Advances in Clinical Chemistry Volume 38

ADVANCES IN CLINICAL CHEMISTRY VOLUME 38 BOARD OF EDITORS Kaiser I. Aziz Galal Ghourab Walter G. Guder E. D. Janus S...

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ADVANCES IN CLINICAL CHEMISTRY VOLUME 38

BOARD OF EDITORS

Kaiser I. Aziz Galal Ghourab Walter G. Guder E. D. Janus Sheshadri Narayanan Francesco Salvatore It-Koon Tan

Milos Tichy Masayuki Totani Casper H. van Aswegen Abraham van den Ende Istvan Vermes Henning von Schenk Zhen Hua Yang

Advances in CLINICAL CHEMISTRY Edited by GREGORY S. MAKOWSKI University of Connecticut Health Center Farmington, Connecticut

Associate Regional Editors Gerard Nowacki Fundacja Rozwoju Diagnostyki Laboratoryjnej Krakow, Poland

Kwang-Jen Hsiao Veterans General Hospital Taipei, Taiwan

VOLUME 38

Elsevier Academic Press 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WC1X 8RR, UK

This book is printed on acid-free paper. Copyright ß 2004, Elsevier Inc. All Rights Reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (www.copyright.com), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2004 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-2423/2004 $35.00 Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (þ44) 1865 843830, fax: (þ44) 1865 853333, E-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting ‘‘Customer Support’’ and then ‘‘Obtaining Permissions.’’ For all information on all Academic Press publications visit our Web site at www.academicpress.com ISBN: 0-12-0103389 PRINTED IN THE UNITED STATES OF AMERICA 04 05 06 07 08 9 8 7 6 5 4 3 2 1

CONTENTS Contributors ................................................................................

ix

Preface ........................................................................................

xi

Lipoprotein Oxidation Products and Arteriosclerosis: Theory and Methods with Applicability to the Clinical Chemistry Laboratory N. Abudu, James J. Miller, and Stanley S. Levinson 1. 2. 3. 4.

Introduction ................................................................................. Radicals, Electrophiles, and Other Reactive Species.................................... Oxidation Products of Lipids and Proteins and Measurement Methods .. ........... Detailed Procedures for Some Methods that Measure Oxidation Products in Plasma.......................................................................... 5. Discussion. ................................................................................... 6. Conclusion ................................................................................... References. ...................................................................................

2 6 8 20 23 26 27

Measurement of Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of Metalloproteinases (TIMP) in Blood and Urine: Potential Clinical Applications Stanley Zucker, Kaushik Doshi, and Jian Cao 1. 2. 3. 4. 5. 6.

Introduction ................................................................................. Background .................................................................................. Biology and Chemistry of MMPs and TIMPs... ........................................ Involvement of MMPs and TIMPs in Pathophysiology of Disease ................... Assays for Measurement of MMPs and TIMPs in Body Fluids. ...................... Blood Levels of MMPs and TIMPs in Physiologic and Disease States (Table 2) .................................................................... 7. Miscellaneous Diseases Associated with Increased Levels of MMPs and TIMPs .......................................................................... 8. MMPs Identified in Urine of Patients with Cancer ..................................... 9. Conclusions .................................................................................. References. ...................................................................................

v

38 38 39 42 45 48 70 71 73 74

vi

CONTENTS

Molecular Method to Quantitatively Detect Micrometastases and its Clinical Significance in Gastrointestinal Malignancies H. Nakanishi, Y. Kodera, and M. Tatematsu 1. Introduction................................................................................. 2. Methodology................................................................................ 3. Quantitative Detection of Micrometastases and its Prognostic Significance .................................................................... 4. General Considerations and Future Directions ......................................... References ...................................................................................

87 89 92 101 103

Zymographic Evaluation of Plasminogen Activators and Plasminogen Activator Inhibitors Melinda L. Ramsby 1. 2. 3. 4. 5.

Introduction................................................................................. Monitoring the PA/PAI System . ......................................................... Materials and Methods for Overlay Zymography ...................................... Results . ...................................................................................... Conclusion .................................................................................. References ...................................................................................

112 113 116 119 124 124

Estrogen Metabolites, Conjugates, and DNA Adducts: Possible Biomarkers for Risk of Breast, Prostate, and Other Human Cancers Eleanor G. Rogan and Ercole L. Cavalieri 1. Introduction................................................................................. 2. Analysis of Estrogens and their Metabolites, Conjugates, and Depurinating DNA Adducts .............................................................. 3. Biomarkers for Increased Risk of Developing Estrogen-Initiated Cancer ................................................................. References ...................................................................................

135 139 144 146

Organophosphates/Nerve Agent Poisoning: Mechanism of Action, Diagnosis, Prophylaxis, and Treatment Jiri´ Bajgar 1. 2. 3. 4. 5. 6.

Introduction................................................................................. Chemistry, Mechanism of Action, and Symptoms ..................................... Cholinesterase Inhibitors and Other Factors Influencing the Activity . .............. Diagnosis .................................................................................... Prophylaxis.................................................................................. Treatment ...................................................................................

152 153 167 176 186 190

CONTENTS 7. Future Trends ............................................................................... 8. Summary ..................................................................................... References. ...................................................................................

vii 196 197 198

The Potential of Protein-Detecting Microarrays for Clinical Diagnostics Alexandra H. Smith, Jennifer M. Vrtis, and Thomas Kodadek 1. 2. 3. 4.

Introduction ................................................................................. Diagnostic Signatures . ..................................................................... Protein-Detecting Microarrays ............................................................ Conclusions .................................................................................. References. ...................................................................................

217 218 225 234 234

Clinical Laboratory Implications of Single Living Cell mRNA Analysis Toshiya Osada, Hironori Uehara, Hyonchol Kim, and Atsushi Ikai 1. 2. 3. 4. 5.

Introduction ................................................................................. AFM.......................................................................................... Manipulations of Biological Material with AFM ....................................... mRNA Extraction from Living Cells ..................................................... Modification of AFM Tips ................................................................ References. ...................................................................................

240 240 242 245 252 255

Letter to the Editor......................................................................

259

Index ...........................................................................................

261

This Page Intentionally Left Blank

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

N. Abudu (1), Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, Kentucky 40292 JirI´ Bajgar (151), Purkyne Military Medical Academy, Hradec Kra´love´, Czech Republic Jian Cao (37), Health Science Center, State University of New York at Stony Brook, Stony Brook, New York 11794 Ercole L. Cavalieri (135), Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198 Kaushik Doshi (37), Veterans AVairs Medical Center, Northport, New York 11768 Atsushi Ikai (239), Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan Hyonchol Kim (239), Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan Thomas Kodadek (217), Department of Internal Medicine and Molecular Biology, Center for Biomedical Inventions, University of Texas Southwestern Medical Center, Dallas, Texas 75390 Y. Kodera (87), Department of Surgery II, Nagoya University School of Medicine, Tsuruma, Showa-ku, Nagoya 466-8550, Japan Stanley S. Levinson (1), Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, Kentucky 40292, and Laboratory Service, VAMC, Louisville, Kentucky 40206 ix

x

CONTRIBUTORS

James J. Miller (1), Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, Kentucky 40292 H. Nakanishi (87), Division of Oncological Pathology, Aichi Cancer Center Research Institute, Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan Toshiya Osada (239), Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan Melinda L. Ramsby (111), Division of Rheumatology, School of Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030 Eleanor G. Rogan (135), Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198 Alexandra H. Smith (217), Department of Internal Medicine and Molecular Biology, Center for Biomedical Inventions, University of Texas Southwestern Medical Center, Dallas, Texas 75390 M. Tatematsu (87), Division of Oncological Pathology, Aichi Cancer Center Research Institute, Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan Hironori Uehara (239), Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan Jennifer M. Vrtis (217), Department of Internal Medicine and Molecular Biology, Center for Biomedical Inventions, University of Texas Southwestern Medical Center, Dallas, Texas 75390 Stanley Zucker (37), Veterans AVairs Medical Center, Northport, New York 11768

PREFACE This volume marks my introduction as series editor. First, I would like to extend my appreciation to the editor emeritus, Dr. Herbert E. Spiegel, for his contributions to the Advances in Clinical Chemistry series over the past twenty years. His foresight has guided the readership during some of the most revolutionary changes in the field of clinical laboratory diagnostics. Dr. Spiegel’s leadership and editorial expertise will certainly be missed, but I will continue to consider him an indispensable resource as we move forward into the twenty-first century of clinical laboratory diagnostics. This volume, number thirty-eight in the series, contains chapters submitted from a diverse field of contributors on various clinical chemistry disciplines and diagnostics (e.g., from basic biochemistry to microarray technology). In keeping with the tradition of the series, I have tried to emphasize novel laboratory advances with application to both clinical laboratory diagnostics and life science studies. I strongly believe that it is through basic fundamental bench top science that the field of clinical chemistry will continue in its evolution to play an integral role in laboratory medicine and clinical diagnostics. As many can appreciate, the submission of a review article is a substantial commitment not easily undertaken. As such, I personally thank each of the contributors for their expertise and willingness in making this volume a reality. I also extend my sincere appreciation to all colleagues who participated in review of this volume for their time, energy, and constructive comments. Their prompt objective attention to the peer review process made this volume even more worthwhile as a clinical laboratory resource. Finally, I would like to acknowledge the help of Elsevier staV, specifically Ms. Netty Vreugdenhil, for her continuous support and guidance throughout the publication of this volume. I hope the readership will enjoy this volume in the series and use it. I actively welcome their comments and participation in making subsequent volumes of the Advances in Clinical Chemistry series of similar high quality. In keeping with Dr. Spiegel’s custom, I would like to dedicate this volume to my daughter Stephanie, the newest member of my family, for her patience and understanding during many quiet hours of concentrated editorship. Gregory S. Makowski xi

ADVANCES IN CLINICAL CHEMISTRY, VOL.

38

LIPOPROTEIN OXIDATION PRODUCTS AND ARTERIOSCLEROSIS: THEORY AND METHODS WITH APPLICABILITY TO THE CLINICAL CHEMISTRY LABORATORY Ntei Abudu,* James J. Miller,* and Stanley S. Levinson*,{ *Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, Kentucky 40292 { Laboratory Service, VAMC, Louisville, Kentucky 40206

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Oxidation Theory of Arteriosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Focus of this Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Radicals, Electrophiles, and Other Reactive Species . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Reactive Oxygen (ROS) and Nitrogen Species (RNS) . . . . . . . . . . . . . . . . . . . . 2.2. Transition Metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Oxidation Products of Lipids and Proteins and Measurement Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Detailed Procedures for Some Methods that Measure Oxidation Products in Plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Total MDA in Plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Plasma Peroxides Using FOX 2 Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Extraction of LDL and IDL by Heparin Gel Affinity Separation for Oxidative Susceptiblility=Resistance Testing . . . . . . . . . . . . . . . 4.4. Baseline Diene Conjugation in LDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 2 4 6 6 7 8 8 16 16 20 21 21 22 22 23 26 27

1 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2423/04 $35.00

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ABUDU ET AL.

1. Introduction 1.1. OXIDATION THEORY OF ARTERIOSCLEROSIS Much evidence links arteriosclerosis with oxidation of lipoproteins, endothelial cell impairment, inflammation, and thrombosis (C3, R11, Z1). Hyperlipidemia, and possibly oxidized (Ox) low density lipoproteins (LDL), appears important in inducing this process leading to plaque formation (C2, C3, S10, S11, W8, W9, Z1). Although arteriosclerosis proceeds within the arterial wall, evidence suggests it is a systemic process proceeding at multiple sites in coronary and peripheral vascular beds (B4, B7, R9, S5, V6). This may be due to a systemic insult such as hypertension, glucose intolerance, smoking, or hypercholesterolemia that damages many sites or due to a generalized facilitation of the oxidative or inflammatory state of individuals. Microcirculatory dysfunction due to this insult allows entry of lipoproteins into the arterial wall, leading to the release of inflammatory mediators that promotes further binding of LDL to the vessel endothelium (R11, Z1). The oxidation hypothesis, illustrated in Fig. 1, proposes that initially minimally modified LDL is formed in the arterial intimal space. Although this LDL can still be taken up by the well-regulated LDL receptor, it is thought that minimally modified LDL promotes release of proinflammatory mediators from leukocytes and endothelial tissue. Minimally modified LDL is thought to be chemotactic for macrophages and monocytes that diVerentiate into macrophages, thus facilitating macrophage recruitment (J4, S8, S9, W9). Macrophages further oxidize LDL, release inflammatory mediators, and rapidly take up lipid to form lipid-laden foam cells, an early event in fatty streak formation and an integral part of the necrotic core within a maturing plaque (J4, W9, Z1). Peroxidation of unsaturated fatty acids gives rise to reactive aldehydes and ketones that may complex positively charged amino acid residues of apo B, the main apolipoprotein found in beta-lipoproteins (H3, Y1). Oxidation also otherwise modifies and fragments apo B (B7, H13). Normally, the LDL receptor binds to apo B in LDL and other beta-lipoproteins and lipoprotein uptake is well regulated, but OxLDL-containing modified apo B, especially lysine adducts, does not recognize the LDL receptor and is taken up in an unregulated way by macrophage scavenger receptors (F1, F4). Phospholipids in OxLDL may also be recognized by scavenger receptors in the absence of adducted apo B (P3). Other proatherogenic eVects of OxLDL and oxphospholipids include the ability to attract monocytes, to inhibit the motility of macrophages, to prevent the release of vasodilatory nitric oxide (NO) from endothelial cells,

LIPOPROTEIN OXIDATION AND ARTERIOSCLEROSIS

3

FIG. 1. Oxidation hypothesis. Proposes minimally modified LDL is formed due to oxidation in the arterial intimal space. This LDL can still be taken up by the LDL receptor, but minimally modified LDL promotes release of proinflammatory mediators from monocytes and acts as a monocyte inhibition factor (MIF), reducing the motility of monocytes and thus leading to recruitment of macrophages (J4, W9). Macrophages further oxidize LDL (OxLDL), release inflammatory mediators, and rapidly take up OxLDL and other lipoproteins via the unregulated scavenger receptor that binds modified apo B to form lipid-laden foam cells. OxLDL is cytotoxic to a variety of cells in culture and may disrupt endothelial tissue, causing the release of inflammatory mediators and the entry of more LDL into the intimal space. Continued accumulation of monocytes and their diVerentiation into macrophages leads to a vicious cycle (J4, W9). Adapted from reference J4.

and to promote abnormal proliferation of vascular smooth muscle cells (C3, P6, W9, Z1). These adverse eVects of OxLDL on coronary artery vasomotion and coagulation pathways may play a role in the latter stages of atherosclerosis leading to acute ischemic syndromes (C3, W9, Z1). As illustrated in Fig. 2, it is further proposed that continued production of oxidation products within the arterial wall promotes a continuing cycle of inflammation (W9, Z1).

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ABUDU ET AL.

FIG. 2. Proposed process of arteriosclerosis leading to ischemic disease. It is proposed that arteriosclerosis is a process of inflammation within the arterial wall that is initiated by arterial injury (endothelial dysfunction), causing the trapping of lipoproteins (R11, Z1). These undergo oxidation as proposed in Fig. 1, leading to foam cells saturated with lipid droplets. Continued accumulation of fatty material within the blood vessel wall promotes a fatty streak. Ultimately, there is muscle cell migration and fibrosis leading to a plaque that consists of a fibrous cap with cholesterol crystals and debris within the deep necrotic layer, while inflammatory cells form a dynamic outer edge. It is thought that oxidized lipoproteins can facilitate many of these processes. Mechanical forces predispose the soft outer layer of the plaque to rupture at sites of structural weakness. Rupture of plaques causes thrombosis and incorporation of thrombi into the plaque. Ultimately, a large thrombus appearing in an obstructed vessel can lead to sudden ischemia and unstable coronary syndromes.

1.2. FOCUS OF THIS CHAPTER Although it is not yet conclusively proven that oxidation is the cause of arteriosclerosis in humans (C3, S10), animal experiments in the form of transgenic and knockout mice models support this hypothesis (C3). OxLDL (N2, Y2) and lipid oxidation products are found in human arteriosclerotic


5

plaques (J1, S7, W2) where the levels of OxLDL are nearly 70 greater than the levels found in plasma (N2). Moreover, persons with arteriosclerosis invariably exhibit increased levels of oxidation products in plasma as compared to nondiseased persons (H7, J9, S12). Besides, elevated LDL cholesterol correlates with elevated levels of lipid oxidation products, and lipid lowering leads to decreased oxidative products and improved endothelial function (A4, D4, R3). Even transplant-associated arteriosclerosis seems dependent on these processes since OxLDL (H8) and OxLDL antibodies appear elevated (A5), and lipid-lowering treatment reduces graft rejection (W6). If oxidation can be shown definitively to be an important prerequisite for arteriosclerosis in humans, it is likely that widespread testing for oxidation products will become standard practice. Native LDL is heterogeneous and contains huge numbers of oxidationsensitive components from which a vast number of oxidation products can be produced. Thus, largely for research purposes, but also to develop approaches for risk assessment, many methods have been developed for measuring oxidation products. Although some of the measurement techniques presently have application for research laboratories only, others are appropriate for use in clinical laboratories and some appear to have adaptability for automation. We focus our discussion on the following products: (1) oxidation products reflecting molecular modifications within the lipids and proteins of LDL; (2) breakdown products of the lipids and proteins; and (3) measurement of whole LDL modified by oxidation. Specificity for lipoproteins can be obtained only by separating the lipoproteins from other oxidizable substances prior to or during measurement. Nevertheless, products measured in the absence of separation can be considered indicative of total body oxidative stress that appears to be correlated with lipoprotein oxidation since plasma levels of many of these substances have been shown to correlate with risk of coronary artery disease (CAD) (H7, J9, S12). Some oxidation products are considered more specific and others less specific. Usually, the degree of specificity is related more to the method of measurement than to the product measured. We discuss these questions of specificity and discuss products measured under each category. We detail some direct spectrophotometric and fluorescence methods and some isolation techniques that can be directly applied to plasma and seem straightforward enough to be adapted for clinical laboratory use. Although more complex methods are also discussed, the procedures will not be detailed but references to the procedures will be given. To encourage better understanding of the oxidation products being measured, the initial portion of the chapter discusses the theoretical bases whereby reactive species are produced and how they give rise to oxidation products.

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2. Radicals, Electrophiles, and Other Reactive Species 2.1. REACTIVE OXYGEN (ROS)

AND

NITROGEN SPECIES (RNS)

Radicals are molecules with an unpaired electron. Radicals, transition metals, and other electrophiles exhibit a capability for oxidizing other molecules (S3). Most redox reactions in organisms are controlled by enzyme-mediated two-electron processes. This ensures that products are closed-shell molecules, avoiding potentially toxic radicals. Nevertheless, as illustrated in Fig. 3A, normally, cells produce superoxide anion radical via NADPH oxidase as a defense against microorganisms and injured cells may release oxidants. Oxidation of lipoproteins within the arterial wall may be mediated by leukocytes, endothelial cells, or transition metals (H2). Radicals produced by leukocytes seem especially important because of the instrumental role the monocyte=macrophage system appears to play in arteriosclerosis. These cells enzymatically generate the ROS superoxide anion radical from oxygen that, in turn, can give rise to the hydroxyl radical, which, although it has a short half life and reacts very close to its site of origin, is the most powerful ROS found in biological systems (G3). Figure 3 illustrates some mechanisms that generate many reactive species. Oxygen itself is a radical (R5) because oxygen contains two unpaired electrons, each with the same spin direction. Due to this spin restriction, it reacts sluggishly since it can only accept unpaired electrons of opposite spin. Although a very weak radical, it can be induced to react with macromolecules via transition to superoxide or by enzymes or transition metals. Neutrophils and monocytes secrete the enzyme myeloperoxidase, which can catalyze the production of the potent oxidant hypochlorous acid from hydrogen peroxide and chloride ion and tyrosyl radical from tyrosine (Fig. 3B) (H2, H3). Endothelial cells produce the weak radical nitric oxide (NO), which is a major regulator of vascular tone. It promotes relaxation of blood vessels, reduces monocyte and leukocyte adhesion to vascular endothelium, decreases platelet adhesion, and inhibits smooth muscle proliferation (H3, M4). Thus, it is potentially antiatherogenic (M3). Normally, NO levels are well regulated. But sustained production of NO by leukocytes and endothelial cells can be induced during inflammation (C1). As illustrated in Fig. 3A, under this situation NO may react with superoxide anion to produce the powerful RNS peroxynitrite. Peroxynitrite can directly promote oxidation of lipoproteins (H2) or give rise to hydroxyl (M5, R8) or longer-lived radicals, such as nitrogen dioxide (E2, R8) and carbonate radical, which can initiate oxidation at sites distant from its origin (R1). Myeloperoxidase from leukocytes can also generate RNS and other reactive species such as those illustrated in Fig. 3B (G1, H3, H11).


7

FIG. 3. Production of reactive species. (A) ROS can be produced from the weak radical oxygen in the mitochondria and endoplasmic reticulum, by various enzymatic reactions, and from oxyhemoglobin. Normally, nontoxic hydrogen peroxide can give rise to the powerful hydroxyl radical in the presence of transition metals (R5). Oxygen can also be induced to react with biomolecules by transition metals and enzymes. RNS can be produced by reaction of superoxide anion radical with the weak radical nitric oxide. These can react to form the powerful oxidant peroxynitrite=peroxynitrous acid, which can cause formation of other radicals, some with longer lives. See the text for details. SOD, superoxide dismutase. (B) Myeloperoxidase in leukocytes can produce the reactive species hypochlorous acid and tyrosyl radical. Unpaired electrons are indicated by the dense dots and paired electrons by the light ones.

2.2. TRANSITION METALS Iron and copper are powerful catalysts of oxidation. As illustrated in Fig. 3A, in the reduced form, these metals can reduce hydrogen peroxide to hydroxyl radical—the Fenton-type reaction. In the oxidized form, they can react with superoxide anion to revert to the reduced form (W4). Thus, in a

8

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biological system when iron or copper is present, there is a potential for the continuous catalytic production of hydroxyl radicals. Importantly, both iron and copper can directly catalyze the peroxidation of lipids in lipoproteins (H2, W5). Transition metals can exist in several spin states, thereby they can arrest the spin restriction of oxygen (B6) and react with oxygen to produce potent metal-containing oxidants (K4). Furthermore, they may allow simultaneous binding or bridging of a biomolecule and oxygen (B6, W5). Lipid peroxides in the presence of oxygen can reduce Cu2þ to Cuþ creating a peroxyl radical. Cuþ can be oxidized to Cu2þ by lipid peroxides to produce an alkoxyl radical, thereby catalyzing a chain of autoxidation (P1). Iron can catalyze lipid peroxidation as well (W5). Enzyme-bound transition metals usually catalyze nontoxic oxidations and iron in the storage form is usually bound as Fe3þ, but reducing agents may convert bound iron to Fe2þ causing its release, whereby it becomes reactive (B6, C4, W5). Free cellular iron may reside in a labile chelatable pool (K1). This pool appears to be regulated by cytoplasmic iron regulatory proteins that modulate production of transferrin and ferritin (C4). Increases in this pool may facilitate oxidation (K1). One of the seven coppers in the acute phase protein ceruloplasmin can catalyze the oxidation of lipoproteins as readily as free copper, and hence is a potentially important physiological prooxidant (F2, M9).

3. Oxidation Products of Lipids and Proteins and Measurement Methods 3.1. FATTY ACIDS Oxidation of polyunsaturated fatty acids (PUFA) in lipoproteins may be mediated by reactive species such as radicals, transition metals, other electrophiles, and by enzymes. Once initiated, oxidation of lipids may proceed by a chain reaction, illustrated in Fig. 4 (R5). In step 1, an oxidant captures an electron from a PUFA to produce a lipid radical. In step 2, after rearrangement, the conjugated diene radical reacts rapidly with singlet oxygen to produce a lipid peroxide radical, which is the kinetically preferred reaction (step 3) (B5). The chain can be terminated if the lipid radical reacts with an antioxidant to produce a stable peroxide (step 4). Otherwise, the peroxyl radical can react with another polyunsaturated fatty acid as shown in step 5 to perpetuate a chain reaction. The chain reaction requires production of lipid peroxides, giving it the name peroxidation. Fatty acids oxidized in the core are largely triglycerides and cholesterol esters, while toward the outer layer fatty acids in phospholipids are oxidized.


9

FIG. 4. Chain reaction of lipid peroxidation. An oxidant removes an electron from a PUFA (step 1) to form a lipid radical. Molecular rearrangement causes formation of a reactive conjugated diene (step 2). This can react with active singlet molecular oxygen (1O2), which is in an excited state rather than the ground state to form a peroxyl radical (step 3). Also, transition metals can react with oxygen to produce potent metal-containing oxidants that may allow simultaneous binding or bridging of a biomolecule and oxygen (B6, K4, W5). The peroxyl radical can be detoxified by an antioxidant to a lipid peroxide (step 4) or the peroxyl radical can act as an oxidant to remove an electron from another PUFA (step 5), eVecting a chain reaction of autooxidation. PUFA, polyunsaturated fatty acid (R5). Dot indicates unpaired electron in radical forms.

The variety of aldehydes, ketones, peroxides, and other oxidation products of fatty acid oxidation will depend on the structures of the fatty acids and on the extent of oxidation. Table 1 lists some products that have been measured, the predominant fatty acid from which the product is derived, and common methods used for measurement. Measurement of these oxidation products has been considered specific or nonspecific, depending on the purity of the product measured and the specificity of the method used. It is also useful to know from which fatty acid which product is predominately produced.

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TABLE 1 MEASUREMENT OF OXIDATION PRODUCT OF POLYUNSATURATED FATTY ACIDS Product

Fatty acid

Method

F2-isoprostanes (PGF)

Arachidonic

GC=MS Immunologically

Hydroxyoctadecadienoic acid (HODEs)

Linoleic

GC=MS

Linoleic Arachidonic acid

HPLC Thiobarbituric-reacting substances

Nonspecific but directly from specimen (TBARS)

1. Spectrophotometrically

Lipid peroxidation-related aldehydes: 4-hydroxynonenals (HNE) Malondialdehyde (MDA)

MDA specific with separation Conjugated dienes

All fatty acids

Peroxides Nonspecific but directly from specimen

All fatty acids

2. Fluorometrically HPLC Spectrophotometric Iodometric Xylenol orange (FOX assay) Methylene blue

Specific with separation

HPLC

TABLE 2 REFERENCE VALUES FOR SOME OXIDATION PRODUCTS IN HUMAN PLASMA Product Lipid peroxides (HPLC) Lipid peroxides using FOX assay 2 with TPP MDA by HPLC F2-isoprostanes Protein carbonyls

Reference range 2.1–4.6 umol=L 1.17–4.87 umol=L 0.36–1.24 umol=L 5–33 ng=L 0.4–1.0 nmol=mg protein

Reference (N4) (N4) (N1) (M8) (D1)

About half of all fatty acids are PUFAs. The major PUFA is linoleic acid (18:3) that is about 7 times more frequent than arachidonic (20:4) or docosahexaenoic acids (22:6) (J9). Many of the products that have been measured are largely from arachidonic acid that represents a minor constituent of the lipoproteins. Each oxidation product listed in Table 1 is considered in the following text, and apparent normal reference ranges for some of them are listed in Table 2 along with the source from which the information was derived.


11

3.1.1. F2-Isoprostanes (PGF) PGF are produced by a nonenzymatic oxidation mechanism from arachidonic acid (J4). They appear to be formed as esters in phospholipids and subsequently released in free form (M8). The F2 class is the most abundant. They were first measured using gas chromatography=negative ion chemical ionization mass spectrometry (GC=MS) after thin layer chromatography and solid phase separations, as described by Morrow et al. (M8). Although several peaks characterize the F2 PGF, a single peak was used for quantification (M8). Later, it was demonstrated that the thin layer chromatography was not necessary (G6). More recently, PGF has been measured by highperformance liquid chromatography (HPLC)=MS, although this technique is not as sensitive as GC=MS (L1). PGF are increased following oxidation of LDL by macrophages, endothelial cells, and copper (G6, G7, L5, P2, P4, R10, W3). PGF have been identified in arteriosclerotic plaques (G4, P4). Increased levels have been identified in persons with hypercholesterolemia and other classical risk factors for CAD (D2, D3, G5, M7, R4) and in persons with peripheral vascular disease (W2). For these reasons, and because they can be measured in urine and plasma, PGF are generally considered to oVer a noninvasive, sensitive, specific direct method for measuring lipid peroxidation in vivo (Y1). The main drawback is that the methods are very complicated, tedious, and not usually available in clinical laboratories. Like other lipid oxidation products, PGF can be generated ex vivo. For this reason, fluids should be preserved with butylated hydroxytoluene (BHT) and EDTA to prevent further oxidation and measured immediately or stored at 70 C (M8). An ELISA has been developed for urine for 8-Iso-PGF2 that is commercially available (O2), but the method is tedious since it requires a column separation prior to ELISA (B1). Also, since PGF are mainly indicators of arachidonic acid oxidation, they do not reflect oxidation of the major PUFA comprising lipoproteins. 3.1.2. Hydroxyoctadecanoic Acid (HODEs) HODEs are primarily C18 oxidation products of linoleic acid (J9). These have not been as widely studied as isoprostanes, but like isoprostanes, these are specific products of nonenzymatic lipid peroxidation that are associated with arteriosclerotic disease and are found in arteriosclerotic plaques (J9, W2). Likewise, they are measured by specific GC=MS techniques that are generally not available in clinical laboratories (J10). They have the advantage that they are products of the major PUFA in lipoproteins—linoleic acid— but they have generally been measured only in lipoproteins extracted from plasma.

12

ABUDU ET AL.

3.1.3. Reactive Aldehydes Malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) are among many reactive aldehydes that are nonenzymatic lipid peroxidation products. These products are illustrated in Fig. 5. Both have been intensively studied as an index of peroxidation (E4). They are not only associated with arteriosclerosis (J9), but are among those electrophilic aldehydes that adduct lysine residues in apo B, leading to uncontrolled OxLDL uptake by macrophages (U1). Moreover, both react with lipid hydroperoxides and decompose them to peroxyl and alkoxyl radicals, which can reinitiate lipid peroxidation (E4, J4, R5, U1, W9). HNE is a 4-hydroxy-2-alkenal (Fig. 5) that is a product of arachidonic and linoleic acid and better represents the mixture of fatty acids in lipoproteins than do some other oxidation products. HNE is cytotoxic to cells and causes the rapid depletion of glutathione, inhibition of DNA, RNA, and protein synthesis and, at high levels, inhibition of many metabolic processes leading to rapid cell death (E4). HNE is a specific product that has been measured by GC=MS and HPLC (E4). HPLC methods may be more accessible to clinical laboratories than GC=MS and have been used both for measuring levels of HNE and HNE protein adducts (U2), but generally they have been measured only in lipoprotein extracts or tissue and not in whole plasma or serum. On the other hand, MDA has been measured in plasma and urine. Because of its relative ease of colorimetric measurement, it is the most widely investigated product of peroxidation (J9). It is largely a product of PUFA with more than two methylene-interrupted double bonds such as arachidonic acid and docosahexaenoic acid. These possess at least three double bonds (C›C C C›CCC›C) so that they can more easily be broken down into w w the small 3-carbon, dicarbonyl MDA than can linoleic acid, which contains only one activated double bond (J2, J9).

FIG. 5. Some reactive aldehydes. MDA is a specific 3-carbon product of arachidonic acid oxidation. 4-hydroxy-2-alkenals is the general class of lipid peroxidation-related aldehydes to which the specific product HNE belongs (U1).


