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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: “anysubstance that, when present in low concentra-tions compared to that of an oxidisablesubstrate, significantly delays or inhibits theoxidation of that substrate”.1 The physiologicalrole of antioxidants, as this definition suggests,is to prevent damage to cellular componentsarising as a consequence of chemical reactionsinvolving free radicals. In recent years, asubstantial body of evidence has developed

supporting a key role for free radicals in manyfundamental cellular reactions and suggestingthat oxidative stress might be important in thepathophysiology of common diseases includingatherosclerosis, chronic renal failure, anddiabetes mellitus. The aim of this review is toconsider mechanisms of free radical formationin 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 chemicalreactions

A free radical can be defined as any molecularspecies capable of independent existence thatcontains an unpaired electron in an atomicorbital.2 The presence of an unpaired electronresults in certain common properties that areshared by most radicals. Radicals are weaklyattracted to a magnetic field and are said to beparamagnetic. Many radicals are highly reac-tive and can either donate an electron to orextract an electron from other molecules,therefore behaving as oxidants or reductants.

As a result of this high reactivity, most radicalshave a very short half life (10−6 seconds or less)in biological systems, although some speciesmay survive for much longer.2 The mostimportant free radicals in many disease statesare oxygen derivatives, particularly superoxideand the hydroxyl radical. Radical formation inthe body occurs by several mechanisms,involving both endogenous and environmentalfactors (fig 1).

Superoxide (O2

−.) is produced by the addi-tion of a single electron to oxygen, and severalmechanisms exist by which superoxide can beproduced in vivo.3 Several molecules, includingadrenaline, flavine nucleotides, thiol com-pounds, and glucose, can oxidise in the

presence of oxygen to produce superoxide, andthese reactions are greatly accelerated by thepresence of transition metals such as iron orcopper. The electron transport chain in theinner mitochondrial membrane performs thereduction of oxygen to water. During thisprocess free radical intermediates are gener-ated, which are generally tightly bound to thecomponents of the transport chain. However,there is a constant leak of a few electrons intothe mitochondrial matrix and this results in theformation of superoxide.4 The activity of several other enzymes, such as cytochromep450 oxidase in the liver and enzymes involvedin the synthesis of adrenal hormones, also

results in the leakage of a few electrons into thesurrounding cytoplasm and hence superoxideformation. There might also be continuousproduction of superoxide by vascular endothe-lium to neutralise nitric oxide,5 6 production of superoxide by other cells to regulate cellgrowth and diV erentiation,7 and the produc-tion of superoxide by phagocytic cells duringthe respiratory burst.8

Any biological system generating superoxidewill also produce hydrogen peroxide as a resultof a spontaneous dismutation reaction. Inaddition, 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

Transitionmetals

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

J Clin Pathol 2001;54:176–186176

Department of ClinicalBiochemistry, Instituteof Clinical Science,Grosvenor Road,

Belfast, NorthernIreland, BT12 6BJ, UK I S Young

Department of Surgery, Royal Freeand University CollegeLondon Medical

School, 67–73 RidingHouse Street, London,W1P 7LD, UK J V Woodside

Correspondence to:Professor Young,

Department of ClinicalBiochemistry, Institute of Clinical Science, RoyalGroup of Hospitals,Grosvenor Road, BelfastBT12 6BJ, UK [email protected]

Accepted for publication5 June 2000

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D-amino acid oxidase, might produce hydro-gen peroxide directly.9 Hydrogen peroxide isnot a free radical itself, but is usually includedunder the general heading of reactive oxygenspecies (ROS). It is a weak oxidising agent thatmight directly damage proteins and enzymescontaining reactive thiol groups. However, itsmost vital property is the ability to cross cellmembranes freely, which superoxide generallycannot do.10 Therefore, hydrogen peroxideformed in one location might diV use a consid-erable distance before decomposing to yieldthe highly reactive hydroxyl radical, which islikely to mediate most of the toxic eV ectsascribed to hydrogen peroxide. Therefore,hydrogen peroxide acts as a conduit to transmitfree 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, areaction that plays an important role in thekilling of bacteria by phagocytes.11

