Biomarkers for Antioxidant Defense and Oxidative Damage

Biomarkers for Antioxidant Defense and Oxidative Damage: Principles and Practical Applications

Biomarkers for Antioxidant Defense and Oxidative Damage: Principles and Practical Applications Edited By

Giancarlo Aldini Kyung-Jin Yeum Etsuo Niki Robert M. Russell

A John Wiley & Sons, Inc., Publication

Edition f rst published 2010 © 2010 Blackwell Publishing Blackwell Publishing was acquired by John Wiley & Sons in F ebruary 2007. Blackwell’s publishing program has been mer ged with Wiley’s global Scientif c, Technical, and Medical business to for m Wiley-Blackwell. Editorial Off ce 2121 State Avenue, Ames, Iowa 50014-8300, USA For details of our global editorial off ces, for customer ser vices, and for infor mation about how to apply for per mission to reuse the cop yright material in this book, please see our Website at www.wiley.com/wiley-blackwell. Authorization to photocopy items for inter nal or personal use, or the inter nal or personal use of specif c clients, is g ranted by Blackwell Publishing, provided that the base fee is paid directl y to the Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been g ranted a photocopy license by CCC, a separate system of pa yments has been ar ranged. The fee code for users of the Transactional Reporting Service is ISBN-13: 978-0-8138-1535-0/2010. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, ser vice marks, trademarks, or registered trademarks of their respecti ve owners. The publisher is not associated with an y product or vendor mentioned in this book. This publication is designed to pro vide accurate and authoritative information in regard to the subject matter co vered. It is sold on the understanding that the publisher is not eng aged in rendering professional ser vices. If professional advice or other expert assistance is required, the ser vices of a competent professional should be sought. Library of Congress Cataloging-in-Publication Data Biomarkers for antioxidant defense and o xidative damage : principles and practical applications / edited by Giancarlo Aldini . . . [et al.]. p. ; cm. Includes bibliographical references and inde x. ISBN 978-0-8138-1535-0 (hardback : alk. paper) 1. Antioxidants. 2. Oxidative stress. 3. Active oxygen. 4. Biochemical markers. I. Aldini, Giancarlo. [DNLM: 1. Biological Markers. 2. Antioxidants–physiology. 3. Oxidative Stress–physiology. QW 541 B6159 2010] RB170.B566 2010 613.2 ′86–dc22 2010011386 ISBN 9780813815350 A catalog record for this book is a vailable from the U.S. Library of Cong ress. Set in 9.5 on 11 pt Times New Roman by Toppan Best-set Premedia Limited Printed in Singapore Disclaimer The publisher and the author mak e no representations or w arranties with respect to the accurac y or completeness of the contents of this w ork and specif cally disclaim all w arranties, including without limitation warranties of f tness for a par ticular purpose. No warranty may be created or extended by sales or promotional materials. The advice and strate gies contained herein ma y not be suitable for every situation. This work is sold with the understanding that the pub lisher is not engaged in rendering le gal, accounting, or other professional ser vices. If professional assistance is required, the ser vices of a competent professional person should be sought. Neither the pub lisher nor the author shall be liab le for damages arising herefrom. The fact that an or ganization or Website is refer red to in this w ork as a citation and/or a potential source of fur ther information does not mean that the author or the pub lisher endorses the infor mation the organization or Website may provide or recommendations it ma y make. Further, readers should be a ware that Internet Websites listed in this w ork may have changed or disappeared betw een when this work was written and w hen it is read. 1 2010

Contents

Preface vii Contributors ix 1.

Antioxidant Activity and Oxidative Stress: An Overview Kyung-Jin Yeum, Robert M. Russell, and Giancar lo Aldini

2.

Enzymatic Antioxidant Defenses 21 Sayuri Miyamoto, Hirofumi Arai, and Junji Terao

3.

Antioxidants as Biomarkers of Oxidative Stress 35 Ikuyo Ichi and Shosuke K ojo

4.

LDL Oxidation as a Biomar ker of Antioxidant Status 51 Mohsen Meydani, EunHee Kong, and Ashley Knight

5.

The Isoprostanes: Accurate Markers and Potent Mediators of Oxidant Injury in Vivo 65 Joshua D. Brooks, Brian E. Co x, Klarissa D. Hardy, Stephanie C. Sanc hez, Sonia Tourino, Tyler H. Koestner, Jocelyn R. Hyman-Howard, and Ginger L. Milne

6.

Hydroxyoctadecadienoic Acid (HODE) as a Mar ker of Linoleic Acid Oxidation 85 Yasukazu Yoshida and Etsuo Niki

7.

Oxysterols: Potential Biomarkers of Oxidative Stress 99 Luigi Iuliano and Ulf Diczfalusy

8.

Lipid Peroxidation Originating α,β-unsaturated Aldehydes and Their Metabolites as Biomarkers 117 Françoise Guéraud

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vi Contents 9.

Oxidative Modif cation of Proteins: An Overview 137 Paul J. Thornalley and Naila Ra bbani

10. Immunochemical Detection of Lipid P eroxidation-specif c Epitopes 157 Koji Uchida 11. Mass Spectrometric Strategies for Identif cation and Characterization of Carbonylated Peptides and Proteins 173 Marina Carini and Marica Orioli 12.

