《食品化学》课程教学资源（文献资料）Antioxidants in health and disease

Downloaded from jcp.bmj.com on February 16,2012-Published by group.bmj.com 7 Clin Pathol20015176-18 Antioxidants in health and disease IS Young,JV Woodside radical production occurs Ho dical night play a adicals and their chemical e da e by defined as any mole the tion orerd the bn in c nce rad or 282014176-180 ther ants o redu In less oxidative stress Road. r.Th mos nd, BT An be “an body nifi the h dd ole of anti as this def nition sugg csts, is produced hu WIP7LD.UK on action t by es and are g 26 diabetes mellitu The s Ho er Endoae0rnalesk Free radical productio rce her CO a tions 02,H202 a and Fe Cu alse OH aitri ction Lipid peroxidation wth and diff Modified DNA bases Protein damage by phag 且Sp m4 Major cao时free radical

Antioxidants in health and disease I S Young, J V Woodside Abstract Free radical production occurs continu￾ously in all cells as part of normal cellular function. However, excess free radical production originating from endogenous or exogenous sources might play a role in many diseases. Antioxidants prevent free radical induced tissue damage by prevent￾ing the formation of radicals, scavenging them, or by promoting their decomposi￾tion. This article reviews the basic chem￾istry of free radical formation in the body, the consequences of free radical induced tissue damage, and the function of anti￾oxidant defence systems, with particular reference to the development of athero￾sclerosis. (J Clin Pathol 2001;54:176–186) Keywords: free radicals; antioxidants; oxidative stress; coronary heart disease; atherosclerosis An antioxidant can be defined as: “any substance that, when present in low concentra￾tions compared to that of an oxidisable substrate, significantly delays or inhibits the oxidation of that substrate”.1 The physiological role of antioxidants, as this definition suggests, is to prevent damage to cellular components arising as a consequence of chemical reactions involving free radicals. In recent years, a substantial body of evidence has developed supporting a key role for free radicals in many fundamental cellular reactions and suggesting that oxidative stress might be important in the pathophysiology of common diseases including atherosclerosis, chronic renal failure, and diabetes mellitus. The aim of this review is to consider mechanisms of free radical formation in the body, the consequences of free radical induced tissue damage, and the function of antioxidant defence systems in health and dis￾ease. Free radicals and their chemical reactions A free radical can be defined as any molecular species capable of independent existence that contains an unpaired electron in an atomic orbital.2 The presence of an unpaired electron results in certain common properties that are shared by most radicals. Radicals are weakly attracted to a magnetic field and are said to be paramagnetic. Many radicals are highly reac￾tive and can either donate an electron to or extract an electron from other molecules, therefore behaving as oxidants or reductants. As a result of this high reactivity, most radicals have a very short half life (10−6 seconds or less) in biological systems, although some species may survive for much longer.2 The most important free radicals in many disease states are oxygen derivatives, particularly superoxide and the hydroxyl radical. Radical formation in the body occurs by several mechanisms, involving both endogenous and environmental factors (fig 1). Superoxide (O2 −.) is produced by the addi￾tion of a single electron to oxygen, and several mechanisms exist by which superoxide can be produced in vivo.3 Several molecules, including adrenaline, flavine nucleotides, thiol com￾pounds, and glucose, can oxidise in the presence of oxygen to produce superoxide, and these reactions are greatly accelerated by the presence of transition metals such as iron or copper. The electron transport chain in the inner mitochondrial membrane performs the reduction of oxygen to water. During this process free radical intermediates are gener￾ated, which are generally tightly bound to the components of the transport chain. However, there is a constant leak of a few electrons into the mitochondrial matrix and this results in the formation of superoxide.4 The activity of several other enzymes, such as cytochrome p450 oxidase in the liver and enzymes involved in the synthesis of adrenal hormones, also results in the leakage of a few electrons into the surrounding cytoplasm and hence superoxide formation. There might also be continuous production of superoxide by vascular endothe￾lium to neutralise nitric oxide,5 6 production of superoxide by other cells to regulate cell growth and diVerentiation,7 and the produc￾tion of superoxide by phagocytic cells during the respiratory burst.8 Any biological system generating superoxide will also produce hydrogen peroxide as a result of a spontaneous dismutation reaction. In addition, several enzymatic reactions, includ￾ing those catalysed by glycolate oxidase and Figure 1 Major sources of free radicals in the body and the consequences of free radical damage. Free radical production Transition metals Fe2+, Cu+ Modified DNA bases Tissue damage Lipid peroxidation Protein damage O2 –., H2O2 OH. Endogenous sources •mitochondrial leak •respiratory burst •enzyme reactions •autooxidation reactions Environmental sources •cigarette smoke •pollutants •UV light •ionising radiation •xenobiotics 176 J Clin Pathol 2001;54:176–186 Department of Clinical Biochemistry, Institute of Clinical Science, Grosvenor Road, Belfast, Northern Ireland, BT12 6BJ, UK I S Young Department of Surgery, Royal Free and University College London Medical School, 67–73 Riding House Street, London, W1P 7LD, UK J V Woodside Correspondence to: Professor Young, Department of Clinical Biochemistry, Institute of Clinical Science, Royal Group of Hospitals, Grosvenor Road, Belfast BT12 6BJ, UK I.Young@qub.ac.uk Accepted for publication 5 June 2000 www.jclinpath.com Downloaded from jcp.bmj.com on February 16, 2012 - Published by group.bmj.com

Downloaded from jcp.bmj.comon February 16.2012-Published by group.bmj.com Amtioridants in health and diseas 177 D-amino acid oxidase,might produce hydr Thenetrestofthereactionseauenceilus elf.but is included Although m nd co pper in th - ca mig oxyl ra cal in v h ab tht diffuse a co he ikel T ydr sa conduit to Dieta tine ent cid nd al p g tr errin. (OF high r radical formatior mino iron av. although 1n ol.In ron th 6 owof the d-blo dic pe y in the DP ATP of is tro to nd ge in the ppe ydroxyl rad n pe he +OH +OH ditions the situ ol alogous tion is e in 1D0 th radical.but Ho that can P t a apid rate e is estab ligh tion o at ar Atmosp and H,0,→oH+oH+O, ng fuid.and this ma .jclinpath.com