The hydroxyl radical (OH.), or a closelyrelated species, is probably the final mediator of most free radical induced tissue damage.12 Allof the reactive oxygen species described aboveexert most of their pathological eV ects bygiving rise to hydroxyl radical formation. Thereason for this is that the hydroxyl radicalreacts, with extremely high rate constants, withalmost every type of molecule found in livingcells including sugars, amino acids, lipids, andnucleotides. Although hydroxyl radical forma-tion can occur in several ways, by far the mostimportant mechanism in vivo is likely to be thetransition 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 transitionmetals. 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 variablevalency, which allows them to undergo reac-tions involving the transfer of a single electron.The most important transition metals inhuman disease are iron and copper. These ele-ments play a key role in the production of hydroxyl radicals in vivo.13 Hydrogen peroxidecan react with iron II (or copper I) to generatethe hydroxyl radical, a reaction first describedby 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 andhydrogen peroxide can react together directlyto produce the hydroxyl radical, but the rateconstant for this reaction in aqueous solution isvirtually zero. However, if transition metal ionsare present a reaction sequence is establishedthat 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 thebody are sequestered in forms that are notavailable to catalyse this reaction sequence, it isstill of importance as a mechanism for the for-mation of the hydroxyl radical in vivo. Theactual reactions, however, may be somewhatmore complex than those described above andit is possible that other reactive intermediates

such as the ferryl and perferryl radicals mightalso be formed.12

Approximately 4.5 g of iron can be found inthe average adult man, most of which iscontained in the haemoglobin molecule andother haem containing proteins. Dietary iron isabsorbed preferentially from the proximal partof the small intestine in the divalent form and istransferred 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. Inaddition, however, it seems likely that a smalliron pool will be maintained as complexes with

a variety of small molecules, such as nucleo-tides and citrate within the cytoplasm andsubcellular organelles.14 This pool is probablycapable of catalysing an iron driven Fentonreaction in vivo. Certainly, these complexes canpromote hydroxyl radical formation in vitro.15

Redox reactive iron can be measured using thebleomycin iron assay,16 although it remainsunclear to what extent iron detected by thisassay correlates with any discrete anatomical orphysiological pool. In normal circumstances,no bleomycin reactive iron is detectable inplasma from healthy subjects, implying thattransferrin or ferritin bound iron is notavailable to drive hydroxyl radical production.17

However, transferrin will release its iron at an

acidic pH, particularly in the presence of smallmolecular weight chelating agents such asADP, ATP, and citrate.15 Such conditions arefound in areas of active inflammation and dur-ing ischaemia reperfusion injury,18 and it istherefore likely that hydroxyl radicals contrib-ute to tissue damage in these settings. Iron isreleased from ferritin by reducing agentsincluding ascorbate and superoxide itself,19 20

and hydrogen peroxide can release iron from arange of haem proteins.21 Therefore, althoughthe iron binding proteins eV ectively chelateiron and prevent any appreciable redox eV ectsunder normal physiological conditions, thisprotection can break down in disease states.

The role of copper is analogous to thatdescribed above for iron.22 23

Although free radical production occurs as aconsequence of the endogenous reactionsdescribed above and plays an important role innormal cellular function, it is important toremember that exogenous environmental fac-tors can also promote radical formation.Ultraviolet light will lead to the formation of singlet oxygen and other reactive oxygenspecies in the skin.24 Atmospheric pollutantssuch as ozone and nitrogen dioxide lead toradical formation and antioxidant depletion inthe bronchoalveolar lining fluid, and this may

Antioxidants in health and disease 177

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exacerbate respiratory disease.25–27 Cigarettesmoke contains millimolar amounts of freeradicals, along with other toxins.28

Various xenobiotics also cause tissue damageas a consequence of free radical generation,including paraquat,29 paracetamol,30 bleomy-cin,31 and anthracyclines.32

Antioxidant defence systemsBecause radicals have the capacity to react inan indiscriminate manner leading to damage toalmost any cellular component, an extensiverange of antioxidant defences, both endog-

enous and exogenous, are present to protectcellular components from free radical induceddamage. These can be divided into three maingroups: antioxidant enzymes, chain breakingantioxidants, and transition metal binding pro-teins2 (fig 2).

THE ANTIOXIDANT ENZYMES

Catalase

Catalase was the first antioxidant enzyme to becharacterised and catalyses the two stageconversion of hydrogen peroxide to water andoxygen:

catalase–Fe(III) + H2O2 → compound Icompound I + H2O2 → catalase–Fe(III) +

2H2O + O2Catalase consists of four protein subunits,

each containing a haem group and a moleculeof NADPH.33 The rate constant for thereactions described above is extremely high(∼107 M/sec), implying that it is virtuallyimpossible to saturate the enzyme in vivo.Catalase is largely located within cells inperoxisomes, which also contain most of theenzymes capable of generating hydrogen per-oxide. The amount of catalase in cytoplasmand other subcellular compartments remainsunclear, 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 oxidationof glutathione at the expense of a hydroperox-ide, which might be hydrogen peroxide oranother species such as a lipid hydroperoxide34:

ROOH + 2GSH → GSSG + H2O + ROHOther peroxides, including lipid hydroperox-

ides, can also act as substrates for theseenzymes, which might therefore play a role inrepairing damage resulting from lipid peroxi-dation. Glutathione peroxidases require sele-nium at the active site, and deficiency mightoccur in the presence of severe seleniumdeficiency.35 Several glutathione peroxidaseenzymes are encoded by discrete genes.36 Theplasma form of glutathione peroxidase isbelieved to be synthesised mainly in thekidney.37 Within cells, the highest concentra-tions are found in liver although glutathioneperoxidase is widely distributed in almost alltissues. The predominant subcellular distribu-tion is in the cytosol and mitochondria,

suggesting that glutathione peroxidase is themain scavenger of hydrogen peroxide in thesesubcellular compartments. The activity of theenzyme is dependent on the constant availabil-ity of reduced glutathione.38 The ratio of reduced to oxidised glutathione is usually keptvery high as a result of the activity of theenzyme glutathione reductase:

GSSG + NADPH + H+→ 2GSH + NADP+

The NADPH required by this enzyme toreplenish the supply of reduced glutathione isprovided by the pentose phosphate pathway.Any competing pathway that utilises NADPH(such as the aldose reductase pathway) mightlead to a deficiency of reduced glutathione andhence impair the action of glutathione peroxi-

dase. Glutathione reductase is a flavine nucle-otide dependent enzyme and has a similartissue distribution to glutathione peroxidase.39

Superoxide dismutaseThe superoxide dismutases catalyse the dismu-tation of superoxide to hydrogen peroxide:

O2

− + O2

− + 2H+ → H2O2 + O2

The hydrogen peroxide must then beremoved by catalase or glutathione peroxidase,as described above. There are three forms of superoxide dismutase in mammalian tissues,each with a specific subcellular location anddiV erent 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 twoprotein 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 amolecular 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 isentirely dissimilar to that of CuZnSOD

Figure 2 Antioxidant defences against free radical attack. Antioxidant enzymes catalysethe breakdown of free radical species,usually in the intracellular environment. Transitionmetal binding proteins prevent the interaction of transition metals such as iron and copper with hydrogen peroxide and superoxide producing highly reactive hydroxyl radicals. Chainbreaking antioxidants are powerful electron donors and react preferentially with free radicalsbefore 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

Transitionmetals

Fe2+

, Cu+

Tissue damage

Repair mechanisms

O2–.

, H2O2

OH.

Enzyme antioxidants •Superoxide dismutases •Catalase •Glutathione peroxidase •Caeruloplasmin

Metal bindingproteins

•Transferrin •Ferritin •Lactoferrin

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and it is not inhibited by cyanide, allowingMnSOD activity to be distinguished fromthat of CuZnSOD in mixtures of the twoenzymes.

(3) Extracellular superoxide dismutase (EC-SOD): EC-SOD was described by Mark-lund in 198242 and is a secretory copperand zinc containing SOD distinct from theCuZnSOD described above. EC-SOD issynthesised by only a few cell types,

including fibroblasts and endothelial cells,and is expressed on the cell surface whereit is bound to heparan sulphates. EC-SODis 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 endothelialderived relaxing factor (nitric oxide or aclosely related compound) is neutralised inthe plasma by superoxide.44

THE CHAIN BREAKING ANTIOXIDANTS

Whenever a free radical interacts with another

molecule, secondary radicals may be generatedthat can then react with other targets toproduce yet more radical species. The classicexample of such a chain reaction is lipidperoxidation, and the reaction will continue topropagate until two radicals combine to form astable product or the radicals are neutralised bya chain breaking antioxidant.45 Chain breakingantioxidants are small molecules that canreceive an electron from a radical or donate anelectron to a radical with the formation of stable byproducts.46 In general, the chargeassociated with the presence of an unpairedelectron becomes dissociated over the scaven-ger and the resulting product will not readily

accept an electron from or donate an electronto another molecule, preventing the furtherpropagation of the chain reaction. Suchantioxidants can be conveniently divided intoaqueous phase and lipid phase antioxidants.

Lipid phase chain breaking antioxidants

These antioxidants scavenge radicals in mem-branes and lipoprotein particles and are crucialin preventing lipid peroxidation. The mostimportant lipid phase antioxidant is probablyvitamin E.47 Vitamin E occurs in nature in eightdiV erent forms, which diV er greatly in theirdegree of biological activity. The tocopherols(, , , and ) have a chromanol ring and a

phytyl tail, and diV

er in the number andposition of the methyl groups on the ring. Thetocotrienols (, , , and ) are structurallysimilar but have unsaturated tails. Both classesof compounds are lipid soluble and havepronounced antioxidant properties.48 Theyreact more rapidly than polyunsaturated fattyacids with peroxyl radicals and hence act tobreak the chain reaction of lipid peroxidation.In addition to its antioxidant role, vitamin Emight also have a structural role in stabilisingmembranes.49 Frank vitamin E deficiency israre in humans, although it might causehaemolysis50 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 humanshas been reviewed by Kayden and Traber,52

although in general the metabolism of vitaminE is not well described. In cell membranes andlipoproteins the essential antioxidant functionof vitamin E is to trap peroxyl radicals and tobreak the chain reaction of lipid peroxidation.53