13

MDA is most commonly measured by reaction with thiobarbituric acid (TBA) after heating at low pH. The 1:2 MDA:TBA adduct is both pigmented and fluorescent so that it can be easily monitored (J2). Although MDA is a specific product, this reaction lacks specificity since many other aldehydes, sugars, and amino acids may react with TBA and peroxides may also be formed during the heating step. It is for this reason that the reaction is generally referred to as thiobarbituric acid-reacting substances (TBARS). The formation of peroxides during the heating step can be eliminated by the addition of BHT to the reagent. The MDA-(TBA)2 adduct has also been measured specifically after separation by HPLC (L4, S2). A direct method for measuring MDA will be detailed. 3.1.4. Conjugated Dienes As illustrated in Fig. 4, formation of conjugated dienes due to molecular rearrangement is a necessary event in the chain reaction of lipid peroxidation (step 2). The formation of conjugated dienes causes a spectral change at a wavelength of 234 nm that can be directly monitored in aqueous solution as oxidation of fatty acids proceeds (E5). The technique was first described by Professor Esterbauer and associates (E5) and has been widely used for continuous monitoring of oxidation kinetics in aqueous solutions when lipoproteins are tested for susceptibility (or the antithesis—resistance) to oxidation. This test is performed by isolating lipoproteins and inducing oxidation in them (usually using iron or copper). It has been demonstrated that susceptibility to oxidation varies according to the individual and to the amount of antioxidant within the lipoprotein particle (D7, J5). Supplementation by antioxidants such as vitamin E or other phenolic nutrients in vitro or by ingestion in vivo decreases the lipoprotein susceptibility to oxidation (D7, J5, J6, V4). Absorbance changes can be divided into three phases (illustrated in Fig. 6). These illustrate the oxidation products that are produced during continuous oxidation of fatty acids. The lag phase is the period during which lipoprotein particles resist oxidation. Resistance is due to antioxidants within the particle, such as vitamin E, and innate resistance properties (M1). This is followed by the propagation phase during which the fatty acids are rapidly oxidized and conjugated dienes are formed. Finally, there is a plateau phase with a dip. The dip represents the time when production of conjugated dienes begins to decrease but other oxidation products that also absorb at 234 nm, such as aldehydes, start to appear. The total diene concentration can be estimated by the maximum 234 nm absorbance using the molar extinction coeYcient of 2.95 104M 1cm 1 (E5). Susceptibility to oxidation is measured by comparing the lag phase for diVerent samples (K3). It is the duration of this phase in minutes that is usually considered a measure of lipoprotein susceptibility

14

ABUDU ET AL.

FIG. 6. Phases of fatty acid oxidation in lipoproteins. Oxidation of LDL was catalyzed by 5 umol=L copper in vitro (K3). LDL, density between 1.020 and 1.063, was isolated by sequential ultracentrifugation in the presence of 100 mmol=L EDTA and frozen at 70 C in aliquots. EDTA was removed by gel filtration chromatography immediately prior to the experiment and oxidation was followed by measuring conjugated diene formation at 234 nm. Propagation phase is the time during which a rapid change in absorbance occurs, which represents the rapid formation of conjugated dienes (see Fig. 4). Lag phase time is determined from the point at which the straight line best fitting the slope of the propagation phase curve crosses the x-axis (indicated by the darker straight line). Plateau phase represents the time when production of conjugated dienes begins to decrease and the dip represents the time other oxidation products that also absorb at 234 nm, such as aldehydes, start to appear. The maximal point, often just before the dip, is an estimation of the total amount of conjugated diene formation.

or resistance to oxidation, although the physiological meaning of the lag phase remains unclear, and it is not certain that the degree of susceptibility of the particle to oxidation is necessarily a predictor of arteriosclerosis (A3, F3,


15

S2). A method that could be applicable for clinical laboratory use measures the baseline diene conjugation (BDC) in LDL (A3). It avoids the use of ultracentrifugation by using heparin to precipitate LDL followed by chloroform–methanol extraction of the lipids. The redissolved lipid is measured at 234 nm and 300 nm, with the diVerence (A234–A300) being converted to molar units using an extinction coeYcient of 2.95 104M 1cm 1. Studies with HPLC and NMR indicate that LDL-BDC is a specific indicator of circulating oxidized LDL. Clinical studies have shown that LDL-BDC has a strong association with various risk factors for CAD, such as obesity and hyperglycemia, and with markers of arteriosclerosis itself, including angiographically documented CAD, arterial elasticity, and carotid intima-media thickness (A2). This method will be detailed below. In order to measure susceptibility to oxidation without the need to isolate the lipoproteins, methods have been developed for oxidizing whole serum or plasma and measuring diene formation (R2, S4). Such approaches may be subject to error as a result of variation in other oxidizable plasma components such as bilirubin, albumin, fibrinogen, and uric acid. A method that uses heparin aYnity chromatography to separate LDL and intermediate density lipoproteins (IDL) from other serum proteins was described by Vinson et al. (V3, V5) and was later better standardized (K3). This approach has been shown to reflect susceptibility to oxidation in animal and human plasma under a variety of conditions (K3, V5). The heparin separation procedure is detailed in the following text. 3.1.5. Peroxides As illustrated in Fig. 4, oxidation of fatty acids cannot occur without the formation of peroxides; therefore, concentrations of lipid peroxides are a measure of oxidative stress. Most tests for lipid peroxides use simple spectrophotometric end points and are applicable for clinical laboratory use. They do not measure specific products but reflect overall oxidation of fatty acids. HPLC can be used to specifically measure individual peroxides (S2). 3.1.5.1. Iodometric. Iodometric measurement of peroxides is one of the oldest techniques. It relies on the capacity of lipid peroxides to convert free I2 to I13 that can be spectrophotometrically measured at 365 nm (S2). Plasma can be directly assayed; however, it is apt to give an overestimation of lipid peroxide concentration because I2 has reactivity toward other compounds, especially molecular oxygen and light (E3, S2). Reaction with oxygen occurs more readily at acid pH (J3). A simple mix-and-read method was available with the color reagents supplied in kit form (E3), but the color reagent was discontinued, which is why it is not listed in detail here. The ingredients in the color reagent have been described (E3), and, for those who wish to try it, the concentration of lipid peroxide can be determined from the molar extinction

16

ABUDU ET AL.

coeYcient of 2.46 104M 1cm 1 (E3). This method that used a pH of 6.2 did not appear to react with molecular oxygen to any appreciable extent and incubation was in the dark. A more detailed discussion of the use of iodometric assays in determination of hydroperoxides can be found elsewhere (J3). 3.1.5.2. Xylenol Orange. Ferrous ion oxidation (FOX) in the presence of xylenol orange is a newer method for measuring lipid peroxides that has been shown to agree well with the iodometric, TBAR, and conjugated diene assays but is simpler to perform (J8). It has been used to assay peroxides in plasma (N4). In this assay, peroxides oxidize Fe2þ to Fe3þ in acid solution and Fe3þ forms a complex with xylenol orange, which absorbs at 560 nm. Two reagents have been described, FOX 1 and FOX 2 (W10). FOX 2 contains methanol that solubilizes lipid peroxides, which is necessary for their measurement. FOX 1, without methanol, measures only hydroperoxides. The FOX 1 reagent also contains sorbitol, which increases the analytical sensitivity by increasing the yield of ferric ions about 15 mol per mol of hydrogen peroxide. Methanol in the FOX 2 reagents replaces sorbitol as an oxygen scavenger, although it has been shown that sorbitol can still improve the analytical sensitivity (D6). The FOX 2 method will be detailed in the following text. 3.2. CHOLESTEROL Most oxysterols are enzyme-induced intermediates produced during conversion of cholesterol to bile acids and some of these intermediates are excreted into the circulation (B3). A variety of oxysterols has been shown to be important in intracellular regulation of cholesterol homeostasis, including 22(R)-hydroxycholesterol, 20(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol (M6). Yet, along with several diverse oxidation products, the only identifiable oxysterol produced from LDL by oxidation with copper and macrophages was 7-ketocholesterol (J5). It remains unclear whether or not 7-keto-, other 7-oxy, or other oxysterols are related to arteriosclerosis. Presently, they do not seem to have much merit compared to other markers of oxidative stress largely because of methodological problems and complexities in measuring them (B3). Measurement of cholesterol ester hydroperoxides by HPLC has been well described (S2). 3.3. PROTEINS 3.3.1. Products of Oxidation Oxidation of the apolipoproteins can produce a vast array of molecular species. Modifications of the protein backbone or modifications of the side


17

FIG. 7. Oxidation products of proteins. The vertical structure in the middle represents the main peptide chain with amino acid side groups extending horizontally (M2). The -carbons in the primary chain can be oxidized to form hydroperoxides. Reactions on the right side near the top exemplify oxidation of the primary chain leading to a peroxyl radical. Side chains represented are lysine, methionine, tyrosine, cysteine, and histidine, top to bottom, respectively. Modifications of the side chains and primary chain lead to carbonyl formation and charge modifications. If these reactions are not detoxified by antioxidants, they may propagate chain reactions within the primary chain, leading to fragmentation of the protein. See the text for details. o, represents reaction with oxygen; RNS, reactive nitrogen species; ROS, reactive oxygen species. Dense dot represents unpaired electron of radical forms.

chains can occur. Although most amino acid side chains can be modified, those containing ring structures, especially aromatic, sulfur-containing, and basic residues are especially prone. Figure 7 illustrates types of reactions that can modify these amino acids exemplified by tyrosine, cysteine and methionine, histidine and lysine, respectively (M2). These modifications lead to carbonyl formation, fragmentation, and charge modifications. As illustrated in Fig. 7, carbonyls and other adducts can be formed by reactive aldehydes such as MDA and HNE binding to basic side chains to form Michael adducts (M2, U1). After reaction with the amino group on lysine, SchiV base adducts can form (M2). RNS and ROS can modify side chains to produce oxidized species such as oxohistidine and 3-nitro-tyrosine, and hypochlorous acid can react to form 3-chlorotyrosine. Sulfhydryl

18

ABUDU ET AL.

TABLE 3 MEASUREMENT OF OXIDATION PRODUCTS OF PROTEINS Product

Species

Method

Relative electrophoretic mobility

apo B

Electrophoresis

Carbonyl groups

apo B

Spectrophotometric HPLC ELISA Western=Dot blot

Malondialdehyde LDL

apo B adduct

ELISA

OxLDL

apo B modification

ELISA

groups can react with reactive aldehydes, RNS, and ROS to form sulfoxides, nitrosothiols, and sulfenic acids. Thiols and other sulfur groups are also susceptible to formation of disulfide bonds. The protein backbone itself can be oxidized by radicals and other electrophiles (D5). Figure 7 illustrates how the main -carbons in the primary chain can be oxidized to form hydroperoxides. If these are not detoxified by antioxidants, they may propagate chain reactions leading to carbonyls and fragmentation of the protein. Some methods for identifying protein oxidation products are discussed and Table 3 lists some assays that are more straightforward or may otherwise have applicability to the clinical laboratory. 3.3.2. Identification of Oxidized Amino Acids by Mass Spectroscopy Work during the late 1990s has explored the technique of isotopic dilution GC=MS for identifying modification in amino acids (H2, H4). These elegant techniques have improved our understanding of the exact species that cause oxidative modifications in proteins and the origins of the species, but are not yet applicable for clinical laboratory use. Catalytic metal-generated oxidation produced ortho-tyrosine and meta-tyrosine, while tyrosyl radical selectively produced o,o0 -dityrosine (L2). The concentrations of ortho-tyrosine and meta-tyrosine were increased in advanced plaques as compared to earlier lesions while o,o0 -dityrosine from early human arteriosclerotic lesions was strikingly increased (L2). This suggests that metal-catalyzed oxidation may not play an important part in directly oxidizing proteins in early arteriosclerosis (H2). Nevertheless, this does not rule out the possibility that oxidation of fatty acids occurs in early arteriosclerosis, leading to protein adducts. Very elevated levels of 3-nitrotyrosine in lesion LDL indicates RNS contribute to plaque oxidation (H2), and the finding that 3-chlorotyrosine and o,o’-dityrosine but not ortho-tyrosine are elevated in LDL from human arteriosclerotic tissue implicates myeloperoxidase as a source of oxidation (H1, H3).


19

3.3.3. Relative Electrophoretic Migration Owing to an increased negativity of the protein as a result of adduct formation or fragmentation, oxidized LDL migrates more rapidly than does native LDL on agarose gel. This technique has been applied for measuring the extent of catalytic metal oxidation of LDL (J7). The application requires that LDL be isolated from other serum proteins. Usually, LDL is isolated by ultracentrifugation but LDL=IDL isolated by heparin aYnity chomatograpy has also been used for this purpose (K2). The extent of oxidation is directly proportional to the distance migrated on an electrophoretic gel. This technique has applicability for clinical laboratories since many routinely perform lipoprotein electrophoresis. 3.3.4. Measurement of Carbonyl Formation As indicated in Table 3, as well as by HPLC (L3), carbonyl formation can be measured using spectrophotometric and ELISA techniques that require only equipment available in many clinical laboratories (D1). Moreover, plasma can be directly measured (B8). For colorimetric or ELISA, proteins are derivatized with dinitrophenylhydrazine (DNPH) and precipitated with trichloroacetic acid. Carbonyl content can be measured in the absorbance range of 355 to 390 nm using a molar absorbance coeYcient of 22,000 M 1 cm 1 (B8, R6) or using a biotinylated anti-DNPH antibody in conjunction with a streptavidin-biotinylated enzyme (B8, W7). Other methods that are applicable to research laboratories include Western and dot blot techniques. Some of these are available as kits and may have applicability to clinical laboratories as well (D1). Although more complicated to perform than ELISA, two-dimensional blotting provides a means for identifying exactly which proteins are aVected by oxidative stress (D1). The apparent normal reference range for carbonyls is listed in Table 2. 3.3.5. Measurement of OxLDL and Malondialdehyde-LDL Both circulating OxLDL and MDA-LDL have been measured in plasma by ELISA. Most commonly, the capture antibody is a monoclonal antibody against OxLDL or MDA-LDL and the detection antibody, a polyclonal or monoclonal antibody directed against apo B (E1, S12, T1). Usually, the capture antibody is developed against chemically modified MDA-LDL or OxLDL but antibody has been developed against homogenate of human arteriosclerotic plaque. This antibody reacted with oxidized phosphatidylcholines but not native LDL, or MDA-LDL (S12). Holvoet et al. have used an ELISA to measure OxLDL and MDA-LDL with an assay based on inhibition of binding of mouse monoclonal antibodies to copper-modified LDL coated on a microtiter plate (H6). The samples

20

ABUDU ET AL.

and standards are incubated with a monoclonal antibody, following which the mixture is added to microtiter plates containing OxLDL-coated wells. The more OxLDL in the sample, the less mouse antibody that will bind to the coated LDL. After washing, the wells are incubated with a rabbit antimouse antibody containing horseradish peroxidase. The reduction of peroxidase reaction absorbance as compared to blank is measured at 492 nm. Two antibodies have been developed, both of which have aYnity for MDAmodified LDL. The antibody used to identify OxLDL has 1000 greater aYnity for OxLDL than native LDL and an equal aYnity for MDA-LDL (H8). The antibody used to detect MDA-LDL has a 500 times greater aYnity for MDA-LDL than native LDL but also a 50 times greater aYnity for MDA-LDL than OxLDL with fragmented apo B (H5). The latter OxLDL ELISA is available in kit form (H10). OxLDL has been found to be elevated in patients with CAD as compared to normal and to be an independent predictor of disease even in the presence of conventional risk factors including lipoprotein lipid markers (E1, H10, S12). The main diVerence between studies of OxLDL is the degree of diVerentiation between persons with CAD from normal. Holvoet et al. found little overlap between groups, including those with heart transplant CAD (H9), while other investigators have found a great deal of overlap (E1, H10, S12). There has also been variance in the exact relationship between MDA-LDL and disease. Holvoet et al. found elevated MDA-LDL to be associated with unstable coronary disease (H5, H9), while others have found it to be a general marker for CAD (T1). There is general agreement that there is a relationship between plasma levels of oxidatively modified LDL and arteriosclerosis (H10) and that its measurement may be a valuable clinical predictor of arteriosclerosis. OxLDL is a complex particle, and monoclonal antibodies react at a single epitope that may not reflect the complexity of the particle; thus, it is not surprising that there are some inconsistencies in the exact results from diVerent studies. Not only does this research seem promising in terms of identifying new markers for predicting CAD, but these types of assays are well within the scope of those that fit into the clinical laboratory for widespread use. It seems that continued studies to determine more exact relationships are well warranted.

4. Detailed Procedures for Some Methods that Measure Oxidation Products in Plasma Although none of the methods described here are simply mix-and-read, they are methods that are straightforward enough to be performed in a clinical laboratory using equipment that is generally available. A diYculty


21

encountered in the past was ex vivo oxidation. This problem has been eliminated by adding BHT to specimens and reagents, including those for OxLDL and MDA-LDL. Except for studying susceptibility to oxidation, we collect specimens in EDTA purple type tubes and bring plasma or serum to 10 umol=L BHT immediately after collection. The BHT is dissolved in methanol. Samples are stored at 70 C. BHT cannot be used for measuring susceptibility of lipoproteins to oxidation because the lipid-soluble substance enters the lipoproteins and cannot be removed by aqueous separation techniques. When measuring susceptibility of lipoproteins to oxidation, the samples are stored at 70 C with 1 mmol=L EDTA that is removed prior to the test by gel filtration, dialysis, or heparin gel aYnity chromatography. 4.1. TOTAL MDA

IN

PLASMA (S2)

1. Mix 200 uL of sample, standards, and controls with 25 uL of BHT solution (54 mmol=L in methanol) and 200 uL orthophosphoric acid solution (200 mmol=L). Mix well. 2. Add 215 uL of TBA reagent (800 mg thiobarbituric acid in 50 mL 0.1 mol=L NaOH). 3. Heat the sample at 90 C for 45 min. 4. Cool to room temperature. 5. Extract TBARS with 500 uL n-butanol containing 50 uL of a saturated NaCl solution. 6. Separate phases by centrifugation (10,000 g for 1 min). 7. Transfer an aliquot of the upper organic phase to a tube and read absorbance at 535 nm or fluorescence at 552 nm. The standard solution is prepared by hydrolysis of 10 mmol=L or 50 mmol=L tetra-methoxypropane with 10 mmol=L HCl for 10 min at room temperature. TBARS can be read in a microtiter plate, in which case absorbance at 572 should be subtracted from 535 to correct for baseline absorption. Some kits are available for MDA (O2, R7). 4.2. PLASMA PEROXIDES USING FOX 2 REAGENT (J8, W10) 1. 10 uL of methanol is added to 90 uL of plasma (Test). (Important note: All methanol must be HPLC-grade to avoid contamination by iron.) 2. 10 uL of 10 mmol=L triphenylphosphine (TPP) in methanol is added to a duplicate plasma (Blank). 3. The samples are mixed and incubated at room temperature for 30 min.

22

ABUDU ET AL.

4. Add 900 uL of FOX 2 reagent consisting of 880 mg BHT, 76 mg xylenol orange, 98 mg of ammonium iron(II) sulfate, and 1 mg=mL EDTA dissolved in 1 L of methanol=acetic acid (1:1 mixture). 5. The samples are incubated for an additional 30 min. 6. The samples are then centrifuged at 10,000 g for 10 min and the supernatants read at 560 nm. 7. Each test sample is subtracted from the corresponding blank and the concentration determined from the diVerence using an extinction coeYcient of 4.3 104 M 1cm 1 or by reference to a hydrogen peroxide standard curve, the latter being desirable when first setting up the test. 4.3. EXTRACTION OF LDL AND IDL BY HEPARIN GEL AFFINITY SEPARATION FOR OXIDATIVE SUSCEPTIBILITY=RESISTANCE TESTING This method, which was previously commercially available as a kit for measuring cholesterol in beta-lipoproteins, has been discontinued (T2). The following approach was developed by JA Vinson (V2) and modified (K3). 1. Pipet 1 mL of heparin aYnity gel (Sigma H6508 stored refrigerated) into a small 1 10 cm column fitted with a fritted cellulose disc at the bottom, and wash with 2 mL of 0.7% NaCl. 2. Pipet 200 uL of serum that was immediately transferred to an EDTAcontaining purple tube after separation from the clot. (Serum can be frozen and thawed once only.) Serum is used rather than plasma because fibrinogen is isolated with the lipoproteins and fibrinogen is a powerful antioxidant (K3, O1). Wash the serum into the column with 50 uL of 0.7% NaCl. 3. After 5 min, wash the column again with 2 mL of 0.7% NaCl. Dispose of the washes that contain serum proteins except beta-lipoproteins. 4. Elute the beta-lipoproteins with 2.5 mL of 2.9% NaCl. 5. Use this material to determine lag time. 6. The columns can be washed with 2.5 mL of 0.7% NaCl and stored at room temperature with 1 mL of saline on top for reuse up to 3 times within a week.

4.4. BASELINE DIENE CONJUGATION IN LDL (A3) 1. Collect blood and immediately transfer the serum to an EDTA-containing purple tube after separation from the clot. 2. LDL are precipitated from 1 mL of the sera by adding 7 mL of buVer containing 0.064 mol=L trisodium citrate adjusted to pH 5.05 with 5 N HCl and containing 50,000 IU=L heparin.


23

3. After vigorous mixing, incubate for 10 min. 4. LDL are sedimented by centrifugation at 1000 xg for 10 min, and the pellet resuspended in 1 mL of 0.1 mol=L of sodium phosphate buVer, pH 8.0 containing saline. 5. Extract the lipids by adding 100 uL of a chloroform–methanol solution (2:1) and evaporate to dryness with nitrogen. 6. Redissolve in cyclohexane and measure the absorbance at 234 nm and 300 nm. 7. Determine the concentration of conjugated dienes from the absorbance diVerence using a molar extinction coeYcient of 2.93 104M 1cm. 1

5. Discussion Much evidence suggests that oxidation of lipoproteins is a cause of human arteriosclerosis. This comprises animal models, including those in which several diVerent antioxidant compounds retarded arteriosclerosis, epidemiological studies (C3, S10), and correlation between oxidation products and CAD in humans (A2, H7, J9). Moreover, evidence shows accumulations of oxidation products in human arteriosclerotic tissue and gruel. These include: (1) ROS such as superoxide radical (W1), (2) Oxidized lipids such as 7-ketostreroids (J1), F2-isoprostanes, and HODEs (W1), and (3) protein products, such as malondialdehyde– and HNE–lysine adducts (Y2), immunologically active LDL (N2, H9), and oxidized forms of tyrosine that suggest oxidation due to RNS either via myeloperoxidase (H2, H3) or induced nitric oxide formation or both (B9). Still, many key questions remain unanswered. Thus, while it is generally agreed that LDL undergoes oxidation in vivo and that OxLDL is found in arteriosclerotic plaques, it is still not known how and where LDL is oxidized nor which of its atherogenic eVects demonstrated in vitro are important in vivo (C3). Besides, it appears that cholesterol accumulation in arteriosclerotic lesions precedes significant amounts of oxidized lipid that would be contrary to the oxidation hypothesis of arteriosclerosis (U3). It was expected that important evidence linking oxidation to CAD would be demonstrated by nutrient antioxidant trials in humans. The antioxidants were expected to retard arteriosclerosis and reduce CAD in treated persons as compared to placebo (C3). But primary and secondary randomized trials with the antioxidants vitamin E, vitamin C, and vitamin A have so far failed to prevent CAD (A1, V7). Moreover, large amounts of vitamin E (A1, N3, U3, V7) and serum protein antioxidants such as albumin and fibrinogen are found in arteriosclerotic tissue that would be expected to retard oxidation-induced disease (B2, V1). These findings do not necessarily negate the oxidation

24

ABUDU ET AL.

hypothesis that is strongly supported by much evidence (S10). But until a firmer link between lipoprotein oxidation and arteriosclerosis in humans is confirmed, it is unlikely that large randomized studies, designed to identify which oxidation products can be used to identify risk, will be conducted. Nor is it likely drug companies will embark on the development of new drugs that can specifically reduce oxidation for the purpose of reducing CAD. Of legitimate concern is the question of whether oxidation products found in blood or plasma are actually artifacts occurring due to atmospheric exposure or due to other sources of ex vivo oxidation during isolation and work-up rather than in vivo oxidation. As has been discussed, this problem has been reduced because current procedures call for adding antioxidants such as BHT and EDTA to blood or plasma immediately upon collection of the sample and addition of these antioxidants to the test reagents during analysis to reduce aberrant oxidation. Moreover, findings showing OxLDL and other oxidation products in human arteriosclerotic lesions and increased concentrations of oxidation products in the blood of patients both at high risk for and with arteriosclerosis, as compared to those without, certainly support the view that oxidation products are not simply an artifact of isolation. As a result, it is generally accepted that, to some extent, these oxidation products are a true manifestation of disease, although it cannot be ruled out that some degree of baseline levels are artifactual. Nor can it be entirely ruled out that oxidation products obtained from arterial tissue and gruel samples are not altered by ex vivo oxidation. Thus, it is important to examine to what degree in vivo studies have taken stringent precautions to avoid inadvertent external oxidation. Even more diYcult to discern is whether oxidation occurred in the artery wall or in the circulation. In the case of identifying those prone to arteriosclerosis, it would seem that measurement of products from the arterial wall would be more specific for identification or prediction of disease. Nevertheless, those with a predisposition to oxidation may exhibit an elevated level of general oxidative stress and may be more prone to oxidation in the arteries as well as other sites. These persons may develop disease more readily. If this is true, the level of oxidative stress as measured in blood may correlate with CAD. This concept will be discussed in more detail below. If a definitive link between lipoprotein oxidation and arteriosclerosis in humans were to be confirmed in the future, it is likely that tests measuring oxidation products will become common in clinical laboratories. For these reasons, it seems worthwhile for clinical laboratorians to be aware of the various tests and the theory under which they function. It also seems worthwhile to consider the possible usefulness of less specific products that may be more easily adapted for widespread use, compared to specific products that are usually measured by specific but tedious techniques. One reason that tests


25

to measure specific products have continued to be developed is because research has failed to identify the exact sites and mechanisms by which oxidation of lipoproteins in humans proceeds, giving rise to a continuing search for methods with increasing specificities that might help identify the sources. As a screening test for risk assessment, specific methods that measure lipid peroxidation products may oVer few advantages over less specific spectrophotometric approaches. This is because as long as inadvertent lipid peroxidation is avoided by the addition of BHT and EDTA (S2), measurement of specific products by HPLC appears to correlate well with simpler spectrophotometric approaches (E3, N4), whether by measurement of conjugated dienes (A2), lipid peroxides (E3, N4), or MDA (J9, L4). In general, less specific automated testing is often more diagnostically sensitive than more specific tests that can be used as follow-up in specialty laboratories to confirm positive screening results when necessary. Measurement of modified proteins may be an alternative or supplement to measuring products of peroxidation. Direct measurement of OxLDL in plasma appears to be a promising avenue for risk assessment in clinical laboratories. This technique utilizes ELISA that lends itself to automation. Studies indicate that elevated levels of OxLDL correlate with CAD and add predictive value to assessment by conventional lipoprotein lipids (E1, H7, H10, S12). Still, basic and clinical research is needed to determine exactly what is being measured, where it originates, and whether or not it is a cause of arteriosclerosis or only secondarily associated with it. Applied research is needed to determine how best to measure and standardize the assays, and randomized clinical studies are needed to determine the exact diagnostic usefulness. OxLDL is very complex with many epitopes altered from that of native LDL. Normally, ELISA is designed to bind homogeneous sites on all molecules so that the binding can be quantified. Thus, normally, apo B concentration can be exactly determined by immunological methods because a monoclonal antibody binds to a single molecular site in each sample and calibrator. But OxLDL contains heterogeneous apo B, where some molecules contain binding epitopes and others do not. These epitopes may be modified by various aldehyde adducts such as MDA or various substitutions, deletions, fragmentations, etc. Thus, it is not surprising that some investigators found little overlap between patients with angina and those without disease (H9), while others found a good deal of overlap (E1). Nor is it surprising that the reference range has been defined diVerently by various investigators as 1.3 0.88 mg=dL using copper-oxidized LDL calibtators (H5, H7), 12.0 6.3 based on abitrary U=mL, and 0.58 0.23 ng=5 ug OxLDL protein based on copper oxidation (E1).

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The source of OxLDL is also unclear. In early disease, it might be due to back diVusion from the vessel wall (H9), while in later disease the source might be rupture or erosions in the plaque (E1, H10). That back diVusion may be a source is suggested by evidence showing OxLDL was elevated very early in the disease process before significant erosion could occur (H10). That plaque erosion is a source is supported by evidence showing patients with acute myocardial infarction exhibited significantly higher levels than did those with angina (E1). Alternatively, since normal persons appear to have some OxLDL in plasma, the origin of minimal oxidation of phospholipids in lipoproteins may be due to leukocytes, endothelial cells, and other sources of oxidation as the lipoproteins proceed through sinusoids in the liver and small tissue capillaries. It is possible that some persons may have more oxidation occurring in the blood at susceptible locations due to various risk factors and that persons with elevated OxLDL are more susceptible to developing disease. This would also explain why OxLDL was elevated in the early disease process (H10). Three specific human monoclonal IgG autoantibodies that recognize oxidized MDA-LDL have been prepared using phage libraries (S6). Such a panel of antibodies may be of value in defining the composition of arteriosclerotic plaques in various stages of development (G2). They may also be directed at cells, lipoproteins, and matrix molecules in a way that can help identify the source of OxLDL in humans. Such human antibodies may also be used in assays. There is still a good deal of research needed to sort out these questions.

6. Conclusion In conclusion, although it is yet to be definitively shown, much evidence supports a link between oxidation of lipoproteins and arteriosclerosis in human beings. If this relationship can be conclusively demonstrated, it is likely there will be a need to measure lipoprotein oxidation products in the clinical laboratory for risk assessment. Establishing a definitive link between oxidation and arteriosclerosis is not a simple task because not only will it be necessary to demonstrate that oxidation is a risk factor for arteriosclerosis in large prospective studies, but it will be important to show it is a marker independent of lipoprotein and inflammatory markers. Furthermore, it would be desirable to identify treatments by which modification of oxidation markers leads to reduction in disease (S1). To date, the failure of randomized trials with antioxidant nutrients to retard arteriosclerosis (A1, Y3) has made it clear that demonstrating a definite relationship in humans may be more diYcult that expected. In the meantime, there is a need to measure these


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products for research purposes. Moreover, a large number of assays are available for products of peroxidation that can be performed with equipment available in many clinical laboratories. Thus, many clinical laboratories may be in a position to collaborate in this eVort. Many of these methods are straightforward enough to be adapted for routine clinical laboratory use should the need arise at a later time and should they prove eVective. Alternatively, ELISA for OxLDL may be valuable for risk assessment, but because of the complexity of OxLDL, applied research is needed to determine how best to use monoclonal antibodies to measure OxLDL and how best to standardize the assays. ACKNOWLEDGMENT This work was supported by the Department of Veterans AVairs, Louisville, KY.

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MEASUREMENT OF MATRIX METALLOPROTEINASES (MMPS) AND TISSUE INHIBITORS OF METALLOPROTEINASES (TIMP) IN BLOOD AND URINE: POTENTIAL CLINICAL APPLICATIONS Stanley Zucker,* Kaushik Doshi,* and Jian Cao{ *Veterans Affairs Medical Center, Northport, New York 11768 { Health Science Center, State University of New York at Stony Brook, Stony Brook, New York 11794 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Background. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biology and Chemistry of MMPs and TIMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Involvement of MMPs and TIMPs in Pathophysiology of Disease . . . . . . . . . . . . . . 4.1. Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Inflammatory Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Liver Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Cardiovascular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Lung Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Central Nervous System Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7. Shock Syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8. Chronic Wounds and Inflammation of the Skin and Oral Cavity. . . . . . . . 5. Assays for Measurement of MMPs and TIMPs in Body Fluids . . . . . . . . . . . . . . 5.1. Pitfalls in Measurement of Blood Levels of MMPs and TIMPs . . . . . . . . . 5.2. Origin of MMPs and TIMPs in Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Blood Levels of MMPs and TIMPs in Physiologic and Disease States (Table 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Plasma MMPs and TIMPs Levels in Pregnancy . . . . . . . . . . . . . . . . . . . . . . . 6.2. Plasma MMPs and TIMPs Levels in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. MMP and TIMP Levels in the Blood of Patients with Inflammatory Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. MMP and TIMP Levels in Liver Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. MMP and TIMP Levels in Diseases of the Nervous System . . . . . . . . . . . . 6.6. MMP and TIMP Levels in Cardiovascular Disease . . . . . . . . . . . . . . . . . . . . 6.7. Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Miscellaneous Diseases Associated with Increased Levels of MMPs and TIMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Diabetes Mellitus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Myeloproliferative Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

38 38 39 42 42 43 43 43 44 44 44 44 45 46 47 48 52 53 59 64 65 67 70 70 70 71


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7.3. Burns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4. Polycystic Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. MMPs Identified in Urine of Patients with Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

71 71 71 73 74

1. Introduction Considerable interest has evolved over the past decade in the measurement of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in blood and urine as an aid in the diagnosis and prognosis of disease. The goal of this chapter is to provide a comprehensive review of the subject with the focus on potential future developments and applications of the technology.

