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
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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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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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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
LIPOPROTEIN OXIDATION AND ARTERIOSCLEROSIS
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).
LIPOPROTEIN OXIDATION AND ARTERIOSCLEROSIS
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
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ABUDU ET AL.
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.
LIPOPROTEIN OXIDATION AND ARTERIOSCLEROSIS
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.
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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.
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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).
LIPOPROTEIN OXIDATION AND ARTERIOSCLEROSIS
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
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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,
LIPOPROTEIN OXIDATION AND ARTERIOSCLEROSIS
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
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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
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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
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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).
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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
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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
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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.
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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.
LIPOPROTEIN OXIDATION AND ARTERIOSCLEROSIS
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
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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
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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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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7.3. Burns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4. Polycystic Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. MMPs Identified in Urine of Patients with Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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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
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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
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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 <10 ng/ml (order of appearance reflects their relative concentrations). Comparison of the sensitivity of ELISAs, gelatin zymography, and radiolabeled substrate degradation assays for measurement of MMP-2 and MMP-9 have been published (Z11). Catterall and Cawston (C3) have recently reviewed methods employed to assay MMPs and TIMPs in biologic fluids. Hanemmaaijer et al. (H1) developed a brilliant immunocapture assay for detection of latent and activated MMP-9 in body fluid samples. The assay
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was made specific for MMP-9 by using an MMP-9 specific monoclonal antibody. Using this antibody, MMP-9 is captured from biological fluids or tissue culture media, and MMP activity of both active and latent MMP-9 is analyzed. In the second step of the procedure, immobilized MMP-9 is incubated with modified pro-urokinase and a chromogenic substrate specific for urokinase; cleavage of this substrate was measured as the end point. Using protein engineering, a modified pro-urokinase reagent was made in which the activation sequence, normally recognized by plasmin, was replaced by an amino acid sequence that is specifically recognized by MMPs. APMA was used to activate proMMP-9 in the specimen, thereby permitting measurement of total MMP-9.
5.1. PITFALLS IN MEASUREMENT OF BLOOD LEVELS OF MMPS AND TIMPS Before proceeding to a discussion of blood levels of MMPs and TIMPs in various diseases, it will be useful for the reader to critically examine the technical limitations of measuring these antigens in blood. An important consideration of measurements of MMPs in disease is the bimodal distribution of blood levels of MMPs in healthy subjects; this is especially noted with plasma MMP-9, where 20% of healthy individuals have MMP-9 levels that are twice that of the remainder of the population (Fig. 2). We have noted that MMP levels are relatively constant in each individual over time. Genetic differences within the population probably account for much of this
FIG. 2. Plasma levels of MMP-9 in healthy patients. Note the non Gaussian distribution of values with a small second peak of normal values.
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variation. Another consideration is MMP differences based on sex; the mean serum level of MMP-3 in men is approximately twice that in women (Z9). Serum levels of MMP-3 have also been reported to increase somewhat with advancing age (M3). Among the difficulties in comparing results using different commercial ELISA kits is the absence of individual purified MMP standards for use in producing calibration curves. Likewise, quantifying MMP measurements can give different absolute results, depending on the specific antibody combinations employed in the assay kit. As a result of these effects, published normal ranges for MMPs and TIMPs vary considerably. Another confounding factor affecting these measurements is that MMPs can bind to connective tissue matrix; hence, increased local release of MMPs in disease may not necessarily be translated into increased plasma levels. Unfortunately, many investigators working in this field and publishing impressive reports of increased levels of serum MMP-9 in disease appear not to have been aware that the measurement of MMP-9 in serum is highly unreliable since it reflects, in large part, the in vitro release of MMP-9 by degranulation of blood neutrophils occurring during the ex vivo blood clotting process (Z3). As a result of MMP-9 release from neutrophils in the test tube specimen, serum concentrations tend to be many fold higher than simultaneously measured plasma levels of MMP-9. Jung et al. (J3) reported that significant differences between patient groups and controls may be lost with serum MMP-9 measurements. The anticoagulant employed for collection of plasma samples can also affect MMP ELISAs; commercial producers of ELISA kits often specify the preferred method of sample preparation. Unfortunately, the package insert of some commercial ELISA kits states that either serum or plasma can be used for the measurement of MMP-9 in blood (M5). Based on these artifacts, we have chosen to exclude from this chapter most reports describing serum MMP-9 measurements. MMP-3 levels have also been reported to be higher in serum than plasma; however, this observation remains of uncertain significance. As a result of platelet release of TIMP-1 during the clotting process, TIMP-1 levels are higher in serum than plasma. Nonetheless, most reports describe the use of serum for TIMP assays. 5.2. ORIGIN OF MMPS
AND
TIMPS
IN
BLOOD
Based on our detection of high levels of MMP-9 and MMP-3 in human cancer tissue and joint tissue and the identification of increased levels of MMP-9 and MMP-3 in the plasma of patients with breast/colon cancer and rheumatoid arthritis, respectively, we postulated that tissue MMPs leach into the bloodstream in increased amounts in patients with biologically aggressive cancer and arthritis (M3). Confirmation of this hypothesis by
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direct experimentation remains to be done. Likewise, the fate of MMPs after entering the circulation has not been examined. Indirect data bearing on this phenomenon comes from an experiment in which low dose bacterial lipopolysaccharide was injected into healthy volunteers to simulate the effects of endotoxin. Plasma levels of MMP-9, presumably released by blood neutrophils, increased 30-fold within 2 hours and fell in 4 to 6 hours; these data suggest that MMPs are removed somewhat rapidly from blood. At this time, it is uncertain whether MMPs are inactivated or complexed, the mechanism(s) remains to be identified (A2). In contrast, the lipopolysaccharide injection did not significantly modify plasma levels of MMP-2 (not stored in neutrophils). In patients with septic shock, Nakamura (N5) reported that plasma MMP-9 concentrations were significantly higher in nonsurviving patients (166 56 ng/ml) as compared to surviving patients (78 16 ng/ ml) and 25 normal controls (32 10 ng/ml). These data suggest that plasma MMP-9 may be a useful prognostic marker in septic shock. Another important misconception in the literature deals with the activation status of MMPs in circulating blood. Moutsiakis et al. (M17) were unable to identify measurable amounts of activated MMP-2/-9 in human plasma, which is consistent with the concept that the molar levels of TIMP-1/ TIMP-2 and 2-macroglobulin in blood are in great excess of plasma level of MMPs. Hence, we postulate that activated MMPs leaching into the bloodstream would rapidly form complexes with inhibitors, leading to removal from the circulation by unidentified mechanisms.
6. Blood Levels of MMPs and TIMPs in Physiologic and Disease States (Table 2) Based on research implicating MMPs in various pathologic processes, many investigators have described the measurement of MMPs and TIMPs in the blood and urine of patients with various diseases. These results have generally taken the form of a comparison of the level (mean standard deviation) of MMPs or TIMPs in a patient population as compared to an age- and sex-matched healthy population. The statement that mean MMP levels are significantly increased in patients with a disease as compared to a healthy population does not indicate the variance in both populations, and hence the overlap between the two groups. It is important to distinguish between this type of comparative data and more detailed analyses (sensitivity, specificity, positive predictive value, negative predictive value) that may have direct applicability to the care of patients. Recent reports have employed Receiver Operating Characteristics (ROC) to describe their data. ROC analysis plots data according to the relationship between sensitivity
TABLE 2 PLASMA, SERUM, AND URINE LEVELS OF MMPs AND TIMPs IN PHYSIOLOGIC AND PATHOLOGIC CONDITIONS Diseases Cancer Lung
Prostate
49 Bladder
Renal Ovarian
Melanoma Gastrointestinal
MMP/TIMP
Type/levels
Diagnostic importance
Prognostic importance
MMP-2 MMP-9
Serum/High Plasma/High
Poor Poor
TIMP-1
Serum/High
Poor
MMP-9 MMP-3 MMP-1 TIMP-1 MMP-9 MMP-2 MMP-2 MMP-3 MMP2:TIMP-2 MMP-1
Plasma/High Serum/high Serum/High Serum/High Urine/High Urine/High Serum/High Serum/High Serum/High Urine/High
MMP-9 MMP-2 MMP-9 MMP-9 TIMP-1 TIMP-2 MMP-2 MMP-9 MMP-2
Urine/þnce Urine/þnce Plasma/High Plasma/High Serum/High Serum/High Serum/High Plasma/High Serum/High
Yes
Poor No Yes Yes Yes
Yes Poor Poor Poor Poor Yes Yes Yes
Poor Poor Poor Poor Poor Poor Poor
Comment
In metastasis (L4) Suggest higher tumor burden, Squamous > 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
51
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 (> 0.5<1). In addition to evaluating single curves, ROC also allows the comparison of ROC curves from independent or separate samples and from correlated or identical samples. Receiver Operating Characteristic Analysis makes it easier to summarize the performance of a test with a manageable number of statistics or to compare the performance of different tests. ROC analysis has been shown to be related in a direct way to cost/benefit analysis of diagnostic decision making (M12) (http://www.accumetric.com). The effect of social habits on MMP levels in blood has received scant attention. Nakamura et al. (N4) reported that plasma MMP-9 concentrations in smoking subjects were significantly higher (50%) than age- and sexmatched nonsmoking subjects. Levels were related to duration of smoking history. After discontinuation of smoking for 6 months, MMP-9 concentrations were found to be significantly lower (17%). In contrast, serum MMP2 concentrations in subjects who smoked were not different from subjects who did not smoke. This data supports the concept that the homeostatic regulation of MMP-9 is different from that of MMP-2. In another fascinating report, Lohmander (L9) reported that serum levels of MMP-3 rose considerably after athletes ran a half marathon. 6.1. PLASMA MMPS
AND
TIMPS LEVELS IN PREGNANCY
It has long been recognized that MMPs serve an important function during fetal implantation, pregnancy, and delivery. Using a relatively insensitive bioassay, Rajabi et al. (R1) demonstrated a rise in gestational serum levels of MMP-1 during term labor, but not earlier. Premature rupture of membranes has long been associated with increased levels of MMPs, particularly MMP-9, and with reduced levels of TIMP-1. MMP-9 concentrations in the amniotic fluid are more elevated in preterm labor resulting in preterm delivery than in term labor (V2). This observation led to speculation that plasma levels of MMP-9 may be useful in identifying preterm labor complications. Tu et al. (T3) reported that plasma MMP-9 levels are elevated approximately 20-fold from week 19 to 36 of pregnancy compared to values obtained in nonpregnant women; during spontaneous labor, plasma MMP-9 levels increase an additional 3-fold. These extreme levels of plasma MMP-9 reflect high production of MMP-9 in the decidua, chorion, and amnion and the ongoing reorganization of extracellular matrix in the uterus and placenta. Clinical correlations, however, failed to show that the plasma MMP-9 level was useful as a biomarker of preterm labor;
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prenatal plasma levels were not different between women delivering preterm or at term. Kolben et al. (K7) also reported that plasma concentrations of MMP-9 rose between the 10th to 40th week of pregnancy. IL-8 was proposed to be responsible for the increased release of MMP-8 and MMP-9; these MMPs were implicated in cervical ripening during labor (O6). Using a prototype sandwich ELISA, Zucker et al. demonstrated that the plasma level of MMP-2 was increased 50% in the second half of pregnancy as compared to early pregnancy or the nonpregnant state (Z6). The relatively high levels of MMP-2 in normal plasma (500 ng/ml) relative to other plasma MMPs (<70 ng/ml) led to assessment of which cells in the body might be responsible for producing MMP-2. Based on in vitro studies of umbilical vein MMP synthesis, Zucker et al. (Z4) proposed that endothelial cells lining blood vessels make a larger contribution to plasma concentrations of MMP-2 than MMP-9, hence the high plasma level of MMP-2 in healthy individuals. These authors suggested that as a consequence of the endothelial contribution to plasma MMP-2, the leaching of lesser amounts of MMP2 from tumors into blood may escape detection. This hypothesis seems to have been borne out by subsequent studies demonstrating increased levels of MMP-2 in the blood of patients with various forms of vascular injury. In contrast to MMPs, serum TIMP-1 levels are low during pregnancy until the 37th week, when the levels rise to the nonpregnant state. Serum TIMP-1 levels then rose during labor and the postpartum period (C7). 6.2. PLASMA MMPS
AND
TIMPS LEVELS IN CANCER
6.2.1. Gastrointestinal Cancer, Breast Cancer, and Head and Neck Cancer Based in large part on the prevailing hypothesis in the 1990s that MMPs were critical factors in cancer invasion and metastasis, many reports appeared in the literature describing increased tissue (P1) and blood levels of MMPs in patients with various forms of cancer and other disease states (Z3). Debate still continues concerning the role of MMPs in metastasis (S10). Employing a sandwich ELISA with two different monoclonal antibodies to MMP-9, Zucker et al. (Z10) demonstrated that plasma MMP-9 was significantly increased in patients with breast cancer and gastrointestinal tract cancer (metastatic and nonmetastatic) as compared to normal subjects. Latent MMP-9:TIMP-1 complexes were significantly increased in the plasma of patients with GI cancer and female genitourinary tract cancer as compared to controls. Some of these patients had increased plasma complexes and normal MMP-9 and vice versa. When the results from both the MMP-9 and complex data were combined, increased levels of either MMP-9 or complexes were found in 36% of patients with GI cancer and 65% of
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women with genitourinary tract cancer. Most importantly, clinical follow-up for 40 months of patients with stage IV (metastatic) GI cancer indicated that the length of survival of patients with elevated levels of either of these plasma MMP-9 measurements was 4 months as compared to patients with normal plasma MMP-9/complex levels who survived 20 months (significance, p < .01). On the basis of these results, Zucker et al. (Z5) suggested that these plasma MMP-9 assays may be clinically useful in predicting survival in subsets of patients with cancer. It was pointed out that although significant differences exist between groups, the bimodal distribution of plasma MMPs and TIMP complexes in the healthy population limits the practical value of these assays for the individual patients since there is considerable overlap in test results between the cancer patient population and the population of healthy individuals. The MMP-9 ELISA was also used to assess the effect of treatment in patients with breast cancer. Both chemotherapy and hormonal therapy resulted in considerable fluctuations in plasma MMP-9 levels, which often showed no correlation with clinical cancer progression (Z3). Ishida et al. (I2) reported that the levels of MMP-9 in portal blood of patients with colorectal cancer and liver metastases correlated with characteristics of the primary tumor. By setting the cutoff ratio of portal to peripheral blood MMP-9 levels at 1.6 in patients subjected to curative resection, elevated ratios predicted subsequent emergence of liver metastases with 78% sensitivity, 81% specificity, and 81% accuracy. The authors concluded that synchronous determination of plasma MMP-9 levels of portal and peripheral blood at curative resection of liver metastases may be useful for selecting colorectal cancer patients at high risk of hepatic recurrence. Oberg et al. (O2) reported that patients with colorectal cancer had significantly higher levels of free serum MMP-2, TIMP-1, and TIMP-2, whereas the level of the MMP-2/TIMP-2 complex was significantly lower than normal. TIMP-1 was significantly higher in Dukes’ D (metastatic) compared to Dukes’ A-C colon cancer. High free serum MMP-2 correlated with poor survival in colorectal cancer. Pellegrini et al. (P2) reported that combining serum TIMP-1 levels with CEA measurements in patients with colorectal cancer was useful to predict prognosis. TIMP-1 levels have been reported to be more than 3-fold elevated in patients with Dukes’ D (stage IV) colorectal cancer as compared to healthy donors (H7). Similar increased levels of plasma TIMP-1 were found in patients with advanced breast cancer. Holten-Andersen et al. (H7) proposed that plasma measurements of TIMP-1 may be of value in the management of cancer patients. Yukawa et al. (Y9) further reported that the plasma concentration of TIMP-1 was increased in colorectal cancer patients with serosal invasion by tumor and metastasis to lymph node and liver.
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55
Several other reports have described MMP and TIMP alterations in the blood of patients with adenocarcinomas originating in other areas of the gastrointestinal tract. Endo et al. (E4) reported that plasma proMMP-9 levels and serum proMMP-2 levels (measured by ELISA) were significantly higher in patients with gastric cancer than in healthy individuals. Similar to the report of Zucker et al. (Z10), there was no significant correlation between CEA and proMMP-9 levels and between CEA and proMMP-2 levels in patients with gastric cancer. Neither proMMP-9 levels nor proMMP-2 levels were significantly related to clinicopathologic features of gastric cancer. In contrast, Fujimoto et al. reported that serum levels of MMP-2 were not increased in stomach and pancreatic cancer, but were increased 30% in patients with hepatocellular carcinoma, hyperthyroidism, and biliary cirrhosis (F7). Yoshikawa et al. (Y7) reported that elevated plasma concentrations of TIMP-1 correlated with serosal invasion and metastasis in patients with gastric cancer. Plasma MMP-9 levels in hepatocellular carcinoma have been reported to be significantly elevated compared with normal controls. Plasma MMP-9 concentration had a sensitivity of 53% and a specificity of 89% for the discrimination between hepatocellular carcinoma and chronic hepatitis or liver cirrhosis. Plasma MMP-9 levels were significantly higher in hepatocellular carcinoma patients with macroscopic portal venous invasion than those without invasion. Plasma MMP-9 levels in cancer patients did not correlate with tumor number, size, volume, or the serum tumor marker -fetoprotein levels (H3). MMP and TIMP measurements have also been made in cancer patients [colorectal cancer (26), mesothelioma (7), renal (6), melanoma (6)] treated with a synthetic MMP inhibitor (MMPI270). A rise in serum TIMP-1 during MMP inhibitor treatment suggested that a reflex increase in TIMP might modify the effects of the drug (L4). Farias et al. (F2) employed a different method for examining plasma levels of MMP-2 and MMP-9 in cancer patients; the euglobulin fraction of plasma was isolated by acid precipitation prior to performing gelatin substrate zymography; results were reported in Arbitrary Units (AU). Using this approach, Farias et al. reported that the median value for plasma MMP-9 activity was significantly increased in breast cancer (Median 1.34 AU/ml plasma, range 0.0 to 7.2) and lung cancer (Median 1.43 AU/ml, range 0.0 to 3.6) compared with the controls (Median 0.48 AU/ml, range 0.0 to 1.8). Multivariate analysis indicated that plasma MMP-9 activity was not predicted by the known clinicopathological parameters such as age, stage, tumor size, number of positive lymph nodes, histological grade, histological type, nuclear grade, or mitotic index. Employing a cutoff value of 1.15 AU/ml of plasma, 59% of breast and 69% lung cancer patients exhibited high values as compared to 24% of healthy controls. The authors proposed that acetic
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acid precipitation of euglobulins may increase MMP activity in plasma, thus revealing subtle differences between patient groups. This laboratory (R2) also reported that the median value for the plasma euglobulin MMP-9 fraction was increased in patients with laryngeal (2.1 AU/ml plasma, range 0.2 to 6.4) and oropharyngeal cancer (2.08 AU/ml plasma, range 0.0 to 5.0) as compared to controls (0.48 AU/ml plasma, range 0.0 to 1.8). In patients with thyroid cancer, Komorowski et al. (K8) reported that plasma MMP-2 levels were higher (605 82 ng/ml versus 149 44 ng/ml), along with TIMP-2 (114 7 ng/ml versus 61 9 ng/ml) than in controls; in contrast, lower levels of MMP-1 (0.70 0.42 ng/ml versus 3.87 0.53 ng/ml) were reported. Increased levels of plasma MMP-3 and MMP-9 were found in patients with medullary thyroid carcinoma. 6.2.2. Lung Cancer In an early well-publicized report, Gabrisa et al. (G5) reported increased serum MMP-2 levels in patients with metastatic lung cancer as compared to localized lung cancer; however, the reported differences were small (<20%). Contrary to the Gabrisa results, Zucker et al. (Z8) reported that plasma MMP-2 was not elevated in patients with advanced cancers as compared to levels in healthy individuals. A major difference in these studies was that Zucker et al. (Z8) also examined plasma levels of MMPs in hospitalized patients without cancer as a second control group; this group served to determine whether blood MMP measurements would be useful as a specific marker of malignancy in a sick patient population. Lizasa (L8) reported that the mean plasma concentration of MMP-9 in patients with nonsmall cell lung cancer was significantly elevated (97%) as compared to healthy volunteers, but the range of values within the cancer population was wide. Plasma MMP-9 concentrations were elevated in 45% of lung cancer patients; however, this elevation did not seem to correlate with MMP-9 production by cancer and stromal cells (immunochemical assessment) identified in patient’s resected tumor specimens. MMP-9 concentrations were significantly higher in patients with squamous cell versus adenocarcinoma of the lung. Elevated plasma MMP-9 levels decreased to levels within the normal range, 4–8 weeks after tumor resection. The authors proposed that measurement of plasma MMP-9 might be a beneficial adjunct for assessing the tumor burden. Serum TIMP-1 levels have also been reported to be higher in lung cancer patients than in control patients, whereas serum TIMP-2 and MMP2:TIMP-2 complex levels were lower in lung cancer patients than in control subjects. High serum TIMP-1 or plasma MMP-9 levels correlated with poor cumulative survival in lung cancer patients (Y5). A rising level of serum TIMP-1 was a significant predictor for risk of death (25% higher risk of
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57
death when TIMP-1 levels increased >100 ng/ml during the course of disease). With multivariate analysis, both serum TIMP-1 and stage of the disease had a statistically significant prognostic value. High serum TIMP-1 (>300 ng/ml) and plasma MMP-9 (>30 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.
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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
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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
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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
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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
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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
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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 had 97% negative predictive value, so this cutoff point might be used in clinical practice as an indicator of a low risk to secondary bleeding. The positive predictive value of MMP-9 >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 (<33 ng/ ml). Previous genetic studies had shown that functional promoter variation of the MMP-9 gene was related to the presence and severity of cardiovascular disease. Blankenberg et al. (B7) demonstrated that the T allele of the C-1562T polymorphism was associated with increased plasma MMP-9 levels in a fairly codominant fashion. Although none of the polymorphisms was significantly related to cardiovascular death, there was a significant association between the R279Q polymorphism and cardiovascular events in patients with stable angina (B7). Combined determination of plasma MMP-9 and IL-18 (a strong inflammatory predictor of cardiovascular risk) identified patients being at very high risk for cardiovascular events. Several groups have measured the level of MMPs in cardiac vessels from blood obtained during cardiac catheterization. Inokubo et al. (I1) reported that plasma levels of MMP-9 and TIMP-1 were increased in the coronary circulation in patients with acute coronary syndrome; they suggested that these elevated levels reflected active plaque rupture. Cedro et al. (C4) reported on the effects of angioplasty on MMP levels. Basal plasma levels of MMP-2 and MMP-9 were measured from blood obtained through a coronary sinus catheter. Two consecutive balloon inflations in patients with stable angina resulted in a significant but transient increase in plasma MMP-9 but not MMP-2. The authors proposed that rapid release of MMP-9 might be reflective of the remodeling occurring in the vascular wall. Hojo et al. (H5) reported that plasma MMP-2 in the coronary sinus was increased significantly 4 and 24 hours after percutaneous transluminal coronary angioplasty; high MMP-2 levels were associated with late restenosis of the coronary arteries. Galley et al. (G4) suggested that the increase in blood levels of MMP-9 occurring after bypass grafting with cardiopulmonary bypass was a component of the inflammatory response during cardiac surgery. Hirohata et al. (H4) reported time-dependent alterations of serum MMP-1 and TIMP-1 levels after successful reperfusion in patients with acute myocardial infarction.
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6.6.2. Congestive Heart Failure Wilson et al. (W3) reported that plasma MMP-9 levels (mean S.D.) were increased in patients with congestive heart failure (72 15 ng/ml) as compared to normal individuals (25 6 ng/ml). Interestingly, plasma levels of MMP-8 were decreased in heart failure patients. The MMP-9:TIMP-1 ratio was increased by 3-fold, whereas the MMP-9:TIMP-2 ratio was increased by 16-fold in the plasma of patients with congestive heart failure. The authors proposed that these changes in MMP:TIMP levels likely reflect the progression and/or acceleration of the left ventricular remodeling process in congestive heart failure. They proposed that serial measurements of plasma MMP:TIMP levels may hold diagnostic/prognostic significance in congestive heart failure (W3). Altieri et al. (A3) reported that the plasma levels of proMMP-9 and activated MMP-9 were approximately twice normal in patients with congestive heart failure, regardless of the phase of heart failure. TIMP-1 and proMMP-2 were also increased in these patients. A positive correlation with left ventricular volume was identified for MMP-2. Schwartzkopff et al. (S4) reported that free MMP-1 and the MMP1:TIMP-1 ratio in serum was higher in patients with dilated cardiomyopathy than in healthy control subjects; they proposed that serum markers of collagen degradation might be valuable markers for progression of left ventricular dilatation. Noji et al. (N9) reported increased circulating levels of MMP-2 and TIMPs in patients with left ventricular remodeling in hypertrophic cardiomyopathy. Marin et al. (M7) reported that patients with atrial fibrillation had lower levels of plasma MMP-1 but increased levels of TIMP-1 and prothrombin fragments F1þ2 (an index of thrombogenesis) and higher ratios of TIMP-1 to MMP-1 as compared to control subjects. The authors proposed that MMP:TIMP measurements in blood might be useful as markers of comorbidity that is associated with increased risk of stroke and thromboembolism in patients with atrial fibrillation. 6.6.3. Heart Transplants Serum MMP-3 and TIMP-2 were reported to be significantly higher in heart transplant recipients than in control subjects. The increase in MMP-3 is consistent with an inflammatory response in transplant recipients (D3). 6.6.4. Aneurysms MMPs have been implicated in the pathogenesis of aneurysm formation. Plasma MMP-9 levels have been reported to be elevated in patients with abdominal aortic aneurysms (86 12 ng/ml) as compared to patients with symptomatic aortic occlusive disease (26 4 ng/ml) or with healthy volunteers (13 2 ng/ml) (M10). Patients with multiple aneurysms have
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higher circulating levels of plasma MMP-9 than patients with an isolated infrarenal aortic aneurysm. McMillan and Pearce (M10) pointed out that the plasma level of MMP-9 may not bear a direct relationship with the size of aneurysm but rather with the extent of the aneurysm. Increased plasma MMP-9 levels were significantly associated with size and expansion of small abdominal aortic aneurysms (L6). Lindholt et al. (L6) proposed that plasma MMP-9 and plasma antitrypsin may predict the natural history of patients with aneurysms. Sangiorgi et al. (S2) described the effect of surgery on plasma MMP levels in patients with aortic aneurysms. After endovascular grafts and open surgery of aortic aneurysms, both plasma MMP-9 and MMP-3 decreased >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.
