilczek, M. Larsson, B.G. Ericzon, Ten years of international experience with Liver Transplantation for familial amyloidotic polyneuropathy: results from the Familial Amyloidotic Polyneuropathy World Transplant Registry, Transplantation 77 (1) (2004) 64–71.

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[141] Y. Takei, T. Gono, M. Yazaki, S. Ikeda, T. Ikegami, Y. Hashikura, et al., Transthyretin‐ derived amyloid deposition on the gastric mucosa in domino recipients of familial amyloid polyneuropathy liver, Liver Transpl. 13 (2) (2007) 215–218. [142] J.D. Gillmore, L.B. Lovat, M.R. Persey, M.B. Pepys, P.N. Hawkins, Amyloid load and clinical outcome in AA amyloidosis in relation to circulating concentration of serum amyloid A protein, Lancet 358 (9275) (2001) 24–29. [143] A. Livneh, D. Zemer, P. Langevitz, J. Shemer, E. Sohar, M. Pras, Colchicine in the treatment of AA and AL amyloidosis, Semin. Arthritis Rheum. 23 (3) (1993) 206–214. [144] K. Tojo, Y. Sekijima, J.W. Kelly, S. Ikeda, Diflunisal stabilizes familial amyloid polyneuropathy‐associated transthyretin variant tetramers in serum against dissociation required for amyloidogenesis, Neurosci. Res. 56 (4) (2006) 441–449. [145] W. Colon, J.W. Kelly, Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro, Biochemistry 31 (36) (1992) 8654–8660. [146] P. Hammarstrom, X. Jiang, A.R. Hurshman, E.T. Powers, J.W. Kelly, Sequence‐ dependent denaturation energetics: a major determinant in amyloid disease diversity, Proc. Natl. Acad. Sci. USA 99 (Suppl. 4) (2002) 16427–16432. [147] M.B. Pepys, J. Herbert, W.L. Hutchinson, G.A. Tennent, H.J. Lachmann, J.R. Gallimore, et al., Targeted pharmacological depletion of serum amyloid P component for treatment of human amyloidosis, Nature 417 (6886) (2002) 254–259. [148] F. Gervais, R. Chalifour, D. Garceau, X. Kong, J. Laurin, R. McLaughlin, et al., Glycosaminoglycan mimetics: a therapeutic approach to cerebral amyloid angiopathy, Amyloid 8 (Suppl. 1) (2001) 28–35. [149] L.M. Dember, P.N. Hawkins, B.P. Hazenberg, P.D. Gorevic, G. Merlini, I. Butrimiene, et al., Eprodisate for the treatment of renal disease in AA amyloidosis, N. Engl. J. Med. 356 (23) (2007) 2349–2360. [150] R. Hrncic, J. Wall, D.A. Wolfenbarger, C.L. Murphy, M. Schell, D.T. Weiss, et al., Antibody‐mediated resolution of light chain‐associated amyloid deposits, Am. J. Pathol. 157 (4) (2000) 1239–1246. [151] W.V. Nikolic, Y. Bai, D. Obregon, H. Hou, T. Mori, J. Zeng, et al., Transcutaneous beta‐ amyloid immunization reduces cerebral beta‐amyloid deposits without T cell infiltration and microhemorrhage, Proc. Natl. Acad. Sci. USA 104 (7) (2007) 2507–2512. [152] M. Mayr, M.J. Dickenmann, [Further evidence for the renoprotective effect of ACE inhibitors: ramipril protects against the progression of chronic renal insufficiency in non‐ diabetic nephropathy with nephrotic proteinuria], Schweiz Med. Wochenschr. 130 (13) (2000) 491. [153] V. Mathew, H. Chaliki, R.A. Nishimura, Atrioventricular sequential pacing in cardiac amyloidosis: an acute Doppler echocardiographic and catheterization hemodynamic study, Clin. Cardiol. 20 (8) (1997) 723–725. [154] C. B. Caputa, A. I. Salama, The Amyloid Proteins of Alzheimer’s Disease as Potential Targets for Drug Therapy (Part 1). Microbiology of Aging, Pergamon Press, 1989, pp. 451–461.

ADVANCES IN CLINICAL CHEMISTRY, VOL. 47

URINARY MARKERS IN COLORECTAL CANCER Bo Feng,1 Fei Yue,1 and Min‐Hua Zheng2 Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

1. 2. 3. 4.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potential Urinary Markers for Colorectal Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analytical Techniques and Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Analytical Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

45 46 47 50 50 52 53 53

1. Abstract Colorectal cancer is one of the most commonly diagnosed cancers and cause of cancer‐related deaths worldwide. Studies have demonstrated that patient outcome is substantially influenced by cancer stage at the time of diagnosis. For example, patients with early stage colorectal have a significant higher 5‐year survival rates compared to patients diagnosed at late stage. Thus, it is important to develop eVective methods for early diagnosis as well as for precise staging of this disease process. Although traditional colonoscopy remains the most eVective means to diagnose colorectal cancer, this approach generally suVers from poor patient compliance. As such, it is imperative to develop accurate and specific tests that utilize a more convenient approaches including urine examination. Urine collection is noninvasive, requires no presampling preparation, and substantially improves compliance. 1 2

These two authors contributed equally to this work. Corresponding author: Min‐Hua Zheng, e‐mail: [email protected] 45

0065-2423/09 $35.00 DOI: 10.1016/S0065-2423(09)47002-1

Copyright 2009, Elsevier Inc. All rights reserved.

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Recent advances in metabolomics, analytical techniques, and data analysis have tremendous potential for application in diagnostic pathology including identification of novel urinary markers in colorectal cancer.

2. Introduction Colorectal cancer is the third most commonly diagnosed cancer and the fourth most common cause of cancer‐related deaths worldwide [1]. In China, the incidence and mortality rates of colorectal cancer continue to increase and are ranked as third and fifth among common cancers, respectively [2]. In Western countries, the incidence and mortality rates of colorectal cancer rank third in both women (after breast and lung cancer) and men (after the prostate and lung cancer) [3, 4]. Based on previous studies, it is generally acknowledged that the outcome of colorectal cancer is significantly influenced by stage of diagnosis. For example, the 5‐year survival rate for 40% of patients diagnosed with localized colorectal cancer is approximately 90% whereas less than 10% of patients with distant metastasis survive for 5 years [5]. Thus, substantial research eVorts have focused on the development of diagnostic and staging tools. Fecal occult blood testing is the most applied strategy for screening and has a tremendous impact on reducing the risk of colorectal cancer‐related death [6]. Despite its positive role, this technique is too nonspecific to guide diagnosis in symptomatic patients. Although traditional colonoscopy still remains the most eVective means diagnose colorectal cancer, there are considerable drawbacks including patient preparation, potential bowel perforation, and general inconvenience [7]. As such, most patients are likely to be noncompliant. Although computerized tomographic colonography and wireless capsule endoscopy might provide alternative testing methods, these novel approaches have not reached widespread use or acceptance within the medical community. Advances in molecular diagnostics as well as the development of genomics, proteomics, and metabolomics, have shown tremendous potential as diagnostic tools in general and as promising alternatives for diagnosis and staging of colorectal cancer specifically [8–10]. In contrast to fecal occult blood testing, multitarget fecal‐based DNA screening could potentially increase sensitivity without sacrificing specificity [11]. However, no direct evidence has shown that the multimarker DNA‐based stool testing reduces mortality of patients with colorectal cancer. In addition, its high cost represents a significant issue for widespread application. Currently, no serum marker has suYcient diagnostic sensitivity or specificity. Although carcinoembryonic antigen (CEA) is still regarded as the classic marker, its use has predominantly prognostic impact [12]. Tissue inhibitor of metalloproteinase type 1

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47

(TIMP‐1) has been proposed as a stage‐independent candidate marker [13], but larger more comprehensive studies are still required. Urine is an ideal sample source which may provide valuable disease related information [14]. Patient compliance is generally not an issue since collection is simple and noninvasive with no presampling preparation. However, not all the diseases present certain molecular markers in the urine especially those outside the urogenital tract. Small low molecular weight molecules including metabolic products can be found in the urine. The metabolomic study of these molecules may lead to the discovery of biomarkers to facilitate diagnosis, staging, and prognosis [9, 15]. This article will review the recent metabolomic studies on urinary markers for colorectal cancer. Literature was obtained from the PubMed database using key words ‘‘metabolomics,’’ ‘‘metabonomics,’’ ‘‘urinary marker,’’ and ‘‘colorectal cancer.’’

3. Potential Urinary Markers for Colorectal Cancer Urinary markers provide important diagnostic information for cancers involving the urinary system [16–18]. However, research has indicated that urine could also provide valuable markers for colorectal cancer. Several studies have demonstrated a correlation between carcinogenesis and increased urinary concentration of modified nucleosides [19, 20]. These RNA‐based markers could result from post‐transcriptional modification by numerous enzymes including methyltransferases and ligases [21]. Modified nucleosides not reused or degraded are excreted in the urine [22]. Metabolomic profile analysis of these urinary substances could potentially lead to identification of biomarkers involved with disease states including colorectal caner. Previously, we reported that 76.9% of colorectal cancer patients could be correctly classified using principal component analysis (PCA) of 14 nucleosides by reversed‐phase high‐performance liquid chromatography (RP‐HPLC). Interestingly, nine of these nucleosides were found to significantly decrease after curative resection thus implying prognostic value [23]. Receiver operator characteristic (ROC) curves were then used to evaluate sensitivity, specificity, and cut‐oV values for two relatively high concentration nucleosides, pseudouridine (Pseu) and 1‐methylguanosine (m1G). At the appropriate cut‐oV concentration, ROC analysis demonstrated substantially improved sensitivity and specificity versus the traditional CEA marker. This preliminary study indicated that the evaluation of both normal and modified urinary nucleosides might provide unique markers in the diagnosis and management of colorectal cancer. Another research group reported that increased urinary concentration of N1,N12‐diacetylspermine was a novel diagnostic marker for colorectal cancer apart from urogenital malignancy [24, 25]. Return of urinary

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diacetylpolyamine to normal levels was indicative of good prognosis [26]. The availability of a commercial highly specific antidiacetylspermine antibody led to the development of immunoassay‐based methods to further study the importance of this biomarker in the screening, diagnosis, and prognosis of colorectal cancer [27]. The chemopreventive eVects of nonsteroidal anti‐inflammatory drugs (NSAID) are speculated to be mediated by their ability to inhibit COX‐ 2 function and prostaglandin production [28–30]. A prospective cohort study found that PGE‐M, the prostaglandin E2 (PGE2) metabolite, was increased in the urine of patients who subsequently developed colorectal cancer (Figs. 1 and 2, Table 1) [31]. Because PGE‐M production is dependent on COX‐2 [32], urinary levels of this metabolite may indicate in vivo COX‐2 activity. Interestingly, COX‐2 is overexpressed in around 90% of colorectal cancers [33–35]. This unique approach may provide a useful tool in the expression patterns of other key enzymes involved with the onset and development of colorectal cancer. In addition to endogenous products, diet‐derived metabolic markers may also provide important diagnostic clues. A cohort study demonstrated that reduced risk of colon cancer in regular tea drinkers was associated with high urinary concentration of epigallocatechin (EGC) and 40 ‐O‐methyl‐ epigallocatechin (40 ‐MeEGC) (Table 2) [36]. A number of in vitro and

Case–control difference in log (PGE-M)

7 6 5 4 3 2

p < 0.001

p = 0.002

p = 0.049

1 +

+

+

Colorectal

Colon

Rectal

0 −1 −2 −3 −4

FIG. 1. Case–control diVerence in log‐transformed urinary prostaglandin E2 metabolite (PGE‐M) levels. The p values were derived from paired t tests [31].

49

URINARY MARKERS IN COLORECTAL CANCER

2.5 Colon Colorectal Rectal

2.0

Log (odds)

1.5

1.0 0.5

0.0 −0.5 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Log (PGE-M) FIG. 2. Association between baseline levels of urinary prostaglandin E2 metabolite (PGE‐M; ng/mg creatinine) and subsequent risk of colorectal cancer [31].

nonhuman in vivo studies have shown that tea‐derived polyphenols act as protective agents against colorectal cancer [37–40]. This report provided the first population‐based evidence that EGC and 40 ‐MeEGC (the methylated metabolite of EGC) was associated with a statistically significant reduction (60%) in risk of developing colon cancer. In contrast, no association was observed between the urinary polyphenols (including their metabolites) and rectal cancer risk. Due to the diVerences in incidence and mortality rates, a comprehensive diet‐based metabolomic profile involving oriental and occidental countries would provide important information related to identifying key substances that play a role in development of colorectal cancer. These studies would likely identify important diet‐derived chemopreventive agents or additional be applied in the study of gastric cancer which has higher incidence in the Eastern countries. It is widely believed that the Western style diet (increased consumption of meat and fat, decreased consumption of vegetables and grains) increases the risk of colorectal cancer [41]. Red meat consumption is associated with increased risk of colon cancer [42]. Lipoperoxides induced by heme yield end‐ products including alkanes, aldehydes, isoprostanes, and 4‐hydroxynonenal. Thus, 1,4‐dihydroxynonane mercapturic acid (DHN‐MA), a major urinary metabolite of 4‐hydroxynonenal [43], could serve as a specific and noninvasive biomarker of lipid peroxidation [44]. Pierre et al. [45] reported that

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TABLE 1 ASSOCIATION OF BASELINE URINARY PGE‐M LEVELS AND SUBSEQUENT RISK OF COLORECTAL CANCER [31] PGE‐M (quartile)

All colorectal cancer (150 pairs) No. of cases No. of controls RR 95% CI Colon cancer (88 pairs) No. of cases No. of controls RR 95% CI Rectal cancer (62 pairs) No. of cases No. of controls RR 95% CI

1

2

3

4

14 37 1.0

28 38 2.5 1.1–5.8

47 37 4.5 1.9–10.9

61 38 5.6 2.4–13.5

7 21 1.0

15 22 2.1 0.7–6.5

29 20 4.8 1.6–14.8

37 25 4.9 1.7–14.7

7 16 1.0

13 16 3.1 0.8–11.6

18 17 4.1 1.0–17.3

24 13 7.2 1.7–3.7

p for trend <0.001

0.009

0.048

Conditional logistic regression models were used to derive p values for linear trends by modeling the log‐transformed urinary PGE‐M levels as continuous variable. PGE‐M, prostaglandin E2 metabolite; case, study participant who had developed colorectal cancer; control, study participant who remained cancer free; RR, relative risk.

urinary DHN‐MA excretion was increased in the rat model and human volunteers as the consequence of high heme diets, implying the potential value of DHN‐MA for the risk determination of preneoplastic lesions in colorectal cancer. These two examples reinforce the need to elucidate key metabolites derived from either protective or hazardous substances for diagnosis, staging, and prognosis in disease states.

4. Analytical Techniques and Data Analysis 4.1. ANALYTICAL TECHNIQUES The field of metabolomics is reliant on robust analytical techniques. The two most predominant information‐rich analytical tools that provide atom‐ specific molecular structural information are nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) [46]. Although NMR analysis is suYciently robust for clinical studies [47], variables in age, gender, and diet may impact its exact application [48, 49]. 1H NMR spectroscopy is

TABLE 2 LEVELS OF URINARY TEA POLYPHENOLS IN RELATION TO RISK OF COLORECTAL CANCER, SHANGHAI COHORT STUDY 1986–2002 [36] Colon cancer No. of cases

No. of controls

32 20 18 13

112 102 95 104

40 ‐MeEGC (mmol/g Cr) First tertile (0–1.49) Second tertile (1.50–7.30) Third tertile (7.31–34.44) Fourth tertile (>34.44) Per unit increment in loge scale p for trend

33 18 15 17

EGC þ40 ‐MeEGC (mmol/g Cr) First tertile (0–2.06) Second tertile (2.07–8.93) Third tertile (8.94–44.02) Fourth tertile (>44.02) Per unit increment in loge scale p for trend

31 19 16 17

Tea polyphenol level EGC (mmol/g Cr) Undetectable (0) First tertile (0.01–1.02) Second tertile (1.03–7.82) Third tertile (>7.82) Per unit increment in loge scale p for trend

Rectal cancer No. of cases

No. of controls

1.00 0.64 (0.33–1.24) 0.60 (0.30–1.20) 0.40 (0.19–0.83) 0.77 (0.63–0.96) 0.02

21 21 13 24

117 0.91 0.97 0.88

98 99 111 105

1.00 0.49 (0.25–0.96) 0.32 (0.16–0.67) 0.41 (0.20–0.84) 0.79 (0.67–0.94) 0.006

23 15 17 24

101 97 107 108

1.00 0.57 (0.29–1.11) 0.39 (0.19–0.80) 0.43 (0.21–0.88) 0.80 (0.68–0.94) 0.007

28 14 15 25

OR (95% CI)

Colorectal cancer No. of cases

No. of controls

OR (95% CI)

1.00 1.22 (0.62–2.49) 0.67 (0.31–1.47) 1.25 (0.62–2.52) 1.05 (0.87–1.26) 0.62

53 41 31 37

229 193 192 192

1.00 0.87 (0.55–1.40) 0.64 (0.39–1.07) 0.76 (0.47–1.24) 0.93 (0.81–1.06) 0.28

104 102 91 96

1.00 0.67 (0.32–1.39) 0.87 (0.42–1.80) 1.03 (0.52–2.06) 1.01 (0.87–1.19) 0.86

56 33 32 41

202 201 202 201

1.00 0.56 (0.34–0.91) 0.55 (0.33–0.90) 0.68 (0.42–1.10) 0.91 (0.81–1.01) 0.09

101 104 95 93

1.00 0.50 (0.24–1.06) 0.64 (0.31–1.30) 0.93 (0.46–1.84) 1.00 (0.87–1.18) 0.90

56 33 31 42

202 201 202 201

1.00 0.54 (0.33–0.86) 0.52 (0.32–0.86) 0.68 (0.42–1.10) 0.91 (0.82–1.02) 0.10

OR (95% CI)

Odds ratios (ORs) were calculated using conditional logistic regression models with matched sets consisting of five control subjects individually matched to the index case patient by date of birth (within 2 years), date of blood collection (within 1 month), and neighborhood of residence at recruitment. All conditional logistic regression models included the following covariates: current smoking status (no, yes), number of cigarettes smoked per day, number of years of smoking, alcohol drinking (no, yes), and number of alcoholic beverages consumed per day; CI, confidence interval.

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especially suited for detection of urinary markers because little or no sample preparation is required. As a result, this technique has become a powerful tool for biofluid analysis including urine. Due to its predominant water composition, the application of a standard water suppression pulse sequence can reduce acquisition time to about 4 min per urine sample. Although 1 H NMR remains the most widely used technique, two‐dimensional (2D) systems have also been reported [50, 51]. 2D J‐resolved spectroscopy could reduce the contribution of macromolecules and provide information on multiplicity and coupling patterns of resonances. Moreover, correlation spectroscopy (COSY) and total correlation spectroscopy (TOCSY) are able to provide 1H‐1H spin–spin coupling connectivities. Heteronuclear multiple quantum coherence (HMQC) technique can even obtain heteronuclear correlations such as 1H‐13C, which can help assign NMR peaks. Sensitivity gain of about 500% is achievable with cryoprobes thus enabling detection in shorter time [46]. In contrast to NMR, MS can be more sensitive and provide lower limits of detection. For example, Fourier transform mass spectrometry (FT‐MS) provides exceptional resolution with a detection mass of 1 ppm or lower for metabolites and peptides [52, 53]. When coupled to the high‐ throughput matrix‐assisted laser desorption/ionization (MALDI), FT‐MS has the advantage of being limited to low charge states, thus improving mass accuracy, sensitivity, and resolution [52, 53]. Prior to analysis, sample fractionation is required and is typically accomplished by LC or HPLC [23, 27, 31, 54]. Although NMR and HPLC are complementary techniques, only a few studies have applied them in combination. When implemented together, the classification of patients with diVerent diseases can be significantly improved [55]. The combination of NMR (nondestructive, relatively quick, and inexpensive) with HPLC (sensitive, rapid, and high‐throughput adaptability) provides an ideal analytical platform for urinary metabolomic studies. 4.2. DATA ANALYSIS Metabolomics is a member of the high‐throughput ‘‘omics’’ study. The massive amount of data should be statistically analyzed and mined by multivariate analysis [9]. PCA has been extensively applied in the dataset analysis, which is a well‐known approach that permits the reduction of the most of variance into a dataset containing less principal components. For example, 44 patients with colorectal cancer and 50 normal controls were clearly identified by PCA when 11 nucleosides were used as data vectors [23]. Hierarchical cluster analysis, soft independent modeling of class analogy (SIMCA), K‐nearest neighbor analysis, and neural networks have been used in data analysis [46]. In contrast to genomics and proteomics, no comparable metabolomic system is available. However, the ‘‘omics space’’ framework has

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been proposed. This approach is described as a dimensionless framework where interactions are defined on the basis of their positions within the coordinate system [56]. In addition, the ‘‘Genome to Phenome Superhighway’’ (GPS) has been proposed to integrate databases and visualize the interactions of ‘‘omics’’ thus paving a ‘‘totalomic’’ approach to system biology. 5. Conclusions Metabolomic research remains the primary means to identify urinary markers in colorectal cancer and has tremendous potential in biochemical biomarker assay development in general [57]. Personalized treatment can be achieved through adjustment of therapeutic regimens according to the metabolic biomarkers in urine. Despite these advances, a number of challenges remain. The selection of a proper urinary marker is of great importance, for many serum markers and tissue markers are not excreted in the urine unless the plasma levels are high enough to overcome renal resorption [58]. Identification of key metabolites is the only one issue. Appropriate research design is imperative. For example, it is inappropriate to use only healthy individuals as controls since this strategy would not eliminate metabolomic profiles mimicked by other diseases and disorders [14]. Standards of metabolomic ontology and experimental reporting have been proposed by the Metabolomics Society [59]. Standardization will facilitate integration of mass data from various studies and enhance reproducibility as well as credibility of metabolomic‐derived data. Data analysis and integration remain considerable major problems in metabolomics. Although much progress has been made, more advanced bioinformatic systems will be needed in the future. As mentioned above, urinary markers identified by various ‘‘omic’’ techniques should be well integrated and validated with larger studies in accordance with the concept of system biology. ACKNOWLEDGMENT The authors would like to express the thanks to Prof. Guowang Xu and Dr. Wenzhao Wang of Dalian Institute of Chemical Physics, Chinese Academy of Science for their helpful revision of the manuscript.

REFERENCES [1] D.M. Parkin, F. Bray, J. Ferlay, P. Pisani, Global cancer statistics, 2002. CA Cancer J. Clin. 55 (2) (2005) 74–108. [2] W.C. You, F. Jin, S. Devesa, et al., Rapid increase in colorectal cancer rates in urban Shanghai, 1972–1997, in relation to dietary changes. J. Cancer Epidemiol. Prev. 7 (3) (2002) 143–146.

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ADVANCES IN CLINICAL CHEMISTRY, VOL. 47

EFFECT OF HORMONE REPLACEMENT THERAPY ON INFLAMMATORY BIOMARKERS Panagiota Georgiadou and Eftihia Sbarouni1 2nd Department of Cardiology, Onassis Cardiac Surgery Center, Athens, Greece

1. 2. 3. 4.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inflammation and Vascular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of Action of HRT in Vascular Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Estrogen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Selective Estrogen Receptor Modulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Phytoestrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Progesterone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. EVects of HRT on Inflammatory Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. C‐Reactive Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Serum Amyloid A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Cell Adhesion Molecules. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Selectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Interleukin‐6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. Monocyte Chemoattractant Protein‐1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7. Tumor Necrosis Factor‐
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

60 60 62 65 65 66 68 69 71 71 75 76 78 80 81 82 82 83

Abbreviations HRT VCAM‐1 ICAM‐1 MCP‐1 IL TNF‐a 1

hormone replacement therapy vascular cell adhesion molecule intercellular adhesion molecule monocyte chemoattractant protein‐1 interleukin tumor necrosis factor‐


Corresponding author: Eftihia Sbarouni, e‐mail: [email protected] 59

0065-2423/09 $35.00 DOI: 10.1016/S0065-2423(09)47003-3

Copyright 2009, Elsevier Inc. All rights reserved.

60 CRP SAA iNFkB NO LDL HDL VLDL ApoE ER SERMS CI IMT

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C‐reactive protein serum amyloid A nuclear factor B nitric oxide low‐density lipoprotein high‐density lipoprotein very low‐density lipoprotein apolipoprotein E estrogen receptors selective estrogen receptor modulators confidence interval intima media thickness 1. Abstract

Cardiovascular disease is the leading cause of death among women. Inflammation plays a central role in the pathogenesis of many forms of vascular disease, including atherosclerosis. Women present with cardiovascular disease a decade after men and this has been attributed to the protective eVect of female ovarian sex hormones. Hormone replacement therapy (HRT), including a variety of estrogen preparations with or without a progestin, has negative eVects on most of these soluble inflammatory markers, including E‐selectin, cell adhesion molecules, monocyte chemoattractant protein‐1, and tumor necrosis factor‐a, inconsistent eVects on interleukin‐6, and stimulatory eVects on vasoprotective cytokine, such as the transforming growth factor‐a. C‐reactive protein, a circulating proinflammatory cytokine produced in both liver and atherosclerotic arteries, increases in response to oral conjugated estrogens but not to transdermal estrogen. Animal and observational studies have shown beneficial eVects of hormone therapy in the perimenopausal period or before the development of significant atherosclerosis, whereas randomized trials in older women have not shown any benefit in either primary prevention or secondary prevention of cardiovascular events. Many important questions about the eVects of ovarian hormones on vascular inflammation and the pathogenesis of vascular disease cannot be answered in human subjects. This review outlines the eVects of HRT on inflammatory biomarkers, summarizes results from observational and randomized trials, and highlights unanswered questions of hormone therapy and cardiovascular risk.

2. Introduction Cardiovascular disease remains the leading cause of death in the twenty‐ first century [1, 2]. Despite the advances in medical therapy and intervention, it is still the main cause of death among women in developed countries.

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Coronary artery disease is responsible for the majority of cardiovascular deaths among women [3]. Women themselves usually underestimate their cardiovascular risk and consider breast cancer as their main threat. Moreover, women have a less favorable prognosis than men with respect to a coronary event [3]. The vast majority of these cardiovascular events occur in postmenopausal women; premenopausal women, although rarely sick, have an even higher mortality compared to men of similar age during acute myocardial infarction and after coronary bypass surgery [4, 5]. Coronary artery disease develops in women, on average, 10 years later in life compared to men; the fact that they are older, and more often hypertensive and diabetics may account for both their higher risk during the course of acute coronary syndromes as well as in elective revascularization procedures (percutaneous transluminal coronary angioplasty and coronary bypass surgery) and their underinvestigation, underdiagnosis, and undertreatment. The 10‐year lag in the occurrence of coronary artery disease in women compared to men has been attributed to the protective eVects of female sex hormones, particularly estrogens, through mechanisms as yet not completely clarified [6]. Initially, data from various observational studies strongly supported a protective cardiovascular eVect of postmenopausal HRT, including diVerent estrogen preparations with or without a progestin (most commonly a synthetic progestin) [7]. However, the results of randomized placebo‐controlled trials in both secondary prevention and primary prevention refute the claims of cardioprotective eVects of HRT based on observational studies [8–10]. The Heart and Estrogen/Progestin Replacement Study (HERS) randomized postmenopausal women with established coronary artery disease to HRT or placebo and failed to show any diVerence in the occurrence of coronary events in midterm [8] or longer term follow‐up [9]. Likewise, estrogen replacement and atherosclerosis (ERA) showed no diVerence in the coronary angiographic findings in women with known coronary artery disease, whether they were treated with HRT or placebo [10]. In addition, the Women’s Health Initiative (WHI), in healthy postmenopausal women, demonstrated that HRT was associated with an initial increased risk of cardiovascular disease [11]. However, many unanswered questions remain. Reasons that may be associated with the discrepancy between older observational studies, of whom the most important is the Nurses Health Study, and the current randomized studies are the following: in majority, randomized placebo‐controlled trials are secondary prevention studies, which included women who were of average 65 years and at least 10 years in the menopause, either with established vascular disease or with subclinical atherosclerosis—in the WHI the majority of ‘‘healthy’’ women were obese and smokers—whereas in the observational studies women were much younger and perimenopausal. In addition, in the observational studies women had a healthy lifestyle, including exercise, moderate alcohol consumption, and regular medical checkup of which

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HRT was part, implying that the recorded benefits in the ‘‘users’’ arm were due to compliance rather than the HRT per se. Less important issues are the type of estrogen administered (unopposed in the observational studies vs. combined with progestins in the randomized placebo‐controlled trials) and the route of administration (oral vs. transdermal). Among the mechanisms that may account for the eVects of sex hormones on cardiovascular risk in women is inflammation. Recently, the role of several biomarkers in the prediction of coronary events has been studied in apparently healthy women, as well as among patients with stable angina and acute coronary syndromes [12, 13]. Clinical studies relating postmenopausal hormone use to vascular inflammation have relied on measurement of such biomarkers, as will be detailed below. This review will discuss the role of inflammation in the pathogenesis of vascular disease and its modulation by HRT, with particular emphasis on circulating biomarkers of inflammation. Misconceptions and controversies generated by the recent body of data will be emphasized in order to shed more light on the complex and confusing area of cardiovascular health and HRT.

3. Inflammation and Vascular Disease Inflammation plays a central role in the pathogenesis of many forms of vascular disease, including atherosclerosis [14]. Inflammatory processes participate all the way from the initial stages of vascular endothelial cell perturbations to the later eventual rupture of the ‘‘vulnerable plaque,’’ which leads to an acute coronary syndrome [15]. Vascular endothelial cell dysfunction begins well before any morphologic manifestations of atherosclerosis are visible, and continues probably waxing and waning throughout the entire course [15, 16]. A hallmark of atherogenesis is leukocyte recruitment and accumulation, which occurs early in atherosclerotic lesion generation. The normal endothelial cell generally resists adhesive interactions with leukocytes. However, very early after initiation of hypercholesterolemia, leukocytes adhere to the endothelium and begin to accumulate lipids and become foam cells. The damaged endothelium expresses certain cellular adhesion molecules, such as vascular cell adhesion molecule (VCAM‐1), intercellular adhesion molecule (ICAM‐1), which tether circulating inflammatory cells (monocyte and T‐lymphocytes) and initiate their rolling across the damaged endothelial surface [17]. Leukocytes are drawn to the vascular endothelium in part as a response to the elaboration of these adhesion molecules and other cells in the vessel wall. Human atherosclerotic lesions demonstrate increased expression of these adhesion molecules in endothelial cells of plaque microvessels or in endothelial cells overlying the

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lipid core, a response that may contribute to further leukocyte recruitment to sites of atherosclerosis [17]. Blocking the action of VCAM‐1 and ICAM‐1 has been shown to protect against the development of atherosclerosis, restenosis, or allograft arteriosclerosis [18, 19]. Studies in rabbits and mice have demonstrated increased expression of VCAM‐1 on endothelial cells overlying very early atheromatous lesions [20, 21]. In genetically altered animal models which do not express VCAM‐1, the atheroma formation was greatly attenuated, confirming the functional role of this adhesion molecule [20]. ICAM‐1 is more promiscuous both in the types of leukocytes it binds and because of its wide and constitutive expression at low levels by endothelial cells in many parts of circulation [22]. Selectins constitute the other broad category of leukocyte adhesion molecules, which mediate the rolling of leukocytes along the endothelium by binding to carbohydrate ligands on the cells [23]. They have been shown to be upregulated in endothelial cells overlying human atheromas, in restenosis, as well as in allograft arteriosclerosis [23–25]. E‐selectin (E stands for ‘‘endothelial’’) is selectively expressed by endothelial cells and preferentially, recruits polymorphonuclear leukocytes—a cell type seldom, if ever, found in early atheroma but an essential protagonist in acute inflammation [23]. Moreover, endothelial cells overlying atheroma do not express high levels of this adhesion molecule. P‐selectin is another member of this family (P stands for ‘‘platelet’’), which may play a greater role in leukocyte recruitment in atheroma, because endothelial cells overlying human atheroma do express this adhesion molecule. In the initiation of atherogenesis, P‐selectin has been shown to precede inflammatory cell infiltration into vessel walls and elimination of P‐selectin with antibodies attenuates both leukocyte rolling and attachment to endothelium, as well as the development of atheromatous and neointimal lesions in damaged vessels [26, 27]. Subsequent migration of inflammatory cells into the subendothelial space requires chemotaxis controlled by chemokines induced by the primary cytokines [28]. Once leukocytes are adherent to the endothelium, they migrate into the vascular wall in response to the influence of diVerent chemoattractant proteins. One such molecule is a monocyte chemoattractant protein‐1 (MCP‐1), which is produced by endothelium in response to oxidized lipoprotein and other stimuli. Human atherosclerotic lesions express increased levels of MCP‐1 compare with uninvolved vessels. Studies conducted with genetically modified mice lacking MCP‐1 or its receptor CCR‐2 have delayed and attenuated atheroma formation when placed on an atherosclerosis‐prone hyperlipidemic genetic background [29]. The activated inflammatory cells replicate and secrete cytokines, such as interleukin‐1 (IL‐1) and tumor necrosis factor‐
(TNF‐
), which, in turn, stimulate endothelial cells and vascular smooth muscle cells [30, 31]. These

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cytokines are also produced by the endothelial cells and vascular smooth muscle cells. Mononuclear cells within the initial infiltrate as well as intrinsic vascular cells subsequently release growth factors that stimulate proliferation of the smooth muscle cells and lead to plaque progression. Recent studies also support a role for interactions between CD40, expressed on lymphocytes, macrophages, endothelial and vascular smooth muscle cells and its ligand in the development of advanced atherosclerotic lesions, as the binding of CD40 to CD40 ligand results in the production of cytokines, matrix metalloproteinases, and adhesion molecules [32]. CD40 ligand can induce tissue factor expression and promote thrombus formation [33]. CD40 L‐null mice have been shown to have smaller atherosclerotic lesions that are less inflammatory [34]. Moreover, the primary proinflammatory cytokines result in the expression of messenger cytokines such as IL‐6, which is released into the systemic circulation and cause the liver to increase production of acute phase reactants, including C‐reactive protein (CRP) and serum amyloid A (SAA) [35]. CRP potently upregulates nuclear factor B (iNFB), a key nuclear factor facilitating transcription of numerous proinflammatory genes [36]. Synthesis of many cytokines such as TNF‐
, IL‐6 and IL‐8, is mediated by iNFB, as is the expression of cyclooxygenase. Cytokine induction leads to increased expression of proinflammatory molecules, increased leukocyte extravasations, and increased inflammation—generating a vicious circle [37, 38]. Furthermore, the dysfunctional endothelium produces less nitric oxide (NO), a leukocyte and platelet inhibitor, and a vasodilator that also helps maintain vascular smooth muscle cells in a nonproliferative state. Decreased NO production is implicated in the clinical course of all known cardiovascular diseases [39]. CRP may itself play a role in repressing the production of NO and diminishing NO bioavailability [39]. Traditional cardiovascular risk factors work in part by undermining the endogenous defenses of the vascular endothelium and contributing to its dysfunctional state. Hypercholesterolemia promotes increased formation of oxidized lipoproteins and foam cells, and reduces intracellular concentrations of NO [39]. The oxidative modification of native low‐density lipoprotein (LDL) to its oxidized form has a number of inflammatory and immunologic implications [40]. Oxidized LDL can increase monocyte adhesion, monocyte and T‐cell chemotaxis, scavenger receptor A expression, foam cell formation, and induce proinflammatory genes through iNFB, AP‐1, and c‐amp [40]. Among other lipoprotein particles, high‐density lipoprotein (HDL) cholesterol has gained significant attention not only for its reverse cholesterol transport properties, but also for its antioxidant activity. HDL particles can transport antioxidant enzymes such as paraoxonase, which can neutralize the proinflammatory eVects of oxidized lipids [41]. Other lipoprotein particles,

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such as very low‐density lipoprotein (VLDL), may have proinflammatory and atherogenic properties [42]. Accumulated evidence has established correlative as well as causative links between chronic inflammation and the insulin resistance of diabetes. Through nonenzymatic glycation of macromolecules, advance glycation end products bind with their receptor. These glycation end products‐ associated modifications can lead to increased expression and production of inflammatory mediators and proinflammatory cytokines [43, 44]. In addition, the diabetic state promotes oxidative stress mediated by reactive nitrogen and oxygen species. In obesity, when adiposity reaches a certain threshold, various factors are released from adipocytes that induce widespread macrophage activation and infiltration and that impair adipocyte insulin sensitivity [45]. Angiotensin II, a vasoconstrictor associated with clinical hypertension, opposes NO action, stimulates production of reactive oxygen species and increases the expression of proinflammatory IL‐6 and MCP‐1, and upregulates VCAM‐1 on endothelial cells [46, 47].

4. Mechanisms of Action of HRT in Vascular Biology 4.1. ESTROGEN As our understanding of the mechanisms by which estrogens aVect the cardiovascular system has increased, it has become clear that the complexity of the biological eVects of estrogens are reflective of quite complex mechanisms of action [48, 49]. The biological actions of estrogen are largely mediated by two distinct estrogen receptors (ERs) isoforms, which are known as ER‐
and ER‐ and both are expressed in cardiovascular cells and tissues [50]. The molecular pathways of ER activation have been deeply investigated. ERs are classically thought of as ligand‐activated transcription factors that reside in the cell nucleus and regulate gene expression in response to hormone binding. Following ligand binding, the ER undergoes conformational changes and biochemical modifications, which induce release of inhibitory proteins (heat shock proteins), receptor dimerization, and interaction with DNA. This mechanism, often referred to as the genomic pathway, likely underlies the longer term eVects of estrogen, such as those on circulating levels of lipids and coagulation factors [50]. More recently, it has become clear that ERs transduce the rapid eVects of estrogen, which occur within minutes and are referred to as nongenomic because they do not depend on changes in gene expression. These seem to be mediated by a receptor isoform, either distinct or identical to cloned ERs, localized at the plasma membrane. These rapid eVects of estrogen are mediated by a subpopulation of ERs

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localized to cell membrane signaling domains called caveolae [51, 52]. The best‐studied example of this nongenomic pathway for estrogen action in the cardiovascular system is the activation of endothelial cells NO synthase, which results in arterial vasodilatation in response to acute administration of estrogen [53]. The role of estrogen in vasculoprotection has been intensely studied in experimental models. Endogenous estrogens protect female rats against neointimal thickening and gonadectomy abolishes the eVect [54]. Estrogen treatment inhibits fatty streak and lesion formation in apolipoprotein E (ApoE)‐deficient mice [55]. Moreover, it inhibits neointimal formation after carotid balloon injury in rabbits, rats, and mice [56, 57]. As noted above, estrogens regulate a variety of systemic or circulating factors, including lipids, inflammatory factors, homocysteine, and members of the coagulation/ fibrinolytic cascades [58–60]. Estrogen can mediate both beneficial and adverse eVects on the cardiovascular system. On the positive side, estrogen has potentially beneficial eVects on lipid parameters, such as reducing total cholesterol and LDL, increasing HDL cholesterol and facilitating NO‐mediated vasodilatation [49]. Recent studies have also demonstrated an antioxidant eVect by estrogen, reducing LDL oxidation in vivo and in vitro. On the negative side, estrogens increase triglycerides [61, 62] and inflammatory markers such as CRP [63, 64]. Estrogens also have many prothrombotic eVects, such as increasing circulating levels of prothrombin and decreasing antithrombin III, contributing to an increased risk of venous thromboembolic events [65, 66]. Importantly, many of these eVects of estrogen are mediated by first‐pass eVects on the liver, and thus result from oral but not transdermal administration of HRT. For example, increased levels of CRP seem to occur only with oral estrogen administration [67, 68]. The extent to which this is associated with an increase in cardiovascular disease risk is uncertain. 4.2. SELECTIVE ESTROGEN RECEPTOR MODULATORS Unlike estrogens, which are uniformly agonists, and antiestrogens, which are uniformly antagonists, the selective estrogen receptor modulators (SERMs) exert selective agonist or antagonist eVects on various estrogen target issues [50, 69, 70]. SERMs act on their target cells by diVusing into the cell and binding to the two ERs with variable aYnities. According to current knowledge, the action of a SERM in a given tissue depends on several factors, such as the diVerential ER expression in the tissue, the ER subtype‐selectivity of the compound and the nature of the target gene promoter [71, 72]. The conformational change that takes place upon binding of the SERM to either of the ERs and particularly, the position of helix 12 in the ligand‐binding domain,

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depends on the SERM involved, and it plays a critical role in the agonist or antagonist configuration of the ER that determines the ability of the ER ligand complex to interact with coregulatory proteins and exert either the estrogen agonist or antagonist response in the tissue [73, 74]. Also, the expression of coregulatory proteins has been shown to vary in diVerent cells and tissues. The SERMs currently in clinical use, namely tamoxifen, raloxifene, and toremifene, are used for the prevention and treatment of breast cancer and postmenopausal osteoporosis. In addition, several promising compounds have been described, including idoxifene and ospemifene [70, 75–77]. The data presently available on the role of SERMs in vasculoprotection are promising. SERM therapy induces a beneficial serum lipid profile. Tamoxifen, toremifene, and raloxifene decrease LDL cholesterol but, unlike estrogen, do not increase triglycerides [78, 79]. We found that raloxifene, administered in postmenopausal women, significantly reduces total and LDL cholesterol as well as lipoprotein (a), although not to the extent that simvastatin does [80]. Toremifene, unlike other SERMs, increases HDL cholesterol [79]. Treatment with SERMs changes blood‐coagulation indexes in the direction of enhanced clotting and except from raloxifene increases indexes of inflammation [79]. Although ospemifene has a cholesterol‐lowering eVect in the rat, it showed a neutral eVect on vascular markers in a recent clinical study [81]. Moreover, in ovariectomized rats, raloxifene was as eVective as estrogen in enhancing NO‐induced coronary artery dilatation and in retarding injury‐induced intimal thickening of the carotid artery [82]. Tamoxifen and raloxifene have been reported to reduce markers of cardiovascular risk and to be favorable in lipid‐induced experimental atherosclerosis although contradictory results also exist [83–85]. The use of tamoxifen for the treatment of breast cancer during 5 years of follow‐up was associated with a reduced risk of acute myocardial infarction in women, suggesting that tamoxifen protects against coronary artery disease [86, 87]. The MORE (Multiple Outcomes of Raloxifene Evaluation) trial, which was designed to determine the eVects of raloxifene on vertebral fractures in women with osteoporosis, showed that 4‐year treatment with raloxifene does not aVect cardiovascular risk in the overall cohort nor in any single year but does so in those women with increased cardiovascular risk at baseline [88]. The RUTH (Raloxifene Use for The Heart) trial was an international randomized clinical trial designed to determine if raloxifene can reduce the incidence of coronary events and invasive breast cancer in postmenopausal women who had cardiovascular disease or who were at high risk of cardiovascular disease [89]. In the raloxifene group, levels of LDL cholesterol declined significantly and levels of HDL cholesterol increased significantly. However, these changes did not correspond to a protective eVect on the heart. Investigators did not see any significant diVerences between the two

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groups in the number of deaths from coronary causes, nonfatal heart attacks, or hospitalizations for an acute coronary syndrome. These results were unaVected by whether women already had cardiovascular disease or were at increased risk for cardiovascular disease. 4.3. PHYTOESTROGENS Phytoestrogens are a group of plant‐derived natural products possessing various degrees of estrogenic activity. They are present in many food products, such as soy and rye, as well as in many food supplements [90–92]. Phytoestrogens are traditionally divided into three main groups, namely isoflavones, lignans, and cumestans, of which isoflavones comprise the most common group [90–92]. The two major isoflavones are genistein and daizein, and their major natural sources are soy and red clover. Phytoestrogens bind to the ERs, and as with SERMs, the conformational change of the receptor, and especially the position of helix 12 of the ligand‐ binding domain, diVers depending on the type of ligand that binds to the receptor [93]. Thus, these compounds could also be called ‘‘phytoSERMs.’’ Genistein has approximately 20 times higher binding aYnity for ER‐ than ER‐
[94]. In micromolar concentrations, genistein also inhibits several enzymes, such as tyrosine kinase, which may contribute to its eVects, particularly its antiproliferative actions, in high doses [95]. Isoflavone and genistein appear to function as a calcium channel blocker in vascular smooth muscle and in cardiac myocytes, demonstrating that the vasodilator eVects are not dependent on the ER‐
, although an eVect of ER‐ could not be excluded. In order for some of these plant‐derived compounds to be biologically active, they must be modified by intestinal bacteria. For instance, isoflavones, including biochanin A, are found in soybeans and chick peas, and have direct biological activity, whereas other isoflavones are modified by the gut flora to produce biologically active compounds such as genistein [96]. Lignans are found in high concentrations in oil seeds and cereals. The most abundant mammalian lignan is enterolactone, which is dependent on the intestinal bacteria for its synthesis from the plant precursor. Flavanoids are often found in a glycoside form. The hypothesis that phytoestrogens have wide‐ranging health benefits, stemmed from several independent lines of evidence [97–99]. First, the phytoestrogens were recognized to exert estrogenic eVects in animals. Second, epidemiological studies, particularly in Asia, suggested that phytoestrogen‐ rich diet is associated with reduced incidence of breast and prostate cancer, cardiovascular disease, osteoporosis, and climacteric symptoms [92, 99]. Third, phytoestrogens were shown to improve plasma lipid levels and to reduce experimental atherosclerosis [100–103]. Several mechanisms were

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postulated, including endothelial vasodilatation, anti‐inflammatory eVects, antioxidant, and antithrombotic eVects [104, 105]. However, the eVects of phytoestrogens on atherosclerosis or cardiovascular disease in humans have not been studied either in clinical trials or in prospective epidemiological studies. In a western population, Van der Schouw et al. found that a high phytoestrogen intake is not associated with decreased incidence of cardiovascular disease [106]. However, they did not include measurements of blood and urine isoflavones and lignans as biomarkers to confirm dietary intake of phytoestrogens and complete information on soy isoflavones in the diet questionnaire [106]. We studied the eVect of genistein on infarct size in oophorectomized female rabbits, both normocholesterolemic and found no protection compared to placebo [107, 108]. During the past 10 years, a large amount of literature has developed on beneficial actions of phytoestrogens, especially as modulators of blood lipid risk factors. Meta‐analyses concluded that soy protein lowers serum cholesterol in moderate to severe hypercholesterolemia but did not have benefits in those with total cholesterol <255 mg/dl [100]. However, there was much variation among the results of individual studies, and the doses of soy protein [109–111]. Overall, the evidence does not support significant lipid eVects by soy isoflavones. Nonetheless, phytoestrogens and other flavonoids remain an interesting group of molecules with biological eVects yet to be fully explored, either as pharmacological agents or as components of the diet. 4.4. PROGESTERONE Progesterone is used as part of HRT in postmenopausal women, usually in combination with estrogens. Progesterone administration is necessary in non‐hysterectomized women since unopposed estrogen promote endometrial hyperplasia. The biological actions of progesterone are mediated by two progesterone receptors isoforms, PR‐A and PR‐B. In tissue culture, PR‐A and PR‐B exhibit diVerent transactivation properties that are specific to particular cell types [112]. Novel mechanisms of signal transduction are being discovered for progesterone receptors in diVerent tissues, some of which are independent of gene transcription regulation, and therefore are ‘‘nongenomic’’ [112]. Furthermore, the contribution to signal transduction of coactivators is currently widely investigated, in order to understand the ways to tissue‐specificity and to engineer new progesterone receptor modulators. The vascular actions of progestins are mediated by progesterone receptors, expressed in endothelial cells and the vascular smooth muscle as well as through downregulation of the estradiol receptor [113]. Progesterone receptors have been identified in the myocardium and in peripheral vascular

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tissues. There is evidence that progesterone is a vasoactive hormone, inhibiting agonist induced vasoconstriction [114]. Administration of progesterone has been shown to lower blood pressure in humans, to blunt the pressor response to angiotensin II in human pregnancy and to inhibit angiotensin II action in rats in some, but not all, reports [114, 115]. These eVects of progesterone cannot be attributed to a genomic eVect and seem to be direct cell‐membrane mediated, not involving a classic steroid/receptor mechanism. Concerning progesterone, evidence suggests that the natural molecule facilitates the inhibitory eVects of estrogen on vascular smooth muscle proliferation and may induce endothelium‐dependent vascular relaxation [116]. In addition, natural progesterone used in HRT appears to preserve the beneficial actions of estrogen [117]. There are studies, however, indicating that progestins may negate estrogen’s beneficial eVects—increase in plasminogen activator inhibitor‐1, decrease in NO, and attenuation of the antiatherogenic eVect of estrogen have been described. We, in an experimental rabbit model of myocardial infarction, found that the addition of progesterone to conjugated equine estrogen does not abolish the protection induced by estrogen on infarct size [118]. Progestins present diVerent pharmacological profiles according to their molecular structure, dosage, and to the presence of comorbidities. As with estrogens, the various progestins used in HRT may diVer significantly as to how closely they mimic their natural counterparts. For instance, progestin molecules with androgenic properties may antagonize estrogen‐dependent beneficial eVects on lipids [119] and a new molecule with antimineralocorticoid activity may reduce blood pressure in postmenopausal women with hypertension [120]. The few epidemiological studies that have investigated the eVect of the addition of a progestogen to estrogen therapy on cardiovascular mortality and morbidity have suggested that this type of HRT may be at least as eVective as estrogen replacement alone in reducing cardiovascular events [7]. Progestins added to estrogen therapy seem to increase inflammatory markers [121]. In addition, medroxyprogesterone acetate associated with conjugated equine estrogens produces no eVects [122] or inhibits endothelium‐dependent vasodilatation stimulated by estrogens [123]. These observations may explain, at least in part, the adverse results observed in the large prospective, randomized, placebo‐controlled trials of combined HRT—the WHI and HERS studies. However, the extent to which findings of these studies of medroxyprogesterone acetate and conjugated equine estrogens apply to other HRT formulations is unclear at present. Notwithstanding the availability of consistent observations on the functional eVects of progestins on the cardiovascular system, the understanding of the molecular basis of progesterone receptor signaling in vascular tissue is of paramount importance for the development of hormonal agents with an optimal cardiovascular profile.