D-amino acid oxidase, might produce hydro￾gen peroxide directly.9 Hydrogen peroxide is not a free radical itself, but is usually included under the general heading of reactive oxygen species (ROS). It is a weak oxidising agent that might directly damage proteins and enzymes containing reactive thiol groups. However, its most vital property is the ability to cross cell membranes freely, which superoxide generally cannot do.10 Therefore, hydrogen peroxide formed in one location might diVuse a consid￾erable distance before decomposing to yield the highly reactive hydroxyl radical, which is likely to mediate most of the toxic eVects ascribed to hydrogen peroxide. Therefore, hydrogen peroxide acts as a conduit to transmit free radical induced damage across cell com￾partments and between cells. In the presence of hydrogen peroxide, myeloperoxidase will gen￾erate hypochlorous acid and singlet oxygen, a reaction that plays an important role in the killing of bacteria by phagocytes.11 The hydroxyl radical (OH. ), or a closely related species, is probably the final mediator of most free radical induced tissue damage.12 All of the reactive oxygen species described above exert most of their pathological eVects by giving rise to hydroxyl radical formation. The reason for this is that the hydroxyl radical reacts, with extremely high rate constants, with almost every type of molecule found in living cells including sugars, amino acids, lipids, and nucleotides. Although hydroxyl radical forma￾tion can occur in several ways, by far the most important mechanism in vivo is likely to be the transition metal catalysed decomposition of superoxide and hydrogen peroxide.13 All elements in the first row of the d-block of the periodic table are classified as transition metals. In general, they contain one or more unpaired electrons and are therefore them￾selves radicals when in the elemental state. However, their key property from the point of view of free radical biology is their variable valency, which allows them to undergo reac￾tions involving the transfer of a single electron. The most important transition metals in human disease are iron and copper. These ele￾ments play a key role in the production of hydroxyl radicals in vivo.13 Hydrogen peroxide can react with iron II (or copper I) to generate the hydroxyl radical, a reaction first described by Fenton in 1894: Fe2+ + H2O2 → Fe3+ + OH. + OH− This reaction can occur in vivo, but the situ￾ation is complicated by the fact that superoxide (the major source of hydrogen peroxide in vivo) will normally also be present. Superoxide and hydrogen peroxide can react together directly to produce the hydroxyl radical, but the rate constant for this reaction in aqueous solution is virtually zero. However, if transition metal ions are present a reaction sequence is established that can proceed at a rapid rate: Fe3+ + O2 − → Fe2+ + O2 Fe2+ + H2O2 → Fe3+ + OH. + OH− net result: O2 − + H2O2 → OH− + OH. + O2 The net result of the reaction sequence illus￾trated above is known as the Haber-Weiss reac￾tion. Although most iron and copper in the body are sequestered in forms that are not available to catalyse this reaction sequence, it is still of importance as a mechanism for the for￾mation of the hydroxyl radical in vivo. The actual reactions, however, may be somewhat more complex than those described above and it is possible that other reactive intermediates such as the ferryl and perferryl radicals might also be formed.12 Approximately 4.5 g of iron can be found in the average adult man, most of which is contained in the haemoglobin molecule and other haem containing proteins. Dietary iron is absorbed preferentially from the proximal part of the small intestine in the divalent form and is transferred to the circulation in which it is car￾ried by transferrin.14 Under most circum￾stances iron remains tightly bound to one of several proteins, including transferrin, lactofer￾rin, haem proteins, ferritin, or haemosiderin. In addition, however, it seems likely that a small iron pool will be maintained as complexes with a variety of small molecules, such as nucleo￾tides and citrate within the cytoplasm and subcellular organelles.14 This pool is probably capable of catalysing an iron driven Fenton reaction in vivo. Certainly, these complexes can promote hydroxyl radical formation in vitro.15 Redox reactive iron can be measured using the bleomycin iron assay,16 although it remains unclear to what extent iron detected by this assay correlates with any discrete anatomical or physiological pool. In normal circumstances, no bleomycin reactive iron is detectable in plasma from healthy subjects, implying that transferrin or ferritin bound iron is not available to drive hydroxyl radical production.17 However, transferrin will release its iron at an acidic pH, particularly in the presence of small molecular weight chelating agents such as ADP, ATP, and citrate.15 Such conditions are found in areas of active inflammation and dur￾ing ischaemia reperfusion injury,18 and it is therefore likely that hydroxyl radicals contrib￾ute to tissue damage in these settings. Iron is released from ferritin by reducing agents including ascorbate and superoxide itself,19 20 and hydrogen peroxide can release iron from a range of haem proteins.21 Therefore, although the iron binding proteins eVectively chelate iron and prevent any appreciable redox eVects under normal physiological conditions, this protection can break down in disease states. The role of copper is analogous to that described above for iron.22 23 Although free radical production occurs as a consequence of the endogenous reactions described above and plays an important role in normal cellular function, it is important to remember that exogenous environmental fac￾tors can also promote radical formation. Ultraviolet light will lead to the formation of singlet oxygen and other reactive oxygen species in the skin.24 Atmospheric pollutants such as ozone and nitrogen dioxide lead to radical formation and antioxidant depletion in the bronchoalveolar lining fluid, and this may Antioxidants in health and disease 177 www.jclinpath.com Downloaded from jcp.bmj.com on February 16, 2012 - Published by group.bmj.com

Downloaded from jcp.bmj.comon February 16.2012-Published by group.bmj.com 178 Free radical production oxide dismutases al binding oxidase 02,H202 ferrin de which migh be Fe,Cu Chain ROOH +2GSH SSG ROH .Consumed during scavenging process fo he Lipid phase OH sulting from active Tissu damag enc thione pero Repa mechanism to mainly the tch as are found in subc ell dist that peroxi is th .25-7 sub ar compart ousn with d gluta of s NAD The ded by acity to react ir ny cellu m as oner 22m of gh hione pe and pre has a stas reins(fi). and transition metal binding pro C IDANT ENZYM The )de to must then be was th ed above e are th form with a sp ellular location and (1)d t超 cforPranmbke plasmand NADPH kDa has tw 10 active ible t the (2) Ma also of the of The of catalas in cyto s of fo abunits,each pro roxisomes ad dur

