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ADVANCES IN CLINICAL CHEMISTRY
VOLUME 38
 BOARD OF EDITORS

Kaiser I. Aziz Milos Tichy

Galal Ghourab Masayuki Totani
Walter G. Guder Casper H. van Aswegen
E. D. Janus Abraham van den Ende
Sheshadri Narayanan Istvan Vermes
Francesco Salvatore Henning von Schenk
It-Koon Tan 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
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PRINTED IN THE UNITED STATES OF AMERICA

04 05 06 07 08 9 8 7 6 5 4 3 2 1
 CONTENTS

Contributors ................................................................................ ix

Preface ........................................................................................ xi

Lipoprotein Oxidation Products and Arteriosclerosis:

Theory and Methods with Applicability to the
Clinical Chemistry Laboratory
N. Abudu, James J. Miller, and Stanley S. Levinson

1. Introduction ................................................................................. 2
2. Radicals, Electrophiles, and Other Reactive Species.................................... 6
3. Oxidation Products of Lipids and Proteins and Measurement Methods .. ........... 8
4. Detailed Procedures for Some Methods that Measure Oxidation
Products in Plasma.......................................................................... 20
5. Discussion. ................................................................................... 23
6. Conclusion ................................................................................... 26
References. ................................................................................... 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. Introduction ................................................................................. 38
2. Background .................................................................................. 38
3. Biology and Chemistry of MMPs and TIMPs... ........................................ 39
4. Involvement of MMPs and TIMPs in Pathophysiology of Disease ................... 42
5. Assays for Measurement of MMPs and TIMPs in Body Fluids. ...................... 45
6. Blood Levels of MMPs and TIMPs in Physiologic and
Disease States (Table 2) .................................................................... 48
7. Miscellaneous Diseases Associated with Increased Levels of
MMPs and TIMPs .......................................................................... 70
8. MMPs Identified in Urine of Patients with Cancer ..................................... 71
9. Conclusions .................................................................................. 73
References. ................................................................................... 74

v
vi CONTENTS

Molecular Method to Quantitatively Detect Micrometastases and

its Clinical Significance in Gastrointestinal Malignancies
H. Nakanishi, Y. Kodera, and M. Tatematsu

1. Introduction................................................................................. 87
2. Methodology................................................................................ 89
3. Quantitative Detection of Micrometastases and its
Prognostic Significance .................................................................... 92
4. General Considerations and Future Directions ......................................... 101
References ................................................................................... 103

Zymographic Evaluation of Plasminogen Activators and

Plasminogen Activator Inhibitors
Melinda L. Ramsby

1. Introduction................................................................................. 112
2. Monitoring the PA/PAI System . ......................................................... 113
3. Materials and Methods for Overlay Zymography ...................................... 116
4. Results . ...................................................................................... 119
5. Conclusion .................................................................................. 124
References ................................................................................... 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................................................................................. 135
2. Analysis of Estrogens and their Metabolites, Conjugates, and
Depurinating DNA Adducts .............................................................. 139
3. Biomarkers for Increased Risk of Developing
Estrogen-Initiated Cancer ................................................................. 144
References ................................................................................... 146

Organophosphates/Nerve Agent Poisoning: Mechanism of Action,

Diagnosis, Prophylaxis, and Treatment
Jirí Bajgar

1. Introduction................................................................................. 152
2. Chemistry, Mechanism of Action, and Symptoms ..................................... 153
3. Cholinesterase Inhibitors and Other Factors Influencing the Activity . .............. 167
4. Diagnosis .................................................................................... 176
5. Prophylaxis.................................................................................. 186
6. Treatment ................................................................................... 190
 CONTENTS vii

7. Future Trends ............................................................................... 196

8. Summary ..................................................................................... 197
References. ................................................................................... 198

The Potential of Protein-Detecting Microarrays for

Clinical Diagnostics
Alexandra H. Smith, Jennifer M. Vrtis, and Thomas Kodadek

1. Introduction ................................................................................. 217

2. Diagnostic Signatures . ..................................................................... 218
3. Protein-Detecting Microarrays ............................................................ 225
4. Conclusions .................................................................................. 234
References. ................................................................................... 234

Clinical Laboratory Implications of Single Living Cell

mRNA Analysis
Toshiya Osada, Hironori Uehara, Hyonchol Kim,
and Atsushi Ikai

1. Introduction ................................................................................. 240

2. AFM.......................................................................................... 240
3. Manipulations of Biological Material with AFM ....................................... 242
4. mRNA Extraction from Living Cells ..................................................... 245
5. Modification of AFM Tips ................................................................ 252
References. ................................................................................... 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