Vitamin E will not prevent the initial formationof carbon centred radicals in a lipid richenvironment, but does minimise the formationof secondary radicals. -Tocopherol is the mostpotent antioxidant of the tocopherols and isalso the most abundant in humans. It quicklyreacts with a peroxyl radical to form a relativelystable tocopheroxyl radical, with the excesscharge associated with the extra electron beingdispersed across the chromanol ring. Thisresonance stabilised radical might subse-quently react in one of several ways.-Tocopherol might be regenerated by reactionat the aqueous interface with ascorbate54 oranother aqueous phase chain breaking antioxi-

dant, such as reduced glutathione or urate.55

Alternatively, two -tocopheroxyl radicalsmight combine to form a stable dimer, or theradical may be completely oxidised to formtocopherol quinone.

The carotenoids are a group of lipid solubleantioxidants based around an isoprenoid car-bon skeleton.56 The most important of these is-carotene, although at least 20 others may bepresent in membranes and lipoproteins. Theyare particularly eYcient scavengers of singletoxygen,57 but can also trap peroxyl radicals atlow oxygen pressure with an eYciency at leastas great as that of -tocopherol. Because theseconditions prevail in many biological tissues,

the carotenoids might play a role in preventingin vivo lipid peroxidation.58 The other impor-tant role of certain carotenoids is as precursorsof 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 polyphenolicantioxidants found in many fruits, vegetables,and beverages such as tea and wine.60–62 Over4000 flavonoids have been identified and theyare divided into several groups according totheir chemical structure, including flavonols(quercetin and kaempherol), flavanols (the cat-echins), flavones (apigenin), and isoflavones(genistein). Epidemiological studies suggest aninverse relation between flavonoid intake andincidence of chronic diseases such as coronaryheart disease (CHD).63–65 However, little is cur-rently known about the absorption and me-tabolism of flavonoids and epidemiologicalassociations might be a consequence of con-founding by other factors. Available evidencesuggests that the bioavailability of manyflavonoids is poor,66–68 and plasma values verylow, although there is some evidence that aug-menting the intake of flavonoids might improvebiochemical indices of oxidative damage.68 69

Apart from flavonoids, other dietary phenolic


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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 eV ective lipid solublechain breaking antioxidant.71 Although presentin lower concentrations than -tocopherol, itcan scavenge lipid peroxyl radicals with highereYciency than either -tocopherol or the caro-tenoids, and can also regenerate membranebound -tocopherol from the tocopheryl radi-

cal.72 Indeed, whenever plasma or isolated lowdensity 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 particularimportance in preventing the propagation of lipid peroxidation.73 However, work to clarifyfurther its role has been hampered by the easewith which ubinquinol-10 becomes oxidisedduring sample handling or analysis.

Aqueous phase chain breaking antioxidants

These antioxidants will directly scavengeradicals 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 forseveral enzymes catalysing hydroxylation reac-tions. In most cases, it provides electrons forenzymes that require prosthetic metal ions in areduced form to achieve full enzymatic activity.Its best known role is as a cofactor for prolyland lysyl oxidases in the synthesis of collagen.However, in addition to these well definedactions, several other biochemical pathwaysdepend upon the presence of ascorbate.75 Inaddition to its role as an enzyme cofactor, theother major function of ascorbate is as a keychain breaking antioxidant in the aqueousphase.76 Ascorbate has been shown to scavenge

superoxide, hydrogen peroxide, the hydroxylradical, hypochlorous acid, aqueous peroxylradicals, and singlet oxygen. During its antioxi-dant action, ascorbate undergoes a two elec-tron reduction, initially to the semidehy-droascorbyl radical and subsequently todehydroascorbate. The semidehydroascorbylradical is relatively stable owing to dispersion of the charge associated with the presence of asingle electron over the three oxygen atoms,and it can be readily detected by electron spinresonance 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. Twomechanisms have been described by whichdehydroascorbate can be reduced back toascorbate; one is mediated by the selenoen-zyme thioredoxin reductase78 and the other is anon-enzyme mediated reaction that uses re-duced glutathione.79 Dehydroascorbate inplasma is probably rapidly taken up by redblood cells before recycling, so that very little,if any, dehydroascorbate is present in plasma.80

Apart from ascorbate, other antioxidants arepresent in plasma in high concentrations. Uricacid 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 asozone.82 Indeed, it has been suggested that theincrease in life span that has occurred duringhuman evolution might be partly explained bythe protective action provided by uric acid inhuman plasma.83 Part of the antioxidant eV ectof urate might be attributable to the formationof stable non-reactive complexes with iron, butit is also a direct free radical scavenger.