exacerbate respiratory disease.25–27 Cigarette smoke contains millimolar amounts of free radicals, along with other toxins.28 Various xenobiotics also cause tissue damage as a consequence of free radical generation, including paraquat,29 paracetamol,30 bleomy￾cin,31 and anthracyclines.32 Antioxidant defence systems Because radicals have the capacity to react in an indiscriminate manner leading to damage to almost any cellular component, an extensive range of antioxidant defences, both endog￾enous and exogenous, are present to protect cellular components from free radical induced damage. These can be divided into three main groups: antioxidant enzymes, chain breaking antioxidants, and transition metal binding pro￾teins2 (fig 2). THE ANTIOXIDANT ENZYMES Catalase Catalase was the first antioxidant enzyme to be characterised and catalyses the two stage conversion of hydrogen peroxide to water and oxygen: catalase–Fe(III) + H2O2 → compound I compoundI+H2O2 → catalase–Fe(III) + 2H2O+O2 Catalase consists of four protein subunits, each containing a haem group and a molecule of NADPH.33 The rate constant for the reactions described above is extremely high (∼107 M/sec), implying that it is virtually impossible to saturate the enzyme in vivo. Catalase is largely located within cells in peroxisomes, which also contain most of the enzymes capable of generating hydrogen per￾oxide. The amount of catalase in cytoplasm and other subcellular compartments remains unclear, because peroxisomes are easily rup￾tured during the manipulation of cells. The greatest activity is present in liver and erythro￾cytes but some catalase is found in all tissues. Glutathione peroxidases and glutathione reductase Glutathione peroxidases catalyse the oxidation of glutathione at the expense of a hydroperox￾ide, which might be hydrogen peroxide or another species such as a lipid hydroperoxide34: ROOH + 2GSH → GSSG + H2O + ROH Other peroxides, including lipid hydroperox￾ides, can also act as substrates for these enzymes, which might therefore play a role in repairing damage resulting from lipid peroxi￾dation. Glutathione peroxidases require sele￾nium at the active site, and deficiency might occur in the presence of severe selenium deficiency.35 Several glutathione peroxidase enzymes are encoded by discrete genes.36 The plasma form of glutathione peroxidase is believed to be synthesised mainly in the kidney.37 Within cells, the highest concentra￾tions are found in liver although glutathione peroxidase is widely distributed in almost all tissues. The predominant subcellular distribu￾tion is in the cytosol and mitochondria, suggesting that glutathione peroxidase is the main scavenger of hydrogen peroxide in these subcellular compartments. The activity of the enzyme is dependent on the constant availabil￾ity of reduced glutathione.38 The ratio of reduced to oxidised glutathione is usually kept very high as a result of the activity of the enzyme glutathione reductase: GSSG + NADPH + H+ → 2GSH + NADP+ The NADPH required by this enzyme to replenish the supply of reduced glutathione is provided by the pentose phosphate pathway. Any competing pathway that utilises NADPH (such as the aldose reductase pathway) might lead to a deficiency of reduced glutathione and hence impair the action of glutathione peroxi￾dase. Glutathione reductase is a flavine nucle￾otide dependent enzyme and has a similar tissue distribution to glutathione peroxidase.39 Superoxide dismutase The superoxide dismutases catalyse the dismu￾tation of superoxide to hydrogen peroxide: O2 − + O2 − + 2H+ → H2O2 + O2 The hydrogen peroxide must then be removed by catalase or glutathione peroxidase, as described above. There are three forms of superoxide dismutase in mammalian tissues, each with a specific subcellular location and diVerent tissue distribution. (1) Copper zinc superoxide dismutase (CuZn￾SOD): CuZnSOD is found in the cyto￾plasm and organelles of virtually all mam￾malian cells.40 It has a molecular mass of approximately 32 000 kDa and has two protein subunits, each containing a cata￾lytically active copper and zinc atom. (2) Manganese superoxide dismutase (MnSOD): MnSOD is found in the mito￾chondria of almost all cells and has a molecular mass of 40 000 kDa.41 It con￾sists of four protein subunits, each prob￾ably containing a single manganese atom. The amino acid sequence of MnSOD is entirely dissimilar to that of CuZnSOD Figure 2 Antioxidant defences against free radical attack. Antioxidant enzymes catalyse the breakdown of free radical species, usually in the intracellular environment. Transition metal binding proteins prevent the interaction of transition metals such as iron and copper with hydrogen peroxide and superoxide producing highly reactive hydroxyl radicals. Chain breaking antioxidants are powerful electron donors and react preferentially with free radicals before important target molecules are damaged. In doing so, the antioxidant is oxidised and must be regenerated or replaced. By definition, the antioxidant radical is relatively unreactive and unable to attack further molecules. Free radical production Chain breaking antioxidants •Directly scavenge free radicals •Consumed during scavenging process Lipid phase •Tocopherols •Ubiquinol •Carotenoids •Flavonoids Aqueous phase •Ascorbate •Urate •Glutathione and other thiols Transition metals Fe2+, Cu+ Tissue damage Repair mechanisms O2 –., H2O2 OH. Enzyme antioxidants •Superoxide dismutases •Catalase •Glutathione peroxidase •Caeruloplasmin Metal binding proteins •Transferrin •Ferritin •Lactoferrin 178 Young, Woodside www.jclinpath.com Downloaded from jcp.bmj.com on February 16, 2012 - Published by group.bmj.com

Downloaded from icp.bmi.com on February 16.2012-Published by aroup.bmi.com 170 peripheral neuropathy that occurs in abetalipo that of CuZnSOD in mixtures of the two The al tra and regulation of OD E cretor SOD trap rad ncuding itial d on the in E will not p it,b ble in ant ant n the of the tocopherol rad pla role ted y factor the h the extra the plm se adical migh coph d by with anothe ueous phase c ain b aking antiox can en re a-to of suc reaction is lipic may be completely oxidised to form the radic are area group of lipid solub of the xidant small tro the ap D with th nd th g product ll not nting the chain e furth smight play the min A aneous nhase and linid nhase antiovidant onoids are a larg group of polyphe anes and lip vegeta d m时 antio int ral gr e ca ring in the Epide logica (g.B.v.and of ch s such mpo nds ou nd rapi ted be a c th of co bility man w,alth ome e th igh hat aug contribute to the Apart from日l onoids.other dietary phenolic