Nitrotyrosine: Quantitative Analysis, Mapping in Pr oteins, and Biological Signif cance 199 José M. Souza, Silvina Bartesa ghi, Gonzalo Peluffo, and Rafael Radi

13. Ubiquitin Conjugates: A Sensitive Marker of Oxidative Stress 219 Fu Shang and Allen Taylor 14. Covalent Modif cations of Albumin Cys34 as a Biomarker of Mild Oxidative Stress 229 Giancarlo Aldini, Kyung-Jin Yeum, and Giulio Vistoli 15. Protein S-glutathionylation and S-cysteinylation 243 Graziano Colombo, Aldo Milzani, Roberto Colombo , and Isabella Dalle-Donne 16. DNA Oxidation, Antioxidant Effects, and DNA Repair Measured with the Comet Assay 261 Mária Dušinská and Andrew R. Collins 17. Hydroxylated Nucleotides: Measurement and Utility as Biomar kers for DNA Damage, Oxidative Stress, and Antioxidant Eff cacy 283 Phyllis E. Bowen 18. Exocyclic DNA Adducts as Biomarkers of Antioxidant Defense and Oxidative Stress 319 Roger W.L. Godschalk Index 333

Preface

Oxidative damage is kno wn to be associated with the aging process and v arious chronic diseases such as atherosclerosis, diabetes, cataract, macular degeneration, and Alzheimer’s disease. Oxidative damage may be either a cause or an effect; therefore, its accurate measurement using sensitive and specif c biomarker is greatly needed by the scientif c community. When oxidative damage acts as a causati ve f actor of disease, its biomark ers can be a useful tool to better understand the pathogenic mechanisms involved, f nd novel drug targets, and evaluate effective defense strate gies such as phar maceuticals, nutraceuticals, or food and deri vatives. When oxidative stress is the disease ’s effect, its measurement could pro vide early prediction of the onset and pro gression for the disease. Hence, o xidative stress biomark ers can be used for diagnosis, prognosis, and treatment eff cacy. Although signif cant methodolo gical adv ances ha ve been made since the de velopment of thiobarbituric acid reactive substances (TBARS) and total carbonyl assays, which were widely used in 1980s and 1990s for measuring o xidative damage, the achievement of a gold standard method to determine antioxidant defense/oxidative damage status is not yet at hand. The development and application of v arious biomark ers for measuring antio xidant defense/o xidative damage is an evolving research area. Biomarkers of oxidative damage can be classif ed as either direct or indirect methods. Direct methods measure the oxidation products involving substrates such as lipids, proteins, and nucleic acids. Indirect methods measure antio xidant status b y analyzing endogenous levels of enzymatic and nonenzymatic antioxidants, as well as the resistance of a biolo gical matrix to an induced o xidative stress. This book describes the methodolo gical principles of cur rent state -of-the-art methods for measuring antio xidant acti vity/oxidative stress and their practical applications. In par ticular, the f rst three chapters describe conventional biomarkers as well as recent advances for measuring antioxidants and antio xidant capacity. Basic concepts and methodolo gies of widel y used assays and their applications are criticall y evaluated. In addition, f actors affecting antioxidant capacity in a biological system as well as the biological relevance of hydrophilic and lipophilic capacity assays are discussed. Determination of o xidative damage using lipid pero xidation products and metabolites is co vered in chapters 4 through 7. In par ticular the follo wing specif c biomark ers for lipid pero xidation are considered: isoprostanes (arachidonic acid C20:4 o xidation products),

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viii Pref

ace

hydroxyloctadecaenoic acid (linoleic acid C18:2 o xidation products), o xysterols (cholesterol oxidation products), and reactive carbonyl species from lipid peroxidation. Chapters 9 through 11 cover an outstanding area in biomark er discovery, that of protein o xidation. After an introduction on oxidative modif cations of proteins, recent advances in the measurement of carbonyls adducted proteins b y antibodies and mass spectrometr y are discussed. These chapters are followed by discussions of the recent de velopment on protein tyrosine nitration (Chapter 12) and ubiquitin -conjugates (Chapter 13) to deter mine mild and se vere o xidative damage. Biomarkers that have recently been developed thanks to cutting -edge technology, which determines oxidative modif cations of protein thiols, are presented in chapters 14 and 15. Well reco gnized biomark ers as w ell as recentl y de veloped biomark ers for DN A o xidative damage and their application in humans are discussed in chapters 16 through 18. In particular, methodological aspects for the measurement of DN A o xidation (comet assa y, h ydroxylated nucleotides, and exocyclic DNA adducts) are described, together with their implication in relation to chronic diseases. We believe that the cur rent book will be of a g reat interest to scientists w ho are in volved in basic research on o xidation, applied scientists e valuating the ef fects of nutraceuticals or pharmaceutical compounds on antio xidant activity/oxidative status, and ph ysicians who want to understand the de gree of oxidative damage in patients with specif c chronic diseases.