2. Background The concept of using the measurements of specific proteins in blood and urine as a window to recognize disease has been around for many decades. Early applications of this concept led to measurements of hormones in patient urine specimens. Bioassay endpoints in mice (changes in end organ function) were employed to identify gonadotrophin hormone indicative of pregnancy and erythropoietin reflecting hormonal stimulation of red blood cell production. Berson and Yalow were acclaimed for their development of the first radioimmune assays for measuring insulin and then multiple other hormones; these methods were later adapted for measurement of serum hormone levels. A decade later, biomarkers (HCG and -fetoprotein) became popularized as tools for classification and monitoring of treatment of testicular cancer. With the advent of immunoassay techniques, it became possible to identify nanomolar concentrations of proteins in body fluids. The enzyme-linked immunosorbent assay (ELISA) system represents the most reliable, sensitive, and widely available protein-based testing platform for detection and monitoring disease states. These tests are robust, linear, and accurate, and have a moderate throughput. Use of an ELISA to test for disease requires a single, validated protein biomarker of disease as well as well-characterized, high affinity antibodies that can bind and detect the protein of interest from serum/plasma specimens. In spite of the many technical advances in clinical immunoassays, the major challenge to these tests is their usefulness in detecting early disease, thereby leading to improvement in treatment outcome. These tests have been used extensively in the cancer field. Serum prostate

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specific antigen (PSA) measurements have been most useful for improving early diagnosis of prostate cancer. Carcinoma embryonic antigen (CEA) has been used to monitor recurrence of colorectal cancer and CA-125 has limited clinical utility in diagnosis of ovarian cancer. More recently, developments in the field of proteomics has led to the identification and quantification of thousands of trace serum components that were previously beyond the range of recognition (W5). Although this technique offers considerable potential for early diagnosis of disease, many technical problems need to be overcome before clinical application can become feasible.

3. Biology and Chemistry of MMPs and TIMPs Interstitial collagenase, the first MMP family member identified, was discovered in experiments designed to explain collagen remodeling in the metamorphosis of a tadpole into a frog (G12). Since collagens represent the major structural proteins of all tissues and the chief obstacle to cell migration, it has long been postulated that collagenolytic enzymes play pivotal roles in facilitating dissemination of cancer and in the pathogenesis of rheumatoid arthritis. A pathological role for MMPs in cancer, arthritis, skin disease, and nonhealing wounds was suggested in the 1980s. Later interest has focused on the role of MMPs in cardiovascular remodeling (such as atherosclerosis, restenosis), aortic aneurysms, congestive heart failure, and diseases of the lung, liver, central nervous system, retina, and kidney (G2). The MMP family is currently composed of 24 related zinc-dependent enzymes that share common functional domains. These enzymes have both a descriptive name typically based on a preferred substrate and an MMP numbering system based on order of discovery (Table 1). MMPs were characterized initially by their extensive ability to degrade extracellular matrix proteins including collagens, laminin, fibronectin, vitronectin, aggrecan, enactin, tenascin, elastin, and proteoglycans (N2). More recently, it has been recognized that MMPs cleave many other types of peptides and proteins and have a myriad of other important functions that are still incompletely understood (O7). MMPs have distinct but often overlapping substrate specificities. The basic structure of MMPs consists of the following homologous domains: (1) a signal peptide which directs MMPs to the secretory or plasma membrane insertion pathway; (2) a prodomain that confers latency to the enzymes by occupying the active site zinc, making the catalytic enzyme inaccessible to substrates; (3) a zinc-containing catalytic domain; (4) a hemopexin domain which mediates interactions with substrates and confers specificity of the enzymes; and (5) a hinge region which links the catalytic and the

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TABLE 1 MATRIX METALLOPROTEINASES (MMPS) INCRIMINATED IN DISEASE PROCESSES: NAMES AND COMMON SUBSTRATES CLEAVED BY THESE ENZYMES Matrix metalloproteinases MMP-1 (Collagenase-1)

MMP-2 (Glatinase A, 72 kDa type IV collagenase)

MMP-3 (Stromelysin-1)

MMP-7 (Matrilysin) MMP-8 (Collagenase 2) MMP-9 (Glatinase B, 92 kDa type IV collagenase) MMP-10 (Stromelysin-2) MMP-11 (Stromelysin-3) MMP-13 (Collagenase-3) MMP-14 (Membrane type-1 matrix metalloproteinase)

Substrates Collagens I, II, III, VII, VIII, X, XI, gelatins, aggrecan, fibronectin, fibrin, fibronogen, entactin, laminin, tenascin, vitronectin Gelatin, collagens IV, V, I, III, VII, VIII, X, XI, aggrecan, decorin, laminin, vitronectin, fibronectin, elastin, fibrin, fibronogen, plasminogen, 1 proteinase inhibitor, proMMP-13, proMMP-9 Perlecan, decorin, aggrecan, laminin, gelatins, collagens III, IV, V, VII, IX, X, XI, fibrin, fibrinogen, fibronectin, proMMP-9, proMMP-1 Aggrecan, decorin, fibronectin, laminin, collagens I, IV, gelatin, elastin, enactin, tinascin, fibrinogen, plasminogen Collagens I, II, III, Clq, aggrecan, 1-protease inhibitor Gelatins, collagen IV, V, XI, XIV, agegrecan, decorin, elastin, fibrin, fibrinogen, plasminogen, -1 proteinase inhibitor Collagens III, IV, V, gelatin, elastin, fibronectin, aggrecan 1 proteinase inhibitor, laminin, fibronectin Collagens I, II, III, IV, VI, IX, X, gelatin, fibronectin, aggrecan, fibronogen, proMMP-9 Fibronectin, collagens I, II, III, gelatin, aggrecan, perlecan, vitronectin, tenascin, fibronectin, 1 proteinase inhibitor, proMMP-2, pro-MMP13

hemopexin domain. The smallest MMP in size, MMP-7 or matrilysin, lacks the hemopexin domain (Fig. 1). Additional structural domains and substrate specificities have led to the division of MMPs into subgroups (Fig. 1). The membrane-type MMPs contain an additional 20 amino acid transmembrane domain and a small cytoplasmic domain (MT1-, MT2-, MT3-, and MT5MMP) or a glycosylphosphatidyl inositol linkage (MT4- and MT6-MMP), which tethers these proteins to the cell surface. MMP-2 and MMP-9 (termed gelatinases based on their substrate preference) contain fibronectinlike domain repeats which aid in substrate binding (Fig. 1). Two sequence motifs are highly conserved in the protein structure of MMPs. The consensus motif HExGHxxGxxH, found in the catalytic domain of all MMPs, contains 3 histidines that coordinate with the zinc ion (Zn) in the active center (B5, S11). The PRCGxPD motif is located in the C-terminal portion of the prodomain of MMPs; coordination of the cysteine residue (C) of this locus with the zinc atom of the active center confers latency to the proenzyme (B5, N2).


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FIG. 1. Domain structure of MMPs. The hemopexin domain has a four-bladed propeller configuration. The pre domain is cleaved before exit from the endoplasmic reticulum. MMP-7 lacks the hemopexin domain. MMP-2 and MMP-9 contain fibronectin-binding domains. MT-MMPs (-1, -2, -3, -5) contain a transmembrane and a cytoplasmic domain.

In Vivo activity of MMPs is under rigid control at several levels. These enzymes are usually expressed in very low amounts and their transcription is tightly regulated either positively or negatively by cytokines and growth factors such as interleukins, transforming growth factors, or tumor necrosis factor alpha (TNF-) (S9, Z2); MMP-2 is the exception to the general rule and is constitutively produced. Some of these regulatory molecules can be proteolytically activated or inactivated by MMPs, providing a feedback effect. Activation of MMPs following secretion from cells depends on disruption of the prodomain interaction with the catalytic site, which may occur by proteolytic removal of the prodomain or conformational change. MMPs that contain furinlike recognition domains in their propeptides (MMP-11, MT-MMPs, MMP-28) are activated intracellularly in the trans Golgi network by members of the subtilisin family of serine proteases (furin). The mechanism for in vivo activation of secreted MMPs is not well understood. Some active MMPs can activate other proMMPs (O3). MT1-MMP plays a central role in the activation of proMMP-2 on the cell surface. Extracellular proteolytic activation of secreted MMPs can be mediated in vitro by serine proteases, e.g. plasmin, which implies an interdependence of these two enzyme systems in ECM remodeling (Z2); the biologic relevance of these in vitro observations remains uncertain. Once activated, MMPs are further regulated by endogenous inhibitors, autodegradation, and selective endocytosis. A cell surface receptor mechanism for endocytosis of MMP-13 (B4) and also MMP-2: thrombospondin complexes through a low density lipoprotein receptor-related protein (LRP) mechanism has been proposed (Y3). The Tissue Inhibitors of MetalloProteinases (TIMP-1, -2, -3, and -4) make up a family of homologous MMP inhibitors widely distributed in tissues (N2). TIMP concentrations in extracellular fluids and tissues generally far exceed the concentration of MMPs, thereby limiting the proteolytic activity of circulating MMPs. In contrast to the inhibitory role of other TIMPs, low concentration of TIMP-2 actually enhances MT1-MMP induced activation of MMP-2 by forming a triplex on the cell surface (S3). In addition, TIMPs have been shown to have growth promoting and inhibitory activities which are independent of their MMP inhibitory function (S5). As noted with

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MMPs, the transcription of TIMPs is regulated by cytokines and growth factors, but specific regulation differs from MMPs. Other endogenous inhibitors of MMPs include the plasma 2macroglobulin and RECK, a recently identified cell surface inhibitor of MMPs (T1).

4. Involvement of MMPs and TIMPs in Pathophysiology of Disease Thousands of papers have been written on the subject of MMPs and TIMPs, dealing with various aspects of their physiologic and pathologic roles in biologic processes. The simplicity of thinking about MMPs solely as extracellular matrix degrading enzymes and TIMPs solely as inhibitors of these processes has recently been eroded by the recognition of numerous other important roles for these proteins (S5, V5). Our understanding of the biology of MMPs and TIMPs has been considerably expanded with the availability of specific MMP and TIMP knockout mice (H6, V4). Experimentation with knockout mice has facilitated a more detailed examination of the functions of MMPs and TIMPs in vivo; this avenue of research is just beginning. 4.1. CANCER Increased tissue levels of MMP-1, -2, -3, -7, -9, -11, -13, and -14, along with TIMP-1 and -2, have been identified in many different types of cancers (E3). Numerous reports have commented on the potential usefulness of these measurements in the management of patients with cancer. The detection of activated MMP-2 and MMP-9 by substrate zymography in human cancer tissue extracts has been proposed as a cancer marker in aggressive breast and colon cancer (B11, D1). Increased ratios of tumor/normal mucosal MMP-9 demonstrated by Northern blot analysis has been shown to correlate with the status of distant metastasis and clinical stage of disease of colorectal cancer (Z1). In some reports, high tumor levels of latent and activated MMPs have been correlated with more aggressive prostate cancer (S8, W4). Of considerable interest are the cell types responsible for producing MMPs in cancer. In situ hybridization studies have demonstrated that most MMPs in tumors are produced by stromal cells rather than the cancer cells themselves: One explanation for this phenomenon is that cancer cells produce Extracellular Matrix Metalloproteinase Inducer (EMMPRIN), a cell surface glycoprotein, which directly stimulates fibroblasts (through direct cell contact) to produce MMP-1, -2, -3, and MT1-MMP (B6). EMMPRIN is also up regulated in inflammatory cells and has been implicated in lung injury. (H2) Nielsen


43

et al. (N7) emphasized that neutrophil and macrophage infiltration of breast and colon cancer tissue are the major sources of MMP-9. The importance of cytokines such as TNF-, interleukin (IL)-1, and IL-6 in stimulating production of MMPs in disease has been emphasized (F3). Increased levels of TIMP-1 and TIMP-2 mRNA have been identified in malignant stromal tissue. A correlation between TIMP-1 levels and the clinical stage of colon cancer has been demonstrated (L11). 4.2. INFLAMMATORY DISEASES A large number of reports have demonstrated increased joint tissue levels of MMP-1 and MMP-3 in rheumatoid joints and joint fluid, (O4) leading to the implication that MMPs contribute to joint injury. MMPs have also been implicated in other types of arthritis. A more important role for aggrecanase, a member of the ADAM family of metalloproteinases, in articular damage has been proposed (A5). 4.3. LIVER DISEASE The characteristic response to liver injury due to chronic excess alcohol intake is increased hepatic fibrosis (cirrhosis). Collagen accumulation in the liver reflects both enhanced synthesis and failure of collagen degradation to keep pace with production. TIMP-1 and MMP-1, -2, -3, and -9 are also produced by the liver in response to injury. MMP-1 levels dropped as the liver fibrosis progressed in cirrhosis (N1). Patients with hepatic fibrosis due to hemochromatosis have an increased TIMP-1/MMP-1, -2, -3 ratio (G7). 4.4. CARDIOVASCULAR DISEASE There has been a long-standing interest in the role of MMPs in cardiovascular disease (D2). Numerous studies have demonstrated increased levels of MMPs, especially MMP-9, at sites of atherosclerosis and aneurysms (G3, V2). The current opinion that the inflammatory process may play a leading role in the development of vascular atherosclerotic plaques has led to the suggestion that secretion and activation of MMPs by macrophages induces degradation of extracellular matrix in the atherosclerotic plaque leading to plaque rupture. Based on these concepts, MMPs have been proposed to represent sensitive markers of inflammation in patients with coronary artery disease. MMP tissue levels have been demonstrated to be increased in the heart in congestive heart failure (C8). Although inhibitors of MMPs have shown value in experimental models of heart disease, uncertainty of overall outcome has dampened enthusiasm for use of MMP inhibitors in heart disease.

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Magid et al. (M1) recently explored the regulation of MMPs in endothelial cells exposed to shear stress. They reported that oscillatory blood flow, but not unidirectional shear, significantly increased MMP-9 mRNA as well as cell secretion of MMP-9 protein. Cell associated TIMP-1 was insensitive to the shear regimen. They demonstrated that the c-Myc transcriptional factor binds specifically to a site in the MMP-9 promoter. This effect may contribute to the progression of atherosclerosis. 4.5. LUNG DISEASES Elevated levels of MMPs have been implicated in the pathophysiology of various lung diseases, including acute respiratory distress syndrome, bronchiectasis, and cystic fibrosis (V2). MMPs, EMMPRIN, and TIMPs are produced by many of the resident cells in the lung, hence complicating the analysis of their role in disease (F5, F6, H2). Potential use of MMP inhibitors for treatment of these disorders remains to be explored. 4.6. CENTRAL NERVOUS SYSTEM DISEASE Following observations of the critical role of MMP-9 in animal models resembling multiple sclerosis and Guillain-Barre’s syndrome, MMPs have been implicated in several different types of neurologic diseases (C9, R4, V2). Treatment with synthetic inhibitors of MMPs has reversed some of the pathology in animal models of brain injury (R4). TIMPs and MMPs have also been implicated in Alzheimer’s disease (P3). 4.7. SHOCK SYNDROMES MMP-8 and MMP-9 are stored in the granules of polymorphonuclear leukocytes. These cells are key effectors in inflammatory and infectious processes. A role for these MMPs in shock is supported by studies in MMP-9 deficient mice that were shown to be resistant to endotoxic shock. Dubois et al. (D4) proposed that specific MMP-9 inhibition constitutes a potential approach for the treatment of septic shock syndromes. 4.8. CHRONIC WOUNDS AND INFLAMMATION OF THE SKIN AND ORAL CAVITY Acute and chronic wounds are associated with high levels of MMP-2 and MMP-9. These observations have led to the suggestion that nonhealing ulcers develop an environment containing high levels of activated MMPs, which results in chronic tissue turnover and failure of wound closure (S1, V2). MMP-9 has been implicated in blistering skin diseases and contact


45

hypersensitivity (L7, V2). MMPs have long been implicated in periodontal disease (G10) and more recently, in inflammatory bowel diseases. (B1)

5. Assays for Measurement of MMPs and TIMPs in Body Fluids Although the first MMP was described in remodeled amphibian tissue in 1962 (G12), it was not until 1986 that MMPs were identified in circulating blood. MMPs were initially recognized in plasma inadvertently, as a contaminant during the chemical isolation of fibronectin (J1). MMP-2 and MMP-9 were subsequently identified as normal components of plasma using gelatin zymography, which depicted these elements as negative staining bands of gelatinolytic activity in gelatin-impregnated SDS polyacrylamide gels following overnight incubation of gels in calcium enriched buffer (V3). Moutsiakis et al. (M17) characterized the spectrum of MMP-2 (gelatinase A) and MMP9 (gelatinase B) and complexes of these MMPs with TIMPs in plasma. MMP activity in plasma has also been measured in physiologic and disease states using different types of collagen substrate assays. Serum MMP-1 levels, measured in a substrate degradation assay, were reported to be increased in pregnancy with ripening of the cervix prior to delivery (G11). During the past decade, immunoassays have been developed to quantify levels of plasma/serum MMP-1, -2, -3, -7, -8, -9, and -13. Immunoassays for serum/plasma TIMP-1 and TIMP-2 have also been developed (B3, F7, O1, Z7). The critical component in the development of a sensitive ELISA is the identification of a capture antibody capable of binding the test antigen to an immobilized surface. Whereas many antibodies function well as detecting antibodies with high specificity, high affinity capture antibodies are much more difficult to produce. Both polyclonal and monoclonal antibodies have been used as capture and detecting antibodies. ELISA kits produced commercially are now available to measure MMPs and TIMPs. Several of these ELISA kits provide good precision and accuracy and appear to be quite reliable when compared to ‘‘homemade’’ ELISAs developed by individual investigators (data not shown). Mean levels of MMP-2, TIMP-1, TIMP-2, MMP-3, MMP-9, MMP-1, and MMP-7 in normal plasma/serum range from 500 ng/ml to Adeno ca (G5, K8, R2) Association with smoking (A2) MMP-9 & TIMP-1 Together—poor prognosis (G5, K8) No correlation with PSA level (Z9) More likely due to inflammatory response (C3, J5) (Z8) More metastasis (L8) Metastatic potential Metastatic potential Poor disease-free survival (Y5) Poor disease-free survival Poor prognosis (Y5) Higher stage with higher level—Poor prognosis (N6) Metastatic potential (U3, Z4) Metastatic potential (E2, U1, U3) Low sensitivity (G8) (G9) Strong predictor of poor survival (G9) (G9) Increased chances of metastasis (J2) (Z4) (Z4) (continues )

TABLE 2 (Continued ) Diseases Breast

Colorectal

Hepatocellular

Head & neck thyroid

50

Medullary thyroid Connective Tissue Disorders Rheumatoid arthritis Systemic lupus erythematosus Scleroderma Psoriatic arthritis Osteoarthritis Gastrointestinal disorders Inflammatory bowel disease Cirrhosis Hepatitis-C Pancreatitis

MMP/TIMP

Type/levels

MMP-9 MMP-9 MMP-2 MMP-9 TIMP-1 MMP-2 MMP-2 MMP-9 TIMP-2 MMP-9 MMP-2 TIMP-2 MMP-1 MMP-3 MMP-9

Plasma/High Urine/þnce Urine/þnce Plasma/High Serum/High Serum/High Serum/High Plasma/High Serum/High Plasma/High Serum/High Serum/High Serum/Low Serum/High Plasma/High

MMP-3 MMP-9 MMP-3

Diagnostic importance

Prognostic importance

Yes Yes Yes

Poor

Comment (Z4) Metastatic potential Metastatic potential Metastasis (P1) High in Duke D—Poor survival (Z10)

Poor Poor Poor Poor Poor Poor Poor Poor Poor Poor Poor Poor

(Y7) (H3) (H3) (H3) (H3) (H3)

Serum/High Plasma/High Serum/High

Poor Poor Poor

More radiological damage (C3, C5, M2) Associated with vasculitis feature (P5) (M8)

MMP-1 TIMP-1 MMP-1 MMP-3

Serum/High Serum/High Serum/High Serum/High

Poor Poor Poor Poor

(Z12) (Z12) (C2) High in debilitating arthritis (K4)

MMP-3

Serum/High

Poor

(L7)

TIMP-1 MMP-3 TIMP-1 MMP-1 MMP1/TIMP-1 complex

Serum/High Serum/High Serum/High Serum/High Serum/High

Yes Yes Yes

(H7)

More fibrosis (Y8)

Poor Poor

Poor prognosis (T2) Poor prognosis (T2)

Cardiovascular Hypertension Atrial fibrillation Congestive heart failure

Dilated cardiomyopathy Aortic aneurysm Unstable angina/ myocardial infarction Stable CAD CNS Stroke

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Alzheimer’s disease Multiple sclerosis Guillian-Barre syndrome Pregnancy Term labor 3rd trimester Labor Miscellaneous Diabetes mellitus

TIMP-1 MMP-1(free) TIMP-1:MMP-1 MMP-9 MMP-8 MMP-9: TIMP-1 MMP-1 MMP-1; TIMP-1 MMP-9 MMP-2

Serum/High Serum/Low Serum/High Plasma/High Serum/High Plasma/High Serum/High Serum/High Plasma/High Serum/High

Yes Yes

MMP-9 MMP-9 TIMP-1

Plasma/High Plasma/High Serum/High

Yes

MMP-9

Plasma/High

MMP-9 TIMP-1 TIMP-2 MMP-9

Plasma/High Serum/High Serum/High Plasma/High

Yes Yes Yes

MMP-1 MMP-9 TIMP-1

Serum/High Plasma/High Serum/High

Yes No Yes

No

MMP-9

Plasma/High

Yes

Yes

Serum/High

Yes

Yes

Detect renal involvement as early as 4 years earlier than microalbuminuria (L6, M10) (S2)

Yes Yes Yes Yes

(L1) (L1) (L1) Poor prognosis (J3)

Essential thrombocytosis/ TIMP-1 polycythemia rubra vera Polycystic kidney disease MMP-9 TIMP-1 MMP-1 Septic shock MMP-9

Plasma/High Serum/High Serum/High Plasma/High

Yes Yes

Yes Yes Poor Yes

More organ fibrosis (D3) (D3) Increased morbidity in stroke (C4, I1)

Poor Poor Poor Poor

Poor prognosis (C4, I1) (H5) (H5) Poor prognosis (A3) (L2)

Yes Yes Yes

Poor prognosis (L2) Poor prognosis (M15) (M15)

Yes

Hemorrhagic stroke—higher the no. poor prognosis (L10, S6) (L10) Helpful for relapse (M18) Helpful for relapse (M18) More severe disease (F6)

Yes

Yes

Good (M12) (N4) Level rise during labor and postpartum period (K7)

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and 1 minus specificity (true positive versus false positive) across all cutpoints of the test and calculates the area under curve (AUC) and its standard error. A test with AUC of 1 is perfectly accurate, whereas one with an AUC of 0.5 is performing no better than chance. Most real tests have AUCs between these values (> 0.530 ng/ml) were associated with poor survival in patients with nonsmall cell lung cancer and potentially might serve as markers to predict aggressive behavior of lung carcinoma. These tests might also be useful in the clinical follow-up of the lung cancer patients. Susskind et al. (S12) recently examined the effect of chest radiotherapy on MMP and TIMP measurements in patients with breast and lung cancer. They reported that lung and breast cancer were associated with high plasma levels of MMP-9 and TIMP-1. High baseline levels of MMP-9 were reduced in the first two weeks of radiotherapy; TIMP-1 levels remained high. Minimal elevations or fluctuations of plasma MMP-3 levels in irradiated patients negated a role for MMP-3 in either the injury or repair response in the lung. The authors concluded that the decrease in plasma MMP-9 after initiation of chest irradiation appeared to reflect a suppressive effect on cancer-induced cellular responses rather than a primary role for MMP-9 in radiationinduced lung injury. The possibility of using plasma MMP-9 measurements to predict cancer recurrence remains unresolved. 6.2.3. Genitourinary Cancers In an early study exploring MMP and TIMP levels in cancer, Baker et al. (B2) reported increased serum levels of MMP-1 and TIMP-1 in patients with prostate cancer as compared to control subjects; levels were highest in patients with metastatic cancer. Jung et al. (J5) confirmed the finding that plasma TIMP-1 concentrations in prostate cancer patients with metastases were significantly higher than those in the control group, patients with benign prostatic hypertrophy, and prostate cancer patients without metastasis. In contrast, serum TIMP-2 levels were normal in patients with bladder cancer. Mean values of plasma MMP-1 and MMP-1:TIMP-1 ratios, however, were not different among cancer patients, healthy subjects, and patients with benign prostatic hypertrophy. Gohji et al. reported that the mean serum MMP-2 and the MMP-2:TIMP-2 ratio in advanced urothelial cancer patients with recurrence were significantly higher (28 and 47%, respectively) than that in patients without recurrence. Levels were less elevated in patients with superficial bladder cancer. The 1and 3-year disease-free survival rates in patients with high serum MMP-2:TIMP-2 ratios were significantly worse than in those patients with normal ratios; this conclusion was verified by univariate and multivariate analyses. (G8) Gohji et al. (G9) later reported increased serum MMP-2 levels in patients with prostate cancer; a correlation with clinical course was claimed. In contrast, Jung et al. reported that prostate cancer patients had

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somewhat lower blood MMP-2 levels in comparison with healthy controls and patients with benign prostatic hypertrophy. (J2) No reasonable explanation is available to account for the different outcomes of these studies in prostate cancer. Jung et al. (J4) also reported increased plasma MMP-3 levels in patients with prostate cancer and metastases. Based on numerous studies in patients with arthritis. (Z9) Zucker et al. proposed that the minimal increase in serum MMP-3 in prostate cancer probably reflects an inflammatory response rather than an effect of cancer. Employing a revised and more sensitive ELISA, Zucker et al. (Z3) reported that plasma MMP-9 levels were increased in the plasma of more than half of patients with prostate cancer (pre- and post-treatment) and bladder cancer, but not gynecologic cancers (ovary, cervix, uterus, vagina) (Fig. 3). In prostate cancer, no correlation was noted between plasma MMP-9 and PSA measurements, which suggests that MMP-9 levels do not correlate with tumor mass. Plasma MMP-9 concentrations have also been reported to be significantly higher in patients with renal cell carcinoma than in healthy controls. However, the sensitivity was only 36% for detecting renal cancer, thus limiting the

FIG. 3. Incidence of increased plasma levels of MMP-9 in patients with various forms of cancer. Some cancer patients had previously received chemotherapy or radiotherapy, but all had evidence of active disease at time of blood procurement. Patients with bladder cancer had the highest incidence of increased levels. Upper limit of normal was based on mean 2 standard deviation of 40 healthy control subjects.


59

clinical usefulness of the test. No correlation was found between pathological TNM staging/histological grade and plasma MMP-9 levels in patients with renal cancer (L3). Tumor tissue levels of MMPs were consistent with the concept that plasma MMP-9 was primarily released from the kidney. In contrast, plasma MMP-2 and TIMP-2 concentrations were actually higher in healthy controls than in renal cancer patients. Zucker et al. reported results of plasma MMP-7 measurements using an ELISA. A bimodal distribution of values was observed in the normal population. Patients with bladder cancer, but not breast, GI, lung, gynecologic, and prostate cancer, demonstrated increased plasma MMP-7 levels (Z3). Manenti et al. (M2) reported that plasma MMP-9, TIMP-1, and TIMP-2 levels were higher in the plasma of ovarian cancer patients than in the plasma of women with nonmalignant disease or healthy women; plasma MMP2 levels were not increased in cancer patients. Of these parameters, only TIMP-1 was associated with a poor survival and mortality risk. They reported that levels of activated MMP-9 (identified by gelatin substrate zymography) were increased in the plasma of patients with either ovarian cancer or nonmalignant ovarian disease as compared to healthy subjects, thus negating the discriminative value of the test for cancer. 6.2.4. Melanoma Based on studies showing that overexpression of MMP-2 protein in tumor tissue was associated with a 5-fold relative risk of dying from melanoma, (V1) studies were subsequently initiated to measure MMP levels in blood. These studies demonstrated that serum MMP-2 is not a prognostic marker in advanced melanoma. However, in serial measurements, median serum MMP-2 concentrations at disease progression was significantly higher than before treatment; (V6) the clinical relevance of this data is doubtful. In contrast, Meric et al. (M11) reported that median total plasma MMP-2 and activated MMP-2 levels as measured by substrate zymography were actually higher in healthy volunteers than in patients with malignant metastatic melanoma; TIMP-1 levels were not increased in these patients.

6.3. MMP

AND

TIMP LEVELS IN THE BLOOD OF PATIENTS WITH INFLAMMATORY DISEASE

Many reports have confirmed increased levels of serum/plasma MMPs (MMP-3, MMP-1, MMP-9) and TIMPs in patients with rheumatoid arthritis and systemic lupus erythematosis (SLE) and to a lesser degree gout, osteoarthritis, and scleroderma (Z9).