and it is not inhibited by cyanide, allowing MnSOD activity to be distinguished from that of CuZnSOD in mixtures of the two enzymes. (3) Extracellular superoxide dismutase (EC￾SOD): EC-SOD was described by Mark￾lund in 198242 and is a secretory copper and zinc containing SOD distinct from the CuZnSOD described above. EC-SOD is synthesised by only a few cell types, including fibroblasts and endothelial cells, and is expressed on the cell surface where it is bound to heparan sulphates. EC-SOD is the major SOD detectable in extracellu￾lar fluids and is released into the circula￾tion from the surface of vascular endothe￾lium following the injection of heparin.43 EC-SOD might play a role in the regula￾tion of vascular tone, because endothelial derived relaxing factor (nitric oxide or a closely related compound) is neutralised in the plasma by superoxide.44 THE CHAIN BREAKING ANTIOXIDANTS Whenever a free radical interacts with another molecule, secondary radicals may be generated that can then react with other targets to produce yet more radical species. The classic example of such a chain reaction is lipid peroxidation, and the reaction will continue to propagate until two radicals combine to form a stable product or the radicals are neutralised by a chain breaking antioxidant.45 Chain breaking antioxidants are small molecules that can receive an electron from a radical or donate an electron to a radical with the formation of stable byproducts.46 In general, the charge associated with the presence of an unpaired electron becomes dissociated over the scaven￾ger and the resulting product will not readily accept an electron from or donate an electron to another molecule, preventing the further propagation of the chain reaction. Such antioxidants can be conveniently divided into aqueous phase and lipid phase antioxidants. Lipid phase chain breaking antioxidants These antioxidants scavenge radicals in mem￾branes and lipoprotein particles and are crucial in preventing lipid peroxidation. The most important lipid phase antioxidant is probably vitamin E.47 Vitamin E occurs in nature in eight diVerent forms, which diVer greatly in their degree of biological activity. The tocopherols (á, â, ã, and ä) have a chromanol ring and a phytyl tail, and diVer in the number and position of the methyl groups on the ring. The tocotrienols (á, â, ã, and ä) are structurally similar but have unsaturated tails. Both classes of compounds are lipid soluble and have pronounced antioxidant properties.48 They react more rapidly than polyunsaturated fatty acids with peroxyl radicals and hence act to break the chain reaction of lipid peroxidation. In addition to its antioxidant role, vitamin E might also have a structural role in stabilising membranes.49 Frank vitamin E deficiency is rare in humans, although it might cause haemolysis50 and might contribute to the peripheral neuropathy that occurs in abetalipo￾proteinaemia.51 The absorption, transport, and regulation of plasma concentrations of vitamin E in humans has been reviewed by Kayden and Traber,52 although in general the metabolism of vitamin E is not well described. In cell membranes and lipoproteins the essential antioxidant function of vitamin E is to trap peroxyl radicals and to break the chain reaction of lipid peroxidation.53 Vitamin E will not prevent the initial formation of carbon centred radicals in a lipid rich environment, but does minimise the formation of secondary radicals. á-Tocopherol is the most potent antioxidant of the tocopherols and is also the most abundant in humans. It quickly reacts with a peroxyl radical to form a relatively stable tocopheroxyl radical, with the excess charge associated with the extra electron being dispersed across the chromanol ring. This resonance stabilised radical might subse￾quently react in one of several ways. á-Tocopherol might be regenerated by reaction at the aqueous interface with ascorbate54 or another aqueous phase chain breaking antioxi￾dant, such as reduced glutathione or urate.55 Alternatively, two á-tocopheroxyl radicals might combine to form a stable dimer, or the radical may be completely oxidised to form tocopherol quinone. The carotenoids are a group of lipid soluble antioxidants based around an isoprenoid car￾bon skeleton.56 The most important of these is â-carotene, although at least 20 others may be present in membranes and lipoproteins. They are particularly eYcient scavengers of singlet oxygen,57 but can also trap peroxyl radicals at low oxygen pressure with an eYciency at least as great as that of á-tocopherol. Because these conditions prevail in many biological tissues, the carotenoids might play a role in preventing in vivo lipid peroxidation.58 The other impor￾tant role of certain carotenoids is as precursors of vitamin A (retinol). Vitamin A also has anti￾oxidant properties,59 which do not, however, show any dependency on oxygen concentra￾tion. Flavonoids are a large group of polyphenolic antioxidants found in many fruits, vegetables, and beverages such as tea and wine.60–62 Over 4000 flavonoids have been identified and they are divided into several groups according to their chemical structure, including flavonols (quercetin and kaempherol), flavanols (the cat￾echins), flavones (apigenin), and isoflavones (genistein). Epidemiological studies suggest an inverse relation between flavonoid intake and incidence of chronic diseases such as coronary heart disease (CHD).63–65 However, little is cur￾rently known about the absorption and me￾tabolism of flavonoids and epidemiological associations might be a consequence of con￾founding by other factors. Available evidence suggests that the bioavailability of many flavonoids is poor,66–68 and plasma values very low, although there is some evidence that aug￾menting the intake of flavonoids might improve biochemical indices of oxidative damage.68 69 Apart from flavonoids, other dietary phenolic Antioxidants in health and disease 179 www.jclinpath.com Downloaded from jcp.bmj.com on February 16, 2012 - Published by group.bmj.com

Downloaded from jcp.bmj.com on February 16.2012-Published by group.bmj.com 8 nigh mall contribu ularly 【ota -10 the reduced form of coer ted tha that has o urred d by u of th a-tocopnerate ght be Indeed.whene a or isolate d lo sed ated in theipd ight play prote ting the of other chai pid peroxi arif chain the r ith which ubinquino-1bcomes oxidised don n thi the in th nt an e is vi In hu an eral ng hy that etic le will inhi of a It i s the main plasma defence aeainst this ide albumin itself is damaged when i on of bumin likely to be gical im can ther poly te th n be Th min ar s and i inin vivo remains duced glutathione (GSH)is so tha partn but An er ant y ging a variety

