Dietary Antioxidants in Health and Disease

PII : S0958-6946(98)00070-3

Int. Dairy Journal 8 (1998) 463—472 ( 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0958-6946/98/$19.00#0.00

Dietary Antioxidants in Health and Disease P. A. Morrissey* and N. M. O’Brien Department of Nutrition, University College, Cork, Ireland ABSTRACT Cellular systems are subject to constant oxidative stress by reactive oxygen species (ROS). Oxidative stress has been implicated as a factor in the aetiology of a variety of degenerative diseases and in the ageing process. ROS are capable of causing adverse modifications of macromolecules including lipids, DNA and proteins. Cellular systems also possess antioxidant defence systems whose role is to minimise adverse oxidative changes. The balance between prooxidant forces and antioxidant defence systems influences the body’s susceptibility to prooxidant damage. A variety of nutrient and non-nutrient dietary constituents, including vitamins C, E and carotenoids, have been shown to affect this prooxidant/antioxidant balance and consequent risk of certain degenerative diseases. Defining optimal intakes of these nutrients and non-nutrients is a key challenge in nutrition research. ( 1998 Elsevier Science Ltd. All rights reserved Keywords: antioxidants; vitamins C, E; oxidative stress; chronic disease

INTRODUCTION

antioxidants in the maintenance of health and prevention of major human diseases.

Under normal physiological conditions, cellular systems are incessantly challenged by stressors arising from both internal and external sources. The most important potential stressors in aerobic organisms are reduced derivatives of oxygen (Cheeseman and Slater, 1993) which are produced as part of normal physiological and metabolic processes, and are classified as reactive oxygen species (ROS). ROS are toxic as they can oxidize biomolecules leading to cell death and tissue injury. Oxidative stress and ROS have been associated with the onset of a variety of chronic disease states in humans including coronary heart disease (CHD), certain cancers, rheumatoid arthritis, diabetes, retinopathy of prematurity, chronic inflammatory disease of the gastrointestinal tract, and diseases associated with cartilage, Alzeimer’s disease, other neurological disorders and also the ageing process (Cheeseman and Slater, 1993; Kehrer and Smith, 1994). Fortunately, evolutionary survival has provided aerobic organisms with a well-balanced protective system to limit inappropriate exposure to ROS (Cheeseman and Slater, 1993; Yu, 1994). These protective components are classified as the ‘antioxidant defence systems’ and consist of an array of enzymes and essential nutrients whose role is to prevent the generation of free radicals and/or to intercept any that are generated. The production of ROS and antioxidant defenses are approximately balanced in vivo. However, it is easy to tip the prooxidant-antioxidant balance in favour of ROS and create the situation of oxidative stress leading to potential tissue damage and onset of chronic disease (Sies, 1991). The objective of this review is to identify the sources of ROS, the consequence of their reactions with cellular components and the roles and functions of natural

SOURCES OF FREE RADICALS Molecular or ‘triplet’ oxygen (the predominant form of the element) is relatively inert, but can be reduced within aerobic cells to produce ROS (Cheeseman and Slater, 1993; Halliwell et al., 1995). ROS include the free radicals which are capable of brief independent existence and contain one or more unpaired electrons (Kehrer and . Smith, 1994). ROS include the superoxide anion (O ~), . 2 hydroxyl radical ( OH), and oxygen centred radicals of . . organic compounds (peroxyl, ROO and alkoxyl, RO ) together with other non-radical reactive compounds such as hydrogen peroxide (H O ) and singlet oxygen 2 2 (1O ). In addition, reactive nitrogen species (RNS) such . . 2 as nitric oxide (NO ) and nitrogen dioxide (NO ) are 2 produced. Other types of radicals produced in cells in. clude carbon-centred radicals in membrane lipids (R ) . and thiol radicals (RS ) formed, for example, in the oxidation of glutathione.