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5. Effects of HRT on Inflammatory Markers 5.1. C‐REACTIVE PROTEIN A feature of most forms of inflammation or tissue damage is the increased serum concentration of acute‐phase reactants such as CRP. Primary proinflammatory cytokines result in the expression of messenger cytokines such as IL‐6, which can travel from local sites of inflammation to the liver, where it triggers a change in the program of protein synthesis characteristic of the acute phase response [35]. Although it is primarily derived from liver, CRP can be produced from vascular walls, particularly in the atherosclerotic intima of human coronary arteries. CRP per se may influence directly vascular pathology through several mechanisms, such as enhanced expression of local adhesion molecules, increased expression of endothelial plasminogen activator inhibitor‐1, reduced endothelial NO bioactivity, and increased LDL uptake by macrophages within atherosclerotic lesions [36, 39]. Moreover, CRP may act as a procoagulant, because it induces the expression of monocyte tissue factor, accelerating intravascular thrombosis and atherogenesis [35]. CRP, a simple downstream marker of inflammation, has now emerged as the best‐characterized biomarker of cardiovascular risk to date. Although the other biomarkers are biologically important, their clinical value has been limited because of inadequate standardization of assay conditions, the instability and short half lives of the proteins, inconsistency of prospective data, or lack of evidence of significant improvement in the prediction of risk over that aVorded by standard lipid screening alone [124]. Large series of prospective studies have demonstrated that CRP, when measured with new high sensitive assays, is an important predictor of risk for future cardiovascular events and death in apparently healthy individuals as well as those with known cardiovascular disease [124]. A prospective study from the Women’s Health Study tested the relative eYcacy of several variables measured at baseline to predict future cardiovascular events (death from coronary heart disease, nonfatal myocardial infarction or stroke, or the need for coronary‐revascularization procedures) in a large cohort of initially healthy middle‐aged women [125]. The relative risk among women in the highest quartile of CRP levels compared with the lowest was 4.4 (95% CI, 2.2–8.9), the highest risk assessment for any of the markers tested, including lipid levels. The only plasma markers that independently predicted future risk were CRP (relative risk, 1.5, 95% CI, 1.1–2.1) and total cholesterol to HDL ratio (relative risk, 1.4, 95% CI, 1.1–1.9), suggesting that the addition of the measurement of CRP to screening based on lipid levels may provide an improved method of identifying women at risk for cardiovascular events [125].

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Elevated CRP levels also appear to predict recurrent coronary events, thrombotic complications after angioplasty, poor outcome in the setting of unstable angina, and vascular complications after bypass surgery [126]. All these data support the concept that inflammation plays a critical role throughout the atherothrombotic process. CRP levels may also guide therapeutic interventions. Both aspirin and statin therapy appear to provide more benefit to those with elevated CRP. In the Physician’s Health Study and the Air Force/Texas Coronary Atherosclerosis Prevention Study (AFCAPS/TexCAPS), lovastatin appeared to lower cardiovascular event rates even for those with below median levels of LDL but above median levels of CRP [127]. Interestingly, statin therapy has been shown to lower CRP in persons with elevated pretreatment levels, suggesting that a component of the vasoprotection conferred by these agents may be related to an anti‐inflammatory eVect [128]. It is important to note, however, that more recent reports indicated that established coronary risk factors are generally stronger predictors than CRP values are [129]. The cut oV value, above which individuals without known disease should be considered at elevated risk and below which level patients with coronary artery disease should be considered at low risk, requires additional evaluation in population studies. Thus, the clinical relevance of CRP measurement in the prediction of the risk of cardiovascular disease remains unproven [130]. Also, it must be acknowledged that in some vascular disorders, CRP levels may remain elevated for years without ever leading to an acute cardiovascular event [131]. A number of investigations have demonstrated that CRP is increased in a rapid and sustained manner after the initiation of oral menopausal hormone therapy (estrogen with or without progestin) therapy. This observation raises the possibility of an upregulation of inflammation among women taking these agents. This issue is of clinical concern because CRP represents a potent independent risk marker for the development of cardiovascular events and such a proinflammatory eVect of HRT may help to explain the increased risk of cardiovascular disease in the first year of treatment with HRT, as observed in the HERS trial [8]. The largest of these, a nested, case‐control study from the WHI observational study, and data from the ERA and PEPI (Postmenopausal Estrogen/Progestin Interventions) trials, showed highly significant 63%, 31%, and 85% increases in CRP with a variety of oral hormone regimens [68, 132, 133]. However, increased CRP levels in these studies were not accompanied by elevations in IL‐6, E‐selectin, fibrinogen, or other acute phase reactants. In fact, decreases in circulating levels of adhesion molecules (E‐selectin, ICAM‐1, VCAM‐1) have been reported with oral menopausal hormone therapy in both healthy postmenopausal women and postmenopausal women with coronary artery disease.

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Furthermore, small studies have confirmed the inconsistent eVect of menopausal hormones on IL‐6 and have found significant reductions in TNF‐
and increases in the vasoprotective cytokine transforming growth factor‐ in postmenopausal women treated with HRT [134, 135]. These findings strongly suggest that the eVects of menopausal hormones on CRP do not represent a generalized proinflammatory eVect mediated through upstream cytokines such as IL‐6, but rather are related to a secondary mechanism. This may be critical, because plasma CRP concentrations are thought to reflect increased hepatic synthesis rather than systemic clearance. However, the clinical significance of these results has not been totally clarified. On the contrary, other trials have shown neutral eVects or a significant reduction in CRP concentrations after HRT therapy. Several trials have compared transdermal delivery to oral administration demonstrating a significant decrease in CRP levels [136–138]. In a cross‐sectional population survey, oral HRT use was associated with increased plasma levels of CRP, whereas no such association was observed in users of transdermal HRT. Wakatsuki et al. have demonstrated that although estrogen increased CRP, medroxyprogesterone acetate attenuated the proinflammatory eVect of estrogen in parallel with medroxyprogesterone acetate dose (2.5 mg vs. 5 mg) [134]. However, in the last study, plasma concentrations of medroxyprogesterone acetate were not measured and a cut oV level where medroxyprogesterone acetate begins to oVset the proinflammatory eVect of estrogen could not be determined. There are several important biological explanations for these variations in the direction of CRP after HRT. First, the finding that transdermal estradiol, unlike oral conjugated estrogen, does not elevate circulating CRP levels suggests that this secondary mechanism may be a first pass eVect of CRP production in the liver after oral estrogen absorption, whereas transdermal delivery bypasses the hepatic first‐pass metabolism eVect. Second, the relative androgenicity of the progestogen seems to be important. Both progestogens and androgens generally display anti‐inflammatory eVects in other tissues. Synthetic progestins such as medroxyprogesterone acetate also have androgenic eVects and medroxyprogesterone acetate may reduce CRP concentration in a similar manner in the liver. There is evidence that a certain polymorphism identifies a group of women (
20%) who have augmented eVects of HRT on levels of HDL cholesterol. Herrington et al. have measured serum levels of soluble E‐selectin and CRP at baseline and 1 year in 264 postmenopausal women randomized to treatment with oral conjugated equine estrogen, estrogen plus progestin, or placebo [139]. Women with the ER‐
IVS1‐401 C/C genotype had greater reductions in E‐selectin but no further increases in CRP with HRT [139]. The C allele produces a functional binding site that, in the presence of B‐myb, is capable of augmenting transcription of a downstream reporter construct

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10‐fold, suggesting that, in some settings, presence of this allele might amplify ER‐
transcription or produce ER‐
isoforms that have diVerent properties than the full‐length gene product. The addition of statin to estrogen administered to postmenopausal women was found to significantly attenuate the increase in CRP levels observed when estrogen was administered alone [140]. This eVect of statin therapy was independent of changes in lipoprotein levels or improvement [141]. These findings may have significant implications in the use of hormone therapy in postmenopausal women by reducing the potentially adverse eVects on inflammation and thrombosis that may result from increasing CRP to the magnitude observed with estrogen alone. In three randomized, placebo‐controlled trials on the eVects of raloxifene on CRP have demonstrated no influence on CRP concentrations compared with HRT and placebo [142–144]. On the other hand, tamoxifen markedly reduces, plasma CRP levels, probably by direct ER antagonism [145]. A nonrandomized trial investigating the eVects of HMR 3339—a newly developed SERM—has shown a dose‐dependent reduction in CRP, implicating a potential beneficial cardiovascular impact [146]. Some inconsistencies exist among studies investigating the eVect of isoflavones on circulating CRP levels. In two studies the use of isoflavonoids in a population of middle‐aged men and women or in postmenopausal women with a history of breast cancer failed to aVect the concentrations of CRP after 1 and 3 month’s therapy, respectively [147, 148]. However, in a randomized, double‐ blind, placebo controlled, crossover dietary intervention trial, isoflavone‐ enriched diet improved CRP concentrations for CRP values >1 mg/l for isoflavone compared with placebo [148]. These conflict results could be explained by the diVerent dose and type of isoflavones used and by the variations in vascular health and daily diets of the study populations. The findings for a beneficial eVect of isoflavones on CRP concentrations suggest that there may be some basis for the recommendation of isoflavone supplements for healthy postmenopausal women for the reduction of inflammatory cardiovascular disease risk factors, although the majority of inflammatory biomarkers were unaVected. Importantly, the clinical significance of hormone‐related increases in CRP is open to question. In the WHI observational study, CRP levels remain independently predictive of subsequent cardiovascular events irrespective of HRT status at baseline [132]. Thus, it would appear that the expressed level of CRP, rather than HRT, is a primary determinant of risk in healthy postmenopausal women. Although hormone therapy was associated with an increase in CRP, this change could not be equated with increased cardiovascular risk. These issues are of particular interest, given the growing body of evidence that markers of inflammation, such as CRP, may be useful for

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targeting preventive therapies, such as aspirin and statins. The fact that the use or non‐use of HRT had less importance than expressed CRP levels in terms of cardiovascular risk assessment also implies that diet, exercise, and smoking cessation are likely to remain the most important interventions for the primary prevention of vascular disease for some time to come.

5.2. SERUM AMYLOID A SAA is another acute‐phase protein that determines long‐term cardiovascular prognosis in women [125]. Data from the Women’s Health Study showed that SAA was among the most significant predictors for vascular events and in a subgroup analysis limited to women with LDL cholesterol <130 mg/dl, remained strongest along with high sensitivity CRP [125]. Like CRP, SAA is produced by the liver and has been shown to directly promote atherosclerosis and vascular inflammation [149]. However, unlike CRP, SAA can form a complex with HDL in the plasma, and the elevated SAA content of the HDL particle has been shown to interfere with antiatherogenic, antioxidative, and anti‐inflammatory HDL function [150]. Although the influence of estrogen on CRP has been studied extensively, eVects of estrogen administration on SAA are still under investigation. Serum levels of amyloid A were measured at baseline and after 12 weeks of treatment with continuous combined HRT containing 1 mg micronized 17‐estradiol and 0.5 mg norethisterone acetate. Despite statistically not significant, the concentration of SAA increased by 9.4% [151]. In a randomized double‐blind trial, 43 healthy women were randomized 6 weeks after surgically induced menopause to receive treatment with either oral or transdermal estradiol over a period of 28 weeks. Levels of SAA did not change significantly after oral or transdermal estradiol [152]. However, in 29 postmenopausal women without coronary heart disease, Abbas et al. have observed that estrogen significantly increased levels of SAA, whereas transdermal estrogen reduced both SAA and SAA content of the HDL, confirming the first‐pass hepatic eVect of oral estrogen [153]. Because elevated SAA levels predict adverse prognosis in healthy postmenopausal women, and elevated SAA content of the HDL particle have been shown to interfere with HDL function, the authors suggest that the route of administration may be an important consideration in minimizing side eVects of estrogen replacement therapy on cardiovascular outcomes. Adding androgens such as medroxyprogesterone acetate—a synthetic progestin—blunted the estrogen‐induced increase in SAA as well as CRP in a concentration‐dependent manner. These findings indicate that androgens may attenuate the proinflammatory eVects of estrogen.

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No significant eVects were seen for men or postmenopausal women, who underwent diets consisting of high‐ and low‐isoflavone soy proteins, in SAA concentration [154]. Similar results on SAA had diets with soy protein or soy nut consumption compared with the control diet in postmenopausal women with the metabolic syndrome. 5.3. CELL ADHESION MOLECULES Cell adhesion molecules, once expressed on the surfaces of endothelial cell or leukocytes after cytokine stimulation, are shed from the surface within 24 h. Several groups have reported the presence of ICAM‐1 and VCAM‐1 in the culture supernatant within 4–6 h of endothelial or leukocyte cell activation and in sera of humans [155]. Although the biological function in sera remains unclear, the clinical relevance of cell adhesion molecules has been suggested by several observational studies. Serum concentrations of cell adhesion molecules have been reported to be higher for patients with coronary artery disease than in healthy subjects [156]. As shown in both the Atherosclerosis Risk in Communities (ARIC) study and the Physicians Health Study, soluble VCAM‐1 levels do not appear to predict future cardiovascular events in healthy persons [157]. However, in persons with established cardiovascular disease, soluble VCAM‐1 is predictive of risk, with a 2‐fold increase in the risk of death in the highest quartile compared with the lowest in a prospective cohort of 1246 patients with coronary artery disease [158]. In contrast to ICAM‐1, which is expressed by many cell types, including circulating leukocytes and fibroblasts, VCAM‐1 expression is localized to the atherosclerotic plaque surface. This restricted pattern of expression may explain its unreliability as a biomarker of cardiovascular risk in healthy persons. In the Physicians’ Health Study, baseline levels of soluble ICAM‐1 were higher among those who had a myocardial infarction than among those who did not; the relative risk for men in the highest quartile was 1.8 times that of the lowest quartile (95% CI, 1.2–2.8, p ¼ 0.02), [159]. The ARIC study showed that the relative risk for coronary artery disease among women and men in the highest quartile of baseline soluble ICAM‐1 was 5.5 times (95% CI, 2.51–12.21) that of the lowest quartile [157]. Similarly, in the Women’s Health Study, the relative risk of a future cardiovascular event for women in the highest quartile of soluble ICAM‐1 at baseline was 2.6 times (95% CI, 1.3–5.1) that of women in the lowest quartile [125]. Concentrations of cell adhesion molecules increase after menopause, whereas HRT has been reported to decrease plasma levels of cell adhesion molecules, which may lead to the reduction in the risk of cardiovascular disease in postmenopausal women. Previous report demonstrated that serum concentrations of ICAM‐1 and VCAM‐1 are higher in postmenopausal

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women with coronary artery disease not on hormone therapy than in postmenopausal women with coronary artery disease on hormone therapy [160]. Several randomized, controlled trials with a pooled population of 364 women demonstrated an overall 10% reduction in these adhesion molecules with hormone therapy. These reductions were statistically significant in all of the trials, which tested a variety of menopausal hormone preparations [161–163]. Similar results were reported by Koh et al. who studied 20 healthy postmenopausal women treated with synthetic or natural progestagen combined with estrogen versus baseline [164]. Furthermore, there were no significant diVerences between therapies with synthetic or natural progestagen. The levels of soluble ICAM‐1 and VCAM‐1 were significantly reduced by HRT in women with diabetes, in patients taking aspirin or statins. These eVects on the levels of cell adhesion molecules may reduce attachment of white blood cells to the vessel wall and inhibit the development of atherosclerosis. However, estrogen therapy alone has also been reported to exert no eVects on the concentrations of ICAM‐1 and VCAM‐1. A possible explanation is that the estrogen‐induced increases in CRP may oVset the favorable eVects of estrogen on cell adhesion molecules, because CRP induces adhesion molecule expression. Nevertheless, the addition of a progestin compound to estrogen, obligatory to all women without previous hysterectomy, may compromise to some degree the eVects of estrogen. Wakakutsi et al. have shown that the addition of medroxyprogesterone acetate tended to decrease the levels of cell adhesion molecules, and use of 5.0 mg medroxyprogesterone acetate showed significant decreases in both cell‐adhesion molecule levels [123]. Thus, medroxyprogesterone acetate‐induced reduction in CRP seems to preserve estrogen’s favorable eVect on cell adhesion molecules [123]. According to Otsuki et al., progesterone but not medroxyprogesterone acetate inhibits VCAM‐1 expression in human vascular endothelial cells. This indicates that medroxyprogesterone acetate anti‐inflammatory eVect, but not its direct eVect, may decrease cell adhesion molecule concentrations [165]. Transdermal HRT decreases serum levels of soluble fragments of ICAM‐1, and VCAM‐1 in postmenopausal women [166]. These findings could indicate that transdermal HRT has a beneficial eVect on the endothelium and thus a potentially modulating eVect on the progression of atherosclerosis in postmenopausal women. Of interest are previous reports regarding the eVects of hormone therapy compared to statins, particularly in hypercholesterolaemic women with established coronary artery disease, since they may be considered for both therapies. Combined HRT or estrogen alone significantly reduced sICAM levels in these patients [167]. In contrast, simvastatin had no significant eVect on cell adhesion molecules, and the combination of HRT and simvastatin was not better than HRT alone. These eVects were associated with

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reductions of lipoprotein (a), which was shown to stimulate the expression of ICAM‐1 in cultured endothelial cells [168]. These findings may be of great importance since lipoprotein (a) is believed to be both atherogenic and antithrombolytic. SERMs are an attractive alternative to estrogen replacement as they obviate the need for a progestin and do not increase CRP levels. Blum et al., in a double‐blind three‐period crossover trial with estrogen, raloxifene versus placebo have shown that both therapies reduced ICAM‐1 levels versus placebo and to a similar degree VCAM‐1 was reduced by 10% with estrogen, but raloxifene did not have an eVect on VCAM‐1 when compared with placebo [142]. In postmenopausal women with hypercholesterolemia and coronary artery disease, raloxifene administration was associated with lower levels of both ICAM‐1 and VCAM‐1 compared to simvastatin [80]. Overall, raloxifene appears to decrease the levels of cell adhesion molecules but does not increase other markers of inflammation associated with estrogen therapy. The biological relevance of these eVects remains to be determined in future vascular studies and clinical trials. Most studies have shown that there is no beneficial eVect of dietary isoflavones on circulating concentrations of ICAM‐1 or VCAM‐1 in postmenopausal women [148, 169]. However, certain subpopulations may respond more beneficially to isoflavone supplementation, as shown, in a recent report, by the decrease in plasma VCAM‐1 concentrations in one of the genotypes of the ER‐ AluI polymorphism [148]. 5.4. S ELECTINS As a mediator of the earliest event of vascular inflammation, P‐selectin was chosen as a potential identifier of persons in the early stages of atherogenesis and, hence, at increased risk of developing clinical cardiovascular disease. A substudy of the Women’s Health Study compared baseline plasma P‐selectin levels from apparently healthy women who subsequently had a cardiovascular event to those from matched control subjects [170]. Participants with plasma P‐selectin levels in the highest quartile had a relative risk of cardiovascular disease 2.2 times higher than those in the lowest quartile ( p ¼ 0.02). This predictive eVect was independent of traditional risk factors. Plasma levels of E‐selectin may serve as a marker of atherosclerosis and as a predictor of coronary heart disease. E‐selectin has been demonstrated in human atherosclerotic arteries by immunohistochemistry. In the ARIC study, higher serum levels of E‐selectin were found in patients with coronary artery disease and carotid artery atherosclerosis than in healthy control subjects [157]. E‐selectin levels may directly contribute to atherosclerosis or reflect regulation of endothelial cell E‐selectin by cytokine stimulation.

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Higher concentrations of adhesion molecules have been associated with risk of first myocardial infarction in two studies, where observed eVects were independent of lipid levels [157, 159]. Apart from reflecting cytokine stimulation of endothelial cells by IL‐1 and TNF, an active role for circulating E‐selectin itself has not been clarified. Possible roles include anti‐inflammatory, proinflammatory, and procoagulant eVects. Seven randomized, controlled trials including 942 participants have demonstrated an overall 15% reduction in soluble E‐selectin levels with menopausal hormone therapy [139, 162–164, 171]. Reductions in soluble E‐selectin were seen with oral conjugated estrogen with or without medroxyprogesterone acetate or progesterone in the PEPI trial. Relative to placebo, when combining active treatment arms, final concentrations of CRP were 85% higher whereas E‐selectin was 18% lower compared with baseline [132]. The opposite eVects of postmenopausal hormones on CRP and E‐selectin may relate to the early increase and subsequent decline in myocardial infarction risk with hormones observed in the HERS trial [8]. This is supported by findings that the risk of myocardial infarction related to higher CRP concentration is greatest for events occurring within 1 year of measurement. On the other hand, the reduction of E‐selectin with treatment may be associated with long‐term physiological changes, which translate to cardioprotection, in a manner similar to that proposed for lipid eVects of hormone use. This idea is suggested because the eVects of therapy on E‐selectin and LDL cholesterol were correlated. Moreover, this finding suggests a hypothesis that part of the influence of hormones on E‐selectin is mediated by lipid eVects. The substudy of the Postmenopausal Hormone Replacement against Atherosclerosis (PHOREA) trial with 17‐estradiol plus progestin and the ERA trial with conjugated estrogen with or without medroxyprogesterone acetate have also highlighted the eVect of hormone therapy on endothelial inflammatory markers, including E‐selectin [139]. Four smaller studies confirm the inhibitory eVect of a variety of menopausal hormone regimens on soluble E‐selectin levels [162–164]. Plasma E‐selectin concentration seems to be reduced by approximately the same extent by high dose and low dose of estrogen, suggesting the favorable eVect of estrogen on cell adhesion molecules is preserved at the lower dosage [172]. Interestingly, participants in ERA trial with a common estrogen receptor polymorphism (ER‐
IVS1‐401 C/C genotype) demonstrated nearly a 2‐fold greater reduction in E‐selectin than those with the C/T or T/T genotypes, resembling closely similar magnitude eVect on HRT‐associated increases in HDL described previously [139]. These findings suggest a subset of women who may be more favorably aVected by hormone therapy with respect to cardiovascular risk.

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In postmenopausal women receiving transdermal HRT plus oral medroxyprogesterone acetate, E‐selectin, decreased significantly in the HRT group whereas did not change in the control group. Furthermore, the change in serum E‐selectin levels correlated positively with the change in carotid artery thickness but there were no significant correlations between the change in carotid artery‐intima media thickness (IMT) and the changes in other vascular inflammatory markers in the HRT group. Although the reason for this finding is unknown, the reduction in carotid artery‐IMT may contribute to the direct eVect of estrogen as well as the decrease in estrogen‐induced serum E‐selectin [173]. In hypercholesterolaemic women with established coronary artery disease, neither the simvastatin nor the combination of HRT and simvastatin had any significant eVect on VCAM‐1 or E‐selectin levels when compared to placebo [168]. Little is known about the eVect of phytoestrogens on E‐selectin, but an abundance of data has shown that diVerent forms of HRT result in decreased circulating levels of E‐selectin [174]. Phytoestrogens may resemble HRT regimens, which are known to reduce the levels of E‐selectin by 18–35%. Nikander et al. have demonstrated a fall by 4% in E‐selectin concentrations during the phytoestrogen regimen, but they were also reduced during the placebo regimen, although to a lesser degree [147]. This is probably the result of normal biological variation. As suggested by the correlation between reductions in E‐selectin levels and elevations in levels of phytoestrogens, the greater reductions in E‐selectin concentrations in women with higher daidzein and genistein levels may suggest that phytoestrogens can reduce the levels of E‐selectin, at least at high concentrations. This reduction may be proved to be in theory vasoprotective. 5.5. INTERLEUKIN‐6 IL‐6 is the principal procoagulant cytokine. It can increase plasma concentrations of fibrinogen, plasminogen activator inhibitor type and CRP, which amplify inflammatory and procoagulant responses. IL‐6 has predictive value in both healthy populations and persons with cardiovascular disease. In the Physicians’ Health Study, men with IL‐6 levels in the highest quartile were at a 2.3‐fold increased risk of future cardiovascular events compared with those in the lowest quartile (95% CI, 1.3–4.3, p ¼ 0.005) [175]. Participants in the Women’s Health Study with IL‐6 levels in the highest quartile suVered a relative risk of future cardiovascular events of 2.2 compared with those in the lowest quartile (95% CI, 1.3–4.7) [125]. Furthermore, in the Fragmin and Fast Revascularization during Instability in Coronary artery disease II trial (FRISC II), elevated IL‐6 was a robust

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predictor of increased mortality rates in participants with acute coronary syndromes and identified those most likely to benefit from an early invasive strategy [176]. IL‐6 has been shown to be modulated by estrogens. A negative correlation has been found between IL‐6 levels and plasma estradiol, and significantly higher IL‐6 levels were observed in postmenopausal women compared with premenopausal women. These findings have been confirmed in the experimental setting, because ovariectomy raised IL‐6 plasma levels in the mice model and HRT reversed this rise. In most studies, HRT increases CRP levels but reduces all other markers of vascular inflammation, including IL‐6. In PEPI trial, no correlation was observed between changes in CRP and IL‐6 levels [132]. Walsh et al. similarly reported that the increase in CRP with oral estrogen may be independent of changes in IL‐6 [177]. In WHI study, the long‐term HRT use was associated with increased CRP levels and with a decrease in IL‐6 and there was no association between HRT and IL‐6 [132]. In this regard, Herrington et al. found that oral estrogen significantly increased IL‐6 levels only in obese women, whereas CRP was significantly increased in both obese and lean women [178]. Adipocytes provide the major source of IL‐6. Therefore, it is unlikely that IL‐6 has a principal role in estrogen‐induced increases in CRP at least at low dosage of estrogen therapy [179]. These results suggest that the HRT‐induced increase in CRP levels may be metabolic and not related to a heightened vascular inflammatory status, particularly in younger postmenopausal women with no coronary risk factors such as diabetes mellitus, obesity, or hypertension. Additional studies of the eVects of diVerent hormone therapies on IL‐6 and other cytokines may shed light on this issue. 5.6. MONOCYTE CHEMOATTRACTANT PROTEIN‐1 MCP‐1 is overexpressed in human and experimental atheroma. It can recruit the mononuclear phagocytes that characteristically accumulate in the nascent atheroma in apolipoprotein(a)‐transgenic mice. Reckless et al. demonstrated that elevated expression of MCP‐1 was correlated with vascular macrophage accumulation. MCP‐1 also potently promotes tissue factor expression, inducing a local procoagulant state [180]. Recent work using compound mutant mice lacking MCP‐1 or its receptor CCR2, and susceptible to atherosclerosis owing to the absence of genes encoding ApoE or the LDL receptor, has shown striking decreases in mononuclear phagocyte accumulation and local lipid levels. Given the multitude of chemokines seen in atherosclerosis, it is truly remarkable that loss of either MCP‐1 or CCR2 can result in a large reduction in atherosclerosis, arguing that at best only partial redundancy occurs in chemokine signaling in atherosclerosis [181].

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Although the biological function in sera remains unclear, one recent study demonstrated that plasma MCP‐1 levels were elevated in patients with acute coronary syndrome and that enalapril therapy significantly reduced plasma MCP‐1 levels compared with placebo [182]. Limited (four trials, 101 pooled participants) trial experience has demonstrated robust and significant decreases (mean, 17.6%) in circulating MCP‐1 levels with hormone treatment, consistent with experimental reports [171, 182, 183]. Transdermal HRT reduces serum levels of MCP‐1 in postmenopausal women. Sumino et al. have demonstrated a significant decrease of MCP‐1 levels along with a decrease in carotid artery thickness [173]. Since the circulating levels of cell adhesion molecules and MCP‐1 are associated with the degree of carotid IMT in various populations, they suggested that this reduction in carotid artery wall thickness may also be attributable to an indirect eVect of estrogen on the artery wall mediated by cell adhesion molecules and MCP‐1 [173]. Estrogen causes an increase in NO production in vessel walls, and the NO, in turn, inhibits MCP‐1 expression in vascular endothelial cells [183]. Thus, it is possible that estrogen indirectly inhibits the expression of MCP‐1 by increasing NO production, and transdermal HRT may exert an antiatherosclerotic eVect by reducing MCP‐1 levels. 5.7. TUMOR NECROSIS FACTOR‐
TNF‐
is a cytokine with a multitude of actions. Originally characterized as an agent able to kill tumor cells, it is also a catabolic hormone (cachectin) and a proinflammatory molecule, acting as a mediator of toxic shock. TNF is synthesized particularly by inflammatory cells of the monocyte/macrophage lineage, but binds to receptors that are widespread. TNF‐
predicts cardiovascular events in both apparently healthy individuals and persons with established cardiovascular disease, as shown in the Cholesterol And Recurrent Events (CARE) trial [184, 185]. Most studies have shown positive influence of HRT on TNF‐
. In addition, Brooks‐Asplund et al. reported that HRT increased mononuclear cell‐derived TNF‐
[172, 186]. Phytoestrogens do not seem to exert changes in levels of TNF‐
.

6. Conclusion The HRT issue is not yet resolved and further experimental and clinical studies need to be done. The clinical studies and animal‐model research are useful for hypothesis generation, but they should never be considered

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adequate to justify a broad‐based pattern of clinical practice, such as the widespread use of HRT that occurred during the last decade or more. Learning how it is that estrogen can have such favorable eVects on lipids, endothelial function, and other aspects of vascular biology and still produce a net increase in clinical cardiovascular events will teach us something fundamentally important about cardiovascular disease that currently remains beyond our grasp. Issues such as lower doses, other routes of administration, treatment of young women in the perimenopause and more research in the genotype regarding which women may benefit more (ER‐
IVS1‐401 C/C genotype, ApoE, HDL receptor SR‐B1) or which are more prone to harm (prothrombin G20210A mutation, factor V Leiden) are still unanswered. Current trials are randomizing young women and the results are eagerly awaited, although the end points, in most cases, are intermediate biological markers and not clinical events; in this respect, these trials are unlikely to change the current practice which is not to prescribe HRT for either primary or secondary prevention. REFERENCES [1] C.J.L. Murray, A.D. Lopez, The Global Burden of Disease, World Health Organization, Geneva, 1996. [2] American Heart Association, Heart Disease and Stroke Statistics—2003 Update, American Heart Association, Dallas, TX, 2002. [3] AHA Scientific Statement 2004, Evidence‐based guidelines for cardiovascular disease prevention in women, Arterioscler. Thromb. Vasc. Biol. 24 (2004) 29–50. [4] V. Vaccarino, S.S. Rathore, N.K. Wenger, et al., Sex and racial differences in the management of acute myocardial infarction, 1994 through 2002, N. Engl. J. Med. 353 (2005) 671–682. [5] V. Vaccarino, Z.Q. Lin, S.V. Kasl, et al., Sex differences in health status after coronary artery bypass surgery, Circulation 108 (2003) 2642–2647. [6] W.B. Kannel, P.W. Wilson, Risk factors that attenuate the female coronary disease advantage, Arch. Intern. Med. 155 (1995) 57–61. [7] F. Grodstein, M.J. Stampfer, J.E. Manson, et al., Postmenopausal estrogen and progestin use and the risk of cardiovascular disease, N. Engl. J. Med. 335 (1996) 453–461. [8] S. Hulley, D. Grady, T. Bush, et al., Heart and Estrogen/Progestin Replacement Study (HERS) research group randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women, JAMA 280 (1998) 605–613. [9] D. Grady, D. Herrington, V. Bittner, et al., Cardiovascular disease outcomes during 6.8 years of hormone therapy: heart and Estrogen/progestin Replacement Study follow‐up (HERS II), JAMA 288 (2002) 49–57. [10] D.M. Herrington, D.M. Reboussin, K.B. Brosnihan, et al., Effects of estrogen replacement on the progression of coronary‐artery atherosclerosis, N. Engl. J. Med. 343 (2000) 522–529. [11] J.E. Rossouw, G.L. Anderson, R.L. Prentice, et al., Risks and benefits of estrogen plus progestin in healthy postmenopausal women principal results from the Women’s Health Initiative randomized controlled trial, JAMA 288 (2002) 321–333.

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ADVANCES IN CLINICAL CHEMISTRY, VOL. 47

PERSONALIZED CLINICAL LABORATORY DIAGNOSTICS Kewal K. Jain1 Jain PharmaBiotech, Basel, Switzerland

1. Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Basic Concepts of Personalized Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Pharmacogenomics and Personalized Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Pharmacogenetics and Personalized Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Role of Pharmacoproteomics in Personalized Medicine . . . . . . . . . . . . . . . . . . 4. Molecular Diagnostic Technologies for Personalized Medicine . . . . . . . . . . . . . . . . . 5. Role of PCR in Development of Personalized Medicine . . . . . . . . . . . . . . . . . . . . . . . . 5.1. SSCP Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Real‐Time PCR Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Combined PCR–Enzyme‐Linked Immunosorbent Assay (ELISA) . . . . . . . . . . . . . . 7. Non‐PCR Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Arrayed Primer Extension (APEX) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Direct Molecular Analysis Without Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. SNP and Personalized Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1. SNP Genotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Genetic Variations in the Human Genome Other Than SNPs. . . . . . . . . . . . . . . . . . . 10.1. INDELs in the Human Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2. Variation in Copy Number in the Human Genome. . . . . . . . . . . . . . . . . . . . . . . 10.3. Structural Variations (SVs) in the Human Genome. . . . . . . . . . . . . . . . . . . . . . . 10.4. Mapping and Sequencing of SV from Human Genomes . . . . . . . . . . . . . . . . . . 11. Role of Biomarkers in Personalized Medicine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Application of Biochip Technology in Developing Personalized Medicine. . . . . . . 12.1. GeneChip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2. AmpliChip CYP450 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3. Protein Biochips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13. Role of Nanobiotechnology‐Based Diagnostics in Personalized Medicine . . . . . . . 13.1. Nanoscale Single Cell or Molecule Identification . . . . . . . . . . . . . . . . . . . . . . . . . 13.2. Nanoparticles for Molecular Diagnostics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author: Kewal K. Jain, e‐mail: [email protected] 95

0065-2423/09 $35.00 DOI: 10.1016/S0065-2423(09)47004-5

Copyright 2009, Elsevier Inc. All rights reserved.

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14. Role of Cytogenetics in Personalized Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.1. Cytomics as a Basis for Personalized Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2. Study of Chromosomes by Atomic Force Microscopy. . . . . . . . . . . . . . . . . . . . 14.3. QD FISH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15. Integration of Molecular Diagnostics and Therapeutics. . . . . . . . . . . . . . . . . . . . . . . . . 16. Concluding Remarks and Future Prospects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Abstract Molecular diagnostics play an important role in development of personalized medicine and may be termed personalized diagnostics. This chapter shows the role of various diagnostic technologies in personalizing treatment. Besides polymerase chain reaction (PCR), several non‐PCR methods, biochips/microarrays, and nanobiotechnologies play an important role. Technologies for biomarkers, single nucleotide polymorphisms (SNPs), and cytogenetics provide basic information for personalized medicine. Finally, integration of diagnostics with therapeutics is an important component of personalized medicine. Future challenges and prospects of personalized diagnostics are discussed. 2. Introduction Personalized medicine, also referred to as individualized therapy, simply means the prescription of specific treatments and therapeutics best suited for an individual taking into consideration both genetic and environmental factors that influence response to therapy [1, 2]. Genomic/proteomic technologies have facilitated the development of personalized medicines but other technologies such as metabolomics are also contributing to this eVort. Any diagnostic procedure that facilitates the development of personalized medicine can be termed ‘‘personalized diagnostics.’’ Molecular diagnostics plays an important role in personalized medicine [3]. Role of molecular diagnostics in personalized medicine and interaction of various technologies is shown in Fig. 1. 3. Basic Concepts of Personalized Medicine 3.1. PHARMACOGENOMICS AND PERSONALIZED MEDICINE Pharmacogenomics applies the large‐scale systemic approaches of genomics to drug discovery and development [2]. It also involves the study of the mechanisms by which drugs change the expression of genes, including drug‐metabolizing enzymes, a phenomenon known as induction. Various

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Personalized medicine

Early diagnosis

Genomics and bioinformatics Molecular diagnostics

Discovery of genes, proteins, and biomarkers

Risk assessment

Reclassification of diseases

Prevention

Genetic screening

Clinical trials

Pharmacogenetics/genomics/proteomics metabolomics

Integrated healthcare

Monitoring of therapy

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Genetics

Toxicology prediction

Identification of drug resistance

Drug development ©

Source: Jain PharmaBiotech FIG. 1. Role of molecular diagnostics in personalized medicine.

technologies enable the analysis of these complex multifactorial situations to obtain individual genotypic and gene expression information. These same tools are applicable to study the diversity of drug eVects in diVerent populations. Pharmacogenomics promises to enable the development of safer and more eVective drugs by helping to design clinical trials such that nonresponders would be eliminated from the patient population and take the guesswork out of prescribing medications. It will also ensure that the right drug is given to the right person from the start. In clinical practice, doctors could test patients for specific SNPs known to be associated with nontherapeutic drug eVects before prescribing in order to determine which drug regimen best fits their genetic makeup. Pharmacogenomic studies are rapidly elucidating the inherited nature of these diVerences in drug disposition and eVects, thereby enhancing drug discovery and providing a stronger scientific basis for optimizing drug therapy on the basis of each patient’s genetic constitution.

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3.2. PHARMACOGENETICS AND PERSONALIZED MEDICINE Pharmacogenetics, a term recognized in pharmacology in the pregenomic era, is the study of influence of genetic factors on action of drugs as opposed to genetic causes of disease [2]. Now it is the study of the linkage between the individual’s genotype and the individual’s ability to metabolize a foreign compound. The pharmacological eVect of a drug depends on pharmacodynamics (interaction with the target or the site of action) and pharmacokinetics (absorption, distribution, and metabolism). It also covers the influence of various factors on these processes. Drug metabolism is one of the major determinants of drug clearance and the factor that is most often responsible for interindividual diVerences in pharmacokinetics. The diVerences in response to medications are often greater among members of a population than they are within the same person or between monozygotic twins at diVerent times. The existence of large population diVerences with small intrapatient variability is consistent with inheritance as a determinant of drug response. It is estimated that genetics can account for 20–95% of variability in drug disposition and eVects. Genetic polymorphisms in drug‐metabolizing enzymes, transporters, receptors, and other drug targets have been linked to interindividual diVerences in the eYcacy and toxicity of many medications. Although interindividual variations in drug response result from eVects of age, sex, disease, or drug interactions, genetic factors represent an important influence in drug response and eYcacy and remain constant throughout life. This has led to the recognition of the discipline ‘‘pharmacogenetics’’ since the 1950s, which can be viewed an as integration of gene profiling and pharmaceutical chemistry. From this initial definition, the scope has broadened so that it overlaps with pharmacogenomics. Pharmacogenomics, a distinct discipline within genomics, carries on that tradition by applying the large‐scale systemic approaches of genomics to understand the basic mechanisms and apply them to drug discovery and development. Pharmacogenomics now seeks to examine the way drugs act on the cells as revealed by the gene expression patterns and thus bridges the fields of medicinal chemistry and genomics. Some of the drug response markers are examples of interplay between pharmacogenomics and pharmacogenetics; both are playing an important role in the development of personalized medicines. The two terms, pharmacogenetics and pharmacogenomics are sometimes used synonymously but one must recognize the diVerences between the two. 3.3. ROLE OF PHARMACOPROTEOMICS IN PERSONALIZED MEDICINE Role of proteomics in drug development can be termed ‘‘pharmacoproteomics’’ [4]. Proteomics‐based characterization of multifactorial diseases may help to match a particular target‐based therapy to a particular marker

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in a subgroup of patients. The industrial sector is taking a lead in developing this area. Individualized therapy may be based on diVerential protein expression rather than a genetic polymorphism. Proteomics will have a great impact on diagnosis during the first decade of the twenty‐first century. By the end of the decade protein chip‐based tests will be available for several diseases. Knowledge gained from genomics and proteomics will be combined to provide optimal detection of disease at an early stage for prevention or early intervention. Proteomics‐based molecular diagnostics will have an important role in the diagnosis of certain conditions and proteomics‐based medicines would be integrated in the total healthcare of a patient. Advantages of application of pharmacoproteomics in personalized medicine are as follows:
Pharmacoproteomics is a more functional representation of patient‐ to‐patient variation than that provided by genotyping.
Because it includes the eVects of posttranslational modification, pharmacoproteomics connects the genotype with the phenotype.
By classifying patients as responders and nonresponders, this approach may accelerate the drug development process.

4. Molecular Diagnostic Technologies for Personalized Medicine Following the discovery of PCR, molecular diagnostics has been expanding rapidly, particularly during the last decade. Several non‐PCR and proteomics‐based tests have joined the list which now runs over several hundred tests. A classification of clinical laboratory tests that are relevant to personalized medicine is shown in Table 1. Tests in these categories may overlap as this classification is a mix of technologies and applications. Some of these technologies will be described briefly in the following text.

5. Role of PCR in Development of Personalized Medicine Several PCR‐based methods are used in the development of personalized medicine. An example is that of single‐strand conformational polymorphism (SSCP) analysis. These can be used alone or in combination with other technologies. PCR‐based methods are time‐consuming and are used mostly in laboratory setting. For the practical development of personalized medicine, point‐of‐care testing is more suitable and rapid results are required such as in the oYce or out‐patient management of infection, where

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TABLE 1 A CLASSIFICATION OF CLINICAL LABORATORY TESTS AND THEIR APPLICATIONS RELEVANT TO PERSONALIZED MEDICINE Technologies Polymerase chain reaction (PCR)‐based methods, for example, single‐strand conformational polymorphism (SSCP) Non‐PCR based methods: for example, direct molecular analysis without amplification Enzyme‐linked immunosorbent assay (ELISA) Peptide nucleic acid (PNA) technology Combined use of nucleic acid amplification and with methods such as immuno‐PCR Proteomics diagnostic technologies Cytogenetics, for example, fluorescent in situ hybridization Biochips and microarrays Nanobiotechnology‐based diagnostics Single nucleotide polymorphisms (SNP) genotyping Gene expression profiling Transcriptome profiling studies Sequencing, for example, multiplex DNA sequencing and whole genome sequencing Applications Biomarker detection and application for diagnostics as well as therapeutics Pharmacogenomic tests Pharmacogenetic tests Pharmacoproteomic tests Pharmacometabolic tests Molecular toxicology: toxicogenomics and toxicoproteomics To understand the molecular mechanisms of disease as basis for rational therapy Discovery and development of personalized medicines Diagnostics as companions to therapeutics Monitoring of therapy as well as determining prognosis of disease # Jain PharmaBiotech.

identification of the microorganism is required before suitable treatment is prescribed and the patient leaves the oYce. 5.1. SSCP ANALYSIS SSCP, an easy and sensitive PCR method, is one of the most popular methods for detection of mutations. The target sequence is amplified and labeled simultaneously by radioactive primers and nucleotides. The amplified product is then heated to dissociate the strands and subjected to nondenaturing polyacrylamide gel electrophoresis. The patient’s DNA is analyzed alongside normal samples on gels and mutations can be detected by diVerences in the respective band patterns seen on autoradiography. Putative mutant PCR products are cloned and sequenced to determine the nature of

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SSCP diVerence. False negative results, however, cannot be excluded. Therefore, the absence of mutations cannot be proven by this technique. Automated capillary electrophoresis (CE) systems have now been introduced for SSCP analysis instead of conventional slab gel electrophoresis. SSCP analysis in combination with CE is a rapid, simple, sensitive, and high‐throughput (HT) mutation‐screening tool, and has been successfully applied for mutation detection involving human tumor suppressor genes, oncogenes, and disease‐ causing genes. The new technique has a great potential for mutation screening of large numbers of samples in clinical diagnosis. The use of SSCP analysis to discover and genotype SNPs has been widely applied to the genetics of hypertension, including both monogenic disorders such as Liddle’s syndrome and polygenic disorders such as essential hypertension [5]. 5.2. REAL‐TIME PCR SYSTEMS Some of the limitations have been addressed in, a number of real‐time PCR systems are now on the market, which oVer many general technical advantages over standard PCR, including reduced probabilities of variability and contamination, as well as online monitoring and the lack of need for postreaction analyses. There are currently five main chemistries used for the detection of PCR product during real‐time PCR: DNA binding fluorophores, 50 endonuclease, adjacent linear and hairpin oligoprobes, and the self‐fluorescing amplicons. Some of real‐time PCR systems were developed with contemporary applications such as quantitative PCR (qPCR), multiplexing, and HT analysis in mind. In real‐time PCR, the amount of product formed is monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into the reaction that is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules is registered. Assuming a certain amplification eYciency, which typically is close to a doubling of the number of molecules per amplification cycle, it is possible to calculate the number of DNA molecules of the amplified sequence that were initially present in the sample. With the highly eYcient detection chemistries, sensitive instrumentation, and optimized assays that are available today the number of DNA molecules of a particular sequence in a complex sample can be determined with unprecedented accuracy and sensitivity suYcient to detect a single molecule [6]. An example of usefulness of real‐time PCR is for identification of pathogens such as methicillin‐resistant Staphylococcus aureus (MRSA) as well as methicillin‐sensitive S. aureus (MSSA) and the accurate determination of antimicrobial sensitivities for prompt optimal patient therapy. Real‐time PCR targeting the S. aureus‐specific thermonuclease nuc gene and the

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staphylococcal methicillin resistance determinant mecA gene has been used as a reliable indicator of the presence of MRSA and detection time for MRSA/MSSA isolates from positive blood cultures was dramatically reduced from 24 to 48 h required for routine tests to approximately 3 h [7]. Quantitative real‐time PCR is useful in detection and staging of lung cancer as well as construction and validation of prognostic and predictive gene expression signatures [8]. Real‐time qPCR can be applied to analysis of clinical samples to help stratification of patients in personalized medicine approach. It enables the measurement of gene expression or DNA copy number in specific cell types that are available only in a small quantity. Limitations of real‐time PCR include that PCR product increases exponentially and variation increases with cycle number. There is an increased variation after transformation to linear values and increased risk of false negative results. 6. Combined PCR–Enzyme‐Linked Immunosorbent Assay (ELISA) PCR is limited with respect to gene quantification because of the exponential amplification of DNA, the tendency to occasionally amplify nonspecific DNA, and the semiquantitative character of such common DNA measurement techniques as Southern blotting or densitometry. Combined PCR–ELISA CR has been developed for overcoming the limitation of quantitative gene analysis. This method combines certain features of PCR and ELISA techniques for accurate, high‐precision measurements of hybridization with sequence‐specific probes. Unlike Southern blotting, the combined PCR–ELISA enables complete removal of competing DNA strands prior to hybridization and does not have the background problems associated with membranes. The quantification of the input DNA using this method is independent of the number of PCR amplification cycles, and can be calculated automatically with a standard laboratory ELISA plate reader. With these advances over current technology, the gene quantification method should find wide application in a variety of diagnostic and research applications in clinical chemistry, microbiology, and genetics. Applications relevant to personalized medicine include:
Diagnosis and typing of infectious diseases.
HLA typing for organ transplantation and autoimmune disease diagnosis.
Cancer diagnosis by oncogene detection.
Diagnosis of genetic diseases.
Monitoring of inflammatory diseases by assaying cytokine gene expression.