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6.3.1. Rheumatoid and Osteoarthritis Arthritis Approximately 80% of patients with rheumatoid arthritis had a 3-fold increase in blood levels of MMP-3 as compared to healthy controls (M4, Z9). Of interest, synovial fluid levels of MMP-3 are several hundred-fold higher than serum levels (Y6). Furthermore, a good correlation has been demonstrated between paired serum and synovial fluid levels of MMP-3. These data are consistent with high level MMP-3 production in the joint space and subsequent leaching into the bloodstream in patients with rheumatoid arthritis (Z3). A correlation between the morphologic appearance of rheumatoid synovium and serum MMP and TIMP profiles has been reported, thus confirming the heterogeneity of rheumatoid arthritis and the possibility that different treatment regimens may be indicated for different histological forms (K5). Discussions have been ongoing during the 1990s over the issue of correlation of blood MMP-3 levels and various parameters of disease activity (M4). Cheung et al. (C5) reported that Health Assessment Questionnaires and Creactive protein levels were highly correlated with serum proMMP-3 levels in patients with rheumatoid arthritis in both early and established disease. Weak but significant relationships were found between serum proMMP-3 levels and disease duration, age, and number of swollen joints. Significant differences in serum proMMP-3 levels were noted between rheumatoid arthritis patients with and without bone erosion. No differences in serum proMMP-3 levels were noted between Rheumatoid Factor positive and negative patients. Serum proMMP-3 levels were significantly higher (>100%) in patients with SEþþþ(Shared Epitope). The highest proMMP3 levels were found in patients with a combination of DR4 and DR1 alleles. Yamanaka et al. (Y1) reported that serum MMP-3 levels at entry into study had a strong correlation with the rheumatoid disease activity (Larsen score) at 6 months and 12 months after entry. Suppression of the serum MMP-3 level in the first year correlated with a decline in joint damage in the second year. The authors concluded that serum MMP-3 is a useful marker for predicting bone damage during the early stage of rheumatoid arthritis. Based on their studies, Roux-Lombard et al. (R5) concluded that serum proMMP-3 correlated closely with C-reactive protein but gave little or no additional clinical information regarding inflammation or radiographic progression of joint destruction in patients with early rheumatoid arthritis. In patients with early rheumatoid arthritis, Posthumus et al. (P5) reported finding no correlation between serum MMP-3 and tender joint count (TJC) or the rheumatoid arthritis index (RAI). In contrast, the radiological score as an outcome measure, especially joint space narrowing, correlated closely with cumulative serum MMP-3. Furthermore, there was no significant


61

difference in serum MMP-3 levels at study entry between patients who developed X-ray damage later in the course of disease compared to patients with X-ray damage at presentation. Thus, high initial serum MMP-3 levels are predictive of the development of radiological damage (P4). Local therapies like intra articular injection of corticosteroid caused significant reduction in serum MMP-3 levels. The authors concluded that serum MMP-3 in early disease is a marker of joint inflammation and joint destruction. The ratios of TIMP-1:MMP-1 and TIMP-1:MMP-3 in blood have been reported to be significantly lower in patients with rheumatoid arthritis versus patients with nonrheumatoid arthritis (C10). In rheumatoid arthritis patients, serum C-reactive protein correlated with MMP-3 and TIMP-1 levels, but not with MMP-1 levels. The number of erosions noted on X-rays correlated with baseline levels of MMP-3, but not TIMP-1. Cunnane et al. (C10) postulated that treatment which inhibits the production and activation of MMP-1 may preferentially limit the formation of new joint erosions and improve the clinical outcome of patients with rheumatoid arthritis. In contrast to circulating levels of MMP-1, Keyszer et al. reported that MMP:TIMP complexes in blood correlate with rheumatoid activity scores (modified Lansbury Index and Keitel Function Index) in rheumatoid arthritis; nonetheless, this relationship to disease activity was weaker than that of MMP-3 or C-reactive protein (K4). Plasma MMP-9 levels have been reported to be significantly higher in patients with rheumatoid arthritis (8-fold higher) as compared to healthy controls. Rheumatoid arthritis complicated by vasculitis was associated with higher MMP-9 levels than in patients without vasculitis (S7). A significant increase in plasma MMP-9 concentrations has also been observed in the plasma of patients with ankylosing spondylitis, but not in the plasma of patients with osteoarthritis. These results suggested that circulating MMP-9 may reflect some degree of neutrophil activation in patients with inflammatory arthritis, especially in those with rheumatoid arthritis complicated by vasculitis. Following treatment of patients with rheumatoid arthritis with low and high dose anti tumor necrosis factor- (TNF) or placebo, serum MMP-1 and MMP-3 levels were assessed by Brennan et al. (B10). In both antibodytreated groups, a significant decrease in serum MMP-3 levels at all time points was observed, reduced maximally to 41% of pre-infusion values at day 7. MMP-1 levels were also reduced, but less dramatically than with MMP-3. While serum MMP-3 levels correlated with C-reactive protein both prior to and following therapy, Brennan et al. concluded that it remains to be demonstrated that serum MMP-3 and/or MMP-1 levels reflect the cartilage and bone resorptive processes which are evident in this disease. Catrina et al. (C2) also examined the effect of anti-tumor necrosis factor- therapy (etanercept) on MMPs. Etanercept therapy downregulated serum

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levels of MMP-3 and MMP-1 in conjunction with reduction in inflammatory mediators (C-reactive protein and erythrocyte sedimentation rate) in patients with rheumatoid arthritis. A clinically relevant issue is whether blood MMP levels can distinguish between different types of arthritis. Serum concentrations of MMP-3 are significantly increased in osteoarthritis, but not to the extent observed in rheumatoid arthritis (Z9). Masuhara et al. (M8) reported that there was no significant difference in the mean serum level of MMP-1, MMP-2, TIMP-1, and TIMP-2 among the patients with rapidly destructive hip osteoarthritis, patients with nonaggressive osteoarthritis, and control groups. In contrast, serum and plasma concentrations of MMP-3 and MMP-9 were significantly higher (>3-fold) in patients with rapidly destructive hip osteoarthritis as compared to the remaining osteoarthritis group or a control group. Plasma MMP-3, TIMP-1 levels, and the MMP-3:TIMP-1 ratios have been reported to be significantly higher in rheumatoid arthritis patients than osteoarthritis patients before hip surgery. In rheumatoid arthritis patients, the plasma MMP-3 and the MMP-3:TIMP-1 ratio decreased after total joint replacement, whereas C-reactive protein and erythrocyte sedimentation rate did not change. Omura et al. (O5) concluded that C-reactive protein and erythrocyte sedimentation rate reflect systemic inflammation. In contrast, plasma MMP-3 and the MMP-3:TIMP-1 ratio reflect inflammation and/or degeneration of the affected joint. Serum and synovial fluid MMP-3 levels have also been reported to be increased in patients with juvenile idiopathic arthridities; higher levels were noted with active versus inactive arthritis (G6). Synovial fluid levels of MMP-3 were 30-fold higher than paired serum levels. Gattorno et al. (G6) proposed that inadequate counterexpression of TIMP-1 may represent a crucial event for the development and perpetuation of tissue damage in juvenile arthritis. Elevated levels of serum MMP-1 and TIMP-1 have also been demonstrated in psoriatic arthritis; these levels were also increased in siblings of patients with psoriatic arthritis, suggesting that genetic factors may be important (M20). 6.3.2. Systemic Lupus Erythematosus Elevated serum levels (3- to 5-fold) of MMP-3 have been demonstrated in patients with systemic lupus erythematosus, the prototype autoimmune disease, as compared to healthy controls. Contrary to anticipated results, serial measurements of MMP-3 in patients with SLE did not correlate with fluctuation in disease activity scores (Z12). Similar results were later reported by Keyszer et al. (K4). These data are consistent with the concept that MMP-3 is more related to the later tissue repair aspect, rather than to the initiating tissue injury process in SLE (Z12). If this assumption is correct, the use of MMP-inhibitory drugs may prove to be detrimental in diseases in which the


63

MMP repair process is beneficial to the host. Thus, the issue of when to use an MMP-inhibitor in a disease process may be critical to the outcome of drug treatment. Kotajima et al. reported high serum levels of MMP-3 levels in 76% of patients with SLE and 82% of patients with rheumatoid arthritis (K9). The serum MMP-3 levels were significantly higher in SLE patients who had a history of the following abnormalities: persistent proteinuria, cellular casts, anti-double stranded DNA antibodies, decreased C3 and C4, decreased creatinine clearance, circulating immune complexes, malar rash, and hypoalbuminemia. They suggested that MMP-3 might be produced predominantly in the diseased kidneys. In agreement with Zucker et al., (Z12) the serum MMP-3 level did not fall with treatment-induced clinical improvement (K9). Serum levels of MMP-1, MMP-2, and TIMP-2 were not increased in patients with SLE (F1, Z12). In patients with IgA-glomerulonephritis and SLE (mesangial proliferative glomerulonephritis), serum MMP-3 and TIMP-2 levels were reported to be significantly higher than in control subjects. On the other hand, in patients with membranous nephritis, serum MMP-2 and TIMP-1 levels were significantly higher than in control subjects (A1). 6.3.3. Scleroderma and Psoriatic Arthritis Toubi et al. (T2) reported a significant association between elevation of both serum MMP-1 and TIMP-1 levels and severity of fibrosis in scleroderma. Those patients who had an increase of more than one MMP and/or TIMP demonstrated life-threatening major organ involvement and poor prognosis. Serum MMP-1 was increased in 19% of patients with scleroderma compared to 7% of patients with rheumatoid arthritis. Serum MMP-3 was elevated in 34% of patients with rheumatoid arthritis as compared to 12% of patients with scleroderma. TIMP-1 was increased in 40% of patients with scleroderma (T2). In contrast, Young-Min et al. (Y8) reported increased levels of serum TIMP-1 especially early in the course of disease, but normal serum MMP-1 and TIMP-2 levels in patients with systemic sclerosis. Yazawa et al. (Y4) reported that serum TIMP-2 levels were elevated in 23% of patients with systemic sclerosis and correlated with severity of disease. Serum MMP-1 and TIMP-1 levels have also been reported to be elevated in patients with psoriatic arthritis and their siblings, thus implicating genetic factors (M20). 6.3.4. Inflammatory Kidney Disease Serum MMP-1 levels in kidney failure patients with chronic transplant nephropathy or during acute kidney rejection have been reported to be significantly higher (250%) than patients with stable graft function and a

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control group. Serum MMP-2 and MMP-3 were also higher (>50%) in patients with chronic transplant nephropathy (R3). 6.3.5. Inflammatory Bowel Disease Elevated serum levels of MMP-3 have also been described in inflammatory bowel disease and graft-versus-host disease, which is consistent with mesenchymal cell production of MMP-3 controlled by cytokines (B1). Thus, high blood MMP-3 levels are not indicative of a single disease, but reflect a more generalized inflammatory response. 6.3.6. Pancreatitis Nakae et al. (N3) examined plasma levels of MMPs and TIMPs in patients with pancreatitis. Increased levels of MMP-1, MMP-1:TIMP-1 complex, and TNF- were reported in patients with pancreatitis and multiorgan dysfunction syndrome; high levels were associated with death.

6.4. MMP

AND

TIMP LEVELS IN LIVER DISEASE

Numerous studies of cirrhotic patients have reported high serum levels of TIMP-1 and a positive correlation between serum TIMP-1 levels and the degree of liver fibrosis in subjects with alcoholic hepatitis and alcoholic cirrhosis (L5). It has been debated whether blood TIMP-1 levels also correlate with the degree of liver inflammation. Plasma TIMP-1 levels were reported to be significantly correlated with the histological activity index, portal inflammation, periportal necrosis, and focal necrosis. Plasma TIMP-2 correlated with fibrosis and confluent necrosis. Receiver Operating Characteristic (ROC) analysis showed significant discriminatory ability of TIMP-1 and TIMP-2 in diagnoses of advanced liver disease (W1). These findings suggest that the measurement of serum TIMP-1 levels in various liver diseases may be useful to estimate the active hepatic fibrogenesis associated with the active inflammatory stage of liver injury (U2). Flisiak et al. (F4) proposed that plasma TIMP-1 and TGF- might also be useful as noninvasive biomarkers of liver fibrosis in patients with chronic hepatitis B and C. Boeker et al. (B8) reported that plasma values of both TIMP-1 and MMP-2 were useful indicators of increasing fibrosis in patients with chronic hepatitis C. Serum TIMP-1 levels correlated with the degree of hepatic fibrosis and inflammation. Serum MMP-1 levels were reported to be decreased in patients with postoperative biliary atresia; TIMP-1/-2 levels were not affected (K6). Koulentaki et al. (K10) reported that the concentrations of MMP-1, -2, -3, and -9 were significantly decreased in the sera of patients with acute hepatitis B. These authors proposed that the decreased


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levels of MMPs may indicate an attempt to limit matrix degradation in early hepatitis. Serum levels of TIMP-2 and proMMP-2 have also been reported to be significantly higher in patients with hepatocelluar carcinoma than in normal controls. Serum levels of TIMP-2 correlated with those of MMP-2 in hepatocellular carcinoma and did not significantly differ from those of patients with chronic hepatitis stage IV. Serum levels of proMMP-2 in patients with chronic hepatitis were strongly correlated with those of type IV collagen (E1). Serum MMP-2 levels also revealed positive relationships with the degree of periportal necrosis, the degree of fibrosis, and total score of the histological activity index. Three research groups (A4, F4, K2) have commented on the usefulness of serum MMP-1 and TIMP-1 measurements in predicting response to interferon therapy. In logistic multivariate regression analysis, the ratio of serum MMP-2:TIMP-1 level, HCV genome subtype, and serum TIMP-2 level were independent predictors for sustained response to interferon therapy (K2). Serum MMP-1 levels are inversely related to the histological degree of periportal necrosis, interlobular necrosis, portal inflammation, and liver fibrosis. Murawaki et al. (M18) proposed that the serum MMP-1 test may be useful to differentiate between active and inactive forms of hepatitis; MMP-1 test was reported to be superior to the serum procollagen type III N-peptide (PIIINP) test in assessing liver necrosis and inflammation (M18). The serum MMP-2 concentration was significantly increased in patients with liver cirrhosis and hepatocelluar carcinoma patients, but not in the patients with chronic hepatitis. In patients with chronic viral liver disease, serum MMP-2 concentrations showed the best correlation with the degree of liver fibrosis and with serum hyaluronate level. Liver MMP-2 content was reported to be markedly increased in cirrhotic livers. In contrast, changes in serum MMP-3 levels are not associated with postnecrotic tissue remodeling and are of little use for assessing ongoing fibrolysis in chronically diseased livers (M19). 6.5. MMP

AND

TIMP LEVELS IN DISEASES OF THE NERVOUS SYSTEM

Based on studies demonstrating increased levels of MMP-9 in the spinal fluid of patients with inflammatory processes involving the nervous system, considerable interest evolved in the measurement of plasma MMPs in various neurologic diseases. Mean plasma levels of MMP-9 in patients with Guillain-Barre syndrome were reported to be five times higher than in healthy subjects or patients with other neurologic diseases. The percentage of plasma MMP-9 in these patients decreased approximately 60% after

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treatment (competed plasma exchange or immunoglobulin administration) (C9). The plasma MMP-9:TIMP-1 ratios were noted to be increased with more severe disease and disability and decreased in late recovery. TIMP-1 also appeared to be increased and served as a disease severity marker in Guillain-Barre syndrome. However, the TIMP-1 findings were not specific as TIMP-1 was also increased in stroke, hydrocephalus, and chronic axonal sensory polyneuropathy. Creange et al. (C9) implicated TGF1 as a potent inducer of TIMP-1 synthesis. Sharshar et al. (S6) demonstrated that circulating levels of plasma MMP-9 (157 141 ng/ml) were higher in demyelinating Guillain-Barre syndrome than in the nondemyelinating syndrome (4845 ng/ml). A role for MMP-9 in the demyelination process was proposed. Plasma levels of MMP-9 have been reported to be elevated more than 2fold in patients with Alzheimer’s disease as compared to control subjects or patients with Parkinson’s disease (L10). Plasma levels of MMP-2, TIMP-1, and TIMP-2 were unchanged. This finding led to the suggestion that circulating levels of MMP-9 may be a contributing factor in Alzheimer’s disease. Lorenzl et al. (L10) suggested that circulating A, proinflammatory cytokines, or oxidative stress may contribute to the increased level of MMP-9 in Alzheimer’s disease. Serum TIMP-1 and TIMP-2 levels were reported to be significantly higher in patients with multiple sclerosis and those with other neurological disease than in healthy controls. TIMP-1 levels were not different during relapse and between relapses (L2). There was a trend for serum TIMP-2 levels to be lower during relapse compared with nonrelapsed periods. Galboiz et al. (G1) reported changes in blood leukocyte mRNA for MMPs in patients with multiple sclerosis responding to interferon-. Montaner et al. (M15) demonstrated high baseline plasma MMP-9 levels (4-fold increase above normal) in patients with stroke who subsequently developed parenchymal hemorrhage. A graded response was found between mean baseline plasma MMP-9 levels on admission to the hospital and the degree of bleeding that subsequently developed as noted by computerized axial tomography (CAT) scan. Baseline plasma MMP-9 was a powerful predictor of parenchymal hemorrhage in multiple logistic regression models. Among cardiovascular risk factors, only patients with diabetes had significantly higher baseline levels of MMP-9. Plasma MMP-2 values were unrelated to any subtype of hemorrhagic transformation. Castellanos et al. (C1) also reported that plasma MMP-9 concentrations on admission were significantly higher in patients who subsequently developed hemorrhagic transformation. Castellanos et al. studied a large number of patients with a hemispheric ischemic stroke of 4–12 h duration; hemorrhagic transformation was seen in 15% of these patients. Of these, 63% had a hemorrhagic infarction and 37% had a


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parenchymal hematoma. Median plasma MMP-9 concentrations measured within 3 hours of symptom onset were 3-fold higher in the hemorrhagic transformation group. A plasma MMP-9 of 140 ng/ml for hemorrhagic transformation was 61% (C1).

6.6. MMP AND TIMP LEVELS IN CARDIOVASCULAR DISEASE 6.6.1. Angina and Myocardial Infarction Serum MMP-2 and plasma MMP-9 levels in patients with unstable angina and acute myocardial infarction on day 0 were reported to be 50 and 250% greater than that in control subjects, respectively (K1). In patients with unstable angina and acute myocardial infarction who underwent medical treatment, the MMP-2 elevation was sustained until day 7; MMP-9 elevations gradually decreased toward normal by day 7. Kai et al. suggested that leakage due to myocardial necrosis was not the main source of MMPs because MMP levels were not correlated with serum myocardial enzyme release; macrophages and leukocytes were proposed as the source of elevated MMPs in acute coronary syndromes. In contrast to that supposition, Bradham et al. (B9) examined plasma MMP and TIMP profiles following alcohol injection into the septal perforator artery in order to induce myocardial injury in patients with hypertrophic obstructive cardiomyopathy. Post induction of septal myocardial infarction, plasma MMP-9 levels increased by over 400% and MMP-8 increased over 100% within 12 hours; plasma TIMP-1 levels were unaffected. A significant linear relationship was observed between cardiac enzyme (CPK-MB1) release and plasma MMP-9 levels. Bradham et al. (B9) proposed that monitoring MMP and TIMP profiles may provide a novel approach to assess wound healing and myocardial remodeling. Noji et al. (N8) demonstrated that mean plasma levels of MMP-9 and TIMP-1 were significantly higher and the mean levels of MMP-2, MMP-3, and TIMP-1 were significantly lower in patients with premature atherosclerosis and stable coronary artery disease; a weak correlation between these MMPs and patient lipid profiles was claimed. Both serum MMP-1 and TIMP-1 showed significant time-dependent alterations after acute myocardial infarction. Serum MMP-1 was more than 1 SD below mean control values during the initial four days, increased thereafter, reaching a peak concentration around day 14, and then returned to the middle control range (H4). Serum TIMP-1 at admission was more than 1 SD below the mean control value, and increased gradually thereafter, reaching a peak

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around day 14. Hirohata et al. (H4) suggested the involvement of MMP-1 in the healing process during cardiac remodeling. Blankenberg et al. (B7) reported that mean plasma concentrations of MMP-9 obtained prospectively beginning at time of hospital admission were significantly higher among patients who subsequently experienced a fatal cardiovascular event (62 ng/ ml) than among those who did not (48 ng/ml); MMP-9 remained independently associated with future cardiovascular death (B7). The crude hazard risk ratio of cardiovascular death associated with increasing quartiles of MMP-9 was significant after adjustment for clinical and therapeutic confounders. Patients in the highest quartile (>72 ng/ml) had a 4-fold increase in cardiovascular mortality compared to patients in the first quartile (50%. The authors proposed that a lack of decrease in plasma MMP levels after endovascular grafting may help to identify patients who will have postoperative endovascular leakage. Chua et al. (C6) reported markedly, but transiently, elevated levels of proMMP-9 and TIMP-1 in the plasma of patients with Kawasaki disease (an acute, self-limiting vasculitis); this data is consistent with MMP-9 involvement in vascular remodeling and an inflammatory response to a microbial agent. Matsuyama et al. (M9) reported that circulating levels of MMPs can be useful in the diagnosis of Takayasu arteritis. 6.7. HYPERTENSION Mean serum baseline free MMP-1 was reported to be decreased 25% and baseline free TIMP-1 was increased 50% in hypertensives compared with normotensive patients. Hypertensive patients with baseline left ventricular hypertrophy exhibited lower levels of free MMP-1 and carboxyl terminal telopeptide of collagen type I and higher values of free TIMP-1 than did hypertensive patients without baseline left ventricular hypertrophy. Laviades (L1) suggested that extracellular digestion of collagen type I is depressed in essential hypertension and may facilitate organ fibrosis in hypertensive individuals.

7. Miscellaneous Diseases Associated with Increased Levels of MMPs and TIMPs 7.1. DIABETES MELLITUS Ebihara et al. (E2) reported that plasma MMP-9 measurements made over a 4-year period in non insulin-dependent diabetics were highly predictive of the development of diabetic nephropathy. Compared with patients with


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normal urinary albumin excretion, patients with microalbuminuria had significantly higher plasma levels of MMP-9 at the second (56 14 ng/ml vs 36 12 ng/ml), third (88 23 ng/ml vs 39 14 ng/ml), and fourth year (117 30 ng/ml vs 44 16 ng/ml), but not initially (34 12 ng/ml vs 33 14 ng/ml); plasma MMP-9 levels of healthy controls were 38 13 ng/ml. The groups did not differ with regard to age, sex, duration of non insulin-dependent diabetes mellitus, blood pressure, or mean glycated hemoglobin. Microalbuminuria was reduced to within the normal range, and plasma MMP-9 concentrations were significantly decreased with ACE inhibitor treatment (5218 ng/ml). The increase in plasma MMP-9 levels preceded the occurrence of microalbuminuria by at least 3 years. The source of increased plasma MMP-9 was postulated to be infiltrated macrophages in the kidney and/or resident renal cells that secrete MMP-9. Uemura et al. (U1) also reported elevated plasma levels of MMP-9 in diabetic patients. 7.2. MYELOPROLIFERATIVE DISEASES Patients with agnogenic myeloid metaplasia, essential thrombocytosis, and polycythemia vera had been reported to have increased plasma TIMP-1 levels. An inverse correlation was demonstrated between marrow fibrosis in patients with agnogenic myeloid metaplasia and plasma MMP-3 levels (W2). 7.3. BURNS Serum MMP-2 and TIMP-1 levels rise within 3 days following extensive burn injury. Ulrich et al. (U3) proposed that the elevated TIMP-1 concentration might contribute to tissue fibrosis. Increased levels of serum TIMP-1, but not MMP-3, have been reported in patients with atopic dermatitis; serum levels of TIMP-1 dropped after conventional therapy (K3). 7.4. POLYCYSTIC KIDNEY DISEASE Elevated levels of serum MMP-1, TIMP-1, and plasma levels of MMP-9 have been reported in patients with autosomal dominant polycystic kidney disease as compared to healthy controls (N6).

8. MMPs Identified in Urine of Patients with Cancer Gelatin zymography has been used to demonstrate the presence of several forms of MMPs in the urine of patients with cancer. The potential for using this test for early diagnosis or staging of cancer has been proposed.

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Based on the molecular weight of MMP-2 and MMP-9 (72 and 92 kDa, respectively) and the glomerular filtration limit of 45 kDa, it was not anticipated that MMPs would be detected in urine. Nonetheless, employing gelatin substrate zymography and immunoassays, Marguilies et al. (M6) demonstrated increased levels of type IV collagenase (MMP-2) in the urine of 70% of patients with transitional cell carcinoma of the bladder. The major form of the enzyme in urine was the amino terminal fragment of MMP2 (45 kDa). A monoclonal antibody (CA-4001) recognized this major fragment of MMP-2 in all patients with transitional cell carcinoma of the bladder, but in none of the control cases. Tissue sections of normal urinary bladder epithelium were negative for immunoreactivity. In contrast, tissue sections of papillary transitional cell carcinoma of the bladder were positive. This result supported the assumption that the source of urinary MMP-2 as well as enzyme inhibitor complexes and enzyme fragments were at least partially derived from the carcinoma itself. In a subsequent study, Moses et al. (M16) reported that MMP-2 and MMP-9 in urine correlated with the presence of malignant disease, not just limited to the genitourinary tract. The presence of biologically active MMP2 or MMP-9 alone in the urine was an independent predictor of organconfined cancer; the higher molecular weight MMP species (>150 kDa) in the urine served as independent predictors of metastatic cancer (M16). The frequency of detection of the three MMP species in the urine was as follows: normal or no evidence of disease (11–20%), cancer (71%), metastatic cancer (90%). The frequency of urinary MMPs was significantly higher in patients with prostate and breast cancer, 75 and 100%, respectively. Odds of metastatic cancer were 30 times greater when the higher molecular weight MMP species were present in urine than when this marker was absent. A 125 kDa MMP was detected only in the urine of patients with breast cancer (M16). Indepth studies by Li et al. (Y2) demonstrated that the 125 and 115 kDa gelatinolytic bands represent complexes of MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). They further demonstrated that lipocalcin serves to protect MMP-9 from degradation, thereby preserving MMP-9 enzymatic activity. Studies by other investigators have confirmed the detection of MMPs in the urine of patients with cancer. In normal subjects, Monier et al. (M13) reported that the level of TIMPs in urine samples was higher than active MMPs (as measured by ELISA). In cancer patients, increased urinary levels of proMMP-9 and active MMP-2 (measured by substrate zymography) with reduced TIMP-2 levels correlated with higher stage and histological grade of urothelial cancers. Contrary to expectation, reduced MMP-9 and NGAL (lipocalin complexes with MMP-9) levels in urine were initial hallmarks of clinical relapse. The imbalance between increased MMP-2 and MMP-9 and


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decreased TIMP-2 levels appears to be linked to tumor stage and grade and, more importantly, to clinical events. Changes in the MMP-9 activation state and a lack of neutrophil-associated lipocalin (NGAL) were proposed as novel markers of tumor progression (M13). Monier et al. (M14) also employed continuous elution electrophoresis for isolating urinary MMPs. Significantly increased levels of MMP-2 and MMP-9 and a high molecular weight MMP-9 band (115 kDa) were detected by zymography in the urine of patients with epithelial cancers including breast, colon, and prostate; even higher enzyme levels were detected in the urine of patients with bladder cancer. Bladder washings revealed increased levels of proMMP-9 but not MMP-2 in patients with evolving bladder cancer as compared to control subjects (M14). Durkan et al. (D5) reported that urinary MMP-1 was detected in a higher percentage of patients with advanced stage (T2–T4) and grade (3) bladder cancers than in patients with early stage (cis/Ta/T1) or grade (1–2) bladder cancer. Patients with increased levels of urinary MMP-1 had higher rates of disease progression and death from bladder cancer. All patient groups also had higher levels of urinary TIMP-1 than the control group. Urine TIMP-1 levels strongly correlated with tumor size. Progression-free survival rates were lower for patients with high urine TIMP-1 concentrations.

9. Conclusions MMPs and TIMPs are involved in numerous physiologic and pathologic processes. In spite of considerable understanding of the chemistry and biology of MMPs, their role in disease processes is incompletely understood. Increased levels of MMPs and TIMPs have been demonstrated in the blood and urine of patients with many different types of disease. A compendium of descriptive reports has documented increased plasma levels of MMP-9 in patients with heart disease, stroke, cancer, skin disease, arthritis, and diabetes. However, the magnitude of increased plasma levels in these diseases is small in comparison to levels documented in pregnancy. Increased serum and plasma levels of MMP-3 have been demonstrated in patients with rheumatoid arthritis, SLE, and debilitating osteoarthritis. Urinary levels of MMP-9 and MMP-2 are increased in patients with cancer. Medical science, employing the technology currently available, appears poised to exploit the use of clinical assays for MMPs and TIMPs in diagnosis and treatment of specific diseases. Future research will need to employ more critical approaches rather than limited descriptive reports to identify practical medical applications for these assays. The possibility that patients with various diseases and increased circulating levels of MMPs reflect a subset of

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patients with unique biological characteristics who might respond selectively to anti-MMP therapy needs to be exploited. A major limitation to practical applications of blood MMP measurements for individual patients is the broad overlap between the ranges of normal and disease populations. Another consideration is whether measurement of blood and urinary levels of MMPs and TIMPs has potential utility in general screening for disease in periodic health examinations. In this instance, comparisons to baseline MMP measurements for single individuals might be useful in detecting development of disease over protracted periods of time, e.g., progressive development of cardiovascular disease. ACKNOWLEDGMENTS This research was supported by a Merit Review Grant and a Research Enhancement Award Program from the Department of Veterans Affairs, a Baldwin Breast Cancer Grant from the Research Foundation, SUNY, Stony Brook, a grant from the American Heart Association, and a grant from the U.S. Army Materiel Command.

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MOLECULAR METHOD TO QUANTITATIVELY DETECT MICROMETASTASES AND ITS CLINICAL SIGNIFICANCE IN GASTROINTESTINAL MALIGNANCIES H. Nakanishi,* Y. Kodera,{ and M. Tatematsu* *Division of Oncological Pathology, Aichi Cancer Center Research Institute, Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan { Department of Surgery II, Nagoya University School of Medicine, Tsuruma, Showa-ku, Nagoya 466-8550, Japan

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Methodology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Molecular Biological Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Quantitative Detection of Micrometastases and Its Prognostic Significance . . . . . 3.1. Free Tumor Cells in Peritoneal Lavage Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Circulating Tumor Cells in Peripheral Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Micrometastasis in Lymph Nodes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. General Considerations and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Recurrence of tumors after curative surgery is a life-threatening event for cancer patients, and the prevention of such recurrence is one of the most important problems to be resolved at the clinical level. The major cause of recurrence after curative resection in cancer patients is considered to be free tumor cells in the body fluid and invisible micrometastases in the distant organs which were already present at the time of removal of the primary neoplasm or had been shed from the primary tumor during surgical manipulation. To prevent recurrence and improve survival rates of cancer patients after curative resection, careful detection and subsequent chemotherapy for micrometastasis may be promising. To date, however, conventional adjuvant 87 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2423/04 $35.00

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chemotherapy has not been applied based on the detection of micrometastasis or objective assessment for recurrence risk. To develop tailor-made therapy against recurrent disease, adjuvant chemotherapy based on the selection of high-risk patients for relapse as well as the evaluation of chemosensitivity of the tumor seem indispensable. ‘‘Micrometastasis’’ has been defined as a minute metastasis measuring less than 2 mm in diameter by UICC (H4, S11). More recently, the term has been classified into two categories: ‘‘isolated tumor cells,’’ which are designated as single tumor cells and a small cell cluster that is no larger than 0.2 mm in greatest diameter, and ‘‘micrometastases’’ larger than 0.2 mm in size (S12). However, isolated tumor cells should be distinguished from micrometastasis since they do not typically show evidence of metastatic activity such as penetration of a vascular or lymph sinus wall, tumor cell growth, or stromal reaction. Micrometastasis is biologically and clinically distinct from macroscopic metastasis at least in the following two ways: (1) Micrometastasis, especially isolated tumor cells, may remain in a dormant state for a long period until they acquire suYcient blood supply to permit growth and invasion (F2, K8) or until they are eliminated by apoptosis and necrosis. In the dormant state, the cytokinetic and apoptotic rate are considered to be equilibrated so that there is no net growth (H7). The most important factor triggering proliferation in dormant micrometastasis may be neovascularization (F1). Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet-derived endothelial growth factor (PD-ECGF) seem to be important players in such angiogenesis. However, the precise mechanism by which the angiogenic switch is turned on remains unclear. (S2) Micrometastasis is chemosensitive to anti-cancer drugs. Since isolated tumor cells or micrometastases appear to be dormant and non-cell cycling (or slow cell cycling) as has been described, it has been speculated that they may be resistant to cell cycle-dependent anti-cancer drugs. In animal experimental study, however, we and other investigators have demonstrated a preferential therapeutic eYcacy for micrometastasis in the lung (K10) and peritoneum (N5) rather than for macroscopic metastasis. Mice bearing micrometastases in the peritoneal cavity survive longer than do those with macroscopic metastases after chemotherapy, and some of them can become pathological CR (complete regression) or be cured. Furthermore, several reports demonstrated that adjuvant chemotherapy (K3) or immunotherapy (S4) leads to a dramatic decrease in the detection rate of circulating tumor cells of cancer patients, suggesting high sensitivity of isolated tumor cells to an anticancer agent in clinical settings. Based on these unique features of micrometastasis, chemotherapy targeting micrometastasis seems to be more advantageous than that targeting macroscopic metastasis. It has thus been proposed as a new therapeutic strategy for preventing recurrence (N5). For

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this purpose, the development of a sensitive and specific detection method for micrometastases is of paramount importance. Historically, detection of micrometastasis began in the 1940s and 1950s with cytologic demonstration of the presence of tumor cells in the peripheral blood and bone marrow in cancer patients without visible metastasis. Since the 1980s, new interest has arisen as a consequence of the introduction of new methods. Schlimok et al. first demonstrated micrometastasis in bone marrow by immunohistochemistry, using an anti-cytokeratin antibody in 1987 (S3). Since then, a number of immunohistochemical studies using antibodies to various epithelialspecific antigens have been performed, and more than the expected distribution of micrometastasis has been demonstrated in the peripheral blood, bone marrow, and lymph nodes of histologically and cytologically tumor-free patients. With the 1990s, advances in molecular biological techniques, including polymerase chain reaction (PCR), made it possible to detect micrometastases more sensitively in the lymph nodes and bone marrow, and disseminated tumor cells in the peripheral blood and peritoneal lavage fluids (G2, M9, N2, S6, S10). With more recent innovations in PCR technology, a method for real-time monitoring of PCR reactions using a fluorescence-labeled probe (L3), and a new generation of thermal cyclers which permit continuous fluorescence monitoring of PCR have been developed (W2, W3). Such real-time fluorescence PCR systems allow accurate quantification of the initial template copy number. This approach became available in clinical settings since 1998–99 and now has been introduced as a practical alternative to conventional RTPCR for assessment of minimal residual disease in hematological malignancies (G1, P4) and micrometastasis in solid tumors (M7, N3). Since comprehensive reviews on the detection and prognostic significance of conventional RT-PCR-based micrometastasis have previously been reported (G6, K2, P2, T3, V1), we here amplified RT-PCR studies by the addition of the quantitative detection of micrometastasis using a real-time PCR technique and its prognostic significance in gastrointestinal malignancies. This chapter, including our own experience, summarizes the literature published mainly between 1997 and 2003 using medical subject headings (PubMed) with cross-referencing from key articles.