PRODUCTION OF FREE RADICALS IN CELLS Free radical production in animal cells can either be accidental or deliberate. Four endogenous sources of ROS appear to account for most of the oxidants produced by cells (Kehrer and Smith, 1994): f During normal aerobic metabolism, mitochondria consume molecular oxygen and reduce it sequentially to produce H O. The inevitable by-products of this . 2. reaction are O ~, H O and OH. It has been cal. 2 2 2 culated that over 2 kgs of O ~ are produced in the 2 human body every year (Halliwell, 1996).

*Corresponding author. Tel.: 00353 21 902406; fax: 00353 21 270244; e-mail: [email protected] 463

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f Peroxisomes which include fatty acyl CoA oxidase, dopamine-b-hydroxylase, urate oxidase and others, produce H O as a by-product, which is then 2 2 degraded by catalase. Some H O escapes degrada2 2 tion and leaks into other compartments and increases oxidative damage. f Cytochrome P-450 mixed-function oxidase system in animals constitutes a primary defence against various xenobiotics and endogenous substances and enhances production of free radicals. . f Some O ~ is produced deliberately. For example 2 phagocytic cells destroy bacteria- or virus-infected . cells with an oxidative burst of O ~, H O , hypo2. 2 2 chlorite (~OCl) and nitric oxide (NO ). Chronic infection by bacteria (e.g. Helicobacter pylori), viruses (e.g. hepatitis B and C), or parasites result in chronic phagocytic activity and consequent chronic inflammation. A number of exogenous sources may also increase the endogenous free radical load. High intakes of iron and copper or ‘misplaced’ iron as a result of tissue breakdown promote the generation of oxidizing radicals from perox. ides (Halliwell, 1987). Cigarette smoke (NO ), radiation (Machlin and Bendich, 1987) and lipid oxidation products in foods (Kubow, 1993) may also contribute an array of free radicals.

oxidant encountered in biological systems (Morrissey et al., 1994). . The fatty acid acyl radical (R ) reacts rapidly with . O to form a fatty acid peroxyl radical (ROO ), which 2 may enter a complex series of reactions in the presence of transition metal ions (e.g. Mn`, Fe2`, Cu`), to yield the . alkoxyl radical (RO ) and a variety of degradation products. #O . . . RH RH# OH "R #H O &&"2 ROO &&" ROOH 2 k k 1 2

ROOH

Mn` &&"

. RO

(1) (2)

. . ROO , RO , ROOH &" products including alkyl rad. icals (R@CH ) a range of al2 dehydes, including hexanal, malondialdehyde and 4-hydroxynonenal, and ethane and pentane. (3) The rate constant k is 3]108 mol s~1 while the rate 1 constant k for the propagation step is relatively low, 2 10 mol s~1 to 102 mol s~1.

PROTEIN AND DNA DAMAGE FREE RADICAL REACTIONS IN BIOLOGICAL SYSTEMS Free radicals are extremely reactive and thus short lived. Consequently, free radical activity is usually assessed by indirect measurements of tissue damage products or ROS ‘footprints’ (Halliwell and Chirico, 1993). All the major macromolecules found within the body are capable of being oxidized or modified and a vast array of changes have been identified that could provide a mechanistic explanation for the resulting cell or tissue damage (Kehrer, 1993). Most interest has centred around the detection of lipid peroxidation products in certain disease states. However, oxidation of nucleic acids, DNA damage and rises in extracellular ‘free’ Ca2` are often more important events in causing cell damage than is the bulk peroxidation of membrane lipids (Halliwell and Chirico, 1993; Holley and Cheeseman, 1993). Lipid peroxidation is often a late event accompanying rather than causing cell damage and death. Nevertheless, oxidative stress is usually monitored by detection of end products of lipid peroxidation because of the ease with which these products can be measured.