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7. Non‐PCR Methods 7.1. ARRAYED PRIMER EXTENSION (APEX) APEX reaction is a straightforward and robust enzymatic genotyping method in which hundreds to thousands of variations in the genome are simultaneously analyzed in a single multiplexed reaction. It diVers from allele‐specific hybridization in that the genotype information in APEX is obtained by single base extension, performed by a specific DNA polymerase, together with four diVerent dye terminators [9]. This approach ensures highly specific discrimination without allele‐specific hybridization, because the primer to be extended anneals just adjacent to the DNA base that needs to be identified. Selection of primers for specific sites or their consecutive placement in tiled format, shifting them by one base, enables SNP analysis, mutation detection, or resequencing of the DNA template. It also permits the analysis of insertions, deletions, splice variants, gene copy numbers, or CpG islands within the genome for gene methylation studies, by performing additional bisulfite reactions. Advantages for this method over usual hybridization strategies are:
Reduced mismatching due to intercession of the polymerase.
Increased resistance to oligonucleotide failure sequences.
Tolerance of a greater range of hybridization conditions. 8. Direct Molecular Analysis Without Amplification Direct analysis of single biological molecules is the key to the next generation of revolutionary technologies. Some technologies can directly analyze individual molecules of DNA, RNA, and proteins, without PCR amplification. They can replace traditional molecular biology techniques, which are hampered by the need for amplification and bulk fluorescence, with the accuracy and sensitivity of direct measurements based on single molecules. Additional advantages of the platform include:
Sensitivity in the femtomolar range.
No need for amplification (e.g., PCR) or enzymatic procedures, eliminating a major source of cost and bias.
Small sample material requirements.
Flexibility across sample types (e.g., DNA, RNA, and protein) and assays. 9. SNP and Personalized Medicine Small stretches of DNA that diVer in only one base are called SNP and serve to distinguish one individual’s genetic material from that of another. SNPs comprise some 80% of all known polymorphisms. Among the roughly

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3‐billion nucleotide base pairs (i.e., the ‘‘letters’’) that make up the genetic code, SNPs occur with a frequency of one per 500 base pairs so that there are approximately 6 million SNPs. Each gene contains approximately 5 coding SNPs, which likely eVect the expression of the currently estimated 20,000– 25,000. Identification of SNPs is important as it helps in understanding the genetic basis of common human diseases. In the absence of functional information about which polymorphisms are biologically significant, it is desirable to test the potential eVect of all polymorphisms on drug response. More than 9 million SNPs have been already generated in public databases using a large number of methods but only a small fraction of these are well characterized and validated [10]. High‐resolution genome‐wide association studies using panels of 300,000 to 1 million SNPs aim to define genetic risk profiles of common diseases. These studies provide an opportunities to explore pathomechanism of human diseases that are unbiased by previous hypotheses or assumptions about the nature of genes that influence complex diseases. Many genetic variants identified as risk factors for diseases by such studies have been localized to previously unsuspected pathways, to genes without a known function. In the absence of functional information about which polymorphisms are biologically significant, it is desirable to test the potential eVect of all polymorphisms on drug response. Potential uses of SNP markers include drug discovery and prediction of adverse eVects of drugs. SNPs have the following relation to an individual’s disease and drug response, which indicates their usefulness for personalized medicine:
SNPs are linked to disease susceptibility.
SNPs are linked to drug response, for example, insertions/deletions of ACE gene determine the response to beta blockers.
SNPs can be used as biomarkers to segregate individuals with diVerent levels of response to treatment (beneficial or adverse) in clinical settings. Genotyping for complex diseases may be insuYcient to predict whether a person is at risk for a particular disease. One tries to associate SNPs with disease, but if no SNP in a certain gene predicts disease, further interest in the gene or protein or enzyme is lost. 9.1. SNP GENOTYPING A classification of technologies used for detection and analysis of SNPs is shown in Table 2. The technologies are described in more detail elsewhere [11]. Desirable characteristics of a genotyping technology are: (1) robust performance and accuracy across a variety of circumstances, (2) HT performance, and (3) low cost. Sequencing oVers the highest degree of specificity

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TABLE 2 CLASSIFICATION OF TECHNOLOGIES FOR SNP ANALYSIS DNA chips and microarrays DNA sequencing Electrochemical DNA detection Fluorescence‐detected 50 ‐exonuclease assays Hybridization assays Mass spectrometry (MS)‐based methods, for example, matrix‐assisted laser desorption ionization time of flight MS (MALDI‐TOF MS) Nanotechnology‐based methods Non‐PCR methods: arrayed primer extension (APEX) PCR‐based methods Peptide nucleic acid (PNA)‐based methods Pyrosequencing Restriction‐fragment‐length polymorphism (RFLP) Single molecular fluorescence technology Zinc finger proteins # Jain PharmaBiotech.

and selectivity. For ten or fewer SNPs and sample numbers in the thousands, the current gold standard is TaqMan real‐time PCR (Applied Biosystems). MassARRAY system (SEQUENOM), a mass spectrometry‐based platform suitable for high throughput and up to 1000 SNPs. Pyrosequencing (Biotage AB), a sequencing‐by‐synthesis method, can be used for up to 100 SNPs. AVymetrix provides the densest coverage at the whole‐genome level with its GeneChip Human Mapping 500K Array Set and AVymetrix GeneChipÒ Scanner 3000 MegAllele, based on Molecular Inversion Probe Technology TM (obtained by acquisition of ParAllele BioScience), enables the highest level of multiplexing that is commercially available as well as increase throughput with low capital investment. Illumina is supplementing its current 100K chip with a 250K chip. Restriction fragment length polymorphism analysis is laborious and hit‐and‐miss as success depends on whether the restriction enzyme recognizes particular SNPs. It is relatively inexpensive, which makes it appropriate for a small number of SNPs and a small number of samples. New methods for SNP genotyping are being investigated.

10. Genetic Variations in the Human Genome Other Than SNPs Although many studies have been conducted to identify SNPs in humans, few studies have been conducted to identify alternative forms of natural genetic variation. These include insertions and deletions (INDELs) as well as copy number variations (CNVs) in the genome.

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10.1. INDELS IN THE HUMAN GENOME A map of 415,436 INDELs have been mapped in the human genome that signal a little‐explored type of genetic diVerence among individuals [12]. INDELs are an alternative form of natural genetic variation that diVers from the much‐studied SNPs. Both types of variation are likely to have a major impact on humans, including their health and susceptibility to disease. Whereas SNPs are diVerences in single chemical bases in the genome sequence, INDELs result from the insertion and deletion of small pieces of DNA of varying sizes and types. If the human genome is viewed as a genetic instruction book, then SNPs are analogous to single letter changes in the book, whereas INDELs are equivalent to inserting and deleting words or paragraphs. INDELs were discovered using a computational approach to reexamine DNA sequences that were originally generated for SNP discovery projects. INDELs are distributed throughout the human genome with an average density of one INDEL per 7.2 kb of DNA. Variation hotspots were identified with up to 48‐fold regional increases in INDEL and/or SNP variation compared with the chromosomal averages for the same chromosomes. The map is being expanded. INDELs already are known to cause human diseases. For example, cystic fibrosis is frequently caused by a 3‐base pair deletion in the CFTR gene, and DNA insertions called triplet repeat expansions are implicated in fragile X syndrome and Huntington’s disease. Transposon insertions have been identified in hemophilia, muscular dystrophy, and cancer. INDEL maps will be used together with SNP maps to create one big unified map of variation that can identify specific patterns of genetic variation to help predict the future health of an individual. The next phase of this work is to figure out which changes correspond to changes in human health and develop personalized treatments.

10.2. VARIATION IN COPY NUMBER IN THE HUMAN GENOME CNV of DNA sequences is functionally significant but has yet to be fully ascertained. A study showing that 12% of human genes vary in the CNV of DNA sequences they contain—a finding that contradicts previous assumptions that the DNA of any two humans is 99.9% similar [13]. The discovery indicates that CNV could play a larger role in genetic disease than previously thought, with broad implications in genetic diagnostic testing. Some CNVs are already known to be associated with disease, including AIDS, inflammatory bowel disease, lupus, cataracts, arterial disease, and schizophrenia. The findings could change the direction of future genetic disease research, which has primarily focused on SNPs. Some diseases are caused by CNV rather than SNPs.

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10.3. STRUCTURAL VARIATIONS (SVS) IN THE HUMAN GENOME Structural changes are extremely common in human populations. Genetic variation among individual humans occurs on many diVerent scales, ranging from gross alterations in the human karyotype to a SNP. More bases are involved in structural changes in the genome than are involved in single‐base‐pair changes. Although the original human genome sequencing eVort was comprehensive, it left regions that were poorly analyzed. Later investigations revealed that, even in healthy individuals, many regions in the genome show SVs, which involve kilobase‐ to megabase‐sized deletions, duplications, insertions, inversions, and complex combinations of rearrangements. A study oVers a new view of what causes the greatest genetic variability among individuals suggests that it is due to the presence of structural changes that cause extended segments of the human genome to be missing, rearranged, or present in extra copies, rather than to single point mutations [14]. This study was designed to fill in the gaps in the genome sequence and to create a technology to rapidly identify SVs between genomes at very high resolution over extended regions.

10.4. MAPPING AND SEQUENCING OF SV FROM HUMAN GENOMES The first high‐resolution map showing the structural variants (SVs) that exists in the human genome has been published [15]. Using a clone‐based method, the complete DNA sequences of eight people of diverse geographic ancestry was examined: four of African descent, two of Asian descent, and two of western European descent. The DNA sequence of those eight people was compared to the DNA sequence derived from the Human Genome Project, which is known as the reference sequence. This map provides a comprehensive picture of the normal pattern of SV present in these genomes, refining the location of 1695 SVs that were more than about 6000 base pairs long; 50% of these were seen in more than one individual and lay outside regions of the genome previously described as SV. The researchers discovered 525 new insertion sequences, ranging in size from a few thousand to 130,000 base pairs, which are not present in the human reference genome, and many of these are variable in copy number between individuals. Complete sequencing of 261 SVs revealed considerable locus complexity and provides insights into the diVerent mutational processes that have shaped the human genome. In various parts of human genome, some people have segments of DNA sequence that other people do not have. Large genetic regions may be flipped in one person compared with another and these diVerences can influence a person’s susceptibility to various diseases. These data provide a standard for

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genotyping platforms and a prelude to future individual genome sequencing projects. The results also indicate that the human genome sequence is still incomplete that sequencing of additional genomes will be required to fill the remaining gaps. The eight people studied are part of a much larger group whose genomes will be sequenced as part of the 1000 Genomes Project, an international eVort to sequence the genomes of people from around the world. In order to understand SV, it is also essential to develop new technologies designed to detect genetic diVerences among people. For example, SNP biochips, whether used in research or in clinical applications, need to reflect this SV to find links between particular gene variants and diseases. Currently available biochips would miss an association for nearly half of these sites. Besides their potential applications, the new results provide a wealth of data to explore hypotheses and make discoveries as we now have eight new reference human genomes.

11. Role of Biomarkers in Personalized Medicine Development of personalized medicine is closely linked to biomarkers, which may serve as the basis for diagnosis, drug discovery, and monitoring of diseases. This process of personalization starts at the development stage of a medicine and is based on pharmacogenomics, pharmacogenetics, and pharmacoproteomics. Biomarkers are becoming more critical in the process of discovering and developing new drugs and in determining additional uses for established drugs. Biomarkers are ushering in the age of personalized medicine. One example is the use of cancer biomarkers in development of personalized therapy of cancer [16]. Identification and characterization of a large number of genetic polymorphisms (biomarkers) in drug metabolizing enzymes and drug transporters in an ethnically diverse group of individuals may provide substantial knowledge about the mechanisms of interindividual diVerences in drug response. Pharmacogenetics is used in preclinical investigations for biomarkers of drug‐response or drug‐induced toxicity, identification of genes with variants that may define patient populations, identification of proteins as potential biomarkers, or the comparison of the response in human and clinical animal models. Application of pharmacogenetic biomarkers should be able to predict adverse reactions in clinical trials. Pharmacogenomics can now be redefined as the study and application of DNA‐, and RNA‐based biomarkers to predict how an individual’s genetic inheritance aVects the body’s response to a drug. Pharmacogenomic biomarkers should be able to predict drug eYcacy in clinical trials. Discovery and

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validation of pharmacogenomics biomarkers can lead to the development of pharmacogenomic tests that can be used to personalize therapy.

12. Application of Biochip Technology in Developing Personalized Medicine Biochip is a broad term indicating the use of microchip technology in molecular biology and can be defined as arrays of selected biomolecules immobilized on a surface. DNA microarray is a rapid method of sequencing and analyzing genes. An array is an orderly arrangement of samples. The sample spot sizes in microarray are usually less than 200 m in diameter. It is comprised of DNA probes formatted on a microscale (biochips) plus the instruments needed to handle samples (automated robotics), read the reporter molecules (scanners), and analyze the data (bioinformatic tools). Microarrays allow a look at very subtle changes in many genes at one time. They provide a snapshot of what genes are expressed or active, in normal and diseased cells. When normal cells or tissues are compared to those known to be diseased, patterns of gene expression can emerge, enabling assessment of the severity of the disease and identification of genes that can be targeted for therapy. This is how microarrays can potentially be used to develop personalized medical treatments. Microarray technology not only helps to make sense of the vast amount of genomic information but also enables its application to the patient by early detection of disease and prediction of drugs response in individuals. Although some problems of standardization and integration with electronic records remain, microarrays are promising for eYcient, cost‐eVective, and personalized approaches to human health care. Numerous biochip technologies are available for clinical applications. The best known are the GeneChip and the AmpliChip CYP450. 12.1. GENECHIP AVymetrix has developed an approach in which sequence information is used directly to design high‐density, two‐dimensional rays of synthetic oligonucleotides. The GeneChip (AVymetrix) probe arrays are made using spatially patterned, light‐directed combinatorial chemical synthesis and contain up to hundreds of thousands of diVerent oligonucleotides on a small glass surface. The arrays have been designed and used for quantitative and highly parallel measurements of gene expression, to discover polymorphic loci and to detect the presence of thousands of alternative alleles HG‐U133 Plus 2.0 Array, gives the protein‐coding content of the human genome on a single piece the size of a fingernail.

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12.2. AMPLICHIP CYP450 AmpliChip CYP450 (Roche Molecular Diagnostics) is used with the GeneChip 3000Dx microarray system of AVymetrix. It was cleared by the regulatory authorities for marketing in the USA and the EU as an in vitro laboratory diagnostic test in December 2004. The microarray chip (also referred to as ‘‘probe microarray’’ contains millions of tiny DNA molecules and the test is performed using DNA that is extracted from a patient’s blood. DNA sequence is determined based on the sequence of the probe molecule to which the DNA is most similar. AmpliChip CYP450 contains more than 15,000 diVerent oligonucleotide probes to analyze both the sense and the antisense strands of an amplified target DNA sample [17]. AmpliChip CYP450 provides comprehensive coverage of gene variations, which play a role in the metabolism of approximately 25% of all prescription drugs. The chip has high built‐in sensitivity for analyzing 29 polymorphisms and mutations for the 2D6 gene and two polymorphisms for the 2C19 gene, thereby increasing the probability of more accurately determining the genotype and phenotype. Accurately genotypes over 99% of the world’s population. AmpliChip CYP450 test is intended to be an aid for physicians in individualizing treatment doses for patients on therapeutics metabolized through these genes.

12.3. PROTEIN BIOCHIPS ProteinChip (Vermillion Inc.) has a role in proteomics comparable to that of GeneChip in genomics. It is based on SELDI (surface‐enhanced laser desorption/ionization) process, which has four parts as applied to patient samples: 1. Patient sample of proteins is processed on the ProteinChip Array. 2. Enhance the ‘‘signal‐to‐noise’’ ratio by reducing chemical and biomolecular ‘‘noise’’ (i.e., achieve selective retention of target on the chip by washing away undesired materials). 3. Read one or more of the target protein(s) retained by a rapid, sensitive, laser‐induced process (SELDI) that provides direct information about the target (molecular weight). 4. Process (characterize) the target protein(s) at any one or more locations within the addressable array directly in situ by engaging in one or more on‐ the‐chip binding or modification reactions to characterize protein structure and function. Software produces map of proteins, revealing expression of marker protein with color change in the patient sample as compared to the control sample.

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ProteinChip system was the first complete tool for disease‐focused protein biology. The ProteinChip system uses small arrays or plates with chemically or biologically treated surfaces to interact with proteins. Unknown proteins are aYnity captured on treated surfaces, desorbed and ionized by laser excitation, and detected according to molecular weight. Known proteins are analyzed using on‐chip functional assays. For example, chip surfaces can contain enzymes, receptor proteins, or antibodies, enabling on‐chip protein‐protein interaction studies, ligand binding studies, or immunoassays. With state‐of‐the‐art ion optic and laser optic technologies, the ProteinChip System detects proteins ranging from small peptides of less than 1000 Da up to proteins of 300 kDa or more and calculates the mass based on time‐of‐ flight. The system includes ProteinChip arrays and reagents consumed in the process, the chip reader, software to analyze results, and proprietary database to enable comparison between phenomic and genomic data. New ProteinChip arrays have been packaged into a series of application‐ specific kits to enhance ease‐of‐use for the biologist performing protein analysis. Vermillion’s new ProteinChip Biomarker System enables clinical researchers to rapidly discover, characterize, and validate predictive protein biomarkers and biomarker patterns in their own laboratories. Protein biochip technology has been used for cytokine profiling to predict responsiveness to etanercept in rheumatoid arthritis [18]. This fulfills a need for predicting a clinical response to a tumor necrosis factor‐alpha (TNF‐) blocking agent. Another example of application is the Evidence Cardiac Panel, an automated protein biochip microarray system used for biomarker‐based diagnosis of acute myocardial infarction, which enables the simultaneous determination of creatine kinase MB, myoglobin, glycogen phosphorylase BB, heart‐type fatty acid‐binding protein, carbonic anhydrase III, and cardiac troponin I [19].

13. Role of Nanobiotechnology‐Based Diagnostics in Personalized Medicine Use of nanobiotechnology in molecular diagnostics and can be termed ‘‘nanodiagnostics.’’ Nanotechnology is defined as the creation and utilization of materials, devices, and systems through the control of matter on the nanometer (one billionth of a meter)‐length scale. Various nanotechnologies and their applications in life sciences are described in detail elsewhere under the term ‘‘nanobiotechnology’’ [20]. Numerous nanodevices and nanosystems for sequencing single molecules of DNA are feasible. Because of the small dimension, some of the applications of nanobiotechnology in molecular diagnostics fall under the broad category of biochips/microarrays but

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are more correctly termed nanochips and nanoarrays. Advances in nanodiagnostics will facilitate the development of personalized medicine by:
Improving the sensitivity and extending the present limits of molecular diagnostics.
Discovery of biomarkers by nanodiagnostics will facilitate clinical trials of personalized medicines and monitoring of eVects of therapy.
Integration of diagnosis and therapy, for example, quantum dots (QDs) for localization of cancer plus delivery of therapeutics. 13.1. NANOSCALE SINGLE CELL OR MOLECULE IDENTIFICATION Nanotechnology has facilitated the development of methods for detection of single cell or a few molecules. Some examples are as follows:
Nanolaser scanning confocal spectroscopy, with capability of single cell resolution, can be used to identify a previously unknown property of certain cancer cells that distinguishes them from closely related normal cells [21].
Nanoproteomics: detection of a single molecule of protein.
Nanoarray devices for detection of a single molecule of DNA.
Biobarcode assays [22].
Carbon nanotube transistors for genetic screening.
Nanopore technology [23]. 13.2. NANOPARTICLES FOR MOLECULAR DIAGNOSTICS 13.2.1. Gold Nanoparticles for Diagnostics Small pieces of DNA can be attached to gold particles no larger than 13 nm in diameter. The gold nanoparticles assemble on to a sensor surface only in the presence of a complementary target. If a patterned sensor surface of multiple DNA strands is used, the technique can detect millions of diVerent DNA sequences simultaneously. The current nonoptimized detection limit of this method is 20 femtomolars. Gold nanoparticles are particularly good labels for sensors because a variety of analytical techniques can be used to detect them. 13.2.2. Quantum Dots QDs are inorganic fluorophores that oVer significant advantages over conventionally used fluorescent markers. Advantages are:





Broad excitation spectra Fluorescence is stable Simple excitation with no need for lasers Simple instrumentation

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Red/infrared colors enables whole blood assays
High sensitivity Applications of QDs for molecular diagnostics include the following:








Cancer Genotyping Whole blood assays Multiplexed diagnostics DNA mapping Immunoassays and antibody tagging Detection of pathogenic microorganisms

The most important potential applications of QDs are for cancer diagnosis. Luminescent and stable QD bioconjugates enable visualization of cancer cells in living animals. QDs can be combined with fluorescence microscopy to follow cells at high resolution in living animals. QDs, coated with a polyacrylate cap and covalently linked to antibodies for immunofluorescent labeling of breast cancer marker Her2 Carbohydrate‐encapsulated QDs with detectable luminescent properties are useful for imaging of cancer. Finally, QDs enable integration of diagnostics with therapeutics. Another application of QDs is for viral diagnosis. Rapid and sensitive diagnosis of respiratory syncytial virus (RSV) is important for infection control and eVorts to develop antiviral drugs. Current RSV detection methods are limited by sensitivity and/or time required for detection, which can take 2–6 days. This can delay eVective treatment. Antibody‐conjugated nanoparticles rapidly and sensitively detect RSV and estimate relative levels of surface protein expression [24]. A major development is use of dual‐color QDs or fluorescence energy transfer nanobeads that can be simultaneously excited with a single light source. A QD system can detect the presence of particles of the RSV in a matter of hours. It is also more sensitive, allowing it to detect the virus earlier in the course of an infection [25]. When an RSV virus infects lung cells, it leaves part of its coat containing F and G proteins on the cell’s surface. QDs have been linked to antibodies keyed to structures unique to RSV’s coat. As a result, when QDs come in contact with either viral particles or infected cells they stick to their surface. In addition, colocalization of these viral proteins was shown using confocal microscopy. 13.2.3. Nanobiotechnology for Molecular Imaging in Living Subjects Nanobiotechnology has been used in various biomedical imaging modalities, that is, optical imaging, computed tomography, ultrasound, MRI, single‐photon‐emission computed tomography, and positron emission

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tomography. Targeting ligands, imaging labels, drugs, and therapeutic agents can all be integrated into nanoparticles to enable targeted molecular imaging and molecular therapy [26]. Iron nanoparticles, 15–20 nm in size, having saturation magnetization, have been synthesized, embedded in copolymer beads of styrene and glycidyl methacrylate (GMA), which were coated with poly‐GMA by seed polymerization [27]. The resultant Fe/St‐GMA/GMA beads had diameters of 100– 200 nm. By coating with poly‐GMA, the zeta potential of the beads changed from 93.7 to 54.8 mV, as measured by an electrophoresis method. This facilitates nonspecific protein adsorption suppression, as revealed by gel electrophoresis method, which is a requisite for nanoparticles to be applied to carriers for bioscreening. Nanoparticles are used as labeling molecules for bioscreening. Superparamagnetic nanoparticles are useful for cell tracking cells and for calcium TM sensing. Ferrofluids (Immunicon’s CellTracks Technology) consists of a magnetic core surrounded by a polymeric layer coated with antibodies for capturing cells. A family of calcium indicators for MRI is formed by combining a powerful superparamagnetic iron oxide nanoparticle‐based contrast mechanism with the versatile calcium‐sensing protein calmodulin and its targets [28]. Superparamagnetic nanoparticles measuring 2–3 nm have been used in conjunction with MRI to reveal small and otherwise undetectable lymph‐ node metastases. Ultrasmall superparamagnetic iron oxide enhances MRI for imaging cerebral ischemic lesions. A dextran‐coated iron oxide nanoparticle enhances intracranial tumors by MRI for more than 24 h.

14. Role of Cytogenetics in Personalized Medicine The term ‘‘cytogenetics’’ has been classically used for studies of the cellular aspects of heredity. It has been used mainly to describe the chromosome structure and identify abnormalities related to disease. Besides clinical diagnostics, cytogenetics has been used for basic genomic research as well. Localizing specific gene probes by fluorescent in situ hybridization (FISH) combined with conventional fluorescence microscopy has reached its limit. The term ‘‘cytogenetics’’ should be replaced by ‘‘cytomics,’’ which means that the structural and functional information is obtained by molecular cell phenotype analysis of tissues, organs, and organisms at the single cell level by image or flow cytometry in combination with bioinformatic knowledge extraction concerning nuclei acids, proteins, and metabolites (cellular genomics, proteomics, and metabolomics) as well as cell function parameters like intracellular pH, transmembrane potentials, or ion gradients. Cytogenetics is

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Other technologies

Cytogenetics

Drug discovery and development

Cytoproteomics

Biomarkers

Personalized medicine FIG. 2. Relation of cytogenetics to personalized medicine.

related to other technologies in the same way as genetics with the diVerence that everything is at cell level. Relation of cytogenetics to personalized medicine is shown in Fig. 2. The broader scope of biology at cell level can be covered by terms such as cytogenomics, cytometabolomics, and cytoproteomics. Because of its important role in diagnosing disease at molecular level, cytogenetics is an important part of molecular diagnostics and can be referred to as molecular cytogenetics. Cytogenetics has been refined with use of nanobiotechnology. 14.1. CYTOMICS AS A BASIS FOR PERSONALIZED MEDICINE Cytomics enables diVerential molecular cell phenotyping between diseased and healthy cells. Molecular pathways can be explored in this way including the detection of suitable target molecules, without detailed a priori knowledge of specific disease mechanisms. This is useful during the analysis of complex diseases such as infections, allergies, rheumatoid diseases, diabetes, or malignancies. The top‐down approach reaching from single cell heterogeneity in cell systems and tissues down to the molecular level seems suitable for a human cytome project to systematically explore the molecular biocomplexity of human organisms. The analysis of already existing data from scientific studies or routine diagnostic procedures will be of immediate value in clinical medicine, for example, as personalized therapy by cytomics [29]. At the single‐cell level in conjunction with data‐pattern analysis, high‐ content screening by image analysis or flow cytometry of clinical cell‐ or tissue‐section samples provides diVerential molecular profiles for the

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personalized prediction of therapy‐dependent disease progression in patients. The molecular reverse‐engineering of these molecular profiles, which is the exploration of molecular pathways, backwards, to the origin of the observed molecular diVerentials, by systems biology has the potential to detect new drug targets in knowledge spaces, typically inaccessible to traditional hypotheses [30]. Furthermore, predictive medicine, by cytomics in stratified patient groups, opens a new way not only for personalized medicine but also for the early detection of adverse drug reactions in patients. 14.2. STUDY OF CHROMOSOMES BY ATOMIC FORCE MICROSCOPY Both AFM and scanning near‐field optical microscopy have been used to obtain local information from G‐bands and chromosomal probes. The final resolution allows a more precise localization compared to standard techniques, and the extraction of very small amounts of chromosomal DNA by the scanning probe is possible. This method is also focused on the combination of biochemical and nanomanipulation techniques, which enable both nanodissection and nanoextraction of chromosomal DNA. 14.3. QD FISH The photostability and narrow emission spectra of nonorganic QD fluorophores make them desirable candidates for FISH to study the expression of specific mRNA transcripts. A method for direct QD labeling of modified oligonucleotide probes using streptavidin and biotin interactions increased sensitivity of multiple‐label FISH [31]. This technique also gives excellent histological results for FISH combined with immunohistochemistry. QD’s broad absorption spectra allowed diVerent colored probes specific for distinct subnuclear genetic sequences to be simultaneously excited with a single excitation wavelength and imaged free of chromatic aberrations in a single exposure. A rapid method for the direct multicolor imaging of multiple subnuclear genetic sequences utilizes novel QD‐based FISH. A Texas red dye gamma‐ satellite probe produces fluorescent foci at the periphery of interphase nucleus and labels every centromere in metaphase chromosomes [32].

15. Integration of Molecular Diagnostics and Therapeutics Integration of molecular diagnostics and therapeutics is an important part of personalized medicine. In a combined system for diagnosis and therapeutics, the term diagnosis will broadly include screening for identification of risk

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factors whereas therapeutics would also include monitoring of therapy. A key factor that will drive the integration of diagnostics and therapeutics is the availability of improved and more precise diagnostic methods, which are easy to perform and are not expensive. As discovery of disease genes progresses, the genes may form the link between diagnosis and gene‐based medicines. Although the companion tests for therapeutic products themselves will be technically simple and most likely test for SNP variants, issues surrounding their development, regulatory approval, marketing and reimbursement remain to be established. Therapy based on diagnosis is applicable to early, acute or chronic stages of a disease. The patient may be treated by a medication determined to be safe and eVective on the basis of molecular diagnostics. Not only would the cause of the illness be better defined by the molecular diagnosis, the most eVective specific medication for a disease in a particular patient can be selected. Appropriate diagnostic tests can facilitate the frequent monitoring of the eVects of therapy to verify the success by objective measurements and to detect the failure of therapy as early as possible so that appropriate changes in treatment can be instituted. Molecular diagnostic methods are an important part of monitoring of therapy. Some companion diagnostics for drugs are already in the market.

16. Concluding Remarks and Future Prospects Gene‐based molecular diagnostics is changing the practice of medicine and will continue to do so for the foreseeable future [33]. Pharmacogenomics and pharmacogenetics are providing the basis for the development of molecular diagnostics to improve drug selection, identify optimal dosing, maximize drug eYcacy, or minimize the risk of toxicity. Rapid advances in basic research have identified many opportunities for the development of personalized treatments for individuals and/or subsets of patients defined by genetic and/or genomic tests. However, the integration of these tests into routine clinical practice remains a major multidisciplinary challenge. A rapid, low‐cost access to each person’s genomic information is the key to enabling molecular diagnostics and, ultimately, personalized medicine. Genomics‐based molecular profiling and related technologies may impact on the delivery of healthcare even before genomics‐based drugs enter the market. Identification of genetic factors aVecting the prognosis of disease is likely to be of most clinical relevance. Relationships of known genes, such as BRCA1 and BRCA2, with risk factors will be clarified; permitting evidence based preventive action in people at high genetic risk and better quantification of risk in family members. Greatest progress will be made in understanding the genetic contribution to the intermediate phenotypes linking genes and disease,

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and thus the biology of the disorder, as in atherosclerotic disease. The greatest impact of personalized medicine will be in the treatment of cancer, infections, cardiovascular diseases, and neurological disorders. Molecular diagnosis of cancer including genetic profiling would be widely used for personalized treatment of cancer by the year 2015. Detection of biomarkers would be greatly improved and facilitate development of diagnostics combined with therapeutics. However, further work is needed to fully optimize some diagnostic technologies for clinical laboratory setting as well as for POC applications. The emerging fields of metabonomics (metabolite profiling to identify genotype‐phenotype associations) and phenomics might oVer solutions to anticipating and decreasing risk for adverse drug reactions in each individual patient but tests based on these approaches are not expected to become generally available to the practicing clinician for at least the next 5 years Within the next decade, measurement devices based on nanotechnology, which can make thousands of measurements very rapidly and very inexpensively, will become available. Future trends in diagnostics will continue in miniaturization of biochip technology to nano range. The most common clinical diagnostic application will be blood protein analysis. Blood in systemic circulation reflects the state of health or disease of most organs. Therefore, detection of blood molecular fingerprints, will provide a sensitive assessment of health and disease. REFERENCES [1] K.K. Jain, Personalised medicine, Curr. Opin. Mol. Ther. 4 (2002) 548–558. [2] K.K. Jain, Textbook of Personalized Medicine, Springer, New York, 2009. [3] K.K. Jain, From molecular diagnostics to personalized medicine, Expert Rev. Mol. Diagn. 2 (2002) 89–91. [4] K.K. Jain, Role of pharmacoproteomics in the development of personalized medicine, Pharmacogenomics 5 (2004) 239–242. [5] Y. Dong, H. Zhu, Single‐strand conformational polymorphism analysis: basic principles and routine practice, Methods Mol. Med. 108 (2005) 149–157. [6] M. Kubista, J.M. Andrade, M. Bengtsson, et al., The real‐time polymerase chain reaction, Mol. Aspects Med. 27 (2006) 95–125. [7] G.M. Hogg, J.P. McKenna, G. Ong, Rapid detection of methicillin‐susceptible and methicillin‐resistant Staphylococcus aureus directly from positive BacT/Alert((R)) blood culture bottles using real‐time polymerase chain reaction: evaluation and comparison of 4 DNA extraction methods, Diagn. Microbiol. Infect. Dis. 61 (2008) 446–452. [8] M. Skrzypski, Quantitative reverse transcriptase real‐time polymerase chain reaction (qRT‐ PCR) in translational oncology: lung cancer perspective, Lung Cancer 59 (2008) 147–154. [9] J. Pullat, A. Metspalu, Arrayed primer extension reaction for genotyping on oligonucleotide microarray, Methods Mol. Biol. 444 (2008) 161–167. [10] S. Kim, A. Misra, SNP Genotyping: technologies and biomedical applications, Annu. Rev. Biomed. Eng. 9 (2007) 289–320.

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[11] K.K. Jain, Molecular Diagnostics: Technologies, Markets and Companies, Jain PharmaBiotech Publications, Basel, (2009) pp. 1–910. [12] R.E. Mills, C.T. Luttig, C.E. Larkins, et al., An initial map of insertion and deletion (INDEL) variation in the human genome, Genome Res. 16 (2006) 1182–1190. [13] R. Redon, S. Ishikawa, K.R. Fitch, et al., Global variation in copy number in the human genome, Nature 444 (2006) 444–454. [14] J.O. Korbel, A.E. Urban, J.P. Affourtit, et al., Paired‐end mapping reveals extensive structural variation in the human genome, Science 318 (2007) 420–426. [15] J.M. Kidd, G.M. Cooper, W.F. Donahue, et al., Mapping and sequencing of structural variation from eight human genomes, Nature 453 (2008) 56–64. [16] K.K. Jain, Cancer biomarkers: current issues and future directions, Curr. Opin. Mol. Ther. 9 (2007) 563–571. [17] K.K. Jain, Applications of AmpliChip CYP450, Mol. Diagn. 9 (2005) 119–127. [18] S. Fabre, A.M. Dupuy, N. Dossat, et al., Protein biochip array technology for cytokine profiling predicts etanercept responsiveness in rheumatoid arthritis, Clin. Exp. Immunol. 153 (2008) 188–195. [19] M.M. Mion, E. Novello, S. Altinier, et al., Analytical and clinical performance of a fully automated cardiac multi‐markers strategy based on protein biochip microarray technology, Clin. Biochem. 40 (2007) 1245–1251. [20] K.K. Jain, Applications of nanobiotechnology in clinical diagnostics, Clin. Chem. 53 (2007) 2002–2009. [21] P.L. Gourlay, J.K. Hendricks, A.E. McDonald, et al., Mitochondrial correlation microscopy and nanolaser spectroscopy—new tools for biphotonic detection of cancer in single cells, TCRT 4 (2005) 585–592. [22] Y.P. Bao, T.F. Wei, P.A. Lefebvre, et al., Detection of protein analytes via nanoparticle‐ based bio bar code technology, Anal. Chem. 78 (2006) 2055–2059. [23] M. Rhee, M.A. Burns, Nanopore sequencing technology: research trends and applications, Trends Biotechnol. 24 (2006) 580–586. [24] A. Agrawal, R.A. Tripp, L.J. Anderson, S. Nie, Real‐time detection of virus particles and viral protein expression with two‐color nanoparticle probes, J. Virol. 79 (2005) 8625–8628. [25] E.L. Bentzen, F. House, T.J. Utley, et al., Progression of respiratory syncytial virus infection monitored by fluorescent quantum dot probes, Nano Lett. 5 (2005) 591–595. [26] W. Cai, X. Chen, Nanoplatforms for targeted molecular imaging in living subjects, Small 3 (2007) 1840–1854. [27] M. Maeda, C.S. Kuroda, T. Shimura, et al., Magnetic carriers of iron nanoparticles coated with a functional polymer for high throughput bioscreening, J Appl. Phys. 99 (2006) 08H103. [28] T. Atanasijevic, M. Shusteff, P. Fam, A. Jasanoff, Calcium‐sensitive MRI contrast agents based on superparamagnetic iron oxide nanoparticles and calmodulin, PNAS 103 (2006) 14707–14712. [29] G. Valet, Cytomics, the human cytome project and systems biology: top‐down resolution of the molecular biocomplexity of organisms by single cell analysis, Cell Prolif. 38 (2005) 171–174. [30] G. Valet, Cytomics as a new potential for drug discovery, Drug Discov. Today 11 (2006) 785–791. [31] P. Chan, T. Yuen, F. Ruf, et al., Method for multiplex cellular detection of mRNAs using quantum dot fluorescent in situ hybridization, Nucleic Acids Res. 33 (2005) e161. [32] L.A. Bentolila, S. Weiss, Single‐step multicolor fluorescence in situ hybridization using semiconductor quantum dot‐DNA conjugates, Cell Biochem. Biophys. 45 (2006) 59–70. [33] J.E. Finan, R.Y. Zhao, From molecular diagnostics to personalized testing, Pharmacogenomics 8 (2007) 85–99.

ADVANCES IN CLINICAL CHEMISTRY, VOL. 47

VERIFICATION OF METHOD PERFORMANCE FOR CLINICAL LABORATORIES James H. Nichols1 Professor of Pathology, Tufts University School of Medicine and Medical Director, Clinical Chemistry, Baystate Health, Springfield, Massachusetts 01199, USA

1. 2. 3. 4. 5. 6.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ISO Quality Management System: The Fundamentals of Quality . . . . . . . . . . . . . . . . Laboratory Quality Standards in Regulations and Accreditation Guidelines . . . . . Comparison of Quality Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Performing Method Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Accuracy or Trueness. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Analytical Measurement Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Reference Interval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. Other Performance Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Abstract Method verification, a one‐time process to determine performance characteristics before a test system is utilized for patient testing, is often confused with method validation, establishing the performance of a new diagnostic tool such as an internally developed or modified method. A number of international quality standards (International Organization for Standardization (ISO) and Clinical Laboratory Standards Institute (CLSI)), accreditation agency guidelines (College of American Pathologists (CAP), Joint Commission, U.K. Clinical Pathology Accreditation (CPA)), and regional 1

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laws (Clinical Laboratory Improvement Amendments of 1988 (CLIA’88)) exist describing the requirements for method verification and validation. Consumers of marketed test kits should verify method accuracy, precision, analytic measurement range, and the appropriateness of reference intervals to the institution’s patient population. More extensive validation may be required for new methods and those manufacturer methods that have been modified by the laboratory, including analytic sensitivity and specificity. This manuscript compares the various recommendations for method verification and discusses the CLSI evaluation protocols (EP) that are available to guide laboratories in performing method verification experiments.

2. Introduction Method verification, a one‐time process to determine performance characteristics before a test system is utilized for patient testing, is confused with method validation, establishing the performance of a new diagnostic tool such as an internally developed or modified method. A number of international quality standards (ISO and CLSI), accreditation agency guidelines (CAP, Joint Commission, U.K. CPA), and regional laws (CLIA’88) exist describing the requirements for method verification and validation. Consumers of marketed test kits should verify method accuracy, precision, analytic measurement range, and the appropriateness of reference intervals to the institution’s patient population. More extensive validation may be required for new methods and those manufacturer methods that have been modified by the laboratory, including analytic sensitivity and specificity. This manuscript compares the various recommendations for method verification and discusses the CLSI EP that are available to guide laboratories in performing method verification experiments. Verification is defined as a ‘‘one‐time process completed to determine or confirm test performance characteristics before the test system is used for patient testing’’ [1]. Validation, on the other hand, is establishing the ‘‘performance of a new diagnostic tool such as an internally developed, analyte‐specific method or reagents or a laboratory developed information system’’ [1]. Manufacturers are required to validate instruments and methods prior to market release, for United States (U.S.) Food and Drug Administration (FDA) approval or European CE mark. Clinical laboratories on purchase of an instrument or new method verify that they can obtain instrument performance within manufacturer’s specifications. Verification is, therefore, the process of characterizing a method’s performance prior to use in patient testing. Good laboratory practice dictates that laboratories verify the performance of a test with the particular instrumentation, staV, and specimens typical of

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that hospital’s patient population. A specific laboratory may diVer from the conditions when the manufacturer validated the method. Variation in operator technique, temperature, humidity, and patient population (including drug, metabolic, and other potential interferences) can aVect assay performance. Verification establishes initial performance expectations for a method prior to analyzing patient specimens. Verification information is useful when interpreting results (i.e., accuracy and precision of the test) and for troubleshooting problems. A number of international standards and guidelines support the requirement for method verification. The ISO recommends performance verification of laboratory equipment as part of a quality management system for clinical laboratories. The CLSI has further organized its standards around the quality systems essentials, and CLSI has a number of guidelines related to both method validation and method verification. These international quality guidelines complement regional quality regulations and accreditation standards throughout the world. Two examples include the U.S. CLIA’88 and U.K. CPA. In the U.S., CLIA’88 [2] is a federal law governing clinical laboratory quality, and sections of the CLIA regulations mandate initial performance verification. The U.K. has adopted the CPA: Standards for the Medical Laboratory [3] that defines the ISO quality management systems for the medical laboratory. The CAP and Joint Commission, organizations providing accreditation of healthcare institutions in the U.S. and internationally, incorporate initial method verification as a part of their accreditation checklists. While a number of international guidelines and accreditation checklists discuss method verification, this review will focus on the general quality recommendations from these organizations; what needs to be verified, and when and how to perform method verification. Readers should refer to local regulations and accreditation guidelines as applicable to their specific laboratory for further information.

3. ISO Quality Management System: The Fundamentals of Quality Quality recommendations for the clinical laboratory trace back to the ISO standards, in particular ISO 15189 Medical Laboratories: Particular Requirements for Quality and Competence [4] (Table 1). ISO has published a set of standards (ISO 9000 [5], ISO 9001 [6], and ISO 9004 [7]) that establish the principles of a quality management system. ISO 9000 provides the definitions and ISO 9001 lists the requirements of quality management systems, while ISO 9004 oVers ideas and suggestions for continual quality

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JAMES H. NICHOLS TABLE 1 COMPARISON OF METHOD VERIFICATION GUIDELINES AND REGULATIONS

Organization ISO

Guideline or standard ISO 9001:2000 Quality Management Systems: Requirements

ISO 15189 Medical Laboratories: Particular Requirements for Quality and Competence CLSI

HS1‐A2 A Quality Management System Model for Health Care

U.S. CLIA

Clinical Laboratory Improvement Amendments of 1988

U.K. CPA

Standards for the Medical Laboratory

CAP

Laboratory General Checklist

Joint Commission

Comprehensive Accreditation Manual for Laboratory and Point‐of‐Care Testing

Relevant sections 7.3.5 Design and Development Verification 7.5.2 Validation of processes for production and service provision 7.6 Control of monitoring and measuring devices 5.3 Laboratory Equipment 5.5 Examination procedures 5.6 Assuring the quality of examination procedures 5.4.2 Equipment Validation 5.6.2 Process Validation or Verification } 493.1253 Establishment and verification of method performance specifications } 493.1255 Calibration and calibration verification procedures D1 Procurement and management of Equipment D3 Management of materials F1 Selection and validation of examination procedures F3 Assuring the quality of examinations GEN.30000‐GEN.30070 Quality management GEN.42020‐GEN.42163 Test method validation EC.6.20 Management of laboratory environment QC.1.70‐QC.1.180 Quality monitoring and control systems

improvement. The ISO 9000 series presents a general approach to quality that is applicable to any industry and any product. ISO 15189 takes the ISO 9001 requirements and defines them for the medical laboratory. So, understanding laboratory method verification requires a familiarity with the fundamentals of a quality management system as described in the ISO 9000 and ISO 9001 standards.