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W3. Wittwer, C. T., Ririe, K. M., Andrew, R. V., David, D. A., Gundry, R. A., and Balis, U. J., The LightCycler: A microvolume multisample fluorimeter with rapid temperature control. Biotechniques 22, 176–181 (1997). W4. Wong, I. H., Yeo, W., Chan, A. T., and Johnson, P. J., Quantitative relationship of the circulating tumor burden assessed by reverse transcription–polymerase chain reaction for cytokeratin 19 mRNA in peripheral blood of colorectal cancer patients with Dukes’ stage, serum carcinoembryonic antigen level, and tumor progression. Cancer Lett. 162, 65–73 (2001). Y1. Yamaguchi, K., Takagi, Y., Aoki, S., Futamura, M., and Saji, S., Significant detection of circulating cancer cells in the blood by reverse transcriptase–polymerase chain reaction during colorectal cancer resection. Ann. Surg. 232, 58–65 (2000). Y2. Yasuda, K., Adachi, Y., Shiraishi, N., Yamaguchi, K., Hirabayashi, Y., and Kitano, S., Pattern of lymph node micrometastasis and prognosis of patients with colorectal cancer. Ann. Surg. Oncol. 8, 300–304 (2001). Y3. Yonemura, Y., Endou, Y., Fujimura, T., Fushida, S., Bandou, E., Kinoshita, K., et al., Diagnostic value of preoperative RT-PCR-based screening method to detect carcinoembryonic antigen-expressing free cancer cells in the peritoneal cavity from patients with gastric cancer. ANZ J. Surg. 71, 521–528 (2001). Y4. Yoshioka, S., Fujiwara, Y., Sugita, Y., Okada, Y., Yano, M., Tamura, S., et al., Realtime rapid reverse transcriptase–polymerase chain reaction for intraoperative diagnosis of lymph node micrometastasis: Clinical application for cervical lymph node dissection in esophageal cancers. Surgery 132, 34–40 (2002).
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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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P4. Piguet, P. F., Vesin, C., Da Laperousaz, C., and Rochat, A., Role of plasminogen activators and urokinase receptor in platelet kinetics. Hematol. J. 1, 199–205 (2000). P5. Plow, E. F., Ploplis, V. A., Carmeliet, P., and Collen, D., Plasminogen and cell migration in vivo. Fibrinol. Proteol. 13, 49–53 (1999). R1. Rakic, J. M., Lambert, V., Munaut, C., et al., Mice without uPA, tPA, or plasminogen genes are resistant to experimental choroidal neovascularization. Invest. Ophthalmol. Vis. Sci. 44, 1732–1739 (2003). R2. Rakoczi, I., Wiman, B., and Collen, D., On the biological significance of the specific interaction between fibrin, plasminogen, and antiplasmin. Biochim. Biophys. Acta 540, 295–300 (1978). R3. Ramsby, M. L., Donshik, P. C., and Makowski, G. S., Ligneous conjunctivitis: Biochemical evidence for hypofibrinolysis. Inflammation 24, 45–71 (2000). R4. Ramsby, M. L., and Kreutzer, D. L., Fibrin induction of tissue plasminogen activator expression in corneal endothelial cells in vitro. Invest. Ophthalmol. Vis. Sci. 34, 3207–3219 (1993). R5. Ramsby, M. L., and Makowski, G. S., Overlay assay. In ‘‘The Encyclopedia of Molecular Biology’’ (T. E. Creighton and L. Hood, eds.), Vol. 3, pp. 1734–1736. John Wiley, New York, 1999. R6. Ranby, M., Sundell, B., and Nilsson, T. K., Blood collection in strong acidic citrate anticoagulant used in a study of dietary influence on basal tPA activity. Thromb. Haemost 62, 917–922 (1989). R7. Redmond, E. M., Cullen, J. P., Cahill, P. A., et al., Endothelial cells inhibit flow-induced smooth muscle cell migration: Role of plasminogen activator inhibitor-1. Circulation 103, 597–603 (2001). R8. Regoeczi, E., Occlusion of plasma proteins by human fibrin: Studies using trace-labelled proteins. Br. J. Haematol. 14, 279–290 (1968). R9. Reilly, J. M., Sicard, G. A., and Lucore, C. L., Abnormal expression of plasminogen activators in aortic aneurismal and occlusive disease. J. Vasc. Surg. 19, 865–872 (1994). R10. Risau, W., Mechanisms of angiogenesis. Nature 386, 671–674 (1997). R11. Robbie, L. A., Dummer, S., Booth, N. A., Adey, G. D., and Bennett, B., Plasminogen activator inhibitor 2 and urokinase-type plasminogen activator in plasma and leucocytes in patient with severe sepsis. Br. J. Haematol. 109, 342–348 (2000). R12. Rosen, S., Wejkum, L., Billing-Claeson, S., et al., Evaluation of a bioimmunoassay for t-PA activity and its relation to PAI-1 activity and antigen levels. Fibrinol. Proteol. 12, 340–346 (1999). R13. Rossignol, P., Ho-Tin-Noe, B., Vranckx, R., et al., Protease-nexin-1 inhibits plasminogen activation-induced apoptosis of adherent cells. J. Biol. Chem. 279, 10346–10356 (2004). S1. Sakharov, D. V., Nagelkerke, J. F., and Rijken, D. C., Rearrangements of the fibrin network and spatial distribution of fibrinolytic components during plasma clot lysis. J. Biol. Chem. 271, 2133–2138 (1996). S2. Samara, G. J., SchaVner, A. D., Eisenstat, J., and Nguyen, H. L., The eVects of the plasminogen pathway on scar tissue formation. Laryngoscope 114, 46–49 (2004). S3. Sappino, A. P., Wohlwend, A., Huarte, J., Belin, D., and Vassalli, J. D., The PAplasmin system during murine embryogenesis. Ann. N. Acad. Sci. 667, 41–47 (1992). S4. Seeds, N. W., Basham, M. E., and Ferguson, J. E., Absence of tissue plasminogen activator gene or activity impairs mouse cerebellar motor learning. J. Neurosci. 23, 7368–7375 (2003). S5. Shak, P. K., Inflammation, metalloproteinases, and increased proteolysis: An emerging pathophysiologic paradigm in aortic aneurysm. Circulation 96, 2115–2117 (1997).
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S6. Shimizu, T., Sato, K., Suzuki, T., Tachibana, K., and Takeda, K., Induction of plasminogen activator inhibitor-2 is associated with suppression of invasive activity in tPA-mediated diVerentiation of human prostatic cancer cells. Biochem. Biophys. Res. Comm. 309, 267–271 (2003). S7. Siconolfi, L. B., and Seeds, N. W., Induction of the plasminogen system accompanies peripheral nerve regeneration after sciatic nerve crush. J. Neurosci. 21, 4336–4347 (2001). S8. Siconolfi, L. B., and Seeds, N. W., Mice lacking tPA, uPA, or plasminogen genes showed delayed functional recovery after sciatic nerve crush. J. Neurosci. 21, 4348–4355 (2001). S9. Siren, V., Kauhanen, P., Carpen, O., et al., Urokinase, tissue-type plasminogen activator and plasminogen activator inhibitor-1 expression in severely stenosed and occluded vein grafts with thrombosis. Blood Coag. Fibrinol. 14, 369–377 (2003). S10. Solberg, H., Rinkenberger, J., Dano, K., Werb, Z., and Lund, L. R., A functional overlap of plasminogen and MMPs regulates vascularization during placental development. Development 130, 4439–4450 (2003). S11. Sporn, L. A., Bunce, L. A., and Francis, C. W., Cell proliferation on fibrin: Modulation by fibrinopeptide cleavage. Blood 86, 1802–1810 (1995). S12. Stefansson, S., and Lawrence, D. A., The serpin PAI-1 inhibits cell migration by blocking integrin v 3 binding to vitronectin. Nature 383, 441–443 (1996). S13. Stefansson, S., McMahon, G. A., Petitclerc, E., and Lawrence, D. A., Plasminogen activator inhibitor-1 in tumor growth, angiogenesis, and vascular remodeling. Curr. Pharm. Des. 9, 1545–1564 (2003). S14. Stibbe, J., Kluft, C., Brommer, E., Gomes, M., de Jong, D., and Nauta, J., Enhanced fibrinolytic activity during cardiopulmonary bypass in open-heart surgery in man is caused by extrinsic (tissue-type) plasminogen activator. Eur. J. Clin. Invest. 14, 375–382 (1984). S15. Sweep, C. G. J., Geurts-Moespot, J., Grebenschikov, N., et al., External quality assessment of trans-european multicenter antigen determination (enzyme-linked immunosorbent assay) or urokinase plasminogen activator (uPA) and its type-1 inhibitor (PAI-1) in human breast cancer extracts. Br. J. Cancer 78, 1434–1441 (1998). T1. Trousseau, A., and Cormack, J. R., Lectures on Clinical Medicine, Delivered at the Hotel-Dieu, Paris (5th Ed) pp. 281–295. United Kingdom, New Sydenham Society, London (1872). V1. Vassalli, J. D., Sappino, A. P., and Belin, D., The plasminogen activator=plasmin system. J. Clin. Invest. 88, 1067–1072 (1991). V2. Veklich, Y., Francis, C. W., White, J., and Weisel, J. W., Structural studies of fibrinolysis by electron microscopy. Blood 92, 4721–4729 (1998). W1. Wagner, O. F., de Vries, C., Hohmann, C., Veerman, H., and Pannekoek, H., Interaction between plasminogen activator inhibitor-1 (PAI-1) bound to fibrin and either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA): Binding of t-PA=PAI-1 complexes to fibrin mediated by both the finger and kringle-2 domain of t-PA. J. Clin. Invest. 84, 647–655 (1989). W2. Watanabe, M., Yano, W., Kondo, S., et al., Up-regulation of urokinase-type plasminogen activator in corneal epithelial cells induced by wounding. Invest. Ophthalmol. Vis. Sci. 44, 3332–3338 (2003). W3. Weckroth, M., Vaheri, A., Myohanen, H., Tukiainen, E., and Siren, V., DiVerential eVects of acute and chronic wound fluids on urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, and tissue-type plasminogen activator
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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.
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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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ADVANCES IN CLINICAL CHEMISTRY, VOL.
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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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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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).
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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 <1). Commercial OP have a ratio of <1 and most have a ratio <0.1. Therefore, among NTE inhibitors, cholinergic toxicity is the factor limiting OPIDN development. Some OP show OPIDN in relatively low doses, e.g., Bromophos, Mevinphos, Phenthion, Parathionmethyl. Another example of OP inducing OPIDN is TOCP mipafox or DFP. Nerve agents such as sarin, soman, and VX show either no potential for inducing OPIDN (VX) in any species (M24) or causing OPIDN in animals when they are treated against the lethal eVect. Sarin at extraordinarily high doses (in excess of 60 LD50) caused delayed neuropathy in hens protected by treatments with atropine and pralidoxime (M24). Thus, for sarin, the ratio LD50/neurotoxic is more than 60. 2.5. LONG-TERM EFFECTS Nerve agent-induced eVects are usually manifested immediately after highlevel or intermediate-level exposure (M2). Nevertheless, there are numerous studies in both human beings and animals showing that survivors of acute poisoning by nerve agents at high-level and possibly intermediatelevel exposure can experience subtle but significant long-term neurological and neuropsychological outcomes that are detectable months, even years, after the recovery from acute poisoning (B37).
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The ongoing debate on a possible relationship between so-called Gulf War Syndrome and accidental exposure to traces of nerve agents in the aftermath of the first Gulf War have made clear that knowledge on the acute and delayed eVects of trace exposure to nerve agents is scarce. From the data published in literature (B37, K17, M2), it can be concluded that exposure to nerve agents may well lead to delayed and persistent adverse eVects, mostly neurophysiological, Indications were obtained for long-term eVects of low-level inhalation exposure of rats to sarin. Some changes in behavioral characteristics, such as decrease in activity and mobility, persisted for 3 to 12 months (K17). Thus, nerve agent poisoning is associated with long-term CNS changes both in experimental animals and in humans, appearing especially following exposure to low concentrations of nerve agents. The book edited by Somani and Romano is of importance in this field (see ref. B31). Though some special changes following diVerent OP pesticide exposure were described (B2), the data available for nerve agents (especially sarin) do not support a hypothesis on carcinogenic, mutagenic, and teratogenic properties of nerve agents.
3. Cholinesterase Inhibitors and Other Factors Influencing the Activity 3.1. CHOLINESTERASES Cholinesterases belong to the group of hydrolases splitting the ester bond, i.e., the esterase subgroup catalyzing the hydrolysis of esters to alcohol and acid. According to the aYnity to substrates and inhibitors, they can be divided into A, B, and C esterases. A-esterases, aromatic esterases, and arylesterases (EC 3.1.1.2) hydrolyze aromatic esters more rapidly than aliphatic esters. They do not split choline esters; they are resistant to OP and eserine. B-esterases (aliphatic esterases, aliesterases, carboxylesterases, CaE) (EC 3.1.1.1) that hydrolyze aliphatic esters more rapidly than aromatic esters, nonhydrolyzing choline esters, and are sensitive to OP and resistant to eserine represent the next group. C-esterases, cholinesterases that hydrolyze choline esters more rapidly than other esters and are more sensitive to OP and eserine than A- and B-esterases, are the most important enzymes from this group. According to the aYnity to natural substrates—choline esters—cholinesterases are divided into AChE and BuChE. AChE, specific or true cholinesterase, the ‘‘e’’ type of cholinesterase (EC 3.1.1.7) has a higher aYnity to acetylcholine than to butyrylcholine, and splits acetyl-beta methylcholine.
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It is inhibited by an excess of substrate. High AChE activity was observed in erythrocytes, the brain, the electric organ of Electrophorus Electricus, and the neuromuscular junction. However, AChE activity was observed in many tissues, including plants, e.g., onion (H1). AChE is composed from subunits. BuChE, pseudocholinesterase, nonspecific cholinesterase, the ‘‘s’’-type of cholinesterase (EC 3.1.1.8) is present in the plasma (serum), pancreas, and liver (where it is synthesized). It is a ubiquitous enzyme present not only in some human and animal tissues but also in many plants and microorganisms. BuChE does not hydrolyze acetyl-beta-methylcholine and has a higher aYnity to butyryl- and propionyl choline in comparison with acetylcholine. Substrate inhibition was not observed. There exist BuChE isoenzymes that are genetically determined (Table 2). Depending on the genetic material, some individuals have a very low or no BuChE activity. A qualitative diVerence between the BuChE of suxamethonium-sensitive individuals and that of other patients was demonstrated by Whittaker (W4). The two types of enzyme hydrolyze the same substrate at diVerent rates and show distinct inhibition with varying concentrations of a suitable inhibitor such as dibucain. These findings were the basis for the hypothesis that the biosynthesis of BuChE is controlled by two allelic genes, Eu1 and Ea1. Individuals with the combination Eu1Eu1 are homozygotes with normal BuChE activity; a combination of Ea1Eu1 (heterozygotes) and Ea1Ea1 (homozygotes) resulted in diminished BuChE activity. The presence of a silent gene (Es1) was also proposed and a fourth gene controlling biosynthesis of BuChE (fluoride resistant, Ef1) was recognized; the hypothesis was, in general, established by family studies. These people with genetically diminished BuChE activity may be at higher risk when exposed to pesticides or suxamethonium (L13). The plasma of individuals with normal BuChE activity hydrolyzes succinylcholine or binds a part of OP pesticide and, therefore, the real dose of these compounds penetrating to the target sites is diminished. In the case of absence of BuChE, the dose administered is not decreased and, therefore, relative overdosage occurred. AChE and BuChE diVer in their enzymatic properties and physiological function (M7, D4). However, there are other types of cholinesterases like benzoylcholinesterase and propionylcholinesterase. AChE splits neuromediator acetylcholine at the cholinergic synapses. It was also observed in erythrocytes but its function here is not yet known in detail. Like the function of BuChE activity in plasma, though, there is evidence that BuChE plays an important role in cholinergic neurotransmission and could be involved in other nervous system functions, in neurological diseases, and in nonspecific detoxification processes (D4). A more detailed knowledge of cholinesterases occurred with the description of the molecular structure of AChE (S26) and it is described in greater
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TABLE 2 DIFFERENCES IN PROPERTIES OF ACHE AND BUCHE Trivial name
Acetylcholinesterase
Other names
specific, true, ‘‘e’’ type cholinesterase
Systemic name
acetylcholineacetylhydrolase EC 3.1.1.7. electric, organ, brain, erythrocytes acetylcholine yes low
Number of the Enzyme Codex Main source Optimal substrate Splitting acetyl- -methylcholine Species diVerences Inhibition by substrate Quaternary ammonium salts Iso-OMPA Phenothiazines Tacrine DMC Ni, Zn Binding Molecular forms Activation Mg, Mn Function
Butyrylcholinesterase cholinesterase, pseudocholinesterase, ‘‘s’’ type cholinesterase acetylcholine-acylhydrolase EC 3.1.1.8. serum, plasma butyrylcholine no high
yes
no
þþþ
þ
þ þþþ þ þþþ þþ complex with lipoprotein subunits Mg > Mn splitting neuromediator acetylcholine
þþþ þ þþþ þ þ glycoprotein; containing sialic acid genetically determined Mg < Mn unknown
detail by many other authors (G2, H2). Like other serine hydrolases, AChE contains a catalytic triad called the esteratic site (Ser200-His440-Glu327) at the bottom of a deep and narrow cavity, known as the ‘‘aromatic gorge,’’ lined with 14 aromatic residues. Acetylcholine must pass down into this gorge and bind to the active site within. Ligands binding to the esteratic site are called acylating ligands (OP, carbamates, alkylsulphonates). Non-acylating ligands are bound to sites other than the esteratic site (anionic sites) (W2) and comprise a large group of chemicals including tetralkylammonium, coumarine, tacrine, and gallantamine. These anionic sites can be responsible (in complexity with the enzyme structure) for AChE allostericity (P1, S26, S30). A schematic structure of AChE is shown in Fig. 7. The crystal structure of human BuChE as a key step for engineering of catalytic scavengers against OP poisoning has also been described (M4).
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FIG. 7. Schematic structure of the active surface of AChE including diVerent binding sites and diVerentiation between acylating and non-acylating inhibitors.
In addition to the catalytic center subsites, AChE possesses one or more additional binding sites for acetylcholine and other quaternary ligands. Such peripheral anionic binding sites are at the lip of this gorge. In BuChE, Trp279, an important component of the peripheral binding site in AChE, is missing. This site is believed to be responsible for substrate inhibition (R1), which is one of the features that distinguishes AChE from BuChE. The comprehensive monograph by Silver (S20) is of continuing interest in the reviews by Massoulie´(M5, M6, M7). 3.2. METHODS FOR DETERMINATION Determination of cholinesterase activity is based on many principles. In general, an enzyme is added to the buVered mixture and the enzymatic reaction is initiated by adding the substrate. DiVerent parts of the reaction mixture are determined (continually or discontinually), i.e., unhydrolyzed substrate or reaction products, both directly or indirectly (A15, H13, W7). The conditions must be chosen very carefully because of diVerent factors influencing the activity (R6). According to the procedure and laboratory instrumentation, the most common methods of cholinesterase determination are as follows: Electrometrical (e.g., M19), titration (e.g., N5), manometric (e.g., W7), colorimetric detection of the unhydrolyzed substrate (e.g., H11), measurement by the change of pH using an indicator (W6), spectrophotometric (e.g.,
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S19, V9, W11), fluorimetric (e.g., S4, K45), radiometric (e.g., I2), calorimetric (K29), polarographic (F4), enzymatic (A1, I2), and others such as near-infrared spectroscopy (D20). These methods are also suitable for the detection of cholinesterase inhibitors using biosensors (B35, C9, D12) or immunochemical assay for detection of chemical warfare agents (L7). A very sensitive and commonly used method for cholinesterase determination was described by Ellman et al. (E2), based on hydrolysis of the thiocholine substrates acetyl- and butyrylthiocholine or others. After enzymatic hydrolysis, the relevant acid and thiocholine are released and thiocholine by its SH-group is detected using 5,50 dithiobis-2 nitrobenzoic acid forming 5-mercapto-2-nitrobenzoate anion determined spectrophotometrically at 412 nm. Sometimes, this method is used with specific inhibitors and there are many modifications described in the literature. This method is in good correlation with other methods. It is suYciently specific and sensitive and it is used for diVerent purposes in many laboratories around the world. Expression of the activity varies greatly, usually as moles of substrate hydrolyzed per min (time) per ml of material examined (e.g., plasma, serum) or per mg of weight tissue (wet, dry, mg of nitrogen, etc.). From these values, the expression of the activity in Units can be derived (it is the quantity of enzyme catalyzing mol of substrate per min at standard conditions). In the clinical laboratory, the activity can be also expressed as catal per liter, i.e., 1 mol of substrate hydrolyzed per sec per liter or kg (cat/l, kg), which is hydrolysis of 1 mol of substrate hydrolyzed per sec per 1 or kg (mol. sec 1 .l 1 or kg 1). There are many publications dealing with the review and modifications of cholinesterase determination. One of the last methodical works improving the Ellman’s method (E2), including a description of the methods, is a paper published by Worek et al. (W11). 3.3. INHIBITORS AND OTHER FACTORS INFLUENCING THE ACTIVITY Inhibition of enzymes can basically be divided into reversible or irreversible. According to inhibition kinetics, it can be divided into three types— competitive, non competitive, and allosteric. Competitive inhibition can be characterized by binding of the inhibitor to the active site of the enzyme (they are structurally similar to substrate) and inhibition can be reversed by substrate access (reversible inhibition). The reaction rate is dependent on the substrate and inhibitor concentrations and their aYnity to the enzyme. Noncompetitive inhibition cannot be reversed by substrate access and the inhibitor reacts with other parts of the enzyme rather than the active site, and it is not structurally similar to the substrate. The enzymatic reaction can be irreversible when the aYnity of the inhibitor to the enzyme is relatively high. Allosteric ligands (inhibitors or activators) are bound to quite another
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part of the enzyme (the allosteric site) and cause a change in conformation of the enzyme molecule. This is a very fine process of enzymatic activity regulation. OP are apparently irreversible inhibitors of cholinesterases (AChE and BuChE) and the reaction can be expressed by a simple scheme (Fig. 8). The principle of this reaction is phosphorylation (phosphonylation) of the serine group on the catalytic triade (active center) of AChE. The rate of spontaneous dephosphorylation is very low and it can be omitted in most cases. However, it can be improved/increased using cholinesterase reactivators (oximes) able to reactivate OP/nerve agent-inhibited AChE. These compounds with an ionized oxime group will break the bond between AChE and OP and restore enzyme activity (reactivation) by the nucleophilic attack on phosphorylated or phosphonylated serine at the active center of the AChE molecule and liberate the free enzyme (K7). This is the limiting factor for therapy with reactivators. The rest of the OP/nerve agent forms a complex with the reactivator (more toxic but less stable), hydrolyzing practically immediately and not having high importance for the course of intoxication (K23). OP inhibits AChE via phosphorylation of the esteratic site. The eYcacy of oxime reactivation is dependent on both oxime and the conjugated phosphonate structure (T3). Simultaneously, the microenvironment of the gorge plays a significant role in determining the selectivity of the substrate and inhibitors for cholinesterases (S8). Depending on the structure of the inhibitor, inhibited AChE is dealkylated (aged) and the
FIG. 8. Schematic representation of OP inhibitor (P) reaction with a living group (p1 – H) and AChE (E). EP – intermediate comples, Kd – dissociation constant, EP1 – phosphorylated complex, EP2 – dealkylated enzyme, ka – bimolecular rate constant, kþ1, k 1, kþ2, kþ3, kþ4 – rate constants. Spontaneous recovery of the enzyme activity is possible forming products (p2-OH) but this reaction rate is very low for OP (modified from A4, A5).