Contributors

Giancar lo Aldini Dipartimento di Scienze Farmaceutiche “Pietro Pratesi ” Università degli Studi di Milano Via L. Mangiagalli 25 20133 Milan, Italy [email protected] Hir ofumi Arai Department of Applied and Environmental Chemistry Kitami Institute of Technology Koen - cho165 Kitami 090 - 8507,Japan [email protected] - it.ac.jp SilvinaBartesaghi Departamento de Histología, Departamento de Bioqu ímica, and Center for F ree Radical and Biomedical Research Facultad de Medicina Universidad de la Rep ública Avda. General Flores 2125 11800 Montevideo, Uruguay [email protected] Ph yllis E. Bowen Department of Kinesiology and Nutrition University of Illinois at Chicago, m/c 994 Chicago, IL 60612 USA pbo [email protected] oshua J D. Brooks Department of Phar macology University of Califor nia San Diego San Diego, CA 92093 USA [email protected]

ix

x Contributors MarinaCarini Dipartimento di Scienze Farmaceutiche “Pietro Pratesi ” Università degli Studi di Milano Via L. Mangiagalli 25 20133 Milan, Italy [email protected] Andr ew R. Collins Department of Nutrition University of Oslo PB 1046 Blinder n 0316 Oslo, Norway a.r [email protected] Gr aziano Colombo Department of Biology University of Milan Via Celoria 26 20133 Milan, Italy [email protected] Rober to Colombo Department of Biology University of Milan Via Celoria 26 20133 Milan, Italy [email protected] BrianE. Cox Division of Clinical Phar macology Departments of Medicine and Phar macology Vanderbilt University School of Medicine Nashville, TN 37232 USA brien.e.co [email protected] Isa bella Dalle - Donne Department of Biology University of Milan via Celoria 26 20133 Milan, Italy [email protected] UlfDiczfalusy Karolinska Institutet Department of Laborator y Medicine Division of Clinical Chemistr y Karolinska University Hospital, Huddinge C1.74 SE -141 86 Stockholm, Sweden [email protected]

Contributors

M á ria Du š insk á CEE Health Effects Group Norwegian Institute for Air Research PB 100 2027 Kjeller , Norway [email protected] and Department of Experimental and Applied Genetics Slovak Medical University Limbova 12 833 03 Bratisla va, Slovakia Ro ger W.L. Godschalk Department of Health Risk Analysis and Toxicology Maastricht University Universiteissingel 50 6200MD Maastricht, The Netherlands R.Godschalk@GRA T.unimaas.nl ran F ç oiseGu é arud INRA Institut National de la Recherche Argonomigue, UMR 1089 X énobiotiques BP 93173 31027 Toulouse Cedex 3, France [email protected] KlarissaD. Hardy Department of Phar macology Vanderbilt University School of Medicine Nashville, TN 37232 USA klarissa.d.hardy@v anderbilt.edu ocelyn J R. Hyman - Ho ward Division of Clinical Phar macology Departments of Medicine and Phar macology Vanderbilt University School of Medicine Nashville, TN 37232 USA [email protected] Ikuy o Ichi Department of Health Chemistr y Graduate School of Phar maceutical Sciences The University of Tokyo Bunkyo - ku,Tokyo 113 - 0033Japan [email protected] - tok yo.ac.jp LuigiIuliano Sapienza University of Rome Department of Experimental Medicine, Unit of Vascular Medicine Vascular Biology and Mass Spectrometry Lab Corso della Republica 79 04100 Latina, Italy [email protected]

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xii Contributors Ashle y Knight Vascular Biology Laboratory Jean Mayer, USDA Human Nutrition Research Center on Aging at Tufts University 711Washington Street Boston, MA 02111 USA knight.ashle [email protected] yler T H. Koestner Division of Clinical Phar macology Departments of Medicine and Phar macology Vanderbilt University School of Medicine Nashville, TN 37232 USA tyler [email protected] Shosuk e Kojo Department of Food Science and Nutrition NaraWomen ’s University Nara 630 - 8506Japan [email protected] - wu.ac.jp EunHeeKong Department of Family Medicine College of Medicine Kosin University Seo - Gu,Busan, South Korea [email protected] MohsenMeydani Vascular Biology Laboratory Jean Mayer USDA—Human Nutrition Research Center on Aging Tufts University 711Washington St. Boston, MA 02111 USA mohsen.me [email protected] Ging er L. Milne Division of Clinical Phar macology Departments of Medicine and Phar macology Vanderbilt University School of Medicine Nashville, TN 37232 USA Ginger [email protected] AldoMilzani Department of Biology University of Milan Via Celoria 26 20133 Milan, Italy [email protected]

Contributors

Sa yuri Miyamoto Departamento de Bioqu í mica Institute de Qu í mica Universidade de S ã oPaulo CP 26077, CEP 05513 -970 S ão Paulo SP, Brazil [email protected] EtsuoNiki Health Research Institute National Institute of Advanced Industrial Science and Technology (AIST) Ikeda, Osaka 563 - 8577Japan etsuo - [email protected] MaricaOrioli Dipartimento di Scienze Farmaceutiche “Pietro Pratesi ” Università degli Studi di Milano Via Mangiagalli 25 20133 Milan, Italy [email protected] GonzaloPeluffo Departamento de Bioqu ímica and Center for F ree Radical and Biomedical Research Facultad de Medicina Universidad de la Rep ública Avda. General Flores 2125 11800 Montevideo, Uruguay [email protected] NailaRabbani Systems Biology, Protein Damage, and Systems Biolo gy Research Group Clinical Sciences Research Institute University of Warwick University Hospital Coventry CV2 2DX, UK N [email protected] RafaelRadi Departamento de Bioqu ímica and Center for F ree Radical and Biomedical Research Facultad de Medicina Universidad de la Rep ública Avda. General Flores 2125 11800 Montevideo, Uruguay [email protected] r Rober t M. Russell Offce of Dietar y Supplements, National Institutes of Health 6100 Executive Blvd. Bethesda, Maryland 20892 USA [email protected] r