Albumin bound bilirubin is also an eYcientradical scavenger,84 and it has been suggestedthat it might play a particularly crucial role inprotecting 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 antioxidantsin 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 afree radical, with the resultant formation of aprotein 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 antioxidantproperties.86 Albumin contains 17 disulphidebridges and has a single remaining cysteineresidue, and it is this residue that is responsiblefor the capacity of albumin to react with andneutralise peroxyl radicals.87 This property isimportant in view of the role albumin plays intransporting free fatty acids in the blood. Inaddition, albumin has the capacity to bindcopper ions and will inhibit copper dependentlipid peroxidation and hydroxyl radical forma-tion. It is also a powerful scavenger of thephagocytic product hypochlorous acid, andprovides the main plasma defence against thisoxidant.88

Because albumin itself is damaged when itacts as an antioxidant, it has been viewed as asacrificial molecule that prevents damageoccurring to more vital species.86 The highplasma 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 proteinthiyl radicals can themselves act as a potentialsource of reactive oxidants. The thiyl radicalcan abstract an electron from a polyunsatu-rated fatty acid to initiate the process of lipidperoxidation,89 a reaction that can be inhibitedby ascorbate and retinol. The antioxidanteV ects of albumin and other proteins have beenshown to decrease at high concentrations and ithas been suggested that this is because thiylradicals can oxidatively damage other mol-ecules. The importance of these findings to theantioxidant role of albumin in vivo remainsunclear.

Reduced glutathione (GSH) is a majorsource of thiol groups in the intracellular com-partment but is of little importance in theextracellular space.90 GSH might functiondirectly as an antioxidant, scavenging a varietyof radical species, as well as acting as an essen-tial factor for glutathione peroxidase (discussedabove). Thioredoxin might also function as a

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key intracellular antioxidant, particularly inredox 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, itis vital to remember that in vivo complex inter-actions between antioxidants are likely to

occur. For instance, it is likely that ascorbatewill recycle the tocopheryl radical at theaqueous–lipid interface, so regenerating toco-pherol.54 This might be crucial in ensuring thattocopherol concentrations are maintained inlipoproteins and membranes. In a similar man-ner, glutathione can regenerate ascorbate fromdehydroascorbate. A complex interplay istherefore likely to exist between antioxidants,making it diYcult to predict how antioxidantswill function in vivo. It therefore becomesmeaningless to ask which antioxidant is mostimportant: the answer will depend on thecircumstances 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 chainbreaking antioxidants is their ability to act aspro-oxidants. In certain circumstances, thepresence of an antioxidant might paradoxicallylead to increased oxidative damage. Forinstance, it has been reported that the adminis-tration of vitamin C can sometimes lead to anincrease in oxidative damage, particularly if iron is also administered.92 Similarly, it hasbeen clearly shown in vitro that tocopherolmight promote LDL oxidation in the absenceof an aqueous phase antioxidant such as ascor-bate.93 Whether these reactions are importantin vivo is as yet unclear. However, thepossibility that antioxidants may have pro-

oxidant eV ects in vivo must be consideredwhen designing and interpreting the results of clinical trials of antioxidant supplementation.

THE TRANSITION METAL BINDING PROTEINS

As discussed above, transition metal bindingproteins (ferritin, transferrin, lactoferrin, andcaeruloplasmin) act as a crucial component of the antioxidant defence system by sequesteringiron and copper so that they are not available todrive the formation of the hydroxyl radical.The main copper binding protein, caeruloplas-min, might also function as an antioxidantenzyme that can catalyse the oxidation of diva-lent iron.94

4Fe

2+

+ O2 + 4H

+→

4Fe

3+

+ 2H2OFe2+ is the form of iron that drives the Fentonreaction and the rapid oxidation of Fe2+ to theless reactive Fe3+ form is therefore an antioxi-dant eV ect.

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 toa wide range of molecular species includinglipids, proteins, and nucleic acids. Lipoproteinparticles or membranes characteristically un-dergo the process of lipid peroxidation, giving

rise to a variety of products including shortchain aldehydes such as malondialdehyde or4-hydroxynonenal,alkanes, and alkenes, conju-gated dienes, and a variety of hydroxides andhydroperoxides. 45 Many of these products canbe measured as markers of lipid peroxidation.Detailed discussion of this complex issue isoutside the scope of this review, but themeasurement of isoprostanes by gas chroma-tography mass spectroscopy is probably the

most specific marker of free radical damage tolipids.95 Oxidative damage to proteins andnucleic acids similarly gives rise to a variety of specific damage products as a result of modifi-cations of amino acids or nucleotides.45 Suchoxidative damage might also lead to cellulardysfunction, and it is this that might contributeto the pathophysiology of a wide variety of dis-eases.