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FIG. 9. Simplified schematic representation of aging (dealkylation) of soman-inhibited AChE (Enzyme). The aging reaction is driven by the interaction of amino acid residues in the active site gorge with the phosphyl moiety but the products of the reaction are phosphylated enzyme and pinacolylalcohol (in case of soman), isopropylalcohol (in case of sarin), and HCN (in case of tabun), respectively. The complex of the enzyme bound to soman on the left side represents EP1 (reactivatable complex), on the right side EP2 (unreactivatable, delkylated, aged complex).
complex formed is resistant to the reactivation eVect (EP1 ! EP2). The molecular mechanism is explained by the splitting of the complex forming the alcohol and unreactivatable enzyme (Fig. 9). This reaction, called aging or dealkylation, is very fast for soman-inhibited AChE (the half-life is about 10 min) and less expressed for sarin (the half-life is about 10 hours); for VX-inhibited AChE, this reaction was not observed within 24 hours (B3, B11, F5, T1). This is one of the reasons for diYcult therapeutic interventions of soman intoxication (B17, W8). Peripheral site ligands may have selective eVects on AChE phosphorylation (R7). The importance of the orientation not only of the OP molecule but also the reactivator has been described by Luo et al. (L18). The reversibility of inhibition is very important for carbamates. They react with AChE in the same manner, forming a carbamylated (inactive) enzyme, preventing phosphylation of the carbamylated portion of the enzyme; however, spontaneous decarbamylation occurs very quickly and the released enzyme serves as a normal enzyme source, provided that no inhibitory concentration of an AChE inhibitor is present anymore. Therefore, reversible inhibitors (mostly carbamates) are used as prophylactics against OP/nerve agents intoxications. The influencing of BuChE activity by gamma-irradiation, stress, gravidity, some neurological and psychiatric disorders, hormones, and medical drugs has been demonstrated (B2, B4, B38). The elevation of BuChE activity is not so frequent; an increase in children with nephritic syndrome has been observed (W4); an elevated ratio of BuChE/LDL cholesterol indicates an increase in the risk of cardiovascular diseases (K46, N4). The involvement of BuChE with the fat (cholesterol) metabolism has been suggested by van Lith et al. (V6, V7). The relationship between BuChE activity and experimentally induced diabetes mellitus in rats was also mentioned (A10). Determination of AChE activity is not so widely used in clinical laboratories. A decrease in red blood cell AChE activity in pernicious anemia has
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been demonstrated; diminished erythrocyte AChE activity is typical for paroxysmal nocturnal hemoglobinemia and ABO incompatibility (R2). AChE activity in the erythrocyte membrane can be considered as an indicator of erythrocyte membrane integrity. Increased AChE activity in rectal biopsy is of great diagnostic significance in Hirschsprung’s disease, especially in the presence of its atypical molecular form (B18, R2). There are other papers demonstrating increased AChE activity in the amniotic fluid during pathologic development of the neural tube (B34). AChE activity in the erythrocytes and cerebrospinal fluid is also diminished in some endogenous depressions and other psychiatric disorders; however, the results presented are not quite clear at present (for a review, see, for example, B2, R2, S21). On the other hand, influencing of the cholinergic nervous system is one of the most important pathological changes in Alzheimer’s disease (A13). A lack of the cholinergic mediator, acetylcholine, was observed (P8). This has led to attempts to correct cholinergic deficiency at various levels of cholinergic functioning: inhibitors of cholinesterases like physostigmine were used; however, physostigmine was not found to be an ideal drug for clinical use because of its short half-life, side eVects, etc. (F7). Clinical study results showed prospective results with the acridine inhibitor of cholinesterase Tacrin (1,2,3,4-tetrahydro-9-aminoacridine) (S28); its 7-methoxyderivative (7-MEOTA) was described as a compound of low toxicity and good therapeutic eVect in experimental intoxication with anticholinergics (F7). Biochemical studies dealing with its eVect on cholinesterases in vivo showed that 7-MEOTA inhibited BuChE in the liver and AChE in brain parts, with the highest sensitivity in the frontal cortex (B22). Current approaches to the treatment of cognitive and behavioral symptoms of Alzheimer’s disease emphasize the use of other cholinesterase inhibitors. Among them, Donepezil has been introduced in clinical practice (D3, W5, B41). There are many cholinesterase inhibitors diminishing both AChE and BuChE activities to a comparable extent. However, there are a number of important exceptions: the selectivity of some OP and carbamates for BuChE has been described by Aldridge (A4). Carbamates belong to a group of insecticides having a large variation in their eVectiveness. They are biologically active because of their structural complementarity to the active surface of AChE and their consequent reaction as substrates with very low turnover numbers (A4, B2). Some carbamates inhibit selectively either AChE or BuChE (B11, B22). The toxicity of carbamates is dependent on their ability to carbamylate AChE in diVerent tissues and on other factors such as distribution, detoxification, and metabolization. 3-Diethylaminophenyl-N-methylcarbamate methiodide ranks among the
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highly toxic carbamates. The in vivo selectivity for both the cholinesterases was not so expressed as was demonstrated in experiments in vitro (B11). Metal cations are an interesting group of compounds modifying cholinesterase activity. Their eVect was studied on relatively simple cholinesterase models. It was demonstrated previously that Hg ions especially diminish AChE activity in low concentrations (B11). The factors influencing cholinesterases are not limited to chemicals only. AChE activity is connected with cholinergic activity in the brain. Using determination of AChE, combined with defined lesions of the diVerent parts of the brain, it was possible to demonstrate cholinergic projections in the central nervous system (B11, H8). AChE shows a polymorphism of quaternary structures, of similar catalytic activity but diVering in their properties. Catalytic subunits, which may vary in glycosylation, can oligomerize into dimers or tetramers, giving rise to the globular (G) forms: G1, G2, and G4. These forms can further be divided depending on their amphiphilicity. Attachment of a collagen-like tail to one, two, or three catalytic tetramers gives the A4, A8, and A12 asymmetric forms, which bind to basal lamina. Tetramers are formed by electrostatic and hydrophobic interaction between two disulphide-bonded dimers (M7). Multiple molecular forms (AChE and BuChE) are also influenced by many factors (B35, R3). The function of these forms is not known at present. There are only scarce data describing the changes of AChE molecular forms following intoxication with highly toxic OP (B13, L6). Some experiments were performed with relatively less toxic OP (M18, B22, B23). From the group of highly toxic OP compounds, sarin, soman, and VX were found to be the most eVective (B13). Molecular forms of AChE showed diVerent sensitivity to inhibitors in vitro (B5, B11, O1, U1) and in vivo (B5, B13, B39, L6). Following DFP (M18, T8) and highly toxic OP (B13, L6), the form with high molecular weight was the most sensitive. Intoxication with Parathion and Neguvon (less toxic OPs) caused medium inhibition of some forms of AChE and BuChE (B13, B39). Subcellular localization of AChE suggested that 2 to 4 AChE forms are present in nerve ending particles and microsomal fractions, while in the mitochondrial fraction, only one was detected (B5). The microsomal form absent in the mitochondrial fraction is most sensitive to OP in vivo. This form of AChE has the lowest Km value (B26) and the greatest decrease in this component after deaVerentation (a selective interruption of aVerent nerve tracts) was also observed (B9). These results suggested that this form of AChE would be very important for normal cholinergic nerve transmission. This fact raises the question of the existence of the forms under physiological conditions. Using thermal denaturation, it was demonstrated that they are not artifacts formed during homogenization or other treatment of the brain
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tissue (B8). The functional role of AChE molecular forms was studied in the rat diaphragm. Apparently, G4 and A12 are the functional AChE forms in the rat diaphragm (B42). The overall data show that the catalytic activity of AChE molecular forms is diVerent and that their inhibition by various inhibitors may be heterogeneous. This heterogeneity was demonstrated for AChE phosphorylating inhibitors as well as for inhibitors with diVerent binding sites for the enzyme. Results with the reversible cholinesterase inhibitor Tacrin and 7-MEOTA fit well with previous findings indicating a greater sensitivity of slowly migrating molecular forms separated by polyacrylamide gel electrophoresis (B11). In fact, it has been demonstrated that slowly migrating forms of cortical AChE correspond to G4 forms separated by sedimentation analysis (V8). The 7-MEOTA data are diVerent from those obtained for DFP and paraoxon but show similar IC50 values for the G4 and G1 forms (V8). These findings have been confirmed for membrane-bound AChE (O1). This is not surprising since the interaction of OP compounds (and physostigmine) with the active site of the enzymatic molecule is diVerent from that for acridine compounds. In fact, OP compounds and carbamates inhibit AChE by phosphorylation/carbamylation of the esteratic serine in the catalytic site. On the other hand, acridine derivatives bind to the hydrophobic area close to the active site of AChE, simultaneously aVecting its catalytic center via an allosteric mechanism (F6, P1). In the case of brain AChE, as has been pointed out (V8), G4 and G1 forms represent distinct pools in the cell, the former being mainly associated with membranes, with its catalytic site exposed to the extracellular space, and the latter confined to the intracellular compartment. Following intoxication with the nerve agents mentioned, the highest sensitivity for the high molecular AChE form was observed (B13). Determination of the whole AChE activity is partly misleading because of the diVerent distribution of AChE molecular forms in the sample. Following determination of the whole activity, a ‘‘mean’’ activity containing the activities of the forms is determined. It can be concluded that in studies requiring high sensitivity (e.g., the studies of antidotal action), AChE molecular forms would be of choice for more detailed information about the functional stage of AChE—an important marker of cholinergic nerve transmission.
4. Diagnosis The eVect of OP/nerve agents is characterized by their interference with cholinergic nerve transmission via inhibition of AChE and BuChE. The cholinergic crisis (accumulation of neuromediator acetylcholine) is
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accompanied by other changes—disturbed membrane permeability, the stressogenic eVect, inhibition of enzymes other than cholinesterases, changes in the cyclic nucleotide levels, oxygen saturation, etc. There are also morphologic and immunologic changes observed soon after the intoxication. Diagnosis of OP poisoning is based on clinical signs and cholinesterase determination using the most suitable material for laboratory diagnostics — the blood. However, there are other changes in the biochemical parameters during the intoxication/exposure to OP, depending on the type of such compound (mutagenicity, delayed neurotoxicity, etc.). 4.1. BASIC METHODS Cholinesterase activity is fundamentally important for the diagnosis of intoxication with cholinesterase inhibitors, including OP and carbamates (B2, C8, M1, M2). On the other hand, the activity depends on many other factors and, therefore, cholinesterase determination is of diagnostic importance in diVerent pathological states, not only intoxications (B2, B11, B35, R2). The activity of these enzymes (AChE and BuChE) is influenced by sex, age, nutrition, hormonal factors, irradiation, etc. (B38, D4, H7, H8, S21, S24, W4). The variation of BuChE activity is greater than that of AChE (B2, B38, K46) and it is genetically determined (B38, W4). Clinical monitoring of intoxication and determination of cholinesterases in the blood are basic methods for the diagnosis and diVerential diagnosis of the intoxication with OP/nerve agents. The criteria elaborated by Namba (N2, N3) were reevaluated later and revised (B29). It is necessary to examine the whole picture of intoxication, i.e., not only biochemical examinations but clinical signs allowing more precise assessment and the prognosis of the intoxication. As for clinical biochemistry, it is necessary to have biological samples, mostly blood and urine. The OP/nerve agents in the urine can be detected; however, their degradation is fast and therefore the time where detection in the urine is possible is short. The detection of metabolites is also possible but limited for such OP metabolizing to the specific products, e.g., para-nitrophenol in the paraoxon poisoning (R10). Therefore, the blood remains the main source of biological material for biochemical examination. Serum biochemical and hematological parameters were examined in rhesus monkeys following acute poisoning with cyclosarin. Significant increase in creatine kinase, lactate dehydrogenase, transaminases (AST, ALT), and potassium ion associated with damage to striated muscle and metabolic acidosis occurred in the treated group (atropine and oxime) two days after exposure. Total protein, albumin, red blood cell count, hemoglobine concentration, and hematocrit were decreased in the treated group at 7 days (Y3).
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The determination of cholinesterases in the blood is the basic method for diagnosis and therapy monitoring for OP poisoning, though some doubt exists, some preferring the clinical signs of poisoning as a leading tool for OP poisoning diagnosis and monitoring (B29). The determination of AChE and BuChE activity in whole blood is possible. The decrease in these activities is a good marker but the diagnostic validity is limited to the statement that some factors causing a decrease in blood cholinesterases are present. In connection with the anamnestic data (exposure to OP), this is important information. The determination of the red blood cell AChE or plasma BuChE is more informative. There are some discussions dealing with AChE and BuChE activity—which is more important for the diagnosis? In general, AChE activity in the red blood cell can be considered to be more important for diagnosis with the nerve agents than plasma BuChE activity. However, there are some discussions dealing with the validity of the BuChE determination. This enzyme was described as a not-very-good marker of OP poisoning and its determination was proposed for exclusion from the recommended biochemical procedures (B28, D21, M21, W15). The temporal profile of BuChE was studied in a cohort of 25 OP-poisoned patients to examine their relationship to the development of intermediate syndrome. The study suggests that BuChE reflects the clinical course of poisoning and that intermediate syndrome may be associated with a persistent BuChE inhibition (K21). Israeli authors also described a direct correlation between the degree of BuChE inhibition levels and the severity of intoxication with OP pesticides (W3). According to Aygun et al. (A16), in the acute phase of OP poisoning, low level of AChE supports the diagnosis of OP poisoning but does not show a significant relationship to the severity of poisoning. The preference of AChE determination has been demonstrated by Worek et al. (W9)—BuChE activity determination for diagnosis and therapeutic monitoring provides no reliable information on AChE status. This is in good agreement with our experimental results (B3). The plasma BuChE activity is, in some cases, a good marker for diagnosis of OP poisoning. It is necessary to exclude a diminishing of BuChE activity caused by other reasons. In all cases, the simple cholinesterase determination gives us information about the decrease of enzyme activity without specification of the factor causing this phenomenon. A more detailed specification is possible using special methods not available in all clinical laboratories. For occupational medicine purposes, the determination of cholinesterases in the blood of workers with OP is obligatory. A decrease of the activity below 70% of normal values is an indicator that the worker should not come into contact with OP. However, the normal values varied within the laboratories, depending on the method of determination. We have experience with determination of cholinesterases in the blood (AChE and BuChE) of workers
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with highly toxic nerve agents for more than 50 years. In the 1960s, the method for determination was based on Hestrin’s method (H11), then colorimetric determination according to Winter (W6). During the last 30 years, Ellman’s modified cholinesterase determination has been used. All the methods mentioned correlated well with them. For practical purposes (individual and interindividual variation), determination of individual norm activity was recommended (this approach is better than that of calculating the decrease from an average value) as well as separate determination of both cholinesterases, the red blood cell AChE, and plasma BuChE. The activity determined in whole human blood corresponds to about 10% of BuChE and 90% of AChE. This is diVerent from rats, where this ratio is 29% of BuChE and 71% of AChE (B12). The erythrocyte AChE activity seems to be more useful for these purposes than BuChE activity in the plasma. In clinical biochemistry, BuChE determination in the plasma or serum is more frequently used than that of AChE in the red blood cells. Except for intoxication with OP or carbamates, a BuChE decrease indicates either a diminishment of the enzyme synthesis or a decrease in the number of production cells in the liver (M3). A special case of diminished BuChE activity is the hereditary aVected presence of atypical variants of BuChE (B38, W4). There are many other factors influencing BuChE activity and the diagnostic importance of diminished BuChE activity is important for the following states—except hereditary decrease of the activity and poisoning with OP/ nerve agents and carbamates—congenital deficiency, liver damage, acute infection, chronic malnutrition, metastasis (especially liver), myocardial infarction, dermatomyositis, intoxication with carbon disulphide or mercury, and obstructive jaundice (B11, B29, K46, M21, W15). There are other biological materials available for special purposes—amniotic fluid, cerebrospinal fluid, and bioptic materials. From these samples, tissue obtained by the rectal biopsy is used most frequently (diagnosis of Hirschsprung’s disease). An elevated AChE activity in the rectal tissue/ homogenate (detected histochemically/biochemically) is one of the good diagnostic markers indicating a need for surgical treatment of Hirschsprung disease and a criterion for diagnosis and management of obstipation (K26). The presence of an unusual AChE band after the electrophoretic separation supports the diagnosis (B18, R2). The same (either AChE elevation or the presence of a new electrophoretic AChE form) in the amniotic fluid can be applied for the diagnosis of malformation of the neural tube development during pregnancy (B34). AChE activity in the cerebrospinal fluid is also changed in some pathological states; however, the diagnostic validity is not high and it can be considered as a complementary examination (K35).
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4.2. OTHER AND SPECIAL METHODS It is necessary to check vital functions (cardiac, ventilation) and other clinical signs and, according to the symptoms, to apply diVerent biochemical examinations and treatment. Diagnostic criteria are mostly based on anamnesis and the state of ventilation (B27, B28, B29). Serum electrolytes (especially potassium), BUN, and creatinine are indicated to assess the degree of volume depletion by the secretory losses. Arterial blood gas, blood pH, glucose, lactate, and pyruvate allow us to assess the degree of hypoxia/ hypercapnia/acidosis in the presence of respiratory distress. The neurological examination involves, for example, recording of the muscle action potential of M. abductor digiti minimi after stimulation of N. ulnaris. A quantitative correlation could be shown between red blood cell AChE activity and paraoxon concentration in the plasma. In these cases, muscle function was severally disturbed when the red blood cell AChE activity was inhibited more than 90% (T5). The direct determinations of the toxic agent (OP or nerve agent) in the circulating system is also possible. However, the parent compound will circulate intact for a short period of time and detection will not be possible for more than hours after exposure. Metabolites circulate for a longer time period and are mostly excreted in urine. A metabolite of sarin (O-isopropyl methylphosphonic acid) could be traced in urine and plasma from victims after the Tokyo subway sarin terrorist attack (N8, N7). For some OP pesticides (parathion, paraoxon), detection of p-nitrophenol in urine is an indicator of exposure (B2). However, the retrospectivity of these methods is limited. Detection using an immunoassay of nerve agents is now in progress. The antibodies against soman may have the appropriate specificity and aYnity for immunodiagnosis of soman exposure (L8, M20). The methods for determination of blood cholinesterases inhibition (AChE and BuChE) do not allow identification of the OP and do not provide reliable evidence for exposure at inhibition levels less than 20%. Moreover, they are less suitable for retrospective detection of exposure due to de novo synthesis of enzymes. A method has been developed which is based on reactivation of phosphylated cholinesterase and carboxylesterase (CaE) by fluoride ions. Treatment of the inhibited enzyme with fluoride ions can inverse the inhibition reaction, yielding a restored enzyme and a phosphofluoridate which is subsequently isolated and quantified by gas chromatography and phosphorus-specific or mass spectrometric detection (D11, P11). Human (and monkey) plasma does not contain CaE but its BuChE concentration is relatively high [70–80 nM (M25, D8)], much higher than the concentration of AChE in blood [ca. 3 nM (H5)]. The plasma of laboratory animals, such as rats and guinea pigs, contains considerable concentrations
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of CaE in addition to cholinesterases. This method allows partial identification of the OP whereas the lifetime of the phosphylated esterase (and, consequently, the retrospectivity of the method) is only limited by spontaneous reactivation, in vivo sequestration, and aging. The rate of the latter process (aging) depends on the structure of the phosphyl moiety bound to the enzyme and on the type of esterase. Phosphylated CaEs generally do not age. Based on this method for retrospective detection of exposure to OP, the exposure of victims of the Tokyo incident to an OP, probably sarin, could be established from analysis of their blood samples (F3, P10, P11). Fluorideinduced reactivation of OP-inhibited AChE is a reliable and retrospective method to establish OP-exposure. It is limited to compounds that regenerated with fluoride ions. A novel and general procedure for diagnosis of exposure to OP, which surpasses the limitations of the fluoride reactivation method, was described (V3). It is based on the rapid isolation of BuChE from the plasma by the aYnity chromatography, digestion with pepsin followed by liquid chromatography, with the mass spectrometric analysis of phosphylated nonapeptides resulting after the digestion of inhibited BuChE with pepsin. The method can be applied for the detection of exposures to various OP pesticides and nerve agents, including soman. This approach is very valuable and represents a new field for the improvement of diagnosis with nerve agents and OP. A comprehensive review of the methods for retrospective detection of exposure to toxic scheduled chemicals has been published by Noort et al. (N6, N8). As was mentioned previously, a decrease in cholinesterase activity is the factor indicating (after the exclusion of other factors) an exposure to OP/nerve agents or other cholinesterase inhibitors. This simple determination does not allow us to make decisions dealing with the antidotal therapy (especially the repeated administration of reactivators) and then have low prognostic validity. Therefore, a new test of the reactivation has been described (B11). The principle of the reactivation test is double determination of the enzyme, the first without and the second one with the presence of a reactivator in the sample. The choice of reactivator is dependent on the availability of the oxime; however, in principle, it is necessary to have in these parallel samples the same concentrations of the reagents. The concentration of the reactivator (usually trimedoxime, but other oximes such obidoxime, pralidoxime, or HI-6 are also possible) must not be higher than the oxime concentration which causes the hydrolysis of the substrate (acetyl- or butyrylthiocholine), i.e., the oxime concentration is lower than 10 3 M because higher concentrations of oximes cause artificial hydrolysis of the substrate (P4). Using this method, reactivation of the whole human blood in vitro inhibited by various nerve agents (VX, sarin, soman) was determined. This reactivation test was used for determination of the reactivatability in
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TABLE 3 REACTIVATION (%) OF HUMAN BLOOD ACHE IN VITRO AND DOG BLOOD ACHE IN VIVO FOLLOWING SARIN, SOMAN, AND VX EXPOSURE (MODIFIED FROM B9, B11) % of reactivation
Compound
(AChE, human blood, in vitro, variation limits for all intervals studied)
(AChE, dog blood, in vivo, means SD, statistical significance p 0.05)
Soman Sarin VX
0–10 15–63 55–85
16.1 6.1 62.7 5.8 87.6 8.8
rats and dogs intoxicated with the same nerve agents (sarin, soman, and VX). The results of the reactivation in vivo in dogs and in vitro in humans are shown in Table 3. From these results, diVerential diagnosis can be derived— in the case of low reactivation (0–10%), soman as the toxic agent is the most probable. A middle reactivation of about 50% indicates sarin intoxication and a high reactivation is typical for VX (B3, B11). The reactivation of AChE in patients poisoned with OP was also determined. Following parathion poisoning, reactivation of erythrocyte AChE was possible up to 7 days after the intoxication but it was not used as a standard method. It is also possible to determine AChE inhibitory activity of the intoxicated patient plasma (T4, T5). An interesting new approach was described by Gopalakrishnakone (G4). The human brain cell lines were exposed to various concentrations of soman for a period of one and two days. A total of 115 and 224 genes involved in signal transduction, metabolism, cell growth, development, apoptosis, and immune response were either up- or down-regulated, respectively. This approach needs to be elaborated in more detail. The delayed neurotoxic eVect can be monitored by the determination of neurotoxic esterase. The determination of this enzyme in the lymphocytes soon after injection of neurotoxicants (15–30 min) permits an assessment of the progress of delayed neurotoxicity (K22). In vitro techniques for the assessment of neurotoxicity have been elaborated by Harry et al. (H3). 4.3. SPECIFICITY AND SENSITIVITY OF DIFFERENT BIOCHEMICAL PARAMETERS The data from biochemical examinations of human intoxications with diVerent OP, including nerve agents, from the literature as well as our data and similar data from animals, were collected (B21). It is not an exhaustive
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sample, however, it allows us to assess the specificity and sensitivity of the parameter determined. Specificity means that the changes are not observed in other intoxication/pathological states; sensitivity means the frequency of these changes in OP poisoning. Fifty human intoxications from the literature, 13 from our database, and hundreds of animal intoxications were analyzed. The following OP-caused intoxications are mentioned: 1. Fenitrothion—O,O-dimethyl O-(3-methyl-4-paranitrophenyl) phosphorothioate, 2. Malathion—O,O-dimethyl S-1,2-bis(ethoxycarbonyl) ethyl phosphorodithioate, 3. Dichlorphos—O,O-dimethyl O-2,2-dichlorovinyl phosphate, 4. Mevinphos—O,O-dimethyl O-2-methoxycarbonyl-1-methylvinyl phosphate, 5. Trichlorphon—O,O-dimethyl 2,2,2-trichloro-1-hydroxyethyl phosphonate, 6. Phosphamidon—O,O-dimethyl O-2-chloro-2-diethylcarbamoyl-1methylvinyl phosphate, 7. Diazinon—O,O-diethyl O-(2-isopropyl-6-methylpyrimidin-4-yl) phosphate, 8. Parathion—O,O-diethyl O-4-nitrophenyl phosphate, 9. Sarin—O-isopropyl methylphosphonofluoridate and 10. Soman—O-pinacolyl methylphosphonofluoridate, respectively. The following biochemical parameters were evaluated: AChE, BuChE, AChE molecular forms, lactate, pO2, pCO2, carboxylesterase (EC 3.1.1.1), alkaline phosphatase (EC 3.1.3.1), aspartate aminotransferase (AST, EC 2.6.1.1), alanine aminotransferase (ALT, EC 2.6.1.2), -glutamyltransferase (-GT, EC 2.3.2.2), lactatedehydrogenase (LDH, EC 1.1.27), number of leukocytes (No leuco), corticosterone (CS), cyclic adenosine and guanosine monophosphate (cAMP, cGMP), OP metabolites (in case of parathion and fenitrothion), phosphonyl moiety attached to the enzyme in case of sarin (phosph. moiety), number of erythrocytes (No RBC), and tyrosine aminotransferase (TAT, EC 2.6.1.5), respectively. The average age of all humans was 37.5 (male) and 35.8 (female) years, the ages varied from 17 to 65 (male) and 18 to 63 (female). The number of males was 35 and females 28. The following number of cases were studied: For OP 1, 12 cases (male/female—5/7); for OP 2, 10 cases (6/4); for OP 3, 3 cases (2/1); for OP 4, 7 cases (5/2); for OP 5, 3 cases (3/4); for OP 6, 8 cases (5/3); for OP 7, 7 cases (4/3); for OP 8, 5 cases (3/2); for OP 9, 2 cases (1/1); and for OP 10, 1 case (0/1).
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According to the severity of poisoning, there were 13/9 characterized as mild, 10/7 as medium, 7/8 as severe, and 5/4 died (the last three groups were used for registration of the intoxication developed). 7/6 were registered from the Czech Republic. Nerve agents represented 4.76% of the total number of intoxications. The complete biochemical spectrum was examined in 4/4 patients, incomplete biochemical parameters were determined in 9/9 patients, and cholinesterase activity was available in only 22/15 patients. The ratio of the number of human/animal was 63/approximately 300. The following biochemical parameters were evaluated and the specificity and sensitivity to these parameters attributed. They are summarized in Table 4. It appears from these data that OP poisoning is very complex and that there exist many biochemical changes to be registered. Though the TABLE 4 SPECIFICITY AND SENSITIVITY OF DIFFERENT BIOCHEMICAL PARAMETERS IN OP/NERVE AGENT POISONING (MODIFIED FROM B21) Specificity/sensitivity (%) Parameter a
AChE forms lactate pCO2 pCO2 AChE BuChE esterasesa APa AST ALT GTb LDH No leuco CSb cGMPa cAMPa OP metabolite phosph.moietyb No RBC TAT a b
Data from animals only. Number of cases <4.