xiii

xiv Contributors StephanieC. Sanchez Division of Clinical Phar macology Vanderbilt University School of Medicine Nashville,TN 37232 - 6602USA Stephanie.sanchez@v anderbilt.edu FuShang Jean Mayer USDA—Human Nutrtion Research Center on Aging Tufts University 711Washington St. Boston, MA 02111 USA [email protected] os J é M. Souza Departamento de Bioqu ímica and Center for F ree Radical and Biomedical Research Facultad de Medicina Universidad de la Rep ública Avda. General Flores 2125 11800 Montevideo, Uruguay [email protected] Allen Taylor Jean Mayer USDA—Human Nutrition Research Center on Aging Tufts University 711Washington Street Boston, MA 02111 USA allen.ta [email protected] unji J Terao Department of Food Science Graduate School of Nutrition and Bioscience Institute of Health Biosciences University of Tokushima Kuramoto - cho3 Tokushima 770 - 8503Japan terao@nutr .med.tokushima - u.ac.jp aul P J. Thornalley Systems Biology, Protein Damage and Systems Biolo gy Research Group Clinical Sciences Research Institute University of Warwick University Hospital Coventry CV2 2DX, UK [email protected] Sonia Tourino Institute for Advanced Chemistry of Catalonia CSIC (ICAQ - CSIC) Barcelona, Spain [email protected]

Contributors

K oji Uchida Laboratory of Food and Biodynamics Graduate School of Bioag ricultural Sciences Nagoya University Nagoya 464 - 8601Japan [email protected] - u.ac.jp Giulio Vistoli GiulioVistoli, Ph.D. Dipartimento di Scienze Farmaceutiche “Pietro Pratesi ” Università degli Studi di Milano Via L. Mangiagalli 25 20133 Milan, Italy [email protected] yKung - JinYeum Jean Mayer USDA—Human Nutrition Research Center on Aging Tufts University 711Washington St. Boston, MA 02111 USA K [email protected] asukazu Y Yoshida Health Research Institute National Institute of Advanced Industrial Science and Technology 2217 - 14Hayashi - cho,Takamatsu 761 - 0395,Japan oyshida - [email protected]

xv

Biomarkers for Antioxidant Defense and Oxidative Damage: Principles and Practical Applications

Chapter1 Antioxidant Activity and Oxidative Stress: An Overview K yung - Jin eYum , RobertM. Russell and ,

Giancar lo Aldini

INTR ODUCTION Oxidative stress is in volved in the process of aging (Kre gel and Zhang 2007) and v arious chronic diseases such as atherosclerosis (F earon and Faux 2009), diabetes (Ceriello and Motz 2004), and eye disease (Li et al. 2009a), whereas fruit and vegetable diets rich in antio xidants such as pol yphenols, vitamin C, and carotenoids are cor related with a reduced risk of such chronic diseases (Christen et al. 2008, Dauchet et al. 2006, Dherani et al. 2008). An excessive amount of reacti ve o xygen/nitrogen species (R OS/RNS) leading to an imbalance betw een antioxidants and oxidants can cause oxidative damage in vulnerable targets such as unsaturated fatty acyl chains in membranes, thiol groups in proteins, and nucleic acid bases in DNA (Ceconi et al. 2003). Such a state of “oxidative stress ” is thought to contribute to the patho genesis of a number of human diseases (Thannickal and F anburg 2000). Sensitive and specif c biomarkers for antioxidant status/oxidative stress are essential to better understand the role of antio xidants and oxidative stress in human health and diseases, thereb y maintaining health and establishing effective defense strategies against oxidative stress. Several assays to measure “total” antioxidant capacity of biolo gical systems ha ve been de veloped to investigate the in volvement of o xidative stress in patholo gical conditions or to e valuate the functional bioavailability of dietary antioxidants. Conventional assays to determine antioxidant capacity primarily measure the antio xidant capacity in the aqueous compar tment of plasma. Consequently, w ater-soluble antio xidants such as ascorbic acid , uric acid , and protein thiols mainly inf uence these assays, whereas fat-soluble antioxidants such as tocopherols and carotenoids show little inf uence over the many results. However, there are new approaches to def ne the total antioxidant capacity of plasma, w hich ref ect the antioxidant network between waterand fat-soluble antioxidants. Revelation of the mechanism of action of antio xidants and their true antioxidant potential can lead to identifying proper strate gies to optimize the antio xidant defense systems in the body . Methodological aspects of v arious antio xidant capacity assa ys ha ve been e xtensively discussed recentl y (Magalhaes et al. 2008). This chapter focuses on impor tant antio xidants in biological systems, f actors affecting bioavailability of antio xidants and, therefore, antio xidant capacity, and basic principles of v arious biomark ers for antio xidant capacity and their applications. Biomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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4 Chapter