JirÍ Bajgar (151), Purkyne Military Medical Academy, Hradec Králové,

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 partici-
pated 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 through-
out 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1. Oxidation Theory of Arteriosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2. Focus of this Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2. Radicals, Electrophiles, and Other Reactive Species . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.1. Reactive Oxygen (ROS) and Nitrogen Species (RNS) . . . . . . . . . . . . . . . . . . . . 6
2.2. Transition Metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3. Oxidation Products of Lipids and Proteins and
Measurement Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.1. Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.2. Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.3. Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4. Detailed Procedures for Some Methods that Measure Oxidation Products
in Plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
4.1. Total MDA in Plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4.2. Plasma Peroxides Using FOX 2 Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4.3. Extraction of LDL and IDL by Heparin Gel Affinity
Separation for Oxidative Susceptiblility=Resistance Testing . . . . . . . . . . . . . . . 22
4.4. Baseline Diene Conjugation in LDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
5. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
6. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Copyright 2004, Elsevier Inc. All rights reserved.

0065-2423/04 $35.00
2 ABUDU ET AL.

1. Introduction

1.1. OXIDATION THEORY OF ARTERIOSCLEROSIS

Much evidence links arteriosclerosis with oxidation of lipoproteins,
endothelial cell impairment, inflammation, and thrombosis (C3, R11, Z1).
Hyperlipidemia, and possibly oxidized (Ox) low density lipoproteins (LDL),
appears important in inducing this process leading to plaque formation
(C2, C3, S10, S11, W8, W9, Z1). Although arteriosclerosis proceeds within
the arterial wall, evidence suggests it is a systemic process proceeding at
multiple sites in coronary and peripheral vascular beds (B4, B7, R9, S5,
V6). This may be due to a systemic insult such as hypertension, glucose
intolerance, smoking, or hypercholesterolemia that damages many sites or
due to a generalized facilitation of the oxidative or inflammatory state of
individuals. Microcirculatory dysfunction due to this insult allows entry of
lipoproteins into the arterial wall, leading to the release of inflammatory
mediators that promotes further binding of LDL to the vessel endothelium
(R11, Z1).
The oxidation hypothesis, illustrated in Fig. 1, proposes that initially
minimally modified LDL is formed in the arterial intimal space. Although
this LDL can still be taken up by the well-regulated LDL receptor, it is
thought that minimally modified LDL promotes release of proinflammatory
mediators from leukocytes and endothelial tissue. Minimally modified LDL
is thought to be chemotactic for macrophages and monocytes that diVerenti-
ate 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).
4 ABUDU ET AL.

FIG. 2. Proposed process of arteriosclerosis leading to ischemic disease. It is proposed that

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

1.2. FOCUS OF THIS CHAPTER

Although it is not yet conclusively proven that oxidation is the cause
of arteriosclerosis in humans (C3, S10), animal experiments in the form of
transgenic and knockout mice models support this hypothesis (C3). OxLDL
(N2, Y2) and lipid oxidation products are found in human arteriosclerotic
 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 com-
pared to nondiseased persons (H7, J9, S12). Besides, elevated LDL choles-
terol 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 prerequi-
site 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 oxidation-
sensitive 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 techni-
ques presently have application for research laboratories only, others are
appropriate for use in clinical laboratories and some appear to have adapt-
ability 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 mea-
surement. 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.
6 ABUDU ET AL.

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 arte-
riosclerosis. 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 oxida-
tion 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
8 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 biomol-
ecule 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 elec-
trophiles, 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 rearrange-
ment, 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.
10 ABUDU ET AL.

TABLE 1
MEASUREMENT OF OXIDATION PRODUCT OF POLYUNSATURATED FATTY ACIDS

Product Fatty acid Method

F2-isoprostanes (PGF) Arachidonic GC=MS

Immunologically
Hydroxyoctadecadienoic Linoleic GC=MS
acid (HODEs)
Lipid peroxidation-related
aldehydes:
4-hydroxynonenals (HNE) Linoleic HPLC
Malondialdehyde (MDA) Arachidonic acid Thiobarbituric-reacting
substances
Nonspecific but directly from 1. Spectrophotometrically
specimen (TBARS)
2. Fluorometrically
MDA specific with HPLC
separation
Conjugated dienes All fatty acids Spectrophotometric
Peroxides All fatty acids
Nonspecific but directly Iodometric
from specimen
Xylenol orange (FOX assay)
Methylene blue
Specific with separation HPLC