Acute
Developed
90/99 20/40 25/50 20/40 90/99 80/98 75/90 70/70 20/60 30/60 20/40 20/20 10/10 40/80 40/70 30/60 99/98 100/99 5/5 60/90
99/90 10/80 25/90 20/90 99/90 90/85 80/80 60/60 20/50 30/50 20/30 20/10 10/10 40/85 40/70 30/60 90/90 100/99 5/5 60/90
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assessment of sensitivity and specificity was rather subjective, it is clear that the most sensitive are two or three parameters: cholinesterase determination (depending on the type of OP, either AChE or BuChE), the possible determination of OP metabolites in the blood, and determination of the phosphonyl moiety on the target enzyme (if possible). In case of developed intoxication when convulsions occurred, tension of the blood gases is also a good marker; however, these changes are not very specific. The same approach can be applied to lactate. This is not surprising because of the existence of convulsions including convulsions of the diaphragm (and thus disturbed ventilation, low oxygen saturation, and an increase in acid metabolites). It should also be mentioned that the validity of these parameters is changed during the intoxication. The changes in transaminases LDH and -GT, indicating liver damage, can be caused by solvents used in commercially available OP insecticides. A low validity in the number of RBC or leucocytes is also indicated. As for CS and TAT-stress markers, it is clear that OP intoxication represents a stress situation. In this connection, an increase in ALT can also be considered as a stress marker and not indicative of liver damage. Determination of AChE or BuChE molecular forms can be interesting and useful for improvement of the diagnosis of OP poisoning. It was demonstrated that these forms are inhibited in diVerent manners, some of the forms are resistant (a low molecular weight) and some of them are very sensitive (a high molecular weight). When the total AChE activity is determined, the value obtained is a ‘‘mean’’ of the activities of these forms. The changes in the cyclic nucleotides are interesting but not very valid for blood. They were determined during animal experiments with toxic OP and are of more interest in connection with the nervous system. Esterases and AP, generally hydrolases, are sensitive but the inhibition potency of diVerent OP is very variable: for nerve agents, these hydrolases are not very valid and, for some OP insecticides like malathion, they are sometimes more sensitive than cholinesterases. In conclusion, diagnosis of OP poisoning represents a serious problem. The development of the new specific methods mentioned (fluoride reactivation, tandem MS analysis of enzymatic digests of BuChE) are of high importance for more precise diagnosis of OP/nerve agent poisoning. An extensive review of Noort et al. (N8) dealing with biomonitoring of exposure to chemical warfare agents (not only nerve agents) can be strongly recommended. From a practical point of view in the clinical laboratory, it is necessary to monitor basic physiological functions, cholinesterases, and other biochemical parameters not only for diagnostic purposes but also preferably for the regulation of treatment.
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5. Prophylaxis The term prophylaxis as used in this chapter is limited to medical countermeasures applied relatively shortly before penetration of a toxic agent into the organism. There is a question what will happen after the administration of the prophylactic drug. When the treatment is unnecessary, it can be described as prophylaxis. However, though successful prophylaxis can be observed for some OP, full protection of the organism without post-exposure treatment, especially for soman poisoning, remains open. When the treatment is involved after exposure, the term ‘‘pre-treatment’’ has become to be accepted. From a practical point of view, it is obvious that when the drug is administered prior to intoxication (either prophylaxis or pre-treatment) with the aim of protecting the organism against the toxic drug, exposure to these agents is expected and, therefore, postexposure therapy can very probably be used, i.e., pre-treatment could be used as the right term. For reasons of simplicity, the term prophylaxis is used in this article. The prophylaxis will be focused on protection of AChE against the inhibition using reversible cholinesterase inhibitors. Diminishing the level of OP using enzymes to hydrolyze these agents or enzymes to bind the agents (to specific proteins or to antibodies) and thus reduce the OP level (and inhibition of cholinesterases) in the organism can be described as detoxification. Another approach to prophylaxis is based on using present antidotes. Unlike other OP, the treatment of soman poisoning is very diYcult and unsatisfactory. This is the reason for intensive studies using pre-treatment/ prophylaxis allowing survival and increasing the resistance of the organism exposed to soman and tabun. The administration of present antidotes (anticholinergics, reactivators, and others) to prevent the eVects of OP is also possible. The problem with this approach is how to achieve suYcient levels of antidotes for a relatively long time. Combinations of these approaches are also possible. 5.1. PROTECTION OF ACHE AGAINST INHIBITION Keeping AChE intact is a basic requirement for eVective prophylaxis, i.e., to change the enzyme in a way that will make it resistant to OP. This can be achieved by using reversible inhibitors, which are able to inhibit AChE reversibly and after spontaneous recovery of the activity, normal AChE serves as a source of the active enzyme. There are many inhibitors of cholinesterases diminishing both AChE and BuChE activities to a comparable extent, as has been described by Aldridge (1969). Carbamates belong to a group of inhibitors having a wide variety in their eVectiveness. They are biologically active because of their structural
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complementarity to the active surface of AChE and their consequent reaction as substrates with very low turnover numbers (A4, A5). The ability of some carbamates to protect an organism poisoned with OP has been known for many years (K36, K28). Physostigmine and neostigmine have been used to protect animals against DFP. The number of OP studied for protection was enlarged, as well as the number of carbamates studied. These studies were performed both in vitro and in vivo. The results are very dependent on experimental conditions; nevertheless, the protective eVect of physostigmine, aminostigmine, pyridostigmine, and others against AChE inhibition caused by diVerent OP (mostly soman) has been demonstrated (P7, M2, T6). Important contributions based on the modeling of diVerent inhibition processes in OP-intoxicated organisms were described by Green (G6, G7) and Maxwell et al. (M11). There have been numerous studies demonstrating the eVectiveness of carbamate pre-treatment/prophylaxis against intoxication with OP. From the results published (and unpublished), it appeared that pyridostigmine was the most promising prophylactic drug, especially against soman poisoning (A9, B10, B16, F8, K8, K14, K15, K16, K17, K18, K19, K33, K37, M2, M10, P5, T10, W8). On the basis of these results, pyridostigmine was introduced into some armies as a prophylactic against nerve agents. Its prophylactic eVect (like the eVects of other carbamates) is limited by its dose. With a higher dose, a higher eYcacy was observed, but the side eVects were more expressed too. This problem can be solved by the adding of pyridostigmine antagonizing drugs—anticholinergics. Many anticholinergics have been tested to protect the organism against intoxication with soman (and other nerve agents) and, on the basis of this research, the prophylactic combination of pyridostigmine with trihexyphenidyle and benactyzine (B16, B17, K5, K8, K14, K15, K18, K19, K20) was introduced into the Czech army. The presence of these two anticholinergics allowed us to increase the pyridostigmine dose and to increase its prophylactic eYcacy. This combination (including follow-up therapy) is not limited to soman, sarin, and VX poisoning but its high eYcacy against tabun (K18), GV (B17), and cyclosarin (K11) intoxications was observed. These nerve agents are also known to be resistant to common antidotal treatment. The prophylactic antidote combination called PANPAL has no side eVects, as has been demonstrated on volunteers: no statistically diVerent changes in the actual psychic state as well as no negative changes in the dysfunction time were observed. An improvement of tapping test following PANPAL administration was demonstrated. A decrease in the heart frequency 60 min following PANPAL administration lasting 480 min and returning to normal values within 24 h was demonstrated (F8). On the basis of the results with the prophylactic eYcacy of diVerent carbamates, aminostigmine was chosen as the most eVective (T6).
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Other carbamates also have a good prophylactic eYcacy, especially physostigmine (due to its central eVect on the contrary to pyridostigmine) (K24, K25, S22, T11, W8). Human study with transdermal physostigmine suggests a serious interest in the prophylactic use of this drug (W1, L9). Mobam and decarboxyfuran were also experimentally considered as potential candidates for prophylaxis. Among other inhibitors, aminophenols and OP were tested but their eVects were lower in comparison with pyridostigmine (M2). Structurally diVerent inhibitors from the carbamate and OP groups were also studied. From these compounds (preferably binding to the AChE anionic site), tacrine, 7-MEOTA, and huperzine A were considered and experimentally studied with respect to prophylaxis in vitro and in vivo (A14, B15, F6, F7, L1, L3, P2, P6, R5). The most interesting results were obtained with huperzine A. It is an inhibitor of the rat brain AChE (the mixed linear competitive type) (M15). Very similar results were obtained with enzymes from other sources (S7). Huperzine A was tested as a potential candidate against OP for its long-lasting eYcacy and relatively low toxicity (G9). However, the results obtained do not support replacement of pyridostigmine by these drugs. 5.2. DETOXIFICATION This principle can be used in two diVerent ways: administration of enzymes splitting the OP or specific enzymes which bind the OP (cholinesterases). OP is bound to the exogenously administered enzyme and thus the OP level in the organism is decreased (it acts as a ‘‘scavenger’’). Enzymes hydrolyzing OP are under research (L10, R4). On the other hand, many studies have been made with cholinesterases as scavengers. BuChE and AChE were observed to be very eVective in protection against OP intoxication (C3, D16, D17, D18, M9, M10, M22, S6). The administration of enzymes as scavengers seems to be very promising: the enzyme is acting at the very beginning of the toxic action, without interaction with the target tissues and without side eVects (C3, D16, D18). All of these features are of great interest and they are yielding practical results—isolation of the enzyme, examination for lack of and autoimmune response, and establishment of pharmacokinetic and pharmacodynamic properties (S5). Moreover, BuChE pretreatment also showed protective eVects on AChE inhibition in the brain parts following low-level sarin inhalation exposure (S9). Given our increasing knowledge in bioengineering and biotechnology, the connection between these two enzymes will be possible with the aim of obtaining a modified enzyme splitting OP and simultaneously reacting with AChE as a scavenger (B36). Antibodies against OP are in the research stage and eVorts are more focused on the detection of OP (L8, M20).
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5.3. THE USE OF STANDARD ANTIDOTES AS PROPHYLACTICS The antidotes currently used for the treatment of OP poisoning can be tested as prophylactics. The aim of this approach is very simple—to achieve a suYcient level of antidotes in the blood vessels before intoxication. Standard antidotes were studied in this respect, i.e., anticholinergics, reactivators, anticonvulsants, and others (P5, S3, M2). The problem with their use is the timing, duration, and achievement of suYcient levels of these antidotes after administration. However, the prophylactic eYcacy is good, as it has been demonstrated in treatment studies—administration of the antidotes mostly takes place very shortly (minutes) after the intoxication. The prolongation of the duration of the antidote eVects by achievement of their suYcient level in the blood by oral administration is not possible (especially reactivators) and therefore it is excluded. It was a reason for searching for other routes of administration. Transdermal administration of one of the most eVective reactivators (HI-6) was shown to be the most realistic approach (D19). The final result was the new prophylactic transdermal antidote TRANSANT. This preparation was clinically tested (including dermal sensitivity) without any harmful eVects and field testing was also successful. The final reports were finished and PANPAL was introduced into the Czech Army. The prophylactic eYcacy of other drugs was studied. As anticonvulsant drugs, benzodiazepines (diazepam, midazolam, alprazolam, triazolam, clonazepam) were studied, but isolated prophylactic administration has not had very good eVects (M2, H6, H9, H10, K40). 5.4. PROPHYLAXIS WITH OTHER DRUGS Prophylactic administration of diVerent drugs (alone or in combination) against intoxication with OP was studied. Calcium antagonists (nimodipine), neuromuscular blockers (tubocurarine), adamantanes (memantine), and the opiate antagonist meptazinol (G1, M2, K4, S27) were also tested with diVerent results but they were not very practical. On the other hand, a positive prophylactic eVect has been demonstrated with procyclidine (antimuscarinic, antinicotinic, and the anti-NMDA receptor drug) (M26, M27). Special importance can be focused on suramine (a protease inhibitor). Administration of this compound prior to soman intoxication (and followed by administration of atropine) showed good prophylactic eVect (C8). However, all these studies are experimental ones and they have not reached the practical output stage. The combinations of various drugs as prophylactics can be of very diVerent character. They can be used simultaneously (a combination of diVerent drugs) or as pre-treatment and following treatment with diVerent antidotes. Administration of pyridostigmine (or other inhibitors) prior to
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intoxication and treatment with diVerent drugs is a typical example (A8, A9, B17, K5, K8, K24, K25, T11). There are other combinations such as the administration of triesterase (T9, T10), procyclidine (K24, K25, M26, M27), clonidine (L14), sustained release of physostigmine, and scopolamine (M16). The results are very dependent on experimental conditions but this approach—administration of diVerent drugs—has yielded some good results, though up to now they have been on an experimental level. Only one prophylactic mixture has been introduced into the army—PANPAL, composed of pyridostigmine, benactyzine, and trihexyphenidyle. It appears from these results that simple prophylaxis (without postexposure treatment) against OP is not suYcient. Therefore, pyridostigmine has importance as a prophylactic drug, especially when it is connected with postexposure antidotal treatment. For further development, it is necessary to search for new prophylactic drugs and new routes of administration. In this connection, preparations of cholinesterases are of special importance for the development of more eVective prophylactics.
6. Treatment 6.1. BASIC PRINCIPLES Based on our knowledge of the mechanism of action, two therapeutic principles are used in the treatment of OP/nerve agent poisoning. The main drugs are anticholinergics that antagonize the eVects of accumulated acetylcholine at the cholinergic synapses (also called symptomatic antidotes) and cholinesterase reactivators (oximes) to reactivate inhibited AChE (causal antidotes). Their eVects are synergistic. Central nervous system antidepressants such as benzodiazepines are also used to treat convulsions (anticonvulsants). Due to the high toxicity of OP/nerve agents, first aid is important for the future fate of the intoxicated organism. It consists of interrupting contacts with the poison (evacuation, protective mask), administration of antidotes if possible, and decontamination. Support of vital functions (the heart, artificial respiration) is necessary. Though administration of the above-mentioned antidotes is recommended, successful therapy of moderate OP/nerve agent and carbamate poisoning has been described using atropinization and the treatment of acidosis with natrium bicarbonate only (B25, O2). 6.2. ANTICHOLINERGICS Of the anticholinergics, atropine is the drug most frequently used for the treatment of human poisoning. This muscarinic cholinergic antagonist acts by blocking the overstimulating eVects of acetylcholine at the muscarinic
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sites and has little eVect at the nicotinic sites. It does not readily cross the blood–brain barrier but it has central ameliorative eVects. In experiments on animals, the good therapeutic eYcacy of benactyzine and biperidene was observed especially against soman poisoning, due to its better central eVect in comparison with atropine (K13). Though some doubts exist about administration of very high doses of atropine, the treatment of human casualties and experimental intoxications is clear (M12): in severe poisoning, both the animal and human data show that very high doses of atropine are life-saving and well tolerated. Animals exposed to 2xLD50 soman were capable of tolerating 0.5 to 3.0 mg/kg, i.v. atropine: this equates to 35 to 210 mg in a 70 kg human. These doses agree with Iranian casualty data. Animal studies show lower doses of atropine (0.1–0.2 mg/kg). Therefore, higher doses of diazepam or more eYcient anticonvulsants are required. Lower atropine doses increase the risk of lethality from poor cardiorespiratory response and a long period of unconsciousness and possibly seizure activity, and, therefore, increase the potential for neurological damage. The further course of OP intoxication is negatively influenced by a low dosage of atropine (B28, B29). High doses of atropine return consciousness more rapidly and support cardiorespiratory eVorts. Aggressive atropinization and prolonged administration of the oxime improved the fate of OP-intoxicated patients (V10). Other anticholinergics may be even more eYcacious. The centrally acting anticholinergics (benactyzine, biperidene) can be very useful in therapy and reduce the necessary amounts of benzodiazepine anticonvulsants (K18, S2). Nerve agents have a long-term eVect on the behavior of experimental animals lasting months after intoxication with low doses of nerve agents (K17) and were eliminated with pharmacological pretreatment followed by antidotal treatment (K39). However, adding diazepam into the therapeutic mixture improved the survival of tabun-intoxicated mice when combined with atropine and methoxime (S10). The anticonvulsant action of some anticholinergics in soman poisoning was demonstrated (C2). A combination of diVerent anticholinergics and other drugs was also successful: A triple therapy consisting of procyclidine, diazepam, and pentobarbital was fully eVective in terminating soman seizure when administered 30 to 40 min after onset (M27). 6.3. REACTIVATORS The current standard treatment with reactivators includes diVerent types of oximes with a similar basic structure diVering by the number of pyridinium rings and by the position of the oxime group in the pyridinium ring. From the common oximes, mono- and bisquaternary pyridinium oximes are frequently used, such as pralidoxime, obidoxime, trimedoxime, methoxime,
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FIG. 10. Structural formulae of some cholinesterase reactivators.
and HI-6 (Fig. 10). Because of some doubts about the use of oximes in the treatment of OP poisoning, Eddleston et al. (E1) published a systematic review of clinical trials dealing with oxime therapy in acute OP poisoning. His generalized statement that pralidoxime should not be used in OP poisoning was not supported by the published results. The use of the reactivators is supported by the observations of OP poisoning (S29, T4, W9, W10). The eVectiveness of antidotal treatment is dependent on the reactivatability of AChE by the reactivator used (B16, B17). Generally, the conventional oximes (pralidoxime or obidoxime) have been considered suYciently eVective against VX, sarin, and GF (D7, K31, K32, K33, C5), and rather ineVective against soman (D7, K31). The diVerences in oxime eYcacy against various nerve agents are mainly due to the various aging rates at which inhibited AChE is converted to a form that can no longer be reactivated by oximes (F5, B32, C7). The reactivation of VX, sarin, or GF-inhibited AChE is still possible hours after the intoxication, while soman-inhibited AChE becomes unreactivatable within minutes and, therefore, renders the treatment of soman poisoning much more diYcult (B11, B16, B17, C7). Some results were achieved with HI-6 (G8). This fact led to the synthesis of a series of bisquaternary oximes, designated as ‘‘H-oximes,’’ that, in combination with anticholinergic drugs, have been relatively successful in antagonizing soman intoxication (S14). Among the H-series oximes, HI-6 has been the best studied and, therefore, seems to be the most promising oxime against soman poisoning (K31, R9). Worek et al. (W14), based on experimental testing of the reactivation potency of
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obidoxime, pralidoxime, HI-6, and HLo-7 in human erythrocyte AChE inhibited by nerve agents, suggested that HLo-7 may serve as a broadspectrum reactivator in nerve agent poisoning at doses therapeutically relevant in humans. The model for an in vitro AChE reactivation was described (W13). However, the results are very dependent on the experimental scheme. The results showing marked diVerence among the species, depending on the inhibitor and on the oxime, indicate that the findings from animal experiments need careful evaluation before extrapolating these data to humans (W12). A comparison of the eYcacy of AChE reactivators by in vitro and in vivo methods against sarin (K10), soman (K9), and cyclosarin (K11) has been demonstrated but the diVerence did not allow recommendation of a universal reactivator. However, in the aforementioned experiments, in vivo tested oxime HLo-7 shows similar or better therapeutic results in comparison with HI-6. A model for the evaluation of eVectiveness should be chosen carefully, depending on the type of nerve agent, reactivator, and experimental animal. To estimate the eVectiveness of treatment with conventional oximes (pralidoxime and obidoxime) and with HI-6 against three common nerve agents, sarin, soman, and GF were used and (K12) the reactivating eYcacy of the oximes for AChE inhibited in vitro was studied in rat brain homogenates. In order to evaluate the therapeutic eYcacy of these three reactivators, they were administered prophylactically. Prophylactic treatment with an oxime should provide much better results than treatment after poisoning and it reduces the agent-specific influence of aging. Big diVerences in eYcacy between HI-6 and conventional oximes have been clearly demonstrated. With therapeutic doses of pralidoxime, it was not possible to rescue rats poisoned with supralethal doses of the nerve agents. Only HI-6 was eVective in human doses in rats poisoned with supralethal doses, regardless of the nerve agent used. The much higher therapeutic potency of HI-6 in comparison with conventional oximes may be caused not only by the higher reactivating properties but also by other antidotal mechanisms based on antimuscarinic, ganglion blocking, and postjunctional nondepolarizing actions as well as cardiovascular and respiratory eVects (R9, S17). Various data suggest that the pharmacological eVects of HI-6 other than the reactivation of AChE are most important for the survival of nerve agent intoxicated animals (V5). The data presented indicate that HI-6 is eVective against supralethal nerve agent intoxication of rats when given in very low doses corresponding to those proposed for humans. On the other hand, pralidoxime and obidoxime are obviously not eVective for the treatment of supralethal intoxication with nerve agents. Similar findings have been presented by many other authors on the basis of their experiments using better animal models for predicting the eVects expected in man—especially guinea pigs and primates (K33, I1,
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L2). Their data also suggest that HI-6 has definite advantages over conventional oximes in the treatment of nerve agent intoxication, with the exception of tabun (K18). If we compare the AChE reactivatability of diVerent oximes and various nerve agents, i.e., the dependence of the percentage of rectivation vs concentration of the oxime (K7), basically, two diVerent types of the curves can be obtained: the first, depending on the oxime concentration, shows an increase with a maximum followed by a decreased part of the curve. The second type is a sigmoid curve reaching to the maximum but the decrease cannot be demonstrated because of too high a concentration of the oxime (very probably it will be the same, i.e., containing a decreasing part). The first type can be represented by HI-6 and HLo-7 for sarin-inhibited AChE (with the reactivation maximum about 10 5 M) and methoxime and HI-6 for cyclosarin-inhibited AChE (the maximum 10 5 and 10 4 M, respectively). HLo-7 has a similar profile in comparison with HI-6 but the AChE reactivation reaches to 50 to 60% only. For soman-inhibited AChE, HI-6 and HLo-7 show the second type of curve, reaching the reactivation maximum at 10 4 to 10 5 M. Pralidoxime and methoxime show a similar profile; however, the lower reactivation (about 30–40%) was observed at the concentration 10 3 M. Obidoxime is ineVective in this case. Obidoxime and pralidoxime are eVective against cyclosarin- and sarin-inhibited AChE at concentrations reaching to 10 3 to 10 2 M (Fig. 11). Therefore, the eVectivity of the oxime in a human can be influenced by the concentration in the target organs, i.e., when administered parenterally, in the dose range of 470 to 2280 mol/kg, the concentration in the brain can be about 10 4 to 10 5 M (K7). These concentrations are able to reactivate suYciently inhibited AChE in the brain, especially in the pontomedullar area (the increase by 10–20%): the minimal level of AChE activity in the pontomedullar area necessary for the survival of nerve agent intoxicated animals was assessed to be about 5% (B19, B20, B24). The crucial question dealing with the reactivator’s eVect on the central nervous system was discussed in the past. Because of their quaternary structure, at intact BBB, the penetration of the reactivators is slow. In order to reach an eVective concentration of the reactivator in CNS, its extremely high plasma concentration is necessary. On the other hand, some authors (E3, F1, H12) have suggested that the central reactivation eVect exists. It is known from other results that the inhibition and reactivation of AChE in the brain is selective for diVerent OP (B6, B7, B33) and following administration of the reactivators to nerve agent-intoxicated animals, reactivation of AChE in diVerent parts of the brain was demonstrated (B19, B20, B24). The ability of oximes to penetrate the blood–brain barrier was confirmed by Sakurada et al. (S1).
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FIG. 11. Hypothetic course of nerve agent-inhibited AChE reactivation by diVerent oximes. DiVerent curves represent the nerve agent–reactivator relationship. Curve 1 – GF-methoxime, sarin-HI-6, HLo-7; curve 2 – GF-HI-6; curve 3 – soman-HI-6, HLo-7; curve 4 – GF-HLo-7; curve 5 – sarin-methoxime, pralidoxime, obidoxime; curve 6 – soman-methoxime, pralidoxime, obidoxime, GF-pralidoxime, obidoxime (modified from K7).
There were and are some attempts to synthesize new reactivators with the aim of making them universal or more eVective, especially against soman- or tabun-inhibited AChE, both in the past (D13, D14, D15) and presently (K41, K42, K43, K44) (for review, see, e.g., P9). A number of alternative oximes have been shown to be significantly more eVective and have a broader spectrum of action than the pralidoxime and several of these may be as or more eVective than HI-6 (K30). However, the results obtained up to now are not of enough interest to introduce them into medical practice. An exhaustive review on cholinesterase reactivators has been published by Kassa (K7). It can be concluded that currently available oximes (pralidoxime, methoxime, obidoxime) are suYcient for therapy of poisoning with OP, but they are not very eVective against nerve agent (especially soman) poisoning. The H-oximes (HI-6, Hlo7; in some cases, methoxime) appear to be very promising antidotes against nerve agents including soman. However, there is no universal oxime suitable for antidotal treatment of poisoning with all OP/nerve agents. 6.4. ANTICONVULSANTS Anticonvulsants were studied empirically, particularly against convulsions caused by OP/nerve agents. These studies were not carried out only for the treatment of seizures. The control of seizures is strongly associated with
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protection against lethality and brain pathology (S13). DiVerent results were obtained using diVerent anticonvulsants such as barbiturates, hydantoins, local anesthetics, calcium channel blockers, sometimes perspective; however, benzodiazepines were chosen as the most eVective (M2). However, some anticholinergics (atropine, scopolamine, biperidene, trihexyphenidyl, procyclidine) also have anticonvulsant eVects (A8, K34, M13, M14, S16). The GABA uptake inhibitor tiagabine, the glutamate receptor antagonists (e.g., memantine, antinicotinic mecamylamine, the alpha(2)-adrenergic agonist clonidine) were not very eVective (S16). Benzodiazepines were eVective against soman-induced seizures, with strong synergistic eVects when combined with centrally active anticholinergic drugs. DiVerent benzodiazepines were tested (avizafon, clonazepam, diazepam, loprazolam, lorazepam, midazolam) and the most pronounced antiseizure activity of diazepam and midazolam was demonstrated. Midazolam may be the most eVective anticonvulsant after nerve agent exposure, but, despite its eYcacy, it has not yet been approved as a drug for OP-induced seizures (R8). Diazepam has been recommended for standard treatment therapy of convulsions caused by OP/ nerve agents; however, midazolam has very similar or better eVects (M13, S13) and these results have led to a study with nasal administration of this drug (G3). Use of diazepam and imidazenil in soman-intoxicated mice has been described and higher or equal anticonvulsive eVect of imidazenil has been reported (R11). All these studies were performed experimentally on animals pretreated with pyridostigmine and treated with atropine and a reactivator (pralidoxime, trimedoxime, HI-6) to eliminate the lethal eVects. A complex therapy including all necessary biochemical examinations is necessary to prevent complications and chronic health disturbance.