compounds might also make a small contribu￾tion to total antioxidant capacity.70 Ubiquinol-10, the reduced form of coen￾zyme Q10, is also an eVective lipid soluble chain breaking antioxidant.71 Although present in lower concentrations than á-tocopherol, it can scavenge lipid peroxyl radicals with higher eYciency than either á-tocopherol or the caro￾tenoids, and can also regenerate membrane bound á-tocopherol from the tocopheryl radi￾cal.72 Indeed, whenever plasma or isolated low density lipoprotein (LDL) cholesterol is ex￾posed to radicals generated in the lipid phase, ubiquinol-10 is the first antioxidant to be con￾sumed, suggesting that it might be of particular importance in preventing the propagation of lipid peroxidation.73 However, work to clarify further its role has been hampered by the ease with which ubinquinol-10 becomes oxidised during sample handling or analysis. Aqueous phase chain breaking antioxidants These antioxidants will directly scavenge radicals present in the aqueous compartment. Qualitatively the most important antioxidant of this type is vitamin C (ascorbate).74 In humans, ascorbate acts as an essential cofactor for several enzymes catalysing hydroxylation reac￾tions. In most cases, it provides electrons for enzymes that require prosthetic metal ions in a reduced form to achieve full enzymatic activity. Its best known role is as a cofactor for prolyl and lysyl oxidases in the synthesis of collagen. However, in addition to these well defined actions, several other biochemical pathways depend upon the presence of ascorbate.75 In addition to its role as an enzyme cofactor, the other major function of ascorbate is as a key chain breaking antioxidant in the aqueous phase.76 Ascorbate has been shown to scavenge superoxide, hydrogen peroxide, the hydroxyl radical, hypochlorous acid, aqueous peroxyl radicals, and singlet oxygen. During its antioxi￾dant action, ascorbate undergoes a two elec￾tron reduction, initially to the semidehy￾droascorbyl radical and subsequently to dehydroascorbate. The semidehydroascorbyl radical is relatively stable owing to dispersion of the charge associated with the presence of a single electron over the three oxygen atoms, and it can be readily detected by electron spin resonance in body fluids in the presence of increased free radical production.77 Dehy￾droascorbate is relatively unstable and hydro￾lyses readily to diketogulonic acid, which is subsequently broken down to oxalic acid. Two mechanisms have been described by which dehydroascorbate can be reduced back to ascorbate; one is mediated by the selenoen￾zyme thioredoxin reductase78 and the other is a non-enzyme mediated reaction that uses re￾duced glutathione.79 Dehydroascorbate in plasma is probably rapidly taken up by red blood cells before recycling, so that very little, if any, dehydroascorbate is present in plasma.80 Apart from ascorbate, other antioxidants are present in plasma in high concentrations. Uric acid eYciently scavenges radicals, being con￾verted in the process to allantoin.81 Urate might be particularly important in providing protec￾tion against certain oxidising agents, such as ozone.82 Indeed, it has been suggested that the increase in life span that has occurred during human evolution might be partly explained by the protective action provided by uric acid in human plasma.83 Part of the antioxidant eVect of urate might be attributable to the formation of stable non-reactive complexes with iron, but it is also a direct free radical scavenger. Albumin bound bilirubin is also an eYcient radical scavenger,84 and it has been suggested that it might play a particularly crucial role in protecting the neonate from oxidative dam￾age,85 because deficiency of other chain break￾ing antioxidants is common in the newborn. The other major chain breaking antioxidants in plasma are the protein bound thiol groups. The sulphydryl groups present on plasma pro￾teins can function as chain breaking antioxi￾dants by donating an electron to neutralise a free radical, with the resultant formation of a protein thiyl radical. Albumin is the predomi￾nant plasma protein and makes the major con￾tribution to plasma sulphydryl groups, al￾though it also has several other antioxidant properties.86 Albumin contains 17 disulphide bridges and has a single remaining cysteine residue, and it is this residue that is responsible for the capacity of albumin to react with and neutralise peroxyl radicals.87 This property is important in view of the role albumin plays in transporting free fatty acids in the blood. In addition, albumin has the capacity to bind copper ions and will inhibit copper dependent lipid peroxidation and hydroxyl radical forma￾tion. It is also a powerful scavenger of the phagocytic product hypochlorous acid, and provides the main plasma defence against this oxidant.88 Because albumin itself is damaged when it acts as an antioxidant, it has been viewed as a sacrificial molecule that prevents damage occurring to more vital species.86 The high plasma concentration of albumin and a rela￾tively short half life mean that any damage suf￾fered is unlikely to be of biological importance. However, in vitro work has shown that protein thiyl radicals can themselves act as a potential source of reactive oxidants. The thiyl radical can abstract an electron from a polyunsatu￾rated fatty acid to initiate the process of lipid peroxidation,89 a reaction that can be inhibited by ascorbate and retinol. The antioxidant eVects of albumin and other proteins have been shown to decrease at high concentrations and it has been suggested that this is because thiyl radicals can oxidatively damage other mol￾ecules. The importance of these findings to the antioxidant role of albumin in vivo remains unclear. Reduced glutathione (GSH) is a major source of thiol groups in the intracellular com￾partment but is of little importance in the extracellular space.90 GSH might function directly as an antioxidant, scavenging a variety of radical species, as well as acting as an essen￾tial factor for glutathione peroxidase (discussed above). Thioredoxin might also function as a 180 Young, Woodside www.jclinpath.com Downloaded from jcp.bmj.com on February 16, 2012 - Published by group.bmj.com

Downloaded from jcp.bmj.comon February 16.2012-Published by group.bmj.com tors. INTERACTIONS BETWEEN CHAIN BREAKING ny ol ca sion mpl comp es by gas c e,it i eryl radical at the eins he nsuring that ic damage cts as ipoproteins and membranes.In as ative damag I to cellul fore likely t making t dif anti oxidan and dis he cond imp of chair the of free ra an a ight par to rer ration of vita ell deat from any caus ly,it his doe ar in th In a n en e the id status at a pre ed dam age errin, ntia he oxidant def tem by s nia is ersally ac as tein ca kthroughs nathero 40,+4→4Fe”+2H,0 ion of Fe he he an an yte is ed LDL but o ca nces of oxidative da ns such radi propagated b nd is ba r by one of mo s including rial wall in a mi muscle cells.mac rophages,and lymphocy