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The ISO 9000 set of standards is based on eight quality management principles as identified by top management that can lead an organization towards improved performance [5]:  Customer focus: Businesses must understand current and future customer needs and strive to meet and exceed customer expectations.  Leadership: Leadership should establish organization focus, direction, and promote an environment for employees to achieve goals.  Involvement of people: All staV should understand their roles, responsibilities, and how they fit into filling the customer needs.  Process approach: Understand all organizational activities and how they relate in a process to achieving the end product.  System approach to management: Managing processes together as a system reduces redundancy and brings organizational focus.  Continual improvement: A goal should be continual improvement of the organization’s overall performance.  Factual approach to decision making: EVective decisions are based on the analysis of data and information.  Mutually beneficial supplier relationships: An organization and its suppliers are interdependent and a mutually beneficial relationship enhances the value of both organizations. These eight quality management principles are interpreted as minimum requirements within the ISO 9001 standard. Section 7.3.5 Design and Development Verification is relevant to method verification and includes the need to verify that the planned development stages of the product meet initial requirements. The design of a product occurs through stages, each with planned requirements that define staV roles and responsibilities in product development. Output of each stage should be verified to ensure that it meets expected and planned requirements and records of the verification results should be maintained. The ISO 9001 Design and Development Verification requirement is applied to clinical laboratories in ISO 15189 as Laboratory Equipment Standards (Section 5.3.2). Equipment shall be shown (upon initial installation and in routine use) to be capable of achieving the performance required [4]. This particular requirement for method verification can thus be traced back through ISO 9001 to the ‘‘process approach’’ and ‘‘value of documentation’’ sections of ISO 9000. General quality principles applicable to any industry (understanding processes and the need for documentation) can thus be applied to a laboratory setting. ISO 15189 emphasizes initial performance verification and the need to establish a program for monitoring and demonstrating proper calibration and function of instruments, reagents, and analytical systems (Table 2). The laboratory should have a documented maintenance and calibration program

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JAMES H. NICHOLS TABLE 2 METHOD VERIFICATION RECOMMENDATIONS

Organization ISO

Guideline or standard ISO 9001:2000 Quality Management Systems: Requirements

ISO 15189 Medical Laboratories: Particular Requirements for Quality and Competence

CLSI

HS1‐A2 A Quality Management System Model for Health Care [20]

U.S. CLIA

Clinical Laboratory Improvement Amendments of 1988

U.K. CPA

Standards for the Medical Laboratory

Verification recommendation summary Product meets functional, performance, and applicable statutory and regulatory requirements Calibrate and verify equipment at specified intervals Equipment can achieve required performance for intended use Calibration, verification, maintenance program Reagent procurement and verification before use Use only validated procedures for analysis Document performance specifications before use to include: linearity, precision, accuracy (uncertainty of measurement), detection limit, measuring interval, trueness, analytical sensitivity, analytical specificity Equipment can achieve required performance for intended use Calibration and calibration verification Waived—follow manufacturer’s instructions Moderate—verify accuracy, precision, reportable range, and reference interval High complexity—verify accuracy, precision, analytical sensitivity and specificity, reportable range, reference interval Calibration verified every 6 months or as needed Equipment appropriate to provide needed service Calibration, verification, maintenance program to manufacturer specifications Internal quality control prior to use, calibration and verification or trueness, assay uncertainty, comparability of results (continues)

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Organization

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(Continued)

Guideline or standard

CAP

Laboratory General Checklist

Joint Commission

Comprehensive Accreditation Manual for Laboratory and Point‐of‐Care Testing

Verification recommendation summary Labs subject to CLIA’88 (prior to patient testing): FDA approved tests: accuracy, precision, reportable range Modified and laboratory developed tests: accuracy, precision, analytic sensitivity, interferences, and reportable range. Labs not subject to CLIA’88 (prior to patient testing): analytic accuracy, precision, analytic sensitivity, analytic specificity (interferences), and reportable range Calibration verified every 6 months or as needed FDA approved tests (prior to patient testing): accuracy, precision, reportable range Modified or laboratory developed tests (prior to patient testing): accuracy, precision, analytical sensitivity, analytical specificity, reportable range, and reference ranges Calibration verification every 6 months or as needed

that meets manufacturer recommendations. Records should be maintained of equipment, sources of reagents and calibrators, equipment performance, maintenance, calibrations, and repairs. Performance records should include dates, times, and results of any calibrations, adjustments, the acceptance criteria, and the frequency of any checks required between maintenance and calibrations. Manufacturer’s recommendations can be utilized to establish acceptance criteria, procedures, and frequency of maintenance or calibration, so the laboratory can utilize package insert recommendations for their minimum acceptance criteria for analytical performance of any particular device. The records of equipment performance should be maintained at

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a minimum for the life of the equipment and longer as required by local regulations [4]. ISO 15189 further recommends that the laboratory should only use validated procedures for tests and if laboratory‐developed procedures are used then they must be validated for their intended use and documented. The extent of procedure validation should be as extensive as necessary to meet clinical needs and test application and the validation should be documented. The method and procedures should be evaluated to give satisfactory results prior to clinical use and each new version of test kits with major changes in reagent or procedure must be checked for performance and suitability for intended use. Procedures must be available at the workstation for staV to use, and any deviation from procedure should be documented. Procedures should include performance specifications for linearity, precision, accuracy expressed as uncertainty of measurement, detection limit, measuring interval (reportable range), trueness of the measurement, and analytical sensitivity and specificity. These performance specifications should relate to the intended clinical use of the test result. The biological reference interval must be established and reviewed periodically, especially with laboratory or reagent changes [4]. ISO 15189 also requires development of a control system to verify the intended quality of results. Focus of this program should be reduction of errors from mistakes in handling, orders and reporting as well as reducing sources of measurement uncertainty like calibration, environmental conditions, and changes in operators and equipment. The laboratory must establish a program for calibration and verification of trueness through periodic analysis of reference materials, method comparison, or participation in external quality control programs (proficiency testing). For tests using multiple instrument, methodologies, or analysis at diVerent sites, the laboratory must periodically verify comparability of results throughout the clinically relevant interval [4]. While the ISO 15189 standard addresses quality management systems in the clinical laboratory, ISO 22870 Point‐of‐Care Testing: Requirements for Quality and Competence [8] similarly interprets quality management systems and method verification for point‐of‐care testing (POCT). A Medical Advisory Committee should define the scope of POCT in an organization and a multidisciplinary POCT management group (the POCT Committee) should be appointed to ensure responsibilities are defined in an organization. This POCT Committee should assist in selecting and evaluating POCT devices in order to define performance criteria for trueness, precision, detection limits, use limits, and interferences. Procedures should monitor ongoing performance and ensure removal of devices from service when performance criteria are not met or safety is a concern [8].

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4. Laboratory Quality Standards in Regulations and Accreditation Guidelines There is significant overlap between the method verification recommendations of the ISO quality management systems and federal CLIA’88 quality laws for the laboratory (Table 2). In the U.S., CLIA’88 has divided laboratory testing into simple (‘‘CLIA’88 waived’’) tests and moderate/high complexity testing categories. Waived tests are approved by the U.S. FDA for home patient self‐testing or are simple tests that do not require sample processing, pipetting, or complicated multistep procedures. Many POCT devices are waived for easier access by clinicians in physician’s oYce laboratories in the U.S. At the other end of CLIA’88 complexities, the high complexity category of tests include those tests that have not been approved for marketing by the U.S. FDA, in‐house or laboratory‐developed tests (sometimes referred to as ‘‘home‐brew’’ tests), or U.S. FDA approved methods that have been modified by a laboratory including use of alternative specimen matrix (e.g., urine rather than serum), antibodies, reagents, timing, or other steps that significantly alter the approved procedure. Other U.S. FDA approved methods that do not fit these categories fall into the moderate complexity category and are test methods utilized without modification by the laboratory. CLIA’88 has diVerent requirements for method verification depending on the complexity category of the test. Waived methods only have to follow manufacturer instructions for use and do not require any specific method verification prior to reporting patient results. However, for moderate complexity methods, the laboratory must verify or establish, for each method, the performance characteristics for accuracy, precision, reportable range, and verify the applicability of the manufacturer’s reference interval to the laboratory’s patient population. For in‐house developed, laboratory‐modified methods, and other high complexity tests, the laboratory must verify or establish performance specifications for reportable range of patient test results, reference interval and any other performance characteristic required in addition to the moderate complexity verification requirements of accuracy, precision, reportable range, and reference interval. Based on the performance specifications, the laboratory must establish calibration and control procedures for patient testing that comply at a minimum with manufacturer’s recommendations. Calibration must be verified at least every 6 months or following a complete change of reagents, after major maintenance, when the controls reflect an unusual trend, or when there is a question regarding instrument performance [2]. The CLIA’88 law sets minimum quality standards for other laboratory inspection and accreditation organizations in the U.S. The CAP [9, 10] and

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Joint Commission [11] both provide laboratory accreditation in the U.S. and internationally. International accreditation uses slightly diVerent guidelines and inspection checklists since CLIA’88 is not applicable outside of the U.S. The basic requirements for quality, however, are similar between the domestic and international CAP and Joint Commission inspection checklists. CAP requires full characterization of a method before use in patient testing. The laboratory must have verification data on every test’s accuracy, precision, analytical sensitivity, interferences, and reportable range (the analytical reportable range and the clinically reportable range as applicable). The analytical reportable range is the limits that the instrument can report a clinically reliable result, while the clinically reportable range is the entire interval where results can be reported for a patient using sample dilution. Unlike ISO 22870, CAP guidelines follow CLIA’88 and do not require performance verification of ‘‘waived’’ testing methods. For unmodified moderate complexity methods, CAP requires verification of accuracy, precision, and reportable range. For high complexity and laboratory‐developed or ‐modified methods, the laboratory must further establish analytical sensitivity and interferences, as applicable [9, 10]. CAP additionally requires calibration verification and result correlation between analyzers or sites at least every 6 months. All tests, including POCT devices must subscribe to an external control or proficiency testing program. Records of method verification and calibration verification must be maintained for at least 2 years after discontinuing the use of an instrument or method. The Joint Commission has similar requirements to CAP for method verification. The laboratory must have an instrument acquisition process that includes initially evaluating the condition and function of the equipment when received and evaluates the training of operators before use of the instrumentation on patients [11]. Test methods must be verified to produce accurate results consistently. The laboratory minimally must verify accuracy, precision, and reportable range for FDA approved tests where the manufacturer has established the performance specifications, and the laboratory must verify the complete range of performance specifications for modified tests including precision, analytical sensitivity, analytical specificity, the reportable range, and the reference interval [11]. The Joint Commission also requires external control or proficiency testing and development of an internal quality control program and criteria that maintains test result performance within required specifications. When diVerent methods produce the same test result, the Joint Commission requires correlation between the methods to ensure applicability of reference ranges at least every 6 months. Calibration and calibration verification is performed at least every 6 months or when there is a complete change of reagents, major maintenance, control results indicate there may be a problem, environmental change, or instrument replacement.

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To contrast the U.S. laboratory regulations, the CPA (U.K.) provides Standards for the Medical Laboratory that defines minimal quality for laboratories in Britain. The CPA standards are derived from the ISO 9000 series and ISO 15189 standards. Specifically, the CPA requires proper procurement and management of equipment to ensure that the laboratory can fulfill the needs of clinicians. The laboratory must ensure that the equipment is appropriate to provide the required service, and that procedures are available for proper operation, maintenance, and calibration. This applies to POCT devices as well as laboratory instrumentation. Each method must be validated for their intended use prior to introduction. The laboratory needs to manage receipt and verification of reagents, controls, and calibrators. CPA requires that the laboratory ensure the quality of results by controlling preanalytical processes, staV training, laboratory environment, use of internal quality control, determination of assay uncertainty, calibration and verification of trueness, comparability of results, and participation in external quality control (proficiency testing). Records of method verification, operation, maintenance, and monitoring of equipment function need to be maintained to document compliance with manufacturer recommendations. The CPA accreditation standards are thus very similar to other quality standards for laboratory testing.

5. Comparison of Quality Requirements A common theme for method verification can be drawn through the laboratory quality documents (Table 2). All methods must be verified for technical performance prior to use in patient testing. For methods that are produced and marketed by a diagnostic manufacturer, the method verification confirms that the laboratory can obtain performance within manufacturer defined specifications. What is required for initial method verification varies in terminology between the documents, but includes accuracy (trueness/result uncertainty), precision, and reportable range at the minimum. Many standards further stress verifying the appropriateness of the manufacturer suggested reference range for the laboratory’s patient population. For laboratory developed methods or modified manufacturer methods, a more extensive method validation is required where the laboratory establishes both the initial performance specifications and also verifies that the method can meet those specifications. These may include analytical sensitivity (detection limit), analytical specificity (interferences), and other performance characteristics as applicable to the assay. The laws and laboratory quality requirements of diVerent countries and accreditation organizations vary. Some countries have not yet adopted specific laws or standards governing laboratory quality.

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The reader is encouraged to refer to local regulations, where applicable, regarding specific requirements for method verification in their region and as required by their laboratory accreditation organizations. Quality standards further stress that laboratories should establish an ongoing means of monitoring assay performance to ensure that the method meets required performance over time. Development of reagent, control and calibrator procurement, and verification procedures ensure traceability of shipments and continuity of results between shipments and lot numbers. Internal control systems, preventive maintenance, and calibration/calibration verification ensures assay performance and result consistency on a periodic or semiannual basis. Subscription to an external control or proficiency testing program correlates the laboratory results with other laboratories performing the same test to verify result comparability. These quality principles are good laboratory practice that applies to laboratory testing whether performed in a core laboratory or point of care.

6. Performing Method Verification While the ISO standards, CLIA’88 regulations, and CAP, CPA, and Joint Commission accreditation guidelines recommend what needs to be verified (e.g., accuracy, precision, etc.) and dictate the frequency of verification (calibration verification every 6 months), these documents do not mandate how to perform method verification. The CLSI guidelines can provide guidance on appropriate protocols for performing method validation experiments. CLSI has published a comparable document to the ISO 9000 series titled, ‘‘Quality Management System Model for Health Care (HS1‐A2).’’ This document, like the ISO 9000 documents, describes quality system essentials for general quality applicable to health care organizations. CLSI recommends that all equipment, instruments, computer systems, and changes need to be validated and verified to ensure that they are capable of achieving the required performance before actual use in patient care. Examples of ways to validate healthcare processes include comparison with another method (old procedure vs. new procedure) or diagnostic utility/clinical relevance. CLSI has a series of EP that can guide laboratories in establishing method performance (Table 3). These EP are applicable to manufacturers, laboratories developing their own methods, and laboratories purchasing test methods without modification. Some of the EP describe validation of performance for newly developed methods, while other EP are intended to verify test performance meets manufacturer specifications. Both types of protocols can be used to meet laboratory quality regulations for method

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TABLE 3 CLINICAL AND LABORATORY STANDARDS DOCUMENTS FOR VERIFYING METHOD PERFORMANCE Document

Performance criteria

EP5‐A2 Evaluation of Precision Performance of Quantitative Measurement Methods EP6‐A Evaluation of the Linearity of Quantitative Measurement Procedures EP7‐A2 Interference Testing in Clinical Chemistry EP9‐A2 Method Comparison and Bias Estimation Using Patient Samples EP15‐A2 User Verification of Performance for Precision and Trueness EP17‐A Protocols for Determination of Limits of Detection and Limits of Quantitation EP21‐A Estimation of Total Analytical Error for Clinical Laboratory Methods C28‐A2 How to Define and Determine Reference Intervals in the Clinical Laboratory

Precision Linearity (analytic measurement range) Interference (analytic specificity) Method comparison (accuracy/ trueness) Precision Trueness (accuracy) Limit of quantitation (analytic sensitivity) Precision Reference intervals

verification, but the manufacturer protocols are larger in size and more involved. 6.1. PRECISION Precision is a measure of assay variability. Precision is defined as ‘‘the closeness of agreement between independent test results from the same sample’’ [1]. A control or stabilized sample that mimics patient specimens is analyzed over several days to estimate within‐run and total imprecision, generally expressed as the coeYcient of variation (the standard deviation divided by the mean). CLSI EP15: User Verification of Performance for Precision and Trueness [12] oVers experiments for determining if a laboratory’s precision meets manufacturer specifications. The laboratory selects a control or stabilized patient sample at a minimum of two diVerent concentrations. These samples are run in triplicate, once a day for 5 days. EP15 presents calculations to estimate total repeatability (precision) and within‐run precision. If the laboratory’s performance is less than or equal to manufacturer claims, then the laboratory has demonstrated performance within manufacturer specifications. If manufacturer specifications or performance claims are not available for an assay, or if the test is newly developed or modified from a marketed method, then other CLSI documents, EP5 and EP9, may be more applicable to defining performance specifications. EP5: Evaluation of Precision Performance of Quantitative Measurement Methods [13] recommends analyzing controls at

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various concentrations in duplicate, twice a day for 20 days. EP9: Method Comparison and Bias Estimation Using Patient Samples [14] estimates method precision from duplicate analysis of a minimum of 40 patient specimens analyzed by the test method and by a reference or comparative method. 6.2. ACCURACY OR TRUENESS Accuracy is the ‘‘closeness in agreement between a test result and the accepted reference method’’ [1]. Selection of an appropriate reference can determine a method’s trueness if the comparative method is an accepted reference method for the given analyte. Glucose methods, for instance, could compare against a laboratory hexokinase or glucose oxidase method, but both of those methods have their own inherent biases. The better choice would be comparison directly to mass spectrometry in a deproteinated specimen, the consensus reference method for international glucose standardization. Unfortunately, most hospitals do not have this method readily available and it can be an expensive analysis for comparing 40 or more specimens. So, laboratories tend to utilize what is available in their laboratory for comparing new test methods. This may be an existing analyzer or method that is being replaced by the new test. CLSI EP15: User Verification of Performance for Precision and Trueness [12] recommends analyzing 20 patient specimens once by the test and comparative method. EP15 gives suggestions for determining whether the laboratory’s performance meets manufacturer performance specifications. For tests without performance criteria, EP9: Method Comparison and Bias Estimation Using Patient Samples [14] can be utilized to establish performance. EP9 recommends analyzing a minimum of 40 patient specimens in duplicate by both test and comparative method. Both EP15 and EP9 documents provide suggestions for plotting the method comparisons and calculating bias statistics. CLSI EP21: Estimation of Total Analytical Error for Clinical Laboratory Methods [15] recommends comparing 120 patient specimens around each of the clinically important decision levels. 6.3. ANALYTICAL MEASUREMENT RANGE The reportable range or measurement interval is the ‘‘interval or range of values over which the acceptability criteria for the method have been met’’ [1]. This is the range of values whose performance characteristics have been verified by a laboratory and can be reported for patient care. CLSI EP6: Evaluation of the Linearity of Quantitative Measurement Procedures; A Statistical Approach [16] provides a means of estimating assay linearity or ‘‘the ability of the assay to provide results that are directly proportional to

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the concentration of analyte in the sample’’ [1]. EP6 recommends analyzing a set of five matrix appropriate samples (to mimic patient specimens) with proportional concentrations, such that the high concentration sample is mixed 1:3, 1:1, and 3:1 with the low concentration to create the set of samples with linear concentration relationship. The concentrations are selected to span the clinically relevant range of values, and each concentration is measured in duplicate. EP6 provides suggestions for spiking samples to obtain suYciently high concentration samples and for plotting and statistical reduction of the experimental data.

6.4. REFERENCE INTERVAL A reference interval is the ‘‘range of test values expected for a given population of individuals’’ [1]. CLSI C28: How to Define and Determine Reference Intervals in the Clinical Laboratory [17] describes both the transference of reference intervals from another method (through method correlation) or the development of a reference interval for new tests or modified methods. An abbreviated protocol can be done using 20 individuals when verifying the appropriate transfer of reference intervals from one method to another. However, developing new reference intervals requires a minimum of 120 individuals. For analytes like creatinine or calcium that may vary by both age and sex, this requires finding a minimum of 120 individuals of each sex categorized by the appropriate age groupings. The key to verifying reference intervals is understanding all of the preanalytic variables that can aVect the test and selecting an appropriate group of individuals that is deemed healthy. Finding age and sex matched, healthy individuals is challenging, given the frequency of over the counter medications, diet and other potential variables that can confound the analysis.

6.5. OTHER PERFORMANCE CRITERIA The need to verify other performance criteria will depend on the accreditation and regional laws governing laboratory quality. EP7: Interference Testing in Clinical Chemistry [18] provides a means of determining assay specificity or the level of assay interference from drugs, lipemia, icterus, or hemolysis or other potential interferents in the sample. Initially, a specimen is spiked with a high concentration of the interfering compound. If no diVerence is noted between the spiked and unspiked sample, then no interference is noted up to the concentration level tested. If a diVerence is noted, then EP7 describes how to create a set of samples with increasing concentration of interferent to determine the level at which clinically significant interference occurs.

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CLSI EP17: Protocols for Determination of Limits of Detection and Limits of Quantitation [19] provides a protocol to determine the limit of detection, the smallest amount that a method can reliably detect as present, and limit of quantitation, the smallest amount the method can reliably measure quantitatively. Some laboratories suggest repeat analysis of a blank sample and estimating the limit of detection as two or three times the standard deviation of the blank and the limit of quantitation as 10 times the standard deviation of the blank. However, EP17 recommends use of both a blank and low concentration samples to not only pinpoint the limit of detection, but also characterize low range performance. EP17 recommends 60 measurements of both a blank and low concentration samples to establish the assay limits, but 20 measurements can suYce when verifying performance to manufacturer specifications. 7. Summary Method verification is the process of characterizing analytic performance prior to patient testing. International standards, laws, and accreditation agencies governing laboratory quality all mandate that laboratories verify that test performance can meet manufacturer’s specifications prior to patient testing. Although the specific performance criteria vary by standard, most require verifying precision, accuracy, reportable range, and the reference interval for the intended patient population. Other aspects of performance, such as analytic sensitivity and analytic specificity may need to be verified if the laboratory developed the method or modified an existing test. None of the quality standards, laws, or accreditation agencies dictates how to verify performance. CLSI has published a number of documents that provide specific protocols and recommendations for both verifying that a laboratory method meets manufacturer specifications or establishing performance criteria for a new or modified method. Laboratory staV involved in installing new equipment, assay development, or other tasks that require characterizing test performance should be familiar with the international quality standards and why method verification is required as well as the CLSI documents recommending how to perform method verification. REFERENCES [1] Clinical and Laboratory Standards Institute, Harmonized terminology database. <www. clsi.org>, (accessed on July 2008). [2] Health and Human Services, Health Care Financing Administration Public Health Service. 42 CFR Part 405, et al., Clinical laboratory improvement amendments of 1988; Final rule. Federal Register, 57 (40) (1992) 7001–7243. Recent revisions available at <www.cms.hhs. gov/CLIA>, (accessed on July 2008).

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[3] Clinical Pathology Accreditation (UK) Ltd, Standards for the Medical Laboratory, CPA, Sheffield, UK, 2007, 57 <www.cpa‐uk.co.uk/>, (accessed on July 2008). [4] International Organization for Standardization, ISO 15189:2007 Medical Laboratories: Particular Requirements for Quality and Competence, ISO, Geneva, Switzerland, 2007, 40. [5] International Organization for Standardization, ISO 9000:2005 Quality Management Systems: Fundamentals and Vocabulary, ISO, Geneva, Switzerland, 2005, 30. [6] International Organization for Standardization, ISO 9001:2000 Quality Management Systems: Requirements, ISO, Geneva, Switzerland, 2000, 23. [7] International Organization for Standardization, ISO 9004:2000 Quality Management Systems: Guidelines for Performance Improvement, ISO, Geneva, Switzerland, 2000, 56. [8] International Organization for Standardization, ISO 22870:2006 Point‐of‐Care Testing: Requirements for Quality and Competence, ISO, Geneva, Switzerland, 2006, 11. [9] Commission on Laboratory Accreditation, Laboratory General Checklist, College of American Pathologists, Northfield, IL, 2007, 130. [10] Commission on Laboratory Accreditation, Chemistry and Toxicology Checklist, College of American Pathologists, Northfield, IL, 2007, 129. [11] Joint Commission, 2009 Comprehensive Accreditation Manual for Laboratories and Point‐ of‐Care Testing, Joint Commission, Oakbrook Terrace, IL, 2008, 440. [12] CLSI, EP15‐A2 User Verification of Performance for Precision and Trueness, Clinical and Laboratory Standards Institute, Wayne, PA, 2005, 49. [13] CLSI, EP5‐A2 Evaluation of Precision Performance of Quantitative Measurement Methods, Clinical and Laboratory Standards Institute, Wayne, PA, 2004, 39. [14] CLSI, EP9‐A2 Method Comparison and Bias Estimation Using Patient Samples, Clinical and Laboratory Standards Institute, Wayne, PA, 2002, 56. [15] CLSI, EP21‐A Estimation of Total Analytical Error for Clinical Laboratory Methods, Clinical and Laboratory Standards Institute, Wayne, PA, 2003, 39. [16] CLSI, EP6‐A Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach, Clinical and Laboratory Standards Institute, Wayne, PA, 2003, 47. [17] CLSI, C28‐A2 How to Define and Determine Reference Intervals in the Clinical Laboratory, Clinical and Laboratory Standards Institute, Wayne, PA, 2000, 38. [18] CLSI, EP7‐A2 Interference Testing in Clinical Chemistry, Clinical and Laboratory Standards Institute, Wayne, PA, 2005, 107. [19] CLSI, EP17‐A Protocols for Determination of Limits of Detection and Limits of Quantitation, Clinical and Laboratory Standards Institute, Wayne, PA, 2004, 39. [20] CLSI, HS1‐A2 A Quality Management System Model for Health Care, Clinical and Laboratory Standards Institute, Wayne, PA, 2004, 79.

ADVANCES IN CLINICAL CHEMISTRY, VOL. 47

INTERPRETING THE PROTEOME AND PEPTIDOME IN TRANSPLANTATION Tara K. Sigdel, R. Bryan Klassen, and Minnie M. Sarwal1 Department of Pediatrics—Nephrology, Stanford University Medical School, Stanford University, Stanford, California 94305, USA

1. Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Protein and Peptide Biomarkers Discovery for Organ Transplantation . . . . . 2.2. Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Peptidomics, a New Approach to Biomarker Discovery . . . . . . . . . . . . . . . . . . . . 2.4. Quantifying Proteomes and Peptidomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Sample Processing for Proteomics and Peptidomics . . . . . . . . . . . . . . . . . . . . . . . . 3. Application of Proteomics and Peptidomics in Transplantation. . . . . . . . . . . . . . . . . . 3.1. Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Heart. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Hematopoietic Stem Cell Transplantation (HSCT): Graft Versus Host Disease (GVHD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Important Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. A Need of Further Sophistication in Proteomic/Peptidomic Methods. . . . . . . 4.2. Analysis Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Sample Size in Clinical Studies and Need of Publicly Available Data Repositories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Development of Diagnostic Platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author: Minnie M. Sarwal, e-mail: [email protected] 139

0065-2423/09 $35.00 DOI: 10.1016/S0065-2423(09)47006-9

Copyright 2009, Elsevier Inc. All rights reserved.

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1. Abstract Publication of the human proteome has prompted eVorts to develop high‐throughput techniques that can catalogue and quantify proteins and peptides present in diVerent tissue types. The field of proteomics aims to identify, quantify, analyze, and functionally define a large number of proteins in cellular processes in diVerent disease states on a global scale. Peptidomics, a newer name in the ‐omics world, measures and identifies naturally occurring low molecular weight peptides, also providing an insight into enzymatic processes and molecular events occurring in the system of interest. One area of major interest is the use of proteomics to identify diagnostic and prognostic biomarkers for diVerent diseases as well as for various clinical phenotypes in organ transplantation that can advance targeted therapy for various forms of graft injury. Outcomes in organ transplantation can be potentially improved by identifying noninvasive biomarkers that will serve as triggers that predate graft injury, and can oVer a means to customize patient treatment by diVerentiating among causes of acute and chronic graft injury. Proteomic and peptidomic strategies can be harnessed for frequent noninvasive measurements in tissue fluids, allowing for serial monitoring of organ disease. In this review, we describe the basic techniques used in proteomic and peptidomic approaches, point out special considerations in using these methods, and discuss their applications in recently published studies in organ transplantation.

2. Introduction A shift has occurred from classic ‘‘hypothesis driven’’ to ‘‘discovery driven’’ investigations: instead of one‐gene one‐protein studies, ‘‘‐omic’’ strategies test thousands of genes or proteins simultaneously (Fig. 1A). This change of paradigm, in concert with the rapid development of bioinformatics, promises to contribute significantly not only to our understanding of the molecular basis of cellular processes but also of health and disease [1]. Examining thousands of genes and proteins has made possible both fast paced biomarker discovery and understanding multifactorial diseases at the cellular and molecular level [2]. Improvement in short term survival has been observed. However, issues such as acute allograft rejection (AR), drug toxicity, and infection still remain primary risk factors for graft functional decline, chronic rejection, and graft loss [3–6]. Absence of specific and sensitive means to monitor diVerent etiologies is a major problem. Improving outcomes in organ transplantation will require (1) the ability to detect early graft injury and monitor

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A Omics-hypothesis generating

Customized application

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FIG. 1. A. The role of high throughput -OMICS methods in biomarker discovery and application. B. Increased application of proteomics and peptidomics in the past 7 years: An increased application of proteomics and peptidomics observed in the period of 2001–2007. The numbers next to the bar are number of publication with proteomics and peptidomics applied. The numbers in the parenthesis are the number of proteomics and peptidomics applied in the field of transplantation. (Source: SciFinder ScholarW, Chemical Abstracts Service, Columbus, Ohio).

triggers that predate chronic graft injury and (2) the means to stratify patient risk to allow for individualizing immunosuppressive drugs. In this situation, proteomics may provide a useful tool to identify biomarkers that can eVectively monitor health of transplanted organs. Genomic and transcriptomic methods are relatively mature, while proteomics and the newly introduced peptidomics are still early in their development process. Proteomics has been aggressively used in basic science as well as translational research, as reflected by the number of publication in the field summarized in Fig. 1B. Peptidomics is being used to study small molecular weight peptides and to search for transplantation biomarkers (Fig. 1B). Early outcomes are encouraging but fall short of translation into clinical application. In this review, we evaluate the availability and application of proteomic and peptidomic techniques, note diYculties encountered in clinical applications and their possible solutions, and survey the current literature of proteomics and peptidomics in organ transplantation.

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2.1. PROTEIN AND PEPTIDE BIOMARKERS DISCOVERY FOR ORGAN TRANSPLANTATION Transplantation is the treatment of choice for end stage organ failure. In the U.S. over the last 10 years, approximately 25,000 transplants have been performed. Even though one‐year graft and patient survival has improved substantially in recent years, grafts remain threatened by chronic rejection, chronic toxicity caused by continuous administration of immunosuppressive drugs, and infection of the graft. Unfortunately, clinical symptoms usually do not appear before significant damage has already occurred, necessitating methods of earlier detection. In addition, current methods of diagnosis are invasive, nonspecific, and prone to complications. Earlier detection of pathological changes should be possible using biomarkers, which might be any easily detected proteins or compounds whose levels indicate the presence or extent of disease. In the case of transplantation, biomarkers associated with immune responses would be especially appropriate for monitoring changes in graft behavior and improving patient outcomes. To find biomarkers, one must sift through a dauntingly large number of candidates. Proteomics and peptidomics are ideal for this task because they are robust and provide high throughput. Successful biomarker identification entails discovery, prevalidation (also called confirmation), and validation. As summarized in Figs. 2 and 3A, the discovery step involves a small patient cohort and is usually performed on pooled samples. This quick and simple assessment of possible protein or peptide biomarkers results in a list of analytes which are then reexamined in a prevalidation step performed on individual samples. This step filters false positives resulting from a disproportionate presence of a candidate analyte in one of the samples in the pool. Biomarker candidates passing the prevalidation step enter the validation phase (with a much larger patient size) and, later, clinical testing (Fig. 2). As described later, proteomic and peptidomic methods have already identified several promising biomarkers for monitoring of allografts. 2.2. PROTEOMICS 2.2.1. Evolution of Proteomic Technologies Since their first use by Schena et al. in 1995, DNA microarrays have revolutionized the way we assay gene transcription level in biological samples [7]. Publication of the complete human genome made high‐ throughput analysis using ‐omic techniques such as transcriptomics, proteomics, metabolomics etc. possible. But the prolific growth and development of genomics notwithstanding, transcripts correlate poorly with levels of their

Number of proteins and peptides

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FIG. 2. Biomarker discovery pathway using proteomics and peptidomics methods.

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FIG. 3. (A) Basic steps for a successful biomarker discovery using proteomics/peptidomics which provide a much needed tool to monitor organ transplantation. (B) Conventional proteomic methods that have been employed for biomarker discovery to monitor organ transplantation. (C) A schematics of DIGE experiment.

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translational product, proteins, which have the prime role in cellular activities. Thus, the direct analysis of proteins more accurately reflects actual cellular events than genomic studies. Proteomic studies have been aided by exciting advances in chromatographic and mass spectrometric methods. Notably, these improvements overcome the limitations of conventional methods that require large quantities of protein or the availability of appropriate antibodies for identification and quantification. To date, only the serum and plasma proteomes have been comprehensively characterized [4]. But this has not discouraged researchers from mining proteomes and peptidomes for biomarkers. High‐throughput analyses have yielded some important findings not discoverable through other ‐omic methods. Using serum, plasma, and some tissue‐specific proteomes, investigators have identified biomarkers in the fields of oncology, diabetes, renal disease, and solid organ transplantation, among others [5–10]. Proteomic research has been particularly vigorous in the search for cancer biomarkers, as reflected by the growing number of publications (Fig. 1B). The current pace of development in proteomic and peptidomic tools suggests that the discovery of biomarkers is becoming increasingly eYcient. 2.2.2. Highlighting Different Proteomic Methods 2.2.2.1. Gel‐Based Methods. In gel‐based proteomics, samples are resolved in a gel and the proteins are qualitatively and quantitatively assayed. Standard 2D gel electrophoresis (2DE) remains popular for its ease of use, but 2D DIGE is increasingly attractive for its ability to run multiple samples on the same gel (Fig. 3B). 2.2.2.2. 2D Gel Electrophoresis. 2DE has evolved significantly since the first report by James O’Farrell in 1975 [8]. In this method, the proteins in a mixture are resolved in two dimensions according to their isoelectric points (pI) and molecular weights. In recent years, the use of immobilized pH gradient (IPG) strips has enhanced reproducibility, while depletion of high‐abundance proteins from blood has improved detection of trace components. In addition, advances in mass spectrometric techniques have greatly speeded protein identification. Proteins of interest can be excised from the gel, digested by proteolytic enzymes (usually trypsin), and finally identified by peptide mass fingerprinting and tandem mass spectrometric analysis. This ease and simple experimental setup continues to make 2DE a workhorse of proteomics [9]. 2.2.2.3. Two‐Dimensional Difference Gel Electrophoresis (2D DIGE). A new variation on conventional 2DE is 2D DIGE. In this method two samples are labeled with diVerent fluorescent dyes (e.g., Cy3 and Cy5). The labeled proteins are mixed with an internal standard and applied to a single 2D gel (Fig. 3C). This approach reduces gel‐to‐gel variability and increases both reproducibility and statistical confidence [10, 11]. With its ability to

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assess diVerential expression or relative abundance of protein in two biological samples (e.g., samples collected from disease and healthy conditions), 2D DIGE is highly promising, but it is severely limited because the enormous dynamic range of protein concentrations makes it diYcult to analyze more than a few hundred proteins. 2.2.2.4. Gel‐free Methods. Several gel‐free platforms are available. Surface‐enhanced laser desorption/ionization time of flight mass spectrometry (SELDI‐TOF, or simply SELDI), which is based on protein‐specific interactions with functionalized surfaces, has been popular in the hunt for biomarkers [12–17], including the search for transplantation biomarkers in humans and model animals [18–22]. The method’s attractions include its speed, easy sample preparation, and uncomplicated data analysis. Although relatively standardized protocols and commercial availability makes SELDI‐ TOF a method of choice, the technique is currently limited to molecules in the range of 4–15 kDa, leaving much of the proteome inaccessible. Liquid chromatography coupled with mass spectrometry (LC‐MS) is also popular because it is very sensitive and covers a wide range of proteins. Preferred ionization methods include electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI). Two strategies, so‐ called ‘‘top‐down’’ and ‘‘bottom‐up,’’ have been used. In the former, which does not involve proteolytic digestion, sample proteins remain intact until ionized and fragmented inside the mass spectrometer. In the latter, proteins are enzymatically digested before analysis by MS [23]. After the resulting peptides have been identified, they may be used to reconstruct the original proteins. For samples that are particularly complex, the MS analysis can be simplified by performing a preliminary 2D liquid chromatographic separation combining strong cation exchange (SCX) chromatography with reverse phase chromatography (RPC). This variation of shotgun proteomics [24] is known as ‘‘multidimensional protein identification technology’’ (mudPIT). A summary of commonly used proteomic methods and their strengths and weaknesses have been summarized in Table 1 and Fig. 3B.

2.3. PEPTIDOMICS, A NEW APPROACH TO BIOMARKER DISCOVERY Unlike proteomics, which focuses on large proteins, the term ‘‘peptidomics’’ refers to the analysis and identification of small molecular weight (<3000 Da) peptide molecules present in biological systems, many of which may be biomarkers [25]. Despite many similarities, peptidomics and proteomics have developed along diVerent paths in biomarker discovery eVorts (Fig. 1B). For example, because proteases and protease inhibitors are active players in ‘‘peptidome’’ dynamics, peptidomics has paid particular

Gel free

Gel based

CE MALDI [36,119]/LC MALDI [120]

LC‐MS (MudPIT with fractionation) [24]

LC‐MS (shotgun: no fractionation)

2D DIGE [11]

2D gel electrophoresis (2 DE) [8]

Method

 Requires relatively more sample than for LC‐MS  Expensive and needs special user expertise

 SuVers from the dynamic range of protein concentration in the sample  Exhaustive (samples once used cannot be revisited)  SuVers from false positive protein IDs  Exhaustive (samples once used cannot be revisited)  Quantitative nature of the assay if the samples are not run immediately after the CE or HPLC run

 Needs relatively less sample compared to 2DE  More sensitive compared to 2DE

 Fast and simple

 Very sensitive and easy protein identification  Samples can be revisited

 Better detection limit and better reproducibility  Quantitative

 Low sensitivity  Poor reproducibility  Poor detection of low abundance proteins  Needs relatively more sample

Weaknesses

 Simple experiment setup  Low cost and easy to run  Easy identification of proteins and run on the western blot

Strengths

TABLE 1 DIFFERENT PROTEOMIC TECHNIQUES AVAILABLE

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attention to their role in generating, maintaining, and degrading peptides as an indicator of diVerent physiological states. Peptidomic applications range widely, including the analysis of neuropeptides and neurohormones [26, 27] and the search for biomarkers in blood [25, 28] and urine [29]. Typically, low molecular weight peptides are extracted and purified before chromatographic separation and MS analysis. The extraction and preparation of peptides is one of the most variable procedures in peptidomic studies. Romanova et al. for example, used 2,5‐dihydroxybenzoic acid (DHB) to extract endogenous signaling peptides from tissue or to preserve tissue samples and extracts, including cell clusters, brain punches, and intact brain regions [30]. But with insect tissues, however, a direct sampling of the tissue onto the MALDI plate and mixing it with the matrix proved adequate [26]. In this way, Predel et al. compared the peptidomes of four related hemipteran species and identified previously unknown sequences for myosuppressin, short neuropeptide F (sNPF), CAPA‐periviscerokinins, and pyrokinins. Currently, the two preferred methods in peptidomics analyses are capillary electrophoresis coupled mass spectrometry (CE MS) and HPLC mass spectrometry (LC‐MS). Because CE MS uses small samples and oVers rapid throughput, it has become a powerful and popular method for peptidomic analysis. Herrero et al. have presented a comprehensive review of this application [31]. CE MS is also adaptable to many specialized applications using proteins and other bioanalytes. LC‐MS has also been extensively used for peptidomics analysis [32–34]. Capillary‐electrophoresis‐coupled mass spectrometric analysis has been used to seek biomarker proteins in urothelial cancer [35] and biomarker peptides for congenital unilateral ureteropelvic junction in newborns [36]. Jurgens et al. studied peptide samples from urine by separating each sample into 96 fractions with reverse phase HPLC and then analyzing them by MALDI TOF. Selected peptides were identified with a nanospray quadrupole TOF (nanoESI‐QTOF). A total of 180 peptides from 69 unique proteins were identified; among these, the most abundant were from collagens and Tamm‐Horsfall protein. Notably, the urinary peptidome and proteome were somewhat complementary as they cover two distinct population of polypeptides. The study demonstrated that plasma peptides were more abundant in hematuria urine than in normal healthy urine; it also revealed that plasma peptides are on the whole more hydrophobic than those of normal urine. By combining peptidomic and proteomic data Zougman et al. identified 563 peptide products from 91 precursor proteins in cerebrospinal fluid (CSF) [37]. Villanueva et al. exploited the capability of peptidomics not only to identify the signature subset of serum peptides for three types of solid tumors, but also to link peptide marker profiles for disease conditions directly to diVerential protease activity [28].

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2.4. QUANTIFYING PROTEOMES AND PEPTIDOMES The distinctiveness of proteomic methods is that they not only identify the proteins and peptides present in a sample but also quantify them. Such quantitation may be relative or absolute; relative quantitation may or may not involve labels (Fig. 5).

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FIG. 4. Depletion of abundant proteins from urine samples: Urinary protein samples from diVerent patients may vary in abundant plasma proteins concentration as seen in immunoaYnity chromatograms of urinary proteins of two renal patients. Two samples of urinary proteins

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Relative quantitation without labels includes both gel and gel‐free methods. In image analysis of gels such as 2D DIGE, the proteins in one sample are marked with Cy3, while those in the other sample are marked with Cy5. For a particular protein of interest, the intensities of the two spots are compared and then normalized by reference to the overall spot intensity of all proteins in the two samples. For gel‐free methods, the area under the peak (i.e., the extracted ion current) for each peptide measures the relative abundance of peptides. Label‐free methods have been used in CE MS to predict the clinical outcome of congenital unilateral ureteropelvic junction obstruction in newborns and in biomarker discovery for urothelial cancer [35, 36]. A similar method, in which the peptides are first fractionated by HPLC followed by MALDI TOF analysis (LC MALDI), has been used to discover oral fluid biomarkers for oral cancer [38]. The beauty of a label‐free method lies in its ability to compare a large number of sample sets; in comparison, labeling methods suVer from either labeling eYciency or limits on the number of labels available to run quantitative analysis on a large sample set. Nevertheless, the success of label‐free methods is sometimes limited by retention time shifts and fluctuations in MS signal intensity. The second strategy in relative quantitative proteomics is stable isotope labeling, in which a labeled moiety is incorporated biosynthetically or chemically. Four methods deserve special note: (1) Stable isotope labeling by amino acids in cell culture (SILAC)—SILAC is based on metabolic incorporation of ‘‘light’’ or ‘‘heavy’’ forms of amino acids into the proteins during their synthesis in two cultures. The labels can then be exploited to measure protein levels in two diVerent cell populations used for MS‐based quantitative proteomics [39]. (2) Isotope Coded AYnity Tags (ICAT) recovered from urines from two renal patients by centrifugal filtration and subjected to immunoaYnity column designed to deplete six most abundant plasma proteins. (A) Chromatogram for Renal patient #1. Peak 1: UV trace at 280 nm for all the proteins except six most abundant plasma proteins and Peak 2: UV trace at 280 nm for six most abundant plasma proteins. (B) Chromatogram for Renal patient #2. Peak 1: UV trace at 280 nm for all the proteins except six most abundant plasma proteins and Peak 2: UV trace at 280 nm for six most abundant plasma proteins. The area under the peaks directly relates to the relative concentration of protein. (C) Depletion of six most abundant plasma proteins from urine protein sample using MARS column (Agilent Technologies, Palo Alto, CA) and resolved by 2D DIGE. Undepleted urinary proteins from a renal transplant patient control (labeled with Cy3 dye—green) and the same sample after depleted (labeled with Cy5 dye—red). The depleted proteins, albumin, antitrypsin, transferrin, and IgG light chain are indicated by arrows (D) urinary protein samples from renal transplant patients with stable graft function (labeled with Cy3—green) and biopsy proven acute rejection patient (labeled Cy5—red). The potential biomarker candidates are indicated by white circles. (E) Depletion of high‐abundance plasma proteins and its impact on the number of observed peptide features. A venndiagram showing number of peptides feature observed when a signal of 70 or more was considered as a positive feature. The diagram shows the number of features from only undepleted and depleted samples. The features common in both are presented as overlapped.

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[40]—ICAT analyzes the relative amounts of cysteine‐containing peptides in enzymatically digested protein samples. The quantity of a protein of interest can then be calculated using the relative abundance of tryptic peptides. It is commercially available as an ICATW kit (Applied Biosystems, Foster City, TM California). (3) iTRAQ Reagents (Applied Biosystems)—This method uses isobaric labels which, upon fragmentation in MS/MS, giving rise to four unique reporter ions (m/z 114–117) [41]. This technique is based upon chemically tagging the N‐terminus of peptides generated from the protein digests with a label consisting of reporter and balancer groups. The reporter is cleaved during collision‐induced dissociation (CID) and then compared to a single peptide of known mass, allowing quantitation of as many as four diVerent samples. This unique ability of analyzing more than two samples has been successfully exploited in clinical studies [42, 43]. A new commercialTM ly available iTRAQ kit analyzes as many as eight samples per experiment. (4) 18O/16O labeling methods use [18O] and [16O] labeled water to compare the tryptic digestion of two samples [44]. In addition to the relative quantitation methods, mass spectrometers can be programmed to monitor selected peptide fragment ions continuously. Only selected spectra are acquired, and for this reason the strategy is termed selected reaction monitoring (SRM) or multiple reaction monitoring (MRM). This approach has been used to identify biomarkers for rheumatoid arthritis [45]. 2.5. SAMPLE PROCESSING FOR PROTEOMICS AND PEPTIDOMICS 2.5.1. Sample Collection and Handling Proteomic and peptidomic eVorts to identify potential biomarkers depend heavily on the quality of resulting data, which in turn depend on the quality of samples used. Biological samples have many intrinsic variables. Sample collection, initial processing, and storage is of paramount importance and has been studied by many groups [20, 46]. Traum et al. refrigerated urine or blood samples for up to 24 h before freezing them at 80  C [46]. Schaub et al. reported no significant changes during long term storage at 70  C or multiple freeze‐thaw cycles [20]. Subsequent extraction of proteins and peptides from complex biological matrices requires rigorous sample preparation and extraction protocols [47–50]. Greater consistency in the protocols used by diVerent groups working in the similar tissue/sample type would increase the utility of such experiments, as it would allow for a comprehensive analysis using individual experiments. 2.5.1.1. Sample Preparation for Urinary Proteomics. Because urine is primarily a mixture of salt, glucose, ketone bodies, and exosomes with little protein, a strategy for enriching urinary proteins irrespective of their relative

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composition is necessary. Apart from very low protein concentrations and intra‐ and interindividual variations in the composition of the urinary proteome, the high salt content in human urine poses a challenge [51, 52]. DiVerent approaches have been used to recover urinary proteins, including desalting by gel filtration [53, 54], centrifugal filtration [52, 55, 56], vacuum dialysis [57], dye precipitation [58], and extraction with organic solvents [48, 59, 60]. Standard protein recovery and processing methods would greatly assist in cross‐experiment data comparisons, particularly for verification studies on the clinical applicability of biomarkers; in such cases, the hundreds of human samples needed will necessitate processing samples at multiple centers [46]. In the analysis of urinary proteins from renal transplant patients, centrifugal filtration was found to be the best for ease and yield consistency [61]. 2.5.2. Depletion of Abundant Proteins and Fractionation: An Approach to Analyze Complex Proteomes 2.5.2.1. Depletion in Plasma and Serum Proteomics. A major hurdle in proteomic research is that the available techniques cannot work in the dynamic range of protein and peptide concentrations found in biological samples. Plasma, perhaps the biofluid most widely used in biomarker discovery eVorts, contains proteins with concentrations varying over twelve orders of magnitude [62, 63]. Depleting the 50–100 most abundant proteins could substantially enhance detection of proteins in the range of pg/ml in blood [32], but even the depletion of only the high‐abundance proteins may contribute to advances in MS‐based proteomics [64]. The usefulness of such a strategy has been tested for potential biomarker discovery [65]. Zolotarjova et al. narrowed the dynamic range of their samples by employing a commercially available immunoaYnity column to deplete the 14 most abundant plasma proteins [66]. Fountoulakis et al. used blue matrix and Protein G‐based aYnity chromatography to deplete high‐abundance albumins and immunoglobulin chains [67]. Others, observing that glycoproteins make up at least 50% of the blood proteome, have used multilectin aYnity chromatography (M‐LAC) [68] to remove glycoproteins and improve dynamic range [68]. This approach has been successfully used in biomarker discovery eVorts [69, 70]. Several commercially available products exist, including Multiple AYnity Removal System (MARS) columns. The Human 14, Human 7, and Human 6 columns from Agilent Technologies (Palo Alto, California) deplete 14, 7, or 6 of the most abundant serum proteins, including albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, a2‐macroglobulin, a1‐acid glycoprotein, IgM, apolipoprotein A‐I, apolipoprotein A‐II, complement C3, and transthyretin (http://www.chem.agilent.com). Qproteome Albumin/IgG Depletion Kit (Qiagen, Valencia, California) removes these two proteins

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eYciently and rapidly from serum and plasma samples. The ProteoPrep 20 ImmunoaYnity Plasma Depletion Kit from Sigma‐Aldrich (St. Louis, Missouri) is designed to eliminate the twenty most abundant proteins from human blood samples (http://www.sigmaaldrich.com). EnchantTM Life Science Kits Albumin Depletion selectively binds albumin using CibracronW Blue‐based support (Pall Corporation, East Hills, New York). ProteomeLab IgY‐12 high‐capacity proteome partitioning kits from Beckman Coulter (Fullerton, California) remove albumin, IgG, transferrin, fibrinogen, IgA, a2‐macroglobulin, IgM, a1‐antitrypsin, haptoglobin, orosomucoid, apolipoprotein A‐I, apolipoprotein A‐II from human serum and plasma. 2.5.2.2. Depletion in Urinary Proteomics. With increasing interest in urinary proteomics and peptidomics, it is important that any confounding eVect due to high‐abundance proteins be minimized. Such high‐abundance plasma proteins, which appear in urine as ultrafiltrates of plasma (e.g., haptoglobin, transferrin, and albumin) or as a result of nonspecific renal injury (albumin), interfere with detection of clinically relevant low abundant proteins. There have been reports of attempts to normalize/enrich urinary protein samples. Pisitkun et al. solubilized Tamm‐Horsfall protein sediment from urinary exosomal extract by DTT in order to analyze urinary exosomes [71]. Thongboonkerd et al. enriched the positively charged fraction of urinary proteins by using ion exchange chromatography [72]. In our urinary proteomic analysis aimed to identify biomarker candidate proteins for acute rejection of renal allograft, we observed significant plasma protein in some renal transplant patients. The quantity of the six most abundant plasma proteins in urinary protein samples from diVerent patients varied, as seen in the immunoaYnity chromatograms of urinary proteins from two renal patients in Figs. 4A and B. Peak 2 corresponds to the relative abundance of the six most abundant plasma proteins after depletion. Because peak area directly correlates with relative concentration of protein, the need to deplete abundant plasma proteins from urinary samples is clear. We used the MARS Human 6 to remove the six most abundant proteins (albumin, IgG, IgA, transferrin, haptoglobin, and antitrypsin) from urine obtained from five renal transplant patients with biopsy proven acute rejection and five patients with stable grafts. DIGE [73] was performed to assess success. When the gel images were assayed by SameSpots (Nonlinear Dynamics), the number of detectable protein spots increased from 858 in the gel with nondepleted urinary protein sample to 1066 (24% increase) in the gel with depleted urinary protein sample, suggesting that depletion helped to detect lower abundance proteins in urine. Out of the 858 detected spots, 43 spots had decreased spot intensity, 486 spots had increased spot intensity, and 329 spots had the same intensity after depletion.