2. Methodology For detection of micrometastasis, immunohistochemical method was first introduced in 1987 (S3) and has been evaluated as a reliable method because the presence of tumor cells can be confirmed visually based on their morphology. To date, this immunohistochemical method is still widely used for detection of micrometastases in the lymph nodes and bone marrow (T3).

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2.1. MOLECULAR BIOLOGICAL METHOD In the past, genetic changes such as p53 and K-ras mutations were used as specific genetic markers for detection of free tumor cells and micrometastasis in the peripheral blood and lymph nodes (H2, H3). However, the positivity rate for such mutations in cancer is generally less than 50%, too low for routine diagnostic use. In addition, this method may detect even dead tumor cells containing the same sequence of interest, leading to false positive results (L1). 2.1.1. Conventional RT-PCR Conventional reverse transcriptase polymerase chain reaction (RT-PCR) assays are sensitive enough to detect 1 tumor cell per 106–107 leukocytes (M9). Therefore, this sensitive and convenient RT-PCR-based method is currently the most widely used for detection of micrometastases. However, there are at least two problems. RT-PCR with widely used markers such as tyrosinase, CEA, and mammaglobin as target genes, the sensitivity varies from 80 to 90%, depending on the tissue origin of cancer cells. However, this sensitivity is still considered to be low or insuYcient. In gastrointestinal and breast malignancies, 10 to 20% of cancer cells express CEA and mammaglobin at a very low level if present and can cause false negative results. For improving sensitivity, a combination assay with multiple markers, for example, CEA and CK 20, may be more sensitive than a single assay (O2). The more important problem with RT-PCR is false positive results. Hematopoietic cells are known to express low levels of CKs and CEA mRNA illegitimately, so any samples contaminated by blood can cause false positive results. Virtually all of the currently used marker genes proved to be more or less leaky expressed by noncancerous cells. For example, weak expression of CK19 mRNA has been demonstrated in the peripheral blood of 20% of healthy volunteers by RT-PCR. Pseudogenes may also give rise to false positive signals (G6). Therefore, the major disadvantage of conventional RT-PCR is the lack of suYcient specificity as a trade-oV for the high sensitivity to detect mRNA derived from tumor cells. One promising approach to reducing false positive results with RT-PCR assay is the introduction of quantitative real-time RT-PCR. In the past, a competitive RT-PCR assay was used for quantification of mRNA expression. However, this method is semiquantitative, and the procedure is laborious and time-consuming. Therefore, it is not practical for clinical diagnosis and now is being replaced by the real-time quantitative RT-PCR method, which will be described. 2.1.2. Real-time Quantitative RT-PCR With the innovations in PCR technology, a method for real-time quantitative PCR using a new-generation PCR system consisting of fluorescence probes and continuous monitoring of PCR reaction has been developed


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(H5). There are at least two types of real-time PCR systems. One is a PCR using a dual-labeled hydrolysis probe (TaqMan PCR) (L3), which is cleaved by the 50 nuclease activity of Taq polymerase (H6) and separates the reporter dye located in the 50 ends, resulting in increased fluorescence based on the Forster-type energy transfer. The other is a PCR using a hybridization probe set, which successfully produces a consistent fluorescence signal based on the fluorescence resonance energy transfer (FRET) resulting from hybridization of the two probes in close proximity (1 base) during the annealing phase (W2, W3). These real-time fluorescence PCR systems allow accurate quantification of the initial template copy number based on the fact that the cycle number at which the sample fluorescence exceeds the background level is correlated with the starting copy number (G1). Thus, this approach has been introduced as a practical alternative to conventional end point PCR. The lower limit of reproducible detection for this assay proved to be at least 1 tumor cell per 1 106 leukocytes, with measurement possible up to 1 106 tumor cells. This indicates that single-round real-time RT-PCR has a sensitivity comparable to conventional (nested) RT-PCR with a wide (1–106) dynamic range. The major advantage of real-time quantitative RT-PCR over conventional RT-PCR is the excellent specificity due to the use of double check probes in addition to specific primer and the continuous monitoring for the exclusion of an abnormal-shaped amplification curve. Introduction of a cutoV value can also serve to discriminate tumor-specific expression from illegitimate expression of nonmalignant cells and contributes to the low incidence of false-positive results with real-time quantitative RT-PCR. We previously reported that 40% of gastric cancer patients with detectable CEA mRNA of more than zero for peritoneal washes were considered to be false-positives based on the cutoV value (N3). The second advantage of quantitative real-time RT-PCR is that it allows cDNA quality assessment on a per-sample basis. DiVerentiation of cDNAs of high and poor quality used to be diYcult since the conventional end point PCR provides positive PCR results after 30 to 40 cycles even for cDNA of rather poor quality. Quality assurance of sample cDNA can be performed by either housekeeping genes such as glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and porphobilinogen deaminase (PBGD)-specific quantitative RT-PCR for large transcripts or quantitative RT-PCR specific to an artificial transcript (internal standard) for a very small amount of transcripts. In the latter, a fixed, usually small, number of stable transfected cells carrying a recombinant vector containing an insert comprising the same primer sites as the wild-type mRNA and a spacer sequence were added to the individual sample prior to sample processing, and are carried throughout the whole process. This internal standard is the most sensitive indicator for high quality

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cDNA and provides information on the false negative results because of the poor cDNA quality (M4). The third advantage of quantitative real-time RT-PCR is that it allows one to monitor the treatment eVect in individual patients as a potential surrogate marker after adjuvant chemotherapy (K3) or immunotherapy (S4) against micrometastatic diseases. For the purpose of monitoring, a relative quantification method which is designed to determine exact, PCR eYciency-corrected mRNA concentration, normalized to a calibrator, might be desirable to overcome the inter-assay variation from run to run (S13). This may make it possible to directly compare the mRNA values at diVerent time points. 2.1.3. Rapid Real-time Quantitative RT-PCR Real-time RT-PCR needs at least 3 to 4 hours for completion of measurement. Therefore, results of conventional RT-PCR and standard real-time RT-PCR are not available during surgery. So far, RT-PCR plays no role in decision-making with regard to various treatment options during interventions such as intraoperative diagnosis of sentinel lymph node micrometastasis and intraperitoneal chemotherapy. To overcome this problem, a rapid quantitative RT-PCR has been developed to enable diagnosis during the actual operation. A combination system with a fully automated mRNA extractor (MagNA Pure LC system) and a real-time one-step RT-PCR with a hybridization probe on the glass capillary (LightCycler) permits the rapid quantification, which allows completion of the entire procedure in approximately 2 hours (M2). The other is based on the loop-mediated isothermal amplification (LAMP) method, a novel method which amplifies DNA with high specificity, eYciency, and rapidity under isothermal conditions. The LAMP method employs DNA polymerase and a set of four specifically designed primers that recognize a total of six distinct sequences on the target DNA (N7). Since the increase in turbidity of the reaction mixture according to the production of by-product correlates with the amount of DNA synthesized, real-time monitoring of the LAMP can be achieved by measuring turbidity (M10). The LAMP method can also be applied for direct amplification of RNA without need of RNA purification or cDNA synthesis. The entire procedure with this technique can be completed within 1 hour after sampling.

3. Quantitative Detection of Micrometastases and Its Prognostic Significance Many reports have been published on the quantitative detection of micrometastasis as determined by real-time RT-PCR in the peripheral blood, bone marrow, lymph nodes, and peritoneal washes, although the reports on its


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prognostic relevance are few. Overviews of currently available results are summarized in Table 1 to Table 3. The evidence to date seems to be more convincing for peritoneal lavage fluids than for peripheral blood and lymph nodes in gastrointestinal malignancies. Thus, this section begins with samples of this anatomic compartment. 3.1. FREE TUMOR CELLS IN PERITONEAL LAVAGE FLUIDS In gastric and ovarian cancers, peritoneal dissemination is the major recurrence pattern after curative resection and is the most important prognostic indicator of patient survival. Peritoneal lavage cytology is reliable and specific, and has been a gold standard for risk assessment of peritoneal recurrence in gastric and ovarian cancer patients, but it lacks sensitivity (B2). Peritoneal recurrence reportedly occurs in 20 to 30% of cytologynegative gastric cancer patients. This low sensitivity of cytology can be improved by more sensitive methods such as immunohistochemistry (S7), RT-PCR, and the quantitative real-time RT-PCR method in gastric cancers. To date, however, this is not the case with ovarian cancers because there are few useful markers capable of detecting a wide spectrum of ovarian cancer cells with heterogenous origins (N4). In addition, there are few reports on the application of RT-PCR in pleural lavage studies, although pleural lavage cytology has been found to be a good prognostic indicator of poor survival of non-small cell lung cancer patients (O3). We first reported the detection of free tumor cells in peritoneal washes and its prognostic value in gastric cancer patients by conventional nested RT-PCR in 1997 (N2) and then quantitative real-time RT-PCR with CEA as a genetic marker in 2000 (N3). The CEA mRNA positivity rate of peritoneal washes by RT-PCR is associated with the depth of invasion (T category): 10% in T1 (cancer confined to mucosa or submucosa), 30% in T2 (muscular or subserosal invasion), 78% in T3–T4 (serosa exposed or invasion to adjacent tissues) stage, compared to 2% in T1, 5% in T2, and 41% in T3–T4 stage in conventional cytology. In quantitative RT-PCR, the respective positivity rate is 4% in T1, 15% in T2, and 67% in the T3–T4 stage, which is intermediate between the other two methods. These results indicate an improvement in specificity without significant loss in sensitivity in quantitative RT-PCR. In fact, the sensitivity and specificity of real-time RT-PCR with an appropriate cutoV value were 85 and 94%, whereas those for conventional cytology were 56 and 91%. In colorectal cancer, peritoneal recurrence is relatively rare as compared with gastric cancers. Broll et al. reported in 2001 that CEA mRNA in the peritoneal fluids was detected at the incidence of 65% in 49 colorectal cancer patients (B3). Guller et al. also reported that the positivity rate of CEA mRNA in peritoneal washes is 0% in stage I–II patients and 33% (6/18) in

TABLE 1 DETECTION AND PROGNOSTIC VALUES OF PERITONEAL LAVAGE FLUIDS BY CONVENTIONAL AND/OR QUANTITATIVE REAL-TIME RT-PCR IN GASTRIC AND COLORECTAL CANCERS Origin

Source of fluids

No. of patients

Positive rate (T stage)

Correlation with stage

48 148 109 230

73% (pT3) 69% (pT3) 67% (pT3) 19% (pT3)

yes yes yes yes

NA Poor prog. NA Poor prog.

N2 K7 N3 Y3

CEA CEA

D and S D and S D and S Preoperative wash D D and S

30 189

45% (pT3) 58% (pT3)

yes yes

S1 K6

cPCR cPCR qPCR qPCR cPCR

CEA CEA CEA CEA/CK20 CEA

P D and S D and S P P

86 136 124 63 75

55% (pT3) 54% (pT3) 71% (pT3) 100% (pT3) 63%

yes yes yes yes yes

NA Independent prog. Poor prog. Poor prog. Poor prog. NA Poor prog.

F3 T2 U1 M2 B3

cRCR qPCR cPCR

CEA/AFP CEA/CK20 CEA/CK20

D P D

64 39 79

19% 25% 19% (pT3)

NA yes yes

NA Poor prog. NA

S5 G8 A3

Authors

Year

PCR

Marker

Nakanishi et al. Kodera et al. Nakanishi et al. Yonemura et al.

1997 1998 2000 2001

stomach stomach stomach stomach

cPCR cPCR qPCR cPCR

CEA CEA CEA CEA

Sakakura et al. Kodera et al.

2001 2002

stomach stomach

cPCR qPCR

Fujii et al. Tokuda et al. Ueno et al. Muratsuka et al. Broll et al.

2002 2003 2003 2003 2001

Schmidt et al. Guller et al. Aoki et al.

2001 2002 2002

stomach stomach stomach stomach colon/stomach/ panc gastrointestinal colorectum colorectum

Prognostic relevance

Ref.

Panc, pancreas; gastrointestinal, colon/stomach/pancreas/hepatocellular/others; cPCR, conventional RT-PCR; qPCR, quantitative RT-PCR; D, Douglas cavity; S, Subphrenic space; P, peritoneal cavity; Preoperative wash, it was done by paracentesis; pT3, tumor penetrates serosa (T category by TNM classification); NA, not assessed; Poor prog., poor prognosticator (Univariate analysis); Independent prog., independent prognosticator (Multivariate analysis).


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stage III colorectal cancer patients by real-time RT-PCR (G8), suggesting that colon cancer cells can be shed from the serosal surface of the primary site and may spread into the peritoneal cavity, much as in the case of gastric cancer. In order to assess the prognostic significance of free tumor cells in peritoneal washes, various clinical follow-up studies have been conducted. All the reports to date on the prognostic significance of CEA mRNA as determined by RTPCR and quantitative RT-PCR in gastric and colorectal cancer patients are summarized in Table 1. A retrospective study using 189 gastric cancer patients by Kodera et al. first demonstrated in 1998 that positive quantitative CEA RT-PCR could identify the patients with reduced overall survival and peritoneal metastasis-free survival and that the quantitative CEA RT-PCR result was an independent prognostic indicator, along with the presence of lymph node metastasis and serosal invasion (K6, K7). Fujii et al. also detected CEA mRNA in 27 out of 49 cases with serosal invasion and without macroscopic peritoneal metastasis. Among them, 15 patients (56%) relapsed with peritoneal metastasis within 12 months after surgery. In contrast, none of the 22 CEA negative cases had peritoneal recurrence (F3). So far, all 6 of the other reports provided similar results (A3, M2, T2, U1, Y3), indicating that CEA mRNA detection by RT-PCR, especially quantitative real-time RT-PCR, is a strong and reliable prognostic indicator. A large-scale multi-center retrospective study based on the standardized RT-PCR protocol and a prospective study using the predetermined cutoV value are currently ongoing by our group to establish its prognostic significance. 3.2. CIRCULATING TUMOR CELLS IN PERIPHERAL BLOOD Since the publication of the original articles by Smith et al. in 1991 on the detection of circulating tumor cells in the malignant melanoma patients (S10), many investigators have analyzed circulating tumor cells in the peripheral blood of patients with a variety of solid tumors by conventional RTPCR. In this section, we first summarize the results from conventional RT-PCR studies and then refer to recent advances in the detection of circulating tumor cells with the quantitative real-time RT-PCR method. We selected melanoma and colorectal cancers, mainly the latter, which is one of the most extensively studied cancers in this compartment. As for malignant melanoma, more than 15 study groups have attempted to detect circulating tumor cells using conventional RT-PCR for melanocytespecific markers, mainly tyrosinase as well as MART-1. Many studies showed a correlation between the detection of tyrosinase RT-PCR positive cells in the peripheral blood and the clinical stage (B4), but not all of the studies (R2). In patients with stage 0, 15 to 30% of the patients were tyrosinase

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RT-PCR positive, whereas in patients with disseminated melanoma (stage IV), RT-PCR positivity remained relatively low (around 50%) with a variation from 23 to 100%. To assess the cause of these discrepancies, the EORTC melanoma group performed a collaborative quality control study, revealing that heterogenous results originate from sample processing (pretreatment of blood, enrichment of tumor cells, RNA extraction, and cDNA synthesis) rather than from PCR amplification procedures (K1). De Vries et al. also carried out a quality control study in which the reason for low detection rates of tyrosinase and MART-1 in stage III–IV patients was analyzed using realtime quantitative RT-PCR for PBGD, a low copy housekeeping gene, as an internal marker (D1). They showed that PBGD mRNA values were not diVerent among samples with 0, 1, 2, 3, and 4 times positive tyrosinase (MART-1) RT-PCR results in a repetitive (quadruplicate) assay. Their results indicated that such a low reproducibility of the RT-PCR assay is not caused by poor mRNA quality, but rather by a small number of target mRNA translated into cDNA samples. Ghossein et al. reported in 1998 that tyrosinase mRNA in peripheral blood is able to predict overall survival and disease-free survival in a statistically significant manner (G3). They also found that tyrosinase RT-PCR positivity in the blood is an independent predictor of disease-free survival. Schmidt et al. also reported that a tyrosinase mRNA determined by the real-time quantitative RT-PCR reflected a trend toward an independent prognostic factor for poor survival (S4). However, Palmieri et al. demonstrated that the presence of RT-PCR positive cells had a significant prognostic value in the univariate analysis, but did not provide additional prognostic information on the stage of disease in multivariate analysis (P1). Therefore, it still remains an open question whether these RT-PCR positive findings are associated with long-term prognosis. A well-designed prospective study using quantitative real-time RT-PCR is required to verify the application of the melanoma marker RT-PCR for routine clinical practice. In colorectal cancer, over 25 RT-PCR studies for detecting circulating tumor cells in the blood have been accumulated (T3). Among the many markers, CK20 and CEA are the most widely used. Many reports have indicated that detection rates of circulating tumor cells in the blood by conventional RT-PCR were significantly correlated with the tumor stages (F5). However, the results from other groups vary with regard to the detection frequency. Some of them report low detection rates ranging from 2 to 15%, as in melanoma cases (H1). Koch et al. showed significantly higher detection rates in the mesenteric venous blood (50%) than in peripheral venous blood (11%). They emphasize the importance of the filter function of the liver for circulating tumor cells in the portal vein and raise doubts as to whether peripheral blood is a suitable compartment for the detection of


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disseminated colorectal cancer cells in blood (K5). Several studies, including our own quantitative real-time RT-PCR study, have demonstrated the enhanced intraoperative tumor cell spread (I1, W1) and decreased detection rate of circulating tumor cells in the peripheral blood after excision of a primary tumor (P3) or preoperative chemoradiation therapy (K3). Therefore, detection of disseminated tumor cells in the peripheral blood samples by the real-time quantitative RT-PCR method can be useful for monitoring such responsiveness to various treatment options, because changes in the copy number of mRNA at diVerent times can be quantitatively compared by this method. Reports on the prognostic significance of CEA mRNA in the peripheral blood as determined by RT-PCR and quantitative RT-PCR in colorectal cancer patients are summarized in Table 2. Hardingham et al. reported in 2000 that preoperative CK19/CK20 mRNA positivity in peripheral blood was significantly associated with shorter survival in colorectal cancer patients (H1). Yamaguchi et al. found that double positive patients with CEA and CK20 mRNA in their drainage blood, but not peripheral blood, have a significantly worse prognosis than that of those who were negative for PCR and the detection of both mRNA together was an independent prognostic indicator (Y1). Taniguchi et al. and Akashi et al. also showed that the prognosis of patients with CEA mRNA positivity in the mesenteric venous blood was significantly worse compared with CEA-negative patients, even if it failed to demonstrate their independent prognostic value (T1). On the other hand, Bessa et al. reported lack of prognostic influence of circulating tumor cells in peripheral blood. Therefore, drainage vein blood again seems to be superior to the peripheral blood for evaluating its prognostic influence on recurrence. Since these studies are relatively small in sample size and short in follow-up period, the evidence is still inconclusive as to whether it is a definite prognostic indicator for survival. Although quantitative real-time RTPCR oVers benefits over conventional RT-PCR (M6, S8), the results on the prognostic significance of circulating tumor cells in the blood are still limited. A large prospective study using the quantitative RT-PCR method and suYcient follow-up time is required to determine the prognostic significance. 3.3. MICROMETASTASIS IN LYMPH NODES In this section, we address micrometastases in the lymph nodes of gastrointestinal malignancies, including esophageal, gastric, and colorectal cancers, as shown in Table 3. To date, micrometastasis in the lymph nodes has been mostly analyzed by the immunohistochemical methods, which showed the presence of lymph node micrometastasis at an incidence of approximately 15 to 70% of esophageal cancer patients in the pNO stage and its association with a higher risk of recurrence and poorer survival (G4, I2). Kijima et al. reported that the

TABLE 2 DETECTION AND PROGNOSTIC VALUES OF CIRCULATING TUMOR CELLS IN PREOPERATIVE BLOOD BY CONVENTIONAL AND/OR QUANTITATIVE REAL-TIME RT-PCR IN COLORECTAL CANCERS Authors

Year

Origin

PCR

Marker

Source of Blood

No. of patients

Correlation with stage

Hardingham et al. Yamaguchi et al.

2000 2000

colorectum colorectum

cPCR cPCR

Multiple CEA/CK20

P D and P

94 52

yes yes

Taniguchi et al. Bessa et al. Wong et al. Ito et al. Guller et al. Miura et al. Akashi et al. Schuster et al.

2000 2001 2001 2001 2002 2003 2003 2004

colorectum colorectum colorectum colorectum colorectum colorectum colorectum colorectum

cPCR cPCR sPCR qPCR qPCR qPCR cPCR qPCR

CEA CEA CK19 CEA CEA/CK20 CEA CEA CEA/CK20

D and P P P P D and P P D P

53 95 33 99 39 36 80 129

yes no yes no no yes no no

Positive rate (stage) 42% (Dukes’ C) 100% (Stage IV) 90% (Dukes’ D) 60% (Stage IV) 42% (Dukes’ C-D) 2% (Stage III) 17% (Stage III) 70% (Dukes’ D) 54% (Stage III) 30% (Stage IV)

Prognostic value Poor prog. Independent prog. Poor prog. No prog. Higher rec. No prog. Poor prog. NA Poor prog. NA

Ref. H1 Y1 T1 B1 W4 I1 G8 M6 A2 S8

cPCR, conventional PCR; sPCR, semiquantitative PCR; qPCR, quantitative PCR; Multiple, CK19/CK20/MUC1/MUC2; P, peripheral blood; D, drainage blood; Poor prog., poor prognosticator (Univariate analysis); Independent prog., independent prognosticator (Multivariate analysis); No prog., no statistically significant prognostic value; Higher rec., higher recurrence; NA, not assessed.

TABLE 3 DETECTION AND PROGNOSTIC VALUES OF LYMPH NODE MICROMETASTASIS BY CONVENTIONAL AND/OR QUANTITATIVE REAL-TIME RT-PCR IN GASTROINTESTINAL MALIGNANCIES Authors

Year

Origin

PCR

Marker

No. of patients

No. of LN examined

Positive rate (N stage)

Prognostic value

Ref.

Kijima et al. Godfrey et al. Raja et al. Yoshioka et al. Okada et al. Kubota et al. Miyake et al. Miyake et al. Noura et al. Rosenberg et al. Merrie et al. Bustin et al.

2000 2001 2002 2002 2001 2003 2000 2001 2002 2002 2003 2004

esophagus esophagus esophagus esophagus stomach stomach colorectum colorectum colorectum colorectum colorectum colorectum

cPCR qPCR qPCR qPCR cPCR qPCR(IHC) qPCR cPCR(IHC) cPCR(IHC) cPCR(IHC) cPCR qPCR

CEA CEA CEA CEA/SCC/Mage-3 CEA/CK20/Mage-3 CEA/CK20 CEA CEA/CK20 CEA CK20 CK20 CK20/CEA/GCC

21 30 23 50 28 21 7 11 64 85 200 42

373 387 37 NS 435 392 102 237 350 NS 2317 302

86% (pN0) 37% (pN0) 17% (pN0) 38% (pN0) 39% (pN0) 20% (pN0) NS 67% (pN0) 30% (pN0) 36% (pN0) 34% (pN0) NS

Higher rec. Independent prog. NA Higher rec. Higher rec. NA NA Higher rec. Independent prog. Independent prog. Independent prog. No prog.

K4 G5 R1 Y4 O2 K9 M7 M8 N8 R3 M5 B5

cPCR, conventional RT-PCR; qPCR, quantitative RT-PCR; (IHC), a comparative study with immunohistochemistry; SCC, squamous cell carcinoma, Mage-3, melanoma antigen-3; GCC, guanylyl cyclase C; pN0, no lymph node metastasis (N category by TNM classification), NS, not stated; Higher rec., higher recurrence; Independent prog., independent prognosticator (Multivariate analysis); NA, not assessed; No prog., no statistically significant prognostic value.

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CEA RT-PCR method has higher sensitivity than the immunohistochemical method and that some patients with a histology-negative, RT-PCR positive node suVered a recurrence (K4). More recently, Yoshioka et al. reported that quantitative real-time RT-PCR using multiple markers such as CEA, SCC, and Mage-3 can improve the detection sensitivity for micrometastasis in the lymph nodes and can predict a subsequent cervical lymph node recurrence in some patients (Y4). With quantitative real-time CEA RT-PCR using RNA extracted from 387 formalin-fixed archival lymph nodes of 30 esophageal cancer patients, Godfrey et al. clearly demonstrated that CEA mRNA positivity results in a significantly lower disease-free and overall survival of node-negative patients, and that quantitative RT-PCR detection is a potent prognosticator independent of other clinicopathological parameters (G5). In gastric cancer, micrometastasis in the lymph node can also be detected by immunohistochemistry at an incidence of 20 to 50% among node-negative patients. However, the prognostic significance of immunohistochemistrydetected micrometastasis in gastric cancer remains controversial (C1, F4, M1, N1). This seems to contrast with esophageal cancer cases, as has been described, and it suggests the existence of biological diVerence in lymph node micrometastasis between esophageal and gastric cancers. Four RT-PCR studies on the lymph node micrometastasis in gastric cancer using CEA, CK19, and CK20 as markers showed up-grading of stages (M3), and 2 of them also reported a poor prognosis in some patients with histology-negative and RT-PCR positive nodes (N6). We also confirmed the higher sensitivity of real-time quantitative CEA RT-PCR over keratin immunohistochemistry for the detection of micrometastasis in lymph nodes (K9). However, the prognostic significance of micrometastasis detected by the RT-PCR method remains still unclear, because previous studies have small sample sizes ranging from 20 to 50 patients and a short follow-up period, if any. In the field of colorectal cancer, there are over 15 reports on the immunohistochemical detection of micrometastasis in the lymph node (A1, O1). Most studies concluded that immunohistochemically detected micrometastasis does not correlate with a poorer outcome. However, the prognostic significance of immunohistochemically detected micrometastases remains still controversial (G7, S2), similar to that for gastric cancer. The majority of previous immunohistochemical studies on the lymph node micrometastasis of gastric and colorectal cancers have only discussed the relationship between the presence or absence of micrometastasis and the prognosis. However, in their comparative study of lymph node micrometastasis between 10 recurrent and 9 nonrecurrent colorectal cancer cases in the pNO stage, Sasaki et al. revealed that the greater the numbers of lymph node metastases and the more distant the lymph node metastases, the higher the recurrence rate, and they proposed the introduction of a concept of


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‘‘threshold’’ for lymph node micrometastasis (S2). In this context, Siewert et al. reported that the presence of three or more tumor cells per lymph node in more than 10% of the sampled lymph nodes was of significant prognostic value in the pN0 gastric cancer cases (S9). The relative abundance of immunohistochemically detected micrometastatic tumor cells (for example, above or below the threshold) rather than the simple presence or absence of micrometastasis may be of importance for the evaluation of prognostic significance. Mori et al. reported the sensitive detection of lymph node micrometastasis with CEA RT-PCR in node-negative colorectal cancer patients in 1995 (M9). Since then, over 13 RT-PCR studies have been reported, but few focused on the prognostic significance of micrometastasis. Liefers et al. reported that detection of lymph node micrometastasis with CEA RT-PCR was significantly associated with an unfavorable outcome (L2). Noura et al. reported a larger-scale comparative study (n ¼ 62) by the CEA RT-PCR method using RNA extracted from archival paraYn blocks and immunohistochemistry. They showed that micrometastasis detected by the RT-PCR method, but not by the immunohistochemical method, can predict a patient’s worse prognosis and is an independent prognostic factor (N8). They speculate that the discrepancy between the RT-PCR and immunohistochemical results were due to the limitations of the immunohistochemical method for diagnosis of lymph node micrometastasis. They previously showed that the detection rate for micrometastasis increases with the slice number examined from one to five, and that at least five slices are needed for a convincing diagnosis of micrometastasis. Yasuda et al. found a high detection rate (more than 76%) of micrometastasis in Duke’s B patients with five sections, suggesting the lack of reproducibility of the immunohistochemical method using one or two slices (Y2). Although Miyake et al. reported the advantages of real-time quantitative RT-PCR over the conventional RT-PCR method for assessment of micrometastasis (M7), quantitative study of the prognostic influence of micrometastasis has still been rather limited. Further large retrospective or prospective studies with the real-time RT-PCR method are required to confirm the prognostic value of the molecular diagnostic method.

4. General Considerations and Future Directions There is increasing evidence to suggest that the presence of micrometastases has prognostic relevance. However, the impact of micrometastasis on the prognosis varies depending on the body compartment (blood, lymph nodes, and peritoneal lavage fluids), tumor origin, and methods used to detect micrometastasis. As for the body compartment and tumor origin,

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the evidence to date seems more convincing for peritoneal lavage fluids in gastric cancer than for peripheral blood and lymph nodes in gastrointestinal malignancies. In gastric cancer, so far virtually all retrospective studies have proved the prognostic value of conventional RT-PCR and real-time RTPCR with CEA as a genetic marker for detecting free tumor cells in the peritoneal cavity. Although peritoneal micrometastasis is not equivalent to gross peritoneal metastasis or positive cytology, RT-PCR positive patients have recurrences in the peritoneal cavity at a higher incidence, suggesting the usefulness of the RT-PCR method for selecting patients with a higher risk of peritoneal relapse. On the other hand, there is still some controversy regarding the detection rate and prognostic relevance of micrometastasis in the blood and lymph nodes. This diVerence in the prognostic significance among diVerent compartments may reflect a higher tumor cell/nontumor cell ratio in the peritoneal washes than those of lymph nodes and blood. Conversely, it indicates the relative diYculty of detecting rare tumor cells among the excess number of contaminated nontumor cells in the peripheral blood and lymph nodes. In addition, sampling error appears to be another problem with lymph nodes and blood. A peritoneal wash can collect tumor cells from the entire peritoneal cavity with minimal loss. However, in the blood, circulating tumor cells distributed nonhomogenously as clumps make the detection of tumor cells a stochastic event, leading to false negative results. The prognostic relevance of circulating tumor cells in the blood of patients with melanoma and colorectal cancer has been demonstrated. However, since reports showing their independent prognostic value are limited, clinical significance has yet to be fully established. Interlaboratory diVerences in the technique used are partly responsible for this uncertainty. Therefore, prospective trials with a standardized detection protocol, including sample pretreatment, tumor cell enrichment, cDNA synthesis, and real-time RT-PCR amplification as well as a large cohort of patients, are needed to clarify the prognostic significance of tumor cells in the blood. In the lymph node micrometastases of gastric and colorectal cancer, there is some discrepancy between the immunohistochemical and RT-PCR results on the prognostic significance of micrometastasis, the latter being possibly more significant. The problem with immunohistochemical study is the possible sampling error due to the examination of only a small number of cutting slices because of practical limitations. It is now becoming apparent that a simple positive or negative method for detection of micrometastasis without quantitative considerations may not always be reliable. On the other hand, previous conventional RT-PCR studies also had qualitative problems with the small sample size ranging from 20 to 50 patients, making suYcient statistical analysis diYcult. In order to reach a consensus on the most adequate and practical detection method for lymph node micrometastasis,


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comparative study of the same samples by the immunohistochemical and real-time RT-PCR methods is of great importance. Although several groups reported such a comparative analysis using formalin-fixed archival tissues, further large prospective studies using fresh tissues are needed to determine the optimal and practical detection method for lymph node micrometastasis. The quantitative real-time RT-PCR technique has great advantages over conventional RT-PCR because of the accurate, convenient quantification and high specificity. The relatively high cost is the only apparent disadvantage. Thus, it now has been adopted as a practical alternative to conventional RT-PCR. Quantitative real-time RT-PCR is a useful guide to identify subgroups of patients with a less favorable prognosis who may benefit from adjuvant chemotherapy. In addition, the potential benefit of quantitative detection of micrometastatic tumor cells lies in the monitoring of the eVectiveness of adjuvant and neoadjuvant therapy. In conclusion, despite the accumulating evidence that micrometastasis might have an impact on survival and relapse rate, there is still no consensus about the best method and the best compartment for detecting micrometastasis in gastrointestinal malignancies. In some fields, explorative clinical trials using molecular diagnostic detection of micrometastasis are now underway. However, standardized and comprehensive methods for all assay steps involved need to be developed before detection of micrometastasis is incorporated into routine clinical practice. Further well-designed prospective randomized trials are necessary, and will lead to improvement in the treatment of gastrointestinal malignancies in the near future.