7. Future Trends It appears from these results that: 1. The mechanism of action needs to be elaborated in a more detailed way, including not only cholinergic but other neurotransmitter systems. 2. A more detailed model for OP/nerve agent intoxication, especially via diVerent routes of administration, needs to be elaborated. 3. The study on binding of diVerent ligands to the molecules of AChE and BuChE, including the molecular forms and receptors with the aim of elucidating cholinergic nerve transmission requires further study. 4. It is necessary to study the relationship between cholinesterases and their functions, including changes in pathological states. 5. The toxicokinetics of OP/nerve agents including stereoisomers at the inhalation and percutaneous routes of administration should be expanded.
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6. There is a need to obtain more information regarding the long-term eVects of low doses (concentrations) of OP/nerve agents. 7. For the treatment of OP/nerve agent intoxication, it is necessary to search out (according to the knowledge of the mechanism of action) new strategies for more eVective treatment. 8. It is desirable to analyze in more detail the eVect of treatment of OP/ nerve agent intoxication without reactivators. 9. The gene expression profile after OP/nerve agent intoxication, which is important for the development of mechanism-based therapies, should be considered.
8. Summary OP/nerve agents are still considered as important chemicals acting on living organisms and are widely used. They are characterized according to their action as compounds influencing cholinergic nerve transmission via inhibition of AChE. Modeling of this action and extrapolation of experimental data from animals to humans is more possible for highly toxic agents than for the OP. The symptoms of intoxication comprise nicotinic, muscarinic, and central symptoms; for some OP/nerve agents, a delayed neurotoxicity is observed. Cholinesterases (AChE and BuChE) are characterized as the main enzymes involved in the toxic eVect of these compounds, including molecular forms. The activity of both enzymes (and molecular forms) is influenced by inhibitors (reversible, irreversible, and allosteric) and other factors, such as pathological states. There are diVerent methods for cholinesterase determination; however, the most frequent is the method based on the hydrolysis of thiocholine esters and subsequent detection of free SH-group of the released thiocholine. The diagnosis of OP/nerve agent poisoning is based on anamnesis, the clinical status of the intoxicated organism, and on cholinesterase determination in the blood. For nerve agent intoxication, AChE in the red blood cell is more diagnostically important than BuChE activity in the plasma. This enzyme is a good diagnostic marker for intoxication with OP pesticides. Some other biochemical examinations are recommended, especially arterial blood gas, blood pH, minerals, and some other specialized parameters usually not available in all clinical laboratories. These special examinations are important for prognosis of the intoxication, for eVective treatment, and for retrospective analysis of the agent used for exposure. Some principles of prophylaxis against OP/nerve agent poisoning comprising the administration of reversible cholinesterase inhibitors such as pyridostigmine (alone or in combination with other drugs),
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scavengers such as preparations of cholinesterases, some therapeutic drugs, and possible combinations are given. Basic principles of the treatment of nerve agent OP poisoning are described. They are based on the administration of anticholinergics (mostly atropine but some other anticholinergics can be recommended) as a symptomatic treatment, cholinesterase reactivators as a causal treatment (diVerent types but without a universal reactivator against all OP/nerve agents) as the first aid and medical treatment, and anticonvulsants, preferably diazepam though some other eVective benzodiazepines are available. New drugs for the treatment are under experimental study based on new approaches to the mechanism of action. Future trends in the complex research of these compounds, which is important not only for the treatment of intoxication but also for the quantitative and qualitative increase of our knowledge of toxicology, neurochemistry, neuropharmacology, clinical biochemistry, and analytical chemistry in general, are characterized. REFERENCES A1. Abernethy, M. H., George, P. M., Herron, J. L., and Evans, R. T., Plasma cholinesterase phenotyping with use of visible-region spectrophotometry. Clin. Chem. 32, 194–197 (1986). A2. Abou-Donia, M. B., Organophosphorus ester-induced delayed neurotoxicity. Ann. Rev. Pharmacol. Toxicol. 21, 511–548 (1982). A3. Abou-Donia, M. B., and Lapadula, D. M., Mechanisms of organophosphorus esterinduced delayed neurotoxicity: Type I and Type II. Ann. Rev. Toxicol. 30, 405–440 (1990). A4. Aldridge, W. N., Organophosphorus compounds and carbamates, and their reactions with esterases. Brit. Med. Bull. 25, 236–239 (1969). A5. Aldridge, W. N., and Reiner, E., ‘‘Enzyme Inhibitors as Substrates.’’ North Holl. Publ. Co., Amsterdam-London, 1973. A6. Altrunas, I., Delibas, N., Demirci, M., Kiline, I., and Tamer, N., The eVects of methidathion on lipid peroxidation and some liver enzymes: Role of vitamins E and C. Arch. Toxicol. 76, 470–473 (2002). A7. Andersen, R. A., Aaraas, I., Gaare, G., and Fonnum, F., Inhibition of acetylcholinesterase from diVerent species by organophosphorus compounds, carbamates, and methylsulphonylfluoride. Gen. Pharmacol. 8, 331–334 (1977). A8. Anderson, D. R., Harris, L. W., Chang, F. C. T., Baze, W. B., Capacio, B. R., Byers, S. L., and Lennox, W. J., Antagonism of soman-induced convulsions by midazolam, diazepam, and scopolamine. Drug Chem. Toxicol. 20, 115–131 (1997). A9. Anderson, D. R., Harris, L. W., Woodard, C. L., and Lennox, W. I., The eVect of pyridostigmine pretreatment on oxime eYcacy against intoxication by soman and VX in rats. Drug Chem. Toxicol. 15, 285–294 (1992). A10. Annapurna, V., Senciall, I., Davis, A. J., and Kutty, K. M., Relationship between serum seudocholinesterase and triglycerides in experimentally induced diabetes mellitus in rats. Diabetologia 34, 320–324 (1991). A11. Antonijevic, B., Stojiljkovic, M. P., Bokonjic, D., Maksimovic, M., and Nedeljkovic, M., EVect of memantine on the permeability of the mice blood–brain barrier in soman poisoning. Toxicol. Lett. 144(Suppl. 1), 121 (2003).
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THE POTENTIAL OF PROTEIN-DETECTING MICROARRAYS FOR CLINICAL DIAGNOSTICS Alexandra H. Smith, Jennifer M. Vrtis, and Thomas Kodadek Department of Internal Medicine and Molecular Biology, Center for Biomedical Inventions, University of Texas Southwestern Medical Center, Dallas, Texas 75390
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Diagnostic Signatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Signature Discovery Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Signature-Detecting Platforms in the Clinic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Protein-Detecting Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Recent Advances in Protein Ligand Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Recent Applications of Protein-Detecting Microarrays . . . . . . . . . . . . . . . . . . . 4. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction The early and accurate detection of disease is an important issue in the development of new medical technology and is crucial for eVective disease treatment. For instance, early stage detection of cancer is crucial for a favorable outcome. In the United States, 72% of lung cancers, 57% of colorectal, and 34% of breast cancers have already metastasized by the time they are detected (G2). Unfortunately, most therapeutics are limited in their eVectiveness once a tumor has invaded surrounding tissue and metastasized throughout the body. Another unsolved problem is to develop diagnostic assays which distinguish diseases with similar symptoms but diVerent pathogenic mechanisms, such as Alzheimer’s disease, Leury-body disease, Creuzfeldt-Jakob disease, frontotemporal dementia (G1), and prognostic subgroups in cancers (J1). 217 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2423/04 $35.00
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Diseases are often diagnosed by measuring an endogenous substance or parameter indicative of the disease process. This substance or parameter, which is present in the blood or some other readily available fluid or tissue, is known as a biomarker of disease. Biomarkers are used to diagnose disease, measure disease progression, and monitor the eVects of treatment. In clinical practice, these biomarkers are generally proteins in blood measured by enzyme-linked immunosorbent assay (ELISA), a highly sensitive method to quantify biomarkers using specific antibodies. A well-known biomarker is prostate-specific antigen, which is elevated in the serum of prostate cancer patients (K5) and is used as a diagnostic tool to detect and monitor the treatment of prostate cancer. Currently, most diagnostic assays detect single biomarkers or a small number of genes or proteins strongly induced in response to disease stimuli, such as cytokines. These biomarkers often lack specificity, which precludes an unequivocal diagnosis. Diseases, even at their early stages, elicit many combinations of slight, but significant changes in protein expression and/or activity (W5). Therefore, measuring a combination of biomarkers (hereafter referred to as a diagnostic signature) should be more eVective than a single biomarker. In order to increase the arsenal of diagnostic assays available in the clinic, two main research approaches are required. First, clinically useful diagnostic signatures for specific diseases should be identified and, second, clinically useful platforms must be designed to measure these signatures.
2. Diagnostic Signatures For diagnostic signatures to be relevant in a clinical setting, a number of factors need to be considered. Ideally, clinically useful diagnostic signatures should be measurable in a readily accessible body fluid such as serum, urine, or saliva, making diagnosis noninvasive. A recognizable signature should be evident prior to the onset of clinical symptoms. Early diagnostic signatures would be valuable for monitoring patients for postoperative infection and for population-screening, as prostate-specific antigen is used to screen for prostate cancer. Furthermore, diagnostically useful signatures should be specific for a given disease. Certain signatures may be common to diseases such as cancers or infections, but useful signatures should distinguish between tumor classes and pathogen types. Lastly, signatures are present in a dynamic biological system and normal variation among healthy individuals must be taken into account. Genetic factors, age, gender, time of day, or environmental conditions such as diet and stress all contribute to variation. This issue presents a serious challenge in recognizing diagnostically useful signatures.
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Researchers must first discover diagnostic signatures which meet these criteria. However, identifying diagnostic signatures will only be useful to the clinician if they can be detected routinely and eYciently. Therefore, an ideal diagnostic signature-detecting system for the clinician’s oYce should allow for on-site analysis for immediate detection or confirmation of a specific disease state, thereby allowing for the necessary targeted treatment. The assay should be sensitive, accurate, and reproducible to prevent false negatives and positives. The system should be inexpensive and easy to use, requiring minimal technical expertise and sample processing to prevent any additional variance. We will focus on progress in detecting clinically useful diagnostic signatures and the development of protein-detecting microarrays to measure these signatures in the clinician’s oYce. 2.1. SIGNATURE DISCOVERY TOOLS The availability of whole genome sequences of organisms starting with a virus (S1), the first bacterium (T4), and the much heralded human genome (L3, V1) has contributed significantly to the understanding of human disease. Analysis of the genome has led to the identification of genetically based diseases and gene variants or polymorphisms that render individuals more susceptible to certain diseases. Depending on developmental stage, age, organ, and environmental factors, a subset of genes is transcribed into messenger RNA (mRNA) that could then be translated into proteins, which are critical for the functional state of a cell. Functional genomics is the study of the transcriptome, i.e., all the genes transcribed into mRNA, while proteomics is the analysis of the proteins expressed under a specific condition, such as disease. Genetic, functional genomic, and proteomic analysis have all contributed to determination of molecular changes related to disease in order to elucidate the cause and develop targeted therapy. In addition, these studies have identified biomarkers/diagnostic signatures that will improve diagnostic accuracy. Diagnostic signatures can be obtained by measuring either mRNA or protein levels in a given sample. It is preferable to measure protein levels, which more accurately describe the conditions in a biological system. Furthermore, mRNA levels are not necessarily correlative of protein levels and activity. In a study comparing mRNA and protein expression in lung carcinomas, only a subset of the proteins studied (17%) exhibited a significant correlation with mRNA levels (C4). Another subset of proteins had a negative correlation, and various protein isoforms had diVerent protein/ mRNA correlations. In addition to measuring protein expression levels, it would be useful to analyze proteins that have undergone post-translational
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modifications, which are crucial for protein activity and function. Clearly, protein diagnostic signatures are significantly diVerent from DNA signatures and would be more specific if certain post-translationally modified and protein isoforms are important disease indicators. Investigations on RNAbased diagnostic signatures are currently more prevalent due to the availability of DNA microarrays. Protein signatures require specialized techniques for the separation and detection of proteins and the technology is less developed for large-scale high-throughput analysis. 2.1.1. DNA Diagnostic Signatures Microarrays represent a new technology that has been used extensively since the first DNA microarray study on diVerential expression of 45 Arabidopsis genes published in 1995 (S2). Many reports have demonstrated the use of DNA microarrays for the investigation of diVerential gene expression in diseased versus healthy tissue. These studies support the idea that obtaining diagnostic signatures for specific disease states is possible. However, very few studies fulfill the criteria outlined for clinically useful diagnostic signatures. Typically, mRNA is isolated from tissue samples rather than readily accessible body fluids, such as urine, plasma, cerebrospinal fluid, and saliva. Most studies compare healthy and diseased tissues rather than comparing diseases to determine whether signatures are specific. Normal variation is rarely reported and most study groups are too small to take the variation of the normal population into account. One study illustrated that blood genomic signatures could be used to distinguish among experimentally induced disease states in rats. The gene expression patterns for 8740 genes in leukocytes was determined on an AVymetrix GeneChip1 24 hours after adult rats were subjected to ischemic strokes, hemorrhagic strokes, sham surgeries, kainite-induced seizures, hypoxia, or insulin-induced hypoglycemia (T3). There were overall similarities in the response patterns in the six experimental conditions compared to the controls, but each disease condition could be identified by unique gene expression patterns. Animal studies are not directly comparable to humans as there are multiple environmental factors and genetic diversity of the human population to take into account. However, this is a significant study as a proof of principle that disease states can be detected and diVerentiated in readily accessible body fluids. Other DNA microarray studies indicate that presymptomatic diagnostic signatures are obtainable. DNA microarray analysis of mRNA samples from a chimpanzee’s liver during acute resolving Hepatitis C virus infection was performed. The study provided insight into the liver response to viral infection. Although the study was not developed to determine a diagnostic
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signature, changes in gene expression could be noticed as early as day 2 post-infection (B4). Early changes in gene expression in a murine model for allogenic bone marrow transplant indicated that acute graft-versus-host disease could be detected before the development of histological changes in the liver (I1). These animal studies in controlled settings indicate that early diagnosis is possible. 2.1.2. Protein Diagnostic Signatures Various methodologies are being developed to detect and quantify the specific combinations of proteins associated with a particular disease. There are significant issues that must be taken into consideration to achieve this goal. The methodology should detect proteins from complex biological samples and should be sensitive to detect low-abundant proteins, which are potentially important diagnostic markers. In addition, other challenges include the solubility of the protein (i.e., membrane proteins have low solubility in aqueous media) and diVerent isoforms and post-translationally modified proteins must be identified. Numerous technologies have been developed to undertake this daunting task, but we focus on reports that are clinically relevant. Mass spectrometry (MS) methodologies have most commonly been used to detect proteins (biomarkers) associated with a particular disease (P2, P5, P7, W5). The most well-established technique for determination of protein biomarkers is two-dimensional polyacrylamide gel electrophoresis coupled with mass spectrometry (2D/MS) (G3, H1). Several thousand proteins can be separated simultaneously according to their charge and molecular mass by 2D electrophoresis and visualized by silver staining. Subsequently, the protein spots of interest are excised from the gel, trypsinized, and analyzed by either matrix-assisted laser desorption/ionization time-of-flight (MALDITOF) MS or electrospray ionization (ESI) MS. Overall, 2D/MS has proven to be a reliable tool to diVerentiate between proteins expressed under diVerent cellular conditions and to detect proteins with various isoforms or posttranslational modifications. Although low abundance proteins, proteins of very low or high molecular weight, and less soluble proteins are not easily detected; thus, information is lost for potentially important diagnostic markers. This methodology has been employed for the identification of potential protein diagnostic signatures. By comparing the protein expression in lung adenocarcinoma tissue samples and uninvolved lung samples by 2D/MS, nine proteins were identified to have increased expression levels (1.4- to 10.6-fold) in lung adenocarcinoma tissue samples (C3). Furthermore, multiple protein isoforms were upregulated for a number of these proteins, but one
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protein isoform for cytosolic inorganic pyrophosphate (P4HB) was significantly overexpressed while another isoform was unchanged relative to normal lung tissue. Thus, this study suggests that 2D/MS is a powerful tool to identify potential biomarkers, including specific isoforms with diagnostic potential. The cited study was done using tissue samples, but potential biomarkers for hepatitis B virus (HBV) infection were determined using serum samples (H2). Although there are a number of biomarkers available for HBV infection, no single serological test can unequivocally diagnose the infection. Therefore, an assay able to detect multiple biomarkers should be more accurate to diagnose HBV. The expression levels of seven proteins were significantly changed in HBV-infected sera as determined by 2D/MS. This protein profile suggests that these serum proteins may be useful in diagnosis, but additional investigations are needed to determine specificity of the pattern with regard to other types of infection and liver inflammation. Inflammatory response markers have been detected with 2D/MS in the urine of stroke-prone rats at least 4 weeks before a stroke occurred and before the appearance of anomalous features could be detected in the brain by MRI (S4). The specificity of inflammatory response markers still needs to be determined; however, this study suggests that proteins in a readily obtainable body fluid can be used as early diagnostic markers. An alternative mass spectroscopy technique that is rapidly gaining recognition for its potential in clinical proteomics is surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF) (I3, I4, W4). Using the SELDI-TOF technology, only a small amount of serum sample (one microliter) is required to provide a diagnostic signature for a particular disease in a relatively short time and therefore a potentially high-throughput clinical proteomic tool. A critical aspect to this technique is that proteins from a serum sample are bound to a ProteinChip1 (Ciphergen Biosystems Inc., Fremont, CA, USA) based upon common physicochemical properties such as charge and hydrophobicity or, more specifically, adhered to the surface via a specific antibody or ligand, while the rest of the sample is washed away. Next, the adhered proteins are ionized and analyzed similar to MALDI-TOF. Since a low-end mass spectrometer lacking MS/MS capabilities is used in SELDI-TOF, the proteins or peptides in the sample are not individually identified; instead, profiles specific to serum samples are compiled using highly sophisticated bioinformatics. Diseased and healthy tissues have been diVerentiated by MALDI-TOF, as illustrated in the MS protein profile for tumor and normal lung tissue samples with the discriminatory peaks in the spectrum marked by an asterisk
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(Fig. 1) (Y1). SELDI-TOF has also been used to distinguish protein profiles. The first reports of using SELDI-MS to reveal diagnostic signatures in readily accessible body fluids were in nipple aspirate fluid to detect breast cancer (P1) and in serum to detect ovarian cancer (P3). To detect ovarian cancer-specific protein profiles, serum proteins were bound to a C16 hydrophobic interaction ProteinChip1 (P3). The mass spectra patterns from 50 ovarian cancer patients were compared to those from 50 unaVected controls. An iterative searching algorithm process identified a small subset of key values that segregated the cancer patients from the unaVected population. The cluster patterns were distinguishable for 50 ovarian cancer cases, including 18 stage I cases and nearly all control samples. This promising study suggests that a readily attainable serum sample could be used for initial screening of patients for ovarian cancer. The identities of the discriminatory peptides in the ovarian cancer sample were not deduced, which illustrates the limitation of the commercial Ciphergen system. The specificity of the protein profile for stage I ovarian cancer still needs to be determined, since the diagnostic signatures could be attributed to general metabolic changes caused by tumors. Combining profiles from additional subset-specific protein chips increased specificity (P4). The Ciphergen system has also been used to determine serum proteomic patterns in other cancers such as prostate cancer (A1, B2), hepatocellular carcinoma (P6), and non-small cell lung cancer (X1). The protein patterns were determined from a combination of data from more than one capture array, thereby increasing the specificity of the diagnostic signature. Another MS-based technique has been used in an attempt to determine the normal peptides present in bodily fluids. Peptides present in a normal urine sample were separated by high-resolution capillary electrophoresis (CE). A peptide pattern was established by analyzing the mass spectra from 18 samples (W3). The patterns contained ion peaks from 247 peptides (out of more than 1000 detected) that were present in more than 50% of the samples. The data was compared to five samples from patients with renal disease and impaired renal function. An alternative pattern was evident for samples from diseased individuals with additional ion peaks in the spectra and the absence of previously observed peaks. Even though a small number of samples were analyzed, valuable information about biological samples was obtained rapidly, illustrating the significance of MS. 2.2. SIGNATURE-DETECTING PLATFORMS IN THE CLINIC DNA microarrays have been a useful research tool. However, there are only a few examples of DNA microarrays used to identify clinically relevant diagnostic signatures, as outlined earlier. The complexity of the technique hinders
FIG. 1. Representative example of potential protein diagnostic profiles obtained by MALDITOF Mass Spectroscopy (MS) from tumor and normal lung tissue samples shown with the molecular weight calculation (m/z values). Asterisks indicate examples of the MS peaks identified by statistical analyses as optimum discriminatory patterns between normal and tumor. Below: hierarchical cluster analysis of 42 lung tumors and eight normal lung tissues in the training cohort according to the protein expression patterns of 82 MS signals. Each row represents an individual proteomic signal and each column represents an individual sample. The dendrogram at the top shows the similarity in protein expression profiles of the samples. Substantially raised (red) expression of the proteins is noted in individual tumor and normal lung tissue
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its use by nonexperts. Detecting diVerential gene expression requires a number of sample preparation steps. Furthermore, significant diVerences in measurable gene expression can be introduced by small variations in sample collection and preservation, RNA quality, cDNA amplification methods, probe labeling, hybridization, and washing conditions. DNA microarrays will be useful to identify individuals susceptible to genetic-based diseases. However, clinical use of DNA microarrays will most likely be done in specialized centers due to the cost and technical expertise required for reproducible results. Mass spectrometry techniques are used to determine diVerential patterns of protein expression but cannot be employed for rapid detection and quantification of specific proteins. Although 2D/MS has been proven to be a powerful tool to analyze protein mixtures, there are limitations that prevent it from use in the clinic. Separation of proteins by 2D is tedious, labor intensive, and not amenable to high-throughput strategies. Mass spectrometry techniques used to determine protein profiles (i.e., SELDI-TOF) have the potential for high-throughput strategies and automation. These methodologies do not require the extensive sample processing required by 2D/MS. However, additional SELDI-TOF studies must be carried out to ascertain its accuracy in detecting positives while reducing the incidence of falsepositives, prior to its use as a clinical diagnostic tool. However, the expense of mass spectrometers may limit their use to specialized centers. Currently, most clinical assays detect protein biomarkers by ELISA. Though ELISA is a well-established technique for diagnostic assays, it is limited to detection of single biomarkers. In the future, we envisage that multiplex ELISAs in the form of protein-detecting microarray-based assays will be used in the clinician’s oYce, or even in the home, for rapid detection of multiple proteins in biological samples. Unlike DNA microarrays, proteindetecting arrays would require minimal sample processing, thus reducing the variability and need for experienced individuals to obtain reproducible and accurate results.
3. Protein-Detecting Microarrays For clinical diagnostics, the goal is to develop protein-detecting microarrays with capture agents/ligands that bind specifically to target proteins in complex biological solutions (Figs. 2 and 3) (K2, K3). The eYciency of a samples. AD ¼ adenocarcinoma, SQ ¼ squamous-cell carcinoma, LA ¼ large-cell carcinoma, META ¼ metastases to lung from other sites, REC ¼ recurrent NSCLC, CAR ¼ pulmonary carcinoid, NL ¼ normal lung.1 1
Reprinted with permission from Elsevier (The Lancet, 2003, 362, 433–439).
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FIG. 2. A protein-detecting microarray. Each square in the grid represents a diVerent feature of the array that would be impregnated with a particular protein ligand (blue shapes). When the sample is applied to the chip, each ligand will capture its target protein (orange and red coils in blow-up). The amount of target protein bound to each feature of the array would be quantitated with probes such as fluorescently labeled antibodies against the captured proteins. A fluoresence scanner would then measure the intensity of fluorescence (diVerently shaded green squares) at each spot, which would reflect the level of captured protein.2 (See Color Insert.)
protein-detecting microarray is dependent on a number of factors. The solid support and surface chemistry should minimize the amount of sample needed and optimize the eYciency of protein detection. Immobilized ligands must be stable and retain activity over extended periods. Ligand-binding capability must be validated to ensure that the working range covers the physiologically relevant concentrations of proteins. In addition, methods for signal detection and quantification should have a large dynamic range 2 Reprinted from Trends in Biochemical Sciences, 27, T. Kodadek, Development of proteindetecting microarrays and related devices, 295–300. Copyright (2002), with permission from Elsevier.