TABLE 2
REFERENCE VALUES FOR SOME OXIDATION PRODUCTS IN HUMAN PLASMA

Product Reference range Reference

Lipid peroxides (HPLC) 2.1–4.6 umol=L (N4)

Lipid peroxides using FOX assay 2 with TPP 1.17–4.87 umol=L (N4)
MDA by HPLC 0.36–1.24 umol=L (N1)
F2-isoprostanes 5–33 ng=L (M8)
Protein carbonyls 0.4–1.0 nmol=mg protein (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 doc-
osahexaenoic acids (22:6) (J9). Many of the products that have been
measured are largely from arachidonic acid that represents a minor constitu-
ent 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.
 LIPOPROTEIN OXIDATION AND ARTERIOSCLEROSIS 11

3.1.1. F2-Isoprostanes (PGF)

PGF are produced by a nonenzymatic oxidation mechanism from arachi-
donic 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 quantifica-
tion (M8). Later, it was demonstrated that the thin layer chromatography
was not necessary (G6). More recently, PGF has been measured by high-
performance liquid chromatography (HPLC)=MS, although this technique is
not as sensitive as GC=MS (L1).
PGF are increased following oxidation of LDL by macrophages, endo-
thelial 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 gener-
ated 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 devel-
oped for urine for 8-Iso-PGF2 that is commercially available (O2), but the
method is tedious since it requires a column separation prior to ELISA (B1).
Also, since PGF are mainly indicators of arachidonic acid oxidation, they do
not reflect oxidation of the major PUFA comprising lipoproteins.

3.1.2. Hydroxyoctadecanoic Acid (HODEs)

HODEs are primarily C18 oxidation products of linoleic acid (J9). These
have not been as widely studied as isoprostanes, but like isoprostanes, these
are specific products of nonenzymatic lipid peroxidation that are associated
with arteriosclerotic disease and are found in arteriosclerotic plaques (J9,
W2). Likewise, they are measured by specific GC=MS techniques that are
generally not available in clinical laboratories (J10). They have the advantage
that they are products of the major PUFA in lipoproteins—linoleic acid—
but they have generally been measured only in lipoproteins extracted from
plasma.
12 ABUDU ET AL.

3.1.3. Reactive Aldehydes

Malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) are among
many reactive aldehydes that are nonenzymatic lipid peroxidation products.
These products are illustrated in Fig. 5. Both have been intensively studied as
an index of peroxidation (E4). They are not only associated with arterioscle-
rosis (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 inves-
tigated 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). Supplemen-
tation 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 parti-
cle, 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 com-
paring the lag phase for diVerent samples (K3). It is the duration of this phase
in minutes that is usually considered a measure of lipoprotein susceptibility
14 ABUDU ET AL.

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

or resistance to oxidation, although the physiological meaning of the lag

phase remains unclear, and it is not certain that the degree of susceptibility of
the particle to oxidation is necessarily a predictor of arteriosclerosis (A3, F3,
 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 chloro-
form–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 angio-
graphically 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 compo-
nents 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 spec-
trophotometric end points and are applicable for clinical laboratory use.
They do not measure specific products but reflect overall oxidation of fatty
acids. HPLC can be used to specifically measure individual peroxides (S2).
3.1.5.1. Iodometric. Iodometric measurement of peroxides is one of the
oldest techniques. It relies on the capacity of lipid peroxides to convert free I2
to I13 that can be spectrophotometrically measured at 365 nm (S2). Plasma
can be directly assayed; however, it is apt to give an overestimation of lipid
peroxide concentration because I2 has reactivity toward other compounds,
especially molecular oxygen and light (E3, S2). Reaction with oxygen occurs
more readily at acid pH (J3). A simple mix-and-read method was available
with the color reagents supplied in kit form (E3), but the color reagent was
discontinued, which is why it is not listed in detail here. The ingredients in the
color reagent have been described (E3), and, for those who wish to try it, the
concentration of lipid peroxide can be determined from the molar extinction
16 ABUDU ET AL.