key intracellular antioxidant, particularly in redox induced activation of transcription fac￾tors.91 INTERACTIONS BETWEEN CHAIN BREAKING ANTIOXIDANTS Although the actions of chain breaking antioxi￾dants have been considered separately above, it is vital to remember that in vivo complex inter￾actions between antioxidants are likely to occur. For instance, it is likely that ascorbate will recycle the tocopheryl radical at the aqueous–lipid interface, so regenerating toco￾pherol.54 This might be crucial in ensuring that tocopherol concentrations are maintained in lipoproteins and membranes. In a similar man￾ner, glutathione can regenerate ascorbate from dehydroascorbate. A complex interplay is therefore likely to exist between antioxidants, making it diYcult to predict how antioxidants will function in vivo. It therefore becomes meaningless to ask which antioxidant is most important: the answer will depend on the circumstances existing in a particular microen￾vironment at a specific time, and on the nature of the oxidant injury taking place. A second important property of chain breaking antioxidants is their ability to act as pro-oxidants. In certain circumstances, the presence of an antioxidant might paradoxically lead to increased oxidative damage. For instance, it has been reported that the adminis￾tration of vitamin C can sometimes lead to an increase in oxidative damage, particularly if iron is also administered.92 Similarly, it has been clearly shown in vitro that tocopherol might promote LDL oxidation in the absence of an aqueous phase antioxidant such as ascor￾bate.93 Whether these reactions are important in vivo is as yet unclear. However, the possibility that antioxidants may have pro￾oxidant eVects in vivo must be considered when designing and interpreting the results of clinical trials of antioxidant supplementation. THE TRANSITION METAL BINDING PROTEINS As discussed above, transition metal binding proteins (ferritin, transferrin, lactoferrin, and caeruloplasmin) act as a crucial component of the antioxidant defence system by sequestering iron and copper so that they are not available to drive the formation of the hydroxyl radical. The main copper binding protein, caeruloplas￾min, might also function as an antioxidant enzyme that can catalyse the oxidation of diva￾lent iron.94 4Fe2+ + O2 + 4H+ → 4Fe3+ + 2H2O Fe2+ is the form of iron that drives the Fenton reaction and the rapid oxidation of Fe2+ to the less reactive Fe3+ form is therefore an antioxi￾dant eVect. Consequences of oxidative damage Oxidative stress, arising as a result of an imbal￾ance between free radical production and anti￾oxidant defences, is associated with damage to a wide range of molecular species including lipids, proteins, and nucleic acids. Lipoprotein particles or membranes characteristically un￾dergo the process of lipid peroxidation, giving rise to a variety of products including short chain aldehydes such as malondialdehyde or 4-hydroxynonenal, alkanes, and alkenes, conju￾gated dienes, and a variety of hydroxides and hydroperoxides.45 Many of these products can be measured as markers of lipid peroxidation. Detailed discussion of this complex issue is outside the scope of this review, but the measurement of isoprostanes by gas chroma￾tography mass spectroscopy is probably the most specific marker of free radical damage to lipids.95 Oxidative damage to proteins and nucleic acids similarly gives rise to a variety of specific damage products as a result of modifi- cations of amino acids or nucleotides.45 Such oxidative damage might also lead to cellular dysfunction, and it is this that might contribute to the pathophysiology of a wide variety of dis￾eases. Oxidative stress and disease A role for oxidative stress has been postulated in many conditions, including atherosclerosis, inflammatory conditions,96 certain cancers,97 and the process of aging.98 In many cases, this follows the observation of increased amounts of free radical damage products, particularly markers of lipid peroxidation, in body fluids. It is important to remember, however, that lipid peroxidation is an inevitable accompaniment of cell death from any cause. In most cases peroxidation is a secondary phenomenon, and this does not therefore directly indicate an important role for oxidative stress in the disease concerned. If a primary role for oxidative stress in a particular setting is to be sustained, there should be a plausible mechanism by which increased free radical production or a decrease in antioxidant defences might occur. In addi￾tion, evidence of oxidative stress should be detectable before the onset of tissue damage and augmentation of antioxidant status at an early stage should either prevent or greatly reduce tissue damage. Atherosclerosis can be taken as an example of a process for which there is substantial evidence of a role for oxidative stress. Hyperc￾holesterolaemia is universally accepted as a major risk factor for atherosclerosis. However, at any given concentration of plasma choles￾terol, there is still great variability in the occur￾rence of cardiovascular events. One of the major breakthroughs in atherogenesis research has been the realisation that oxidative modifi- cation of LDL might be a crucially important step in the development of the atherosclerotic plaque.99 100 The formation of foam cells from monocyte derived macrophages in early atherosclerotic lesions is not caused by native LDL but only after the modification of LDL by various chemical reactions such as oxidation. Oxidation of LDL is a process initiated and propagated by free radicals or by one of several enzymes,101 and is believed to occur mainly in the arterial wall in a microenvironment where antioxidants may become depleted. All the cells of the vessel wall—endothelial cells, smooth muscle cells, macrophages, and lymphocytes— Antioxidants in health and disease 181 www.jclinpath.com Downloaded from jcp.bmj.com on February 16, 2012 - Published by group.bmj.com

Downloaded from jcp.bmj.com on February 16.2012-Published by group.bmj.com 8 he rith CHD ca e radi the proc rea ars to be th uch as t most effectiv tion by h i ind s inhi antiox rdise (CVD hat of ant mmun ng cas be n to be (MD. association has b of LDL had a 37 isk ofch d of after adius for ae 45 promote atherogen apar had a 41 nts fo Oxi DL are This after che and the use c the endo nd the cyto lial ase in lthough。 LDL CVD one than is im nun wa not been fo nd the alt rp8 ria study in ing rgely dse of vitamin C in is in of 160 ectly in with in aged zyme with m ith higher te of ors ome indic hanis with ing othelial fun h ally redu B-ca pre upplementation in nimal models of ather ated a compared with those in the low own that