LIPID PEROXIDATION Lipid oxidation is a free-radical-mediated chain reaction, which is initiated by the abstraction of a labile hydrogen from a methylene group adjacent to a double bond of a polyunsaturated fatty acid (RH), by the action . of the highly reactive radical ( OH), or certain Fe—O complexes, such as ferryl or perferryl radicals (Halliwell . and Chirico, 1993; Morrissey et al., 1994). The OH radical is remarkably effective in bringing about initiation of lipid peroxidation, because it is the most potent

Proteins and nucleic acids are less susceptible than lipids to free radical attack as there is less possibility of rapidly progressing, destructive chain-reactions being initiated. The amino acid composition of proteins exert a considerable influence on susceptibility to ROS. Proteins containing significant levels of aromatic and sulphur-containing amino acids are most vulnerable to ROS-induced damage (Lunec et al., 1985). Enzymes whose active sites involve these amino acids may have their activity modulated by ROS and the UV fluorescence patterns of the aromatic amino acids, tryptophan and tyrosine, are altered by ROS attack (Griffiths et al., 1988). However, convincing evidence for the involvement of free radical damaged proteins in the aetiology of a human disease process is lacking. ROS modification of proteins can also modulate their degradation by intracellular proteolytic systems (Davies and Delsignore, 1987). ROS-mediated oxidation of proteins may also be involved in the aetiology of inflammatory conditions, such as rheumatoid arthritis which involves oxidation of IgG (Jose et al., 1987). ROS-induced damage to key enzymes, e.g. antioxidant enzymes, may also result in significant pathophysiological effects. Oxidative stress resulting from UV-light exposure has been reported to result in a decrease in catalase and superoxide dismutase activities in skin cells (Shindo and Hashimota, 1997; O’Connor and O’Brien, 1997). Metalspecific binding sites on proteins are also vulnerable. In . this case, the metal reacts with H O and generates OH 2 2 which react at, or near the metal binding site, giving rise to ‘site-specific’ damage (Stadtman and Oliver, 1991). Modification of nucleic acids by oxidative attack can significantly alter cell function and is potentially carcinogenic. Components of DNA, for example, can be at. tacked by OH (Inouye, 1984). ROS give rise to a broad spectrum of DNA damage including modification of all bases, production of base-free sites, deletions, frame

Antioxidants, health and disease

shifts, strand breaks, DNA-protein crosslinks and chromosomal rearrangements (Halliwell and Arouma, 1991). Approximately 20 known products of oxidative damage to DNA have been identified including thymine-, thymidine- and cytosine-glycols, 8-hydroxy-2-deoxyguanosine and 5-hydroxymethyluracil. Known RNA damage products include 8-hydroxyguanosine (Simic, 1992). Cathcart et al. (1984) estimated that there may be a total of 104 oxidative hits per day to DNA in each of the human body’s 6]1013 cells. Cells, however, do possess a wide range of enzymes which recognise abnormalities in DNA and remove them by excision, resynthesis and rejoining DNA strands (Halliwell and Aruoma, 1993; Demple and Harrison, 1994). However, depending upon the oxidant/antioxidant balance in the cellular milieu, these defense mechanisms can be overwhelmed and mutagenesis can and does occur. ANTIOXIDANT DEFENCE SYSTEM Since free radicals and other oxygen-derived species are constantly produced in biological systems as a result of internal and external stressors, the cell has evolved a powerful and complex antioxidant defence system to limit inappropriate exposure to these stressors. The complex network consists of primary or preventative antioxidants which limit the initial formation of oxygen centred radicals of organic compounds (peroxyl and alkoxyl radicals). Secondary scavenging or chain-breaking antioxidants are present to trap intermediate reactive oxygen species and thus break the chain reaction. A third line of defence consists of repair systems for damaged nucleic acids, proteins and lipids (Yu, 1994; Halliwell, 1996). Living organisms have an array of enzymes which . serve to minimise production of OH. Superoxide dis. mutase (SOD) enzymes serve to remove O ~ by acceler2 ating the formation of H O . Mammalian cells have 2 2 a SOD enzyme containing active site manganese (MnSOD) in mitochondria. SOD with active site copper and zinc (CuZu SOD) are present largely in the cytosol (Fridovich, 1986). SOD enzymes work in collaboration with H O -removing enzymes (catalase and glutathione 2 2 peroxidase). The latter enzyme contains active site selenium. An additional important primary antioxidant defence is the presence of metal ion storage and transport proteins (Halliwell, 1989). Examples are tranferrin, lactoferrin, haptoglobin, ceruloplasm, metallothionein (Thurnham, 1990) and carnosine (Chan and Decker, 1994). In addition, vitamin A may also contribute to antioxidant defence in limiting the decompartmentalisation of highly catalytic iron through the maintenance of cellular integrity. Much of the work on antioxidant defence has been confined to studies on the chain-breaking antioxidants vitamins C and E and the carotenoids (especially b-carotene). Lutein and other carotenoids (Chopra and Thurnham, 1993) ubiquinol-10, protein thiols and uric acid (Stocker et al., 1991) also have been shown to have chain-breaking antioxidant properties. a-Tocopherol (TOH) is quantitatively the most important antioxidant in plasma and low density lipoproteins (LDL) because it is present in concentrations at least 15-times higher than