coeYcient of 2.46  104M 1cm 1 (E3). This method that used a pH of 6.2
did not appear to react with molecular oxygen to any appreciable extent
and incubation was in the dark. A more detailed discussion of the use of
iodometric assays in determination of hydroperoxides can be found
elsewhere (J3).
3.1.5.2. Xylenol Orange. Ferrous ion oxidation (FOX) in the presence
of xylenol orange is a newer method for measuring lipid peroxides that has
been shown to agree well with the iodometric, TBAR, and conjugated diene
assays but is simpler to perform (J8). It has been used to assay peroxides in
plasma (N4). In this assay, peroxides oxidize Fe2þ to Fe3þ in acid solution
and Fe3þ forms a complex with xylenol orange, which absorbs at 560 nm.
Two reagents have been described, FOX 1 and FOX 2 (W10). FOX 2 con-
tains 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 con-
version 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, includ-
ing 22(R)-hydroxycholesterol, 20(S)-hydroxycholesterol, and 24(S),25-epox-
ycholesterol (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. Pres-
ently, 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
 LIPOPROTEIN OXIDATION AND ARTERIOSCLEROSIS 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 repre-
sented 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 methio-
nine, histidine and lysine, respectively (M2). These modifications lead to
carbonyl formation, fragmentation, and charge modifications.
As illustrated in Fig. 7, carbonyls and other adducts can be formed
by reactive aldehydes such as MDA and HNE binding to basic side chains
to form Michael adducts (M2, U1). After reaction with the amino group on
lysine, SchiV base adducts can form (M2). RNS and ROS can modify side
chains to produce oxidized species such as oxohistidine and 3-nitro-tyrosine,
and hypochlorous acid can react to form 3-chlorotyrosine. Sulfhydryl
18 ABUDU ET AL.

TABLE 3
MEASUREMENT OF OXIDATION PRODUCTS OF PROTEINS

Product Species Method

Relative electrophoretic mobility apo B Electrophoresis

Carbonyl groups apo B Spectrophotometric
HPLC
ELISA
Western=Dot blot
Malondialdehyde LDL apo B adduct ELISA
OxLDL apo B modification ELISA

groups can react with reactive aldehydes, RNS, and ROS to form sulfoxides,
nitrosothiols, and sulfenic acids. Thiols and other sulfur groups are also
susceptible to formation of disulfide bonds. The protein backbone itself can
be oxidized by radicals and other electrophiles (D5). Figure 7 illustrates how
the main -carbons in the primary chain can be oxidized to form hydroper-
oxides. 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 other-
wise 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 arterioscle-
rosis (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’-dityr-
osine but not ortho-tyrosine are elevated in LDL from human arteriosclerotic
tissue implicates myeloperoxidase as a source of oxidation (H1, H3).
 LIPOPROTEIN OXIDATION AND ARTERIOSCLEROSIS 19

3.3.3. Relative Electrophoretic Migration

Owing to an increased negativity of the protein as a result of adduct
formation or fragmentation, oxidized LDL migrates more rapidly than
does native LDL on agarose gel. This technique has been applied for mea-
suring 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 electropho-
retic 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 homoge-
nate 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 antibo-
dies to copper-modified LDL coated on a microtiter plate (H6). The samples
20 ABUDU ET AL.

and standards are incubated with a monoclonal antibody, following which

the mixture is added to microtiter plates containing OxLDL-coated wells.
The more OxLDL in the sample, the less mouse antibody that will bind to the
coated LDL. After washing, the wells are incubated with a rabbit antimouse
antibody containing horseradish peroxidase. The reduction of peroxidase
reaction absorbance as compared to blank is measured at 492 nm. Two
antibodies have been developed, both of which have aYnity for MDA-
modified 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 diVer-
entiation 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
 LIPOPROTEIN OXIDATION AND ARTERIOSCLEROSIS 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 tech-
niques. When measuring susceptibility of lipoproteins to oxidation, the
samples are stored at 70 C with 1 mmol=L EDTA that is removed prior
to the test by gel filtration, dialysis, or heparin gel aYnity chromatography.

4.1. TOTAL MDA IN PLASMA (S2)

1. Mix 200 uL of sample, standards, and controls with 25 uL of BHT
solution (54 mmol=L in methanol) and 200 uL orthophosphoric acid
solution (200 mmol=L). Mix well.
2. Add 215 uL of TBA reagent (800 mg thiobarbituric acid in 50 mL
0.1 mol=L NaOH).
3. Heat the sample at 90 C for 45 min.
4. Cool to room temperature.
5. Extract TBARS with 500 uL n-butanol containing 50 uL of a saturated
NaCl solution.
6. Separate phases by centrifugation (10,000  g for 1 min).
7. Transfer an aliquot of the upper organic phase to a tube and read
absorbance at 535 nm or fluorescence at 552 nm.
The standard solution is prepared by hydrolysis of 10 mmol=L or
50 mmol=L tetra-methoxypropane with 10 mmol=L HCl for 10 min at
room temperature. TBARS can be read in a microtiter plate, in which case
absorbance at 572 should be subtracted from 535 to correct for baseline
absorption.
Some kits are available for MDA (O2, R7).