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Quantitative proteomics

Relative quantitation (label free)

Relative quantitation (stable)

Absolute quantitation (SRM and MRM)

Samples

Isotopic labeling of separate protein mixtures

Samples

Digestion, separate LC separation-XIC data

Digestion of the protein and LC separation

Digestion of protein, LC separation

MS-MS analysis

MS-MS analysis

Database searching

Database searching

Detection of preprogrammed peptide, MS-MS analysis

Relative protein abundance from MS data

Relative protein abundance from MS data

Absolute protein abundance form MS data

*XIC-extracted ion current, **SRM single reaction monitoring, **MRM multiple reaction monitoring

FIG. 5. A summary of available quantitative proteomic strategies and steps involved in each approach.

Excluding the high‐abundance proteins that were targeted for depletion, a large number of identifiable protein spots increased in intensity after the depletion. Some of these proteins bear biological relevance based on their function and/or previous associations with renal injury and the depletion process may thus augment detection of these potentially valuable biomarker proteins [50]. We also evaluated the eVectiveness of depletion by shotgun proteomics with label‐free MALDI. The tryptic peptides of the depleted fraction were analyzed by injecting the peptides on a 0.1 mm  150 mm C18 reverse phase column (Michrom) and eluted with a gradient of 5–55% acetonitrile over 50 min using a Michrom MS4 HPLC. A total of 100 fractions were collected and analyzed by LC‐MALDI. Although more than 10,000 peptide mass features were present in both undepleted and depleted samples, depletion enhanced some weak peaks while eliminating many intense peaks attributed to the abundant proteins (3860 peaks were enhanced; 6231 peaks disappeared; see Fig. 4E). After deisotoping the MALDI peaks, we noted 155 peptides unique to the undepleted sample. Among 10 identified peptides present only in the depleted samples, none of them originated from the six most abundant plasma proteins, indicating the eYcacy of the depletion experiment (Sigdel and Sarwal, unpublished data).

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2.5.2.3. Fractionation of Complex Protein/Peptide Mixture. The presence of an overwhelming number of proteins/peptides in biological samples is another issue that needs to be addressed for successful outcome. Whereas depletion helps to unmask low abundance proteins by depleting high‐ abundance proteins, complex protein/peptide mixtures need to be fractionated. Several strategies have been tested, ranging from 1D fractionation (using either SDS‐PAGE gel or chromatography) to multidimensional fractionation (using 2D gel or mudPIT). Because of its ability to fractionate small proteolytically digested peptides, mudPIT has become a popular proteomic method [74]. In this method, peptides are fractionated by a 2D LC separation using SCX in the first dimension followed by a RPC in the second. Among other methods, isoelectric focusing (IEF) [75] and, free‐flow electrophoresis (FFE) [76] have been used to fractionate peptides. Other ways of sample enrichment include enriching specific peptides such as cysteinyl [77] and glycopeptide [78]. In a unique attempt Righetti et al. attempted a democratic way of reducing protein concentration diVerences by using a library of combinatorial ligands [79]. The so‐called ‘‘Protein Equalizer Technology’’ is reported to equalize protein concentration diVerence by reducing high abundance proteins and enriching low abundace proteins.

3. Application of Proteomics and Peptidomics in Transplantation Many biomarkers currently used to monitor organ transplants were originally identified by research stemming from mechanism‐driven hypotheses. For example, serum creatinine is normally used in renal transplants as a surrogate biomarker for underlying graft injury [80]. Serum creatinine, however, lacks high specificity and sensitivity, making it a poor marker. False positives may be found in patients with greater body mass, who tend to have higher values even in the absence of graft injury; conversely, smaller or younger patients may produce false negatives. The limitations of biomarkers such as serum creatinine may be overcome by using biomarkers selected for their strong empirical correlations with disease. Such correlations may be found by using high‐throughput omic screens to link biomarkers with particular transplantation outcomes, such as the onset of acute or chronic graft rejection or prediction of the injury response to immunosuppressive medications (Fig. 1A). Peptidomic and proteomic methods may also help to customize immunosuppressive therapy for the individual patient—that is, to find a threshold between rejection and infection in order to avoid allorecognition yet retain normal infectious immunity. For example, patients with spontaneous

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operational tolerance [81, 82] possess a highly regulated gene set in peripheral blood that may guide the design of customized patient immunosuppression. Blood and urine will remain popular sources of possible biomarkers for their easy collection and noninvasive nature, although in some cases tissue collection will still be necessary, despite the need for invasive procedures. 3.1. KIDNEY Despite improvements in immunosuppression regimens, renal AR remains a major cause of graft loss in kidney transplantation. The diagnostic gold standard for AR is renal biopsy, yet it is both invasive and nonspecific. The need for noninvasive and specific biomarkers has provided the impetus for several genomic [61, 83, 84] and proteomic studies [19, 85, 86]. Vidal et al. have proposed criteria for an ideal biomarker for renal tubular injury [87]. An ideal biomarker should be easy to collect (e.g., urine or blood); sensitive to early detection during acute injury; specific in distinguishing among tubular, perirenal, and glomerular injury; and expressed in the kidney. Because urine is the ideal noninvasive specimen, the number of proteomic studies of urine has surged [88–93]. Urinary proteins may arise from several sources including filtration of plasma proteins, secretion from nephrons, proteolytic degradation products, secretion from the lower urinary tract, and physiological and/or pathological cell death. Using SELDI‐TOF MS, Clarke et al. identified two protein peaks (3.4 and 10.0 kDa) as biomarkers for AR of renal allografts [94]. The same platform was used by Schaub et al. to identify rejection‐specific peptide patterns [85] in the nontryptic cleavage products of b2‐microglobulin [19]. Finally, O’Riordan et al. reported that a group of seven peptide masses distinguish acute rejection patients from stable patients [86]. A follow‐up study using tandem MS and ProteinChip immunoassays identified two of these peptides as human beta‐defensin‐1 (4.7 kDa) and a‐1‐antichymotrypsin (4.4 kDa) [95]. Levels of the former were reduced in AR, while levels of the latter were increased. We are optimistic that biomarker for renal transplant dysfunction will not only help to monitor renal grafts, but also will help to predict outcomes and therapeutic responses. The current status of the field of biomarker discovery in renal transplant is summarized in Table 2. 3.2. LIVER In the liver, a serious complication of transplantation is ischemia– reperfusion injury. Molecular mechanisms of the resulting graft dysfunction are poorly understood [96]. Emadali et al. [97] studied liver biopsies from early phases of organ procurement (ischemia) and transplantation

TABLE 2 A LIST OF PROTEIN BIOMARKERS IDENTIFIED BY THE USE OF VARIOUS PROTEOMIC APPROACHES Organism Human

Mouse

Disease

Specimen

Biomarkers

Techniques

[110] [82]

2004

[91]

2007

[111]

Ischemia–reperfusion injury (liver) Ischemia–reperfusion injury (liver)

Liver biopsy

26 proteins identified aB‐crystallin and tropomyosin IQGAP1

Liver biopsy

NCK‐1

Urine serum

Merpin‐1‐a Haptoglobin

DIGE 2DE/MS

2007 2004

[112] [116]

Liver biopsy

Cyclophilin

DIGE MS

2007

[89]

SDS‐PAGE, LC‐MS SDS‐PAGE, LC‐MS

Rat

De novo autoimmune hepatitis

Liver biopsy

Carbonic anhydrase III, glutathione S‐transferase (GST), and b‐1 subunit of proteasome

2DE

Human

AR for renal transplant

Urine

SELDI, MS/MS

BK virus‐associated renal allograft nephropathy (BKVAN) Acute rejection of renal graft

Urine

Mouse

2006 2006

Urine Heart biopsy

Acute rejection of renal graft Chronic lung allograft rejection

Urine Bronchoalveolar lavage fluid (BALF)

b‐Defensin‐1 (4.7 kDa) and a‐1‐antichymotrypsin (4.4 kDa) m/z values of 5.872, 11.311, 11.929, 12.727, and 13.349 kDa Peptides with m/z values of 6.5, 6.7, 6.6, 7.1, 13.4, 10.0, and 3.4 kDa b 2‐Microglobulin Human neutrophil peptides (HNP)

Acute rejection of skin graft

Serum

A number of peptide masses

Urine

Gel‐based method

Reference

Kidney injury AR of heart transplant

Acute renal failure Liver transplant tolerance Sepsis‐induced acute renal failure

2DE/MS 2DE

Year

[113]

Gel‐free method

2007

[87]

SELDI

2006

[114]

SELDI

2003

[86]

SELDI MALDI MS, MS/MS

2005 2005

[27] [115]

SELDI

2006

[26]

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(reperfusion). Global expression and phosphorylation patterns of cytoskeleton‐related proteins suggest that IQGAP1, a Cdc42/Rac1 eVector, regulates actin cytoskeleton remodeling and maintenance of bile canaliculi (BC) integrity [97]. In order to identify early targets of oxidative ischemia–reperfusion injury during liver transplantation, Avellini et al. applied a diVerential proteomics approach to HepG2 human liver cells and liver needle biopsies after cold and warm ischemia [98]. The cell line experiment revealed a specific modification of the peroxiredoxins active site thiol into sulfinic and/or sulfonic acid. Proteomic analysis of liver needle biopsy revealed immunophenotypic expression of APE1/Ref‐1. The authors report hyperoxidation of peroxiredoxin family of hydroperoxide scavengers, PrxI, PrxII, and Prx VI occurred during I/R of liver transplantation. Because hyperoxidation depended on the duration of warm ischemia, these findings may improve graft preservation and outcomes in liver transplants [98]. The mechanism of tolerance of transplanted organs remains a subject of discussion and research. Proteomics/peptidomics may probe tolerance specific changes in proteins that signal an appropriate time to wean from immunosuppressive drugs, thereby avoiding the possible complications of immunosuppressive drug. Hsu et al. studied an orthotopic liver transplant patient who had been out of immunosuppression for 5 years following posttransplant lymphoproliferative disease (PTLD) [99]. They found twelve proteins whose levels were significantly increased in the tolerant patient. Notably, haptoglobin was increased, consistent with its reported immunosuppressive activity [100, 101]. The current status of the field of biomarker discovery in liver transplant is summarized in Table 1. 3.3. HEART Several high‐throughput proteomic techniques have been used in heart transplantation research [102]. Changes in protein levels in heart tissue from patients with dilated cardiomyopathy (DCM) to ischemic heart disease (IHD) were first studied by Corbett et al. [103]. The authors applied 2DE method to 28 DCM heart biopsies, 21 explanted hearts from IHD patients, and 9 donor heart biopsies. Levels of 88 proteins diVered in tissues from DCM and IHD patients, including myosin light chain 2 and a group of proteins identified as desmin. The investigators suggested that significant diVerences in protein quantity from DCM heart may result from altered protease activity. Meirovich et al. used plasma from 16 transplant patients and observed that levels of regulated‐on‐activation, normal T‐expressed and secreted (RANTES), neutrophil‐activating protein‐2 (NAP‐2) and insulin growth

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factor binding protein‐1 (IGFBP‐1) correlated significantly with brain natriuretic peptide (BNP) plasma levels during Grade 3A (Grade 2 revised [2R]) or above rejection as diagnosed by endomyocardial biopsy score according to the International Society for Heart and Lung Transplantation (ISHLT) grading system [104]. The study demonstrated a correlation between BNP plasma levels observed during acute rejection episode of heart transplant and cytokines other than proinflammatory cytokines. The report further suggested that p38 signaling is required for stimulation of BNP secretion from cardiocytes, indicating to unifying mechanism relating inflammation with cardiac hormone production in acute AR of heart transplant [104]. Cardiac allograft vasculopathy (CAV) is one of the major risk factors influencing graft loss and patient survival [105]. Studies have suggested that CAV is prevented by protective genes. Searching for protector proteins, De Souza et al. applied 2DE followed by MS and identified heat shock protein (HSP27) as a marker in 4/6 biopsies from patients without CAV but absent from 5/6 patients with CAV [106], a finding later validated by immunohistochemistry. They concluded that diphosphorylated HSP27 is a marker of healthy blood vessels that is depleted in vessels of patients with graft vasculopathy. The current status in the field of biomarker discovery in heart transplant is summarized in Table 1.

3.4. HEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT): GRAFT VERSUS HOST DISEASE (GVHD) HSCT continues to face complications from GVHD [107]. Currently, diagnosis of GVHD is primarily based on clinical tests and biopsies. Proteomic analysis by using signature pattern of peptides has shown some promise in early diagnosis of GVHD. Using CE followed by ESI‐TOF MS approach to analyze urine from allogenic stem cell transplantation (SCT), autologous SCT, and five patients with sepsis, Kaiser et al. identified 16 unique peptides that discriminated SCT with GVHD from SCT without GVHD [108]. They also identified two peptides from leukotriene A4 hydrolase and a peptide from serum albumin that might serve as biomarkers for GVHD in SCT patients. Srinivasan et al. investigated 88 serum samples from HCT patients using SELDI‐TOF method [109]. Their peptide pattern analysis distinguished GVHD samples from both posttransplant non‐GVHD samples and pretransplant samples with high accuracy (100% specificity and 100% sensitivity). The current status in the field of biomarker discovery in GVHD is summarized in Table 1.

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4. Important Issues 4.1. A NEED OF FURTHER SOPHISTICATION IN PROTEOMIC/PEPTIDOMIC METHODS Advances in protein and peptide sample fractionation techniques coupled with mass spectrometric analyses have significantly enhanced our abilities to gather large datasets and to discover clinically useful biomarkers, although such analysis is less well‐developed than microarray analysis. Biomarkers are especially useful when their levels correlate with a particular state of health or disease. Recent improvements in MS instrumentation themselves demand more sophisticated analytical methods. 4.2. ANALYSIS ISSUES In proteomic methods, changes in protein level between experimental and control samples are compared and presented in terms of fold change without estimates of false positive and false negative rates. A need to use more rigorous statistical analysis approaches has been realized: to analyze arthritis antigen microarrays, Hueber et al. used significance analysis of microarray (SAM) to analyze secreted proteins in early rheumatoid arthritis [110]. Oberg et al. recently demonstrated how proteomic data obtained from multiple experiments with iTRAQ labels can be successfully analyzed using ANOVA [111]. Roxas and Li applied a SAM method to normalize and analyze LC‐MS data to produce protein diVerential profiles and estimate false discovery rates [112]. 4.3. SAMPLE SIZE IN CLINICAL STUDIES AND NEED OF PUBLICLY AVAILABLE DATA REPOSITORIES Limited availability of samples often hinders translational research from producing statistically significant results that can be taken it to the next level of validation. When sample sizes at a single location are too small, groups working in diVerent clinics and hospitals may join together their eVorts and collaborate. For such collaborative multicenter studies, sample collections will have to follow a common protocol for processing, storage, and shipment. Within the larger medical research community, developing and following standard protocols for processing and even proteomic analyses of diVerent tissue types would enhance quality and cross‐study comparisons of data. It would also permit the research community to integrate studies in comprehensive analyses for a meaningful outcome. Another way to expand the usefulness of data generated by proteomic and peptidomic methods is to create public repositories and encourage

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researchers to deposit the raw data in a standardized format. In transcriptomics, this practice already exists in the form of the Minimum Information about Microarray Experiment (MIAME) guidelines. The MIAME guidelines outline the minimum information that should be included in describing a microarray experiment, and several repositories such as the Gene Expression Omnibus (GEO) and the Stanford Microarray Database (SMD) require researchers to deposit data in compliance with MIAME. These public repositories, with their collected MIAME compliant microarray data, have encouraged several integrated approaches to mining the deposited data, with early success [113, 114]. Integrating the high volume of proteomic and peptidomic data remains a challenge, because various groups have chosen diVerent algorithms for sample analysis and data handling [115–117]. To reconcile these disparate approaches, a proteomic of MIAME called Minimum Information about a Proteomics Experiment (MIAPE) has been established [118]. Universal adoption of these guidelines would promote deposition of MIAPE‐compliant data into public repositories, thereby permitting more eVective use of experimental data. 4.4. DEVELOPMENT OF DIAGNOSTIC PLATFORMS A search for biomarkers that relies only on global proteome profiling suVers from challenges in quantitation and sensitivity. The goal of high‐ throughput proteomic and peptidomic techniques in the discovery phase should therefore be able to identify potential biomarker molecules that could be verified by more sensitive and quantitative methods in the preclinical validation phase. The value of such validation methods is that the candidate molecules can be validated in an independent and larger cohort of patients in a relatively short period of time. Usually, the quantitative methods that can individually measure biomarker proteins or peptides have been used for these validation steps. Assays such as ELISA and MRM are now being used for such purposes. ELISA remains popular and with a small number of biomarker molecules in hand, a multiplexed assay based on ELISA is possible. When appropriate purified antigen‐antibody pairs are not commercially available, however, the time and eVort required to obtain them makes MRM an attractive alternative. MRM uses triple‐ quadrupole mass spectrometers in a robust strategy providing suYcient throughput, specificity, and quantitation for candidate biomarker validation. With this method, measurements can be substantially multiplexed to provide a minimum of 30–100 specific protein assays in a single LC‐MS analysis. Combined with appropriate stable isotope‐labeled internal standards, the resulting ion current can accurately measure protein concentrations or changes in relative abundance. Once validated through preclinical

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steps such as MRM, a very small number of proteins or peptides can then be used to develop an ELISA assay. Because MRM requires a sophisticated mass spectrometer, however, it may be inconvenient in clinical settings for outpatient testing purpose.

5. Conclusion Unlike the genome, the proteome is metastable. It is cell specific, has a wide dynamic range concentration, and can be changed by several physiological and environmental factors. Because of its highly diverse and complex of proteins and peptides, obtaining comprehensive information with an approach that uses a single platform is not easy. As a result, despite numerous studies of human tissues, no comprehensive proteomic map has been published except for human serum and plasma. Yet proteomic methods promise to discover clinically relevant biomarkers for existing health problems including dysfunction of transplanted organs. Fractionation strategies will help to simplify the complex nature of the samples and get quantitative information about low abundant proteins. In addition to proteomics of high MW proteins and naturally occurring small MW peptides, the study of diVerent compartments of the proteome, such as glycoproteomes, phosphoproteomes, membrane proteomes, exosomes, etc. will help build a detailed and comprehensive proteomic database for each tissue and organ type. The study of clinical samples is particularly diYcult because of the challenge in obtaining well‐annotated clinical samples from a large cohort of patients, and because of the likelihood of continuous disease progression. For these reasons collaboration among diVerent researchers using a larger cohort of patients and consideration of physiological and clinical data along with the proteomic and peptidomic outcome will be essential [1]. The translation of potential biomarkers from the lab bench to the clinical bedside will therefore not be easy and will require the concerted eVort of immunologists, molecular biologist, transplantation specialists, geneticists, and bioinformatics experts. Rigorous studies, using larger patient samples and exploiting sophisticated proteomic, peptidomic, and bioinformatics tools— including quantitative methods—will provide more reliable results. Such developments will lay the cornerstone for a new age of translational research and eventually contribute to the replacement of nonspecific, invasive diagnostic tests with noninvasive, specific tests for organ transplant monitoring.

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ADVANCES IN CLINICAL CHEMISTRY, VOL. 47

BIOMARKERS IN LONG‐TERM VEGETARIAN DIETS Iris F.F. Benzie1 and Sissi Wachtel‐Galor Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Kowloon, Hong Kong

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Possible Nutritional Deficiencies in Association with Long‐Term Vegetarian Diets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Protein and Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Essential Fatty Acids and Long‐Chain PUFAs of Omega‐3 Series . . . . . . . . . . 2.4. Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Minerals and Trace Metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Biomarkers of Oxidant/Antioxidant Balance in Association with Vegetarian Diets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Introduction: Concepts of Antioxidants and Oxidative Stress and Suggested Role of Dietary Antioxidants in Health Promotion. . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Biomarkers of Antioxidant Status and Oxidative Stress in Vegetarians and Nonvegetarians: Implications for Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Biomarkers that Reflect Lower Risk of Disease in Long‐Term Vegetarians . . . . . . 4.1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. CVD and Type 2 Diabetes: Background and Biomarker Studies in Relation to Vegetarian Diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Bone Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Renal Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Immune Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Biomarkers to DiVerentiate the Vegetarian from the Nonvegetarian . . . . . . . . . . . . . 6. Summary and Recommendations for Clinical Chemistry. . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Vegetarianism is an increasingly common lifestyle choice. It is estimated that >5% of the UK adults,
2.5% of US adults, 4% of adult Canadians, 5% of Germans, and 3% of adults in The Netherlands are vegetarian, and the figure is higher for adolescent girls [1–5]. A vegetarian diet is often understood to be one that does not include food of animal, fowl, or fish origin. However, vegetarian diets range from the vegan (the most extreme form of which is the uncooked vegan ‘‘living food’’ diet), in which tissues and products of animals, fowl, and fish/seafood are strictly excluded, through the pescovegetarian, in which fish is eaten, to the lacto‐ovo‐vegetarian, in which eggs, milk, cheese, and other dairy products are eaten [1, 3, 6]. Some self‐described vegetarians eat fish or chicken, and many ‘‘semivegetarians’’ eat meat or chicken and fish occasionally. Therefore, there is no practical agreement on what the term ‘‘vegetarian’’ means. Unless otherwise specified, in this article the term vegetarian is used to describe a diet that is composed entirely of plant‐based foods. This is often referred to as a vegan diet and is followed by around 1% of the adult population in the UK and US [1, 2]. There are various reasons why a vegetarian diet is adopted and maintained, including religious requirements, cultural habits, and personal beliefs, mainly in regard to animal welfare. However, an increasing trend is seen in the number of people who follow a vegetarian diet because of its health benefits. There is a substantial and convincing body of evidence that people who eat a plant‐rich diet enjoy better health and have lower rates of many chronic, age‐related diseases, including age‐related maculopathy, cancer, dementia, diabetes, coronary artery disease, and stroke [1, 5–15]. Further, evidence supports a nutritionally adequate plant‐only diet as most beneficial to health [13, 16]. The common age‐related diseases, such as cancer, type 2 diabetes, and heart disease, are multifactorial, and the benefits of vegetarian diets in relation to these are likely to be mediated through various mechanisms [6–8, 15]. One of the ways in which plant‐based diets benefit health is believed to be owing to the high antioxidant content of plant‐based foods. High antioxidant intake may decrease oxidative changes to protein, lipid, and DNA (‘‘oxidative stress’’). Oxidation of key biomolecules is believed to be a key mechanism in ageing, and increased oxidative stress is found in all age‐related diseases [8, 12, 17]. In addition to higher antioxidant intake, vegetarians have higher potassium intake and better calcium balance than those who eat meat, and bioactive phytochemicals (‘‘nutraceuticals’’) in plant‐based foods may have anti‐inflammatory, anticarcinogenic, immunomodulating, lipid lowering, and endocrine eVects that benefit health in the long term [6, 17–23]. The benefits of vegetarianism are likely also to stem

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from what is not eaten, or is eaten is smaller amounts [15]. For example, there is no heme‐based iron in a meat‐free diet, and sodium, saturated fat, total fat, and total caloric intake are generally lower in the plant‐based diet. Carcinogenic heterocyclic amines in fried or barbequed meat are largely avoided, as is intake of meat‐derived hormones, parasites, and infectious agents. Furthermore, the gut microflora, which have a large impact on genotoxic and carcinogenic eVects of heterocyclic amines, may be diVerent in vegetarians, and it has been suggested that heterocylic amines are less carcinogenic in those whose diet is largely plant based [19, 23]. However, while the long‐term health benefits of vegetarian diets are varied and significant, these could be outweighed in the shorter term if the diet is not nutritionally adequate in terms of protein or selected amino acids or in micronutrients that are in short supply or are less bioavailable in plant‐based foods [1]. In this paper, four aspects of eVects of a long‐term vegetarian diet on health and on biomarkers (a term we use here to mean biochemical markers in body fluids or cells that reflect body status in terms of nutrition, antioxidant balance, physiological function, and pathological change) are presented and discussed. These four aspects are:  Biomarkers that reflect possible nutritional deficiency in association with long‐term vegetarian diets.  Biomarkers that reflect improved oxidant/antioxidant balance in association with vegetarian diets.  Biomarkers that reflect lower risk of disease in long‐term vegetarians.  Biomarkers that can diVerentiate the vegetarian from the nonvegetarian. Finally, some recommendations are presented in regard to how the Clinical Chemistry laboratory can advance its contribution to health assessment, as an alternative or additional paradigm to the diagnosis and monitoring of established disease.

2. Possible Nutritional Deficiencies in Association with Long‐Term Vegetarian Diets 2.1. INTRODUCTION Several nutrients (carnitine, carnosine, creatine, omega‐3 polyunsaturated fatty acids (PUFA), taurine, vitamin B12, vitamin D) are found primarily in animal tissues and have been termed ‘‘carninutrients’’ [24]. Other nutrients, such as protein and trace metals, may be less bioavailable in a plant‐based diet. Therefore, those who follow a strictly vegetarian diet for a prolonged

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period of time are at risk of specific nutritional deficiencies, most commonly of iron and vitamin B12 [1, 24–27]. In addition, in certain circumstances or age groups, for example, in pregnancy, during lactation, in childhood and adolescence, and in athletic training, there is greater demand for certain nutrients, as well as for relatively greater caloric intake [1, 27, 28]. In this section, the eVect of long‐term vegetarian diet in relation to biomarkers of nutritional status are presented and discussed briefly. 2.2. PROTEIN AND AMINO ACIDS The digestibility score of protein from plant foods such as nuts, beans, and seeds is reported to be only 76% that of protein from animal source or fish [26]. Therefore, intake of protein from plant‐based foods needs to be
25% greater than in a diet that contains meat, fish, and milk and supplying around 60 and 75 g/day, respectively, for vegetarian women and men [26, 29, 30]. Provided there is adequate protein intake, plasma albumin is maintained at normal levels. In a study by Haddad and Tanzman, the daily intake of protein of 25 vegans (average duration of plant‐only diet 4.2 years, range 1–37 years) was
20% lower than that of 20 age‐matched nonvegetarians, but the plasma albumin was significantly higher (P < 0.05) in vegans than the nonvegetarians studied; mean (SD) plasma albumin of the vegans was 49.3 (2.9) g/l and that of the nonvegetarians was 46.9 (3.8) g/l [3]. Urinary nitrogen has been used as biomarker of protein intake [31], and was found to be
20% lower in vegetarians (n ¼ 23) than in those on a mixed diet (n ¼ 34). Interestingly, in both vegetarians and nonvegetarians, daily protein intake from food records was underestimated by
14% as compared to that using urinary nitrogen excretion [31]. There are nutritionally significant diVerences in the amino acid content of animal and plant proteins [29]. Lysine intake is the major diVerence, but sulfur‐containing amino acids are low in soy protein, cassava has low threonine content, and maize has low content of tryptophan [29]. In addition, taurine (a ‘‘carninutrient’’) is found almost exclusively in animal products. Humans have limited ability to convert cysteine to taurine, and the plasma and urine of vegetarians is reportedly low in taurine [24, 32]. Low taurine status has been hypothesized to be responsible for the increased ex vivo platelet aggregability seen in vegetarians compared to omnivores [24]. There is also lower taurine content in breast milk of vegetarians, although this is not reported to lead to nutritional deficiency or health problems in the young child [33]. It is diYcult to assess amino acid requirements or status, and there are currently no routinely available biomarkers of amino acid status. Traditional methods of assessing nitrogen balance studies are regarded as unreliable.

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Plasma lysine and other amino acids can be measured, but low levels may not occur unless there is very low levels of intake or dietary protein is in the form of low digestible millet or sorghum (found in yam) [29]. However, insuYcient protein intake overall or inadequate intake of a specific amino acid may occur in two main situations in the absence of generalized malnutrition: one is in children in developing countries where the staple food is of poor protein quality; the other is in those taking a diet limited in protein, such as vegetarian diets, and who also have lowest energy requirement and whose overall caloric intake is low, such as the sedentary elderly [30]. A biomarker of interest in relation to protein status is transthyretin (prealbumin), which is rich in tryptophan [29]. Serial measurement of plasma transthyretin is reported to be a sensitive and useful marker of whole body protein nutritional status [34]. Tryptophan is an indispensable dietary‐ derived amino acid (an ‘‘essential’’ amino acid) and there is a very small reserve in higher vertebrates, therefore synthesis of tryptophan‐rich transthyretin is low when dietary supplies of tryptophan are low [34, 35]. To date there are very few studies that have measured plasma transthyretin, but this biomarker oVers the possibility to assess actual protein status and to monitor response to dietary change. An amino acid of special interest in relation to vegetarians is homocysteine. This amino acid is from endogenous synthesis, not dietary intake, and is higher in vegetarians than nonvegetarians [36]. Elevated plasma homocysteine is a risk factor for cardiovascular disease (CVD), and this creates an apparent paradox in terms of the clear benefits of a well‐balanced vegetarian diet [37]. The mechanism for increased homocysteine in vegetarians is commonly believed to be related to deficiency of vitamin B12 (as described later), however, it is suggested that the root of the problem is chronic deficiency of nitrogen and methionine (a sulfur‐containing amino acid) in the plant‐based diet [30, 35]. 2.3. ESSENTIAL FATTY ACIDS AND LONG‐CHAIN PUFAS OF OMEGA‐3 SERIES Humans cannot synthesize fatty acids with double bonds (counting from the N‐terminus) between carbons 3 and 4 (‘‘omega‐3 fatty acids’’) and between carbons 6 and 7 (‘‘omega‐6 fatty acids’’). Linoleic acid (LA) and alpha linolenic acid (ALA) are regarded as the ‘‘parent’’ essential fatty acids (EFA) of arachadonic acid (AA) an important omega‐6 PUFA (n‐6 PUFA) synthesized from LA, and eicosopentaenoic acid (EPA) and its metabolite docosahexaenoic acid (DHA), which are of the n‐3 PUFA series synthesized from ALA [38]. Endogenous synthesis of AA is eYcient and tightly regulated, while dietary input is more important for EPA and DHA as their synthesis is limited [39, 40]. These long‐chain PUFAs can be

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metabolized for energy, but are largely conserved for more specialized purposes. When incorporated into membrane phospholipids they confer the characteristics of fluidity, flexibility, and permeability, and DHA in retina and other neural tissue is needed for neurotransmission. AA and EPA are precursors of eicosanoids (the leukotrienes, thromboxanes, prostacyclins, and prostaglandins) which are important in the regulation of inflammation, blood pressure, and gene expression [38]. Eicosanoids originating from EPA often have the opposite eVect to those from AA. Meat, eggs, and fish have a high content of preformed AA, and fish oils and seafood are rich in DHA and EPA [39–41]. Intake of AA, EPA, and DHA from plant‐based foods is low, although a suitable dietary source of DHA for vegetarians is microalgae [42]. The AA status of vegetarians is reported to be similar to that of omnivores, but the DHA status is much lower, indicating that humans can synthesize AA quite well from its parent fatty acid (LA), but that DHA is poorly synthesized from its parent (ALA) [38, 43]. This view is supported by studies that show that DHA status is not appreciably improved by dietary supplementation with ALA, and that there is strong correlation (r ¼ 0.80) between dietary intake of DHA and adipose content of this omega‐3 PUFA [38, 39, 44]. The poor DHA status of vegetarians has raised concerns in relation to pregnancy and the developing fetal brain. DHA synthesis in the fetus is unclear, but there is preferential placental transfer of DHA from the maternal to the fetal circulation. Therefore, if fetal supply is compromised due to poor maternal DHA status there could be consequences in terms of intelligence and visual acuity of the child [33]. In this regard, placental and cord blood EPA and DHA levels are lower in vegetarians than omnivores, and increased intake of these long‐chain PUFA in pregnant women who follow a plant‐based diet may be advisable [45]. Furthermore, the DHA content of maternal milk is determined by dietary intake. Low omega‐3 long‐chain PUFA intake in the newborn causes a delay in neurodevelopment, and this correlates with biomarker evidence of poor DHA status [45]. Increased intake of long‐chain omega‐3 PUFA is associated with improved neonatal neurodevelopment and lower risk of CVD in later life [38]. Increased platelet aggregation is found in association with low EPA and DHA, and vegetarians are reported to show enhanced platelet aggregability [46]. Ten healthy subjects who had followed a lacto‐ovo vegetarian diet for at least 2 years had significant (P < 0.05) lowering of platelet function after 8 weeks supplementation with 700 mg/day of DHA and the same of EPA [46]. Another bioactive dietary‐derived fatty acid of interest is conjugated linoleic acid (CLA) [39, 47]. This is an isomer of LA, diVering in the position of the two double bonds. CLA is converted from LA by rumen bacteria [39]. The main dietary sources of CLA are milk‐ and fat‐rich dairy products such

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as yoghurt and butter, foods that are not eaten by vegetarians. The interest in this fatty acid stems mainly from its antitumorigenic eVects, but it also suppresses synthesis of AA and, thereby, production of proinflammatory eicosanoids of the E2 series [39, 47]. Fatty acids can be measured in plasma, saliva, erythrocyte cell membranes, platelets, and in adipose tissue using high‐performance liquid chromatography (HPLC), gas–liquid chromatography (GLC), and gas chromatography and mass spectrometry (GC‐MS) [39, 43–45, 47, 48]. For PUFA analysis, samples have to be isolated or lipids extracted and the material protected from oxidation immediately after collection. Adipose tissue reflects long‐ term dietary fatty acid profile better than other types of samples, but adipose tissue from diVerent sites gives diVerent results [39]. Furthermore, sampling of adipose tissue is invasive and not commonly performed. In summary, humans are poor synthesizers of DHA, plant foods (with the exception of microalgae) are low in DHA, and subclinical DHA deficiency in unsupplemented vegetarians and their children is likely to be quite common. Measurement of DHA, and other long‐chain PUFA, can be performed by GC or HPLC, but is complicated by postsampling oxidation and degeneration, and by the nature of the sample required (adipose tissue) to assess long‐term dietary intake. Therefore, while a biomarker of DHA status may be a useful addition to the Clinical Chemistry health assessment profile, more work is needed to develop a relatively simple, reliable, and noninvasive method. 2.4. VITAMINS 2.4.1. Introduction Preformed vitamin A (retinol) can be obtained from animal liver and fish oils, but vitamin A can be made within the human body from a dietary‐ derived precursor, b‐carotene found in orange‐colored fruits and vegetables [5, 8]. Vitamin C (ascorbic acid) and vitamin E (a group of eight tocopherols and tocotrienols the most abundant of which in human tissues is a‐tocopherol) are vitamins of plant origin [8, 49, 50]. Therefore, there is negligible risk of inadequate status of vitamins A, C, or E in association with a long‐term vegetarian diet per se. However, a plant‐based diet is low in vitamin D and one of the B group vitamins, vitamin B12 (cobalamin), and vegetarians may be deficient in these, as discussed below. 2.4.2. Vitamin B12 (cobalamin) Dietary sources of vitamin B12 are of animal origin, and vegetarians are known to be at risk of vitamin B12 deficiency [51]. If severe, deficiency of vitamin B12 can cause irreversible degeneration of the spinal cord [25].

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The biologically active fraction of vitamin B12 in plasma is known as holotranscobalamin II. In this form, the vitamin can be delivered to all nucleated cells where it plays an important role in cell division [52]. Severe deficiency of vitamin B12 causes neurological damage, but this degree of depletion occurs only in association with severely restricted diet, malabsorption, or hyperemesis gravidarum (an extreme form of pregnancy‐associated nausea and vomiting). However, marginal depletion of vitamin B12 is a well‐recognized problem in vegetarians, and is of particular concern in vegetarian pregnant women and children, whose requirements are relatively high [51–56]. Measurement of plasma total vitamin B12 (by immunoassay) can reveal overt deficiency and, using this biomarker, young healthy Taiwanese (n ¼ 37) who had been vegetarian for at least 1 year were found to have mean plasma vitamin B12 concentrations of 192 pmol/l compared to 311 pmol/l in healthy nonvegetarians of similar age and body mass (n ¼ 32) [54]. It was reported also that 52%, 29%, and 1% of those on, respectively, an entirely plant‐based diet (n ¼ 29), a lacto‐ovo‐ or lacto‐vegetarian diet (n ¼ 66) and an omnivorous diet (n ¼ 79) had low concentrations (<156 pmol/l) of plasma vitamin B12 [52]. Interestingly, 29% of those on a plant‐based diet reported using vitamin B12 supplements, and all those who did not and even some who did take supplements had low plasma vitamin B12 concentrations [52]. Measurement, by immunoassay, of plasma holotranscobalamin II, the biologically active fraction of vitamin B12, is a more sensitive and specific index of vitamin B12 status than measuring the B12 itself. At high plasma vitamin B12 concentrations, the correlation between the vitamin and holotranscobalamin II concentrations is strong (r ¼ 0.769; P < 0.001), but at low B12 concentrations the correlation with the active fraction, while still statistically significant, is much weaker (r ¼ 0.403; P < 0.05) [52]. When comparing holotranscobalamin II concentrations in groups of diVerent vitamin B12 concentrations (normal (n ¼ 69), marginal (n ¼ 20), and depleted (n ¼ 44)), the median plasma vitamin B12 concentrations in the diVerent groups were 291, 206, and 151 pmol/l, but the plasma holotranscobalamin concentrations in these groups (respectively, 58, 26, 11 pmol/l) showed a much greater relative diVerence, indicating its superiority in assessing functional B12 status [52]. Other useful biomarkers of vitamin B12 status are plasma concentrations of total homocysteine (tHcy) and methylmalonic acid (MMA) [51, 52, 57]. These biomarkers are very sensitive to vitamin B12 status, and can be taken to reflect the metabolic action of vitamin B12. Homocysteine is remethylated in vivo to methionine by methionine synthase, which requires vitamin B12 as coenzyme and 5‐methyl‐tetrahydrofolate as a methyl donor [52, 58]. In vitamin B12 deficiency, homocysteine accumulates. Furthermore, a phenomenon referred to as the ‘‘folate trap’’ occurs, in which folate (of which

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there is an abundance in the vegetarian diet) is trapped as 5‐methyl‐ tetrahydrofolate. This means that the normal or high plasma folate concentrations seen in vegetarians may not reflect accurately an adequate intracellular suYciency of ‘‘active’’ folate [52]. Marginal depletion of active vitamin B12 is associated with, firstly, reduced plasma concentration of holotranscobalamin II, and then functional changes manifested as increases in plasma MMA and tHcy and, ultimately, the advanced deficiency state characterized by megaloblastic anemia, DNA hypomethylation, and disturbed neurotransmission [52]. Such overt clinical deficiency is rare. However, chronic subclinical deficiency, and low plasma holotranscobalamin II and increased plasma MMA and tHcy concentrations are common in vegetarians [56]. Measurement of plasma vitamin B12 alone may not reveal marginal deficiency, and biomarker assessment of functional vitamin B12 status should include measurement of holotranscobalamin II and tHcy and, if possible MMA [59]. Detection of subclinical deficiency is important. Elevated tHcy is an independent risk factor for coronary heart disease (CHD) [37]. Therefore, chronic, marginal vitamin B12 deficiency may significantly increase CVD risk through its eVect on homocysteine, and this could oVset some of the reduction in other CVD risk factors, such as blood pressure, inflammation, and lipids (see Section 4). Elevated plasma tHcy concentrations in vegetarians can be normalized by vitamin B12 administration [46]. However, response to oral supplementation may be poor. It is noted also that, while therapy to decrease severe hyperhomocysteinaemia leads to clear benefit in terms of adverse vascular events, the clinical benefit of lowering mildly elevated homocysteine is not clear [37]. Homocysteine is elevated in renal dysfunction, and recent evidence indicates that the epidemiological association between mild elevations of homocysteine and CVD may be mediated by renal dysfunction, not homocysteine itself [37]. Nonetheless, a sensitive biomarker of functional vitamin B12 status, such as holotranscobalamin II or MMA, would be useful item on the Clinical Chemistry test menu for nutritional and health assessment in those who adhere to a vegetarian diet. 2.4.3. Vitamin D There is increasing awareness that vitamin D is crucial to health and to the function of many body systems through both endocrine ‘‘calciotropic’’ eVects and autocrine eVects on cell proliferation and diVerentiation [60]. Through these combined eVects, vitamin D plays an important role in the musculoskeletal, immune, reproductive, and cardiovascular systems, and in the prevention of cancer, and is important also in host defense against tuberculosis [60, 61].

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The active form of the vitamin is 1,25‐dihydroxycholecalciferol (1,25‐ dihydroxy D; 1,25‐DHD), which is formed by hydroxylation of a precursor, 25‐hydroxyD (25(OH)D), by the action of a mitochondrial enzyme [60]. Activation of 25(OH)D to 1,25‐DHD is well known to occur in renal tissue but occurs in other tissues also, where local autocrine eVects may be more pronounced [60]. There are two sources of 25(OH)D. One is skin, where the action of ultraviolet blue light from sunlight converts cholecalciferol (7‐hydrocholesterol; D3) to 25(OH)D by cleavage of a steroid ring [62]. The amount synthesized by this route depends upon the degree of exposure to sunlight and the degree of pigmentation of the skin. Food is the other source of 25(OH)D, and the main sources are oily fish, meat, and fortified foods, with milk and margarine being the main fortified food sources [62, 63]. The minimum daily dietary requirement of 25(OH)D is diYcult to assess because of the unknown and variable contributions from skin. Currently, the recommended intake from birth to 50 years is 200 IU/day (5 mg/day), with a doubling of this recommended for those aged 51–70 years, and 600 IU/day (15 mg/day) recommended for those aged 70 years and over [60]. Provided skin is exposed to UVB at times, and fish, meat, and fortified foods feature regularly in the diet, overt vitamin D deficiency is unlikely to develop. However, a vegetarian who eats no meat, fish, or dairy products and who has low exposure to sunlight (either because of geographical location or cultural habits) may develop 25(OH)D deficiency. While the combined daily supply from intake and skin synthesis is diYcult to assess, plasma concentrations of 25(OH)D can be measured by radioimmunoassay and by liquid chromatography‐tandem mass spectrometry (LC‐MS) [64]. Plasma 25(OH)D concentrations of >20 nmol/l are adequate to prevent rickets, but a conservative cutoV of 37.5 nmol/l has been suggested for vitamin D suYciency, and concentrations of 80 nmol/l or higher may be needed to support both the endocrine and autocrine functions of 25(OH)D [60, 63, 65]. Even the lower level of adequacy may be diYcult to attain in people with a restricted diet, the elderly and those who have severely limited exposure to sunlight [62, 66, 67]. Such severe deficiency is likely to aVect not only calcium balance and bone health, but increase risk of other diseases. For example, a study of 210 tuberculosis patients from diVerent ethnic and religious groups in the UK [61] showed that >75% had low (<22 nmol/l) or undetectable (<13 nmol/l) plasma concentrations of 25(OH)D, and the great majority of these patients were Indian, Pakistani, Afghanistani, or Somalian. Many of these ethnicities follow a habitual vegetarian diet. However, skin pigmentation is also an important determinant of vitamin D status. Lower serum 25(OH)D concentrations were found in black American women compared to white American women with similar vitamin D intakes [66]. At low intake, black American women had
50% lower serum 25(OH)D

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concentrations (
40 vs.
80 nmol/l) than white American women. This diVerence presumably reflects diVerences in cutaneous synthesis, and highlights the importance of this in maintaining vitamin D status. Interestingly, young Canadian women taking the recommended 200 IU/day (5 mg/day) cannot maintain their plasma 25(OH)D concentrations above threshold during winter when sunlight for cutaneous synthesis is lacking [67]. Interestingly also, even in women who took supplements of >400 IU/day (10 mg/day), black American women had
25% lower serum 25(OH)D concentrations, and it was only in those black American women who took >400 IU/day in supplemental vitamin D that serum concentration approached the suggested desirable threshold of 80 nmol/l [66]. Even those black women who took three or more servings of fortified food per week had serum concentrations of 25(OH)D that fell significantly short of this threshold, averaging only
50 nmol/l [66]. This suggests that certain groups, such as those with deeply pigmented skin, or in those who are deprived of sunlight, may require even higher doses of dietary or supplemental vitamin D to attain the desirable threshold because of low cutaneous synthesis. For those vegetarians with dark skin and who for cultural or geographical reasons have low exposure of their skin to sunlight, the risk of vitamin D deficiency may be particularly high. When serum 25(OH)D concentrations of UK tuberculosis patients were analyzed by religion (as a possible surrogate for dietary custom), significantly lower (P < 0.01) plasma concentrations were found in Hindu patients, with high odds of having undetectable 25(OH)D in plasma [61]. While lack of exposure to sunlight plays its part, the problem is undoubtedly exacerbated by the vegetarian Hindu diet, which is characterized by both a low 25(OH)D content and a high content of phytate, which increases vitamin D catabolism [68]. The combination of low cutaneous synthesis and inadequate intake of 25(OH)D is a scenario that is likely to arise in many long‐term vegetarians in northern latitudes, at least in winter. However, the prevalence and severity overall in vegetarians or the population at large is unknown because currently measurement of plasma 25(OH)D is not performed as a routine biomarker of health assessment in the Clinical Chemistry test menu.