465

any of the other lipid-soluble antioxidants (Burton et al., 1983). TOH is an indispensable component of biological membranes with membrane-stabilizing properties. The hydrophobic tail is the means by which TOH inserts into lipoproteins or anchors into membranes next to unsaturated fatty acids (Diplock, 1985). The chromanol nucleus lies at the surface of a lipoprotein or at the surface of membranes, and it is the phenolic hydroxyl group which quenches free radicals (Packer, 1993). TOH is only active during the propagation phase of lipid peroxidation, that is, the consumption of this lipid-soluble antioxidant is associated with the formation of lipid hydroperoxides. When the chromanol phenolic group of TOH encoun. ters a peroxy radical (ROO ), it forms a hydroperoxide . and in the process a tocopheroxyl radical (TO ) is formed: . . TOH#ROO P ROOH#TO .

(4)

The rate-constant (k ) for this chain-inhibition reaction is 3 8]104 mol s~1 (Morrissey et al., 1994). Since, the rate constant (k ) for the chain propagation (eqn (1); approx2 imately 102 mol s~1) is much lower than for chain-inhibition k , TOH may outcompete the propagation reaction . 3 and scavenge the ROO about 104 times faster than RH . reacts with the ROO . Thus, the kinetic properties of antioxidants, and in particular TOH, require that only relatively small amounts be present for them to act as effective antioxidants. L-Ascorbic acid, present as ascorbate, is considered the most important antioxidant in extracellular fluids. As. . corbate efficiently scavenges O ~ , H O , ~OCl, OH, . 2 2 2 ROO radicals and RNS (Sies et al., 1992). According to Frei (1991), ascorbic acid is reactive enough to effectively intercept oxidants in the aqueous phase before they can attack and cause detectable oxidative damage to lipids. It has been postulated that ascorbate can also restore the antioxidant properties of vitamin E. Packer and Kagan (1993) concluded that the unique ability of low concentrations of vitamin E to act as an efficient antioxidant in biological systems is due to its ability to be re-reduced from its chromanoxyl radical form to its native state by intracellular reductants such as ascorbate. However, as a reducing agent ascorbate has the ability to reduce Fe3` to Fe2` and Cu2` to Cu`, thereby increasing the prooxidant activity of these metals and . . generating O ~ , H O and OH (Buettner and Jur2 2 2 kiewicz, 1996). Thus, ascorbate can serve as both a prooxidant and an antioxidant. In general, at low concentrations, ascorbate is prone to be a prooxidant, and at high concentrations, it will tend to be an antioxidant. Hence, there is a crossover effect and the position of this crossover is a function of the catalytic metal ion concentration (Buettner and Jurkiewicz, 1996). Of the approximately 600 naturally occurring carotenoids that have been characterised, approximately 40 are regularly consumed by humans (Bendich, 1993). There are five well characterised carotenoids in most human blood samples, namely b-carotene, lutein, lycopene, b-cryptoxanthin and a-carotene (Cantilena et al., 1992). The antioxidant nature of carotenoids has been attributed to their unique structure, an extended system of conjugated double bonds (Stahl and Sies, 1996). The radical quenching and antioxidant activity of carotenoids have been extensively reviewed in the literature (Krinsky, 1989; Sies and Stahl, 1995).