1

OXIDATIVE STRESS AND ANTIOXIDANTS IN A BIOLOGICAL SYSTEM ROS are continuousl y generated b y nor mal metabolism in the body (Gate et al. 1999) and these ROS are necessary to maintain biological homeostasis through various functions such as vasoregulation and v arious cellular signal transduction (Hensle y and Flo yd 2002). However, overproduction of these ROS can also cause damage to the macromolecules necessar y for cell structure and function. Cellular production of ROS such as superoxide anion (O2•−), hydroxyl radical (HO• ),peroxyl radical (R OO•), and alk oxyl radical (R O•) occurs from both enzymatic and non -enzymatic reactions. Mitochondria appear to be the most impor tant subcellular site of R OS production, in particular of O 2•− and H 2 O2 in mammalian organs. The electron transfer system of the mitochondrial inner membrane is a major source of supero xide production when molecular oxygen is reduced b y a single electron. Supero xide can then dismutate to for m h ydrogen pero xide (H2 O2), and then can fur ther react to for m the hydroxyl radical (HO •) and ultimately water. In addition to intracellular membrane-associated oxidases, soluble enzymes such as xanthine oxidase, aldeh yde o xidase, dih ydroorotate deh ydrogenase, f avoprotein deh ydrogenase, and tryptophan dioxygenase can generate R OS during catal ytic c ycling. Auto-oxidation of small molecules such as dopamine, adrenaline (epinephrine), f avins, and quinols can be an important source of intracellular ROS production as well. In most cases, the direct product of such auto oxidation reactions is the supero xide anion (Thannickal and F anburg 2000). Any compound that can inhibit oxidation of external oxidants is considered to be an antioxidant. This is a relati vely simple def nition but, at times, it becomes v ery diff cult to e valuate whether a compound actuall y has an antio xidant action, par ticularly in vivo . It is still not clear w hat kinds of R OS play a role in the patho genesis of human disease and where the major sites of R OS action occur. There is, ho wever, convincing evidence that lipid peroxidation is related to human patholo gy, such as in atherosclerosis (V alkonen and K uusi 1997). The actions of antio xidants in biolo gical systems depend on the nature of o xidants or ROS imposed on the systems, and the acti vities and amounts of antio xidants present and their cooperative/synergistic interactions in these systems. Numerous epidemiological studies have indicated that diets rich in fr uits and vegetables are correlated with a reduced risk of chronic diseases (Czer nichow et al. 2009, Hung et al. 2004, Liu et al. 2001, Liu et al. 2000). It is probab le that antioxidants, present in the fr uits and vegetables such as polyphenols, carotenoids, and vitamin C, prevent damage from harmful reactive oxygen species, w hich either are continuousl y produced in the body during nor mal cellular functioning or are deri ved from exogenous sources (Gate et al. 1999). The possible protective effect of antioxidants in fruits and vegetables against ROS has led people to consume antio xidant supplements such as β - carotene,α-tocopherol, and/or multi vitamins. It is not sur prising to note that more than 11% of US adults age 20 y ears or older consume at least 400 IU of vitamin E per da y from supplements (F ord et al. 2005). However, inter vention studies ha ve failed to show a consistent benef cial effect of antioxidant supplements such as vitamin E (Lee et al. 2005) or β-carotene (Baron et al. 2003, Omenn et al. 1996) against chronic diseases. How can we explain these apparent contradictory results between observational studies and intervention trials? It is interesting to note that although se ven and a half years of supplementation with a combination of antio xidants (vitamin C, β-carotene, zinc, and selenium) did not af fect the risk of metabolic syndrome, baseline concentrations of ser um vitamin C and β - carotenewere negatively associated with metabolic syndrome in a generall y well-nourished population (Czernichow et al. 2009). It is probab le that the generall y w ell-nourished population maintains optimal ranges of antio xidants through a balanced dietar y fr uit and v egetable intak e. However, high doses of a single or limited mixture of antioxidant supplements may not affect the already saturated in vivo antioxidant network, but rather could result in an imbalance in the antio xidant

Antioxidant Activity and Oxidative Stress: An Overview

5

network and could possib ly even act as pro -oxidants. A recent prospecti ve study sho wing an inverse association of baseline plasma antioxidant concentrations with the risk of heart disease and cancer also suppor ts the benef cial effect of a balanced antio xidant status, w hich can be attained by eating diets high in fr uits and vegetables (Buijsse et al. 2005).