2.5. MINERALS AND TRACE METALS 2.5.1. Iron Vegetarian diets can contain as much or even more dietary iron that nonvegetarian diets, however, the bioavailability of iron in plant‐based diets is less than from meat‐based diets. Absorption of iron from vegetarian diet is estimated at 10%, compared to 18% from a mixed diet [69]. The lower

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absorption is due mainly to the form of iron and to the high intake of phytate in plant‐based diets [70]. Heme‐iron, which is found in meat, poultry, and fish, is better absorbed than the nonheme form of iron found in plants, although nonheme iron absorption is better regulated [70, 71]. Other dietary components also aVect iron absorption, including polyphenols and soy protein, which inhibit absorption of nonheme iron, and ascorbic acid (vitamin C) and carotenes, which enhance absorption [70]. Vegetarian diets are likely to be rich in both enhancers and inhibitors of absorption of nonheme iron. Nonetheless, the enhancing eVect is likely to be more than oVset by the inhibiting eVect of high consumption of inhibitors and the low solubility of nonheme iron. Long‐term vegetarians, particularly women, have lower iron stores, and may also have lower hemoglobin concentrations. A study from UK reported that 30% of vegetarian girls (up to 18 years of age) had low hemoglobin concentration [72]. No distinction was made between those who were long‐ term vegetarians and those who had recently adopted that lifestyle. In teenage girls, individual (as opposed to a family based) lifestyle change involving vegetarianism may well be associated with poor diet overall, and it is of interest that this study also showed that those with higher vitamin C intake (which can be used as a surrogate for fruit and vegetable intake and a healthy diet) had higher hemoglobin concentrations [72]. Interestingly also, the prevalence of low iron status (
20%) was found to be similar in vegan and omnivore females of average age 17 years, and it was suggested that low iron status was more of a ‘‘female rather than a vegan problem’’ [73]. However, low iron status does not equate to iron deficiency, and long‐term vegetarians in Western countries do not in general have higher prevalence of iron‐deficiency anemia than omnivores [70]. Iron deficiency anemia is more common in Chinese and Indian female vegetarians. However, this may be related also to the characteristic phytate‐rich diets and perhaps to lower quantity of food taken by these groups. Assessing iron stores well is not simple [74]. Serum iron concentration can be measured, but is not a reliable index of iron status as serum iron shows very high intraindividual variation and samples are easily contaminated during collection and processing. Low hemoglobin concentration is not itself specific for iron deficiency, and iron‐deficiency anemia is a late manifestation of chronically inadequate iron intake. Other, more sensitive, biomarkers of iron status exist, including serum ferritin concentration, degree of saturation of transferrin in serum, the total iron binding capacity (TIBC) of serum, and the serum concentration of soluble transferrin receptor fragments (sTfr) [71, 74, 75]. Generally, several biomarkers are measured in assessing iron status. Serum ferritin concentration, measured by immunoassay, is directly related to body iron stores, and low concentration in association with low

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hemoglobin denotes iron‐deficiency anemia. Bone marrow can also be tested for stainable iron, but this is not a routine procedure [75]. Vegetarians have been reported to have lower concentrations of serum ferritin than meat eaters, although as noted most studies do not show increased prevalence of iron deficient anemia in vegetarians [70, 71]. It is noted also that ferritin is an acute phase protein, which aVects its reliability in assessing iron stores when inflammation or infection is present [71]. The sTfr concentration can be measured by immunoassay, and its concentration increases as iron stores decrease. This biomarker is not aVected by acute phase response, making it more specifically related to iron status. Concentrations of sTfr are reported to correlate with mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) better than do bone marrow iron stores, and sTfr is currently the most useful biomarker of iron deficient erythropoiesis [74, 75]. However, at this time there is no generally accepted reference method for measuring sTfr, and there is no consensus on cut oV values [74]. While many vegetarians may have lower iron stores than meat eaters, low iron stores without low hemoglobin have not been associated to date with obvious deleterious eVects on health. Indeed, low iron stores have been suggested to protective against oxidative stress by decreasing the chance of Fenton chemistry producing highly reactive oxygen species (ROS) in vivo [17]. In epidemiological study, high iron stores have been reported to be associated with increased risk of CHD and cancer [76, 77]. In addition, low iron status, whether induced by a lacto‐ovo vegetarian diet or by phlebotomy, is associated with enhanced insulin sensitivity and glucose disposal, independent of body mass index (BMI) [78, 79]. This finding is supported by a Taiwanese study of 49 long‐term vegetarians (mainly Buddhist nuns whose diet contains no animal or fish products) and 49 age‐matched omnivores [80]. Iron status was not measured, but all biomarkers of insulin sensitivity measured (fasting plasma insulin, glucose, insulin:glucagon ratio) as well as the calculated homeostasis model assessment‐insulin resistance (HOMA‐IR) were significantly (P < 0.001) lower in the vegetarian group [80]. The mechanism of the suggested insulin sensitizing eVect of decreased iron stores is unknown, but has clear implications for a novel treatment—or even preventive—approach for diabetes. This could be a rewarding area of research into possible benefits of low iron stores. However, poor iron status without anemia has been reported to aVect aerobic adaptation to exercise [81] and poor iron status may increase susceptibility to toxicity of lead or other heavy metals [70, 82]. In summary, long‐term vegetarians have lower iron stores than omnivores due to a combination of ingestion of poorly soluble nonheme iron from plant foods and high intake of inhibitors of iron absorption. To date, low iron

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stores without low hemoglobin concentration does not appear to present a problem for health under normal circumstances, although there may be increased susceptibility to heavy metal exposure. Indeed, there may be health benefits to having low, but adequate, iron stores in terms of improved insulin sensitivity and decreased oxidative stress. Biomarkers of body iron stores are becoming more sensitive and reliable with the increased availability of sTfr measurements, as well as ferritin and the more ‘‘traditional’’ but less reliable measurements of serum iron and TIBC. Using the modern day biomarker panel for iron status along with novel, validated biomarkers of oxidative stress and insulin sensitivity allows further study of mechanisms by which low iron status per se and vegetarian diets overall help determine long‐term health. 2.5.2. Zinc Zinc is an essential trace mineral that is incorporated into various enzymes and therefore needed for normal growth and metabolic control [83]. As is the case with iron, zinc content and bioavailability of plant‐based diets are likely to be lower than in meat‐based diets, although the form of zinc is not diVerent in diVerent foods [70, 84]. Legumes, whole grains, nuts, and seeds have high content of zinc, but also have high content phytate, a zinc (and iron) absorption inhibiting agent. However, unlike iron, severe deficiency of which leads to anemia, there is no clear clinical outcome of zinc deficiency, although it is suggested to lead to poor wound healing, loss of sense of smell and taste, increased susceptibility to oxidative stress, stunting of growth, depressed immune function, and increased risk and severity of infection [83, 85]. The zinc status of long‐term vegetarians is likely to be lower than that of omnivores, but there is no accepted biomarker of body zinc status. Zinc can be measured in plasma, urine, saliva, and hair by atomic flame spectroscopy. Vegetarian diets are reported to be associated with a decrease in plasma zinc in the few weeks following the change from a meat‐based diet, but the concentration plateaus at a lower but normal value [70]. Lower hair and salivary zinc levels are reported in vegetarians, but the relationship between this and body zinc status is unclear. Furthermore, plasma zinc concentrations are aVected by stress, infection, and other factors [85]. In studies comparing omnivores and vegetarians, no significant diVerences in serum zinc were found, despite lower zinc intake by the vegetarians in both studies [83, 86]. Interestingly, severe zinc depletion caused decreased plasma zinc concentration and decreased urinary excretion of zinc, along with decreased plasma ALP activity, but these biomarkers did not change with marginal zinc depletion [85]. Therefore, while long‐term vegetarians may have marginally lower zinc status than meat eaters, zinc status cannot be reliably assessed at this time, and long‐term health eVects of lower zinc status are not known.

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2.5.3. Copper Vegetarian diets contain more copper than nonvegetarian diets, so that even though the relative absorption is less in the vegetarian diet (33% in a study of lacto‐ovo‐vegetarians compared to 42% in a nonvegetarian diet using stable copper isotope absorption) the total amount of dietary copper absorbed is higher [87]. Plasma concentrations of copper (measured using atomic absorption spectroscopy) and the copper‐containing protein caeruloplasmin (measured by a colorimetric method or by immunoassay) have been compared in nonvegetarians and lacto‐ovo‐vegetarians, but no diVerences were found [87]. No changes in these putative biomarkers of copper status were seen in a study of a wide range of controlled copper intake (15–150 mg/kg of body weight for 2 months) [85, 88]. Nor was there any eVect of copper intake on erythrocyte or white cell copper content, and the intracellular content did not correlate with serum copper of caeruloplasmin concentrations [88]. Therefore, as is the case with zinc, there is no reliable biomarker of copper status at this time, and the eVect of long‐term vegetarian diet on copper status is not known. 2.5.4. Calcium The calcium content of plant‐based diets can be low, though is not necessarily so as some plants, for example, broccoli, are high in calcium. If intake is low, increased secretion of parathyroid hormone (PTH) stimulated by negative calcium balance increases calcium absorption by up to 40% and decreases renal loss. These homeostatic mechanisms are eVective, and poor calcium balance is not a general feature of a vegetarian diet [1, 26]. 2.5.5. Iodine Seafood is naturally rich in iodine, and meat, dairy products, and eggs are also good sources when animal fodder is supplemented with iodine [89]. However, vegetarians, especially those who live in an area where the soil is deficient in iodine, are at risk of developing iodine deficiency as their iodine intake is very limited [90, 91]. Table salt is iodinized in many countries, but health conscious vegetarians may avoid adding salt to food, and so may miss this means of adding to their dietary intake of iodine. The issue of iodine deficiency and its eVects on thyroid hormone balance is a public health concern because pregnant and lactating vegetarian women may have insuYcient iodine intake to meet the needs of the fetus and breast‐fed baby [92]. In a review of studies of thyroid function in vegetarians [92], plasma thyroid‐ stimulating hormone (TSH) and T4 were normal in two studies, but one showed high TSH in the plasma of 5 of 48 vegetarians. However, it was noted that highest TSH values were found in individuals who usually took

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iodine‐rich kelp (seaweed). Plasma and urine iodine concentrations were not measured in these subjects but were likely to be high, and this may have caused an iodine‐induced depression of thyroid function, with concomitant increase in TSH secretion [92]. Measurement of urinary iodine excretion has been used a biomarker for iodine deficiency [93], and results show very wide variation in vegetarians. Therefore, while there is a need for more awareness that vegetarians are at risk of iodine deficiency, there is also a need to increase awareness that use of iodine‐containing dietary supplements, such as kelp, may result in excessive iodine intake. Both severe deficiency and excess intake of iodine can result in hypothyroidism, and this can have serious consequences, particularly for the developing and growing child. However, the long‐term eVects of a habitual diet that is marginally low in iodine are not known at this time.

3. Biomarkers of Oxidant/Antioxidant Balance in Association with Vegetarian Diets 3.1. INTRODUCTION: CONCEPTS OF ANTIOXIDANTS AND OXIDATIVE STRESS AND SUGGESTED ROLE OF DIETARY ANTIOXIDANTS IN HEALTH PROMOTION ROS are partially reduced species of molecular oxygen, and include superoxide, hydrogen peroxide, hydroxyl radical, peroxyl radical, nitric oxide, peroxynitrite, and hypochlorous acid [17]. ROS are ubiquitous and unavoidable within the human body, some being produced for a purpose, such as intracellular communication, microbial killing, or control of blood pressure, while others are produced as by‐products of aerobic respiration, during inflammation or through detoxification of xenobiotics. Additional sources of ROS include air pollution, cigarette smoking, and ultraviolet or X‐ray irradiation [17]. ROS cause oxidative damage to protein, lipid, and DNA (‘‘oxidative stress’’) and such damage is found in all of the major age‐related diseases and is believed to be a key player in the ageing process itself [12, 17]. A direct causal link between oxidative stress and disease has not been confirmed but, conceptually at least, increased antioxidant defense will oppose oxidative damage, minimize oxidative stress, and promote healthy ageing. ROS are many and varied, and a diverse and interactive array of antioxidants is needed to remove, inactivate, or destroy them. The antioxidants of the human body are of two types, endogenous, which include the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), metal binding proteins such as transferrin, and the small molecular weight scavengers or reducing antioxidants glutathione and uric acid [17, 49]. There

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are also antioxidants within the human body that are exclusively of dietary origin. The major dietary‐derived antioxidants are the water‐soluble vitamin C (ascorbic acid) and the lipid‐soluble vitamin E, which is a family of eight isomers, and the most abundant of which in human plasma and membranes is a‐tocopherol [8, 17, 49, 50, 94–98]. The human body cannot synthesize vitamin C or vitamin E, and regular, adequate intake is required to avoid deficiency [50, 99–102]. While supplementation studies have not yielded expected benefits [103, 104], diets rich in these antioxidants do benefit health [7–11, 94, 95, 105–108]. ‘‘Optimal intake’’ of antioxidant vitamins, as opposed to adequate intake to avoid simple deficiency, is now a concept advanced for health maintenance and promotion, although what is ‘‘optimal’’ is not yet agreed, and the significance of other dietary antioxidants, such as polyphenols is still not established [8, 50, 95, 100, 102, 106, 109]. Nonetheless, because of the clear health benefits of diets rich in vitamins C and E, the US recommended daily intake (RDI) was revised upwards in 2000, from 30 mg/day vitamin C to 75 mg/day for nonpregnant and 100 mg/day for men and pregnant women [95, 102]. The RDI for vitamin E was also increased to 15 mg/day, though most people do not meet this [50]. The major source of vitamins C and E is plant‐based food [8, 50, 96, 101]. Plants synthesize these antioxidants for the defense and growth of their own cells [96]. Plants also synthesize a huge range of other antioxidant compounds, mainly of the carotenoid and flavonoid families [8, 17–29, 96, 109]. It is these that give fruits and vegetables their colors of blue, purple, red, orange, and yellow and that, possibly, confer their benefits to human health. It is worth noting here that results of primary and secondary disease prevention trials with ‘‘pure’’ antioxidant supplementation have not shown the expected benefits, and supplementation with some antioxidants may increase mortality [103, 104]. In meta‐analysis, vitamin C supplements appear to have no eVect on mortality, while b‐carotene and vitamin E supplementation were shown to increase relative risk of mortality by, respectively, 7% and 4% (P < 0.05) [103]. Nonetheless, while antioxidant supplementation trials have produced no evidence of benefit overall, apparently healthy people who have high concentrations of ascorbic acid in their fasting plasma have significantly less mortality, cancer, heart disease, and stroke in the ensuing years than apparently healthy people with lower ascorbic acid concentrations [11, 94, 106–110]. In a study of almost 3000 apparently healthy subjects in the UK who were followed up for an average of 6 years [11], those in the highest quartile of plasma ascorbic acid at baseline had an Odds Ratio (OD) for future coronary artery disease of 0.67 (95% confidence intervals (CI) 0.52, 0.87) (Fig. 1). This relationship between plasma ascorbic acid and future coronary events was independent of sex, age, diabetes, smoking, BMI, lipids, blood pressure, and hsCRP, and raises the interesting possibility that plasma

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1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Quartile Mean (SD)

1st

2nd

3rd

4th

27.6(9.7)

47.3(3.2)

58.5(3.7)

77.1(11.5)

Plasma ascorbic acid (mmol/l) FIG. 1. Risk of future coronary artery disease in 2773 subjects by quartile of plasma ascorbic acid at entry: dotted line ¼ unadjusted OR; solid line ¼ OR adjusted for smoking, diabetes, BMI, blood pressure, lipids, and hsCRP. Follow‐up period averaged 6 years (data from Ref. [11]).

ascorbic acid concentration may be a useful additional biomarker of cardiovascular risk. It is noted that, while higher plasma ascorbic acid concentrations indicate higher dietary intake of vitamin C, lower mortality and disease risk may not be driven directly by ascorbic acid. It is possible that ascorbic acid may be a coincidental cotraveller with other, as yet unknown but key constituent(s) of plant foods. This could account for the paradoxical findings that vitamin C supplements do not appear to benefit health (unless a deficiency was corrected) but that diets rich in vitamin C clearly do. Antioxidant intake in a vegetarian diet is high, but vegetarians do not necessarily have higher or more diverse antioxidant intake than nonvegetarians who follow general dietary recommendations to eat an abundance fresh fruit and vegetables, and who eat chocolate, or drink tea, coVee, and red wine. All of these dietary agents are very rich in antioxidants, some with high vitamin C content, others high in flavonoids, some rich in carotenoids or other antioxidants [8, 96, 111–115]. Health eVects may be related to one or more of these, or to antioxidant cooperation and synergy in vivo [97, 98]. Plasma vitamin C concentration is clearly related to intake of fruits and vegetables [116], but does always significantly correlate with estimated intake of vitamin C, as relative absorption decreases with dose, and plasma concentration is influenced also by turnover rate and, possibly, by its

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60.00

#

Change in FRAP (mM)

40.00

#

20.00 0.00 −20.00

0

45

90

150

*

−40.00 −60.00 −80.00

Min (post ingestion)

FIG. 2. Postingestion changes in plasma antioxidant capacity of healthy adults (n ¼ 6) and expressed as the FRAP value, after intake of 300 ml of coVee (triangles), cocoa (squares), and cow’s milk (as control; circle) in controlled cross‐over study. Results are mean  SEM; *significantly diVerent from baseline (P < 0.05); #significantly diVerent from time‐matched result after control (P < 0.05).

recycling of other antioxidants such as vitamin E in vivo [101, 102, 117–119]. Furthermore, the bioavailability of plant‐based phenolic antioxidants is poor, so that even if intake is high, plasma levels are very low (<1 mmol/l) and their bioactivity and physiological role are still not clear [8, 109]. Nonetheless, high intake of antioxidants from plant‐based foods might be expected to enhance antioxidant status and decrease oxidative stress, and there is evidence that plasma ‘‘total antioxidant capacity’’ increases after ingestion of antioxidant‐rich foods and beverages, including tea, wine, chocolate, and herbs [113, 120–123] (Fig. 2). It is noted that some of this increase may be due to an increase in plasma uric acid after ingestion of fructose‐ containing foods (fruits), and this should be corrected for [124, 125]. CoVee is reportedly the major contributor (>60%) to total dietary antioxidant intake, but total ‘‘noncoVee’’ antioxidant intake correlates with plasma concentrations of the carotenoids lutein and b carotene (P < 0.01; n ¼ 2672) [126]. In this section, the relatively few studies that have assessed biomarkers of antioxidant status and oxidative stress in vegetarians and nonvegetarians will be reviewed briefly. It is noted that currently, measurement of antioxidant status and oxidative stress biomarkers is not performed in the routine Clinical Chemistry laboratory. The most commonly used biomarkers of antioxidant status in research studies are plasma ascorbic acid (by HPLC or a specific enzyme‐linked colorimetric assay referred to as ‘‘FRASC’’)

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[127, 128], plasma a‐tocopherol (by HPLC) [50] and the plasma ‘‘total antioxidant capacity.’’ There are several methods available for measuring total antioxidant capacity of plasma (and other biological fluids and foods), but a relatively simple, robust and widely used test is the ferric‐reducing/ antioxidant power (FRAP) assay [127, 129, 130]. Plasma carotenoids and flavonoids can be measured by HPLC [126, 131]. Plasma uric acid can be measured by commercial test kits employing uricase, but the relevance of uric acid in antioxidant defense is still not clear [125]. It is not currently measured as part of an antioxidant status biomarker panel, but perhaps should be to correct the total antioxidant capacity for the contribution of uric acid [124, 125, 127]. Commonly used biomarkers of oxidative stress include plasma malondialdehyde (MDA, by HPLC), which is a lipid peroxidation product, and oxidative damage to DNA in lymphocytes (usually as single strand breaks measured using the comet assay) [17, 132, 133]. Less widely used biomarkers of oxidative stress include plasma allantoin (by HPLC), nitrotyrosine (by immunoassay), plasma and urine F2 isoprostanes (by immunoassay or LC‐MS), and urine 8‐hydroxy‐2 deoxyguanosine (8‐OHdG, by immunoassay or LC‐MS) [17, 134–136]. However, to date there are no published data on most of these biomarkers in relation to long‐term vegetarian diets. In addition, we find that healthy subjects have undetectable nitrotyrosine concentrations in plasma, at least when measured by immunoassay (our unpublished results). 3.2. BIOMARKERS OF ANTIOXIDANT STATUS AND OXIDATIVE STRESS VEGETARIANS AND NONVEGETARIANS: IMPLICATIONS FOR HEALTH

IN

As reviewed by Rauma and Mykka¨nen, vegetarians have higher plasma concentrations of vitamin C, vitamin E, and carotenoids than omnivores [137]. Haldar et al. reported on the antioxidant status in healthy nonsmoking vegetarians and omnivores aged 18–64 years [138]. Most of the 31 ‘‘vegetarians’’ reported eating meat, chicken, or fish, though rarely (no more than six times per year), and only 6 completely excluded animal flesh, fish, eggs, and dairy products from their diet. Antioxidant supplements were used by 13 of the 31 vegetarians and by 11 of the 58 omnivores. All subjects had followed their selected diet for at least 3 years. The age, BMI, total energy intake, total fruit intake, zinc, and folate intakes were not significantly diVerent between the ‘‘vegetarian’’ group (n ¼ 31) and the omnivore group (n ¼ 58). No data were shown for the true vegetarian (‘‘vegan’’ group) alone. When antioxidant supplement users were excluded, few significant diVerences were seen between the groups (Table 1). However, plasma levels of the dietary‐derived carotenoids (which include lutein, cryptoxanthins, and lycopene, as well as a‐ and b‐carotene) and ascorbic acid were higher in the

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BIOMARKERS IN LONG‐TERM VEGETARIAN DIETS TABLE 1 ANTIOXIDANT STATUS OF VEGETARIANS AND OMNIVORES (NONE TAKING ANTIOXIDANT SUPPLEMENTS); RESULTS ARE MEAN (SD)

Erythrocyte GPxˆ (U/g Hb) Erythrocyte SODˆ (U/g Hb) Erythrocyte glutathione (GSH)ˆ (mmol/g Hb) Plasma a‐tocopherol# (mmol/l) Plasma g‐tocopherol# (mmol/l) Plasma lutein# (mmol/l) Plasma a‐cryptoxanthin# (mmol/l) Plasma b‐cryptroxanthin# (mmol/l) Plasma lycopene# (mmol/l) Plasma a‐carotene# (mmol/l) Plasma b‐carotene# (mmol/l) Plasma ascorbic acid# (mmol/l) Plasma uric acidˆ (mmol/l) Plasma total antioxidant capacity (as FRAP value) (mmol/l) Plasma nonuric acid FRAP (mmol/l)

‘‘Vegetarian’’ group (n ¼ 17)

Omnivore group (n ¼ 47)

61 (11) 1409 (175) 5.6 (1.0) 38 (8) 2.2 (0.8) 0.50 (0.19)* 0.14 (0.06)* 0.29 (0.12) 1.24 (0.37) 0.26 (0.22) 0.95 (0.61) 76 (24) 262 (58)* 1076 (137)*

69 (16) 1368 (220) 5.3 (0.9) 38 (8) 2.4 (1.0) 0.41 (0.18) 0.11 (0.05) 0.31 (0.28) 1.07 (0.41) 0.23 (0.12) 0.79 (0.46) 69 (27) 292 (65) 1153 (173)

552 (57)

566 (77)

‘‘Vegetarian’’ group contained subjects who sometimes ate meat, chicken, or fish (data from Ref. [138]). # Exclusively derived from the diet, and main sources are plant‐based foods; ˆ endogenous antioxidants; * significant diVerence between the groups (P  0.05).

vegetarian group [138], consistent with their higher intake of plant‐based foods, which are rich in these antioxidant compounds [8, 96, 116]. In a study by Sˇebekova´ et al., oxidative stress biomarkers were measured in 90 healthy lacto‐ovo‐vegetarians and 46 of their omnivorous family members [139]. Apart from plasma b‐carotene, which was significantly (P < 0.01) higher in the vegetarians, antioxidant status was similar in the two groups. The lipid peroxidation marker measured (plasma MDA) was no diVerent across the groups, but vegetarians had higher (P < 0.01) advanced oxidation protein products in plasma. The authors suggested these originated from increased phagocyte activation in the vegetarians [139]. However, leukocyte number and C‐reactive protein (CRP) concentrations were no diVerent in the vegetarians, and the relevance of the increase in the biomarker of phagocyte activation‐related oxidative stress is unclear. Manjari et al. compared plasma MDA and erythrocyte SOD and GPx in healthy vegetarians (10 women and 20 men) and nonvegetarians (14 women and 30 men) in southern India [43]. Results showed significantly (P < 0.05)

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lower plasma MDA in both male and female vegetarians compared to their nonvegetarian counterparts. Erythrocyte SOD was significantly (P < 0.05) higher in vegetarian women compared to nonvegetarian women, but there were no significant diVerences in SOD between male vegetarians and nonvegetarians, whose values were similar to those of nonvegetarian women. No diVerences were seen in erythrocyte GPx in the diVerent dietary groups for either gender. Oxidative stress may increase with age, although whether this is due to ageing itself, to a poorer diet in the elderly, or is a sign of undiagnosed disease in older subjects is not clear. A vegetarian diet may prevent age‐related increases in oxidative stress, as reported in a Slovakian study of young (20–30 years) and old (60–70 years) ovo‐lacto‐vegetarian and nonvegetarian women [140]. No diVerences in oxidative stress biomarkers were seen across the younger age dietary groups, but in the 60–70 years age groups, vegetarians (n ¼ 33) showed lower oxidative stress and higher antioxidant status than the age‐matched nonvegetarians (n ¼ 34) [140]. DNA strand breaks caused by presence of oxidized purines or pyrimidines, and fatty acid conjugated dienes were measured as markers of oxidative stress and were significantly lower (P < 0.05) in the old vegetarian women compared to old nonvegetarian women. Furthermore, unlike the nonvegetarians, the old vegetarian women did not show higher oxidative stress compared to younger vegetarian group. Protein carbonyls, another biomarker of oxidative stress, were also lower in the old vegetarians compared to the old nonvegetarians, but the diVerence did not reach statistical significance. Plasma vitamin C and b‐carotene concentrations were higher (P < 0.01) in the old vegetarians. Plasma vitamin E was slightly lower in the vegetarians, but it is noted that the total, rather than the lipid standardized, vitamin E was reported [140]. Vitamin E is carried in the plasma within lipoproteins, and unless reported corrected for cholesterol and triglycerides, diVerences in lipid concentrations confound results [141]. Szeto et al. also found lower vitamin E in association with a long‐term vegetarian diet in a study that compared biomarkers of antioxidant status in 30 long‐term (5–55 years duration) Chinese vegetarians and 30 age‐ and sex‐ matched Chinese nonvegetarians in Hong Kong [142]. The vegetarians studied followed the Taoist religion, which is related to Buddhism, and they ate no flesh, but occasionally some took eggs or milk in small amounts. Results showed that the vegetarians had fasting plasma ascorbic acid concentration
50% higher than the nonvegetarians (mean (SD) 91 (21.0) mmol/l and 62 (17) mmol/l, respectively; P < 0.01) [142]. However, the lipid standardized a‐tocopherol concentration was significantly lower in the vegetarians: mean (SD) 3.76 (0.57) mmol/mmol total cholesterol (TC) plus triglycerides compared to 4.23 (0.58) mmol/mmol in the nonvegetarian group (P < 0.01) [142].

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The reasons for lower vitamin E may be due to lower intake of the vitamin, as most Taoists eat no nuts or seeds, to less vitamin E absorption due to poor zinc status (most dietary zinc is from meat), or to high phytic acid intake. The plasma concentration of MDA was slightly but not significantly lower in the vegetarians [142]. The plasma total antioxidant capacity (as the FRAP value [127, 129] was not significantly diVerent between the groups (mean (SD) 1028 (180) and 1040 (170) mmol/l in vegetarians and nonvegetarians, respectively), but the contribution of ascorbic acid to this was markedly greater in the vegetarians [142]. This was partly due to their higher ascorbic acid concentration, but also because vegetarians had lower plasma uric acid than nonvegetarians (mean (SD) 239 (88) and 306 (68) mmol/l, respectively; P < 0.01) [142]. Uric acid is regarded as an endogenous antioxidant, but is also an independent risk factor for CHD, and lower uric acid may be a sign of improved CHD risk [125]. Interestingly, hsCRP, a ‘‘predictor’’ biomarker for CHD [11], was significantly (P < 0.01) lower in the vegetarian group, and significant (P < 0.05) inverse correlations were seen overall between ascorbic acid and hsCRP, and between ascorbic acid and uric acid [142]. This supports a cardioprotective role for vitamin C or something very closely related to it in the diet, and supports the suggestion that plasma ascorbic acid measurement may be useful in assessing health status. This requires further study, but the compelling evidence from prospective observational trials that plasma ascorbic acid concentration of apparently healthy subjects is related to subsequent health status and mortality [9, 12, 104, 107] provides a strong rationale for such study, and suggests that it may be timely to consider the inclusion of plasma ascorbic acid measurement as a routine test in Clinical Chemistry laboratories as part of a health assessment biomarker profile. Oxidative DNA damage, using the comet assay [132], was found to be significantly lower (P < 0.010) in long‐term (5–26 years duration) vegetarians (n ¼ 24) compared to nonvegetarians (n ¼ 24) [143]. Both single strand breaks and oxidized purines in lymphocytic DNA were lower in the Slovakian vegetarians studied, but other markers of genomic stability were not diVerent between the groups [143, 144]. In summary, vegetarians have higher ascorbic acid status than omnivores, but vitamin E status is more variable and can be low. Plasma ‘‘total’’ antioxidant capacity is not necessarily higher in vegetarians, but the relative contributions to this by ascorbic acid and uric acid are diVerent (higher and lower, respectively). It has been suggested that the plasma nonuric acid FRAP value (i.e., the total antioxidant capacity corrected for the contribution of uric acid) may be a more useful index of antioxidant status than the total value [124, 125]. More work is needed in relation to antioxidant status and long‐term health, but evidence points to a role for plasma ascorbic acid concentration, and perhaps the nonuric acid total antioxidant capacity of

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plasma, as potentially useful biomarkers of health status for the routine Clinical Chemistry health assessment test menu. In relation to biomarkers of oxidative stress, plasma allantoin can be measured by HPLC [134], and the increasing availability of LC‐MS in Clinical Chemistry opens up the prospect of a more routine assessment of oxidative stress using specific markers such as urinary F2 isoprostanes and 8‐OHdG [135, 136, 145]. The comet assay is a relatively simple procedure, although scoring the cells for damage is quite time consuming, and oVers a robust and well validated biomarker of DNA damage for further assessment of oxidative stress levels in biomonitoring studies [132, 133, 136].

4. Biomarkers that Reflect Lower Risk of Disease in Long‐Term Vegetarians 4.1. INTRODUCTION Vegetarians have lower risk for many age‐related diseases, including diabetes, heart disease, stroke, osteoporosis, rheumatoid arthritis, and cancer [13, 146–148]. These diseases are multifactorial, but diet plays an important role in determining risk. However, many of the biomarkers that are used to assess risk or detect disease in its early stages are generally not specific for one disease. For example, atherosclerosis underlies both heart disease and stroke, and atherosclerosis is related to lipid levels, antioxidant status, blood pressure, inflammation, iron status, homocysteine concentrations, and others, while Type 2 diabetes mellitus is associated with insulin resistance, increased oxidative stress, dyslipidaemia, and central obesity and greatly increases risk of CVD [149–155]. Osteoporosis is caused by calcium imbalance, but biomarkers of bone health (plasma calcium, PTH, and vitamin D) are not used in predicting risk or diagnosing osteoporosis, which needs radiographic measurement of bone mineral density measurement rather than laboratory biochemical assessment [156]. Indeed, for many diseases there are no validated biochemical markers of risk, although some genetic factors have been identified. Cancer is caused by mutations in key genes controlling cell proliferation and diVerentiation and DNA repair, and some inherited or sporadic mutations present at birth greatly increase certain types of cancer, such as retinoblastoma, breast cancer, and colon cancer [157]. The APOE 4 allele increases risk of both heart disease and Alzheimer’s disease [158]. However, there are no sensitive and specific nongenetic biomarkers for cancer risk, and none for cognitive decline, although increased oxidative stress, though increased oxidative damage to DNA is likely to increase risk of cancer, and oxidative damage to lipid‐rich structure of the brain is found in Alzheimer’s

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disease, other dementias and Parkinson’s disease [12, 17, 158, 159]. Decline in immune surveillance, found with age and in association with certain drugs and lifestyle factors, including diet [160, 161] is also associated with increased cancer risk, but assessing immune status is not currently used as a tool for cancer risk assessment. In this section, the eVects of a vegetarian diet on biomarkers associated with common diseases are presented and briefly discussed. There are several studies that have investigated eVects of short‐term change to a vegetarian diet, and some that have investigated eVects of increased intake of fruits and vegetables within an omnivorous diet. Some of these studies are discussed briefly, but the main focus is on studies that have compared biomarkers in long‐term vegetarians and omnivores. In addition, the emphasis is on CVD and diabetes, as most published studies on eVect of vegetarian diets relate to these. EVects on variables or body functions that currently cannot be assessed using a biomarker approach are not discussed.

4.2. CVD AND TYPE 2 DIABETES: BACKGROUND AND BIOMARKER STUDIES IN RELATION TO VEGETARIAN DIET 4.2.1. Background and Biomarker Studies in Relation to Vegetarian Diet CVD is the main cause of death worldwide, causing 30% of all global deaths [149, 153]. The clinical manifestations of CVD include peripheral vascular disease (PVD), angina, myocardial infarction (MI), transient cerebral ischaemic attacks, and stroke (cerebrovascular accident; CVA) [149, 151, 153]. The underlying cause of CVD is atherosclerosis, a chronic inflammatory process that is influenced by various factors, including blood pressure, tobacco use, physical activity, diet, dyslipidaemia, iron status, elevated uric acid, hyperhomocysteinaemia, antioxidant status, oxidative stress, and genetic factors [149, 151–154]. The presence of several risk factors compounds overall CVD risk, and there is strong epidemiological evidence that combining risk factors into scores is capable of predicting an individual’s total cardiovascular risk with reasonable accuracy [151]. Type 2 diabetes greatly increases CVD risk [150, 154, 155]. Diabetes aVects 150 million people worldwide, and of these >90% have type 2 diabetes and more than two‐thirds will die from MI or stroke [150, 154, 161]. Patients with type 2 diabetes mellitus die of CVD at rates two to four times higher than patients without diabetes but with similar demographic characteristics, and overall mortality from heart disease is twice as great in diabetic men and is four to five times higher in diabetic women compared to those without diabetes [154, 155].

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The huge global impact of CVD and type 2 diabetes underlines the need for healthcare to move in the direction of risk assessment and modulation, rather than focusing on confirmation of advanced disease. Clinical Chemists need to identify, evaluate, and oVer a new generation of biomarkers that can assess CVD risk at an early or predictive stage, thereby opening the door to an era of targeted preventive care. Therefore, before focusing on eVects of vegetarian diets, it is worth recapping briefly the biomarkers used to assess risk or management of these common diseases. There are some biomarkers about which there are enough data to form a general consensus to their correlation with development of CVD. Limiting these to biochemical markers (as opposed to physiological markers such as blood pressure and waist:hip ratio), elevated concentrations of plasma TC, low‐density lipoprotein cholesterol (LDL‐C), triglycerides, and glucose are well‐established risk factors [149, 151–153]. There is also increasing evidence for elevated plasma hsCRP, uric acid, and homocysteine concentrations being useful biomarkers for CVD risk assessment or prediction, while others, including plasma creatinine, uric acid, lipoprotein (a), lipoprotein‐associated phospholipase A2 (Lp‐PLA2), fibrinogen, C‐peptide, soluble adhesion molecules, sialic acid, biomarkers of iron status, antioxidant status (especially ascorbic acid), oxidative stress, cystatin C, and urine albumin (microalbuminuria), and MMA are still under debate [17, 37, 56, 125, 149–154, 162–169]. Inflammation plays a key role in the pathophysiology and development of the atheromatous plaques, and is correlated with endothelial dysfunction and insulin resistance [153]. Several inflammatory biomarkers can be measured in plasma, including hsCRP, phospholipase A2, TNF‐alpha and interleukins (e.g., IL‐6, IL‐18) [153, 163, 164]. In type 2 diabetes, higher glucose and glycosylated hemoglobin (HbA1c) levels are predictive of CVD [154]. In addition, diabetes is often associated with hypertriglyceridemia, elevated TC, LDL‐C, and depressed high‐density lipoprotein cholesterol (HDL‐C) [149–151, 155]. Oxidative stress is also involved in the development of CVD, and is unquestionably elevated in diabetes [17, 134, 170, 171], although a direct link between acute increases in plasma glucose and oxidative stress is not well established [172, 173]. Oxidative stress has an eVect on LDL oxidation, vascular endothelial dysfunction, and gene regulation [17, 170, 174, 175]. For example, cholesterol has been shown to activate the metabolism of the arachidonic acid pathway which is associated with NAD(P)H oxidase activation, and F2 isoprostanes (a specific biomarker of lipid peroxidation) are elevated in the arteries of hypercholesterolaemic animals [174, 175]. In relation to diet and CVD, WHO and AHA guidelines advise strongly that total fat intake should be reduced to
30% of calories, saturated fat intake should be <10% of calories, and trans‐fatty acids should be avoided

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[149, 150]. Most dietary fat should be polyunsaturated (up to 10% of calories) or monounsaturated (10–15% of calories). Salt intake should be <5 g (90 mmol)/day. The diet should contain at least 400 g/day of a range fruits and vegetables, and contain whole grains and pulses. Well planned vegetarian diets meet these recommendations and therefore are expected to show beneficial eVects on biomarkers of CVD risk. In addition, vegetarian diets are generally lower in calories and simple sugars, which helps prevent type 2 diabetes [150, 176]. 4.2.2. Short‐Term Intervention Studies In a 4‐month cross‐over study (2 months each treatment) in the US, 35 healthy premenopausal women followed a low‐fat vegetarian diet deriving
10% of energy from fat, or the subject’s regular diet taken together with a placebo pill [177]. Mean serum LDL‐C, HDL‐C, and TC concentrations decreased 16.9%, 16.5%, and 13.2%, respectively, from baseline ( p < 0.01) with the intervention diet, mean serum triglycerides concentrations increased by 18.7%, most likely due to the increased carbohydrate intake in the low‐fat diet. No change in the ratio of LDL‐C to HDL‐C was seen [177]. These workers also reported [178] on eVects of a low‐fat vegan diet in type 2 diabetes patients (n ¼ 49) in comparison with eVects of a diet following the American Diabetes Association guidelines (n ¼ 50) [150]. The intervention lasted 22 weeks, and significant changes from baseline in both groups were seen in fasting plasma glucose, 24 h urinary albumin excretion, TC, HDL‐C, TC/HDL‐C, LDL‐C and triglycerides, but the TC and LDL‐C decreases were statistically (P < 0.05) greater in those on the low‐fat vegan diet whose medication remained unchanged, compared to those on the ADA diet [178]. However, no significant changes in plasma lipids (or insulin sensitivity as HOMA‐IR) were seen in a study of 80 overweight and obese type 2 diabetic subjects in the US who were assigned to a low‐fat lacto‐ovo‐ vegetarian diet for 18 months [179]. 4.2.3. Effect of Long‐Term Vegetarian Diet In a study by Szeto et al., in Hong Kong, 30 vegetarian subjects who had been vegetarian for at least 5 years we compared with an age‐ and sex‐ matched omnivore control group [142]. The vegetarian group had significantly (P < 0.01) lower TC (mean (SD) 4.8 (1.1) vs. 4.9 (1.3) mmol/l), triglycerides (1.06 (0.45) vs. 1.35 (0.57) mmol/l), uric acid (239 (88) vs. 306 (68) mmol/l), and hsCRP (0.77 (1.29) vs. 1.30 (1.38) mg/l). Similar results were shown in a study that assessed 109 long‐term (mean (SD) duration 8 (5) years) Chinese vegans and lactovegetarians and 107 matched omnivores in Taiwan [180]. The plasma levels of TC and LDL‐C (but not HDL‐C) were significantly lower in the vegetarian group than the

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matched group. Also, lag time of in vitro conjugated diene formation was longer and TBARS formation was lower in oxidized LDL from the vegetarian group. This implies that the LDL in the vegetarians was less susceptible to oxidation than that from omnivores, although interestingly, the dietary vitamin C and E intakes were no diVerent between the groups, the LDL a‐tocopherol content was slightly lower in the vegetarians, and the intake of PUFA was higher in the vegetarians [180]. Therefore, while the increased resistance to oxidation in the LDL from the vegetarians in this study may reflect a cardioprotective eVect of a vegetarian diet, the cause is not clear. A lower CVD risk biomarker profile of vegetarians compared with omnivores was also noticed in a study of 67 vegetarian (49 lacto‐ovo vegetarian, 9 vegans, 7 pescovegetarian, and 2 lactovegetarian) and 134 omnivorous subjects in Brazil [148]. The mean (SD) duration of vegetarianism was 19 (10) years. Plasma TC, HDL‐C, LDL‐C, VLDL‐C, triglycerides, glucose, urea, uric acid, and urinary sodium and potassium were measured, and vegetarians showed significantly lower values in all parameters except for HDL‐C where no significant diVerence was found [148]. In a German study of 201 adherents to a mainly raw food diet, plasma cholesterol, HDL‐C, and LDL‐C were significantly lower with increasing proportion of consumed raw food [181]. Lower lipids were also noticed in an African study where vegans (V, n ¼ 8) and lacto‐ovo‐vegetarians (LOV, n ¼ 28) showed lower (P < 0.05) cholesterol and triglycerides concentrations than the nonvegetarian group ((NV, n ¼ 40) [182]. Mean (SEM) mmol/l results for cholesterol and triglycerides in the V, LOV and NV groups were 4.5 (0.1) V, 4.8 (0.2) LOV, 5.3 (0.2) NV, and 0.9 (0.1) V, 1.2 (0.1) LOV, 1.4 (0.1) NV groups. No significant diVerences were seen in blood glucose concentrations [182]. The same group showed also significantly (P < 0.001) lower plasma fibrinogen and higher fibrinolytic activity in vegetarians compared to nonvegetarians [183]. CVD incidence is higher in women after the menopause. EVects of long‐ term vegetarian diets on CVD biomarkers were investigated in healthy postmenopausal women in Taiwan [184]. A total of 35 healthy vegetarian (of 2–35 years duration) and 35 omnivore women were examined. None of the women was on hormone replacement therapy. The vegetarian diet was similar to that in the study of Szeto et al. [142], that is, some subjects occasionally consumed small amounts of eggs and milk. The vegetarians had statistically (P < 0.05) lower TC, LDL‐C, triglycerides, and fasting glucose [184]. Uric acid levels averaged
10% lower in the vegetarian, but the diVerence did not reach statistical significance [184]. Su et al. studied circulating vascular adhesion molecules, lipids, homocysteine, and glucose in 57 healthy postmenopausal vegetarians and 61 matched healthy omnivorous controls in Taiwan [164]. The vegetarian diet was exclusively plant‐based and had been followed for at least 5 years. Fasting

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plasma TC, HDL‐C, LDL‐C, and glucose were significantly (P < 0.05) lower in the vegetarians. Interestingly, sVCAM‐1 was higher (P < 0.05), as was homocysteine; however, the sVCAM‐1 was found to be positively associated with age, not vegetarianism in this group [164]. Nonetheless, and as noted in an earlier section, inadequate vitamin B12 is a common finding in plant‐based diets, and this can cause elevations in plasma homocysteine, which in turn may attenuate the cardiovascular benefits of vegetarian diets. sICAM‐1 was no diVerent between vegetarian and nonvegetarian groups, and no diVerences were seen in plasma creatinine [164]. In an Australian study of 18 vegans, 43 ovolactovegetarians, 60 moderate meat eaters, and 18 high meat eaters (all male), meat eaters had a significantly higher cluster of cardiovascular risk factors, including TC, LDL‐C, triglycerides, LDL‐C:HDL‐C ratio, and Factor VII activity [185]. However, there was no diVerence in platelet aggregation between the groups. Similar to the findings in other studies, the HDL‐C in the vegetarians was lower; however, the LDL‐C:HDL‐C ratio was also lower in the vegetarians, indicating improved lipid balance [185]. A study in Taiwan compared lipids, uric acid, homocysteine, white cell count, glucose, and hsCRP in 99 healthy lacto‐ovo‐vegetarians and 99 healthy omnivores [186]. The vegetarians had adhered to their dietary pattern for at least 1 year prior to enrolling in the study. The groups were matched for age, but there were more women (n ¼ 65) in the vegetarian group than in the omnivore group (n ¼ 46). The vegetarian group had significantly (P < 0.01, and by
10%) lower plasma TC, LDL‐C, uric acid. Plasma hsCRP concentrations were also lower (P < 0.05) in the vegetarians. No significant diVerences were seen in white cell count or plasma glucose, HDL‐C or triglycerides, but once again, the vegetarian group had slightly but significantly higher plasma homocysteine concentration than the omnivores: mean (SD) 10.97 (6.69) versus 8.44 (2.50) mmol/l; P < 0.001 [186]. The significance of slight elevation in plasma homocysteine in regard to CVD risk is not clear [37], but elevated homocysteine in vegetarians is reported to be normalized by administration of vitamin B12 [46]. Vegetarians may show increased platelet aggregation due to dietary lack of n‐3 fatty acids, and this may increase CVD risk [46]. Supplementation with 700 mg/day of EPA and DHA fatty acids for 8 weeks in 10 vegetarians led to increased incorporation of these fatty acids into plasma lipids, with a reduction of platelet aggregation with all the agonists tested (ADP, epinephrin, collagen, arachidonic acid). However, no significant change in bleeding time after supplementation was seen [46]. In a review, which summarized five cross sectional studies on the eVects of vegetarian diet on hemostasis and thrombosis [187], the author concluded that overall there is a favorable impact of vegetarian diet on hemostasis.

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This benefit was shown in terms of either a lower concentration of coagulation factors or an increase in fibrinolysis [187]. However, a cautionary point is the finding of downregulation of IGF (insulin‐like growth factor)‐1 activity in association with a very low‐fat vegan diet, and this is hypothesized to have implications for risk of stroke [188]. IGF‐1 promotes endothelial health through eVects on nitric oxide synthase and this is believed to have especial relevance to the cerebral vasculature. It is not suggested that plant‐based diets per se increase risk of stroke, but the reported IGF‐1 decrease in a very low‐fat vegan diet [188] highlights the fact that vegetarian diets must be balanced and well planned, and even then cannot be regarded as a panacea. In relation to diabetes, of interest is an observational study that followed 37,309 healthy women (45 years) for 8.8 years in which it was reported that a significant increased diabetes risk was most pronounced in those who with frequent consumption of total processed meat [189]. Intake of cholesterol, animal protein, and heme iron were also significantly associated with a higher risk of type 2 diabetes, while intake of fruit and vegetables decreased risk [189]. In a meta‐analysis of large nine studies that included 91,379 men and 129,701 women, the risk of CVD was decreased by 4% and 7% respectively for each additional portion or fruit and vegetable [105]. Fruit and vegetable consumption was found to be inversely associated with the risk of CVD and beneficial eVects of intake of fruits and vegetables were also seen in the diabetic population [105]. Advanced glycosylation endproducts (AGEs) are found in increased amounts in plasma in diabetes [17]. Plasma AGEs (measured by fluorescence and as carboxymethyl‐lysine using an ELISA method after proteinase K digestion) in healthy subjects were compared between omnivores (n ¼ 19) and aged‐matched long‐term vegetarians (9 vegans, 19 lacto‐ovo veg, and 14 semivegetarians, with duration of vegetarianism over 7 years on average in each group) [190]. Biomarkers of kidney function (plasma creatinine, cystatin C, glomerular filtration rate (GFR)) and inflammation (hsCRP) were examined, and fasting glucose was also measured. No significant diVerences were found between the four groups except for AGEs, the level of which was significantly (P < 0.05) higher in the vegetarians [190]. The authors suggested that the elevated plasma AGEs was due to enhanced dietary intake in the vegetarians, as pathogenic factors, such as increased oxidative stress, diabetes, renal of liver dysfunction, were unlikely contributors in this case or had been ruled out by further study [190]. However, it is noted that elevated AGEs in vegetarians was not seen in a follow‐up study by this group [139]. Nonetheless, this serves to remind that some biomarkers associated with disease may be aVected directly, as well as indirectly, by diet, and that correct interpretation of biomarker data for health and disease risk assessment requires knowledge of biological (including dietary‐driven) as well as pathological factors.

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Insulin resistance is a fundamental abnormality in type 2 diabetes. Plasma glucose and insulin resistance in normal weight, healthy long‐term lacto‐ovo vegetarians (n ¼ 95, mean (SD) duration of vegetarianism, 10.2 (0.5) years) and an omnivore control group (n ¼ 107) were compared [191]. Fasting glucose (4.47 (0.05) vs. 4.71 (0.07) mmol/l), insulin (4.96 (0.23) vs. 7.32 (0.41) mU/l) and insulin resistance (as HOMA‐IR: 0.99 (0.05) vs. 1.59 (0.10)) were all significantly (P < 0.05) lower in the vegetarian group [191]. Significantly lower insulin resistance (i.e., higher insulin sensitivity) was found also in 49 lacovegetarian premenopausal women as compared with 49 omnivores, as evidenced by lower fasting plasma insulin, glucose, and insulin: glucagon ratio [80]. 4.2.4. Summary In summary, various studies of diVerent design and locale provide strong and convincing evidence that a vegetarian diet can confer cardiovascular benefits through beneficial eVects on lipids, uric acid, glycaemic control, and inflammation, and lower risk of diabetes and its cardiovascular complications. As noted earlier, ascorbic acid status is higher, oxidative stress is lower, and iron status lower (though not inadequate) in a well‐balanced, plant‐based diet, and this is likely also to confer benefit. However, lack of ‘‘carninutrients’’ may partially counter balance benefits through elevated plasma homocysteine. Therefore, selected supplements, notably vitamin B12, may be advisable adjuncts to a plant‐only diet to fully realize its health benefits. 4.3. BONE HEALTH The primary bone disease aVecting the developed world is osteoporosis, an age‐related disease characterized by low bone mineral density and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture [156, 192]. The reference method to assess bone mass is dual‐energy X‐ray absorptiometry, but there are several biochemical biomarkers for bone health and calcium metabolism, include urinary and plasma calcium and phosphate, urinary hydroxyproline, plasma 25‐hydroxyvitamin D, calcitropic hormones, such as plasma parathyroid hormone‐1‐a‐hydroxylase axis (PTH) and follicle‐stimulating hormone (FSH), plasma C‐telopeptide of type I collagen, bone‐specific ALP, urinary deoxypyridinoline excretion (a biochemical indicator of bone resorption), plasma prostaglandin E2 (a lipid mediator of bone remodeling), osteocalcin, procollagen type I intact N‐terminal propeptide (P1NP), osteoprotegerin, free pyridinolines and free deoxypyridinolines, and plasma estrogen concentration is also a factor that impacts on bone health [156–196].