Oxidative stress and diseaseA role for oxidative stress has been postulatedin many conditions, including atherosclerosis,

inflammatory conditions,

96

certain cancers,

97

and the process of aging.98 In many cases, thisfollows the observation of increased amountsof free radical damage products, particularlymarkers of lipid peroxidation, in body fluids. Itis important to remember, however, that lipidperoxidation is an inevitable accompaniment of cell death from any cause. In most casesperoxidation is a secondary phenomenon, andthis does not therefore directly indicate animportant role for oxidative stress in the diseaseconcerned. If a primary role for oxidative stressin a particular setting is to be sustained, thereshould be a plausible mechanism by whichincreased free radical production or a decreasein antioxidant defences might occur. In addi-

tion, evidence of oxidative stress should bedetectable before the onset of tissue damageand augmentation of antioxidant status at anearly stage should either prevent or greatlyreduce tissue damage.

Atherosclerosis can be taken as an exampleof a process for which there is substantialevidence of a role for oxidative stress. Hyperc-holesterolaemia is universally accepted as amajor 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 themajor breakthroughs in atherogenesis researchhas been the realisation that oxidative modifi-

cation of LDL might be a crucially importantstep in the development of the atheroscleroticplaque.99 100 The formation of foam cells frommonocyte derived macrophages in earlyatherosclerotic lesions is not caused by nativeLDL but only after the modification of LDL byvarious chemical reactions such as oxidation.Oxidation of LDL is a process initiated andpropagated by free radicals or by one of severalenzymes,101 and is believed to occur mainly inthe arterial wall in a microenvironment whereantioxidants may become depleted.All the cellsof the vessel wall—endothelial cells, smoothmuscle cells, macrophages, and lymphocytes—


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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 reactiveoxygen species by enzymes such as myeloper-oxidase and lipoxygenase, and direct modifica-tion by reactive nitrogen species. Becauseoxidation of LDL is primarily a free radicalmediated 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 stainatherosclerotic lesions but not normal arterialtissue.105 LDL extracted from animal andhuman lesions has been shown to be oxidisedand is rapidly taken up by macrophage scaven-ger receptors.106 In young survivors of myocar-dial infarction (MI), an association has beendemonstrated between increased susceptibilityof 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 thatpromote atherogenesis, apart from its rapiduptake into macrophages via the scavengerreceptor. Oxidised forms of LDL are chemo-tactic for circulating macrophages and smoothmuscle cells and facilitate monocyte adhesionto the endothelium and entry into the suben-dothelial space.109 Oxidised LDL is alsocytotoxic towards arterial endothelial cells110

and inhibits the release of nitric oxide and theresulting endothelium dependent vasodila-tion.111 Therefore, there is a potential role foroxidised LDL in altering vasomotor responses,perhaps contributing to vasospasm in diseasedvessels. In addition, oxidised LDL is immuno-

genic; autoantibodies against various epitopesof oxidised LDL have been found in humanserum.112 113 and immunoglobulin (IgG) spe-cific for epitopes of oxidised LDL can be foundin lesions.114 Oxidised LDL can induce arterialwall cells to produce chemotactic factors,adhesion molecules, cytokines, and growthfactors 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 recognisedthat reactive oxygen and nitrogen speciesdirectly interact with signalling mechanisms inthe arterial wall to regulate vascular function.117

The activities of oxidant generating enzymes in

the arterial wall are regulated by both receptoractivation and by non-receptor mediated path-ways. The eV ects of antioxidants on theseprocesses are complex but provide alternativemechanisms by which antioxidant supplemen-tation might ameliorate vascular pathology, forinstance by improving endothelial function.

Evidence that antioxidant micronutrientspotentially reduce the risk of CHD comes fromfour major sources. First, studies of antioxidantsupplementation in animal models of athero-sclerosis have generally shown a reduction indisease.118 119 Second, many studies have nowshown that antioxidant supplementation in

healthy subjects or patients with CHD canreduce levels of free radical damage productsand protect LDL against oxidation.120 121 Vita-min E appears to be the most eV ective antioxi-dant; both -carotene and vitamin C have pro-duced extensions in lag time to oxidation onlyin a few studies, although it remains possiblethat they might have a beneficial eV ect in indi-viduals with poor baseline status. Third, largescale epidemiological studies generally show

that low intakes of antioxidants are associatedwith 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 longitudinalstudies in the USA examined the associationbetween antioxidant intake and the risk of CHD. In a group of 39 910 male healthprofessionals, men who took vitamin E supple-ments in doses of at least 100 IU/day for overtwo years had a 37% lower relative risk of CHDthan those who did not take vitamin E supple-ments, after adjustment for age, coronary riskfactors, and intake of vitamin C and-carotene.126 In the nurses’ health study of

87 245 female nurses, women who tookvitamin E supplements for more than two yearshad a 41% lower relative risk of major coronarydisease.127 This eV ect persisted after adjust-ment for age, smoking, obesity, exercise, bloodpressure, cholesterol, and the use of postmeno-pausal oestrogen replacement, aspirin, vitaminC, and -carotene. High vitamin E intakesfrom dietary sources were not associated with asignificant decrease in risk, although even thehighest dietary vitamin E intakes were far lowerthan intakes among supplement users.