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FIG. 3. A bead-based format for the parallel detection of proteins. Each bead displays a diVerent binding agent directed against a specific protein target (blue shapes). Each bead is colorcoded by covalent linkage of two dyes (red and orange shapes) at a characteristic ratio, allowing for uniquely coded beads. Only two beads are shown for clarity. Upon application of the biological sample, the target protein binds to the capture agents. A mixture of secondary binding ligands (in this case, antibodies) conjugated to a fluorescent tag (green) is applied to the mixture of beads. The beads are then passed through a detector where two lasers ‘‘read’’ the ratio (n:m, x:y) of dyes and thus identify the bead, while the fluorescence intensity is read to quantitate the amount of labeled antibodies present (which will reflect the analyte level).2 (See Color Insert.)
appropriate for biological samples. Various aspects of protein arrays, from surface chemistry to detection systems, have been reviewed (C6, E1, F2, K2, K3, M1, T1, Z1). We will focus on the progress in ligand isolation, which is the most crucial feature for the development of clinical protein-detecting microarrays. 3.1. RECENT ADVANCES IN PROTEIN LIGAND ISOLATION Currently, antibodies are most often used as protein ligands because of their high specificity and aYnity (KD in the nM range) for a target protein. However, traditional methods of antibody production are not amenable to high-throughput isolation. Generally, the production of polyclonal antibodies takes 2 to 4 months and requires about 0.2 to 2 mg of purified antigen. It will take another 2 to 4 months to then produce a monoclonal antibody for the particular antigen. Moreover, the high-throughput production of purified antigen is challenging because purifying protein antigens is labor intensive and conditions generally need to be optimized for each protein. Recently, a more high-throughput method has been developed to generate polyclonal antibodies in mice (C2), which uses genetic immunization (T2) rather than purified antigen. Genetic immunization involves directly transfecting antigen-presenting cells with genes to express the antigen. Antibody response is enhanced by codon optimization of genes and addition of various elements to enhance antigenicity, such as plasmids encoding genetic adjuvants. Using this method, polyclonal antibodies can be produced within 4 to 8 weeks, even for antigens that failed to produce a response in protein
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form. The exact aYnities of the polyclonal antibodies have not been determined, but are thought to be comparable to other antibodies. Antibodies may not be optimal ligands for protein-detecting arrays even with high-throughput antibody production. Most commercially available antibodies were found to be unsuitable for microarray-based analysis of cellular lysates (M1). A crucial drawback is that any type of antibody or folded protein is prone to loss of activity upon immobilization and storage. In contrast, small synthetic ligands are more stable and can be produced and purified economically and eYciently in bulk. These synthetic ligands are typically protein aptamers (antibody mimics), peptides, peptide-mimics, and small organic molecules. Various molecular biology techniques are available to screen for protein aptamer and peptide ligands for specific proteins. Phage display technology, introduced in 1985 (S5), has been used for the isolation of peptide (B5, F1, L5) and antibody fragment (G4) ligands for specific proteins. A 2002 review focuses on the principle of phage display technology and methods for the construction and bio-panning of phage libraries (A3). Libraries are constructed in vitro by inserting foreign DNA into specific locations of the genome of filamentous phage. The encoded protein or peptide is displayed on the surface as a fusion protein with one of the phage coat proteins, generally pIII, which displays five copies. Ligands bind to the protein of interest, which is immobilized on a plate. Bound phage are then eluted and amplified for more stringent rounds of panning. The amino acid sequence of the selected ligand can readily be determined by sequencing inserted DNA in the phage genome. Antibody fragment libraries from immunized and nonimmunized sources can be used in phage display and peptide libraries are commercially available. An alternative technique is to design protein aptamers that consist of a stable protein scaVold on which random peptides are displayed. An example of protein aptamers are aYbodies, which present a library of 13 randomized amino acids on the Z domain of Staphylococcus aureus protein A. Crystal structure studies indicate similarity in the binding of an aYbody to its target to protein–antibody interactions. However, aYbodies have a dissociation constant of approximately 1 M compared to antibody–antigen complexes of 1 nM or less (H3, R1, W1). The larger the library, the greater the probability of selecting rare highaYnity ligands. Phage display libraries typically contain 108–9 peptides with the limiting factor being the transformation eYciency of bacteria (L6). The in vitro techniques, ribosome and mRNA display, overcome this limitation since more complex libraries up to 1013 can be prepared (R2, W2). During in vitro transcription–translation of random DNA libraries, the encoded peptide remains associated with its mRNA. Either a ribosome complex is formed noncovalently by stalling the ribosome or the peptide is covalently linked to the mRNA through puromycin. Additional advantages of these
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techniques are that binding of the ligand is monovalent and aYnity maturation can be achieved over several rounds of screening by error-prone PCR or DNA shuZing (A3). This technique has been used successfully to isolate ligands that retain their high-aYnity binding properties when immobilized on a protein microarray. An mRNA library of antibody-mimics was prepared by randomizing three exposed loops on a stable, soluble protein, the tenth fibronectin type III domain. After 10 selection rounds, high-aYnity ligands for TNF- were isolated with dissociation constants between 1 and 24 nM. These ligands were further optimized by random mutagenesis to provide a ligand with a KD of 20 pM (X2). Disadvantages of both phage and mRNA display are the requirement for numerous rounds of selection and amplification, as well as the need to express and purify target proteins. Selectively infective phage (J3) and bacterial (H4, J2) and yeast (Y2) two-hybrid methods can overcome these obstacles because they are one-step screening assays with in vivo expressed target proteins. For the selectively infective phage technique, the N-terminal domains of the pIII coat protein is replaced with peptides from a ligand library, resulting in noninfective phage particles. To restore phage infectivity, adaptor molecules consisting of the target protein coupled to the missing N-terminal domains are required. These adaptor molecules can be expressed and exported to the periplasm in E. coli, eliminating the need for purified protein. Interaction between the fused peptide expressed on the phage coat and the adaptor molecule restores infectivity, allowing ligand selection in a single round. Although this method appears to have potential for ligand isolation (I2), few protein ligands are reported in the literature. This technology may be less successful for ligand isolation because of the potential for false positives (I3) or the size restriction of the target or ligand (C1). The yeast two-hybrid system detects protein–protein or protein–peptide interactions in vivo. The target or ‘‘bait’’ protein and the ligand library are fused to either the DNA-binding domain or the transcription activation domain. Yeast cells are transformed with both plasmids and only the transformants expressing the protein–ligand interaction are selected (Y2). The main advantage is the one-step in vivo screening; however, the library size is limited to about 107 because of the transformation eYciency of the cells. A variation on the yeast two-hybrid technique is the bacterial two-hybrid system. In this case, the target protein and the library-encoded peptide are each fused to a monomer of the DNA binding domain. Only if the target and ligand interact will the DNA binding domain form an active dimeric repressor. An activated repressor results in cells immune to phage infection, allowing for one-step selection of immune cells. This method was shown to be
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capable of selecting a peptide that specifically bound to its target protein with a KD in the micromolar range and inhibited the activity of the target protein in vivo (Z2). Although specific peptide ligands have been selected by both yeast and bacterial two-hybrid methods, there is little documented evidence for the identification of high-aYnity ligands. An alternative to the biological methodologies for screening protein ligands is the synthetic combinatorial library approach. Chemical libraries are prepared on a solid-support, usually on bead or a microarray format, and encompass a variety of synthetic molecules such as peptides, peptide mimics, and small organic molecules. For feasibility reasons, the libraries are usually limited to a size of 105 to 106, which is several-fold less than libraries developed from other techniques. However, an appealing feature is that a synthetic library is not limited to the 20 natural amino acids, thereby allowing for the inclusion of a variety of chemical properties. One such example are peptoids, N-substituted oligoglycines, which are structurally similar to peptides but are resistant to proteolytic cleavage, easily synthesized on resin, and which have diverse chemical side chains on the nitrogen of the peptoid backbone (A2, F3). Synthetic ligands from a combinatorial library are amenable to high-throughput screening and can easily be prepared in large quantities with little variability. Numerous protein ligands and inhibitors have been identified using combinatorial libraries. A comparison of combinatorial peptide library approaches has been outlined in a recent review (L7). The one-bead-onecompound (OBOC) approach entails the synthesis of thousands or millions of random compounds on bead. Small molecules as well as peptide mimetics have been identified as ligands to cellular proteins such as protein kinases and intracellular signaling proteins using an OBOC approach (L2). Protein ligands have also been isolated from biased libraries, which include a structural motif or derivatives of initial leads. By incorporating a consensus sequence into a peptide library, ligands were discovered to bind to the SH3 domain of phosphatidylinositol 3-kinase with modest aYnity (C5). Inhibitors of aspartyl proteases have been isolated from a peptide library which incorporated chemical functional groups known to interact with essential active site residues (L4). Bead-based libraries are most commonly used for the development of protein ligands. However, a combinatorial small-molecule library on a microarray format was screened and included an inhibitor of the transcription factor Hap3p (K4). Both the biological methods and the chemical combinatorial libraries usually yield low to modest aYnity protein-ligands (KD in micromolar range), which are insuYcient to capture low abundance proteins from complex biological mixtures. Rather than designing and synthesizing larger libraries, an alternative approach is to synthesize multivalent ligands. Two
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modest aYnity ligands can be linked to yield a new ligand with an aYnity that theoretically equals the product aYnity for the two individual molecules. One example of this ‘‘pincer’’ strategy was demonstrated when nuclear magnetic resonance was used to identify small molecules that bind to diVerent surfaces on FK506-binding protein and determine the appropriate linker for the two compounds. The chimeric molecule of the individual FK506binding protein binders had a KD of 19 nM (S3). A potent inhibitor of cSrc kinase was designed using a similar strategy. Initial lead molecules for kinase inhibition were identified from aldehyde-derived oxime compounds. Screening of a small library of chimeric compounds of the initial hits yielded a much more eVective kinase inhibitor with an inhibition constant, KI, of 60 nM. The pincer approach oVers a unique opportunity for creating chemically diverse protein-ligands, though designing optimal linkers for the pincer molecule requires some experimental eVort. Instead of linking two solution binders, protein ligands can be immobilized onto solid support, providing a wide variety of combinations of the two ligands. Appropriately positioned ligands will bind diVerent surfaces of the same protein, increasing the overall aYnity. Therefore, two noncompetitive, modest-aYnity ligands can be synthesized on solid support without a linker to provide a high-aYnity chimeric molecule, also known as the mixed-element capture agent (MECA) (Fig. 4) (B1). To demonstrate this, a MECA of two specific protein ligands was synthesized on resin. Each peptide of the MECA was specific for monomeric protein, either MBP or Mdm2. The MECA was determined to have a slightly higher aYnity for the MBP–Mdm2 fusion protein as compared to the individual peptide ligands in solution, but was a much more eVective capture agent on solid support. Using a similar idea, high-aYnity protein capture agents can be designed simply by immobilizing modest-aYnity ligands for multimeric proteins.
FIG. 4. Schematic representation of the MECA concept. Two noncompeting ligands (red and blue shapes) could be immobilized individually (left) or as a linear fusion (right), allowing for two appropriately positioned molecules to cooperate in the capture of the target protein. Reprinted with permission from (B1). Copyright (2003) American Chemical Society.
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Some fractions of these ligands should be appropriately oriented on the surface to promote binding of multiple ligands to one protein (i.e., one ligand bound per monomer). High-aYnity capture agents were created by synthesizing peptide ligands to dimeric proteins on Tentagel resin (N1). These immobilized ligands dramatically increased the half-life of the peptide–target protein complex when compared to random peptide ligands. The creation of high-aYnity capture agents from modest aYnity solution binders suggests that protein-detecting arrays may be more readily available than previously expected. 3.2. RECENT APPLICATIONS OF PROTEIN-DETECTING MICROARRAYS Due to the limited availability of well-characterized ligands, proteindetecting arrays are not ideal as a signature/biomarker discovery tool. However, protein-detecting microarrays are being developed as research tools and for diagnostics (L1). The first generation of protein microarrays are constructed with antibodies as capture agents. Although antibodies are less stable, very few synthetic ligands are currently available. An array of 368 antibodies was developed to identify the proteins present in the tissue of a single case of oral cavity cancer (K1). Antibody suspensions were spotted onto a thin film of nitrocellulose bonded to a glass slide. Protein lysates were biotinylated and bound protein was detected and quantified by an enzymelinked colorimetric assay. The antibody arrays were capable of detecting cancer-related proteins, as three of the eleven proteins detected were previously identified in tissue culture models of oral cavity cancer. Although this is the largest array to date, the assay needs to be validated, since the antibodies were not characterized with respect to aYnities, concentration, and cross-reactivity. Furthermore, the detected proteins were in non-native form. In another study, a screen of potential serum biomarkers of human prostrate cancer identified five proteins with significantly diVerent expression levels between 33 prostate cancer samples and 20 healthy controls (M3). These proteins were detected by antibodies spotted on either microscope slides coated with poly-L-lysine/N-hydroxysuccinimide-4-azidobenzoate (HSAB) or acrylamide-based HydrogelTM-coated slides. One hundred and eighty-four antibodies to target serum proteins and intracellular proteins were spotted in quadruplicate. The sample proteins were labeled directly with fluorescent tags and compared to a reference sample consisting of equal volumes of all serum samples. Labeling the sample eliminates the need for paired antibodies able to detect noncompeting epitopes of a protein, but can lead to bias. To control for labeling bias, the samples and reference were alternately labeled with diVerent fluorescent tags (reverse labeling). Labeling bias due to diVerent fluorophores can be controlled this way, but
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bias will be introduced if the presence of any label interferes with binding of the labeled protein to its specific ligand. Hydrogels were considered superior to the poly-L-lysine/HSAB-coated slides because of the lower background and more antibodies with a measurable signal (78 compared to 23). A number of companies have developed antibody arrays for research purposes. For example, a bead-based assay to screen up to 17 cytokines is available from Bio-Rad Laboratories (Hercules, CA, USA). In addition to the bead-based assays, Zyomyx, Inc. (Hayward, CA, USA) oVers a Human Cytokine Biochip for profiling of 30 biologically relevant cytokines. BD Biosciences Clontech (Palo Alto, CA, USA) has developed the largest commercially available antibody array for research purposes. This array includes over 500 antibodies with aYnity for proteins involved in a range of biological functions such as signal transduction, cancer, cell cycle regulation, cell structure, apoptosis, and neurobiology. Furthermore, the company reports that proteins present in the low pg/ml range can be detected in complex protein mixtures. The first antibody array with diagnostic potential was produced for immunotyping of leukemia (B3). Sixty antibodies were adhered to a film of nitrocellulose bound to a glass slide. Leukocytes from leukemia patients and healthy controls were incubated on arrays and bound leukocytes were visualized by dark-field microscopy. Relative densities of subpopulations of cells with distinct immunophenotypes were determined by eye. Distinctive and reproducible patterns were obtained for five leukemia types, indicating the potential for accurate diagnosis. Flow cytometric analysis of samples from two patients with chronic lymphocytic leukemia correlated closely with the array analyses for antigens expressed at high levels. A comparison of samples from 20 patients with chronic lymphocytic leukemia and 20 healthy controls indicated that leukocyte expression levels for 7 of the 60 cell-surface antigens could discriminate between the two sets. Although the results are only semiquantitative, this study suggests that leukemia types can be diVerentiated rapidly using a simple technique without specialized, expensive equipment. An alternative to the antibody array is the immobilization of proteins and detection of specific antibodies in sera. These antibody-detecting arrays will probably be the first to be routinely available in the clinic for serodiagnosis of autoimmune and infectious diseases. Serum samples from 60 individuals were tested with an array of microbial antigens printed on silanized glass microscope slides (M2). Anti-human IgG and IgM detection antibodies were labeled with fluorophores and quantified using confocal scanning microcopy. Comparison with commercially available ELISAs indicated that the microarray assay could identify positive and negative sera with similar eYciency. In this experiment, only 5 microbial antigens were arrayed.
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However, it is conceivable that arrays could be developed with ligands for a range of infectious disease agents and microbial toxins, allowing for rapid serodiagnosis in a clinical setting.
4. Conclusions Protein-detecting microarrays have great clinical diagnostic potential for monitoring the level and molecular state of native proteins in readily accessible body fluids such as serum, urine, and saliva. There are a number of significant hurdles that need to be overcome before these protein-detecting microarrays will be routinely accessible. First, diagnostic signatures specific to certain disease states need to be identified. MS-based signature discovery tools will play an extensive role in identifying the set of useful biomarkers for the development of disease-specific protein-detecting arrays. The ideal protein microarray will be capable of detecting all proteins, isoforms, and post-translationally modified proteins essential to diagnose disease. Even simple protein profiles may consist of hundreds to thousands of proteins. We hypothesize that within complex signatures there are some proteins that are more informative. As a first step toward clinically relevant protein-detecting microarrays, smaller arrays of protein-ligands should be useful to identify diagnostic signatures. Antibody microarrays for research purposes have been developed, but ideally arrays will consist of synthetic capture agents, which are more stable and amenable to highthroughput isolation and production. The next hurdle, therefore, will be the isolation of high specificity and aYnity ligands for clinically relevant proteins and any significant post-translational states and isoforms. REFERENCES A1. Adam, B. L., Qu, Y., Davis, J. W., et al., Serum protein fingerprinting coupled with a pattern-matching algorithm distinguishes prostate cancer from benign prostate hyperplasia and healthy men. Cancer Res. 62, 3609–3614 (2002). A2. Alluri, P. G., Reddy, M. M., Bachhawat-Sikder, K., Olivos, H. J., and Kodadek, T., Isolation of protein ligands from large peptoid libraries. J. Am. Chem. Soc. 125, 13995–14004 (2003). A3. Azzazy, H. M., and Highsmith, W. E., Jr., Phage display technology: Clinical applications and recent innovations. Clin. Biochem. 35, 425–445 (2002). B1. Bachhawat-Sikder, K., and Kodadek, T., Mixed-element capture agents: A simple strategy for the construction of synthetic, high-aYnity protein capture ligands. J. Am. Chem. Soc. 125, 9550–9551 (2003). B2. Banez, L. L., Prasanna, P., Sun, L., et al., Diagnostic potential of serum proteomic patterns in prostate cancer. J. Urol. 170, 442–446 (2003).
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CLINICAL LABORATORY IMPLICATIONS OF SINGLE LIVING CELL mRNA ANALYSIS Toshiya Osada, Hironori Uehara, Hyonchol Kim, and Atsushi Ikai Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. AFM. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Manipulations of Biological Material with AFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. A Protein-Unfolding Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. AFM Images of Subcellular Structures in Rat Vomeronasal Epithelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. The Distribution of Terminal GalNAc on Vomeronasal Epithelial Sections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. A Quantitative Measurement of Adhesion Force between Cell Adhesion Molecules and Living Cells with AFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. mRNA Extraction from Living Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Preparation of Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. AFM Setup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Pretreatment of AFM Instrument and Tip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. AFM Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Expression of -Actin mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Modification of AFM Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Modification of AFM Tips with Amino Groups. . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Modification of AFM Tips with Cross Linkers. . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Modification of AFM Tips with Proteins or DNA . . . . . . . . . . . . . . . . . . . . . . 5.4. A Microbead as an AFM Tip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction The selection of medical treatment is based mainly on clinical laboratory diagnoses analyzed by mass standardized levels. Each patient’s own bias must be ignored. It is necessary to tailor treatments based on individual problem analyses. Knowledge of human genetic codes oVers great promise for individualized tailor-made therapy. Recent advances in genomics and proteomics have led to new possibilities in the prediction of therapeutic eVects and risks of adverse reactions based upon the individual genotype. It may be possible in the future to monitor living cells from patients using genomic and proteomic biomarkers. Individual living cells should be examined before and after pharmacological treatment to determine appropriate doses of drugs. To achieve these examinations, extraction of biomolecules such as mRNAs, proteins, and lipids from living cells should be performed without severe damage to the cells. An atomic force microscope (AFM) is a powerful candidate for the manipulation of biomolecules because the AFM tip makes direct contact with the sample surface in liquid with high positional accuracy. These techniques may lead to tailor-made treatment of individual cells.
2. AFM The AFM, which was invented by Binnig et al. in 1986 (B1), is a kind of scanning probe microscope and is a powerful instrument for studying structures and properties of nanoscale molecules. The AFM is constructed with several components, as shown in Fig. 1. An AFM tip is positioned at the top of the cantilever, which is a tiny sheet spring. The most popular mode to obtain topographical images of samples is the optical lever method, in which the deflection of the cantilever is determined by monitoring a shift of a laser spot at a photo detector. The shift is transferred to the feedback loop, and is recovered by the elongation or shrinkage of a piezoelectric micropositioner to keep the deflection of the cantilever constant. Because of the mechanism of signal detection, the AFM does not require electrical conductivity on the surface of the sample and can be operated in a liquid environment as well as under ambient conditions. Because of these advantages, the AFM has been frequently applied to the study of biological systems and high-resolution images of DNA, proteins, and cells have been taken (C2, F1, L2, M3–M5, S1, U2, V1). As the tip of the AFM makes direct contact with the sample, the physical properties as well as topography of the surface can be examined. For example, the AFM can be used as a mechanical nano-indentor. Young’s moduli of a living cell were measured with high spatial resolution (C1, H1, M1, S2).
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FIG. 1. (A) A schematic image of an atomic force microscope. A laser beam is reflected at the back of the triangular cantilever. (B) A picture of an atomic force microscope which is combined with an optical microscope. The white box indicated by an arrow consists of the AFM unit including the piezo. A step motor causes the box to move up and down.
The AFM can also be used to measure adhesive forces between the probe tip and the sample surface by monitoring the continuous swing of the distance between the AFM tip and the sample surface. This movement is captured as a relationship between the Z position of the tip and the deflection that is referred to as the ‘‘force curve,’’ and the force is usually calculated from the curve (Fig. 2). When the tip is adhered to the sample surface, the cantilever
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FIG. 2. Schematic drawing of the force curve. (A) When the AFM tip does not bind to the sample surface, a retract follows the same path of approach. (B) When the tip binds to the sample surface, the retraction does not follow the same path of approach, because the tip bends in the direction of the sample surface. The bend is suddenly recovered by the detachment of the tip from the surface, and the binding force is calculated from the deflection of the cantilever.
bends toward the sample surface following the shrinkage of the piezo scanner, and the bend is suddenly recovered by the detachment of the tip from the surface (Fig. 2B). Since the cantilever obeys Hooke’s law for small displacements, the interaction force between the tip and the sample can be calculated. Forces are required to separate a single pair of molecular complexes, such as those formed between antigens and antibodies, which have been measured by immobilizing the molecules to the AFM tip and a surface, and are discussed from the point of view of thermodynamics (D1, E1, G1, S3).
3. Manipulations of Biological Material with AFM Recent progress in the field of AFM has enabled us to perform direct manipulations of biological material. We introduce our representative works for AFM applications to biological material in Fig. 3. 3.1. A PROTEIN-UNFOLDING EXPERIMENT A protein was covalently sandwiched through cross-linking reactions between the silicon substrate and the AFM tip (I1, I2). The sandwiched protein was mechanically unfolded by lowering the sample stage. The extension of the
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FIG. 3. An application of AFM to biological samples.
protein (E ) was determined by subtracting the cantilever deflection (d ) from the distance covered by the piezo movement (D). The tensile force was calculated by multiplying the cantilever deflection (d ) by the cantilever spring constant (k) and is expressed as a function of the elongation (E ). E¼D d F ¼ kd This technique uses an extremely sensitive AFM force sensor that is able to detect forces in the range of pico to nano Newtons. The relationship between
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the tensile forces versus the extension of the protein molecule is obtained in terms of a force-extension (F–E) curve. The force required to stretch a target protein leading to a complete breakdown of its native conformation into an unfolded one is then analyzed from the F–E curve. 3.2. AFM IMAGES OF SUBCELLULAR STRUCTURES IN RAT VOMERONASAL EPITHELIUM For AFM observations, the sample surface should be fairly smooth, with gently undulating hills corresponding to the structure of the surface. Although epoxy resin sections used for TEM (transmission electron microscope) have a smooth surface, they do not have sharp relief. There is a need to develop new methods to improve the resolution of surface structures (O1). The electron beam radiation method we have developed engraves the epoxy resin deeply, resulting in improved resolution of the subcellular structures (Fig. 4). After electron beam treatment, the resolution of the AFM images was greatly improved. Most of the subcellular structures observed in the TEM images, including the inner membrane of the mitochondria, ciliarystructure precursor body, junctional complexes between neurons and supporting cells, and individual microvilli, were now visible in the AFM images. The electron beam treatment appeared to melt the embedding resin, bringing subcellular structures into high relief. The result of this study suggests that electron beam etching of histological samples may provide a new method for the study of subcellular structures using AFM. 3.3. THE DISTRIBUTION OF TERMINAL GALNAC ON VOMERONASAL EPITHELIAL SECTIONS An AFM can also be used to measure forces between the probe tip and the sample surface. The distribution of sugar chains on the tissue section of rat vomeronasal epithelium, and the adhesive force between the sugar and its specific lectin were examined by AFM (O2). AFM tips were modified with a lectin, Vicia villosa agglutinin, that recognizes terminal N-acetyl-D-galactosamine (GalNAc). When the modified tip scanned the luminal surface of the sensory epithelium, adhesive interactions between the tip and the sample
FIG. 4. The electron beam etching method.
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surface were observed. Most of these adhesive interactions disappeared when GalNAc was added to the solution, which proved that the adhesion forces observed were related to the binding force between the lectin and the sugars distributed across the vomeronasal epithelium. The final rupture force between the lectin and the sugar chain was calculated to be about 50 pN, based on the spring constant of the AFM cantilever. The distribution patterns of sugar chains obtained by mapping the force were very similar to those observed with an optical microscope using fluorescence-labeled lectin staining, and the resolution was comparable or better. 3.4. A QUANTITATIVE MEASUREMENT OF ADHESION FORCE BETWEEN CELL ADHESION MOLECULES AND LIVING CELLS WITH AFM A microbead was used as an AFM tip to extend the contact area about 40 times larger than the conventional AFM tip, and it was modified with fibronectin. Based on the force curve measurement, the adhesion force between fibronectin and mouse fibroblast cells was calculated to be 400 to 500 pN. In the presence of 0.1 mg/ml gelatin in the scanning solution, the number of force curves with large adhesion force decreased. No large adhesion forces between the unmodified microbead and cells were observed. These results indicated that the adhesion force was due to specific interaction between the fibronectin and cell surface. We also carried out force curve mapping with a microbead. The pattern of adhesion force mapping was reproducible on six repeated runs. These results indicated that the system developed here would be a promising method to evaluate a quantitative analysis of cell adhesion (K1–K3).