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Other nonbiochemical factors that are associated with bone health are BMI, alcohol intake, smoking, physical activity and, importantly, diet [192, 194–196]. Calcium and vitamin D are crucial for both accruing and maintaining skeletal mass [196, 197]. Fiber, potassium, and trace elements also show an eVect [195]. Vitamin K has a positive role in bone health [192]. Protein is also important and of concern are several epidemiologic studies that demonstrate reduced bone density and increased rates of bone loss and fractures in individuals habitually consuming low‐protein [194] or high‐protein diets [195]. Adequate dietary protein may help in healing fractures and preventing bone loss following fracture [194]. Animal‐ or plant‐based proteins may release sulfur‐containing amino acids, causing acid overload, with consequent release of calcium and phosphate for maintenance of pH balance, suggesting that osteoporosis may, in part, be caused by acid‐induced ‘‘leaching’’ of calcium phosphate from bone for acid–base balance if protein intake is high [197–199]. There is indication that vegetarianism can be associated with several factors which are likely to have a detrimental impact on bone health. This includes lower circulating levels of estrogen, lower average BMI, and deficiencies in calcium and vitamin D [196–199]. However, as noted above, well‐balanced vegetarian diets are not necessarily deficient in calcium or vitamin D, and increased intake of fruits and vegetables may have beneficial eVects on bone health [192, 198, 200]. In a review by New [200] of more than 27 studies over 20 years, it is concluded that vegetarians do not have diVerent bone mass compared to omnivores. In a study that included 76 postmenopausal Korean vegetarian women who had maintained a vegetarian diet for over 20 years and 76 aged‐ matched nonvegetarian controls, the bone mineral density of the spine and femoral neck BMDs were not significantly diVerent [201]. The author concluded that it may be due to the higher consumption of soybean‐containing isoflavones in addition to more fruits and vegetables. However, the vegetarian group had significantly lower mean (SD) serum levels of ferritin (63 (32.7) vs. 93.5 (49.5) pmol/l), zinc (11.7 (1.5) vs. 14.3 (2.7) mmol/l), copper (14.7 (2) vs. 16.2 (3.5) mmol/l), and urinary deoxypyridinoline (6.82 (2.0) vs. 7.45 (2.3) nmol/mmol creatinine) than the controls [201]. In a recent study that looked at 1865 adult males and females, no diVerences in bone mineral density were observed between vegetarians and nonvegetarians of either sex; however, no biomarkers were measured [202]. In the EPIC‐Oxford study which included 7947 men and 26,749 women aged 20–89, the fracture risk was examined [91]. Out of the total number of subjects, 19,249 were meat eaters, 4901 fish eaters, 9420 vegetarians, and 1126 vegans. Fracture incidence (self reported) was followed over a period of 5.2 years. Fracture rates were similar for meat eaters, fish eaters, and

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vegetarians. However, fracture risk was 30% higher among vegans for both men and women [91]. This was halved in magnitude by adjustment for energy and calcium intake and no diVerence was seen when the analysis was restricted to subjects who consumed at least 525 mg/day calcium. The authors concluded that higher fracture rate among vegans appears to reflect their markedly lower mean calcium intake. The similarity therefore according to the authors, in fracture rates among meat eaters, fish eaters, and lacto‐ovo vegetarians may reflect the similarity in mean calcium intakes in these three groups [91]. Similar results were reported in a study of 18 vegan subjects who consumed a raw food vegetarian diet for a mean of 3.6 years and a matched omnivorous group [203]. Body composition, bone mineral content and density, 25‐hydroxyvitamin D, hsCRP, IGF, and bone turn over markers (C‐telopeptide of type I collagen, bone‐specific ALP) were examined. The results showed that a raw food vegetarian diet is associated with low bone mass (at lumbar spine, hip, femoral neck, trochanter), but no biomarker evidence of increased bone turnover or impaired vitamin D status was found [203]. However, impaired bone turnover was seen in a Polish study of vegetarian children aged between 2 and 10 years [204]. Serum 25‐OHD of vegetarian children (n ¼ 50) was around half that of nonvegetarian children (n ¼ 50), and serum osteocalcin, C‐telopeptide, and bone‐specific ALP were, respectively, 20%,15%, and 10% lower in the vegetarian children. However, it is noted that the vegetarian children studied had dietary intakes of calcium and vitamin D that were only half that of the nonvegetarian children, and this accounts for the diVerence in bone biomarkers between the two groups [204]. Nonetheless, a review of the literature [199] concluded that vegans do have lower bone mineral density than their nonvegan counterparts, and the author highlighted that a vegan diet eliminates all dietary food sources of vitamin D. Therefore, vegetarian diets can be associated with increased risk of osteoporosis if the diet is not balanced for calcium intake and if vitamin D balance is poor, for example, due to lack of cutaneous UV exposure as well as low intake (see Section 2.4.3). 4.4. RENAL FUNCTION Renal function can be assessed by renal plasma flow as well as GFR, as creatinine clearance or estimated (eGFR), urinary protein excretion, and urine albumin excretion [205]. Normal GFR varies according to age, sex, and body size, and declines with age [205]. Chronic kidney disease is associated with higher levels of inflammatory biomarkers, as well as elevated plasma urea, creatinine, and cystatin C and micro‐ and macroalbuminuria [205, 206]. Dietary factors related to renal function are sodium intake and protein intake [1]. High salt intake can cause hypertension, which is deleterious

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to renal health. Vegetarian diets are generally low in sodium. High protein diets have been shown to accelerate renal deterioration in subjects with even mildly reduced kidney function [206]. Most omnivorous diets are high in animal protein and relatively low in plant protein sources such as legumes. Animal proteins contain more cysteine and methionine (both sulfur‐containing amino acids) than plant protein; however, the total sulfur‐ containing amino acid content of animal protein is similar to that of plant proteins [1]. Excessive sulfur‐containing amino acid consumption is detrimental to renal function whatever the source because their catabolism increases the amount of sulfate to be excreted. However, protein intake is generally lower (but adequate) in a well‐balanced vegetarian diet [1]. In a study where a vegetarian diet was alternated with a conventional low‐protein diet for patients with chronic renal failure (n ¼ 20), the vegetarian diet was found beneficial in relation to cholesterol level, urea excretion, and protein catabolic rate [207]. Long‐term vegetarian diet was examined in 13 vegetarian and 20 omnivorous young healthy females [208]. Calcium excretion in the omnivorous group was significantly higher, even though dietary intake was not higher, and may be a result of a higher protein intake in the meat‐containing diet [208]. Acid–base status did not show any significant diVerence between the groups with respect to blood PCO2, plasma bicarbonate, or base excess [208]. Renal health can be aVected also by formation of kidney stones. Urolithiasis has been progressively increasing over the past century, and this has been attributed to changes in lifestyle and dietary patterns. Nutrition is one of the most important prerenal risk factors in urinary stone formation, since urinary composition is strongly related to various dietary components. Acute vitamin B6 deficiency increases urinary oxalate excretion, but marginal vitamin B6 deficiency, which can occur in diets with no animal foods, is unlikely to be a cause of calcium oxalate kidney stones in young healthy population [209]. In a small supplementation study, 10 healthy male subjects followed diVerent diets that included or excluded meat for 5 days [210]. Urine volume, pH, creatinine, chloride, sodium, sulfate, phosphate, ammonia, and uric acid were measured, and uric acid crystallization risk was calculated. The vegetarian diet showed a significantly lower (93%) risk of uric acid crystallization compared to the meat‐containing diet due to lower uric acid excretion and higher urinary pH when the vegetarian diet was followed [210]. 4.5. CANCER Cancer is a group of diseases characterized by uncontrolled growth and spread of abnormal cells caused by mutations in key genes [157]. Cancer is the second leading cause of mortality in the developed world, following

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CVD, and in some countries is the main cause of death [7, 157]. There are a range of mechanisms that are linked to cancer, such as DNA damage and methylation, inherited mutations, stimulation of cell proliferation, hormonal balance, obesity, inflammation, immune status, apoptosis, exposure to carcinogens, and diet [7, 12, 17, 157, 159, 211–213]. Cancer incidence across time and place can vary in genetically similar populations that migrate from their native countries, suggesting that patterns of cancer are altered by diet and environmental factors, and it has been estimated that 30–40% of all kinds of cancer can be prevented with a healthy lifestyle and dietary measures [7, 157, 211–213]. Case control and epidemiological studies show that intake of fruits and vegetables is associated with a significant reduction in the risks of some cancers [7, 8, 157, 212–216]. Elements in a cancer‐preventive diet include high levels of selenium, folic acid, vitamin B12, vitamin D, chlorophyll, fiber, and antioxidants [211], while high intake of red meat, total calories, and salt increase the risk of some cancers [7, 211, 212]. With the exception of vitamin D, intake of which is low in plant‐based food, the vegetarian diet contains all the elements of a cancer‐preventive diet. A biomarker approach which examines plasma levels of dietary micronutrients in relation to cancer has been employed in some observational studies. Vegetarians, and others who have high intake of fruits and vegetables, have higher levels of some micronutrients (such as folate and antioxidants) and to have lower incidence of cancer of various sites [7, 8, 211–216]. Hormonal and growth factors play an important role in cancer development, and mean serum IGF‐1 was 9% lower in 233 vegan men compared to 226 meat eaters [216]. In vegan women (n ¼ 92), the IGF‐I concentration was 13% lower compared with to meat eaters (n ¼ 99). Concentration of IGF‐binding protein‐1 and 2 were 20–40% higher in vegan women [216]. However, while lower IGF‐1 concentration may be associated with lower cancer risk (and CVD risk, but, in contrast, increased risk of stroke [188]), there are insuYcient data to support its use as a biomarker of cancer risk. Vegetarian diets are often high in soy foods which are rich in phytoestrogens, and this may reduce breast cancer risk. Isoflavones intake and breast cancer risk in a cohort of 37,643 British women participating in the European Prospective Investigation into Cancer and Nutrition was investigated; however, no evidence for a strong association between vegetarian diets in either the pre‐ or post‐postmenopausal period, or dietary isoflavone intake and risk of breast cancer was seen [214]. Even though the exact etiology of cancer is not fully understood, there are some factors such as genomic stability that aVect the development of the disease. In a study that examined 13 lacto‐ovo‐vegetarians (average length of diet 10.8 years), 11 lacto‐vegetarians (average length of diet 8.2 years) and 24 healthy omnivorous controls, chromosome aberrations, micronuclei,

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and DNA damage (as strand breaks measured using the comet assay) were examined in peripheral blood lymphocytes [143]. Vegetarians showed slightly lower levels of oxidative DNA damage in lymphocytes, but genomic stability was not aVected [143]. The eVect of a vegetarian diet was investigated also in regard to DNA methylation, which is involved in gene regulation. Hypo‐ and hypermethylation can be found in diVerent cancer types and there is some indication of relation between B12 deficiency, homocysteine levels, and methylation reactions [212]. No diVerence in DNA methylation was found in 48 lacto‐ovo vegetarians compared to 23 vegans [212]. In summary, there is epidemiological evidence of lower cancer risk in those whose diet is rich in plant‐based foods, but not all studies agree, and the impact of a vegetarian diet on cancer is not clear, even though it would appear to mirror (with the exception of vitamin D) ‘‘cancer‐preventive’’ dietary guidelines. There is a lack of reliable biomarkers for cancer risk, and so there is a paucity of biomarker data in relation to cancer risk in those who adhere to a long‐term vegetarian diet. 4.6. IMMUNE STATUS The immune system is an eVective defense system that is designed to recognize and destroy foreign, infected and changed cells. Immune surveillance to detect and remove somatic cells at an early, precancerous stage is an important anticancer strategy, and a decline in immune status and response is associated with increased risk of cancer, as well as increased susceptibility to infection and lower response to vaccination [160, 161]. Immune status declines with age and in association with certain disease states, stress, and dietary factors [160, 161]. Assessment of immune status involves a range of biochemical and cell‐ based tests and includes total white blood cell count and diVerential (though this is most commonly performed in the hematology laboratory), white cell subset testing (using flow cytometry), and plasma immunoglobulin and complement testing. White cell response to activating factors, and plasma levels of inflammatory markers, such as CRP and interleukins, can also be measured. Diets lacking suYcient protein are associated with impaired cell‐mediated immunity, complement activity, phagocyte function, IgA antibody concentrations, and cytokine production, and deficiency of single nutrients such as iron, copper, zinc, selenium, vitamins A, C, E, and B6, and folic acid (vitamin B9) also reduces immune responses [160]. Haddad et al. examined immune status in 25 vegans and 20 nonvegetarian subjects [86]. The vegan group had significantly lower numbers of leukocytes, lymphocytes, and platelets and lower complement factor 3, but there was no significant diVerence between the groups in natural killer (NK) cell activity or in the mitogen stimulation index [86].

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Inflammation, which is involved in many acute and chronic diseases, is closely linked with the immune system, and can be aVected by diet [163]. Rheumatoid arthritis is an inflammatory disease, and in a Finnish study, changing the diet of rheumatoid arthritis patients into a ‘‘living food’’ (uncooked vegan, with high intake of flavonoid‐rich berries) diet for 3 months resulted in decreased joint stiVness and pain by subjective and objective assessment, however, no biomarker data was presented [217]. In a Norwegian study, 7–10 days of fasting followed by 3.5 months of a gluten‐free vegan diet and then 9 months of a lactovegetarian diet resulted in less pain, less stiVness and swelling, and more movement in the joints of 27 rheumatoid arthritis patients compared to 26 control patients who followed their usual omnivorous diet throughout the course of the trial [218]. In terms of inflammation biomarker data, erythrocyte sedimentation rate (ESR), plasma CRP, total white cell count were all significantly (P < 0.001) lower in the vegetarian diet intervention group, the changes in most cases being most marked in the first month, that is during the vegan diet phase [218]. Interestingly, the favorable clinical eVects were found to be associated with changes in fecal microflora and in the antibody activity against Proteus mirabilis, and the author suggested that the benefit of the dietary change may have been mediated by alteration in fecal microflora [218]. Interestingly, a strong and direct correlation (r ¼ 0.935; P < 0.01) has been reported between animal food (meat and oVal fat) and prevalence of rheumatoid arthritis across eight countries across the developed world [219]. This could be due to nitrite and iron intake, as suggested by the author [219], but could also be due to dietary influences on gut microflora. Regardless of whether it is iron, nitrite, microflora changes, or other eVects of meat, it is possible to suggest that a plant‐only diet may lower risk of rheumatoid arthritis and perhaps other diseases that have an inflammatory basis, including CVD.

5. Biomarkers to Differentiate the Vegetarian from the Nonvegetarian Many of the biomarkers that are aVected by vegetarian diet are aVected by age and by other lifestyle factors, such as exercise and cigarette smoking. In addition, the foods eaten by the vegetarian are generally eaten also by omnivores. Therefore, it is not possible to use any of the biomarkers currently available to diVerentiate the vegetarian from the nonvegetarian. Unquestionably, plasma concentrations of plant‐derived compounds, including ascorbic acid, folate, carotenoids, catechins, and other polyphenols, are likely to be higher in long‐term vegetarians, and vitamins B12 and D may be low. However, the diVerence in most is a matter of degree, and low levels of

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vitamins B12 and D are not a necessary consequence of following a vegetarian diet. Plasma levels of carotenoids are related to fruit and vegetable intake [116, 126]. However, fruit and vegetable intake is high in many meat eaters who achieve the ‘‘5 or more a day’’ diet recommended for health [7, 145, 146]. Plasma concentrations of salicylic acid are related to fruit and vegetable consumption [220], but again the diVerence between plasma levels in vegetarians and omnivores is a matter of degree. Plasma concentrations of phytoestrogen are 5–50 times higher in vegetarians than in nonvegetarians, and in many nonvegetarians are undetectable [221]. However, the variation among diVerent subjects and regions is substantial [221]. Therefore, measuring the concentration of salicylic acid, flavonoids, or other phytochemicals cannot identify the long‐term vegetarian with any reliable index of discrimination. Measurement of urine pH has been suggested as a way to monitor change in fruit and vegetable, as a significant (P < 0.001) trend was seen in relation to increasing pH of urine with increasing daily intake of fruits and vegetables [222]. However, while an increase in urine pH may reflect higher intake of plant‐based foods, urine pH cannot distinguish between the vegetarian and nonvegetarian. Looking at the question of how to diVerentiate the vegetarian from the nonvegetarian from another angle, is there a biomarker of intake of meat or animal products? In this regard, plasma phytanic acid may have potential, as this has been reported to be almost sevenfold higher in meat and dairy product eaters than in plant eaters [223]. Phytanic acid is a C20 branched chain fatty acid found mainly in red meat and dairy products. It is formed from phytol, a breakdown product of chlorophyll, and this process occurs in the ruminant but not in the human gastrointestinal system. Plasma phytanic acid was measured by GC‐MS in female vegans (n ¼ 46), lacto‐ovo‐vegetarians (n ¼ 25), and meat eaters (n ¼ 25) [223]. The geometric means (95% CI) for plasma phytanic acid concentration (mmol/l) in these three groups were: 0.86 (0.70–1.05) for vegans, 3.93 (3.00–5.15) for lacto‐ovo‐vegetarians, 5.77 (4.40–7.57) for meat eaters (P < 0.000). Therefore, plasma phytanic acid may act as a biomarker for intake of meat or dairy products. Interestingly, phytanic acid upregulates the activity of an enzyme (a‐methylacyl‐coenzyme A racemase: AMACR) which is needed for b‐oxidation of phytanic acid. AMACR is overexpressed in cancers of prostate, breast, and colon, forming a suggested link between high intake of phytanic acid from red meat and/or dairy products and cancer development [223] and could explain at least part of the decreased risk of these cancers in vegetarians. Other diVerences between vegetarians and meat eater is in the fatty acid profile of cell membranes, and in the amino acid profile in plasma and, possibly, in hair. The abundance of the stable isotope 15N in tissue proteins increases with the food chain. Animal proteins contain more of this

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stable isotope than do plant protein, and hair keratins, which are not metabolized, are reported to provide a reliable record of dietary habits over the previous months [224]. However, biomarker‐based diVerentiation between vegetarians and nonvegetarians is neither possible nor needed at this time. On point of note that does need attention is the possibility that a diVerent reference interval for some biomarkers in vegetarian subjects may be needed. For example, increased rates of false positive Down syndrome screening results have been reported in vegetarian mothers because of elevated maternal mid‐trimester serum free b‐human chorionic gonadotropin concentration [225]. This was related to low serum vitamin B12, which as noted above is a common finding in those who adhere to a plant‐based diet. There is a report also that the estimated GFR (eGFR) is decreased significantly (but with no changes in plasma cystatin C concentration) after intake of cooked meat [226]. The eGFR decrease was due to increases in plasma creatinine following meat ingestion, and this could lead to error in diagnosis or monitoring of chronic kidney disease.

6. Summary and Recommendations for Clinical Chemistry Vegetarianism has become an increasingly popular lifestyle choice, mainly because of the perceived and actual health benefits of a plant‐based diet [227, 228]. Benefits include lower plasma lipids, lower inflammatory status, decreased thrombotic index, better immune status, improved calcium balance, enhanced antioxidant status, decreased oxidative stress, and improved insulin sensitivity. The plant‐based constituents that confer these benefits are not entirely clear, but higher intake of plant‐based antioxidants may underlie many of the eVects. In addition, some benefit is undoubtedly due to lower intake of cholesterol, sodium, heme‐iron, saturated fat, and total calories in the vegetarian diet. It is not currently possible—nor is there a need—to distinguish the long‐term vegetarian from the omnivore using a biomarker approach. However, a vegetarian diet must be nutritionally adequate in order for the long‐term benefits to be realized. Nutritional and antioxidant/ oxidant balance assessments are something that most Clinical Chemistry laboratories do not currently oVer. However, as ever larger numbers in the population choose vegetarianism as lifestyle, it is timely to add biomarker profiling for nutritional status to the routine Clinical Chemistry test menu. It should be noted also that it is not only the long‐term vegetarian who can develop nutritional deficiency of key micronutrients. Furthermore, with the ageing of the global population and the increased awareness of dietary influences and oxidative stress on health, it is timely for Clinical Chemistry to advance more rapidly into the arena of nutritional and

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oxidant/antioxidant assessment, and to oVer measurement of biomarkers that assess health, as opposed to solely focusing on those that aid diagnosis and management of established disease. More awareness of the direct and indirect influence of diet on biomarkers of health and disease is needed as well. In this regard, some biomarkers deserve more widespread use and attention in Clinical Chemistry. Biomarkers that reflect vitamin B12 status may be useful due to the impact of low vitamin B12 on homocysteine metabolism and CVD risk. Increasing evidence points to vitamin D as having a key and multifaceted role in health maintenance, and a biomarker of vitamin D status may be a useful addition to the Clinical Chemistry test menu. Antioxidant status and oxidative stress assessment is currently limited to research laboratories, but biomarkers of antioxidant balance and oxidative stress oVer potentially valuable and novel biomonitoring tools to assess health, investigate gene/nutrient/ environment interactions, and to detect pathological changes at an early stage. The strong relationship seen in epidemiological trials between higher plasma ascorbic acid concentration and lower mortality in ensuing years indicates that plasma ascorbic acid may be a useful biomarker of health status, a ‘‘vitality index,’’ even if it is not ascorbic acid itself that directly mediates the eVect. The antioxidant capacity of plasma, corrected for uric acid, may also be a useful biomarker, and its assessment is simple and rapid. With more widespread availability of HPLC and LC‐MS equipment it is timely also to consider the inclusion of sensitive and specific biomarkers of oxidative stress, such as plasma allantoin and urinary F2 isoprostanes and 8‐OHdG in the Clinical Chemistry health assessment biomarker profile. Concepts of oxidants and antioxidants are changing, but their role in cellular signaling, ageing, and health is widely recognized as a crucial one, albeit as yet unclear. Only by oVering more widespread assessment can we gain further insight into their role in health and disease, and only by such insights can Clinical Chemistry move towards adopting the paradigm of assessing and promoting health, as opposed to simply aiding in the diagnosis and monitoring of disease. ACKNOWLEDGMENT The authors are grateful to The Hong Kong Polytechnic University for supporting this work.

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EFFECT OF CALORIC RESTRICTION ON OXIDATIVE MARKERS ˇkrha1 Jan S Laboratory for Endocrinology and Metabolism and 3rd Department of Internal Medicine, 1st Faculty of Medicine, Charles University in Prague, U Nemocnice 1, 128 08 Prague 2, Czech Republic

1. 2. 3. 4. 5.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Foods and ROS Generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondria as a Source of Reactive Oxygen and Nitrogen Species. . . . . . . . . . . . . Caloric Restriction and Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. ROS Reduction by Caloric Restriction or Moderate Exercise . . . . . . . . . . . . . . 5.2. Gender DiVerences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Fasting and ROS Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Oxidative Stress Markers by Caloric Restriction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Reactive Oxygen or Nitrogen Species (ROS, RNS) . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Oxidative Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Antioxidant Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Nonenzymatic Scavengers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. Other Markers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Data Interpretation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

224 224 225 226 229 229 230 231 232 232 233 235 237 240 240 241 242

Abbreviations AGE Cu, Zn SOD GPx GR

advanced glycation endproducts copper, zinc superoxide dismutase glutathione peroxidase glutathione reductase

1

Corresponding author: Jan Sˇkrha, e‐mail: [email protected] 223

0065-2423/09 $35.00 DOI: 10.1016/S0065-2423(09)47008-2

Copyright 2009, Elsevier Inc. All rights reserved.

224 GSSG GSH HPLC MnSOD mtDNA mtNOS NADPH2 NO NOS PON1 RNS ROS TBA TBARS UCP3 8‐OH‐dG

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oxidized form of glutathione reduced form of glutathione high‐performance liquid chromatography manganese superoxide dismutase mitochondrial deoxyribonucleic acid mitochondrial nitric oxide synthase reduced form of nicotinamide adenine dinucleotide phosphate nitric oxide nitric oxide synthase paraoxonase 1 reactive nitrogen species reactive oxygen species thiobarbituric acid thiobarbituric acid‐reacting substances uncoupling protein 3 8‐hydroxy‐deoxyguanosine

1. Abstract Caloric restriction is associated with a decreased level of oxidative stress. Reactive oxygen species (ROS) generated predominantly in mitochondria are attenuated by decreased caloric intake. On the other hand, antioxidative mechanisms are frequently accelerated by increased gene expression or activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, paraoxonase, etc.). Measurement of diVerent oxidative stress markers in relationship to caloric restriction is therefore important in experimental as well as clinical studies. Estimation of ROS in tissues and fluids is typically performed by measurement of oxidant products (i.e., malondialdehyde, F‐2‐isoprostanes, nitrotyrosine) and markers of antioxidant system (enzymes, glutathione, alpha‐tocopherol, ascorbic acid, ubichinone, etc.). Because both components are critical to objectively understand the oxidative stress state, tangible biochemical data is required in order to comprehensively elucidate pathobiologic mechanisms and potential therapeutic regimes involving lifestyle changes that include caloric restriction or moderate physical activity. 2. Introduction Biologic systems create ROS like superoxide anion, hydroxyl radical, hydrogen peroxide, and nitrogen oxide radical that are deleterious for cells or tissues. ROS production is quenched by a scavenger system involving either

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endogenous or exogenous substances. Both processes, ROS generation and inhibition, are balanced in healthy organisms, but impairment may arise if either ROS production is increased or defensive mechanisms are ineVective. Overproduction of ROS has been repeatedly described in a variety of pathologic disorders including inflammation, diabetes, atherosclerosis, and oncogenesis. In addition, one the most important theories concerning pathogenesis of aging is based on ROS generation and oxidative stress [1–3]. This hypothesis was formulated more than a decade ago on the observation that: (a) overexpression of antioxidative enzymes retard the age‐related oxidative damage and extend life span in experimental animals; (b) variations in longevity among species inversely correlate with mitochondrial generation of superoxide radical or hydrogen peroxide; and (c) restriction of caloric intake decreases levels of oxidative stress, retards the age‐related changes, and extends life‐span in mammals [1]. 3. Foods and ROS Generation Pandemia of obesity in developed countries has caused an extensive research of its risk factors and their consequences at molecular level. Modern lifestyle involving low physical activity as well as overeating characterized by high caloric intake are leading factors in overweight or obesity development. Changes in the ROS production and antioxidant mechanisms have been observed in obese persons and the possible role of both exogenous factors, physical inactivity, and overeating has been intensively studied. Results obtained both in animal and human studies have repeatedly demonstrated that physical exercise and decreased food intake bring favorable eVects to their subjects. The eVect of ingested calories on ROS production has been confirmed in experimental and clinical studies (Fig. 1). Overeating

+

Aging

Caloric restriction −

+

+ −

Antioxidant enzymes

Diabetes, cancers, inflammation, .... +

ROS production

+ −

Non-enzymatic antioxidants

FIG. 1. Factors influencing reactive oxygen species (ROS) production and antioxidant system.

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Although higher caloric intake generates more ROS in general, food composition and method of preparation may significantly modify ROS expression specifically [4]. Exogenous advanced glycation endproducts (AGEs) produced in greater amount by broiling and frying food preparation methods result in increased plasma AGEs and act as contributor of ROS [5]. Foods containing fats showed the highest amount of AGE content [4]. Acute vascular dysfunction due to consumed AGEs has been described postprandially [6]. Excessive AGE consumption represents an independent risk factor for oxidant stress and may promote premature development of complex pathologic states (diabetes and cardiovascular disease) typically associated with adulthood. Dietary restriction is the most powerful modulator of the aging process in diverse groups of organisms. Its multifaceted eVects are achieved by potentiating immune responses, lowering oxidative stress, acting as a neuroprotector, and attenuating inflammatory processes [3, 7]. Dietary restriction has, therefore, robust eVects on delaying mortality, increasing the lifespan (and life quality), and attenuating chronic diseases of aging.

4. Mitochondria as a Source of Reactive Oxygen and Nitrogen Species Mitochondrial dysfunction is an important component of aging, type 2 diabetes, cancer, and neurodegenerative disorders such Alzheimer or Parkinson disease [8, 9]. Oxidative stress has been considered a main pathogenic process associated with mitochondrial dysfunction [3]. Superoxide and hydrogen peroxide are two main ROS produced in mitochondria. The majority of mitochondrial superoxide (70–80%) is released to mitochondrial matrix whereas the remaining 20–30% are released into the intermembrane space [3]. Intramitochondrial manganese superoxide dismutase (MnSOD) catalyzes superoxide transformation into hydrogen peroxide inside the mitochondrial matrix. Hydrogen peroxide production is modulated by the mitochondrial metabolic state and by the intramitochondrial concentration of nitric oxide (NO). Rates of hydrogen peroxide production are aVected by ion movements through the inner mitochondrial membrane. Nitric oxide is produced by NO donors or by nitric oxide synthase (NOS) localized within the mitochondria (mtNOS) although there is still some controversy concerning its existence [3]. The enzymatic reaction requires arginine, nicotinamide adenine dinucleotide phosphate (NADPH2), and O2 as substrates and produces citrulin, NO, and H2O (Fig. 2). NO inhibits complex III electron transfer and increases superoxide and hydrogen peroxide production. NO is transformed to peroxynitrite which is strong

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O2

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NADPH NOS

L-arginine

Citrulline NADH

NO• O2− OONO−

Superoxide Peroxynitrite

H+ HO• + NO2− Tyrosine Nitrotyrosine FIG. 2. Nitric oxide and nitrotyrosine generation.

oxidant and inhibitor of both complexes I and III. Peroxynitrite remains in the intramitochondrial space and leads to mitochondrial dysfunction and apoptosis. It is the source for nitration of tyrosine residues in proteins and peptides. Nitrotyrosine may be detected as the resulting product. Oxidative damage is induced by ROS produced primarily as a by‐product of mitochondrial oxidative phosphorylation which is responsible for 85–90% of cellular oxygen consumption [10]. Mitochondrial ROS can cause damage to mitochondrial DNA (mtDNA), proteins, and membrane lipids and thus contributes to functional and morphological changes observed in pathological states. This process, in addition, has self‐perpetuating cycle character because increased ROS production leads to incremental damage and further ROS generation [11]. Main‐free radical source is the mitochondrial respiratory chain where oxygen radical generation has been attributed to complex I and III (Fig. 3) [12–14]. The importance of complex I for the ROS production in comparison of short‐lived and long‐lived species has been repeatedly documented [15, 16]. The ROS generator was localized within the FeS clusters placed in the hydrophilic matrix domain of the complex I [13, 16] whereas ROS generation in the complex III was directed to the cytosolic site [17]. DiVerent localization may explain that the mtDNA oxidative damage is more common when the ROS are generated in the complex I. The previous hypothesis that ROS production depends on oxygen consumption was not confirmed. There is clear evidence that mitochondrial

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Creating H+ gradient H+

Decreasing H+ gradient

H+

H+

H+

H+ UCP

III

I

Q−

e−

II

cyt c

e−

IV e−

e− NAD+

NADH

Succinic acid

ATP

H2O

Fumaric acid

Energy ADP + P

O2

Mn-SOD H2O + O2

O•2−

O2

FIG. 3. Superoxide radical generation in the respiratory chain of mitochondria. Complexes I, II, III, and IV, UCP, uncoupling protein, MnSOD, manganese superoxide dismutase.

membrane potential is a major factor that determines ROS production [18]. Membrane potential may be lowered in the presence of mitochondrial uncouplers or inhibitors. Increased mitochondrial uncoupling protein (UCP3) content was accompanied by lower rate of ROS production [19]. Localization of ROS production within mitochondria explains why mtDNA is more susceptible to damage versus nuclear nucleic acid [20]. Increased rate of oxygen radical attack on mtDNA contributes to diVerences between short‐ and long‐lived animals [21]. A higher rate of oxidative attack in mtDNA was found in short‐lived animals. Repair mechanisms were similarly aVected in short‐lived organisms. Oxidative damage of mtDNA may be assessed by measurement of 8‐hydroxy‐20 ‐deoxyguanosine (8‐OH‐dG), a marker that has been inversely correlated to avian and mammalian longevity [20]. This relationship was not, however, found for nuclear DNA. Mutations in mtDNA caused by ROS are deleterious to cells. These include depressed respiration, enhanced radical formation, increased susceptibility to oxidative‐stress‐triggered apoptosis, accumulation of mutant mitochondria inside the cells, and ROS secretion by mutated cells. These changes observed in short‐lived animals provide convincing evidence of the role of mtDNA mutations in aging acceleration [22]. Increased aging rate due to frequent mtDNA mutations has been directly demonstrated in mice [23]. Impaired lysosomal degradation of oxidatively damaged mitochondria can also

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contribute to the aging process [24]. The low rate of mitochondrial ROS production accompanied by low levels of oxidatively damaged mtDNA may therefore delay aging. This was demonstrated in diVerent organs including brain, heart, and liver.

5. Caloric Restriction and Oxidative Stress Extensive clinical and experimental data obtained over the last decade has demonstrated that overfeeding accelerates ROS production and consequently increases a variety of vascular complications typically associated with obese subjects. Overfeeding also increases aging rate and lowers longevity. On the other hand, restriction of energy intake has been accepted as the main therapeutic principle oVering better prognosis to obese persons. Animals under caloric restriction without malnutrition maintain most physiological functions in a youthful state at more advanced ages. Caloric restriction retards age‐related diseases, such as diabetes, cardiomyopathy, nephropathy, cancer, and hypertension‐related diseases [25]. Beneficial eVects can be observed when caloric restriction is initiated not only in young age but in middle age or later [26]. Ongoing studies have demonstrated that caloric restriction may reduce aging rate in rodents [27] as well as primates [28]. These preliminary results have prompted further initiatives to elucidate the exact molecular mechanisms involved with this protective process. 5.1. ROS REDUCTION BY CALORIC RESTRICTION OR MODERATE EXERCISE The protective eVects of caloric restriction on oxidative damage via reduction of mitochondrial free radical generation has been well demonstrated in rat liver [29], heart [30], skeletal muscle [31], and brain [32]. In many experimental studies, a 40% reduction in caloric intake was suYcient to demonstrate the beneficial eVects of reduced ROS production in as few as 7 weeks [33]. Decreased mitochondrial ROS production within complex I of the respiratory chain was found in a variety of studies. More detailed studies have conclusively shown that decreased mitochondrial ROS production was related to diet composition. While protein restriction caused a significant decrease in ROS generation and oxidative mtDNA damage, no such eVects were found with carbohydrate‐ or fat‐restricted diets [34–37]. Lowered protein intake, not caloric intake, was found to be responsible for decreased mitochondrial ROS generation. Interestingly, methionine restriction appeared to be specifically responsible for decreased ROS production in caloric‐restricted diet [34]. Because 40% caloric reduction may be considered too excessive, the eVects of lower‐restricted diets (8.5% and 25%) have been

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investigated. Unfortunately, these lower‐restricted diets were generally ineVective in changing rates of ROS production or oxygen consumption in rat liver mitochondria [38]. A 25% caloric‐restricted diet appears to have opposing eVects on respiratory chain complex I and III activities. Decreased complex I activity tended to decrease mitochondrial ROS production, whereas increased complex III activity tended to increase ROS generation. These observations clearly indicate that greater than 25% caloric reduction is required to decrease mitochondrial oxygen radical generation due to significantly reduced complex I activity within the respiratory chain activity. However, energy intake is not the only possible modulator of oxidative stress in experimental animals or humans. Physical exercise was found to influence both ROS production and antioxidant defense mechanisms. It has been shown that exercise decreased ROS production as demonstrated by lowered malondialdehyde concentration [39], increased ROS generation as confirmed by higher plasma carbonyl derivatives [40], or has a neutral eVect [41]. Activation of antioxidant defense systems was substantiated by increased superoxide dismutase activity or plasma vitamin E concentration [39, 40, 42]. However, decreased protective eVects of antioxidant mechanisms have also been observed [43, 44]. These controversial results were recently reviewed [45]. Changes in oxidative stress were followed during moderate physical exercise by the increased median lifespan, decreased oxidative damage, and prevention in the decline of cytochrome oxidase activity [46]. Regular exercise appears to retard accumulation of cell damage and physiologic dysfunction [47]. Moderate exercise activates DNA repair and increases resistance against oxidative stress [46]. Positive eVects of moderate exercise have been observed in organs of experimental animals. On the other hand, the high‐intensity or long‐duration exercise accelerates oxidative stress and decreases reduced glutathione (GSH) and oxidized glutathione (GSSG) ratio (GSH/GSSG) [48–50]. The resultant oxidative stress level is dependent on exercise intensity. Moderate and chronic physical activity may decrease oxidative stress levels whereas acute and intense exercise accelerates ROS production and may decrease antioxidant enzyme activity [51, 52]. Caloric restriction in combination with moderate physical exercise may be protective against oxidative stress [53]. Lifestyle modification based on both regimes (lower caloric intake and moderate physical activity) has beneficial outcomes associated with oxidative stress reduction [54]. 5.2. GENDER DIFFERENCES The link between gender and cardiovascular disease is well documented in human and animal studies [55, 56]. Although protection of females against cardiovascular complications has been primarily attributed to sex hormones

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[56], the role of mitochondrial respiratory chain in this process has only been examined over the last few years [57]. Clear gender diVerences in mitochondrial energy metabolism (and likely ROS production) has been reported in rat liver, skeletal muscle, and adipose tissue [58–60]. To further investigate this finding, the eVect of caloric restriction on ROS generation in cardiac muscle tissue was compared in female and male rats [57]. Female cardiac muscle exhibited lower mitochondrial content without any loss of functionality. The caloric restriction eVect on decreased mitochondrial hydrogen peroxide production appeared to be related to lower activity of respiratory chain complexes I and III and not increased antioxidant activity. Greater mitochondrial diVerentiation with higher oxidative‐phosphorylation eYciency in female cardiac muscle has been proposed. Estrogens may, however, have positive modulating influence. For example, increased mitochondrial hydrogen peroxide generation was found in the liver and brain from ovariectomized female rats, whereas hydrogen peroxide production reverted to normal levels when these rats were estrogen supplemented [61]. Estrogen appears to have a protective eVect on lipoprotein oxidation in supplemented versus nonsupplemented postmenopausal women. SuYcient estrogen levels may decrease lipid peroxidation. As such gender should therefore be taken into account when comparing the eVects of caloric restriction on oxidative stress. 5.3. FASTING AND ROS PRODUCTION Caloric restriction without malnutrition decreases ROS production and promotes the beneficial eVects of improved longevity and delayed onset of several diseases. Although very short fasting has some benefits, prolonged fasting over 24 h may be harmful [62]. Severe food deprivation promotes oxidative stress by increased mitochondrial free radical generation and increased sensitivity of hepatic membranes to oxidative damage (lipid peroxidation). The oxidative changes are induced either by reactive carbonyl compounds or through amino acid oxidation [62]. Starvation induces superoxide anion release from hepatocytes with paralleled GSH decrease [63]. Depletion of liver antioxidant stores and release of hepatic oxygen free radicals may cause organ damage and increase morbidity in malnourished individuals. Nevertheless, intermittent fasting has been shown to exert similar beneficial eVects to caloric restriction, decreasing risk factors for cardiovascular disease and increasing life span [64]. Both caloric restriction and intermittent fasting may be related to decreased production of free radicals and improved activity of protective mechanisms [65]. In addition, increased resistance to oxidative stress during intermittent fasting could contribute to beneficial eVects of this regimen [66].

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6. Oxidative Stress Markers by Caloric Restriction The level of oxidative stress depends on ROS production and the eYciency of antioxidant system to scavenge it. As such, it is important to analytically evaluate both mechanisms in the tissue/organ or appropriate body fluid to ascertain actual oxidative state. Measurement of specific analytes for confirming oxidative stress level may be useful clinically for at least two reasons. Stress markers indicate actual risk of oxidative damage and may predict eYcacy of therapeutic approach. As such, measurement of these biomarkers in plasma or urine may be useful clinically. Although direct measurement of ROS production may be accomplished under experimental conditions, it is seldom performed in clinical practice. Alternatively, products of lipid or protein oxidation may be measured as evidence of oxidative stress level. The protective barrier of antioxidant mechanisms involving enzymatic or nonenzymatic compounds may characterize inherent ability to mitigate oxidative stress.

6.1. REACTIVE OXYGEN OR NITROGEN SPECIES (ROS, RNS) Mitochondria is the main ROS generator producing, most importantly, superoxide and hydrogen peroxide [3]. NO is generated from arginine by mtNOS and may be subsequently transformed to peroxynitrite. Hydrogen peroxide is the source of harmful hydroxyl radical whereas peroxynitrite increases protein nitration. These metabolites are prooxidants that accelerate oxidative stress. Overfeeding‐induced higher ROS production may be confirmed by increased levels of lipid or protein oxidation products. Food or caloric restriction is an eVective modulator of oxidative stress. Reduced ROS generation in mitochondria was repeatedly demonstrated following restricted diet. 6.1.1. Hydrogen Peroxide Hydrogen peroxide (H2O2) is usually produced by reduction of superoxide via the reaction catalyzed by superoxide dismutase. Interestingly, hydrogen peroxide has been found in the skeletal muscle of a unique strain of rats characterized by a self‐low‐caloric intake, fed a high‐carbohydrate diet [67]. H2O2 was restored when a high‐fat diet was consumed [67]. Caloric restriction (40%) did not alter proton leak or H2O2 production in rat liver [68]. The observed diVerences in results may be partly explained by the various experimental conditions used in these studies. Due to its inconvenience, direct H2O2 measurement as a marker of oxidative stress has not, however, been utilized in clinical practice.

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6.1.2. NO and Its Reactive Compounds Although, NO and related metabolites accelerate oxidative stress, their direct measurement has been limited in clinical studies. Reactions producing peroxynitrite and consequently protein and lipid nitration have been studied in experimental conditions [3]. Reactive intermediates are not reliable as markers of oxidative stress in human studies due to their short half‐life and inherent instability. Nitrotyrosine is better indicator of protein nitration and thus can be used in studies evaluating the role of NO in oxidative and/or nitrative stress.

6.2. OXIDATIVE PRODUCTS Several markers that indicate oxidative stress level have been used in both experimental and human studies. These typically represent molecules derived by ROS or reactive nitrogen species (RNS) in tissue or body fluids. Oxidized plasma lipids or susceptibility of plasma lipids to in vitro oxidation and subsequent measurement of conjugated dienes are examples of such oxidative products [45]. Oxidized low‐density lipoprotein (LDL) is strongly immunogenic and autoantibodies produced against oxidized LDL may be used as a biomarker to confirm these oxidative changes in LDL molecules. 6.2.1. Malondialdehyde and thiobarbituric acid‐reactive substances (TBARS) Lipid peroxidation results in malondialdehyde production which can be measured spectrophotometrically via reaction with thiobarbituric acid (TBA) or by high‐performance liquid chromatography (HPLC). Nonspecific reaction products in biological fluids when TBA reacts with other compounds like saccharides or bilirubin are described as TBARS. Measurement of malondialdehyde demonstrated that hypercaloric diet caused an increase in ROS production. Caloric restriction was associated with decreased plasma concentration of oxidized LDL [69]. Significantly lower urinary malondialdehyde levels have been found after short fasting in healthy women [70]. A nonsignificant decrease in plasma malondialdehyde was found after very low caloric diet (600 kcal) for 8 days in type 2 diabetic patients. In contrast, significantly decreased malondialdehyde levels found in healthy persons support a suspicion on the influence of insulin resistance which may reduce the eVect of caloric restriction [71]. Similarly malondialdehyde was unchanged after caloric restriction in streptozotocin‐induced diabetic rats whereas a small decrease was observed in nondiabetic animals [72]. Chronic undernutrition like in marasmic children increased oxidant

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status and decreased antioxidant mechanisms as demonstrated from increased malondialdehyde and lowered antioxidant potential [73]. In summary, malondialdehyde may be useful as a simple oxidative stress biomarker for clinical and experimental studies that evaluate caloric impact. Although its plasma concentration may be increased acutely or as a consequence of oxidative stress activation, its concentration may decrease if experimental conditions favor decreased lipid peroxidation. As such, diVerentiation of acute and chronic phases is important to properly evaluate the overall oxidative stress state. 6.2.2. F‐2‐Isoprostanes F‐2‐isoprostane, an analyte derived from peroxidation of arachidonic acid, has been associated with intensity of lipid peroxidation in biologic fluids and tissues. Measurement of 8‐isoprostaglandin F‐2 alpha is considered as a sensitive marker of oxidative stress [74]. Interestingly, increased F‐2‐isoprostanes plasma concentration were correlated to increased lipid oxidation in aging of rats [75]. Caloric restriction decreased plasma as well as liver and kidney concentrations [76]. In addition F‐2‐isoprostanes significantly correlate with 8‐oxodeoxyguanosine, an indicator of DNA oxidation. Short‐term fasting reduces lipid peroxidation products as demonstrated by decreased urinary 8‐isoprostaglandin F‐2 alpha and malondialdehyde in healthy women [70]. Combination of exercise with either high‐calorie or low‐calorie diet was associated with significant decrease of serum F‐2‐ isoprostanes [77]. Exercise was, therefore, considered as the main factor contributing to reduced lipid peroxidation, independent of caloric intake. Lifestyle modification characterized by dietary and exercise intervention may ameliorate factors associated with atherosclerosis [54]. A significant decrease in the concentration of 8‐isoprostaglandin F‐2 alpha confirmed a reduction in ROS production [54]. Because F‐2‐isoprostanes are sensitive markers of lipid peroxidation, they may be used as one of the best indicators of oxidative stress. To comprehensively evaluate divergencies in the role of F‐2 isoprostanes, urine and plasma levels should be assessed simultaneously [78]. 6.2.3. Nitrotyrosine Formation of peroxynitrite from NO and superoxide may induce nitration in tyrosine residues to nitrotyrosine. Caloric restriction has been associated with decreased nitrotyrosine levels in brain [79, 80]. Nitrotyrosine concentration was lower in skeletal muscle of rhesus monkeys fed a calorie‐ restricted diet [81]. This study demonstrated that caloric restriction may attenuate aging by reducing oxidative stress. Rats fed a low‐fat complex‐ carbohydrate diet reduced nitrotyrosine accumulation versus rats fed a

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high‐fat sucrose diet [82]. Accelerated oxidative stress in high‐fat diet was confirmed by increased plasma malondialdehyde concentration which was reduced by low‐fat diet. Unfortunately, only limited data are available on nitrotyrosine in human dietary studies. Additional studies are clearly indicated. 6.2.4. 8‐Hydroxydeoxy‐Guanosine mtDNA damage is related to accelerated ROS production. 8‐Hydroxydeoxy‐guanosine, a marker of DNA damage due to oxidative stress, has been correlated to F‐2‐isoprostanes or malondialdehyde, that is, markers of ROS generation [76]. The association of increased oxidative stress with mtDNA damage was demonstrated in mice heterozygous for the SOD2 gene which encodes mitochondrial Mn‐superoxide dismutase (MnSOD). Oxidative mtDNA damage has been correlated to increased 8‐hydroxydeoxy‐guanosine in nuclear DNA [83]. Caloric restriction was associated with reduced ROS production and decreased DNA damage as measured by 8‐hydroxydeoxy‐ guanosine [76]. However, short‐term fasting, despite decreased lipid peroxidation, did not reduce DNA damage as measured by this marker [70]. Decreased 8‐hydroxydeoxy‐guanosine concentration was not consistently correlated to ROS generation. As such, DNA damage should be evaluated independently when changes in oxidative stress are observed. 6.3. ANTIOXIDANT ENZYMES Enzymes that detoxify free oxygen radicals and their related molecules play an important role in the balance of ROS production and elimination. Enzyme activity significantly impacts intracellular oxidative stress level. Outcomes are frequently dependent on cooperation of several enzymes that catalyze transformation of ROS intermediates to stable molecules. As such, evaluation of oxidative stress should be based on determination of enzyme activity that influence these cascade reactions of intermediate ROS molecules. In addition, it is important to note that genetic and environmental factors may also influence enzyme activity and hence their eYciency to catalyze these detoxifying reactions. 6.3.1. Superoxide Dismutase Superoxide is generated by cytoplasmic Cu, Zn‐SOD, and mitochondrial MnSOD. Overeating associated with high energy intake may cause dramatic increase of substrates for metabolic pathways that subsequently accelerate the mitochondrial respiratory chain. High superoxide generation is often combined with increased SOD activity. Reduced energy intake produces fewer free radicals leading to less oxidative damage. It could, therefore, be

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interesting to determine if caloric restriction can influence SOD activity. Interestingly, experimental studies using calorie‐restricted diets showed increased [84], decreased [85], or no changed [72, 86, 87] SOD activity. In another study, MnSOD mRNA was specifically increased following short‐term calorie restriction [88]. Actual MnSOD level was not, however, increased. Increased myocardial SOD was explained by synergistic action of olive oil added to calorie‐restricted diet [89]. Short‐term very low calorie diet induced a significant increase of SOD activity in both diabetic and control subjects [71]. Aging was associated with increased ROS generation, whereas combination with food restriction lead to attenuated aging. SOD activity, while increased with aging, were found to be substantially attenuated by food restriction [90].