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Research on the antioxidant properties of carotenoids was stimulated by Burton and Ingold (1984) who reported that, in a model system containing methyl linoleate in chlorobenzene, b-carotene was an antioxidant, particularly at low O pressures (15 torr) found 2 physiologically. Under these physiological conditions, . they suggested that the lipid peroxyl radical (ROO ) attached to the conjugated b-carotene chain and the unpaired electron was delocalized through the chain. These workers also suggested that other carotenoids would exhibit antioxidant properties. Earlier work by Foote and Denny (1968) had described the 1O quench2 ing properties of carotenoids. Quenching involves the transfer of excitation energy from 1O to the carotenoid 2 resulting in ground state oxygen and excited triple state carotenoid. This energy is then dissipated through rotational and vibrational interactions between the excited carotenoid and the surrounding solvent to yield the ground state carotenoid and thermal energy (Stahl and Sies, 1996). A number of studies of the antioxidant nature of carotenoids have been reported. Kennedy and Liebler (1992) reported that b-carotene protected against oxidation in a liposomal model system at low O partial pressure 2 (15 torr) with little protection at higher pressures (750 torr). Recent work in our laboratory has demonstrated the increased protective effect of b-carotene at low partial pressures (7.5 torr) compared to its protective effect at atmospheric partial pressures of oxygen (150 torr) in a cellular model using chicken embryo fibroblasts (CEF) which were oxidatively stressed with paraquat (Lawlor and O’Brien, 1997). Thurnham (1994) suggested that lutein, lycopene and b-cryptoxanthin were . superior to b-carotene in quenching ROO radicals and more efficient in vitro than TOH. Tinkler et al. (1994) also investigated the protective effects of carotenoids against singlet-oxygen mediated damage in human lymphoid cells in vitro. The highest protection was provided by lycopene followed by astaxanthin and b-carotene, only little protective effects were observed with canthaxanthin. It has also been reported that when human skin was subjected to oxidative stress by irradiation with UV light, more skin lycopene was destroyed than b-carotene (Ribaya-Mercado et al., 1995). These authors suggested a preferential protective role of lycopene in the process of radical quenching. Palozza and Krinsky (1992) demonstrated the antioxidant properties of astaxanthin in vitro by exposing rat liver microsomal membranes to either chelated iron (Fe3`/adenosine 5@-diphosphate) and a reducing component NADPH or to the water soluble azo-initiator (2,2@-azobis-2-aminopropane) and measuring lipid peroxidation. Astaxanthin strongly suppressed malondialdehyde formation and produced a distinct induction period superior to b-carotene and comparable or superior to TOH. Kurashige et al. (1990) reported that astaxanthin was 100—500-fold more effective than TOH in inhibiting Fe3`-induced lipid peroxidation in liver mitochondria isolated from vitamin E-deficient rats. Dietary administration of astaxanthin (1 g kg~1 diet) protected against xanthine/xanthine oxidase and iron-induced lysis of erythrocyte ghosts from vitamin E deficient rats (Miki, 1991). We have also highlighted the potent antioxidant properties of astaxanthin using the CEF model system. Astaxanthin was a superior antioxidant

compared to TOH in this model (Lawlor and O’Brien, 1994, 1995).