The evidence linking the water soluble vita-min C with CVD is less strong than for vitaminE. In the physicians’ follow-up study, a high

intake of vitamin C was not associated with alower risk of CHD in men, whereas in womenfrom the nurses’ health survey, an initial eV ectwas attenuated after adjustment for multivita-min use. Only one prospective study involving11 348 adults demonstrated an inverse relationbetween vitamin C intake and overall cardio-vascular mortality.128 This eV ect resultedlargely from the use of vitamin C in supple-ments and might have been caused by otherantioxidant vitamins in multivitamin prepara-tions. A prospective population study of 1605healthy men aged 42, 48, 54, or 60 years inFinland has recently shown that men who hadvitamin C deficiency had a relative risk of MI of 2.5 compared with men with higher plasmavitamin C concentrations, after adjustment forother risk factors.129 There is also some indica-tion that increased dietary intake of -caroteneis associated with reduced risk of CHD,although again the evidence is less convincingthan that for vitamin E. In the prospectivenurses’ health survey, consumption of vitaminsA and -carotene in food and supplementsweakly predicted the incidence of CHD;Gaziano and Hennekens calculated a 22% riskreduction for women in the highest quintile of -carotene compared with those in the low-est.130

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Thus, there is a plausible case supported byexperimental studies, animal experiments, andepidemiology linking oxidative stress andatherosclerosis. The key test of such a hypoth-esis is whether increased antioxidant intake canbe shown to prevent the clinical manifestationsof atherosclerosis in humans. Several publishedrandomised studies have now considered thisissue, and others are currently ongoing. Earlyresults have not been encouraging.

The -tocopherol,-carotene cancer preven-tion trial (ATBC), conducted among 29 133male heavy smokers in Finland, found noreduction in CHD morbidity or mortality dur-ing five to eight years of treatment with vitaminE (50 mg daily) or -carotene (20 mg daily).131

Those assigned vitamin E had no significantdecrease in deaths from ischaemic heartdisease (IHD),but a 50% excess of deaths fromcerebral haemorrhage, whereas those assignedto -carotene experienced an 11% increase indeaths from IHD. In a further analysis, asubgroup of the original subjects who had suf-fered a previous MI were considered.132 Theendpoint of this substudy was the first major

coronary event after randomisation. The pro-portion of major coronary events did notdecrease with either -tocopherol or-carotene supplements. In fact, -caroteneconferred an excess of fatal IHD (75% increasein risk). There was a beneficial eV ect of vitaminE on non-fatal MI with a risk reduction of 38%. By contrast, in the Chinese cancerprevention study conducted among 29 584poorly nourished residents of Linxian, China,those randomised for 5.25 years to a combinedregimen of 15 mg/day -carotene, 30 mg/dayvitamin E, and 50 µg/day selenium had asignificant 9% reduction in total mortality, asignificant 21% decrease in stomach cancer

deaths, and a non-significant 10% decrease incerebrovascular mortality.133 However, the wis-dom of generalising these findings to wellnourished populations remains uncertain.

The -carotene and retinol eYcacy trial(CARET), designed to test the eV ects of acombined supplement of 30 mg -caroteneand 25 000 IU retinol daily among 18 314cigarette smokers and individuals with occupa-tional asbestos exposure, was ended early whenresearchers recognised a raised risk of deathfrom lung cancer in those receiving -caroteneand, again, no beneficial eV ect on CVD wasfound.134 For CVD mortality, there was a non-significant 26% increase in the treated group(p = 0.06).

The physicians’ health study followed morethan 22 000 US male doctors treated with50 mg -carotene or placebo every other dayfor an average of 12 years. The trial appears tohave been conducted meticulously and itsresults seriously question any beneficial eV ectwith such supplementation on CVD in wellnourished populations. There were no signifi-cant eV ects on individual outcomes, or on acombined endpoint of non-fatal MI, non-fatalstroke, and cardiovascular death, for which therelative 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 eV ects could not be ruled out.

Greenberg et al studied the eV ect of -carotene supplementation (50 mg/day) in1720 male and female subjects for a medianperiod of 4.3 years with a median follow up of 8.2 years.136 Subjects whose plasma values of -carotene were in the highest quartile at thebeginning of the study had the lowest risk of death from all causes compared with those inthe lowest quartile. However, supplementationhad no eV ect on either all cause or cardiovas-cular mortality. Thus for -carotene supple-mentation, it would appear that there are nooverall benefits among those individuals with agood nutritional status who are at low or aver-age risk of developing CHD. The situationmight be diV erent, however, for those with aprevious history of such disease.