4. mRNA Extraction from Living Cells Most cells can change their patterns of gene expression in response to extracellular stimuli. For example, fibroblast cells at G0 state change their gene expression pattern in response to various growth factors for cell proliferation. Relative mRNA levels of more than 500 genes change in response to stimulation (I3). The expression pattern of each gene shows diVerent profiles. For example, the mRNAs for immediate early genes are induced within 15 min after stimulation and are terminated in 20 min. In another case, nerve growth factor (NGF) can change the gene expression pattern of rat adrenal pheochromocytoma PC12 cells and induce morphological changes accompanying neurite outgrowth. Many events are common to cells before and after extracellular stimuli, which indicates many proteins are common to cells at diVerent stages. Some proteins are specifically induced with extracellular stimuli and determine the properties of individual cells. The diVerent combinations
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of protein expression result in structural and functional changes of individual cells. Although most of these events depend on diVerences in gene expression, no method is available to examine time-dependent gene expression of individual living cells. We have developed a novel method to examine gene expression of living individual cells using an AFM that was used as a manipulator to extract mRNAs from cells. The obtained mRNAs with AFM were analyzed with RT-PCR, nested PCR, and quantitative PCR. This method enabled us to examine time-dependent gene expression of individual living cells without serious damage to the cells. 4.1. PREPARATION OF CELLS Adherent cells were maintained in DMEM/F-12 culture medium supplemented with 10% heat-inactivated fetal bovine serum and 100 U/ml penicillin-100 g/ml streptomycin at 37 C and under 5% CO2. In our experiments, VNOf90 cells derived from rat vomeronasal organs were used. Two days before use, cells which were in a confluent state with a density of about 105/cm2 were released from wells using 0.25% trypsin-1 mM EDTA. A drop of cell solution (about 103 of the cells) was plated on the center of 35 mm tissue culture dishes and incubated for about an hour. After 1 h incubation, cells were weakly attached on dishes and the dish was then filled with culture medium. The cells were only located at the center of the dish when we performed mRNA extraction with the AFM. This procedure is quite important to decrease the probability of mRNA contamination, because dead or floating cells are one of the causes of contamination. Two hours before use, the cells were rinsed with culture medium without FBS two or three times, and the dish was filled with FBS-free culture medium. 4.2. AFM SETUP The experiments were carried out by an AFM (NVB-100, Olympus, Inc., Tokyo, Japan). The use of an AFM combined with an optical microscope and CCD camera is recommended, because these instruments greatly reduce the diYculties of the experimental procedure. For example, it is possible to easily find a target cell, mount an AFM tip onto the cell, and perform tip penetration by monitoring the CCD camera. 4.3. PRETREATMENT OF AFM INSTRUMENT AND TIP For the mRNA extraction experiment, one of the most important points is to prevent contamination, such as RNase or RNA molecules. To overcome this problem, careful washing of the instruments is critically important during the preparation of the experiment. The procedures are as follows.
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First, all the instrument parts were carefully wiped with absolute ethanol. Next, the AFM head was immersed into DNAZap (Ambion, Inc., Austin, TX), which destroys all of the DNA and RNA, for 15 min followed by washing with pure water (Fluka, Buchs SG, Switzerland) and ethanol at least two times, respectively. The AFM tips (NP, Digital Instruments, Santa Barbara, CA) were also washed with pure water and ethanol. 4.4. AFM OPERATION For the extraction of mRNAs from a living cell, the AFM was used as a nano-manipulator (O3). As described previously, the AFM tip makes direct contact with the sample, and thus it is possible to insert the tip into a cell body by increasing applied force. For the penetration, there are two kinds of methods: 1. An application of general force measurement 2. The use of a step motor. The former is useful for detailed quantitative analysis, such as the estimation of the amount of cell deformation; however, the applicable force is restricted by the range of piezo movement. To apply a stronger force, the latter method is more useful. A step motor is a coarse adjustment in the Z direction of an AFM, and is generally used for the approach of an AFM tip to the sample surface before imaging of the sample or measuring interaction forces using a piezo microscanner. In this study, we used the latter method, mainly to penetrate the AFM tip into a cell body to lift out mRNAs, and the former method was used to help with the analysis. The procedures of the step motor method are as follows: 1. An AFM tip is positioned on a cell. 2. The tip is approached using the step motor. 3. When the tip attaches to the cell surface, an AFM cantilever is deflected and the position of laser on the photo detector shifts. 4. The step motor stops at a suitable position where an appropriate loading force is applied. 5. After the adequate incubation time, the tip is retracted. A loading force can be estimated with continuous monitoring of the shift of the laser position on the photo detector. The detailed procedures are as follows: First, the AFM tip was washed with ethanol and water, and positioned on a target cell by monitoring the CCD camera that was combined with the AFM, as has been described (Fig. 5A). After locating the target cell, the AFM tip was carefully approached on the cell using the step motor. The
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FIG. 5. An AFM tip was positioned on a target cell by monitoring the CCD camera (A). After the appropriate incubation time, the tip was retracted from the cell. The arrow indicates a position where the AFM tip was inserted (B).
pitch of the step motor was about 0.5 m. When the tip approached the cell surface, the AFM cantilever was deflected. This was detected by monitoring the position of the laser on the optical detector. The tip penetrated into the cell body by applying additional force, and the penetration was stopped at a suitable position when the appropriate loading force was applied. The typical loading force was about 150 to 200 nN. This force was strong enough to reduce the possibility of a miss of penetration. After a suitable incubation time, the tip was retracted from the cell (Fig. 5B). The typical incubation time was 1 min, and the tip was immediately placed into a PCR tube. 4.5. PCR For the detection of rat -actin mRNA, we performed the following procedures: RT-PCR was performed with a one step RT-PCR kit (Qiagen, Valencia, CA), according to the kit’s instructions, with 0.2 l of each primer in a 50 l reaction volume. The sequences of the PCR primer pairs (50 to 30 ) that were used are as follows: rat -actin, 50 -primer 50 -TTGTAACCAACTGGGACGATATGG-30 and 30 -primer 50 -GATCCTTGATCTTCATGGTGCTAGG30 . First-strand cDNA synthesis was performed at 50 C for 30 min, at which time the reaction was heated to 95 C for 15 min to activate HotStrTaq DNA polymerase. The amplification reaction was carried out for 30 or 35 cycles, and each cycle consisted of 94 C for 45 sec, 55 C for 45 sec, and 72 C for 1 min, followed by a final 10 min elongation at 72 C. In single cell assay, RTPCR reaction buVer contains an AFM tip (NP, Digital Instruments, Santa Barbara, CA) (Fig. 6). The NP tip itself showed no eVect against reaction for
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FIG. 6. Principal features of the experimental procedure. A target locus of a cell was positioned underneath the AFM tip, and then the AFM tip was lowered onto the cell and inserted into it. It was then held for approximately 60 s to allow the tip to bind the cell ingredient containing mRNA with physical absorption. The tip was then lifted oV the cell and placed into a PCR tube.
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RT-PCR. In addition, the AFM tip contains not only cell components but also a small amount of culture medium. This also does not aVect RT-PCR. Nested PCR was carried out for 30 cycles, and each cycle consisted of 94 C for 45 sec, 55 C for 45 sec, and 72 C for 1 min, followed by a final 10 min elongation at 72 C. A 5 l volume of the reaction product from the first round was transferred to 45 l of a second round mix for the 2nd round. The sequences of the PCR primer pairs (50 to 30 ) that were used are as follows: rat -actin, 50 -primer 50 -AAGATTTGGCACCACACTTTCTAC-30 and 30 -primer 50 ACACTTCATGATGGAATTGAATGT-30 . PCR products were visualized on ethidium bromide-stained 1% agarose gels and then photographed. Quantitative PCR was performed with an Applied Biosystem Prism 7000 sequence detection system and the SYBR Green I PCR Mastermix (Qiagen, CA, USA), according to the kit’s instructions, with 0.6 l of each primer in a 50 l reaction volume. A 1 l volume of the reaction product from the first round was transferred to 49 l of the second-round mix for the 2nd round. The sequences of the PCR primer pair (50 to 30 ) that were used are as follows: rat -actin, 50 -primer 50 -GTAGCCATCCAGGCTGTGTT-30 and 30 -primer 50 -CCCTCATAGATGGGCACAGT-30 . The PCR cycling conditions were 50 C for 2 min, 95 C for 15 min, and 40 cycles of 94 C for 15 sec, 55 C for 30 sec, and 72 C for 45 sec. Standard curves were generated by using DNA fragments from rat -actin (Toyobo, Osaka, Japan) at 4-fold intervals. Amplification plots and predicted Ct values from the exponential phase of the PCR were analyzed with ABI Prism 7000 SDS software. 4.6. EXPRESSION OF -ACTIN mRNA In our previous paper (O3), we examined the -actin and fibronectin mRNAs expression of rat fibroblastlike VNOf90 cells. We also examined time-dependent gene expression of individual living cells. The response of rat VNOF90 cells to serum has been used as a model for studying the changes in c-fos gene expression (Fig. 7). These results demonstrated that time-dependent gene expression patterns in single cells are not always similar to those in large cell populations. At the single-cell level, gene expression patterns sometimes fluctuate up and down. In another paper (U1), we examined the distribution of -actin mRNA in VNOf90 cells. The number of -actin mRNA in the cell was estimated to be about 12,000 copies per cell. AFM tips inserted into the single living cells were covered with cell ingredients under SEM observation. If we inserted an AFM tip into the vicinity of the nucleus, we could always detect PCR products of -actin mRNA from the inserted tips. The -actin mRNA was only detected in the peripheral region of the cells when they migrated
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FIG. 7. The single cell analysis. Time dependent mRNA expression of single living cells was examined in two diVerent conditions (a and b). The mRNA expression of three individual cells was examined in each condition. The ordinate represents the number of initial mRNA copies of c-fos divided by those of -actin. The data shown here were modified with the data from our previous paper (28).
FIG. 8. The detection of -actin mRNA from several loci of a single cell. The cells shown in (a) and (b) were migrating. -actin mRNA was detected in the peripheral region as well as in the vicinity of the nucleus. -actin mRNA of the cell that was not migrating (c) was detected only in the vicinity of the nucleus. White arrows indicate loci where we could detect -actin mRNA. Black arrows indicate loci where we could not detect -actin mRNA.
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(Fig. 8) -actin mRNAs are reported to be localized to the leading edge of the cells when the cells start to migrate (K4, L1).
5. Modification of AFM Tips Our method using an AFM can be applied to extracting other biomolecules as well as mRNA from living cells. When we try to identify specific biomolecules on the cell surface, we can use modified AFM tips that interact with our interest. We introduced here the method for AFM tip modification (Fig. 9). 5.1. MODIFICATION OF AFM TIPS WITH AMINO GROUPS 1. The silicon or silicon nitride tips were cleaned with ethanol for 5 min, followed by rinsing with Milli-Q water for 5 min. 2. They were treated with a freshly prepared acidic mixture (H2O2:H2SO4 (v/v) ¼ 7:3) for 15 min, followed by rinsing with Milli-Q water for 5 min. 3. They were dried in a vacuum for 10 min to remove the water layer on the surface. 4. They were treated with 2% 3-aminopropyltriethoxysilane (APTES) in dry toluene for 2 h. 5. They were rinsed with toluene, ethanol, and Milli-Q water for 3 min in each case. 6. They were dried in a vacuum for 1 h and then stored in a desiccator until used. APTES reacts with-OH groups and introduces-NH2 groups on the tip surface, as shown in Fig. 10. The APTES-tip can interact with negatively charged molecules and also can be used for further modification of the tip. When other groups on the tip are needed, another silanization reagent can be chosen. For example, 3-mercaptopropyltriethoxysilane (MPTS) introduces-SH groups on the tip surface. 5.2. MODIFICATION OF AFM TIPS WITH CROSS LINKERS Amino-reactive AFM tip with Disuccinimidyl suberate (DSS): 1. The APTES-modified AFM tip (APTES-tip) was treated with 2 mM DSS dissolved in DMSO (Dimethyl Sulfoxide)/ethanol (1:1) for 1 hour. 2. It was rinsed with ethanol and Milli-Q water for 3 min in both cases.
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Amino-reactive AFM tip with glutaraldehyde: 1. The APTES-modified AFM tip (APTES-tip) was treated with 1% glutaraldehyde for 10 min. 2. It was rinsed with Milli-Q water for 5 min. DSS is a homobifunctional cross linker which has two NHS ester groups reactive to the amino group ( NH2). One of the NHS ester groups is used to attach to the NH2 group on the tip and the second one is for the NH2 group on the biomolecules. Glutaraldehyde is also a bifunctional cross linker. It reacts with the SH group as well as the NH2 group. The SH reactive AFM tip with Sulfosuccinimidyl 6-[30 -(2-pyridyldithio)-propionamido] hexanoate (Sulfo-LC-SPDP): 1. The APTES-modified AFM tip (APTES-tip) was treated with 2 mM Sulfo-LC-SPDP dissolved in DMSO (Dimethyl Sulfoxide)/ethanol (1:1) for 1 h. 2. It was rinsed with ethanol and Milli-Q water for 3 min in both cases. Sulfo-LC-SPDP is a heterobifunctional cross linker which has two reactive groups for the NH2 and SH groups. 5.3. MODIFICATION OF AFM TIPS WITH PROTEINS OR DNA 1. A DSS- or glutaraldehyde-modified AFM tip was treated with 10–100 g/ml proteins or 1–10 g/ml NH2-oligoDNA solution for 1 h. The sample was dissolved in PBS. It was necessary that the sample solution contained no NH2 groups. 2. It was rinsed with PBS for 10 min and then reacted with 20 mM glycine in PBS for 1 h to block reactive cross linkers on the tip. 3. It was rinsed with PBS for 10 min. Since there is the possibility that some of the reactive groups of the cross linkers on the tip do not attach to the protein and remain intact, we had to eliminate their reactivity. In order to do this, we used glycine (containing NH2 groups) solution as blocking reagent. 5.4. A MICROBEAD AS AN AFM TIP To increase the contact area between the sample and the tip of the AFM, a microbead was used as an AFM tip. We introduced here the preparation of the microbead attached to the AFM cantilever. In our study, a carboxylated polystyrene microbead (Polybead Carboxylate Microsphere, r ¼ 5 m, Polyscience, Inc., Warrington, PA) was used.
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This bead is ideal for the immobilization of proteins or as a size reference, because the bead is a series of monodispersed polystyrene particles, and carboxyl groups are exposed on the bead surface. 5.4.1. Preparation of Microbeads A drop of the microbead solution was diluted with water and gently mixed to wash the beads. The beads were collected using a centrifuge with the force of about 50 g. This procedure was repeated three times. After washing, beads were suspended in ethanol solution. 5.4.2. Mounting of the Microbead on the AFM Cantilever A hot plate was warmed up to 70 C and three glass plates were put onto the hot plate. An AFM cantilever, epoxy resin (Yuka-Shell, Inc., Japan, melting point: 64 C), and a drop of microbead solution were put onto the warmed glass plates, respectively. Ethanol of the bead solution was immediately evaporated and epoxy resin was melted. A small amount of
FIG. 9. Modification of AFM tips.
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FIG. 10. Silane coupling method; R: functional group; OX: hydrolyzable group forming covalent bond with hydroxyl group.
molten epoxy was taken by immersing a glass pipet (GD-1, Narisinge Co.), which was mounted on micromanipulators (MMB-1, MMO-202ND, and MN-153, Narisinge Co., Tokyo, Japan), into the melted epoxy, and placed on the end of the cantilever. A microbead was picked up using another glass rod and placed on the epoxy-coated spot on the cantilever, and the temperature was allowed to fall to room temperature. 5.4.3. Immobilization of Proteins on the Microbead Protein was covalently modified to the microbeads as described here: The carboxylated surface of the microbeads reacted with 10 mg/mL 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, Pierce, Rockford, IL) and 10 mg/ml N-hydroxysulfosuccinimide (Pierce) in pH 6.0 PBS for 15 min by covering the beads with the solutions. In this step, hydrophobic dishes (for example, an Iwaki 35 mm nontreated dish, Scitech, Chiba, Japan) were used to make a small drop of solution. After washing with PBS at pH 6.0, the beads reacted with our choice of protein (e.g., antibody) in pH 7.4 PBS for 2 h and then washed with pH 7.4 PBS. A typical concentration of the protein was 0.1 mg/ml. After the reaction, 20 mM of glycine in pH 7.4 PBS was mounted on the beads and incubated for 30 min to block the cross linkers on the beads.
REFERENCES B1. Binnig, G., Quate, C., and Gerber, C., Atomic force microscope. Phys. Rev. Lett. 56, 930–933 (1986). C1. Charras, G. T, Lehenkari, P. P., and Horton, M. A., Atomic force microscopy can be used to mechanically stimulate osteoblasts and evaluate cellular strain distributions. Ultr‘amicroscopy 86, 85–95 (2001). C2. Chen, C. H., and Hansma, H. G., Basement membrane macromolecules: Insights from atomic force microscopy. J. Struct. Biol. 131, 44–55 (2000). D1. Dammer, U., Hegner, M., Anselmetti, D., et al., Specific antigen/antibody interactions measured by force microscopy. Biophys. J. 70, 2437–2441 (1996).
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E1. Evans, E., and Ritchie, K., Strength of a weak bond connecting flexible polymer chains. Biophys. J. 76, 2439–2447 (1999). F1. Fotiadis, D., Scheuring, S., Muller, S. A., Engel, A., and Muller, D. J., Imaging and manipulation of biological structures with the AFM. Micron. 33, 385–397 (2002). G1. Gergely, C., Voegel, J., Schaaf, P., et al., Unbinding process of adsorbed proteins under external stress studied by atomic force microscopy spectroscopy. Proc. Natl. Acad. Sci. USA 97, 10802–10807 (2000). H1. Haga, H., Sasaki, S., Kawabata, K., Ito, E., Ushiki, T., and Sambongi, T., Elasticity mapping of living fibroblasts by AFM and immunofluorescence observation of the cytoskeleton. Ultramicroscopy 82, 253–258 (2000). I1. Ikai, A., Nanomechanics of proteins with applications. Superlattices and Microstructures 31, 43–62 (2002). I2. Ikai, A., Idiris, A., Wang, T., et al., Nanotechnology and protein mechanics. J. Biol. Phys. 28, 561–572 (2002). I3. Iyer, V. R., Eisen, M. B., Ross, D. T., et al., The transcriptional program in the response of human fibroblasts to serum. Science 283, 83–87 (1999). K1. Kim, H., Arakawa, H., Osada, T., and Ikai, A., Quantification of cell adhesion interactions by AFM: EVects of LPS/PMA on the adhesion of C6 glioma cell to collagen type I. Appl. Surf. Sci. 188, 489–492 (2002). K2. Kim, H., Arakawa, H., Osada, T., and Ikai, A., Quantification of fibronectin and cell surface interactions by AFM. Colloid Surf. B. Biointer. 25, 33–43 (2002). K3. Kim, H., Arakawa, H., Osada, T., and Ikai, A., Quantification of cell adhesion force with AFM: Distribution of vitronectin receptors on a living MC3T3-E1 cell. Ultramicroscopy 97, 359–363 (2003). K4. Kislauskis, E., Zhu, X., and Singer, R., Beta-actin messenger RNA localization and protein synthesis augment cell motility. J. Cell. Biol. 136, 1263–1270 (1997). L1. Lawrence, J., and Singer, R., Intracellular localization of messenger RNAs for cytoskeletal proteins. Cell 45, 407–415 (1986). L2. Lin, H., Lal, R., and Clegg, D. O., Imaging and mapping heparin-binding sites on single fibronectin molecules with atomic force microscopy. Biochemistry 39, 3192–3196 (2000). M1. Marshall, B. T., Long, M., Piper, J. W., Yago, T., McEver, R. P., and Zhu, C., Direct observation of catch bonds involving cell-adhesion molecules. Nature 423, 190–193 (2003). M2. McElfresh, M., Baesu, E., Balhorn, R., Belak, J., Allen, M. J., and Rudd, R. E., Combining constitutive materials modeling with atomic force microscopy to understand the mechanical properties of living cells. Proc. Natl. Acad. Sci. USA 99, 6493–6497 (2002). M3. Muller, D. J., Fotiadis, D., Scheuring, S., Muller, S. A., and Engel, A., Electrostatically balanced subnanometer imaging of biological specimens by atomic force microscope. Biophys. J. 76, 1101–1111 (1999). M4. Muller, D. J, Hand, G. M., Engel, A., and Sosinsky, G. E., Observing structure, function, and assembly of single proteins by AFM. Prog. Biophys. Mol. Biol. 79, 1–43 (2002). O1. Osada, T., Arakawa, H., Ichikawa, M., and Ikai, A., Atomic force microscopy of histological sections using a new electron beam etching method. J. Microsc. 189, 43–49 (1998). O2. Osada, T., Takezawa, S., Itoh, A., Arakawa, H., Ichikawa, M., and Ikai, A., The distribution of sugar chains on the vomeronasal epithelium observed with the atomic force microscope. Chem. Sens. 24, 1–6 (1999).
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O3. Osada, T., Uehara, H., Kim, H., and Ikai, A., mRNA analysis of single living cells. J. Nanobiotechnol. 1, 2 (2003). S1. San Paulo, A., and Garcia, R., High-resolution imaging of antibodies by tapping-mode atomic force microscopy: Attractive and repulsive tip-sample interaction regimes. Biophys. J. 78, 1599–1605 (2000). S2. Sasaki, S., Monimoto, M., Haga, H., Kawabata, K., Ito, E., Ushiki, T., Abe, K., and Sambongi, T., Elastic properties of living fibroblasts as imaged using force modulation mode in atomic force microscopy. Arch. Histol. Cytol. 61, 57–63 (1998). S3. Schwesinger, F., Ros, R., Strunz, T., et al., Unbinding forces of single antibody–antigen complexes correlate with their thermal dissociation rates. Proc. Natl. Acad. Sci. USA 97, 9972–9977 (2000). U1. Uehara H, Osada T. and Ikai A. Quantitative measurement of mRNA at diVerent loci within an individual living cell. Ultramicroscopy, 2004 (in press). U2. Umemura, K., Komatsu, J., Uchihashi, T., et al., Atomic force microscopy of RecA–DNA complexes using a carbon nanotube tip. Biochem. Biophys. Res. Comm. 281, 390–395 (2001). V1. Vie, V., Giocondi, M. C., Lesniewska, E., Finot, E., Goudonnet, J. P., and Le Grimellec, C., Tapping-mode atomic force microscopy on intact cells: Optimal adjustment of tapping conditions by using the deflection signal. Ultramicroscopy 82, 279–288 (2000).
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LETTER TO THE EDITOR Dear Sir, I read with great interest an article by Dr. Bartosz published in the recent issue of the Journal (B2). This review article covers in detailed and comprehensive manner the contribution of exo- as well as endogenous substances to the total antioxidant capacity (TAC). Although excellent in many respects, this article doubts and misinterprets the role of increased TAC caused by elevated serum bilirubin levels in subjects with Gilbert syndrome (GS) characterized with mild benign hyperbilirubinemia. First, as a consequence of decreased bilirubin conjugation in the liver tissue, unconjugated bilirubin is the only diVerent primary biochemical parameter in GS. For a long time bilirubin was believed to be only a waste product, however, this bile pigment exhibits potent antioxidant capacity. Indeed, bilirubin was demonstrated in in vitro study by Wu and colleagues to be almost 30 times more eYcient in preventing LDL oxidation than Trolox, a water-soluble analogue of alpha-tocopherol (W1). Benign hyperbilirubinemia was recently proved to be associated with protection against development of coronary heart disease (V1). Although increased TAC may have under specific conditions a negative prognostic value with respect to the risk of atherosclerosis as described for example in patients with subclinical carotid atherosclerosis hyperuricemia (N1), the elevation of serum bilirubin in GS subjects is primary and not secondary to oxidative stress burden such as in patients with elevation of serum uric acid (N1). What is thus the relationship between TAC and elevated serum bilirubin levels? In particular, it should be noted that elevation of TAC by increased bilirubin levels is several times higher than might be simply expected from molar ratio. We have proved in our in vitro study (V1) that addition of unconjugated bilirubin to serum with defined TAC results in 3,7-6,8 fold higher molar increase of TAC depending on bilirubin concentration. These in vitro results are in accord with data by Gopinathan et al. who revealed similar relationship in their in vivo study on newborn hyperbilirubinemia (G1). More interestingly, the role of bilirubin as an antioxidant of physiological importance was suggested by results of Baranano and colleagues who demonstrated that as little as 10 nM bilirubin may protect neuronal cultures against almost 10000-fold higher concentration of H2O2, presumably due to amplification of biliverdinbilirubin redox cycle (B1,D1). 259
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Probably the most important argument in discussion about bilirubin and atherosclerosis is based on results of our recent meta-analytic study on the relation between serum bilirubin levels and ischemic heart disease (N2). Using three diVerent statistical approaches we were able to demonstrate unanimous inverse relation between both variables. In general, the role of bilirubin in the pathogenesis of oxidative stressmediated disease is underestimated. It is certain that further studies are needed to elucidate all molecular mechanisms of bilirubin-mediated protection against these disorders as well as its epidemiological consequences. References B1. Baranano, D. E., Rao, M., Ferris, C. D., Snyder, S. H., Biliverdin reductase: A major physiologic cytoprotectant. Proc. Natl. Acad. Sci. USA 99, 16093–16098 (2002). B2. Bartosz, G., Total antioxidant capacity. Adv. Clin. Chem. 37, 219–292 (2003). D1. Dore, S., Takahashi, M., Ferris, C. D., Zakhary, R., Hester, L. D., Guastella, D., Snyder, S.H., Bilirubin, formed by activation of heme oxygenase-2, protects neurons against oxidative stress injury. Proc. Natl. Acad. Sci. USA 96, 2245–2250 (1999). G1. Gopinathan, V., Miller, N. J., Milner, A. D., Rice-Evans, C. A., Bilirubin and ascorbate antioxidant activity in neonatal plasma. FEBS Lett. 349, 197–200 (1994). N1. Nieto, F. J., Iribarren, C., Gross, M. D., Comstock, G. W., Cutler, R. G., Uric acid and serum antioxidant capacity: A reaction to atherosclerosis? Atherosclerosis 148, 131–139 (2000). N2. Novotny´, L., Vitek, L., Inverse relationship between serum bilirubin and atherosclerosis in men: A meta-analysis of published studies. Exp. Biol. Med. 228, 568–571 (2003). V1. Vı´tek, L., Jirsa, Jr., M., Brodanova´, M., et al. Gilbert Syndrome and Ischemic Heart Disease: A Protective EVect of Elevated Bilirubin Levels. Atherosclerosis 16, 449–456 (2002). W1. Wu, T. W., Fung, K. P., Yang, C. C., Unconjugated bilirubin inhibits the oxidation of human low density lipoprotein better than Trolox. Life Sci. 54, 477–481 (1994).