MARKERS OF ANTIOXIDANT CAPACITY IN A BIOLOGICAL SYSTEM Several human studies ha ve f ailed to sho w a direct cor relation between the ph ysiologic consumption of dietar y f at-soluble antio xidants and subsequent changes in antio xidant capacity (Castenmiller et al. 1999, Pellegrini et al. 2000). For example, it has even been suggested that carotenoids may not act as antio xidants in vivo (Rice - Ev ans et al. 1997 ).These suggestions derive from the lack of proper analytical methods for measuring antioxidant capacity. Inasmuch as conventional methods, such as total radical trapping antio xidant parameter (TRAP), oxygen radical absorbance capacity (ORA C), etc., use primaril y h ydrophilic radical generators and measure primarily antioxidant capacity in the aqueous compartment of plasma, they are unable to deter mine the antio xidant capacity of the lipid compar tment (Cao et al. 1993, Lussignoli et al. 1999). Therefore, it is not surprising that most of the methods used to measure pur ported “total antio xidant capacity ” of plasma are not af fected b y lipophilic antio xidants, such as carotenoids (Cao et al. 1998b, Castenmiller et al. 1999, Pellegrini et al. 2000). This can be e xplained by the f act that plasma carotenoids, w hich are deepl y embedded in the core of lipoproteins, are not a vailable for reaction with aqueous radical species or fer ric complexes used in these assa ys. In addition, an assa y to measure total antio xidant capacity in a biolo gical sample such as plasma must consider the hetero geneity of the sample, w hich consists of both h ydrophilic and lipophilic compar tments that contain w ater-soluble and f atsoluble antioxidants, respectively. Possible cooperative/synergistic interactions among antioxidants in biological samples should not be o verlooked. Azo initiators are a class of radical inducers (w hich contain the –N= N –group) widely used in experiments in vitro to generate radical species. The azo initiators decompose at a temper ature-controlled rate to give carbon -centered radicals, which react rapidly with O 2 to yield the peroxyl radical (ROO• ). R − N = N − R → N 2 + 2R i R i + O 2 → ROOi Peroxyl radicals deri ved from azo initiators can induce the lipid pero xidation cascade and can also damage proteins. Depending on the lipophilicity of the azo initiators [2,2 ′ - azobis - (2 amidinopropane) dihydrochloride (AAPH) is w ater soluble whereas 2,2 ′ - azobis(2,4 - dimeth ylvaleronitrile (AMVN) and ,2 ′ - azobis(4 - metho xy - 2,4 - dimeth ylvaleronitrile) (MeO - AMVN)are lipophilic], the pero xyl radicals are generated in the aqueous or lipid phase of the sample, respectively. The choice of the site of radical generation is of g reat impor tance, because the activities of antio xidants present in both the lipid and aqueous compar tments depend on the localization of the attacking radical species (Y eum et al. 2003). Table 1.1 shows the cur rently available assays to deter mine antioxidant capacity in h ydrophilic and lipophilic en vironments in biolo gical samples such as plasma. When used alone, those assays (Cao et al. 1993, Valkonen and Kuusi 1997) that use hydrophilic radical initiators and probes are insuff cient for deter mining the antio xidant activity of carotenoids, w hich are deeply embedded in the lipoprotein core of biolo gical samples. There have been attempts to determine the activity of fat-soluble antioxidants by measuring the antioxidant activity of lipid extracts dissolv ed in an or ganic solv ent (Prior et al. 2003). This approach, ho wever, cannot appreciate the possible interactions between the fat-soluble and water-soluble antioxidants. The alternative approach of producing radicals in the lipid compar tment of w hole plasma and monitoring lipid pero xidation by a lipophilic probe (Aldini et al. 2001) allows measurement

Table 1.1. Assays to determine antioxidant capacity in biological systems.

6 Assa y

Radical inducer

Oxidizab le substrate (probe)

W avelength

Plasma susceptibility against exogenous pro-oxidant induced oxidation (hydrophilic assay) TRAP AAPH DCFH λ ex = 480, λ em = 526 R - Ph ycoerythrin λ ex = 495, λ em = 595 ORA C Crocinbleaching

AAPH AB AP

R - Ph ycoerythrin

λ ex = 495, λ em = 595 445nm

Plasma quenching ability of stable/pre-formed radicals (hydrophilic assay) TEA C ABTS+ • 734nm FRAP

F e3+

593nm

Plasma susceptibility against exogenous pro-oxidant induced oxidation (lipophilic assay) LipophilicORAC AAPH Fluorescein λ ex = 485, λ em = 520 Lipophilicantioxidant AAPH DPHPC λ ex = 354, λ em = 430 activity T AP MeO - AMVN BODIPY581/591 λ ex = 500, λ em = 520 TRAP:Total radical - trappingantioxidant parameter. ORA C: Oxygen radical - absorbingcapacity. TEAC: Trolox equivalent antioxidant capacity. FRAP:Ferric - reducingability of plasma. T AP: Total antioxidant performance. AAPH,ABAP: 2,2′ - Azobis - (2 - amidinopropane)dih ydrochloride. ABTS:2,2′ - Azinobis(3 - ylbenzothiazoline eth 6 - sulphonate). AUC: Area under the cur ve. MeO - AMVN: 2,2′ - Azobis(4 - metho xy - 2,4 - dimeth ylvaleronitrile). DCFH:2′ ,7′ - Dichlorodih ydrof uorescein. DPHPC:1 - P almitoyl - 2 - ((2 - (4 - (6 -ylphen - trans - 1,3,5 -xatrienyl)phenyl)ethyl) he - carbon yl - sn -ycero gl - 3 - phosphocholine. BODIPY581/591: 4,4 - Difuoro - 5 - (4 - phen yl - 1,3 - butadien yl) - 4 - bora - 3a,4a - diaza - s - indacene - 3 - undecanoic acid. Modifed from Yeum et al. 2009b.