can modify LDL in vitro.102–104 Several mecha￾nisms are likely to be involved, including tran￾sition metal ion mediated generation of hydroxyl radicals, the production of reactive oxygen species by enzymes such as myeloper￾oxidase and lipoxygenase, and direct modifica￾tion by reactive nitrogen species. Because oxidation of LDL is primarily a free radical mediated process that is inhibited by antioxi￾dants, antioxidant depletion might be a risk factor for cardiovascular disease (CVD). Evidence for LDL oxidation in vivo is now well established. In immunocytochemical stud￾ies, antibodies against oxidised LDL stain atherosclerotic lesions but not normal arterial tissue.105 LDL extracted from animal and human lesions has been shown to be oxidised and is rapidly taken up by macrophage scaven￾ger receptors.106 In young survivors of myocar￾dial infarction (MI), an association has been demonstrated between increased susceptibility of LDL to oxidation and the degree of coronary atherosclerosis,107 whereas the pres￾ence of ceroid, a product of lipid peroxidation, has been shown in advanced atherosclerotic plaques.108 Oxidised LDL has several properties that promote atherogenesis, apart from its rapid uptake into macrophages via the scavenger receptor. Oxidised forms of LDL are chemo￾tactic for circulating macrophages and smooth muscle cells and facilitate monocyte adhesion to the endothelium and entry into the suben￾dothelial space.109 Oxidised LDL is also cytotoxic towards arterial endothelial cells110 and inhibits the release of nitric oxide and the resulting endothelium dependent vasodila￾tion.111 Therefore, there is a potential role for oxidised LDL in altering vasomotor responses, perhaps contributing to vasospasm in diseased vessels. In addition, oxidised LDL is immuno￾genic; autoantibodies against various epitopes of oxidised LDL have been found in human serum.112 113 and immunoglobulin (IgG) spe￾cific for epitopes of oxidised LDL can be found in lesions.114 Oxidised LDL can induce arterial wall cells to produce chemotactic factors, adhesion molecules, cytokines, and growth factors that have a role to play in the develop￾ment of the plaque.115 116 Apart from the atherogenic consequences of LDL oxidation, it is increasingly recognised that reactive oxygen and nitrogen species directly interact with signalling mechanisms in the arterial wall to regulate vascular function.117 The activities of oxidant generating enzymes in the arterial wall are regulated by both receptor activation and by non-receptor mediated path￾ways. The eVects of antioxidants on these processes are complex but provide alternative mechanisms by which antioxidant supplemen￾tation might ameliorate vascular pathology, for instance by improving endothelial function. Evidence that antioxidant micronutrients potentially reduce the risk of CHD comes from four major sources. First, studies of antioxidant supplementation in animal models of athero￾sclerosis have generally shown a reduction in disease.118 119 Second, many studies have now shown that antioxidant supplementation in healthy subjects or patients with CHD can reduce levels of free radical damage products and protect LDL against oxidation.120 121 Vita￾min E appears to be the most eVective antioxi￾dant; both â-carotene and vitamin C have pro￾duced extensions in lag time to oxidation only in a few studies, although it remains possible that they might have a beneficial eVect in indi￾viduals with poor baseline status. Third, large scale epidemiological studies generally show that low intakes of antioxidants are associated with increased cardiovascular risk after correct￾ing for other risk factors.122–125 The epidemio￾logical evidence is strongest in the case of vita￾min E. In particular, two large longitudinal studies in the USA examined the association between antioxidant intake and the risk of CHD. In a group of 39 910 male health professionals, men who took vitamin E supple￾ments in doses of at least 100 IU/day for over two years had a 37% lower relative risk of CHD than those who did not take vitamin E supple￾ments, after adjustment for age, coronary risk factors, and intake of vitamin C and â-carotene.126 In the nurses’ health study of 87 245 female nurses, women who took vitamin E supplements for more than two years had a 41% lower relative risk of major coronary disease.127 This eVect persisted after adjust￾ment for age, smoking, obesity, exercise, blood pressure, cholesterol, and the use of postmeno￾pausal oestrogen replacement, aspirin, vitamin C, and â-carotene. High vitamin E intakes from dietary sources were not associated with a significant decrease in risk, although even the highest dietary vitamin E intakes were far lower than intakes among supplement users. The evidence linking the water soluble vita￾min C with CVD is less strong than for vitamin E. In the physicians’ follow-up study, a high intake of vitamin C was not associated with a lower risk of CHD in men, whereas in women from the nurses’ health survey, an initial eVect was attenuated after adjustment for multivita￾min use. Only one prospective study involving 11 348 adults demonstrated an inverse relation between vitamin C intake and overall cardio￾vascular mortality.128 This eVect resulted largely from the use of vitamin C in supple￾ments and might have been caused by other antioxidant vitamins in multivitamin prepara￾tions. A prospective population study of 1605 healthy men aged 42, 48, 54, or 60 years in Finland has recently shown that men who had vitamin C deficiency had a relative risk of MI of 2.5 compared with men with higher plasma vitamin C concentrations, after adjustment for other risk factors.129 There is also some indica￾tion that increased dietary intake of â-carotene is associated with reduced risk of CHD, although again the evidence is less convincing than that for vitamin E. In the prospective nurses’ health survey, consumption of vitamins A and â-carotene in food and supplements weakly predicted the incidence of CHD; Gaziano and Hennekens calculated a 22% risk reduction for women in the highest quintile of â-carotene compared with those in the low￾est.130 182 Young, Woodside www.jclinpath.com Downloaded from jcp.bmj.com on February 16, 2012 - Published by group.bmj.com

Downloaded from jcp.bmj.comon February 16.2012-Published by group.bmj.com Amtioridants in health and diseas 183 Thus,there is a plausible case supported by harm(or benefit)amo ong the 11%of partici ld not be ruled ou ir nedian fol ngoing.Ean P udy h rote 2g3 ed with th Finlan us for o upp enehits among se in oping CH of uch dis Hodis in CAn poi was nts of 107% the wel ns un g a myo t th in 25000 18314 nded of se en rom lung cancer in th he eff E mig thes 0.06 b 2 p ula ats ith e eith oint of nor had 0.91t01.09.a ry car There was also no