Libor Vı´tek, MD, PhD Institute of Clinical Biochemistry and Laboratory Diagnostics, and 4th Department of Internal Medicine 1st Medical Faculty, Charles University, Prague Czech Republic e.mail:
[email protected]
INDEX A AChE, see also Cholinesterase brain, 176 BuChE and, 168–169 deakylated inhibited, 172–173 determination of activity of, 173–174 erythrocyte activity of, 174, 179 functional role of, 176 inhibition of, 164–165, 168 inhibition of brain and, 158–160 inhibitors on, 174–175 nerve agent/OP primary action site as, 156 prophylaxis protecting against inhibition of, 186–188 reactivation of OP-inhibited, 181–183 reactivity, 193–194 schematic structure of, 169, 170 subcellular localization of, 175–176 AFM, see Atomic force microscope Allosteric ligands, 171–172 Alzheimer’s disease, 174, 217 Amino acids excitatory, 158 mass spectroscopy identified oxidides, 18 Aneurysms, MMPs/TIMPs in, 69–70 Antibodies array with diagnostic potential, 233 against OP, 188 OxLDL identification and, 26 polyclonal, 227–228 as protein ligands, 227 as protein microarrays’ capture agents, 232 rheumatoid arthritis patients treated with, 61–62 against soman, 180 Anti-cancer drugs, micrometastases as chemosensitive to, 88 Anticholinergies, for OP/nerve agent poisoning treatment, 190–191 Anticonvulsants, 195–196 Apolipoproteins, oxidation of, 16–17
Arteriosclerosis lipoproteins’ oxidation as cause of human, 23–25 oxidation as cause of, 4–5 oxidation theory of, 2, 3–4 OxLDL and, 20 sites of, 2 Arthritis, see also Osteoarthritis; Psoriatic arthritis; Rheumatoid arthritis; Scleroderma arthritis blood MMP levels distinguishing types of, 62 Atomic force microscope (AFM) adhesion force between cell adhesion molecules/living cells with, 245 amino groups modification of tips of, 252 biological material manipulations with, 242–245 biomolecules manipulation and, 240 construction of, 240, 241 cross linkers modification of tips of, 252–253 fine curve of, 241–242 gene expression of living cells using, 246–249, 250, 251, 252 microbead mounting on cantilerer of, 254–255 microbeads as tip of, 253–254 microbeads’ preparation for, 254 mRNA extraction from cells pretreatment of, 246–247 operation of, 247–248 protein-unfolding experiment with, 242–244 protiens/DNA modification of tips of, 253 subcellular structures images, 244 terminal GalNAc distribution and, 244–245 tips modification, 252–255 use of, 240–242 261
262
INDEX B
Baseline diene conjugation (BDC), in LDL, 15, 22–23 Basic fibroblast growth factor (bFGF), angiogenesis role of, 88 BDC, see Baseline diene conjugation bFGF, see Basic fibroblast growth factor BHT, see Butylated hydroxytoluene Biomarkers ELISA detected protein, 225 estrogens as possible, 136, 138, 144–146 HBV, 222 signature discovery tools for, 219–220 Biomolecules, AFM for manipulation of, 240 Blood cholinesterases determination in, 178–179 circulating tumor cells in peripheral, 95–97 MMPs/TIMPs origin in, 47–48 tyrosinase mRNA in peripheral, 96 Body fluid(s), see also Urine assays for MMPs/TIMPs measurement in, 45–48 free tumor cells in, 87 Breast cancer estrogen levels of women with, 139, 140, 141, 142 estrogens in etiology of human, 135–136 LC/MS/MS techniques and, 146 plasma MMPs/TIMPs levels in, 53–56 Burns, 71 Butylated hydroxytoluene (BHT), 11 C Cancers, see also Breast cancer detecting, 223 early stage detection of, 217 EMMPRIN produced by, 42–43 estrogen initiated, 138, 143–144 estrogens as possible biomarkers for susceptibility for, 136, 138, 144–146 MMP levels in, 42–43 MMP-1/TIMP-1 and, 57–58 MMP-2 levels in patients with, 56, 59
MMP-7 and, 59 MMP-9 levels in patients with, 56 MMPs identified in urine of patients with, 71–73 peritoneal dissemination as prognostic indicator of survival of patients with, 93, 95 plasma MMP-9 levels of patients with, 54–55 plasma MMPs/TIMPs levels in, 53–59 TIMP levels and, 43 TIMP-1 levels of patients with, 54 tumor recurrence after curative surgery for patients with, 87–88 Carbamates, 174–175 Carbonyls formation of, 17–18 measurement of formation of, 18, 19 Carcinoma embryonic antigen (CEA), 39 Cardiac disease, 69–70 Catechol estrogens, see Estrogen(s) CEA, see Carcinoma embryonic antigen Chemotherapy, see also Anti-cancer drugs adjuvant, 88 micrometastases targeted by, 87–89 Cholesterol, oxidation and, 16 Cholinesterase, see also AChE AChE/BuChE division of, 167–169 activity decrease of, 181 determination in blood, 178–179 inhibition of, 164, 172 inhibitors, 167–176 inhibitors diminishing AChE/BuChE activities, 174–175 intoxication with OP/nerve agents diagnosis methods monitoring, 177–179 metal cations monitoring activity of, 175 methods for determination of activity of, 170–171 OP inhibition by, 154–157 Ciphergen system, 223 Collagens, disease role of, 39 Congestive heart failure, MMP/TIMP levels in, 69 Conjugated dienes, formation of, 9, 13 Coronary artery bypass graft (CABG), fibrin zymography of PAs from patients of, 123–124 Curative surgery, tumor recurrence after, 87–88
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INDEX D Diagnostic signatures clinically useful, 218–223, 224, 225 detection platforms in clinic, 223, 225 DNA, 220–221 ELISA quantified, 218 obtaining, 219 peptides and, 223 protein, 220–223 recognizing, 218–219 SELDI-TOF for, 222 signature discovery tools for, 219–220 Dinitrophenylhydrazine (DNPH), 19 Diseases Alzheimer’s, 174 biomarkers for diagnosing, 218 cardiovascular, 43–44, 67–70 detection of, 217–218 diagnostic signatures for diagnosing, 218 ELISA testing of, 38–39 genome analysis and, 219 inflammatory, 43, 59–64 liver, 43 lung, 44 MMP-3 levels and, 60 MMP-inhibitory drugs for, 62–63 MMPs/TIMPs in blood levels of, 48, 49–51, 57–70 MMPs/TIMPs increased levels associated with, 70–71 myeloproliferative, 71 of nervous system, 65–67 pathophysiology of, 42–45 protein measurement for recognition of, 38 proteins associated with, 221–223 Disuccinimidyl suberate (DSS), 252–253 DNA AFM tips modified by, 253 diagnostic signatures, 220–221 microarrays, 220–221, 223, 225 DNPH, see Dinitrophenylhydrazine Donepezil, 174 DSS, see Disuccinimidyl suberate
EMMPRIN, see Extracellular matrix metalloproteinases inducer Endothelial cells arteriosclerosis and impairment of, 2 NO produced, 6 vasiodilatory NO released by, 2–3 Enzyme-linked immunosorbent assay (ELISA) biomarkers quantified by, 218 development of, 45 disease testing by, 38–39 MMP-9, 54 PA/PAI system monitored by, 113–114 protein biomarkers detected by, 225 prototype sandwich, 53 Enzymes inhibition of, 171 of MMP family, 39, 40 OP detoxification with, 188 Estrogen(s) 4-hydroxylated estrogens role in genotoxic properties of, 136 analysis of metabolites/conjugates/ depurinating DNA adducts of, 139, 141 as biomarkers for cancer susceptibility, 136, 138, 144–146 breast cancer patients’ levels of, 139, 140, 141, 142 cancer initiated by, 138, 143–144 catechol, 141, 142 metabolism of, 136, 137, 138 MS/MS techniques and, 144–146 tumor initiation mechanisms by, 135–136 Estrogen-DNA adducts, depurinating, 143–146 Estrogens, analysis of metabolites/conjugates/ depurinating DNA adducts of, 139, 140, 141–146 Extracellular matrix metalloproteinases inducer (EMMPRIN) cancer cells producing, 42–43 lung disease and, 44 F
E Electrophiles, molecules oxidized by, 6 ELISA, see Enzyme-linked immunosorbent assay
F2-Isoprostanes (PGF), 11 Fatty acids oxidation products of, 9–10 phases of oxidation of, 14
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INDEX
Ferrous ion oxidation (FOX) FOX 2 reagent, 21–22 in xylenol orange’s presence, 16 Fibrin deposition, wounds’ normal healing controlled by, 112 Fibrin overlay zymography, see Overlay zymography FOX, see Ferrous ion oxidation (FOX) FOX 2 reagent, plasma peroxides using, 21–22
Interstitial collagenase, identification of, 39 Intoxication of OP, 152–153 OP’s intoxication’s toxicoynamics/ toxicokinetics, 155–161 symptoms of OP, 164–166 Isolated tumor cells, see also Micrometastases micrometastases v., 88 L
G Gastrointestinal cancer, plasma MMPs/ TIMPs levels in, 53–56 Genetic codes, individualized tailor-made therapy/knowledge of human, 240 Genetic immunization, process of, 227–228 Genitourinary cancers, plasma MMP/TIMP levels in, 57–59 Glucose intolerance, arteriosclerosis and, 2 Gulf War Syndrome, 167 H HBV, see Hepatitis B virus Head cancer, plasma MMPs/TIMPs levels in, 53–56 Heart transplants, MMPs/TIMPs in, 69 Hepatitis B virus (HBV), 222 Hydroxyoctadecanoic acid (HODEs), measuring, 11 Hypercholesterolemia, arteriosclerosis and, 2 Hypertension arteriosclerosis and, 2 MMP/TIMP levels in, 70 I IDL, see Intermediate density lipoproteins Immunoassays for MMPs/TIMPs measurement in body fluids, 45–48 techniques, 38–39 Immunochemistry, micrometastases in lymph nodes detected by, 89–90, 100–101 Inflammatory bowel disease, 64 Inflammatory disease, see also Diseases MMP/TIMP levels in blood of patients with, 59–64 Intermediate density lipoproteins (IDL), 15
LDL, see Low density lipoproteins Leukocytes, radicals produced by, 6 Linoleic acid, see PUFA Lipids oxidation products of, 8–20 peroxidation of, 9, 11, 13, 25, 157 Lipoproteins, see also Oxidized low density lipoproteins arteriosclerosis and oxidation of, 2, 23–27 mediation of oxidation of, 6 peroxidation of lipids in, 8 PUFA oxidation in, 8, 9 specificity for, 5 Living cells, mRNA extraction from, 245–249, 250, 251, 252 Low density lipoproteins (LDL) arteriosclerosis and, 2 BDC in, 15, 22–23 extraction of, 22 migration of, 19 native, 5 in oxidation, 2, 3 proathergenic eVects of, 2–3 Lung cancer, plasma MMPs/TIMPs levels in, 56–57 Lymph nodes micrometastases detected by immunochemistry in, 89–90, 100–101 micrometastases in, 97, 100–103 M Macroscopic metastasis, micrometastases v., 88–89 Malodialdehyde (MDA) measuring, 12–13 as nonenzymatic lipid proxidation products, 12
INDEX Malondialdehyde-LDL (MDA-LDL), measurement of, 19–20 Mass spectroscopy (MS) MALDI-TOF, 222, 224 oxidized amino acids/identified, 18 proteins associated with diseases detected by, 221 Matrix metalloproteinases (MMPs) activation of, 41 in angina/myocardial infarction, 67–68 anti-tumor necrosis factor- therapy on, 61–62 arthritis types distinguished by blood levels of, 62 biology/chemistry of, 39–42 blood levels in physiologic/disease states of, 48, 49–51, 52–70 in body fluids assays for measurement, 45–48 cancer and levels of, 42 cardiovascular disease’s levels of, 67–70 cardiovascular disease’s role of, 43–44 in demyelination process, 66 diseases associated with increased levels of, 70–71 enzymes of, 39, 40 gelatin zymography and, 71 in inflammatory disease patient’s blood, 59–64 inflammatory/liver disease and, 43 inhibitors of, 41–44 inhibitory drugs, 62–63 levels in liver disease, 64–65 lung/nervous system diseases and, 44 measurement of, 38, 55 in nervous system diseases, 65–67 origin in blood of, 47–48 in pathophysiology of disease, 42–45, 73–74 pitfalls in measurement of blood levels of, 46–47 plasma levels of, 55–56 plasma measurement of, 65–66 pregnancy/levels of plasma, 52–53 radiotherapy on measurements of, 57 sequence motifs in structure of, 40 serum/plasma, 59 social habits eVect on blood levels of, 52 structure of, 39–40, 41
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in urine of cancer patients, 71–73 in vivo activity of, 41 wounds and, 44–45 MDA-LDL, see Malondialdehyde-LDL MECA, see Mixed-element capture agent Melanoma, see also Cancers MMPs/TIMPs and, 59 Metabolism of estrogen, 136, 137, 138 nerve agents influence on oxidative, 156–157 Metastasis, see also Macroscopic metastasis MMPs’ role in, 53 Microarrays antibodies as capture agents for protein, 232 clinical diagnostic potential of proteindetecting, 234 DNA, 220–221, 223, 225 protein-deleting, 225–227 protein-detecting, 225–234 recent applications of protein-detecting, 232–234 Micrometastases, see also Isolated tumor cells body compartment/tumor origin of, 101–102 as chemosensitive to anti-cancer drugs, 88 chemotherapy targeting, 87–89 defining, 88 detection of, 89 immunochemical method for detection of, 89–90, 100–101 in lymph nodes, 97, 100–103 macroscopic metastasis v., 88–89 methodology of detection of, 89–92 molecular biological method for detection of, 90–92 prognostic significance of quantitative detection of, 92–93, 94, 95–97, 98–99, 100–101 real-time RT-PCR detection of, 92–93, 94 tumor recurrence after curative surgery and, 87 Mixed-element capture agent (MECA), 231 MMPs, see Matrix metalloproteinases mRNA -actin, 250, 251, 252 CEA, 97 cell preparation for extraction from, 246 diagnostic signatures obtained by measuring, 219
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INDEX
mRNA (cont.) extraction from living cells, 245–249, 250, 251, 252 tyrosinase, 96 MS, see Mass spectroscopy Myeloperoxidase, as oxidation source, 18 N Neck cancer, plasma MMPs/TIMPs levels in, 53–56 Nerve agents AChE as primary site of action of, 156 anticholergenics for treatment of poisoning with, 190–191 antidotal treatment resistance of, 187 eVects of, 166–167 future trends for, 196–197 oxidative metabolism influenced by, 156–157 reactivators treatment for poisoning with, 191–194 treatments for poisoning with, 190–196 Nervous system diseases cholinergic, 174 MMPs/TIMPs in, 44, 65–67 Nitric oxide (NO) endolethial cells producing, 6 endolethial cells release of vasiodilatory NO, 2–3 as vascular tone regulator, 6 Nitrogen species (RNS), production of, 6, 7 O OPIDN, see Organophosphate Induced Delayed Neurotoxicity Organophosphate Induced Delayed Neurotoxicity (OPIDN), 165–166 Organophosphates (OP), see also Phosphorous AChE and poisoning of, 185 AChE as primary site of, 156 AChE reactivation in patients poisoned with, 182 AChE/BuChE and poisoning of, 178 antibodies against, 188 anticholergenics for treatment of poisoning with, 190–191 antidotes for poisoning with, 189
basic actions of, 158, 159 biochemical changes and poisoning of, 184–185 carbonates protection of organism poisoned with, 187–188 chemistry of, 153–155 cholinesterase inhibition of, 156–157 cholinesterase inhibitors’ compound groups, 154–155 as cholinesterases inhibitors, 172 compound groups of, 153 detoxification of, 188 diagnosis of poisoning of, 176–179 direct determinations of, 180 eVects of, 152 exposure to, 181 future trends for, 196–197 inhibition eYcacy/toxicity of diVerent, 164 intoxication symptoms of, 164–166 intoxications, 152–153 LD50 and, 161–162, 163 lipid peroxidation influenced by, 157 metabolites, 185 as mutagenic/carcinogenic, 160 nerve agent poisoning and, 197–198 OPIDN and, 165–166 oximes in treatment of poisoning of, 192 pesticides of, 154, 180 poisoning’s severity, 184 prophylaxis for, 186 prophylaxis with other drugs treating poisoning of, 189–190 reactions of, 156 reactivators treatment for poisoning with, 191–194 specificity/sensitivity of intoxication with, 182–185 toxicity/poisoning, 157–158, 161–164 toxicoynamics/toxicokinetics of intoxication of, 155–161 treatments for poisoning with, 190–196 Osteoarthritis, MMP/TIMP levels in blood of patients with, 60–62 Overlay zymography advantages of, 115–116 fibrin, 115, 118–119 fibrin indicator gel preparation for, 117, 118 of fibrin matrices, 124 materials/methods for, 116–119 PAs and fibrin, 115
INDEX PAs’ fibrin, 119, 123–124 sample preparation for, 116 usefulness for PA/PAI detection of fibrin, 119–124 Oxidation, see also Fatty acids; Ferrous ion oxidation absorbance changes’ phases during, 13–15 arteriosclerosis caused by, 4–5 of arteriosclerosis/lipoproteins link, 26–27 CAD and, 23–24 catalytic metal-generated, 18 cholesterol and, 16 fatty acids products of, 9–10 LDL in, 2, 3 measuring plasma’s products of, 20–23 measuring products of, 5 measuring susceptibility in, 15 myeloperoxidase as source of, 18 peroxides and, 9, 15–16 PGF in, 11 products of, 24 products of lipids’/proteins’, 8–20 proteins and products of, 16–18 of PUFA, 8, 9 by radicals, 6 resistance to, 13 transitional metals and, 7–8 Oxidative metabolism, OP/nerve agents influence on, 156–157 Oxidized (Ox) low density lipoproteins (LDL) antibodies and identification of, 26 ischemic syndromes role of, 3, 4 measurement of, 19–20, 25 proathergenic eVects of, 2–3 source of, 26 OxLDL, see Oxidized low density lipoproteins Oxphospholipids, proatherogenic eVects of, 2–3 Oxygen as radical, 6, 7 ROS produced from, 6 Oxysterols, 16 P Pancreatitis, MMP/TIMP levels in patients with, 64 PA/PAI system densitometric analysis of activity of, 119–120, 121
267
ELISA and, 113–114 fibrin zymography of PAs in, 119–120, 123–124 fibrin zymography’s usefulness for detection of, 119–124 molecular methods for assessing, 114 monitoring, 113–116 overlay zymography advantages and, 115–116 in pathophysiology, 112–113 reverse fibrin zymography for PAIs in, 121, 122, 123 substrate gel eletrophoresis as detecting, 114 in wounds’ normal healing, 112 Pathophysiology, PA/PAI system in, 112–113 PD-ECGF, see Platelet-derived endothelial growth factor Peptides, diagnostic signatures and, 223 Peritoneal dissemination, as prognostic indicator of cancer patient survival, 93, 95 Peritoneal lavage fluids body compartment/tumor origin and, 101–102 free tumor cells in, 93, 95 Peroxidation of lipids, 8, 9, 11, 13, 25 OP influence of lipids’, 157 Peroxides HPLC measured, 15 iodometric measurement of, 15–16 oxidation and, 9, 15–16 oxidation of fatty acids and, 9, 15 xylenol orange and, 16 Peroxynitrite, 6 PGF, see F2-Isoprostanes Phosopholipids, fatty acids oxidized in, 8 Phosphorous biological properties of organic compounds of, 152 role of, 152 Physiologic states, MMPs’/TIMPs’ levels in blood and, 48, 49–51, 52–70 Plasma BuChE in, 180–181 measuring oxidation products of, 20–23 OxLDL in, 26 peroxides using FOX 2 Reagent, 21–22 total MDA in, 21
268
INDEX
Plasminogen activity/plasminogen activity inhibitor system, see PA/PAI system Platelet-derived endothelial growth factor (PD-ECGF), angiogenesis role of, 88 Polyunsaturated fatty acids (PUFA), 9–10 MDA as product of, 12 oxidation of, 8, 9 Pregnancy, plasma MMPs/TIMPs levels in, 52–53 Prophylaxis AChE protected against inhibition by, 186–188 carbamates as, 188 for OP, 186 standard antidotes used as, 189 use with other drugs, 189–190 Prostate-specific antigen, 218 Protein antigens, 227 Proteins 2D/MS for analyzing, 225 AFM tips’ modification with, 253 AFM use for experiment unfolding, 242–244 C-reactive, 61 disease associated, 221–223 DNPH derived, 19 immobilization of, 233 measurement of modified, 25 MECA and, 231 microarrays detecting, 225–234, 226–227, 231 on microbead immobilization, 255 MS methodologies detecting disease associated, 221 oxidation products of, 8–20 oxidative modifications in, 18 phage display technology and, 228–229 synthetic combinatorial library approach and, 230 Proteomics, 39 Psoriatic arthritis, MMP/TIMP levels in patients’ blood with, 63 PUFA, see Polyunsaturated fatty acids R Radicals hydroxl, 8 molecules oxidized by, 6
Radiotherapy, on MMP/TIMP measurements, 57 Reactive aldehydes, 12–13 Reactive oxygen (ROS), superoxide anion radical produced from oxygen, 6 Receiver Operating Characteristics (ROC), 48, 52 Reverse transcriptase polymerase chain reaction (RT-PCR) rapid real-time quantitative, 90 real-time quantitative, 90–92 tumor cell detection by, 90–92 Reverse zymography, 119 for PAIs, 121, 122, 123 Rheumatoid arthritis antibody treated patients with, 61–62 MMP/TIMP levels in blood of patients with, 60–62 proMMP-3 and, 60 RNS, see Nitrogen species ROC, see Receiver Operating Characteristics ROS, see Reactive oxygen RT-PCR, micrometastases’ quantitative detection by real-time, 92–93, 94 S Sarin detoxifying, 160 toxicity of, 158–160 Scleroderma arthritis, MMP/TIMP levels in patients’ blood with, 63 Seizures control of, 195–196 Shock syndromes, MMPs role in, 44 SLE, see Systemic lupus erythematosis Soman antibodies against, 180 detoxifying, 160 intoxication, 173 toxicity of, 158–160 Substrate gel electrophoresis, PA/PAI system detected by, 114 Systemic lupus erythematosis (SLE), 59 MMP-3 levels in patients with, 63 MMP/TIMP levels in patients’ blood with, 62–63
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INDEX T TBA, see Thiobarbituric acid Thiobarbituric acid (TBA), 13 TIMPs, see Tissue inhibitors of metalloproteinases Tissue inhibitors of metalloproteinases (TIMPs) in angina/myocardial infarction, 67–68 biology/chemistry of, 39–42 in body fluids assays for measurement, 45–48 cancer and, 43 cardiovascular disease’s levels of, 67–70 diseases associated with increased levels of, 70–71 in inflammatory disease patient’s blood, 59–64 inhibitory role of, 41–42 levels in liver disease, 64–65 liver disease and, 43 measurement of, 38, 55 in nervous system diseases, 65–67 origin in blood of, 47–48 in pathophysiology of disease, 42–45 physiologic/disease states of, 48, 49–51, 52–70 pitfalls in measurement of blood levels of, 46–47 pregnancy/levels of plasma, 52–53 radiotherapy on measurements of, 57 serum/plasma, 59 Toxicity acute, 161 LD50 and, 161–162, 163 of OP, 157–158, 161–164
Transition metals molecules oxidized by, 6 oxidized form/reduced form of, 7–8 spin states of, 8 Tumor cells adjuvant chemotherapy/immunotherapy on, 88 circulating, 95–97, 102 peritoneal lavage fluids and free, 93, 95 Tumors chemosensitivity of, 88 estrogen’s mechanisms of initiation of, 135–136 recurrence after curative surgery of, 87–88 U Urine, MMPs identified in cancer patients’, 71–73 V V compounds, toxicity of, 160 Vascular endothelial growth factor (VEGF), angiogenesis role of, 88 VEGF, see Vascular endothelial growth factor W Wounds MMPs and, 44–45 PA/PAI system in normal healing of, 112 X Xylenol orange, FOX in presence of, 16
SMITH ET AL., FIG. 2. A protein-detecting microarray. Each square in the grid represents a diVerent feature of the array that would be impregnated with a particular protein ligand (blue shapes). When the sample is applied to the chip, each ligand will capture its target protein (orange and red coils in blow-up). The amount of target protein bound to each feature of the array would be quantitated with probes such as fluorescently labeled antibodies against the captured proteins. A fluoresence scanner would then measure the intensity of fluorescence (diVerently shaded green squares) at each spot, which would reflect the level of captured protein.
SMITH ET AL., FIG. 3. A bead-based format for the parallel detection of proteins. Each bead displays a diVerent binding agent directed against a specific protein target (blue shapes). Each bead is color-coded by covalent linkage of two dyes (red and orange shapes) at a characteristic ratio, allowing for uniquely coded beads. Only two beads are shown for clarity. Upon application of the biological sample, the target protein binds to the capture agents. A mixture of secondary binding ligands (in this case, antibodies) conjugated to a fluorescent tag (green) is applied to the mixture of beads. The beads are then passed through a detector where two lasers ‘‘read’’ the ratio (n:m, x:y) of dyes and thus identify the bead, while the fluorescence intensity is read to quantitate the amount of labeled antibodies present (which will reflect the analyte level).