4.2. PLASMA PEROXIDES USING FOX 2 REAGENT (J8, W10)

1. 10 uL of methanol is added to 90 uL of plasma (Test). (Important note:
All methanol must be HPLC-grade to avoid contamination by iron.)
2. 10 uL of 10 mmol=L triphenylphosphine (TPP) in methanol is added to
a duplicate plasma (Blank).
3. The samples are mixed and incubated at room temperature for
30 min.
22 ABUDU ET AL.

4. Add 900 uL of FOX 2 reagent consisting of 880 mg BHT, 76 mg

xylenol orange, 98 mg of ammonium iron(II) sulfate, and 1 mg=mL
EDTA dissolved in 1 L of methanol=acetic acid (1:1 mixture).
5. The samples are incubated for an additional 30 min.
6. The samples are then centrifuged at 10,000  g for 10 min and the
supernatants read at 560 nm.
7. Each test sample is subtracted from the corresponding blank and the
concentration determined from the diVerence using an extinction coeY-
cient 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 EDTA-
containing 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-con-
taining 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, epidemio-
logical 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-ketos-
treroids (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 arterio-
sclerosis (U3).
It was expected that important evidence linking oxidation to CAD would
be demonstrated by nutrient antioxidant trials in humans. The antioxidants
were expected to retard arteriosclerosis and reduce CAD in treated persons as
compared to placebo (C3). But primary and secondary randomized trials with
the antioxidants vitamin E, vitamin C, and vitamin A have so far failed to
prevent CAD (A1, V7). Moreover, large amounts of vitamin E (A1, N3, U3,
V7) and serum protein antioxidants such as albumin and fibrinogen are found
in arteriosclerotic tissue that would be expected to retard oxidation-induced
disease (B2, V1). These findings do not necessarily negate the oxidation
24 ABUDU ET AL.

hypothesis that is strongly supported by much evidence (S10). But until a

firmer link between lipoprotein oxidation and arteriosclerosis in humans is
confirmed, it is unlikely that large randomized studies, designed to identify
which oxidation products can be used to identify risk, will be conducted. Nor
is it likely drug companies will embark on the development of new drugs that
can specifically reduce oxidation for the purpose of reducing CAD.
Of legitimate concern is the question of whether oxidation products found
in blood or plasma are actually artifacts occurring due to atmospheric
exposure or due to other sources of ex vivo oxidation during isolation and
work-up rather than in vivo oxidation. As has been discussed, this problem
has been reduced because current procedures call for adding antioxidants
such as BHT and EDTA to blood or plasma immediately upon collection of
the sample and addition of these antioxidants to the test reagents during
analysis to reduce aberrant oxidation. Moreover, findings showing OxLDL
and other oxidation products in human arteriosclerotic lesions and increased
concentrations of oxidation products in the blood of patients both at high
risk for and with arteriosclerosis, as compared to those without, certainly
support the view that oxidation products are not simply an artifact of
isolation. As a result, it is generally accepted that, to some extent, these
oxidation products are a true manifestation of disease, although it cannot
be ruled out that some degree of baseline levels are artifactual. Nor can it be
entirely ruled out that oxidation products obtained from arterial tissue and
gruel samples are not altered by ex vivo oxidation. Thus, it is important to
examine to what degree in vivo studies have taken stringent precautions to
avoid inadvertent external oxidation. Even more diYcult to discern is wheth-
er 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 worth-
while 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
 LIPOPROTEIN OXIDATION AND ARTERIOSCLEROSIS 25

to measure specific products have continued to be developed is because

research has failed to identify the exact sites and mechanisms by which
oxidation of lipoproteins in humans proceeds, giving rise to a continuing
search for methods with increasing specificities that might help identify
the sources.
As a screening test for risk assessment, specific methods that measure lipid
peroxidation products may oVer few advantages over less specific spectro-
photometric approaches. This is because as long as inadvertent lipid peroxi-
dation is avoided by the addition of BHT and EDTA (S2), measurement of
specific products by HPLC appears to correlate well with simpler spectro-
photometric 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 mole-
cules so that the binding can be quantified. Thus, normally, apo
B concentration can be exactly determined by immunological methods be-
cause 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 substitu-
tions, 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).
26 ABUDU ET AL.

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 dis-
ease. This would also explain why OxLDL was elevated in the early disease
process (H10).
Three specific human monoclonal IgG autoantibodies that recognize oxi-
dized MDA-LDL have been prepared using phage libraries (S6). Such a
panel of antibodies may be of value in defining the composition of arterio-
sclerotic 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
 LIPOPROTEIN OXIDATION AND ARTERIOSCLEROSIS 27

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. Alterna-
tively, 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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