Calculation Lagtime A UC Absorbance

Absorbance

Reference V alkonen and Kuusi 1997 Ghiselli et al. 1995 Caoet al. 1995 T ubaro et al. 1998 Kampa et al. 2002

Absorbance

Milleret al. 1993 Re et al. 1999 Benzieand Strain 1996

A UC Lagtime

Prioret al. 2003 Ma yer et al. 2001

A UC

Aldiniet al. 2001

Antioxidant Activity and Oxidative Stress: An Overview

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of the actual “total” antio xidant acti vity including possib le interactions among antio xidants located in the h ydrophilic and lipophilic compar tments, because the interference of lar ge amounts of protein (e.g. albumin) in the h ydrophilic compar tment can be o vercome b y this approach. HYDR OPHILIC ANTIOXIDANT CAPACITY ASSAYS There are mainly two hydrophilic approaches to deter mine the antioxidant capacity in plasma. The f rst approach measures the antioxidant capacity in plasma using hydrophilic assays in the presence of o xidants that act as pro -oxidants. These assa ys deter mine the susceptibility of plasma against o xidation induced b y added pro -oxidants (radical inducers) and monitored b y an exogenous oxidizable substrate (probe). The oxidation of the probe is theoretically inhibited by the antioxidants present in plasma during the induction period. The TRAP and ORAC assays are presently the most widel y used methods for measuring antio xidant capacity in biolo gical systems such as ser um and tissues. Dichlorof uorescein - diacetate,phycoerythrin (R - P e), and crocin-based assays also are included in this category of assays. Specif cally, plasma or serum, when challenged with a h ydrophilic radical inducer such as 2,2 ′ - azobis(2,4 - amidinopropane) dihydrochloride (AAPH), can be monitored b y a h ydrophilic o xidizable substrate such as 2′ ,7′ - dichlorodih ydrof uorescein (DCFH) (V alkonen and K uusi 1997), crocin (Kampa et al. 2002, Tubaro et al. 1998), or R-Pe (Cao and Prior 1999). Antioxidant capacity can be expressed in various ways such as lag phase, area under the cur ve, or competition kinetics. AAPH is a hydrophilic azo-compound that spontaneously decomposes at 37°C with a known rate constant ( Ri = 1.36 × 10−6 [AAPH] mol/liter/sec), gi ving rise to carbon -centered radicals that then react with oxygen, yielding the corresponding peroxyl radicals. DCFH, which can be oxidized to highl y f uorescent (Exc 480 nm, Em 526 nm) dichlorof uorescein by peroxyl radicals, is used as an o xidizable substrate in the TRAP assay (Valkonen and K uusi 1997). R -Pe is a protein isolated from Corallina off cinalis, and is used as the o xidizable substrate in the TRAP (Ghiselli et al. 1995) and ORA C (Cao and Prior 1999) assays. R -Pe is a f uorescent protein that emits in the visib le region (Exc 495 nm, Em 595 nm) and is characterized b y f uorescence quenching upon reaction with pero xyl radicals. Crocin, isolated from saf fron and characterized by a polyene chain with a high extinction coeff cient, has been used as an oxidizable substrate in the assa y developed by Tubaro (Tubaro et al. 1998) and then automated b y Kampa (Kampa et al. 2002) in the crocin b leaching assay. The reaction of crocin with pero xyl radical leads to a loss of the double bond conjugation and hence to bleaching that can be readily monitored at 445 nm. The second approach to measure antio xidant capacity in plasma using a h ydrophilic assay is to quench a stab le and pre -formed radical that does not act as a pro -oxidant. The trolo x equivalent antio xidant capacity (TEA C) assa y, w hich w as repor ted b y Miller et al. (1993), determines the antio xidant capacity of plasma b y measuring the ability of plasma to quench the radical cation of 2,2′ - azinobis(3 - ylbenzothiazoline eth - 6 - sulfonate) (ABTS). The quenching reaction is monitored by measuring the decay of the radical cation at 734 nm. The ferric reducing ability of plasma (FRAP) assa y has received a g reat deal of attention because of its quick and simple methodolo gy (Benzie and Strain 1996). The FRAP assa y measures the reduction of the fer ric ion to fer rous ion at lo w pH, w hich causes a colored fer rous-tripyridyltriazine complex to form. FRAP values can be obtained by comparing the absorbance change at 593nm in test reaction mixtures with those containing the fer rous ion in a kno wn concentration. LIPOPHILICANTIOXIDANT CAPACITY ASSAYS Two decades ago, Niki (1990) introduced AAPH and AMVN as the sources of w ater- and lipid-soluble pero xyl radicals respecti vely. As sho wn in the w ork of Massaeli et al. (1999), where preincubation of LDL with f at-soluble antio xidants increased the protecti ve ef fect