REFERENCES A1. Adell, G., Boeryd, B., Franlund, B., Sjodahl, R., and Hakansson, L., Occurrence and prognostic importance of micrometastases in regional lymph nodes in Dukes’ B colorectal carcinoma: An immunohistochemical study. Eur. J. Surg. 162, 637–642 (1996). A2. Akashi, A., Komuta, K., Haraguchi, M., Ueda, T., Okudaira, S., Furui, J., et al., Carcinoembryonic antigen mRNA in the mesenteric vein is not a predictor of hepatic metastasis in patients with resectable colorectal cancer: A long-term study. Dis. Colon Rectum 46, 1653–1658 (2003). A3. Aoki, S., Takagi, Y., Hayakawa, M., Yamaguchi, K., Futamura, M., Kunieda, K., et al., Detection of peritoneal micrometastases by reverse transcriptase–polymerase chain reaction targeting carcinoembryonic antigen and cytokeratin 20 in colon cancer patients. J. Exp. Clin. Cancer Res. 21, 555–562 (2002). B1. Bessa, X., Elizalde, J. I., Boix, L., Pinol, V., Lacy, A. M., Salo, J., et al., Lack of prognostic influence of circulating tumor cells in peripheral blood of patients with colorectal cancer. Gastroenterology 120, 1084–1092 (2001).

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B2. Boku, T., Nakane, Y., Minoura, T., Takada, H., Yamamura, M., Hioki, K., et al., Prognostic significance of serosal invasion and free intraperitoneal cancer cells in gastric cancer. Br. J. Surg. 77, 436–439 (1990). B3. Broll, R., Weschta, M., Windhoevel, U., Berndt, S., Schwandner, O., Roblick, U., et al., Prognostic significance of free gastrointestinal tumor cells in peritoneal lavage detected by immunocytochemistry and polymerase chain reaction. Langenbecks Arch. Surg. 386, 285–292 (2001). B4. Brossart, P., Keilholz, U., Willhauck, M., Scheibenbogen, C., Mohler, T., and Hunstein, W., Hematogenous spread of malignant melanoma cells in diVerent stages of disease. J. Invest. Dermatol. 101, 887–889 (1993). B5. Bustin, S. A., Siddiqi, S., Ahmed, S., Hands, R., and Dorudi, S., Quantification of cytokeratin 20, carcinoembryonic antigen, and guanylyl cyclase C mRNA levels in lymph nodes may not predict treatment failure in colorectal cancer patients. Int. J. Cancer 108, 412–417 (2004). C1. Cai, J., Ikeguchi, M., Maeta, M., Kaibara, N., and Sakatani, T., Clinicopathological value of immunohistochemical detection of occult involvement in pT3N0 gastric cancer. Gastric Cancer 2, 95–100 (1999). D1. de Vries, T. J., Fourkour, A., Punt, C. J., van de Locht, L. T., Wobbes, T., van den Bosch, S., et al., Reproducibility of detection of tyrosinase and MART-1 transcripts in the peripheral blood of melanoma patients: A quality control study using real-time quantitative RT-PCR. Br. J. Cancer 80, 883–891 (1999). F1. Folkman, J., and Klagsbrun, M., Angiogenic factors. Science 235, 442–447 (1987). F2. Foulds, L., The experimental study of tumor progression: A review. Cancer Res. 14, 327–339 (1954). F3. Fujii, S., Kitayama, J., Kaisaki, S., Sasaki, S., Seto, Y., Tominaga, O., et al., Carcinoembryonic antigen mRNA in abdominal cavity as a useful predictor of peritoneal recurrence of gastric cancer with serosal exposure. J. Exp. Clin. Cancer Res. 21, 547–553 (2002). F4. Fukagawa, T., Sasako, M., Mann, G. B., Sano, T., Katai, H., Maruyama, K., et al., Immunohistochemically detected micrometastases of the lymph nodes in patients with gastric carcinoma. Cancer 92, 753–760 (2001). F5. Funaki, N. O., Tanaka, J., Ohshio, G., Onodera, H., Maetani, S., and Imamura, M., Cytokeratin 20 mRNA in peripheral venous blood of colorectal carcinoma patients. Br. J. Cancer 77, 1327–1332 (1998). G1. Gerard, C. J., Olsson, K., Ramanathan, R., Reading, C., and Hanania, E. G., Improved quantitation of minimal residual disease in multiple myeloma using real-time polymerase chain reaction and plasmid-DNA complementarity determining region III standards. Cancer Res. 58, 3957–3964 (1998). G2. Gerhard, M., Juhl, H., KalthoV, H., Schreiber, H. W., Wagener, C., and Neumaier, M., Specific detection of carcinoembryonic antigen-expressing tumor cells in bone marrow aspirates by polymerase chain reaction. J. Clin. Oncol. 12, 725–729 (1994). G3. Ghossein, R. A., Coit, D., Brennan, M., Zhang, Z. F., Wang, Y., Bhattacharya, S., et al., Prognostic significance of peripheral blood and bone marrow tyrosinase messenger RNA in malignant melanoma. Clin. Cancer Res. 4, 419–428 (1998). G4. Glickman, J. N., Torres, C., Wang, H. H., Turner, J. R., Shahsafaei, A., Richards, W. G., et al., The prognostic significance of lymph node micrometastasis in patients with esophageal carcinoma. Cancer 85, 769–778 (1999). G5. Godfrey, T. E., Raja, S., Finkelstein, S. D., Gooding, W. E., Kelly, L. A., and Luketich, J. D., Prognostic value of quantitative reverse transcription–polymerase chain reaction


G6. G7.

G8.

H1.

H2.

H3.

H4. H5.

H6.

H7.

I1.

I2.

K1.

K2. K3.

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ZYMOGRAPHIC EVALUATION OF PLASMINOGEN ACTIVATORS AND PLASMINOGEN ACTIVATOR INHIBITORS Melinda L. Ramsby Division of Rheumatology, School of Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. The PA=PAI System in Normal Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . 1.2. The PA=PAI System in Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Monitoring the PA=PAI System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Enzyme-Linked Immunosorbent Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Molecular Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Substrate Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Fibrin Overlay Zymography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Advantages of Overlay Zymography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Materials and Methods for Overlay Zymography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. SDS-PAGE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Preparation of the Fibrin Indicator Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Fibrin Zymography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Reverse Zymography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Fibrin Zymography of PAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Densitometric Analysis of PA Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Fibrin Zymography of PA Induction in Cell Culture . . . . . . . . . . . . . . . . . . . . 4.4. Reverse Fibrin Zymography for PAIs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Fibrin Zymography of PAs from CABG Patients . . . . . . . . . . . . . . . . . . . . . . . 5. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction 1.1. THE PA=PAI SYSTEM IN NORMAL WOUND HEALING Normal wound healing is controlled by fibrin deposition and its subsequent removal via a highly regulated complex of proteases and protease inhibitors, i.e., the plasminogen activator=plasminogen activator inhibitor (PA=PAI) system (A7, G3, H8, I1, L4, L7, S2, W3, W7, X2). Fibrin degradation is mediated by the activity of plasmin generated from its zymogen, plasminogen, by tissue type plasminogen activator (tPA), or urokinase type plasminogen activator (uPA) (F7, H6, R2, V1). PA activity is balanced through its specific interaction with fibrin as well as its endogenous inhibitors, plasminogen activator inhibitor-1 (PAI-1), and PAI-2 (A3, B3, B4, K5, K7, W1). Although the PA=PAI system serves the primary role in mediating fibrinolysis, other proteases (elastase, cathepsin) and serpin-type protease inhibitors (1-antiplasmin, C1 inhibitor) appear to be involved in this process, albeit less specifically (A6, B4, V1). In addition to its ability to bind PAs, fibrin acts as a reserve of other enzymes that participate in its deposition (thrombin), stabilization (transglutaminase), and resolution (plasminogen) (F7, H6, R2, V1). Fibrin also provides a structural framework for wound stabilization as well as a functional matrix for cell migration and subsequent collagen elaboration (K8, S1, V2). The action of its associated PA=PAI system result in the production of fibrin-derived peptides that can further regulate wound healing, i.e., neutrophil-derived chemotaxis, elaboration of cytokines from inflammatory cells, and adhesion and migration of fibroblasts, via remodeling of the extracellular matrix (B7, B8, F5, G11, H1, L5, S11). Research suggests that the PA=PAI system itself serves a much more pleiotropic role in nature. The PA=PAI system has been implicated in a wide variety of important physiologic processes including cell migration=adhesion (B7, D7, P5, S12, W4), tissue regeneration and healing (C9, D3, D9, G1, H2, X1), corneal repair (W2), platelet activation (P4), angiogenesis (D6, F4, L3, R7, R10), growth and development (F2, L10, O1, S3, S10) and nerve regeneration (S7, S8). Interestingly, recent research has found that the PA=PAI system may also play a role in learning and memory function (S4). 1.2. THE PA=PAI SYSTEM IN PATHOPHYSIOLOGY Trousseau was first to describe the association between thrombosis and malignant disease over 100 years ago (T1). Despite this finding, the exact biochemical mechanisms responsible for this pathophysiological correlation still remain to be characterized. Recent research has focused on the PA=PAI system due to its central role in hemostasis and the biochemical similarities

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between tumor growth and wound healing (D12, D13, D14). Numerous studies have supported a role for PA=PAI system in malignancy, either directly or indirectly, and specifically in promoting tumor invasion and metastasis (A2, A5, B1, B5, D1, D2, D4, D8, D10, D11, Z1, G6, G7, G8, J1, K1, M8, N1, S6, S13, W6), the main causes of morbidity and mortality in cancer patients. The PA=PAI system has also been implicated in an ever-expanding number of pathophysiological conditions including renal dysfunction (G9, H3, H7, K4), arthritis (K6), endometriosis (G4, L6), sepsis (R11), hepatic disease (F3), ligneous conjunctivitis (B6, C7, R3), apoptosis (R13), and periodontal disease (L8). The PA=PAI system has also been implicated in many vascular pathologies including stroke (M5), retinopathy (P3, R1), vascular aging (J2, J3), atherosclerosis (A8, P1, S9), and aneurysm (A4, F1, L9, R9, S5). Interestingly, it has been reported that circulating PAI-1 levels may also influence thrombolytic therapy (N2, S9). Evidence has suggested a neurodegenerative role for the PA=PAI during amyloid deposition (M7) and in Alzheimer’s disease (M6). Given its multifaceted role in both physiologic and pathophysiologic processes, the PA=PAI system remains the subject of considerable interest and intense scrutiny by investigators involved in basic fundamental research as well as clinical science.

2. Monitoring the PA/PAI System The following section provides a brief description of analytical systems typically used in the qualitative and quantitative measurement of PAs and PAIs. The advantages and limitations of each technique are discussed. It should be noted, however, that the usefulness of any of these techniques is dependent on the specific application in the biological system of interest. 2.1. ENZYME-LINKED IMMUNOSORBENT ASSAY Although enzyme-linked immunosorbent assays (ELISAs) are commercially available and provide a convenient method for monitoring the PA=PAI system, their discriminating ability for individual PA and PAI components has been questioned (C4, L11, N3, N4). Both mass- and activity-based (immunofunctional) ELISAs have been developed in an eVort to circumvent this limitation (C4, L11, M1, N3, N4, R12). Despite this progress, ELISA-based assays continue to be problematic due to variable antibody specificity with PAs and PAIs, especially those in PA=PAI complexes (i.e., PAI-1, C1 inhibitor, antiplasmin), and lack of appropriate calibration material (C4, D5). Furthermore, these activity-based assays typically require optimization to reduce bias and improve correlation for the biological system being investigated (C4, D5). Because of these problems, it has been suggested that assay specific reference

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ranges be used for each method (C4). Several focused studies have, however, generated favorable interlaboratory variability for ELISA-based methodology for uPA and PAI-1 (B2, S15), thus supporting their usefulness in clinical laboratory diagnostics (D11). In general, researchers typically resort to a combination of ELISA techniques, i.e., mass- and activity-based, to accurately assess the PA=PAI system in biological systems. 2.2. MOLECULAR METHODS Molecular methods provide a highly accurate means to assess gene expression for individual components of the PA=PAI system. Northern blot analysis has been widely used in the semiquantitative evaluation of mRNA PA=PAI gene expression (A1, C2, D6, F2, L8, L10, R7, S7, S8). A 2002 article has described a sensitive reverse transcription-polymerase chain reaction (RT-PCR) method for uPA and PAI-1 mRNA quantitation and demonstrated its usefulness for evaluation of primary breast cancer (C1). Despite the advantages of molecular technology, quantitative results of gene expression typically require correlation to actual protein mass concentration and=or protein functional activity. 2.3. SUBSTRATE GEL ELECTROPHORESIS Proteases such as the PAs may also be detected by substrate gel electrophoresis, a technique in which the desired substrate is co-polymerized, i.e., ‘‘fixed’’ in the polyacrylamide gel prior to electrophoresis (G10, H4, H5). Following electrophoresis, SDS is removed with a nonionic detergent (C8) and the substrate gel developed in an appropriate buVer solution specifically designed to activate proteases of choice (G2, G10, L1). Subsequent degradation of substrate results in ‘‘cleared’’ degraded regions that are visualized by staining the undegraded background. Substrate gel electrophoresis is a convenient method to detect both free and complexed PAs since PA=PAI complexes are SDS stable (G10). The use of substrate gel electrophoresis for detection of PAs was originally described using a gelatin as a nonspecific substrate incorporated into the polyacrylamide gel with plasminogen supplementation. (H5) In contrast to gelatin, which binds tightly to acrylamide and is irreversibly incorporated into the polyacrylamide matrix, other substrates, such as casein or albumin, are poorly incorporated (G10, H4, H5). This eVect results in decreased background staining, which lowers the overall sensitivity of the assay. DiVusion of protease activity into the incubation buVer additionally contributes to this problem. Although several studies by a group of researchers have demonstrated that fibrin may also be copolymerized into the polyacrylamide matrix (C6, K2, K3), the usefulness of this technique for the specific detection of uPA or tPA has not been evaluated.


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2.4. FIBRIN OVERLAY ZYMOGRAPHY Fibrin overlay zymography is an agarose-based enzymatic assay used for detection and semi-quantitative analysis of proteases in biological samples, specifically plasminogen activators (PAs) such as urokinase-type PA (uPA) and tissue-type PA (tPA) (G10, R5). Fibrin zymography is performed using an overlay technique in which samples are first electrophoretically fractionated under nonreducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (M2). Following removal of SDS (C8), the polyacrylamide gel is overlaid on an agarose indicator gel forming a ‘‘sandwich.’’ Because the indicator gel contains plasminogenenriched fibrin, subsequent diVusion of PAs into the indicator gel results in activation of plasminogen to plasmin and degradation of the fibrin substrate. Following incubation, the sandwich is separated and the fibrin indicator gel is then stained to demonstrate zones of proteolysis that appear as cleared bands on a dark background. Proteolytic zones can then be identified by comparison to commercially available PA standards electrophoresed on the same polyacrylamide gel. Like substrate gel electrophoresis, fibrin zymography is a highly adaptable technique since acrylamide gel pore size or resolving gel buVer composition may be modified to optimize resolving power (M2).

2.5. ADVANTAGES OF OVERLAY ZYMOGRAPHY Overlay enzyme methods, such as fibrin zymography, confines enzymatic activity within the gel sandwich. This property is advantageous since it greatly diminishes the diVusional loss of protease activity to the surrounding incubation buVer typically observed with substrate gel electrophoretic assay techniques (G10, H4, H5, R5). Furthermore, overlay enzyme assays are highly desirable for the analysis of complex proteolytic systems since various PAs and PA=PAI complexes can be electrophoretically resolved and simultaneously evaluated. A variety of zymogens, co-factors, co-substrates, and inhibitors may also be incorporated singly or as multiple components into the indicator gel, thereby increasing the flexibility of this technique. The generation of overlay zymograms under near-physiologic conditions results in retention of many biologic properties and activities of materials incorporated within the matrix. Because overlay zymograms are not subjected to electrophoresis, the incorporated substrate is well retained within the agarose matrix. In contrast, substrate gels are prone to loss of incorporated substrate during exposure to electrophoretic fields for up to 2 to 3 hours in the presence of high concentrations of the protein solubilizing detergent SDS.

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In this chapter, the utility of fibrin zymography to functionally assess the PA=PAI system in several biological systems is reviewed. The usefulness of this technique is specifically demonstrated to address an unexplained phenomenon, increased fibrinolytic activity in patients undergoing coronary artery bypass graft (CABG) surgery (C3, P2, S14).

3. Materials and Methods for Overlay Zymography The following section gives a brief description of the materials and methods required to perform fibrin overlay zymography. For more comprehensive and illustrative details, the reader is referred to the accompanying references. 3.1. MATERIALS Most materials (technical or electrophoretic grade) used in this study were available from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Some materials were specifically obtained as follows: bovine fibrinogen (plasminogen-rich) from Organon Teknika, Holland; SeaKem agarose and Gelbond support backing from FMC BioProducts (Rockland, ME); human uPA and tPA from American Diagnostica, Inc. (Greenwich, CT); and 3.2% sodium citrate anti-coagulated Vacutainer tubes (Becton-Dickinson, Franklin Lakes, NJ). All reagents were prepared using ultra-pure (double-distilled, deionized) water. Reagents were stored as required by the manufacturer. 3.2. SAMPLE PREPARATION Samples were prepared, as appropriate, in 2X or 5X Laemmli nonreducing sample buVer containing the anionic detergent sodium dodecylsulfate (SDS) without heating (L2). Sample preparation in the absence of reducing agent is required since SDS may cause artifactual activation of thiol-dependent proteases (such as lysosomal cathepsins B, H, L) as well as inactivation of disulfide-stabilized proteases including PAs (R5). Similarly, heating is avoided to preclude artifactual aggregation, activation, or inactivation of thermally sensitive proteases. Once prepared, samples can be stored frozen at 60 C. Samples obtained from patients undergoing CABG were collected in 3.2% sodium citrate anti-coagulated Vacutainer tubes. Following centrifugation (1600g, 15 min), the plasma was carefully removed and stored at 60 C. Stabilization of PAs may be achieved under acidified conditions (C5). The use of citrate-anticoagulant is suggested for collection of human plasma to reduce contaminating PAI-1, which can increase from 10- to 20-fold following release from platelets (R6).


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3.3. SDS-PAGE Samples were electrophoresed on 10% discontinuous SDS-PAGE (0.75 mm thick slab gels; 13 mm resolving gel height) with a vertical slab gel apparatus (Shadel, Inc., San Francisco, CA) at 20 mA constant current (Hoefer Scientific Instruments, Inc., San Francisco, CA) as previously described (M2, R5). The electrophoretic apparatus was disassembled and the polyacrylamide gels removed and notched for later orientation purposes. The polyacrylamide gels were transferred to an appropriately sized plastic container containing 200 ml of 2.5% (v=v) Triton X-100 and washed twice (200 ml=gel, 30 min each) on an orbital platform rotator at room temperature to remove SDS, thereby allowing proteins to renature and regain activity. Wash solution volume and length of time should be accurately monitored to avoid excessive leaching of protease and to assure interassay reproducibility. Following renaturation, the polyacrylamide gels were oriented correctly and carefully overlaid on indicator gels (see Section 2.5). 3.4. PREPARATION OF FIBRIN INDICATOR GEL The fibrin indicator gel was prepared as outlined in the schematic (Fig. 1) and as described previously (R4, R5). Briefly, plasminogen-rich fibrinogen was slowly solubilized at a concentration of 5 mg=ml in 0.85% (w=v) saline (prewarmed to 37 C) with gentle mixing (inversion several times over 1.5– 2 h) in a temperature-controlled water bath at 37 C. Slow solubilization of fibrinogen was necessary to prevent protein flocculation. Agarose was separately prepared as a 1% (w=v) solution in phosphate-buVered saline (PBS, pH 7.4) by thorough heating in a boiling water bath. The water bath was turned oV and the dissolved agarose allowed to slowly cool to approximately 55 to 60 C. At this time, 30 l of a thrombin stock solution (100 U=ml) was mixed with 5 ml fibrinogen solution and then added to the hot agarose (25 ml) in rapid succession and with constant swirling. The agarose–fibrin overlay solution was quickly layered onto the hydrophilic side of a Gelbond support film (13 17 cm) using a wide-bore 25 ml glass pipet in a continuous sweeping motion from left to right. This technique results in the formation of an agarose gel of uniform thickness. The Gelbond support film had been previously stabilized on a glass plate using a small drop of water. The agarose indicator gel was allowed to solidify for at least 30 min at room temperature prior to overlay with the washed renatured acrylamide gel (see Section 2.5). Alternatively, the agarose indicator gel may be stored refrigerated (4–8 C) in a closed humidified container for several days. Following solidification, the agarose indicator gel may be easily handled through manipulation of the plastic Gelbond support membrane. The composition of the final overlay indicator gel was 0.8% (w=v) agarose, 0.8 mg=ml plasminogen-rich

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FIG. 1. Preparation of fibrin agarose indicator overlay gel. Schematic outlining sequence of steps for preparation of indicator gel is shown. For methodological details, see Section 2.4 (R5).

fibrinogen, and 0.1 U=ml thrombin (R4, R5). The indicator gel was notched for orientation purposes. 3.5. FIBRIN ZYMOGRAPHY The washed polyacrylamide gel was carefully oriented and overlaid on the fibrin indicator gel forming a ‘‘sandwich.’’ A small pipet was gently rolled over the sandwich to extrude any trapped air bubbles that would prevent contact, i.e., diVusion, between the agarose and polyacrylamide overlay. The sandwich was then placed in a plastic container containing water-moistened paper towels and sealed to provide a humidified atmosphere. During incubation, electrophoretically resolved PAs diVuse into the indicator gel that contains plasminogen (zymogen) and thrombin-catalyzed fibrin (protein substrate). PAs cleave plasminogen to form plasmin, a serine protease that degrades fibrin. Following incubation in a temperature-controlled water bath (37 C, 18–20 h), the acrylamide gel was carefully removed and the agarose indicator gel was briefly stained (about 1 min) with 100 ml of 0.1% (w=v) amido black, 70% (v=v) methanol, 10% (v=v) acetic acid, and destained with several changes of 70% methanol, 10% acetic acid. Following destaining, the agarose overlay gel was allowed to dry at room temperature overnight. Once


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dried, agarose indicator gels are very stable and may be stored indefinitely at room temperature. Gels may be photographed with background lighting or digitally scanned for reproduction purposes. Following construction of an appropriate set of calibration standards, semiquantitative data may also be obtained from fibrin zymograms by densitometric analysis. 3.6. REVERSE ZYMOGRAPHY For detection of plasminogen activator inhibitors (PAIs), uPA was incorporated into the indicator agarose gel at a concentration of 0.8 U=ml (E1, R4). Under these conditions, lysis occurs everywhere except where inhibitor has diVused from the polyacrylamide gel into the agarose indicator gel. Thus, lytic-resistant zones such as PAIs will appear as dark bands against a cleared background (i.e., reverse zymography).

4. Results The following section demonstrates the usefulness of fibrin overlay zymography for the detection of PAs and PAIs in complex biological systems. Despite the generally qualitative nature of overlay zymography, semiquantitative results may be obtained by densitometric analysis of PA=PAI calibration standards appropriate to the specific application. 4.1. FIBRIN ZYMOGRAPHY OF PAS Plasminogen activators (PAs) can be readily analyzed by fibrin zymography using a gel overlay assay (Fig. 2A). As can be seen, uPA (45-kDa), tPA (70-kDa) as well as the tPA=PAI-1 complex (110-kDa) were all well resolved using 10% polyacrylamide gels. The ability to detect fibrinolytic activity at the molecular weight corresponding to migration of the PA=PAI-1 complex results from Triton X-100 mediated dissociation of the protease inhibitor complex (G5, W5). These PA standards can be used in the construction of a calibration curve (Fig. 2B). Protease and protease inhibitor identification can be verified by parallel zymograms in which indicator agarose overlay gels contain PA-specific antibodies or inhibitors such as amiloride to inhibit uPA and erythina to inhibit tPA, as has been described elsewhere (R4). 4.2. DENSITOMETRIC ANALYSIS OF PA ACTIVITY Fibrin zymograms may be subjected to densitometric analysis to obtain quantitative information about PA activity (Fig. 3). As can be seen, good calibration curves were obtained for both uPA and tPA over an approximately

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FIG. 2. Fibrin zymography of PA standards. (A) Fibrin zymogram demonstrating electrophoretic migration of uPA (45-kDa), tPA (70-kDa), and higher molecular weight tPA=PAI-1 complex (110-kDa). Fibrin indicator gels were stained with amido black (see Section 3.5). Samples 1–5 correspond to PAs and PAIs of various compositions as described (R4, R5). (B) Calibration curve obtained with PA standards. Linear regression analysis was performed and correlation coeYcient (r) is shown (r ¼ 1.000, perfect correlation).

10-fold range in PA concentration. For quantitative purposes, standard curves should be constructed to determine dose–time relationships with respect to size of the lytic zone for each test system. Generally, overlay assay of PAs requires incubation for 6 to 18 hours to obtain an appropriately sized zone of lysis. Overlay indicator assay incubation (37 C) should be performed in a thermally controlled water bath (1 C) since PA activity is temperature-dependent (R5).

4.3. FIBRIN ZYMOGRAPHY OF PA INDUCTION IN CELL CULTURE Fibrin zymography is a convenient research tool for the detection of PA induction in conditioned media from cell and tissue culture (Fig. 4). As can be seen, zymographic analysis of media obtained from culture bovine endothelial cells demonstrated elaboration of uPA (45-kDa), tPA (70-kDA), and the tPA=PAI-1 complex (110-kDa) in a time- and dose-dependent fashion, as previously described (R4). All three PA forms were well resolved and demonstrated variable fibrinolytic activity.


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FIG. 3. Densitometric analysis of plasminogen activators. Various concentrations of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) were electrophoresed on 10% polyacrylamide gels and subjected to fibrin overlay zymography. The indicator gels were densitometrically scanned as described (M3). Data is shown as mean with error bars indicating standard deviation (SD). Linear regression analysis was performed and correlation coeYcient (r) is shown (r ¼ 1.000, perfect correlation).

4.4. REVERSE FIBRIN ZYMOGRAPHY FOR PAIS Incorporation of a plasminogen activator such as uPA in the indicator gel allows for the detection of PAIs in test samples (Fig. 5). DiVusion of PAI into the indicator gel results in PA=PAI complex formation and subsequent inhibition of fibrinolysis, i.e., increased staining. Because tPA=PAI-1 complexes are SDS stable (R5), it is likely that the PAI-1 identified on this reverse zymogram represents free noncomplexed inhibitor. Unfortunately, the incorporation of a PA into the indicator gel also results in an overall decrease in background intensity due to activation of the plasminogen-rich fibrin (ogen) (see Section 2.3). Despite this limitation, it is still possible to detect

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FIG. 4. Fibrin zymography for detection of PA induction in cell culture media. Samples 1–7 were from conditioned media of bovine corneal endothelial cells at diVerent growth stages and in the presence of various PA inducers (R4).

FIG. 5. Reverse fibrin zymography for detection of PAIs. The fibrin overlay zymogram was supplemented with uPA (see Section 3.6). Samples 1–5 were from conditioned media of bovine corneal endothelial cells at diVerent growth stages and in the presence of various PA inducers (X2). Increased amido black staining indicating position of PAI-1 (55-kDa) is shown (lanes 1 and 2). A small amount of uPA (45-kDa) and tPA (70-kDa) in lanes 3–5 is also noted.


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sample-specific PAs, such as uPA (45-kDa) and tPA (70-kDa), on reverse zymograms as hydrolyzed ‘‘cleared’’ regions of relatively low intensity (see lanes 3–5, Fig. 5). 4.5. FIBRIN ZYMOGRAPHY OF PAS FROM CABG PATIENTS Increased fibrinolytic activity is a common, yet unexplained, phenomenon in patients undergoing coronary artery bypass graft (CABG) surgery (C3, P2, S14). A series of citrate anticoagulated samples were collected from patients prior to, during, and post CABG surgery, as previously described (M4). Following recalcification of plasma, paired fibrin matrices were generated by thrombin (R8) and assessed for spontaneous fibrinolysis (SDSPAGE) and PA activity (fibrin zymography) (Fig. 6). As can be seen, increased fibrinolysis, i.e., loss of characteristic - dimers and -monomers (F6), was found in fibrin matrices generated from plasma collected from the CABG patient during and immediately post bypass. In contrast, fibrin

FIG. 6. PA activity associated with increased fibinolysis during CABG. Citrate anticoagulated samples were obtained from a patient undergoing coronary artery bypass graft (CABG) surgery. Fibrin matrices were generated in duplicate by addition of thrombin to recalcified plasma diluted 1:20 to minimize artifactual trapping (R8). (A) One matrix was placed in incubation buVer and spontaneous fibrinolysis monitored by SDS-PAGE under reducing conditions. (B) The second matrix was loaded directly on polyacrylamide gels and fibrin overlay zymography was performed to detect PAs. As can be seen, fibrinolysis as measured by loss of characteristic - dimers and -monomers (F6) was associated with increased fibrin binding activity of PA=PAI-1. Plasma corresponds to samples collected prior to (samples 1, 2, and 3), during (sample 4), immediately post (sample 5), and 40 h post (sample 6) CABG. Reproduced with permission (M4).

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matrices generated from samples collected prior to or 40 h post bypass were very resistant to spontaneous fibrinolysis. Overlay zymography of the fibrin matrices revealed that fibrinolysis during and immediately post bypass was associated with increased PA activity with an electrophoretic migration consistent with PA=PAI-1 (110-kDa). No tPA (45-kDA) or uPA (70-kDa) was evident in any of the fibrin matrices subjected to fibrin overlay zymography.