Thus, there is a plausible case supported by experimental studies, animal experiments, and epidemiology linking oxidative stress and atherosclerosis. The key test of such a hypoth￾esis is whether increased antioxidant intake can be shown to prevent the clinical manifestations of atherosclerosis in humans. Several published randomised studies have now considered this issue, and others are currently ongoing. Early results have not been encouraging. The á-tocopherol, â-carotene cancer preven￾tion trial (ATBC), conducted among 29 133 male heavy smokers in Finland, found no reduction in CHD morbidity or mortality dur￾ing five to eight years of treatment with vitamin E (50 mg daily) or â-carotene (20 mg daily).131 Those assigned vitamin E had no significant decrease in deaths from ischaemic heart disease (IHD), but a 50% excess of deaths from cerebral haemorrhage, whereas those assigned to â-carotene experienced an 11% increase in deaths from IHD. In a further analysis, a subgroup of the original subjects who had suf￾fered a previous MI were considered.132 The endpoint of this substudy was the first major coronary event after randomisation. The pro￾portion of major coronary events did not decrease with either á-tocopherol or â-carotene supplements. In fact, â-carotene conferred an excess of fatal IHD (75% increase in risk). There was a beneficial eVect of vitamin E on non-fatal MI with a risk reduction of 38%. By contrast, in the Chinese cancer prevention study conducted among 29 584 poorly nourished residents of Linxian, China, those randomised for 5.25 years to a combined regimen of 15 mg/day â-carotene, 30 mg/day vitamin E, and 50 µg/day selenium had a significant 9% reduction in total mortality, a significant 21% decrease in stomach cancer deaths, and a non-significant 10% decrease in cerebrovascular mortality.133 However, the wis￾dom of generalising these findings to well nourished populations remains uncertain. The â-carotene and retinol eYcacy trial (CARET), designed to test the eVects of a combined supplement of 30 mg â-carotene and 25 000 IU retinol daily among 18 314 cigarette smokers and individuals with occupa￾tional asbestos exposure, was ended early when researchers recognised a raised risk of death from lung cancer in those receiving â-carotene and, again, no beneficial eVect on CVD was found.134 For CVD mortality, there was a non￾significant 26% increase in the treated group (p = 0.06). The physicians’ health study followed more than 22 000 US male doctors treated with 50 mg â-carotene or placebo every other day for an average of 12 years. The trial appears to have been conducted meticulously and its results seriously question any beneficial eVect with such supplementation on CVD in well nourished populations. There were no signifi- cant eVects on individual outcomes, or on a combined endpoint of non-fatal MI, non-fatal stroke, and cardiovascular death, for which the relative risk was 1.0 (95% confidence interval, 0.91 to 1.09).135 There was also no evidence of harm (or benefit) among the 11% of partici￾pants who were current smokers at baseline, although small eVects could not be ruled out. Greenberg et al studied the eVect of â-carotene supplementation (50 mg/day) in 1720 male and female subjects for a median period of 4.3 years with a median follow up of 8.2 years.136 Subjects whose plasma values of â-carotene were in the highest quartile at the beginning of the study had the lowest risk of death from all causes compared with those in the lowest quartile. However, supplementation had no eVect on either all cause or cardiovas￾cular mortality. Thus for â-carotene supple￾mentation, it would appear that there are no overall benefits among those individuals with a good nutritional status who are at low or aver￾age risk of developing CHD. The situation might be diVerent, however, for those with a previous history of such disease. Hodis et al have shown a reduction in CAD progression (as measured angiographically) in men given 100 IU vitamin E daily, although no benefit was found for vitamin C.137 Singh et al found that a combination of vitamins A, C, E, and â-carotene administered within a few hours after acute MI and continued for 28 days led to significantly fewer cardiac events and a lower incidence of angina pectoris in the supplemented group.138 The Cambridge heart antioxidant study (CHAOS), a trial of vitamin E supplementation on 2002 patients with angiographic evidence of coronary disease, was carried out with a mean treatment duration of 1.4 years.139 It was found that this short term supplementation with á-tocopherol (268 or 537 mg/day) reduced CHD morbidity in pa￾tients, in that patients had a significantly (77%) decreased risk of subsequent non-fatal MI. However, no benefit was found in terms of cardiovascular mortality, with a non-significant excess among vitamin E allocated participants. The GISSI-P study randomised 11 324 men surviving a myocardial infarction to 300 mg vitamin E, 1 g n-3 polyunsaturated fatty acids (PUFAs), both, or neither in a randomised, placebo controlled trial.140 Results suggested a beneficial eVect of n-3 PUFAs but no benefit with vitamin E (p = 0.07). However, further analysis of secondary endpoints suggested some beneficial eVects of vitamin E. In addition, the eVect of vitamin E might have been ameliorated by the Mediterranean diet of the subjects. Neither of these qualifications holds true for the HOPE study,141 which recruited over 9000 subjects likely to be eating a typical northern European diet, who were at high risk for cardiovascular events because they had CVD or diabetes in addition to one other risk factor. Subjects were randomly assigned according to a two by two factorial design to receive either 400 IU of vitamin E daily from natural sources or matching placebo, and either an angiotensin converting enzyme inhibitor (ramipril) or matching placebo for a mean of 4.5 years. Vitamin E supplementation had no eVect on primary or secondary cardiovascular endpoints. Antioxidants in health and disease 183 www.jclinpath.com Downloaded from jcp.bmj.com on February 16, 2012 - Published by group.bmj.com

Downloaded from jcp.bmj.comon February 16.2012-Published by group.bmj.com 8 defence from the ATBC which shou in ma icatin CHAOS study alth suf y the e of CHD.atte to inter gave a borde ine eas the HOP o far been of the bioche L A we int th disco trial future therapeutic advance ort to sho tha IM E with 9 values and dietar ntak and mal B the al Pro the 品 high in fru ine:the role of the re 10 ical (su ction able H. 262:64 88- 135 HA ugh ther t of asc ide in the CHD 17 C.Q nd 18 Pep y b ph ts are lir 19 198 en speees b an inte nd ChI have in to control for in cohort analyses wer of LEC rats vidence that cal and thath and

Thus, for vitamin E in Western populations, the only available trial data in primary preven￾tion are from the ATBC trial, which show no eVect. In secondary prevention, the accumulat￾ing trial data for vitamin E are less consistent, although not particularly encouraging. The CHAOS study was positive, although it suVers from design limitations. The GISSI-P study gave a borderline result, whereas the HOPE study was unequivocally negative. How should we interpret the discordance between data from cohort studies and the results so far available from clinical trials? In general, it might be that the duration of clinical trials is too short to show a benefit, and that antioxidant intake over many years is required to prevent atherosclerosis. Thought needs to be given to trial design, with dose, duration of treatment and follow up period, initial antioxi￾dant values and dietary intake, and extent and distribution of existing atherosclerosis being taken into consideration. Animal models have nearly always tested the eVects of antioxidants on the early atherosclerotic lesions. Whether or not antioxidants have inhibitory eVects on the later stages remains to be seen. In addition, the complex mixture of antioxidant micronutrients found in a diet high in fruit and vegetable intake might be more eVective than large doses of a small number of antioxidant vitamins. It could be that several of these compounds work together but have no eVect individually, or that other dietary components (such as trace elements) might be eVectors of antioxidant action. The trial evidence available so far relates only to á-tocopherol and â-carotene. Although eVective at protecting against lipid peroxidation, these antioxidants have little eVect on arterial endothelial function. Ascor￾bate, in contrast, seems more eVective in improving endothelial function, although there is less epidemiological support for a protective eVect of ascorbate. Alternatively, the significant results linking antioxidant intake with CHD risk observed in cohort studies might be the result of confound￾ing with other lifestyle behaviours. Slattery et al examined dietary antioxidants and plasma lip￾ids in the coronary artery risk development in young adults (CARDIA) study and found that a higher intake of antioxidants was associated with other lifestyle factors such as physical activity and non-smoking.142 Plasma concen￾trations of antioxidants are linked with social class, being higher in more aZuent groups. Although these variables can be individually controlled for in analyses, it might be that a complex lifelong behaviour pattern needs to be studied before conclusions regarding antioxi￾dants and CHD can be made. For example, passive smoking has recently been shown to have an atherogenic eVect on LDL,143 yet exposure to smoke is a diYcult lifestyle variable to control for in cohort analyses. Conclusions There is overwhelming evidence that oxidative stress occurs in cells as a consequence of normal physiological processes and environ￾mental interactions, and that the complex web of antioxidant defence systems plays a key role in protecting against oxidative damage. These processes appear to be disordered in many conditions, and a plausible hypothesis may be constructed implicating oxidative stress as a cause of tissue damage. However, as illustrated by the example of CHD, attempts to intervene therapeutically by using antioxidant supple￾ments have so far been largely unsuccessful. A more complete understanding of the biochemi￾cal events occurring at a cellular level to influ￾ence oxidative damage is required to guide future therapeutic advances. 1 Halliwell B, Gutteridge JC. The definition and measure￾ment of antioxidants in biological systems. Free Radic Biol Med 1995;18:125–6. 2 Halliwell B; Gutteridge JM. Free radicals in biology and medi￾cine, 2nd ed. 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