8 Chapter

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against free radicals w hile preincubation with w ater-soluble antio xidants did not sho w an y effect, the importance of lipophilicity vs. hydrophilicity in antioxidants and free radical generating systems for deter mining antio xidant capacity has been reco gnized. It has also been demonstrated (Yeum et al. 2003) that the acti vities of antio xidants present in both the lipid and aqueous compar tments depend on the localization of the attacking radical species. In an effort to understand the biological signif cance of lipophilic antioxidants, several recent studies ha ve paid attention to the antio xidant capacity in the lipid compar tment of plasma. Mayer et al. (2001) proposed a continuous spectroscopic method using selecti ve f uorescence markers to monitor the aqueous and lipid phases in human ser um. In par ticular, diphen ylhexatriene-labeled proprionic acid w as used as an appropriate probe for the aqueous phase because it preferentially binds to albumin, w hile diphenylhexatriene-labeled phosphatidylcholine, which incorporates into lipoproteins, monitors the lipid compartment oxidizability. AAPH was selected as the radical inducer for both compar tments. By using this method, the authors reported that supplementation of human serum with quercetin, rutin, vitamins E and C, or total apple phenolics in vitro led to a decrease in oxidizability depending on the o xidation mark er and the h ydrophobicity of the antio xidant. That is, f atsoluble antioxidants such as quercetin and vitamin E sho wed higher protective effects against lipoprotein oxidation, whereas water-soluble lutin and vitamin C more eff ciently protected the aqueous phase. An improved TEAC assay has been repor ted b y Re et al. (1999). By using a pre -formed radical mono -cation of ABTS and an appropriate solv ent system, the assa y is applicab le to both hydrophilic and lipophilic systems. The ORAC assay has also been e xpanded to ref ect lipophilic antioxidants by using randoml y methylated β-cyclodextrin (RMCD) as a solubility enhancer, AAPH as a radical initiator , and f uorescein as an oxidizable substrate (Huang et al. 2002). Recently, this updated ORA C assay was applied to human plasma (Prior et al. 2003) and the authors repor ted that lipophilic antio xidants represent less than 30% of the total antioxidant capacity of the protein -free plasma. For the lipophilic ORAC assay, lipophilic antioxidants w ere e xtracted b y he xane, dried , and resuspended in 7% RMCD solution (50% acetone/50% w ater, v/v). Ho wever, this assa y, w hich par titioned h ydrophilic and lipophilic antioxidants, may not be rele vant to a tr ue biological system in w hich active communication occurs among hydrophilic and lipophilic antio xidants. Aldini et al. (2001) repor ted a method that measures antio xidant capacity in both the hydrophilic and lipophilic compar tments of plasma and allo ws for interaction betw een the antioxidants in the two compartments. A lipophilic radical generator coupled with a selective f uorescent probe capab le of detecting lipid pero xidation w as used to measure the lipid compar tment. 2,2 ′ - azobis(4 - metho xy - 2,4 - dimeth ylvaleronitrile) (MeO - AMVN),which decomposes at 37 °C, w as selected as a lipid -soluble radical inducer , and 4,4 -dif uoro - 5 - (4 phenyl - 1,3 - butadien yl) - 4 - bora - 3a,4a - diaza s - indacene - 3 - undecanoic acid (BODIPY581/591) was used as a selective lipophilic oxidizable substrate (Drummen et al. 2002, Pap et al. 1999). The signif cantly higher rate constant of MeO -AMVN as compared to that of AMVN allows it to easily achieve lipid peroxidation in a biological system (Aldini et al. 2001). An oxidationsensitive f uorescent probe, BODIPY 581/591, w hich has a high quantum yield and readil y enters membranes (Dr ummen et al. 2002), provided the sensiti ve and selecti ve measurement of o xidation in the lipid compar tment of plasma. The selecti ve incor poration of BODIPY 581/591 into the indi vidual lipoprotein fractions, VLDL, LDL, and HDL, of human plasma has been further conf rmed (Yeum et al. 2003). A signif cant correlation (p < 0.0001) between plasma carotenoid concentration and antio xidant capacity deter mined by this assay was found in subjects w ho participated in a dietar y intervention trial with high fr uit and v egetable diets (Yeum et al. 2005). It is interesting to note that a high amount of single antio xidant ( > 15 mg of α - tocopherol) has been reported to be required to show a difference in antioxidant capacity, whereas less than a half serving of fruits and vegetables resulted in signif cant difference in antioxidant capacity

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Antioxidant Activity and Oxidative Stress: An Overview

in a recently reported cross-sectional study (Talegawkar et al. 2009). This observation supports the impor tance of syner gistic action among the numerous antio xidants found in foods vs. a single antio xidant supplement. Another notab le impro vement of this assa y is that it requires a much lo wer dilution of plasma (5 to 10 × dilution) as compared to those of pre viously reported assays, which require 100 ×, 150 ×, and 250 × dilutions for the FRAP (Benzie and Strain 1996), ORAC (Cao et al. 1995), and TRAP (Ghiselli et al. 1995) assays, respectively. One of the dra wbacks of con ventional assa ys to measure antio xidant capacity has been the high dilution of plasma resulting in v ery low concentrations of antio xidants in the reaction mixtures. APPLICATION OF HYDROPHILIC AND LIPOPHILIC ANTIOXIDANT CAPACITY ASSAYS When hydrophilic assays are applied , the majority of the antio xidant capacity of plasma can be accounted for b y protein (10% to 28%), uric acid (7% to 60%), and ascorbic acid (2% to 27%), whereas the ef fect of vitamin E (