5. Conclusion The plasminogen activator=plasminogen activator inhibitor (PA=PAI) system is of considerable fundamental importance in elucidating the biochemical mechanisms of many physiological and pathophysiological processes. Although many techniques are available to assess the PA=PAI system, including ELISA and molecular assays, fibrin overlay zymography provides a highly versatile tool for the detection and semi-quantitative investigation of PAs, PAIs, and PA=PAI complexes. By combining one-dimensional electrophoresis under nondenaturing conditions, fibrin zymography allows for the simultaneous separation and evaluation of individual components of the PA=PAI system. Although two-dimensional PAGE, either isoelectric focusing (IEF) or non-equilibrium pH gradient electrophoresis (NEpHGE), may be envisioned to increase resolution of specific PAs and PAIs isoforms, this technique has not yet been described for overlay zymography to the author’s knowledge. As can be seen, a variety of samples can be used in fibrin overlay zymography, including those obtained from clinical studies as well as samples obtained in basic research, i.e., cell culture media. Unique to the overlay method is the opportunity to incorporate into the indicator gel multiple components including activators, inhibitors, antibodies, zymogens, cofactors, and allosteric modifiers. This enables highly sensitive and specific assays to be developed, and thus aVords extensive biochemical investigation of diverse proteolytic components including those of the PA=PAI system. REFERENCES A1. Akai, T., Niiya, K., Sakuragawa, N., Iizuka, H., and Endo, S., Modulation of tissuetype plasminogen activator expression by platelet activating factor in human glioma cells. J. Neurooncol. 59, 193–198 (2002). A2. Akai, T., Niiya, K., Sakuragawa, N., Iizuka, H., and Endo, S., Modulation of tissuetype plasminogen activator expression by platelet activating factor in human glioma cells. J. Neurooncol. 59, 193–198 (2002). A3. Alessi, M. C., Juhan-Vague, I., Declerck, P. J., Anfosso, F., Gueunoun, E., and Collen, D., Correlations between t-PA and PAI-1 antigen and activity and t-PA=PAI-1


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in cultured human keratinocytes and fibroblasts. Wound Repair Regen. 9, 314–322 (2001). Weiss, E., Yamaguchi, Y., Falabella, A., Crane, S., Tokuda, and Falanga, V., Un-crosslinked fibrin substrates inhibit keratinocyte spreading and replication: Correction with fibronectin and factor XIII cross-linking. J. Cell Physiol. 174, 58–65 (1998). Willems, P. K. A., Gils, A., Aertgeerts, K., De Ranter, C. J., and Declerck, P. J., Induction of conformational changes within crystals of plasminogen activator inhibitor1 (PAI-1). Fibrinol. Proteol. 13, 203–207 (1999). Wojtukiewicz, M., Sierko, E., Zacharski, L. R., Rozanska-Kudelska, M., and Imnoch, L., Occurrence of components of fibrinolytic pathways in situ in laryngeal cancer. Semin. Thromb. Hemost. 29, 317–320 (2003). Wysocki, A. B., Kusakabe, A. O., Chang, S., and Tuan, T. L., Temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor, and gelatinase-B in chronic wound fluid switches from a chronic to acute wound profile with progression to healing. Wound Repair Regen. 7, 154–165 (1999). Xia, W., de Bock, C., Murrell, G. A., and Wang, Y., Expression of urokinase-type plasminogen activator and its receptor is up-regulated during tendon healing. J. Orthop. Res. 21, 819–825 (2003). Xiao, Li, H., Bunn, C., and Bartold, P. M., The expression of plasminogen activator system in a rat model of periodontal wound healing. J. Periodontol. 72, 849–857 (2001). Zetter, B., Angiogenesis and tumor metastasis. Ann. Rev. Med. 49, 407–414 (1998).



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ESTROGEN METABOLITES, CONJUGATES, AND DNA ADDUCTS: POSSIBLE BIOMARKERS FOR RISK OF BREAST, PROSTATE, AND OTHER HUMAN CANCERS Eleanor G. Rogan and Ercole L. Cavalieri Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Mechanisms of Tumor Initiation by Estrogens . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Metabolism of Estrogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Estrogens as Possible Biomarkers for Risk of Developing Cancer . . . . . . . . . 2. Analysis of Estrogens and Their Metabolites, Conjugates, and Depurinating DNA Adducts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Analysis of Estrogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Analysis of Catechol Estrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Analysis of Estrogen Metabolites and Conjugates . . . . . . . . . . . . . . . . . . . . . . . 2.4. Analysis of Depurinating Catechol Estrogen-DNA Adducts . . . . . . . . . . . . . . 3. Biomarkers for Increased Risk of Developing Estrogen-Initiated Cancer . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

135 135 136 138 139 139 141 141 143 144 146

1. Introduction 1.1. MECHANISMS OF TUMOR INITIATION BY ESTROGENS Various types of evidence have implicated estrogens in the etiology of human breast cancer (C5, C9, C10, F1, L5, L6). They are generally thought to cause proliferation of breast epithelial cells through estrogen receptormediated processes (F1). Rapidly proliferating cells are susceptible to genetic errors during DNA replication, which, if uncorrected, can ultimately lead to malignancy. While receptor-mediated processes may play an important role in the development and growth of tumors, accumulating evidence suggests 135 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2423/04 $35.00

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that specific oxidative metabolites of estrogens, if formed, can be endogenous ultimate carcinogens that react with DNA to cause the mutations leading to initiation of cancer (C5, C9, C11). Thus, estrogen metabolites, conjugates, and DNA adducts could serve as biomarkers for increased susceptibility to breast, prostate, and other human cancers. One hypothesis on the etiology of breast cancer has been that its induction is caused by a covalent bond of 16-hydroxyestrone (16-OHE1), a metabolite of E1, with the estradiol (E2) receptor. This receptor modification would result in permanent, uncontrolled stimulation of cell proliferation by receptor-mediated processes (B7, S1, S6). This hypothesis implies a correlation of high levels of 16-OHE1 with induction of breast cancer. Over the years, however, this hypothesis has never been substantiated. Several lines of evidence, including metabolism and carcinogenicity studies by Liehr and coworkers, led to the recognition that the 4-hydroxylated estrogens play a major role in the genotoxic properties of estrogens (L5–L7). We have hypothesized that the estrogens E1 and E2 initiate breast cancer by reaction of their electrophilic metabolites, catechol estrogen-3,4-quinones [E1(E2)-3,4-Q], with DNA to form depurinating adducts (C5, C9, C10). These adducts generate apurinic sites, leading to mutations that may initiate breast, prostate, and other human cancers (C5, C9, C11). 1.2. METABOLISM OF ESTROGENS The estrogens E1 and E2 are obtained via aromatization of 4-androstene-3, 17-dione and testosterone, respectively, catalyzed by cytochrome P450(CYP) 19, aromatase (Fig. 1). E1 and E2, which are biochemically interconvertible by the enzyme 17-estradiol dehydrogenase, are metabolized to the 2-catechol estrogens, 2-OHE1(E2), and 4-OHE1(E2), predominantly catalyzed by the activating enzymes CYP1A1 (S4) and 1B1 (H2, S2–S4), respectively, in extrahepatic tissues. The estrogens are also metabolized, to a lesser extent, by 16-hydroxylation (not shown). The catechol estrogens are further oxidized to the catechol estrogen quinones, E1(E2)-2,3-Q and E1(E2)-3,4-Q (Fig. 1). In general, the catechol estrogens are inactivated by conjugating reactions, such as glucuronidation and sulfation. A common pathway of inactivation in extrahepatic tissues, however, occurs by O-methylation catalyzed by the ubiquitous catechol-O-methyltransferase (COMT) (B1). If formation of E1 or E2 is excessive, due to overexpression of aromatase and/or the presence of excess sulfatase that converts the stored E1 sulfate to E1, increased formation of catechol estrogens is expected. In particular, the presence and/or induction of CYP1B1 and other 4-hydroxylases could render the 4-OHE1(E2), which are usually minor metabolites, as the major metabolites. Thus, conjugation of 4OHE1(E2) via methylation in extrahepatic tissues might become insuYcient,

FIG. 1. Formation, metabolism, conjugation, and DNA adducts of estrogens.

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and competitive catalytic oxidation of 4-OHE1(E2) to E1(E2)-3,4-Q could occur (Fig. 1). Protection at the quinone level can occur by conjugation of E1(E2)-Q with glutathione (GSH), catalyzed by S-transferases (Fig. 1). A second inactivating process for E1(E2)-Q is their reduction to catechol estrogens by quinone reductase. If these two inactivating processes are not eVective, E1(E2)-Q may react with DNA to form stable and depurinating adducts (C5, C8–C10, D5, L2, S5). We hypothesize that imbalances in estrogen homeostasis, that is, the equilibrium between activating and protective enzymes with the scope of avoiding formation of catechol estrogen semiquinones and quinones, can lead to initiation of cancer by estrogens. 1.3. ESTROGENS AS POSSIBLE BIOMARKERS FOR RISK OF DEVELOPING CANCER Based on these considerations, the identification and quantification of estrogen metabolites, conjugates, and depurinating DNA adducts in human specimens could provide early diagnostic tools for determining the risk of developing breast, prostate, and other human cancers. These possible biomarkers could include the estrogens E1 and E2 themselves, the catechol estrogens, methoxy catechol estrogens, catechol estrogen-GSH conjugates and/or their derivatives, catechol estrogen-cysteine (Cys) and catechol estrogen-N-acetylcysteine (NAcCys) conjugates, as well as the depurinating DNA adducts 4-OHE1(E2)-1-N3adenine (Ade), 4-OHE1(E2)-1-N7guanine (Gua), and 2-OHE1(E2)-6-N3Ade (Fig. 2). The CE-GSH conjugates generally undergo further metabolism by the mercapturic acid synthesis pathway to generate catechol estrogen-Cys and, subsequently, catechol estrogenNAcCys conjugates (Fig. 3) (B6).

FIG. 2. Depurinating DNA adducts, 4-OHE1(E2)-1-N3Ade, 4-OHE1(E2)-1-N7Gua, and 2-OHE1(E2)-6-N3Ade.

ESTROGEN BIOMARKERS FOR CANCER RISK

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FIG. 3. GSH conjugation with the electrophilic compound RX, followed by mercapturic acid biosynthesis to yield the NAcCys conjugate as the final product.

2. Analysis of Estrogens and Their Metabolites, Conjugates, and Depurinating DNA Adducts 2.1. ANALYSIS OF ESTROGENS Investigators have measured the levels of estrogens in women with and without breast cancer since the 1970s (C1, D1). During the 1990s, estrogens in human blood and tissues have been analyzed in a variety of ways. Some of these analyses have been conducted on untreated blood serum samples (B3, D4, K1, T4), but others have purified a fraction containing the estrogens, usually by organic extraction of serum, followed by column chromatography (C4, H1, H3, T2). Radioimmunoassays have been conducted to analyze E1, E2, and other estrogens in both untreated and purified serum samples. In nine diVerent studies (B2, B3, C4, D4, H1, H3, K1, T2, T4) that were combined for meta-analysis (T1), the level of E1 in serum from women with and without breast carcinoma ranged from approximately 50 to 150 fmol/ml and the level of E2, from 20 to 150 fmol/ml. The relative risk of breast cancer for women whose E2 levels were in the top quintile was twice that of women whose E2 levels were in the bottom quintile. Since 2000, the estrogens E1 and E2 have been analyzed in tissues from a variety of laboratory animal models. The estrogens were extracted from the tissues and analyzed by HPLC with multichannel electrochemical detection

TABLE 1 ANALYSIS OF ESTROGEN METABOLITES AND CONJUGATES IN HUMAN BREAST TISSUE FROM WOMEN WITH AND WITHOUT BREAST CANCER pmol/g tissuea

Breast tissue

E1(E2)

2-OH-E1(E2)

4-OH-E1(E2)

16-OHE1(E2)

2-MethoxyE1(E2)

4-MethoxyE1(E2)

Quinone Conjugatesb

Controls – noncancer subjects (49) Breast cancer cases (28) pc

4.1 3.0 (43)

5.4 5.1 (24)

3.4 2.7 (10)

2.8 1.2 (33)

3.5 2.8 (16)

4.1 2.6 (27)

2.6 1.5 (29)

8.0 6.8 (46)

4.5 4.9 (46)

13.3 13.2 (54)

3.5 2.7 (18)

1.9 1.1 (29)

3.2 2.4 (39)

8.2 7.0 (57)

n.s.

n.s.

0.01

n.s.

n.s.

n.s.

0.003

Number in parentheses presents the percentage of positive samples (i.e., frequency of detection, %). n.s. ¼ Statistically nonsignificant diVerences from controls. a Values are mean S.D. of the positive samples. b Quinone conjugates are 4-OHE1(E2)-2-NAcCys, 4-OHE1(E2)-2-Cys, 2-OHE1(E2)-(1+4)-NAcCys, and 2-OHE1(E2)-(1+4)-Cys. c Statistically significant diVerences (compared to controls) were determined using the Wilcoxon rank sum test.


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(C7, C8, D2, L2). This methodology was also used to analyze the levels in human nontumor breast tissue. In women without breast cancer, E1 and E2 combined together equaled 4 pmol/g, whereas the tissue from women with breast carcinoma contained 8 pmol/g (Table 1) (R1). These tissue levels are one to two orders of magnitude higher than the levels in blood. 2.2. ANALYSIS OF CATECHOL ESTROGENS The catechol estrogens, 2-OHE1(E2) and 4-OHE1(E2), have been analyzed in a variety of samples, along with the hydroxylated estrogen 16-OHE1. These hydroxylated estrogens were first considered ‘‘unusual’’ estrogens and were found at elevated levels in urine samples from women with breast cancer (C1). The catechol estrogens and 16-OHE1 were analyzed as the trimethylsilylethers in human breast tissue and breast cyst fluid by using gas chromatography/mass spectrometry (GC/MS) after initial purification by reversed-phase HPLC (C2). The catechol estrogens were analyzed in the rat brain following a more complex procedure that involved extraction, formation of the acetate derivatives, and several purification steps before analysis by liquid chromatography-atmospheric pressure chemical ionization-ion trap tandem mass spectrometry (MS/MS) (M1). In a study in 2002, the catechol estrogens plus 16-OHE1 were analyzed in normal and tumor tissue from human breast by using GC/MS coupled with HPLC (C3). In addition, catechol estrogens, 16-OHE1, and methoxy catechol estrogens have been determined in nontumor breast tissue from women with and without breast cancer (R1) and in selected tissues of laboratory animals (C7, C8, D2, L2) by using HPLC coupled with multichannel electrochemical detection (Fig. 4). The level of 4-OHE1(E2) was significantly higher in breast tissue from women with breast carcinoma than in tissue from women without breast cancer (Table 1) (R1), and tissue from women with breast cancer contained significantly more 4-OHE1(E2) than 2-OHE1(E2), as seen previously in much smaller studies (C2, L4). The level of 16-OHE1 was about the same in breast tissue from women with and without breast cancer (Table 1), but slightly more methoxy catechol estrogens than unmodified catechol estrogens were found in breast tissue from women without breast cancer, suggesting that methylation of catechol estrogens to protect them from oxidation to E1(E2)-Q happens more often in women without breast cancer. 2.3. ANALYSIS OF ESTROGEN METABOLITES AND CONJUGATES Estrogen metabolites and conjugates have been analyzed in tissues from laboratory animals and in human breast tissue by using HPLC with multichannel electrochemical detection. A total of 31 compounds, including the

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FIG. 4. Multichannel electrochemical response from HPLC of standard mixture of estrogens, estrogen metabolites, estrogen conjugates, and estrogen-DNA adducts. The peak numbers correspond to the compounds as follows: (1) 2-OHE2-1-SG, (2) 2-OHE2-4-SG, (3) 4-OHE2-2SG, (4&9) 2-OHE2-1(&4)-Cys, (5) 2-OHE1-1(þ4)-SG, (6) 4-OHE2-1-N7Gua, (7) 4-OHE1-2-SG, (8) 4-OHE2-2-Cys, (10) 4-OHE1-1-N7Gua, (11) 2-OHE2-1-NAcCys, (12) 16-OHE2, (13) 2-OHE2-4-NAcCys, (14) 4-OHE1-2-Cys, (15) 2-OHE1-1(þ4)-Cys, (16) 4-OHE2-2-NAcCys, (17) 2-OHE1-1(þ4) NAcCys, (18) 4-OHE1-2-NAcCys, (19) 16-OHE1, (20) 4-OHE2, (21) 2-OHE2, (22) 2-OHE1, (23) 4-OHE1, (24) E2, (25) 4-OCH3E2, (26) 2-OCH3E2, (27) E1, (28) 4-OCH3E1, (29) 2-OH-3-OCH3E2, (30) 2-OCH3E1, (31) 2-OH-3-OCH3E1.

estrogens E1 and E2 themselves, the catechol estrogens and methoxy catechol estrogens, and conjugates formed by reaction of E1(E2)-Q with GSH have been detected in one HPLC run (Fig. 4). Chronic treatment of male Syrian golden hamsters with 4-OHE2 induces kidney tumors (L1, L3). When hamsters were intraperitoneally injected with 4-OHE2 and the kidney tissue analyzed 1 to 24 h later, the GSH, Cys, and NAcCys conjugates of 4-OHE1 and 4-OHE2 were identified in the picomole range, with 4-OHE22-Cys predominating (D3). Urine collected for 24 h following treatment of hamsters with 4-OHE2 contained both the methoxy catechol estrogens that arise from methylation of catechol estrogens and the Cys and NAcCys conjugates derived from reaction of E1(E2)-Q with GSH (T3). Treatment of hamsters with E2 also resulted in the formation of catechol estrogens and methoxy catechol estrogens in both the liver and kidney within 4 h, with the liver containing higher levels of the compounds, especially 2-OHE1(E2) and 2-methoxyE1(E2) (C8). If the hamsters were injected with L-buthionine (S, R) sulfoximine to deplete the cellular levels of GSH, followed by E2 2.5 h later, the GSH conjugates were virtually nondetectable in both the liver and kidney, but the kidney now had detectable levels of the 4-OHE1(E2)-1-N7Gua adducts (C8). Prostate carcinomas arise in Noble rats several months after injection with E2 and implantation with testosterone (B5). When Noble rats were treated with 4-OHE2 or E2-3,4-Q for 90 min and their prostates removed, dissected,


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and analyzed, 4-OHE1(E2), 4-methoxyE1(E2), the GSH conjugates (or the derivative Cys and NAcCys conjugates) were detected in the four lobes of the prostate (C7). The levels of these compounds suggest that areas of the prostate susceptible to induction of carcinoma have less protection of catechol estrogens by COMT, GSH, and quinone reductase, favoring reaction of E1(E2)-3,4-Q with DNA. In addition to studies of laboratory animals treated with estrogens, analyses have been conducted on tissue from untreated animals susceptible to mammary tumors. A novel model of breast cancer was established by crossing mice carrying the Wnt-1 transgene (100% of females develop spontaneous mammary tumors) with the estrogen receptor- knock-out (ERKO) mouse line (B4). Mammary tumors develop in these mice despite the lack of functional estrogen receptor-. Extracts of hyperplastic mammary tissue contained the 4-catechol estrogens, but not the 4-methoxy catechol estrogens or the 2-catechol estrogens and 2-methoxy catechol estrogens (D2), which typically predominate in normal tissue of laboratory animals and humans. In addition, the 4-catechol estrogen-GSH conjugates and their hydrolytic conjugates with Cys or NAcCys were detected, demonstrating formation of E1(E2)-3,4-Q in this tumor-prone tissue (D2). Catechol estrogen-GSH conjugates and their hydrolytic Cys or NAcCys conjugates were identified and quantified in breast tissue from women with breast carcinoma at significantly higher levels than in breast tissue from women without breast cancer (Table 1) (R1). This finding demonstrates that the E1(E2)-Q are present in human breast tissue, suggesting that the quinones may react with DNA to generate mutations leading to breast cancer. 2.4. ANALYSIS OF DEPURINATING CATECHOL ESTROGEN-DNA ADDUCTS The quinones E1(E2)-2,3-Q and E1(E2)-3,4-Q can react with DNA to form very small amounts of stable adducts and larger amounts of six depurinating adducts: 4-OHE1(E2)-1-N3Ade, 4-OHE1(E2)-1-N7Gua, and 2-OHE1(E2)-6N3Ade (Fig. 2) (C6, C10, L2, S5). The depurinating adducts derived from reaction of E1(E2)-3,4-quinone are far more abundant than those coming from E1(E2)-2,3-quinone (C6). The apurinic sites resulting from loss of the depurinating Ade adducts have been hypothesized to generate the mutations initiating cancer (C5, C9, C11). Thus, depurinating catechol estrogen-DNA adducts are very promising biomarkers for susceptibility to estrogen-initiated cancer. When hamsters were intraperitoneally injected with 4-OHE2 and the kidney tissue analyzed 1 to 24 h later, both the 4-OHE1-1-N7Gua and 4-OHE21-N7Gua adducts were identified by mass spectrometry (D3). The N7Gua

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adducts were also detected by mass spectrometry in 24-h urine samples collected from these hamsters (T3). As noted in Section 2.3, the 4-OHE1-1N7Gua and 4-OHE2-1-N7Gua adducts were not detectable in the hamster kidney 2 h after injection of E2. The adducts were detected in the kidney by HPLC coupled with electrochemical detection when the hamsters had been pretreated with L-buthionine(S,R)sulfoximine to deplete GSH (C8). Equimolar amounts of the DNA adducts 4-OHE2-1-N3Ade and 4-OHE21-N7Gua (12 mol/mol DNA-P) have been found in the skin of female SENCAR mice 4 h after topical treatment with E2-3,4-Q (C11). Higher amounts of these adducts have been detected in the mammary glands of female ACI rats following intramammillary injection of E2-3,4-Q (C12). In both cases, the adducts were identified and quantified by HPLC coupled with multichannel electrochemical detection.

3. Biomarkers for Increased Risk of Developing Estrogen-Initiated Cancer Many types of markers are being evaluated to estimate risk of developing breast cancer. These include breast density; body mass index; expression of BRCA1 and/or BRCA2 gene; cytological changes in breast epithelial cells collected by ductal lavage; and levels of estrogens and androgens in serum, breast tissue, or nipple aspirate fluid. Some studies suggest that levels of selected estrogen metabolites, estrogen conjugates, and/or depurinating estrogen-DNA adducts in breast ductal fluid, collected either by nipple aspiration or ductal lavage, could prove to be early biomarkers of susceptibility to breast cancer. For example, in the recent study of estrogen metabolites and conjugates in breast tissue, the levels of both 4-OHE1(E2) and the combined 4-catechol estrogen-GSH, Cys, and NAcCys conjugates were significantly higher in women with breast carcinoma than in women without breast cancer (Table 1) (R1). Breast fluid is particularly attractive as a source of biomarkers because the estrogen metabolites are more concentrated in breast fluid (C2) and it can be collected by noninvasive means. In the future, analysis of small molecules, such as estrogen metabolites, estrogen conjugates, and depurinating estrogen-DNA adducts as biomarkers can be accomplished by using liquid chromatography/mass spectrometry. Especially promising is capillary HPLC through a short column, such as a guard column, preceding MS/MS analysis. This approach is being used now to analyze serum for a variety of small molecules, and it should also be very eVective to analyze breast fluid. Estrogen metabolites with identical molecular weights, such as 2-OHE1(E2) and 4-OHE1(E2), can readily be distinguished by MS/MS (Fig. 5). Thus, if high levels of 4-OHE1(E2) turn out to


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FIG. 5. MS/MS of 2-OHE2 (top) and 4-OHE2 (bottom). The 2 catechol estrogens have the same molecular weight, but are distinguishable by the primary daughter: fragment m/z ¼ 147 for 2-OHE2 and m/z ¼ 161 for 4-OHE2.

be a biomarker of elevated risk of breast cancer, as suggested by the data in Table 1, these estrogen metabolites can be analyzed by known MS/MS techniques. Similarly, the conjugates formed by reaction of E1(E2)-Q with GSH (Table 1) may be found to be biomarkers of susceptibility to breast cancer. The depurinating estrogen-DNA adducts, which are a direct measure of DNA damage, may ultimately be definitive biomarkers of risk of

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FIG. 6. LC/MS/MS of 4-OHE1(E2)-1-N7Gua and 4-OHE1(E2)-1-N3Ade. The adducts are identified by the transition of parent ion to daughter ion after application of collision energy.

developing breast cancer. These adducts can be analyzed and distinguished by using HPLC with MS detection (LC/MS) with a limit of detection of approximately 100 femtomoles (Fig. 6). Application of LC/MS/MS techniques coupled with minimal cleanup of samples is a promising approach for analyzing biomarkers of breast cancer susceptibility. The best biomarkers need to be identified, but a number of estrogen metabolites, estrogen conjugates, and depurinating estrogen-DNA adducts are likely candidates. Ongoing comparisons of women with and without breast cancer will allow us to identify the most promising candidate biomarkers of susceptibility. The most useful biomarkers will be validated through prospective studies that follow development of breast cancer in selected populations of women. ACKNOWLEDGMENTS We thank Dr. Sandra J. Gunselman for mass spectra. This research was supported by U.S. Public Health Service grants P01 CA49210 and R01 CA49917 from the National Cancer Institute. Core support in the Eppley Institute is provided by grant P30 CA36727 from the National Cancer Institute.

REFERENCES B1. Ball, P., and Knuppen, R., Catechol oestrogens (2- and 4-hydroxyestrogens): Chemistry, biogenesis, metabolism, occurrence, and physiological significance. Acta Endocrinol. (Copenhagen) 93(Suppl. 232), 1–127 (1980). B2. Barrett-Connor, E., Friedlander, N. J., and Khaw, K. T., Dehydroepiandrosterone sulfate and breast cancer risk. Cancer Res. 50, 6571–6574 (1990). B3. Berrino, F., Muti, P., Micheli, A., et al., Serum sex hormone levels after menopause and subsequent breast cancer. J. Natl. Cancer Inst. 88, 291–296 (1996). B4. Bocchinfuso, W. P., Hively, W. P., Couse, J. F., Varmus, H. E., and Korach, E. S., A mouse mammary tumor virus-Wnt-1 transgene induces mammary gland hyperplasia and tumorigenesis in mice lacking estrogen receptor. Cancer Res. 59, 1869–1876 (1999).


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B5. Bosland, M. C., Ford, H., and Horton, L., Induction at high incidence of ductal prostate adenocarcinoma in NBL/Cr and Sprague-Dawley Hsd:SD rats treated with a combination of testosterone and estradiol-17 or diethylstilbestrol. Carcinogenesis 16, 1311–1317 (1995). B6. Boyland, E., and Chausseaud, L. F., The role of glutathione and glutathione S-transferases in mercapturic acid biosynthesis. Adv. Enzymol. 32, 173–219 (1969). B7. Bradlow, H. L., Hershcopf, R. J., Martucci, C. P., and Fishman, J., Estradiol 16-hydroxylation in the mouse correlates with mammary tumor incidence and presence of murine mammary tumor virus. A possible model for the hormonal etiology of breast cancer in humans. Proc. Natl. Acad. Sci. USA 82, 6295–6299 (1985). C1. Castagnetta, L., D’Agostino, G., LoCasto, M., Traina, A., and Leake, R. E., Breast cancer: A comparison of response to endocrine therapy and oestrogen excretion patterns including unusual metabolites. Br. J. Cancer 44, 670–674 (1981). C2. Castagnetta, L. A., Granata, O. M., Arcuri, F. P., Polito, L. M., Rosati, F., and Cartoni, G. P., Gas chromatography/mass spectrometry of catechol estrogens. Steroids 57, 437–443 (1992). C3. Castagnetta, L. A., Granata, O. M., Traina, A., et al., Tissue content of hydroxyestrogens in relation to survival of breast cancer patients. Clin. Cancer Res. 8, 3146–3155 (2002). C4. Cauley, J. A., Lucas, F. L., Kuller, L. H., Stone, K., Browner, W., and Cummings, S. R., Elevated serum estradiol and testosterone concentrations are associated with high risk for breast cancer. Study of Osteoporotic Fractures Research Group. Ann. Intern. Med. 130(4 Pt. 1), 270–277 (1999). C5. Cavalieri, E., Frenkel, K., Liehr, J. G., Rogan, E., and Roy, D., Estrogens as endogenous genotoxic agents: DNA adducts and mutations. In ‘‘JNCI Monograph 27: Estrogens as Endogenous Carcinogens in the Breast and Prostate’’ (E. Cavalieri and E. Rogan, eds.), pp. 75–93. Oxford Press, 2000. C6. Cavalieri, E., Kohli, E., Zahid, M., and Rogan, E., Greater reactivity of estradiol3,4-quinone vs estradiol-2,3-quinone with DNA in the formation of depurinating DNA adducts. Proc. Amer. Assoc. Cancer Res. 44(2nd ed.), 180 (2003). C7. Cavalieri, E. L., Devanesan, P., Bosland, M. C., Badawi, A. F., and Rogan, E. G., Catechol estrogen metabolites and conjugates in diVerent regions of the prostate of Noble rats treated with 4-hydroxyestradiol: Implications for estrogen-induced initiation of prostate cancer. Carcinogenesis 23, 329–333 (2002). C8. Cavalieri, E. L., Kumar, S., Todorovic, R., Higginbotham, S., Badawi, A. F., and Rogan, E. G., Imbalance of estrogen homeostasis in kidney and liver of hamsters treated with estradiol: Implications for estrogen-induced initiation of renal tumors. Chem. Res. Toxicol. 14, 1041–1050 (2001). C9. Cavalieri, E. L., Rogan, E. G., and Chakravarti, D., Initiation of cancer and other diseases by catechol ortho-quinones: A unifying mechanism. Cell & Mol. Life Sci. 59, 665–681 (2002). C10. Cavalieri, E. L., Stack, D. E., Devanesan, P. D., et al., Molecular origin of cancer: Catechol estrogen-3,4-quinones as endogenous tumor initiators. Proc. Natl. Acad. Sci. USA 94, 10937–10942 (1997). C11. Chakravarti, D., Mailander, P., Li, K.-M., et al., Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H-ras gene. Oncogene 20, 7945–7953 (2001). C12. Chakravarti, D., Mailander, P. C., Higginbotham, S., Cavalieri, E. L., and Rogan, E. G., The catechol estrogen-3,4-quinone metabolites induce mutations in the mammary gland of ACI rats. Proc. Amer. Assoc. Cancer Res. 44(2nd ed.), 180 (2003).

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D1. Dao, T. L., Metabolism of estrogens in breast cancer. Biochim. Biophys. Acta 560, 397–426 (1979). D2. Devanesan, P., Santen, R. J., Bocchinfuso, W. P., Korach, K. S., Rogan, E. G., and Cavalieri, E. L., Catechol estrogen metabolites and conjugates in mammary tumors and hyperplastic tissue from estrogen receptor-knock-out (ERKO)/Wnt-1 mice: Implications for initiation of mammary tumors. Carcinogenesis 22, 1573–1576 (2001). D3. Devanesan, P., Todorovic, R., Zhao, J., Gross, M. L., Rogan, E. G., and Cavalieri, E. L., Catechol estrogen conjugates and DNA adducts in the kidney of male Syrian golden hamsters treated with 4-hydroxyestradiol: Potential biomarkers for estrogeninitiated cancer. Carcinogenesis 22, 489–497 (2001). D4. Dorgan, J. F., Longcope, C., Stephenson, H. E., Jr., et al., Relation of prediagnostic serum estrogen and androgen levels to breast cancer risk. Cancer Epidemiol. Biomarkers Prev. 5, 533–539 (1996). D5. Dwivedy, I., Devanesan, P., Cremonesi, P., Rogan, E., and Cavalieri, E., Synthesis and characterization of estrogen 2,3- and 3,4-quinones. Comparison of DNA adducts formed by the quinones versus horseradish peroxidase-activated catechol estrogens. Chem. Res. Toxicol. 5, 828–833 (1992). F1. Feigelson, H. S., and Henderson, B. E., Estrogens and breast cancer. Carcinogenesis 17, 2279–2284 (1996). H1. Hankinson, S. E., Willett, W. C., Manson, J. E., et al., Plasma sex steroid hormone levels and risk of breast cancer in postmenopausal women. J. Natl. Cancer Inst. 90, 1292–1299 (1998). H2. Hayes, C. L., Spink, D. C., Spink, B. C., Cao, J. Q., Walker, N. J., and Sutter, T. R., 17-estradiol hydroxylation catalyzed by human P450 1B1. Proc. Natl. Acad. Sci. USA 93, 9776–9781 (1996). H3. Helzlsouer, K. J., Alberg, A. J., Bush, T. L., Longcope, C., Gordon, G. B., and Comstock, G. W., A prospective study of endogenous hormones and breast cancer. Cancer Detect. Prev. 18, 79–85 (1994). K1. Kabuto, M., Akiba, S., Stevens, R. G., Nerishi, K., and Land, C. E., A prospective study of estradiol and breast cancer in Japanese women. Cancer Epidemiol. Biomarkers Prev. 9, 575–579 (2000). L1. Li, J. J., and Li, S. A., Estrogen carcinogenesis in Syrian hamster tissue: Role of metabolism. Fed. Proc. 46, 1858–1863 (1987). L2. Li, K-M., Todorovic, R., Devanesan, P., et al., Metabolism and DNA binding studies of 4-hydroxyestradiol and estradiol-3,4-quinone in vitro and in female ACI rat mammary gland in vivo. Carcinogenesis 25, 289–297 (2004). L3. Liehr, J. G., Fang, W. F., Sirbasku, D. A., and Ari-Ulubelen, A., Carcinogenicity of catecholestrogens in Syrian hamsters. J. Steroid Biochem. 24, 353–356 (1986). L4. Liehr, J. G., and Ricci, M. J., 4-Hydroxylation of estrogens as marker of human mammary tumors. Proc. Natl. Acad. Sci. USA 93, 3294–3296 (1996). L5. Liehr, J. G., and Roy, D., Free radical generation by redox cycling of estrogens. Free Rad. Biol. Med. 8, 415–423 (1990). L6. Liehr, J. G., Genotoxic eVects of estrogens. Mutat. Res. 238, 269–276 (1990). L7. Liehr, J. G., Is estradiol a genotoxic mutagenic carcinogen? Endocr. Rev. 21, 40–54 (2000). M1. Mitamura, K., Yatera, M., and Shimada, K., Studies on neurosteroids. Part XIII. Characterization of catechol estrogens in rat brains using liquid chromatography-mass spectrometry-mass spectrometry. Analyst 125, 811–814 (2000). R1. Rogan, E. G., Badawi, A. F., Devanesan, P. D., et al., Relative imbalances in estrogen metabolism and conjugation in breast tissue of women with carcinoma: Potential biomarkers of susceptibility to cancer. Carcinogenesis 24, 697–702 (2003).