Hodis et al have shown a reduction in CADprogression (as measured angiographically) inmen given 100 IU vitamin E daily, although nobenefit was found for vitamin C.137 Singh et al

found that a combination of vitamins A, C, E,and -carotene administered within a fewhours after acute MI and continued for 28 daysled to significantly fewer cardiac events and alower incidence of angina pectoris in thesupplemented group.138 The Cambridge heartantioxidant study (CHAOS), a trial of vitaminE supplementation on 2002 patients withangiographic evidence of coronary disease, wascarried out with a mean treatment duration of 1.4 years.139 It was found that this short termsupplementation with -tocopherol (268 or537 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-significantexcess among vitamin E allocated participants.

The GISSI-P study randomised 11 324 mensurviving a myocardial infarction to 300 mgvitamin E, 1 g n-3 polyunsaturated fatty acids(PUFAs), both, or neither in a randomised,placebo controlled trial.140 Results suggested abeneficial eV ect of n-3 PUFAs but no benefitwith vitamin E (p = 0.07). However, furtheranalysis of secondary endpoints suggestedsome beneficial eV ects of vitamin E. Inaddition, the eV ect of vitamin E might havebeen ameliorated by the Mediterranean diet of the subjects. Neither of these qualificationsholds true for the HOPE study,141 which

recruited over 9000 subjects likely to be eatinga typical northern European diet, who were athigh risk for cardiovascular events because theyhad CVD or diabetes in addition to one otherrisk factor. Subjects were randomly assignedaccording to a two by two factorial design toreceive either 400 IU of vitamin E daily fromnatural sources or matching placebo, and eitheran angiotensin converting enzyme inhibitor(ramipril) or matching placebo for a mean of 4.5 years. Vitamin E supplementation had noeV ect on primary or secondary cardiovascularendpoints.


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Thus, for vitamin E in Western populations,the only available trial data in primary preven-tion are from the ATBC trial, which show noeV ect. In secondary prevention, the accumulat-ing trial data for vitamin E are less consistent,although not particularly encouraging. TheCHAOS study was positive, although it suV ersfrom design limitations. The GISSI-P studygave a borderline result, whereas the HOPEstudy was unequivocally negative.

How should we interpret the discordancebetween data from cohort studies and theresults so far available from clinical trials? Ingeneral, it might be that the duration of clinicaltrials is too short to show a benefit, and thatantioxidant intake over many years is requiredto prevent atherosclerosis. Thought needs to begiven to trial design, with dose, duration of treatment and follow up period, initial antioxi-dant values and dietary intake, and extent anddistribution of existing atherosclerosis beingtaken into consideration. Animal models havenearly always tested the eV ects of antioxidantson the early atherosclerotic lesions. Whether ornot antioxidants have inhibitory eV ects on the

later stages remains to be seen. In addition, thecomplex mixture of antioxidant micronutrientsfound in a diet high in fruit and vegetableintake might be more eV ective than large dosesof a small number of antioxidant vitamins. Itcould be that several of these compounds worktogether but have no eV ect individually, or thatother dietary components (such as traceelements) might be eV ectors of antioxidantaction. The trial evidence available so farrelates only to -tocopherol and -carotene.Although eV ective at protecting against lipidperoxidation, these antioxidants have littleeV ect on arterial endothelial function. Ascor-bate, in contrast, seems more eV ective inimproving endothelial function, although there

is less epidemiological support for a protectiveeV ect of ascorbate.

Alternatively, the significant results linkingantioxidant intake with CHD risk observed incohort 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 inyoung adults (CARDIA) study and found thata higher intake of antioxidants was associatedwith other lifestyle factors such as physicalactivity and non-smoking.142 Plasma concen-trations of antioxidants are linked with socialclass, being higher in more aZuent groups.Although these variables can be individually

controlled for in analyses, it might be that acomplex lifelong behaviour pattern needs to bestudied before conclusions regarding antioxi-dants and CHD can be made. For example,passive smoking has recently been shown tohave an atherogenic eV ect on LDL,143 yetexposure to smoke is a diYcult lifestyle variableto control for in cohort analyses.

ConclusionsThere is overwhelming evidence that oxidativestress 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 rolein protecting against oxidative damage. Theseprocesses appear to be disordered in manyconditions, and a plausible hypothesis may beconstructed implicating oxidative stress as acause of tissue damage. However, as illustratedby the example of CHD, attempts to intervenetherapeutically by using antioxidant supple-ments have so far been largely unsuccessful. Amore complete understanding of the biochemi-

cal events occurring at a cellular level to influ-ence oxidative damage is required to guidefuture therapeutic advances.

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