6.3.2. Catalase Catalase, a ubiquitous heme protein that catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen, occupies an important downstream position relative to SOD. It would therefore be expected that this close interplay would contribute to a synergistic relationship [84–86]. Caloric restriction was associated with increased [84], decreased, or unchanged catalase activity. Increased SOD activity sometimes occurs with decreased catalase activity [85], but with increased glutathione peroxidase (GPx) activity. An oscillatory relationship between catalase and GPx activity has also been proposed [85].

6.3.3. Glutathione Peroxidase GPx detoxifies peroxides with GSH which acts as an electron donor thus producing GSSG. DiVerent GPx isoenzymes have been described in the cytosolic membrane or extracellular compartments [91]. GPx may also share the same molecular substrate, hydrogen peroxide, with catalase. Accelerated ROS production has been associated frequently with increased GPx activity [84, 86]. Enzyme activity was decreased in diabetic animals fed ad libitum, but was increased by caloric restriction in both diabetic and control animals [86]. A significant decrease in GPx activity may suggest enzyme inactivation by increased ROS or decreased substrate (GSH) availability. In such cases, an oscillatory increase in catalase activity has been observed [86]. Significantly increased GPx activity together with increased GSH/GSSG ratio in mouse kidney was found after caloric restriction [87]. These results likely indicate increased antioxidant capacity.

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6.3.4. Glutathione Reductase Glutathione reductase (GR) plays a pivotal role in antioxidant defense mechanisms by recycling GSH from GSSG. Increased GR as well as GPx activity have been found in experimental animals subjected to caloric restriction [85]. This finding may thus explain the eVect of diet on increased capacity of antioxidative system and reduced oxidative stress. However, other experiments did not demonstrate any changes in liver GR activities in rats subjected to a 30–40% restriction in caloric intake [92, 93]. Improved antioxidative state was associated with changes of other components of oxidative stress (i.e., decreased ROS production and increased activity of other scavenger enzymes). 6.3.5. Paraoxonase 1 Paraoxonase 1 (PON1), a gender‐dependent enzyme specifically associated with high‐density lipoprotein (HDL), has been shown to hydrolyze lipid peroxides present in LDL. PON1 activity has been shown to be sensitive to diet as well as other factors [94]. Although, short‐term diet restriction was associated with decreased serum PON1 activity, hepatic PON1 mRNA levels were unchanged [94]. In rats, fasting was shown to specifically induce (within the first hours) increased serum PON1 activity [95]. This phenomenon was subsequently followed by progressively decreased PON1 activity and significantly decreased hepatic PON1 mRNA levels. Fasting resulted in decreased GPx activity possibly due to depletion of hepatic GSH and increased lipid peroxides. Deleterious eVect of prolonged fasting may, therefore, be explained by accelerated oxidative stress in association with decreased antioxidant defense mechanisms. Other conditions, like acidosis or production of ketone bodies, during prolonged fasting may further contribute to decreased PON1 activity by accelerating oxidative stress [96]. Early increase of oxidative stress during the initial hours of fasting may stimulate antioxidant response activation via increased PON1 activity [97]. The above results demonstrate that the low‐intensity oxidative stress that results from short periods of fasting would elicit a defense response via enhancement of protective antioxidant enzyme mechanisms [94]. 6.4. NONENZYMATIC SCAVENGERS The human body contains endogenous compounds which have protective role and act as antioxidants. Both high (transferin, ceruloplasmin, albumin, metalothioneins, or chaperons) or low molecular weight compounds (GSH, ascorbic acid, alpha‐tocopherol, coenzyme Q, lipoic acid, uric acid, bilirubin) play an essential role in maintaining ROS homeostasis. Although several low

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molecular weight compounds (uric acid and bilirubin) have utility in evaluating the antioxidative defense system, these are seldomly used in this capacity clinically.

6.4.1. Glutathione As described above, GSH is an important redox buVer due to its role in suppressing the oxidative factors and maintaining the normal reduced state of the cell. Structurally, GSH is a unique tripeptide composed of ‐glutamyl‐ cysteinyl‐glycine. GSH and GSSG conversion is regulated by ROS production and the antioxidant enzymes GPx and GR. GSH/GSSG ratio and determination of enzyme activity is useful to assess oxidative stress. A lower GSH/GSSG ratio is associated with increased oxidative stress. Animals overfed or fed ad libitum demonstrated decreased GSH and increased GSSG [85, 86]. In contrast, caloric restriction increased GSH levels and decreased GSSG. As animals age, mitochondrial GSH redox potential within diVerent organs becomes less negative and more prooxidant [98, 99]. A calorie‐restricted diet may thus attenuate GSH decline and therefore retard age‐related degenerative processes influenced by increased oxidative stress. Due to their important redox role, GSH and GSSG may thus be used as reliable markers in oxidative stress. Subsequent correlation to scavenger enzyme activity oVers important information on oxidative stress defense mechanisms physiologically and pathophysiologically.

6.4.2. Ascorbic Acid Ascorbic acid reduces both organic and inorganic radicals. It is involved in regeneration of tocopheryl radical to alpha‐tocopherol, a reaction that produces ascorbyl radical as a source of dehydroascorbic acid. Ascorbic acid regeneration plays an important role to decrease the prooxidative properties of dehydroascorbic acid. Regeneration of ascorbic acid and other compounds cycling between the reduced and oxidized forms occurs via electron transport mechanisms (Fig. 4). Ascorbic acid may be decreased by aging or oxidative stress [99]. In this study, caloric restriction attenuated the aging process and also slowed the decrease in ascorbic acid level in rat retina. Although short‐term very low calorie diet induced ascorbic acid increases in healthy persons, this phenomenon was not found in type 2 diabetic patients [71]. The mechanism responsible for this observation was unclear, but may be related to antioxidative mechanism abnormalities in diabetes.

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a-tocopherol

Dehydroascorbic acid

GSSG

a-lipoic acid

NAD+

Red.a-tocopherol

Ascorbic acid

2 GSH

Dihydrolipoic acid

NADH

FIG. 4. Cycling in the scavenger system.

6.4.3. Alpha‐Tocopherol Alpha‐tocopherol is lipophilic antioxidative compound that preserves lipid membranes from the eVects of peroxidation. Alpha‐tocopherol transforms fatty acid peroxyl radicals to hydroperoxides which are further acted upon by GPx. Tocopherol is transformed to tocopheryl radical which may be partly reduced by ascorbic acid. Aging is associated with increased oxidative stress characterized by increased lipid peroxidation markers, protein carbonyls, or nitrotyrosine and decreased antioxidant defenses. A positive association was found between plasma SOD, alpha‐tocopherol, and survival in a longitudinal study [100]. The highest survival was observed in patients with high serum alpha‐tocopherol and low plasma malondialdehyde concentrations [100]. Caloric restriction attenuates a decline of alpha‐tocopherol content in plasma membrane caused by aging [101, 102]. Caloric restriction was associated with decreased oxidative stress markers and increased activity of enzymes that protect cells against age‐related oxidative stress [102]. However, mitochondrial alpha‐tocopherol content was diminished by calorie‐restricted diet in rats [103]. Short‐term very low calorie diet resulted in decreased serum alpha‐tocopherol levels [71]. Unfortunately, plasma membrane content has not, however, determined. Alpha‐tocopherol changes may contribute to accelerated oxidative stress when production of tocopheryl radicals is not suYciently reduced. Supplementation of alpha‐tocopherol under conditions of increased oxidative stress may induce adverse reactions and further promote the oxidative state [104]. 6.4.4. Coenzyme Q (Ubichinone) Coenzyme Q (ubichinone) is another lipophilic antioxidant that cooperates with alpha‐tocopherol by influencing the transmembrane redox system and thus protects the cell membrane against lipid peroxidation. Coenzyme Q has three functions in mitochondria: (a) transfer of reducing equivalents in the electron transport chain (Fig. 3), (b) generation of superoxide anion radical, and (c) free radical quenching. As with alpha‐tocopherol, coenzyme

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Q decreases with aging. Although dietary supplementation with coenzyme Q augments endogenous mitochondrial content in various tissues, it had no significant eVect on the main antioxidant defenses or prooxidant generation [105, 106]. Caloric restriction has been shown to increase coenzyme Q and attenuated aging [80, 101, 103]. 6.5. OTHER MARKERS The association of fluorescent oxidation products with several indicators of oxidative stress suggests that this finding could be a global oxidative stress marker [107]. Unfortunately, there is no data that specifically evaluates caloric restriction. However, fluorescent oxidative products appear highly correlated with increased oxidative stress associated with smoking. DiVerent biochemical markers have been used to create an OXY‐SCORE index that reflects both oxidant and antioxidant markers [108]. Damage score was based on plasma free and total malondialdehyde, GSH/GSSG, and urine isoprostanes protective score whereas protective score was based on GSH, alpha‐ and gamma‐tocopherol levels, and antioxidant capacity. Although diVerences in damage and protection scores were related to age and gender, more data is clearly needed to comprehensively evaluate this index.

7. Data Interpretation Evaluation of oxidative stress is diYcult because many factors influence its final state. As such, markers of both processes, ROS generation and antioxidant defense systems, have to be determined. The ROS system is usually measured by production of oxidative products like malondialdehyde or F‐2‐ isoprostanes, that is, lipid peroxidation. The antioxidant system is evaluated by assessment of enzymes such as SOD, Catalase, GPx, and GR. Increased ROS generation is associated with increased concentration of oxidative products, whereas antioxidant enzymes may be often increased due to a secondary eVect of higher oxidative stress. Unchanged or decreased enzyme activities in the tissues or body fluids may also be detected. This phenomenon may be a consequence of enzyme inhibition due to specific condition, for example, diabetic hyperglycemia. Determination of enzyme gene expression may help to elucidate stimulation or depression of antioxidant enzymes. It is important to note that caloric restriction and prolonged fasting are diVerent. In the former, the lower energy consumption or decreased protein intake attenuate ROS generation and decrease oxidative products. Although changes in antioxidant enzymes may be induced by experimental conditions, it can be generally assumed that increased GSH attenuates oxidative stress due

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to its inherent reducing capacity. Advanced oxidative stress is frequently associated with decreased GSH and therefore decreased GSH/GSSG ratio. Monitoring oxidative markers is dependent on the experimental conditions. For example, initial changes characterized by enhanced ROS production (increased malondialdehyde concentration) may be followed by depressed ROS generation and therefore decreased malondialdehyde or F‐2‐isoprostanes. Acute and chronic phases can diVerently influence changes in the oxidative stress. Thus, oxidative markers need to be repeatedly determined during follow‐up. Oxidative products and antioxidant system must be rigorously monitored throughout the entire experimental process to properly assess impact. Prolonged fasting stimulates ROS production. Oxidative products are usually increased, whereas antioxidant enzymes may be time dependent. Longer fasting may depress enzyme activities as well as the plasma alpha‐ tocopherol. Similar diVerences have been observed between moderate and highly intensive physical activity. Depressed oxidative stress with lowered ROS production and frequently increased antioxidant enzymes have been found in moderate physical exercise. This finding contrasts with accelerated oxidative stress following intensive exercise. Because changes in oxidative markers are dynamic, repeat and appropriately timed measurements are necessary and essential. 8. Conclusions Oxidative stress is a powerful process modifying cellular reactions both in health and disease. Accurate evaluation is essential to a comprehensive understanding to influence therapeutic or experimental procedures. Prolonged complete fasting as well as intensive exercise can initiate oxidative stress with harmful ROS production whereas caloric restriction, short intermittent fasting or moderate physical activity may lower ROS generation. The results suggest that caloric restriction to lower oxidative stress shifts the balance between ROS generation and antioxidant defense systems and thus has substantial and beneficial eVects on aging and metabolic disorders like obesity or diabetes mellitus. Clearly, more research is warranted to properly evaluate the impact of caloric restriction in combination with other modalities including moderate physical activity. ACKNOWLEDGMENT This work was supported by a research project of the Ministry of Education in the Czech Republic, MSM0021620807.

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INDEX A Accreditation guidelines and laboratory quality standards, 129–131. See also Clinical laboratories, method verification Advanced glycation endproducts, 226 Advanced glycosylation endproducts, 200 AFCAPS/TexCAPS. See Air Force/Texas Coronary Atherosclerosis Prevention Study AGEs. See Advanced glycation endproducts; Advanced glycosylation endproducts Air Force/Texas Coronary Atherosclerosis Prevention Study, 72 ALA. See Alpha linolenic acid Allograft rejection (AR), 140 Alpha linolenic acid, 175 Alpha‐tocopherol, role of, 239 AmpliChip CYP450, applications, 110. See also Personalized medicine Amyloid aggregation mechanisms, 6–9 deposition and organ dysfunction, 13 fibril of amyloid A and Val30Met TTR, composition of, 6 and microenvironment, interactions, 9–12 structure of, 3–6 typing of, 17–18 Amyloid A fibril, composition of, 6 Amyloidogenic protein production mechanism of, 8–9 treatment for, 28 Amyloidosis classification, clinical presentation and prognosis of familial amyloidosis, 23–25 localized amyloidosis, 26–28 primary amyloidosis, 20–21

secondary amyloidosis, 21–23 senile amyloidosis, 25–26 diagnosis, 14–20 historical perspectives of, 2–3 pathogenesis of amyloid aggregation mechanisms, 6–9 amyloid fibril structure, 3–6 amyloid proteins, tropism, 12–13 microenvironment and amyloid fibril, interactions, 9–12 organ dysfunction, mechanisms, 13 therapies for amyloid fibril destabilizing agents, 33–34 amyloidogenic protein native structure, stabilization of, 32 amyloid precursors production, treament, 28–32 immunologic therapy, 34–35 localized amyloidosis, treatment of, 35–36 supportive care, 35 Amyloid P component protein, 10 Angiotensin II, role of, 65 Antioxidants. See also Biomarkers in health promotion, concepts of, 186–190 status, in vegetarians and nonvegetarians, 190–194 APEX. See Arrayed primer extension ApoE. See Apolipoprotein E APOE 4 allele, role of, 194 Apolipoprotein E, 66 Arachadonic acid (AA), 175 ARIC study. See Atherosclerosis Risk in Communities study Arrayed primer extension, 103 Arterial vasculature, amyloid deposition in, 13 Ascorbic acid regeneration, role of, 238 Atherosclerosis Risk in Communities study, 76 249

250

INDEX B

Bile canaliculi (BC), 158 Biochip technology, in personalized medicine development, 109–111 Biomarkers discovery pathway, in organ transplantation, 142–144 (see also Human organ transplantation) of oxidant/antioxidant, in vegetarian diets antioxidant status and oxidative stress in, 190–194 dietary antioxidants, role of, 186–190 in personalized medicine, 108–109 in vegetarian and nonvegetarian diet diVerentiation, 207–209 in vegetarians disease detection, 194–195 (see also Vegetarian diets) bone health, 201–203 cancer, 204–206 CVD and type 2 diabetes, 195–201 immune status, 206–207 renal function, 203–204 Biomedical imaging modalities, nanobiotechnology, 113–114 BNP. See Brain natriuretic peptide Body mass index (BMI), 183 Bone health detection, biomarkers, 201–203. See also Vegetarians disease detection, biomarkers Brain natriuretic peptide, 159 C Calcium content, vegetarian diets, 185. See also Minerals and trace metals, vegetarian diets Caloric restriction data interpretation, 240–241 and oxidative stress gender diVerences, 230–231 ROS reduction, 229–230 oxidative stress markers antioxidant enzymes, 235–237 biochemical markers, 240 nonenzymatic scavengers, 237–240 oxidative products, 233–235 reactive oxygen and nitrogen species, 232–233

Cancer diagnosis biomarkers in, 204–206 (see also Vegetarians disease detection, biomarkers) QDs applications for, 113 CAP. See College of American Pathologists Capillary electrophoresis coupled mass spectrometry, 148 Carcinoembryonic antigen, 46 Cardiac allograft vasculopathy, 159 Cardiac amyloidosis, measurement of, 21 Cardiac MRI, in tissue amyloid imaging, 19 Cardiovascular disease (CVD) epidemiology and clinical studies, 60–62 and gender, 230–231 HRT, mechanisms of action estrogens, 65–66 phytoestrogens, 68–69 progesterone, 69–70 selective estrogen receptor modulators, 66–68 and inflammation, 62–65 risk factors of, 175, 179 and type 2 diabetes detection, biomarkers, 195–201 (see also Vegetarians disease detection, biomarkers) CARE trial. See Cholesterol And Recurrent Events trial Carninutrients, definition of, 173. See also Vegetarian diets Catalase protein, role of, 236 CAV. See Cardiac allograft vasculopathy CEA. See Carcinoembryonic antigen Cell adhesion molecules, 76–78 CE MS. See Capillary electrophoresis coupled mass spectrometry Cerebrospinal fluid, 148 Cerebrovascular accident, 195 Cholesterol And Recurrent Events trial, 82 CID. See Collision‐induced dissociation CLA. See Conjugated linoleic acid CLIA’88. See Clinical Laboratory Improvement Amendments of 1988 Clinical laboratories, method verification, 122–123 accreditation guidelines and laboratory quality standards, 129–131 ISO quality management system, 123–128 performing method verification, 132, 136 accuracy, 134

INDEX analytical measurement range, 134–135 precision, 133–134 reference interval, 135 quality requirements, comparison of, 131–132 Clinical Laboratory Improvement Amendments of 1988, 122 laboratory testing by, 129 quality standards for laboratory inspection, 129 role of, 123 Clinical Laboratory Standards Institute, 121, 132 Clinical Pathology Accreditation, 121 derivation of, 131 role of, 123 standards for the medical laboratory, 131 CLSI. See Clinical Laboratory Standards Institute CLSI C28: How to Define and Determine Reference Intervals in the Clinical Laboratory, importance of, 135 CLSI EP21: Estimation of Total Analytical Error for Clinical Laboratory Methods recommendations of, 134 CLSI EP6: Evaluation of the Linearity of Quantitative Measurement Procedures recommendations of, 134–135 CLSI EP17: Protocols for Determination of Limits of Detection and Limits of Quantitation, role of, 136 CLSI EP15: User Verification of Performance for Precision and Trueness recommendations of, 134 role of, 133 CNVs. See Copy number variations Cobalamin content, vegetarian diets, 177–179. See also Vitamins content, vegetarian diets Coenzyme Q (ubichinone), role of, 239–240 College of American Pathologists guidelines of, 130 and Joint Commission, role of, 129–130 Collision‐induced dissociation, 151 Colorectal cancer analytical techniques and data analysis, 50, 52–53 epidemiology of, 46 urinary markers for, 47–50

251

Combined PCR–enzyme‐linked immunosorbent assay, application of, 102 Conducting system, amyloid deposition, 13 Congo red staining, amyloidosis diagnosis, 16 Conjugated linoleic acid, 176 Continuous ‐sheet helix, 3–4 Copper content, in vegetarian diets, 185. See also Minerals and trace metals, vegetarian diets Copy number variations, 105–106 Coronary artery disease, women, 61 Correlation spectroscopy, 52 COSY. See Correlation spectroscopy CPA. See Clinical Pathology Accreditation 11 C‐PIB. See Pittsburg compound B, in tissue amyloidosis imaging C‐reactive protein, 64, 71–75, 191 Cross‐ spine. See Continuous ‐sheet helix CRP. See C‐reactive protein CSF. See Cerebrospinal fluid Cutaneous amyloidosis, 27. See also Amyloidosis CVA. See Cerebrovascular accident Cystic fibrosis, INDELs role, 106 Cytogenetics, in personalized medicine, 114–116 Cytomics and personalized medicine, 115–116 D DCM. See Dilated cardiomyopathy 2D DIGE. See Two‐dimensional diVerence gel electrophoresis 2DE. See 2D gel electrophoresis 2D gel electrophoresis, 145 DHA. See Docosahexaenoic acid DHB. See 2,5‐Dihydroxybenzoic acid 1,25‐DHD. See 1,25‐ Dihydroxycholecalciferol DHN‐MA. See 1,4‐Dihydroxynonane mercapturic acid Dietary antioxidants in health support, role of, 186–190 Dietary restriction, of aging process, 226 Diet‐based metabolomic profile, colorectal cancer development, 49 Diet‐derived metabolic markers, colorectal cancer, 48–49

252

INDEX

Diflunisal, in familial amyloidosis treatment, 32 2,5‐Dihydroxybenzoic acid, 148 1,25‐Dihydroxycholecalciferol, 180 1,4‐Dihydroxynonane mercapturic acid, 49 Dilated cardiomyopathy, 158 2D J‐resolved spectroscopy, role of, 52 DNA microarray method, 109 Docosahexaenoic acid, 175 E Echocardiography, tissue amyloid imaging, 18–19 EFA. See Essential fatty acids EGC. See Epigallocatechin eGFR. See Estimated GFR Eicosopentaenoic acid, 175 Electrospray ionization, 146 EP. See Evaluation protocols EPA. See Eicosopentaenoic acid Epigallocatechin, 48 EP7: Interference Testing in Clinical Chemistry, role of, 135 EP17, recommendations of, 136 ERA. See Estrogen replacement and atherosclerosis Erythrocyte sedimentation rate, 207 E‐selectin, role of, 63, 78–79 ESI. See Electrospray ionization ESR. See Erythrocyte sedimentation rate Essential fatty acids, 175–177 Estimated GFR, 209 Estrogen replacement and atherosclerosis, 61 Estrogens eVect, cardiovascular system, 65–66 Etanercept, amyloidosis treatment, 29–30 Evaluation protocols, 122 Evidence Cardiac Panel, application of, 111 F Familial amyloidosis classification, clinical presentation and prognosis of, 23–25 transplantation for, 30–31 Familial amyloidotic polyneuropathy, 23 FAP. See Familial amyloidotic polyneuropathy Fast Revascularization during Instability in Coronary artery disease II trial, 80

Fatty acids, measurement of, 177 Fecal occult blood testing, colorectal cancer, 46 Federal CLIA’88 quality laws, 129 Ferric‐reducing/antioxidant power, 190 Ferritin content, vegetarians, 183. See also Minerals and trace metals, vegetarian diets FFE. See Free‐flow electrophoresis Fibrilogenesis, oligomers, 6–7 Fibrinogen A‐chain amyloidosis, 25 FISH. See Fluorescent in situ hybridization F‐2‐isoprostane markers, application of, 234 Fluorescent in situ hybridization, 114 Fourier transform mass spectrometry, 52 FRAP. See Ferric‐reducing/antioxidant power Free‐flow electrophoresis, 155 FRISC II trial. See Fast Revascularization during Instability in Coronary artery disease II trial FT‐MS. See Fourier transform mass spectrometry G Gas chromatography and mass spectrometry, 177 Gas–liquid chromatography, 177 Gastrointestinal involvement, in amyloidosis, 14 GC‐MS. See Gas chromatography and mass spectrometry Gel‐based methods, organ transplantation, 145. See also Proteomics Gel‐free methods, organ transplantation, 146. See also Proteomics Gene‐based molecular diagnostics, future perspectives, 117 GeneChip, clinical applications, 109. See also Personalized medicine Genetic diseases, CNV role, 106 Genome to Phenome Superhighway, 53 Genomics‐based molecular profiling, future perspectives of, 117 Genotyping technology, characteristics, 104–105 GFR. See Glomerular filtration rate GLC. See Gas–liquid chromatography Glomerular filtration rate, 200

INDEX Glutathione peroxidase (GPx), 186, 236 Glutathione reductase (GR), 237 Glycidyl methacrylate, 114 Glycosaminoglycans, role of, 12 GMA. See Glycidyl methacrylate GNNQQNY peptide, structure of, 5 Gold nanoparticles, molecular diagnostics, 112 GPS. See Genome to Phenome Superhighway GPx. See Glutathione peroxidase Graft versus host disease, 159 GSH. See Reduced glutathione GSSG. See Oxidized glutathione GVHD. See Graft versus host disease H HDL. See High‐density lipoprotein HDL‐C. See High‐density lipoprotein cholesterol Heart, amyloid deposition, 13 Heart and Estrogen/Progestin Replacement Study, 61 Heart transplantation, 158–159. See also Human organ transplantation Heat shock protein, 159 Hematopoietic stem cell transplantation, 159 Heme‐iron, sources of, 182 Hemoglobin concentration and vegetarian diets, 182. See also Minerals and trace metals, vegetarian diets HERS. See Heart and Estrogen/Progestin Replacement Study Heteronuclear multiple quantum coherence technique, 52 HG‐U133 Plus 2.0 Array, 109 High‐density lipoprotein, 21, 64 High‐density lipoprotein cholesterol, 196 High‐performance liquid chromatography, 177, 233 High‐resolution genome‐wide association studies, 104 High‐resolution map, for structural variants, 107 HMQC technique. See Heteronuclear multiple quantum coherence technique Holotranscobalamin II, definition of, 178. See also Vitamins content, vegetarian diets

253

HOMA‐IR. See Homeostasis model assessment‐insulin resistance Homeostasis model assessment‐insulin resistance, 183 Hormone replacement therapy, 60 eVects on inflammatory markers cell adhesion molecules, 76–78 C‐reactive protein, 71–75 Interleukin‐6, 80–81 monocyte chemoattractant protein‐ 1, 81–82 selectins, 78–80 serum amyloid A, 75–76 tumor necrosis factor‐, 82 mechanisms of action, cardiovascular disease estrogens, 65–66 phytoestrogens, 68–69 progesterone, 69–70 selective estrogen receptor modulators, 66–68 HPLC. See High‐performance liquid chromatography HPLC mass spectrometry, 148 HRT. See Hormone replacement therapy HSCT. See Hematopoietic stem cell transplantation HSP. See Heat shock protein Human amyloid disease, proteins in, 7 Human genome, genetic variations in, 105–108 Human Genome Project, 107 Human organ transplantation issues in, 160–162 outcomes in, 140–141 peptidomics in, 146–148 protein and peptide biomarkers discovery for, 142 proteomes and peptidomes, quantification of, 149–151 proteomics and peptidomics application of, 155–159 sample processing for, 151–155 proteomics role in, 141–146 Hydrogen peroxide (H2O2) measurement, 25‐HydroxyD, 180 8‐Hydroxy‐2 deoxyguanosine, 190 8‐Hydroxy‐20‐deoxyguanosine, 228 8‐Hydroxydeoxy‐guanosine marker, 235 Hypercholesterolemia, role of, 64

254

INDEX I

ICAM‐1. See Intercellular adhesion molecule ICAT. See Isotope Coded AYnity Tags IEF. See Isoelectric focusing IGFBP‐1. See Insulin growth factor binding protein‐1 IHD. See Ischemic heart disease IL‐6. See Interleukin‐6 Immobilized pH gradient, 145 Immune status, biomarkers, 206–207. See also Vegetarians disease detection, biomarkers Immunohistochemical analysis, for amyloidosis, 16 IMT. See Intima media thickness INDELs. See Insertions and deletions Individualized therapy. See Personalized medicine Inflammation and vascular disease, 62–65 Inflammatory markers, HRT eVects cell adhesion molecules, 76–78 C‐reactive protein, 71–75 Interleukin‐6, 80–81 monocyte chemoattractant protein‐1, 81–82 selectins, 78–80 serum amyloid A, 75–76 tumor necrosis factor‐, 82 Insertions and deletions, 105–106 Insulin growth factor binding protein‐ 1, 158–159 Intercellular adhesion molecule, 62 Interleukin‐6, 80–81 International Society for Heart and Lung Transplantation, 159 Intima media thickness, 80 Iodine content, vegetarian diets, 185–186. See also Minerals and trace metals, vegetarian diets IPG. See Immobilized pH gradient Iron content, vegetarian diets, 181–184 Ischemic heart disease, 158 ISHLT. See International Society for Heart and Lung Transplantation ISO 9000 importance of, 123–124 quality management principles of, 125 ISO 9001 design and development verification of, 125 role of, 123–124

ISO 15189 quality management systems in, 128 recommendations of, 128 role of, 125 Isoelectric focusing, 155 ISO 15189 Medical Laboratories, 123–124 ISO 22870 point‐of‐care testing, 128 8‐Isoprostaglandin F‐2 alpha, measurement of, 234 ISO quality management system, 123–128. See also Clinical laboratories, method verification ISO 9004, role of, 123–124 Isotope Coded AYnity Tags, 150–151 J Joint Commission and CAP, 129–130 K Kidney, amyloid deposition in, 13 Kidney transplantation, 156. See also Human organ transplantation L Laboratory quality standards and accreditation guidelines, 129–131 Lacto‐ovo‐vegetarians, 198 LC‐MS. See HPLC mass spectrometry; Liquid chromatography coupled with mass spectrometry LDL‐C. See Low‐density lipoprotein cholesterol LDL. See Low‐density lipoprotein Lenalidomide, in amyloidosis treatment, 29 Linoleic acid (LA), 175 Lipoprotein‐associated phospholipase A2, 196 Liquid chromatography coupled with mass spectrometry, 146 Liver transplantation, 156–158. See also Human organ transplantation Localized amyloidosis, 26–28, 35–36. See also Amyloidosis LOV. See Lacto‐ovo‐vegetarians Low‐density lipoprotein, 64, 233

INDEX Low‐density lipoprotein cholesterol, 196 Lp‐PLA2. See Lipoprotein‐associated phospholipase A2 M MALDI. See Matrix‐assisted laser desorption/ionization Malondialdehyde (MDA), 190, 233–234 Manganese superoxide dismutase, 226 MARS. See Multiple AYnity Removal System Matrix‐assisted laser desorption/ ionization, 52, 146 MCHC. See Mean corpuscular hemoglobin concentration MCP‐1. See Monocyte chemoattractant protein‐1 MCV. See Mean corpuscular volume Mean corpuscular hemoglobin concentration, 183 Mean corpuscular volume, 183 Medin associated aortic medial amyloid, 28. See also Amyloidosis 40‐MeEGC. See 40‐O‐methylepigallocatechin Melphalan and prednisone, amyloidosis treatment, 29 Metabolomic profile analysis, urinary substances, 47 Methicillin‐resistant Staphylococcus aureus, 101 Methicillin‐sensitive Staphylococcus aureus, 101 Methylmalonic acid (MMA), 178 MIAME. See Minimum Information about Microarray Experiment MIAPE. See Minimum Information about a Proteomics Experiment Microarray chip, 110 Microarray technology, application of, 109. See also Personalized medicine Microextraction techniques, amyloid fibrils, 17 Micropurification techniques, amyloid fibrils, 17 Minerals and trace metals, in vegetarian diets copper and calcium, 185 iodine, 185–186 iron, 181–184 zinc, 184

255

Minimum Information about a Proteomics Experiment, 161 Minimum Information about Microarray Experiment, 161 Mitochondrial dysfunction and reactive oxygen species, 226–229 Mitochondrial energy metabolism, gender diVerences, 231 M‐LAC. See Multilectin aYnity chromatography MnSOD. See Manganese superoxide dismutase Molecular diagnostics and therapeutics integration, personalized medicine, 116–117 Molecular diagnostics role, personalized medicine, 97 Monocyte chemoattractant protein‐1, 63, 81–82 MORE trial. See Multiple Outcomes of Raloxifene Evaluation trial MRM. See Multiple reaction monitoring MRSA. See Methicillin‐resistant Staphylococcus aureus MSSA. See Methicillin‐sensitive Staphylococcus aureus MudPIT. See Multidimensional protein identification technology Multidimensional protein identification technology, 146 Multilectin aYnity chromatography, 152 Multimarker DNA‐based stool testing, 46 Multiple AYnity Removal System, 152 Multiple Outcomes of Raloxifene Evaluation trial, 67 Multiple reaction monitoring, 151 Myocardial infarction (MI), 195 N NADPH2. See Nicotinamide adenine dinucleotide phosphate Nanobiotechnology, in personalized medicine, 111–114 NanoESI‐QTOF. See Nanospray quadrupole TOF Nanolaser scanning confocal spectroscopy, 112. See also Personalized medicine Nanoparticles, molecular diagnostics, 112–114. See also Personalized medicine

256

INDEX

Nanoscale single cell, molecule identification, 112. See also Personalized medicine Nanospray quadrupole TOF, 148 NAP‐2. See Neutrophil‐activating protein‐2 Neutrophil‐activating protein‐2, 158 Nicotinamide adenine dinucleotide phosphate, 226 Nitric oxide and nitrotyrosine, generation of, 226–227 Nitrotyrosine concentration, measurement of, 234–235 Nodular primary localized cutaneous amyloidosis, 27 Nonsteroidal anti‐inflammatory drugs, 48 Nonvegetarian diets antioxidant status and oxidative stress, 190–194 and vegetarian diets, biomarkers to distinguish, 207–209 NPLCA. See Nodular primary localized cutaneous amyloidosis n‐6 PUFA. See Omega‐6 PUFA NSAID. See Nonsteroidal anti‐inflammatory drugs Nuclear magnetic resonance (NMR) spectroscopy, for urinary markers detection, 50, 52 O Obesity and ROS generation, 225–226 25(OH)D. See 25‐HydroxyD 8‐OHdG. See 8‐Hydroxy‐2 deoxyguanosine 8‐OH‐dG. See 8‐Hydroxy‐20‐deoxyguanosine Omega‐6 PUFA, 175 Omega‐3 series content, vegetarian diets, 175–177. See also Vegetarian diets 40‐O‐methylepigallocatechin, 48 Organ dysfunction, amyloid role in, 13 Oxidative damage, mitochondrial DNA, 228 Oxidative stress and caloric restriction gender diVerences, 230–231 ROS reduction, 229–230 data interpretation, 240–241 markers, by caloric restriction antioxidant enzymes, 235–237 biochemical markers, 240 nonenzymatic scavengers, 237–240

oxidative products, 233–235 reactive oxygen and nitrogen species, 232–233 in vegetarians and nonvegetarians, 190–194 (see also Biomarkers) Oxidized glutathione, 238 Oxidized low‐density lipoprotein, usage of, 233 P Paraoxonase 1, 237 Parathyroid hormone, 185 PCA. See Principal component analysis PEPI trials. See Postmenopausal Estrogen/ Progestin Interventions trial Peptide biomarker, organ transplantation, 142. See also Human organ transplantation Peptidomics, human organ transplantation, 141, 146–148 heart, 158–159 HSCT and GVHD, 159 kidney, 156 liver, 156–158 Peripheral nerve involvement, amyloidosis, 14 Peripheral vascular disease, 195 Personalized medicine biochip technology, application in, 109–111 biomarkers role in, 108–109 combined PCR–ELISA, 102 cytogenetics role in, 114–116 definition of, 96 future perspectives of, 117–118 molecular diagnostics and therapeutics, integration of, 116–117 molecular diagnostics techniques for, 99 nanobiotechnology in, 111–114 non‐PCR methods and direct molecular analysis, 103 PCR‐based methods in development of, 99 real‐time PCR systems, 101–102 SSCP analysis, 100–101 and pharmacogenetics, 97–98 and pharmacogenomics, 96–97 pharmacoproteomics in, 98–99 and SNP, 103–105 PET. See Positron emission tomography

INDEX Pharmacogenetics application of, 108 biomarkers, application of, 108–109 and personalized medicine, 97–98 Pharmacogenomics and personalized medicine, 96–97 Pharmacoproteomics role, personalized medicine, 98–99 PHOREA trial. See Postmenopausal hormone replacement against atherosclerosis trial Phytoestrogens eVect, on cardiovascular system, 68–69 PhytoSERMs, definition of, 68 Pittsburg compound B, in tissue amyloidosis imaging, 19–20 Plasma concentrations biomarker, in vitamin B12, 178. See also Vegetarian diets Plasma uric acid, measurement of, 190 P1NP. See Procollagen type I intact N‐terminal propeptide POCT. See Point‐of‐care testing Point‐of‐care testing, 128 Polyunsaturated fatty acids, 173 PON1. See Paraoxonase 1 Positron emission tomography, 19 Postmenopausal Estrogen/Progestin Interventions trial, 72 Postmenopausal hormone replacement against atherosclerosis trial, 79 Posttransplant lymphoproliferative disease, 158 Primary amyloidosis. See also Amyloidosis classification, clinical presentation and prognosis of, 20–21 diagnosis of, 15 therapies for, 29–36 Primary proinflammatory cytokines, 71 Principal component analysis, 47 Probe microarray. See Microarray chip Procollagen type I intact N‐terminal propeptide, 201 Progesterone eVect, on cardiovascular system, 69–70 Protein and amino acids content, vegetarian diets, 174–175. See also Vegetarian diets Protein biochip technology, applications, 111. See also Personalized medicine

257

Protein biomarker, organ transplantation, 142. See also Human organ transplantation ProteinChip biomarker system, applications, 110–111 Proteolytic cleavage, amyloid formation, 9 Proteomes and peptidomes, quantification of, 149–151 Proteomics based molecular diagnostics, role of, 98–99 evolution of, 142–145 in human organ transplantation, 141, 155–156 heart, 158–159 HSCT and GVHD, 159 kidney, 156 liver, 156–158 methods in, 145–146 and peptidomic, sample processing for abundant proteins and fractionation, depletion of, 152–155 sample collection and handling, 151–152 Proteus mirabilis, 207 Protofibrils, 8 P‐selectin, 63, 78 PTH. See Parathyroid hormone PTLD. See Posttransplant lymphoproliferative disease PUFA. See Polyunsaturated fatty acids PVD. See Peripheral vascular disease Q Quality Management System Model for Health Care, 132 Quantum dots (QDs) based FISH, 116 for molecular diagnostics, 112–113 R Radioiodinated amyloid P component scan, in tissue amyloidosis imaging, 19–20 Radionuclide imaging techniques, tissue amyloid imaging, 19 Raloxifene Use for The Heart trial, 67 RANTES. See Regulated‐on‐activation, normal T‐expressed and secreted RDI. See Recommended daily intake Reactive nitrogen species, 233

258

INDEX

Reactive oxygen species (ROS), 183, 224–225. See also Caloric restriction data interpretation, 240–241 factors aVecting, 225 and mitochondrial dysfunction, 226–229 oxidative stress measurement antioxidant enzymes, 235–237 biochemical markers, 240 nonenzymatic scavengers, 237–240 oxidative products, 233–235 reactive oxygen and nitrogen species, 232–233 production and fasting, 231 reduction, by caloric restriction and exercise, 229–230 Real‐time PCR systems, applications of, 101–102 Receiver operator characteristics, 47 Recommended daily intake, 187 Reduced glutathione, 238 Reference interval, definition of, 135. See also Clinical laboratories, method verification Regulated‐on‐activation, normal T‐expressed and secreted, 158 Renal function detection, biomarkers, 203–204. See also Vegetarians disease detection, biomarkers Renal involvement, amyloidosis, 14 Respiratory syncytial virus, 113 Reversed‐phase high‐performance liquid chromatography, 47 Reverse phase chromatography, 146 RNS. See Reactive nitrogen species ROC. See Receiver operator characteristic RPC. See Reverse phase chromatography RP‐HPLC. See Reversed‐phase high‐ performance liquid chromatography RSV. See Respiratory syncytial virus RUTH trial. See Raloxifene Use for The Heart trial S SAA. See Serum amyloid A protein SAM. See Significance analysis of microarray SAP. See Serum amyloid P protein Scanning near‐field optical microscopy, applications, 116 SCT. See Stem cell transplantation SCX. See Strong cation exchange

Secondary (AA) amyloidosis. See also Amyloidosis classification, clinical presentation and prognosis of, 21–23 screening for, 15 therapies for, 29–36 SELDI process. See Surface‐enhanced laser desorption/ionization process SELDI‐TOF. See Surface‐enhanced laser desorption/ionization time of flight mass spectrometry Selected reaction monitoring, 151 Selectins, 63, 78–80 Selective estrogen receptor modulators, 66–68 Senile amyloidosis, 14, 25–26. See also Amyloidosis SERMs. See Selective estrogen receptor modulators Serum amyloid A protein, 4, 21, 64, 75–76 Serum amyloid P protein, 9, 33 Short neuropeptide F, 148 Significance analysis of microarray, 160 SILAC. See Stable isotope labeling by amino acids in cell culture SIMCA. See Soft independent modeling of class analogy Single biological molecules, direct analysis of, 103 Single nucleotide polymorphisms, 96, 103–105 Single proton emission computed tomography, 19 Single‐strand conformational polymorphism analysis, 99–101 Skin pigmentation and vitamin D status, 180–181. See also Vitamins content, in vegetarian diets SNP biochips, application of, 108 sNPF. See Short neuropeptide F SNPs. See Single nucleotide polymorphisms SOD. See Superoxide dismutase Soft independent modeling of class analogy, 52 Soluble transferrin receptor fragments, 182 SPECT. See Single proton emission computed tomography SRM. See Selected reaction monitoring SSCP analysis. See Single‐strand conformational polymorphism analysis Stable isotope labeling by amino acids in cell culture, 150

INDEX Stem cell transplantation, 30, 159 sTFr. See Soluble transferrin receptor fragments Strong cation exchange, 146 Structural variations (SVs), human genome, 107–108 Subcutaneous fat aspirate, amyloidosis diagnosis, 17 Superoxide dismutase, 186, 235–236 Surface‐enhanced laser desorption/ionization process, 110 Surface‐enhanced laser desorption/ionization time of flight mass spectrometry, 146 T Tamm‐Horsfall protein, 148, 153 TBARS. See Thiobarbituric acid‐reactive substances Thalidomide, amyloidosis treatment, 29 tHcy. See Total homocysteine Thiobarbituric acid‐reactive substances, 233–234 Thyroid stimulating hormone, 185 TIBC. See Total iron binding capacity TIMP‐1. See Tissue inhibitor of metalloproteinase type 1 Tissue biopsy, amyloidosis diagnosis, 15 Tissue inhibitor of metalloproteinase type 1, 46–47 TNF‐. See Tumor necrosis factor‐ TOCSY. See Total correlation spectroscopy Total correlation spectroscopy, 52 Total homocysteine, 178 Total iron binding capacity, 182 Tracheobronchial tree and larynx, amyloidosis, 27. See also Amyloidosis Transdermal HRT application, 77 Tryptophan, vegetarian diets, 175 TSH. See Thyroid stimulating hormone TTR familial amyloidosis, 23, 30–31 Tumor necrosis factor‐, 82 Two‐dimensional diVerence gel electrophoresis, 145–146 U UCP3. See Uncoupling protein Uncoupling protein, 228

259

Urinary bladder, localized amyloidosis, 26–27. See also Amyloidosis Urinary iodine excretion, measurement, 186. See also Iodine content, vegetarian diets Urinary markers, for colorectal cancer, 47–50 V Vaccination, for amyloidosis treatment, 34–35 Val30Met TTR fibril, composition of, 6 Vascular cell adhesion molecule, 62 Vascular disease and inflammation, 62–65 Vasculoprotection estrogen in, 66 SERMs role in, 67 VCAM‐1. See Vascular cell adhesion molecule Vegetarian diets biomarkers of oxidant/antioxidant balance in antioxidant status and oxidative stress in, 190–194 dietary antioxidants, role of, 186–190 clinical chemistry in, 209–210 lower risk of disease in, 194–195 bone health, 201–203 cancer, 204–206 CVD and type 2 diabetes, 195–201 immune status, 206–207 renal function, 203–204 and nonvegetarian, biomarkers to distinguish, 207–209 nutritional deficiencies in association with, 173–174 essential fatty acids and omega‐3 series, 175–177 minerals and trace metals, 181–186 protein and amino acids, 174–175 vitamins, 177–181 worldwide scenario and benefits of, 172–173 Vegetarians disease detection, biomarkers, 194–195. See also Vegetarian diets bone health, 201–203 cancer, 204–206 CVD and type 2 diabetes, 195–201 immune status, 206–207 renal function, 203–204 Very low‐density lipoprotein, 65

260

INDEX

Viral diagnosis, QDs applications for, 113 Vitamin B12, sources and role of, 177–179. See also Vitamins content, vegetarian diets Vitamin C, source of, 187 Vitamin D, sources and role of, 179–181. See also Vitamins content, vegetarian diets Vitamin E, source of, 187 Vitamins and bone health, 202. See also Bone health detection, biomarkers in Vitamins content, vegetarian diets vitamin B12, 177–179

vitamin D, 179–181 VLDL. See Very low‐density lipoprotein W WHI. See Women’s Health Initiative Women’s Health Initiative, 61 Z Zinc content, vegetarian diets, 184. See also Minerals and trace metals, vegetarian diets

115 Å 24 b-strands

PLATE 1

A

Gln5

B

Gly1

Asn3

Asn2

Tyr7

Gln4

Asn6

Asn3 Gln5

4.87 Å

a

b

Gln5 Asn3

c

Asn2

C

Gln5

Asn3

b

Tyr7

Gln4

c

D

a

a

c

c

Wet interface Dry interface

E

Asn6

Asn2

Gln4

Gly1 Gln5

Asn3

2.9

2.9

3.0 2.8

2.9

2.9

3.0 2.9 2.8

2.9

2.8 2.9

3.1

2.8

3.1

2.9

Tyr7

3.2 2.8

2.8 3.0 2.9 2.7 3.2 2.9

2.9

2.8

2.8 3.0 2.9 2.7 3.2 2.9

2.9

3.2

b

2.8

a

PLATE 2

Model class

Native protein

Intermediate

Refolding

Natively disordered

Gain-ofinteraction

PLATE 3

PLATE 4

Fibril

A

B

Sample collection, initial processing and storage

C

Conventional proteomic methods Gel free

Gel based

Study sample

Protein prefractionation

Control sample

Label with Cy 5

Protein extraction, normalization/depletion Combine

Quantitative approach to identify candidate biomarkers and identification

Validation through quantitative proteomics/ELISA on larger cohort of patients

DIGE gel

Clinical trial

PLATE 5

Label with Cy 3

Absorbance 280 nm (mAU)

B

Absorbance 280 nm (mAU)

A Peak 1

Peak 2

Peak 1

Peak 2

Retention time

Retention time

C

D

50

50

MW (kDa)

100 75

MW (kDa)

100 75

25 20

15

25 20

15 pl

3

10

pl

3

E

6231 Undepleted only

3860 Depleted only

10,451 Overlapped

Undepleted

Depleted

PLATE 6

10