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S1. Schneider, J., Kinne, D., Fracchia, A., et al., Abnormal oxidative metabolism of estradiol in women with breast cancer. Proc. Natl. Acad. Sci. USA 79, 3047–3051 (1982). S2. Spink, D. C., Hayes, C. L., Young, N. R., et al., The eVects of 2,3,7,8-tetrachlorodibenzop-dioxin on estrogen metabolism in MCF-7 breast cancer cells: Evidence for induction of a novel 17-estradiol 4-hydroxylase. J. Steroid Biochem. Mol. Biol. 51, 251–258 (1994). S3. Spink, D. C., Spink, B. C., Cao, J. Q., et al., Induction of cytochrome P450 1B1 and catechol estrogen metabolism in ACHN human renal adenocarcinoma cells. J. Steroid Biochem. Mol. Biol. 62, 223–232 (1997). S4. Spink, D. C., Spink, B. C., Cao, J. Q., et al., DiVerential expression of CYP1A1 and CYP1B1 in human breast epithelial cells and breast tumor cells. Carcinogenesis 19, 291–298 (1998). S5. Stack, D., Byun, J., Gross, M. L., Rogan, E. G., and Cavalieri, E., Molecular characteristics of catechol estrogen quinones in reactions with deoxyribonucleosides. Chem. Res. Toxicol. 9, 851–859 (1996). S6. Swaneck, G. E., and Fishman, J., Covalent binding of the endogenous estrogen 16-hydroxyestrone to estradiol receptor in human breast cancer cells: Characterization and intranuclear localization. Proc. Natl. Acad. Sci. USA 85, 7831–7835 (1988). T1. The Endogenous Hormones and Breast Cancer Collaborative Group, Endogenous sex hormones and breast cancer in postmenopausal women: Reanalysis of nine prospective studies. J. Natl. Cancer Inst. 94, 606–616 (2002). T2. Thomas, H. V., Key, T. J., Allen, D. S., et al., A prospective study of endogenous serum hormone concentrations and breast cancer risk in post-menopausal women on the island of Guernsey. Br. J. Cancer 76, 401–405 (1997). T3. Todorovic, R., Devanesan, P., Higginbotham, S., et al., Analysis of potential biomarkers of estrogen-initiated cancer in the urine of Syrian golden hamsters treated with 4-hydroxyestradiol. Carcinogenesis 22, 905–911 (2001). T4. Toniolo, P. G., Levitz, M., Zeleniuch-Jacquotte, A., et al., A prospective study of endogenous estrogens and breast cancer in postmenopausal women. J. Natl. Cancer Inst. 87, 190–197 (1995).



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ORGANOPHOSPHATES/NERVE AGENT POISONING: MECHANISM OF ACTION, DIAGNOSIS, PROPHYLAXIS, AND TREATMENT Jirı´ Bajgar Purkyne Military Medical Academy, ´ love ´ , Czech Republic Hradec Kra

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Chemistry, Mechanism of Action, and Symptoms . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Toxicodynamics and Toxicokinetics of Intoxication. . . . . . . . . . . . . . . . . . . . . . 2.3. Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Symptoms of Intoxication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Long-Term Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cholinesterase Inhibitors and Other Factors Influencing the Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Cholinesterases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Methods for Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Inhibitors and Other Factors Influencing the Activity . . . . . . . . . . . . . . . . . . . . 4. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Basic Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Other and Special Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Specificity and Sensitivity of Different Biochemical Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Prophylaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Protection of AChE Against Inhibition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Detoxification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. The Use of Standard Antidotes as Prophylactics . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Prophylaxis with Other Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Basic Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Anticholinergics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Reactivators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Anticonvulsants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Future Trends. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

152 153 153 155 161 164 166 167 167 170 171 176 177 180 182 186 186 188 189 189 190 190 190 191 195 196 197 198


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1. Introduction Organic compounds of phosphorus show a broad variety of biological properties. They can be irritating (some derivatives of phosphine, some organophosphorus insecticides), mutagenic (triethylphosphoramide), teratogenic or carcinogenic (cyclophosphamide), nephrotoxic (dimethylphosphate), myelotoxic, or pneumotoxic (hexymethylenephosphoramide). Some of these compounds damage the pancreas and testes (tri-O-cresyl phosphate), others can influence the nervous system psychotomimetically (psylocibine) or in a depressive manner (triethylphosphate), or they have delayed neurotoxic eVects (tri-O-cresyl phosphate). Phosphorus plays a very important role in living organisms, e.g., in photosynthesis, metabolism, synthetic reactions, nucleic acids, coenzyme systems, and transmission of signals. Organic phosphates are involved in energetic metabolism (ATP, phosphorylated saccharides) and influence the action of hormones or neuromediators (c-AMP, c-GMP). From a practical point of view and taking into consideration their biological eVects, organophosphorus inhibitors of cholinesterases (commonly called organophosphates, OP) are the most important chemicals in this group. These compounds produce some of the eVects mentioned but most of them are covered by their acute eVect, which is characterized by influencing cholinergic nerve transmission. The importance of this eVect is significant and, therefore, toxicological research of this eVect is as important as technological research. These compounds are used in industry as softening agents, hydraulic liquids, lubricant additives, plasticizers, antioxidants, and for antiflammable modifications. They are also used in veterinary or human medicine as drugs or chemicals for the study of nervous functions and, last but not least, these compounds are, unfortunately, usable (and used) for military purposes as chemical warfare agents and as poisons used by terrorists. The first such attack with these compounds occurred in Matsumoto in 1994 and one year later in the Tokyo subway. Sarin was used by the Aum Shinrikyo terrorist sect. Thousands of people were aVected and dozens of people died (M23, N1, O2, Y1, Y2). The broadest spectrum of these compounds (OP) are used as pesticides, insecticides, acaricides, etc. These compounds are commercially available and are used in agriculture, which leads to professional, suicidal, or accidental intoxications. According to the World Health Organization, more than one million serious accidental and two million suicidal poisonings with insecticides occur worldwide every year, and of these approximately 200,000 die, mostly in developing countries (J1). A similar situation is observed in some other countries (E1, K3). The OP intoxications comprise approximately 1/3 to 1/2 of all intoxications. The mechanism of action, diagnosis, and treatment of intoxications with OP and

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nerve agents is a very hot topic at present. Moreover, some principles of the eVects, diagnosis, and therapy are very similar for OP and highly toxic nerve agents and, therefore, the principles described in this chapter can be applied in general for both groups—OP and nerve agents. There are many thousands of articles, books, and reports dealing with OP/nerve agents. However, according to this author’s opinion, it is important to remember Koelle’s fundamental work on these topics (K27). Even though many other publications are more updated, Koelle’s book continues to be the classic source of basic information.

2. Chemistry, Mechanism of Action, and Symptoms 2.1. CHEMISTRY OP include a large variety of compounds with diVerent physical, chemical, and biological properties, including toxicity. Although their synthesis was first described in the nineteenth century and their biological eVects were observed in the twentieth century, interest in their more detailed study had begun in the 1930s and continues to the present day. OP are liquids of diVerent volatility, soluble or insoluble in water, organic solvents, etc., and diVer in toxicity from practically nontoxic chemicals (malathion) to highly toxic agents such as VX and other nerve agents. The most important group having a significant biological eVect include compounds of the general formula

where R1–2 are hydrogen, alkyl (including cyclic), aryl and others, alkoxy, alkylthio, and amino groups. R3 is a dissociable group, e.g., halogens, cyano, alkylthio group, and the rest of inorganic or organic acid. They can be distinguished by: compounds substituted by halogen or cyanogroup in R3, phosphoramides where R1 or R2 is represented by NR2, phosphate esters, OP containing P ¼ S bond, dithiophosphates, alkylphosphonates, and trialkylphosphates, anhydrides of phosphorus containing acids.

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FIG. 1. Structural formulae of some OP

Chemical formulae of some OP pesticides are shown in Fig. 1. In the case of highly eVective OP cholinesterase inhibitors (nerve agents), there are four groups of compounds as follows:


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Typical representatives of group I are sarin, soman, and cyclosarin; group II is represented by VX and diVerent V-compounds; for group III, tabun is typical, while group IV is represented by GV compounds. The structural formulae of some highly toxic nerve agents are shown in Fig. 2. From the many sources in the literature, a book by Fest and Schmidt (F2) seems to be very useful for orientation in chemistry and the biological action of OP in general. 2.2. TOXICODYNAMICS AND TOXICOKINETICS OF INTOXICATION The toxicodynamics (mechanism of action) of OP are known: the action is based on apparently irreversible acetylcholinesterase (AChE, EC 3.1.1.7) inhibition at the cholinergic synapses. The resulting accumulation

FIG. 2. Structural formulae of some highly toxic nerve agents.

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of acetylcholine at the synaptic junctions overstimulates the cholinergic pathways and subsequently desensitizes the cholinergic receptor sites. The evidence supporting AChE as the primary site of both OP and nerve agent action has been summarized by many authors (B2, B11, L15, M1, M2, T2). It includes the following observations: symptoms of OP poisoning are similar to those of the AChE inhibitor physostigmine; the in vivo LD50 value for a variety of OP correlates well with the inhibition eYcacy to AChE determined in vitro; and cholinesterase reactivators (e.g., oximes), anticholinergics (e.g., atropine), and spontaneously reactivating AChE inhibitors (e.g., carbamates) can reduce OP toxicity. However, there is a variety of documented data showing that AChE inhibition is not the only important biochemical change during intoxication. These data have described many other changes accompanying the development of intoxication that might contribute to OP toxicity. They have included changes of other enzymes, neurotransmitters, immune changes, anaphylactoid reaction, and changes in behavior. The evidence includes the data indicating that prophylactic/ therapeutic drugs might also have multiple sites of action similar to those observed during intoxication (B3, B11, B29, C8, K6). Nevertheless, the first reaction of OP is interaction with cholinesterases in the bloodstream (B2, B11, B31) and then in the target tissues—the peripheral and central nervous system (B2, B11, B29, G6, G7, M1, M2). The delayed neurotoxic eVect is caused by a reason other than cholinesterase inhibition. The neurotoxic esterase has been described as the target site for this symptom; however, only some OP are neurotoxic in that sense (A3, L16, J2, J3, J4). The mechanism of AChE inhibition for the all OP and nerve agents is practically the same—the inhibition via phosphorylation or phosphonylation of the esteratic site of AChE. However, reactivation of inhibited AChE by oximes is diVerent for diVerent nerve agents: phosphorylated but reactivatable AChE is changed to a nonreactivatable complex. The half-times for this reaction described as dealkylation (F5) are diVerent for various OP/nerve agents (B3, B11). Thus, the basic trigger mechanism for nerve agents, as for other OP, is an intervention into cholinergic nerve transmission via an inhibition of AChE and other hydrolases (B3, M1, M2, M8). Monitoring the cholinesterase changes is at present the best reflection of the severity of OP poisoning as well as a reaction to antidotal therapy. Many kinds of specific and nonspecific eVects have been demonstrated using animal experiments. They involve cholinesterase inhibition with subsequent changes of the neurotransmitters including acetylcholine and catecholamines, changes in membrane permeability, and other metabolic imbalances (B3, K6) (Fig. 3), e.g., changes in the brain energy metabolism during soman intoxication (H4). OP/nerve agents influence the oxidative


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FIG. 3. Schematic representation of possible complex eVects of OP/nerve agents (modified from B3, K6).

metabolism (lipid peroxidation in the cerebral hemispheres). The protective eVects of antioxidants against soman- and malathion-associated lipid peroxidation have been demonstrated (T7). Lipid peroxidation is influenced by the administration of the OP pesticide methidathion. A single-dose treatment with a combination of antioxidants (vitamin C and E) after the administration of OP can reduce lipid peroxidation (A6). Increased depolarization induces a great increase in the ATP level in the brain (G10). It can also influence the blood–brain permeability by either toxic agents or therapeutic drugs (A11, R3). An interesting hypothesis was suggested by Cowan et al. (C8): acetylcholine acts as an agonist of autoacid release, and autoacids such as histamine can augment soman-induced bronchial spasm. With respect to the demonstrable critical role of cholinergic crisis in OP/nerve agent toxicity, the precepts of neuroimmunology indicate that secondary adverse reactions encompassing anaphylactoid reactions may complicate OP toxicity. Shih et al. (S14) have demonstrated that soman-induced convulsions are associated with postexposure brain pathology. These findings lead to the hypothesis that central cholinergic mechanisms are primarily involved in eliciting convulsions following exposure to highly toxic OP such as soman and the subsequent recruitment of other excitatory neurotransmitter system. Loss of inhibitory control may be responsible for sustaining these convulsions and for producing the subsequent brain damage. The important role of glutamate and its transporters (glutamate transporters are plasma membrane proteins which actively pump glutamate from the extracellular to the intracellular side of the membrane) has been demonstrated during soman poisoning (D2, L5).

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It has been described that excitatory amino acids play an important role during OP poisoning. After AChE inhibition and increase in the acetylcholine level in the nervous system, the excess of acetylcholine triggers seizure activity. Once the seizures are initiated, the noncholinergic systems are progressively recruited and the seizures become refractory to the muscarinic receptor antagonists and cause the release of excessive amounts of glutamate, damaging neighboring neurons. This may lead to death through activation of NMDA receptors, calcium accumulation, dearrangement of cellular activity, activation of catabolic enzymes, and cellular death (S25). On this basis, the good protective activity of adenosine receptor agonists has been demonstrated (V4). Both the toxicodynamics and toxicokinetics of OP/nerve agents can be explained by their biochemical characteristics of interacting with cholinesterases and other hydrolases. A scheme containing four basic actions (absorption, transport, metabolization, and the toxic eVect) is presented here (Fig. 4). The absorption is accomplished by penetration of OP through biological barriers into the blood representing the transport system. The losses originate either physically or biologically. This part of OP (reacting by this mechanism) is screened out from toxic action. The losses in the transport system originate from detoxification and nonspecific binding to proteins and enzymes—esterases, AChE, and butyrylcholinesterase (BuChE). Binding to plasma proteins is included also. Inhibition of cholinesterases in the blood is practically the first target for OP, according to the principle ‘‘first come, first served’’ (B31). The OP is carried out at the sites of metabolic and toxic eVects. However, there are diVerences, especially in the detoxification of highly toxic nerve agents: G-agents like sarin and soman are detoxified but compounds containing the P-S bond (V-agents) are not detoxified (A12, B11). The toxic eVect site is a multicompartmental system, minimally the central and peripheral nervous systems. In these places, OP reacts with cholinesterases—AChE and BuChE. Inhibition of cholinesterases is a trigger mechanism for the toxic action of OP. Important nerve agents soman and sarin are rapidly absorbed at all routes of administration, including inhalation, percutaneous, and oral administration (B3, B11), and inhibit cholinesterases (preferably AChE) in the central and peripheral nervous system. Because of soman’s high lipophility, it possesses a high aYnity to the brain AChE (A9, B3, B11). Sarin is less lipophilic; however, its aYnity to the brain AChE is also very high (B3, P3). Inhibition of the brain AChE by G compounds (sarin and soman) is very fast, reaching 50% activity within minutes. For VX, there is a delay and a decrease in AChE activity was observed after more than 20 minutes, probably caused by a more diYcult absorption in comparison with sarin and soman. The half-lives are very dependent on the dose of the agent


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FIG. 4. Schematic representation of four basic actions of OP (absorption, transport, metabolization, toxic eVect) and possible reactions of OP in the organism (modified from B2, B14).

administered, on the species, and other factors and therefore it is diYcult to compare diVerent results. In general, inhibition of AChE in vivo is faster for G-compounds in comparison to V-compounds (A7, B3, C6). From the point of view of pharmacodynamics and therapeutic possibilities, soman represents the most serious poison. Its toxicity is comparable to that of sarin and VX

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(B3, B11, C4, C5, S12), but the therapeutic eYcacy of the antidotal treatment with current and perspective drugs is not good enough (B40, D7, K2, K38, M8). This is probably a reason for intensive research dealing with soman intoxication and treatment. Soman and sarin are detoxified in the liver, plasma (B11, J5, S23), and, according to some authors, also in the lungs (S11); therefore, this part is excluded from the toxic eVect. The parent compounds can be monitored in the bloodstream as well as in metabolites which are excreted in urine (B30, B31, N7, N8). Binding to nonspecific esterases also causes losses of G-compounds in the organism and this part of soman and sarin does not have a toxic eVect. It was assessed that only 1 to 3% of the dose administered inhibited AChE in the brain, i.e., 1 to 3% of the dose administered caused the basic toxic eVect (B11, K1, L11, S11). Another factor (until now, not very elucidated) influencing soman and sarin poisoning is the existence of a depot in the organism from which the nerve agent can be released and then cause a new attack of intoxication. This depot has been described for the skin, erythrocytes, muscles, and lungs (K1). Bearing in mind the very low portion of the dose administered causing the basic toxic eVect, it is clear that the release of a very small quantity of sarin and soman can significantly influence the survival or death of the intoxicated organism independently of the treatment. On the other hand, V compounds are not detoxified in the organism (B11). This is probably the reason for the higher toxicity of V compounds in comparison with G-compounds. The eVect of V-compounds (especially VX) is prolonged in comparison with sarin and soman (V1). The toxicokinetics of diVerent nerve agents, including stereoisomers, have also been described (B30, V2). The mechanism of action for VX is inhibition of AChE, preferably in the peripheral nervous system (B3, B6, B7, M2). However, inhibition of AChE in the brain parts was described as being selective and most marked in the pontomedullar area of the brain (B6, B7, B12). Detoxification of OP with lower toxicity is also important. Moreover, for some OP, especially those containing the P ¼ S bond, oxidation giving rise to more toxic products is observed (P ¼ S ! P ¼ O). This reaction, called ‘‘lethal synthesis,’’ is typical, for example, for malathion (oxidized to malaoxon) or parathion (oxidized to paraoxon). Oxo-derivatives (more toxic) are released into the transport system and can cause a new attack of intoxication. A similar reaction can be observed after releasing the OP from the depot, mostly from fat tissue (D6, D21). In place of the toxic eVect (nervous system), the reaction with enzymes is important, though some other direct interactions with receptors have been described and nonspecific reactions (the stressogenic eVect) have been also observed. Some OP can be mutagenic or carcinogenic (B2, F2).


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A delayed neurotoxic eVect is caused by inhibition of a quite diVerent enzyme from cholinesterases—neurotoxic esterase. Depending on the target, acute, intermediate, chronic, or delayed eVects are manifested (A3, L17). 2.3. TOXICITY Toxicity of chemicals is one of the basic characteristics for chemical compounds. Depending on the conditions of its determination, diVerent types (acute, subchronic, chronic) of toxicity are diVerentiated. Acute toxicity is mostly characterized by LD50. The LD50—in its simplest form—is the dose of a compound that causes 50% mortality in a population. It is the statistically derived dose of a substance that can be expected to cause death in 50% of the animals. Derived expressions are also used, e.g., the dose causing a given eVect like incapacitation (IC50), or the dose causing 50% of enzyme inhibition in vivo (ID50). Though the expressions can be diVerent, it needs to be exactly (and, if possible, quantitatively) defined. The value of LD50 is not a constant, rather, it is a statistical term designed to describe the lethal response of a compound in a particular population under defined experimental conditions. However, this information is only one of many indices for assessing acute toxicity. The slope of the dose–response curve, the time to death, the signs of poisoning, and other parameters are very important, especially in the case of highly toxic OP such as nerve agents. Acute toxicities vary greatly among diVerent species; they are dependent on many factors (sex, age, genetic disposition, body weight, diet, hormonal factors). Especially in case of nerve agents, it can be of importance. These agents should be regarded as ‘‘hit-and-run poisons’’ (B31) and, therefore, time of the onset of convulsions (convulsive time, CT) or death (lethal time, LT) is very valuable information. When we compare the toxicity of soman and one representative of V compounds (O-isopropyl 2-S-dimethylaminoethyl methylphosphonothiolate, iPr-Me) (Table 1), the toxicity is practically the same (i.m. LD50 for soman—70–80 g/kg, for iPr-Me 80–100 g/kg). The CT and LT values are diVerent: CT and LT values for soman are 3–4 min and 8–12 min; for iPr-Me, these values are 8–12 min and 20–25 min, respectively. The route of administration is also of great importance. There are diVerent methods for determining the LD50 values; however, probit-logarithmical extrapolation of the dose–response reaction is the common method for LD50 determination. In case of OP and especially nerve agents where the mechanism of action is generally known and is limited to one main factor (cholinesterase inhibition), the slope of the dose–response curve is very strong. In this case (for the same compound), the slopes of the curves are dependent on the route of administration in order i.v.>i.m. (s.c.)>i.p.>p.o.>p.c. A typical example of the LD50 values at diVerent

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TABLE 1 TOXICITIES OF DIFFERENT OP/NERVE AGENTS FOR RATS (EXPERIMENTALLY DETERMINED) AND HUMAN (ASSESSED) Toxicity (LD50) a

Compound

i. m., rat (g/kg)

a

Et – Me Et–i Pr (VX) i Pr–i Pr Et–Et i Pr–Me sarin soman GV DFP TEPP parathion paraoxon DDVP trichlorfon systox dimethoate malathion

25–30 12–16 40–50 20 70–100 200 70–80 17 800 850 500–600 300–500 17 440 230 000 3110 1000–2000 –

0.1–0.2 0.08–0.09 0.1 0.2 0.8 0.7–0.9 0.5–0.6 0.19 1–13 2–15 6–7 3 62 625 – 215–270 800–1200

a b c

p.o., rat (mg/kg)

b

p. o., human (mg/70kg) 6–10 5 – – – 8–12 7–12 8 20–80 30–100 50–200 30–50 500–1000 grams – 1–2 g grams

c

i. m., human (g/kg) 3–4 20–25 60–70 110–130 130–150 – – 20–25 40–50 – 2800–3000 300–350 150–200 – 4000 – –

Experimental data from literature (B2, B3, B11, F2, C4, C6, D17, K2, K12, M2, M8, S12, V1). Assessed data from literature (B2, B11, M2). Assessed data from Fig. 6.

routes of administration for O-ethyl S-2-dimethylaminoethyl methyl phosphonothiolate (Et-Me) is given in Fig. 5. The toxicities of diVerent OP/nerve agents are shown in Table 1. V-compounds are designated by the abbreviation of the oxyalkyl group on the phosphorus head and by the alkyls on the nitrogen atom, e.g., VX is designated as Et-iPr. The data represent the values of LD50 (oral, p.o., and intramuscular, i.m. administration) experimentally obtained in rats and assessed oral doses for humans (LD50 for 70 kg weight) from literature sources. Another assessment of human LD50 (i.m.) is based on experiments in vitro (determination of pI50, negative decadic logarithm of the inhibitor concentration causing 50% of brain AChE inhibition) and in vivo toxicities (LD50 for i.m. administration) in diVerent species, as is demonstrated in Fig. 6. Many experiments have been conducted on the modeling of OP poisoning. Investigations have included among other things, the symptomatologic assessment of OP-induced lethality in mice (L12), expressing OP poisoning through mathematical equations (G6, G7), and evaluating a model for


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FIG. 5. Toxicities of O-ethyl S-2-dimethylaminoethyl methyl phosphonothiolate at diVerent routes of administration in rats. An example of real experiments. LD50 (with 95% confidence limits), g/kg: i.v. 20.3 (18.5–22.3); i.m. 27.4 (23.9–31.4); i.p. 48.2 (37.7–61.7); p.c. 76.1 (60.1–96.4).

FIG. 6. Summarization of results correlating inhibition eYcacy (pI50) and toxicity (log LD50) for some OP and nerve agents. Equation: y ¼ 9.87 1.26x; p < 0.01; rxy ¼ 0.9489. The lines indicate experimentally determined pI50 (human brain AChE) values (axe y) or extrapolated values (axe x) of LD50 for systox and VX. Each point represents the value of pI50 corresponding to LD50 value for rabbit, rat, guinea pig, mouse, and dog. The compounds under code are designated by the abbreviation of the oxyalkyl group on the phosphorus head, and by the alkyl on the nitrogen atom – e.g. VX is designated as Et-iPr (modified from B11, B14 and P3).

carbamate and OP-induced emesis in humans (D1). Predicting toxicokinetic parameters in humans from the toxicokinetic data acquired from three small mammalian species was the aim of another study (B1). Similarly, possible rat models for minimal brain dysfunction have been presented (L4). Other

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studies correlating structure vs activity of both OP and their antidotes have been presented (C1, D5, G5, M11). A very interesting approach described by Maxwell et al. (M11) using the multiple regression model for in vivo rate cholinesterase inhibition contained three independent variables (blood flow, carboxylesterase, and cholinesterase) and this could account for 94% of the observed variation. A theoretical expression for the protection associated with stoichiometric and catalytic scavengers in a single compartment model of OP poisoning has been described (S31). In our previous paper (B2), we described the scheme of the multiple eVects of OP including the influence on cholinesterases and other enzymes, detoxification, and the possibility of metabolization. These studies were elaborated with the aim of extrapolating the data from animals to humans. Though the inhibition of cholinesterases is the trigger mechanism of action of OP/nerve agents, a simple correlation of toxicity and inhibition eYcacy was not linear and statistically significant. A good correlation was achieved when the toxicity data was expressed as logarithm and the inhibition eYcacy as a negative decadic logarithm of the I50 value (pI50). The value of pI50 for human brain AChE interaction with OP allows us to extrapolate the corresponding toxicity data for humans (B11, B14). However, this extrapolation is possible for the highly toxic OP where the inhibition is the most important (Fig. 6). This correlation is closer in that the inhibition potency is expressed as the inhibition rate in vivo and correlated with the toxicity data (B11, B14). These results dealing with the relationship between the inhibition eYcacy and the toxicity of diVerent OP showed a good correlation between these two parameters. It is diYcult to compare these results with the literature. Nevertheless, there are some data dealing with i.v. toxicity of the VX (S18): in healthy volunteers, the value of i.v. LD50 was assessed to be 10 g/kg or 7 g/kg (M2). It appears from our results that the calculated i.m. LD50 value was 20 g/kg. These data are in good agreement because the percentage of the i.v. dose is about 40 to 60% of the i.m. dose (B11). It can be concluded that this methodical approach at least allows us to assess the toxicity of some OP to humans without experiments on volunteers, which has been considerably hazardous for that particular group of substances. 2.4. SYMPTOMS OF INTOXICATION Dominating signs of poisoning with OP and nerve agents are caused by hyperstimulation of the cholinergic nervous system due to an elevated level of acetylcholine caused by inhibition of AChE (acute cholinergic crisis).


165

According to type and localization, peripheral and central muscarinic and nicotinic symptoms are observed. Peripheral muscarinic symptoms are observed in the exocrine glands— nasal mucosa (rhinorrhea), bronchial mucosa (bronchorrhea), sweat (sweating), lacrimal, and salivary glands (lacrimation, salivation). An elevated level of acetylcholine in the smooth muscles causes miosis (iris), failure of accommodation (ciliary muscle), abdominal cramps, diarrhea (gastrointestinal tract), micturition, increased frequency of urination (bladder), and bradycardia (heart). Peripheral nicotinic symptoms due to accumulation of acetylcholine include sympathomimetic eVects, pallor, tachycardia, hypertension (autonomic ganglia) and muscular weakness, fasciculations and convulsions, and later paralysis (skeletal muscles including diaphragm and intercostal muscles). Central (muscarinic and nicotinic) symptoms are not very specific and include giddiness, anxiety, restlessness, headache, tremor, confusion, failure to concentrate, convulsions, and respiratory depression. These eVects are called the cholinergic eVects. OP/nerve agents have many other eVects which influence various organs and systems. They are called nonspecific (noncholinergic) eVects. These eVects are usually registered later, after the manifestation of the cholinergic eVects. Therefore, the OP/nerve agent poisoning can be divided into three phases (S15): cholinergic phase characterized by cholinergic eVects (also called acute cholinergic crisis), transitional phase characterized by mixed cholinergic and noncholinergic eVects, and noncholinergic phase characterized by the predominance of nonspecific eVects. The intermediate syndrome in OP poisoning is clinically characterized by weakness in the territory of cranial nerves, weakness of respiratory, neck and limb muscles, and depressed deep tendon reflexes. It occurs between the acute cholinergic crisis and the usual onset of OP-induced delayed neuropathy (D9, D10). Postexposure changes of neurological character have also been observed (B37). It was demonstrated that low doses of nerve agents also caused long-lasting changes in behavior and neuroexcitability in experimental animals (K17). The time course of poisoning is dependent on the type of agent, the dose incorporated, and the route of exposure. Symptoms appeared minutes after inhalation of nerve agents and minutes to hours after incorporation of OP pesticides. Death can be observed (without treatment) within minutes after nerve agent inhalation and within hours to days after OP pesticide exposure. Description of the course of poisoning with OP and nerve agents can be found in many publications, either national or international, and it is mentioned in various diVerent publications (B29, M1, M2, L15, and others). The delayed neurotoxic eVect, also called OrganoPhosphate Induced Delayed Neurotoxicity (Neuropathy) (OPIDN), is characterized by sensoric and motoric disturbances of the peripheral nervous system (degeneration of

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axons and myeline and inhibition of so called ‘‘neurotoxic esterase’’). OPIDN is manifested following OP exposure (sometimes it is not accompanied by acute syndromology) during days (weeks) after the exposure. It is characterized in 30 to 40% by acute intoxication and is manifested as nausea, headache, and other nonspecific symptoms. After a latent period (1–4 weeks), cholinergic irritation can be observed in about 30% of patients (increased salivation, nose secretion, pharyngitis, laryngitis). Paralysis of the leg muscles follows these symptoms for 1 to 2 weeks, persisting 1 to 2 months without significant changes of sensitive innervation. Then denervation and atrophy of the leg muscles is observed. Partial restitution is possible; however, convalescence is long, abnormal reflexes being observed for years. Tri-O-cresyl phosphate (TOCP) has been reported as the typical compound producing OPIDN (A2, A3, L16, L17, M17) due to inhibition of the neurotoxic esterase (NTE) (J2, J3, J4, L16). High inhibition NTE in the nervous system, measured within hours after dosing, correlates with the delayed onset of clinical signs 10 to 20 days later. From the practical point of view, it is important how the dose causing OPIDN compares with that causing acute cholinergic toxicity. A ratio LD50/neurotoxic >1 discriminates OP causing OPIDN at doses which do not cause cholinergic toxicity from those which cause it only if animals are treated against cholinergic symptoms (ratio

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