OXIDATIVE STRESS AND DISEASE A number of lines of evidence suggest that oxidative processes are implicated in the pathogenesis of numerous disorders. However, oxidative stress may be a secondary phenomenon and not the primary cause of the disease (Gutteridge, 1993). The following discussion summarizes some of the data supporting a contributory role of free radicals in some diseases. Cancer 1. Clinical and epidemiological data, together with data derived from experimental models, support a role for the involvement of free radicals throughout the cancer process. Nutrient and non-nutrient free-radical scavengers and inhibitors have been shown to protect against cancer development in animal models and may be chemoprotective in humans (Guyton and Kensler, 1993). The intake of metals such as iron, nickel and chromium, which facilitate the production of ROS, is correlated with cancer development in humans and animals (Nelson, 1992; Stevens and Nerishi, 1992). Chronic inflammatory conditions with related increases in oxidative stress have been associated with increased incidence of certain cancers (Weitzman and Gordon, 1990). Many chemical carcinogens have been reported to act through free radical metabolities or processes (Trush and Kensler, 1991). Goldstein and Witz (1990) list a number of lines of evidence that suggest that free radicals are involved in tumour promotion and progression. These include: (a) Free-radical generating compounds, such as organic peroxides are tumour promotors and progressors. (b) Chemical promotors (e.g. phorbol esters, benzoyl peroxide) stimulate the endogenous producton of ROS. (c) ROS generating systems mimic the action of tumour promoters in cell culture. (d) Tumour promotors provoke rapid and sustained changes in cellular antioxidant enzyme activities. (e) Antioxidants inhibit tumour promotion and progression. While definitive proof of a role for free radicals and ROS in tumour development, particularly in humans is still lacking, the cumulative evidence suggesting a causative role is compelling. Comprehensive reviews of the role of ROS in the initiation, promotion and progression of cancer are available (Guyton and Kensler, 1993; Wiseman and Halliwell, 1996). Coronary heart disease Oxidation of LDL, a major cholesterol-carrying protein in human blood plasma, has been implicated as an initiator of atherosclerosis (Steinberg et al., 1989; Witztum and Steinberg, 1991; Ross, 1993). LDL are not only rich in cholesterol, but also contain high levels of linoleic, arachidonic and docosahexaenoic acid; about 1300 molecules of RH are associated with LDL particles of approximately 2.5 MDa (Esterbauer et al., 1992). These RH

Antioxidants, health and disease

are highly susceptible to peroxidation by oxidative attack through oxygen radicals. It has been shown that oxidized LDL exist in vivo; lipoproteins having all the chemical, physical and biological porperties of oxidized LDL have been isolated from atherosclerotic lesions (Rosenfeld et al., 1990; Jialal and Devaraj, 1996). Oxidative modification of LDL, whether induced by incubation with cells or as a result of autoxidation by transition metal ions, is linked to oxidation of its polyunsaturated fatty acids (Esterbauer et al., 1992). When LDL is depleted in antioxidants, the rate of lipid peroxidation rapidly accelerates, with oxidation of PUFA, generation of cholesterol oxidation products (Kritharides et al., 1993) and derivitization of e-amino groups of lysine (probably with aldehydes) in apolipoprotein B (apoB; Steinbrecher et al., 1989). The oxidatively modified LDL no longer binds to the LDL receptor, but to the scavenger receptor of macrophages, and this leads ultimately to the formation of lipid-laden foam cells. ANTIOXIDANTS AND DISEASE SITUATIONS Antioxidants and low-density lipoprotein LDL is protected by TOH (about 6 (range 3—15) mol per mol LDL). Also, present are c-tocopherol, b-carotene, lycopene, a-carotene, b-cryptoxanthin, lutein, zeaxanthin, cantaxanthan and phytofluene (Esterbauer et al., 1992). When isolated LDL was exposed to oxidant stress, the lipid-soluble antioxidants became progressively depleted in the order: TOH, c-tocopherol, and then the carotenoids (first lycopene first followed by b-cryptoxanthin, lutein, and finally b-carotene). While lipid soluble antioxidants are still present in LDL, only minimal oxidation of LDL occurs (Esterbauer et al., 1992, 1993). Several studies have shown that supplementation with TOH enhances the ability of LDL to withstand oxidative stress in vitro (Dieber-Rotheneder et al., 1991; Esterbauer et al., 1992, 1993; Fuller et al., 1996; Princen et al., 1992, 1995; Abbey et al., 1993, 1995; Reaven & Witztum, 1993). Esterbauer et al. (1993) observed a highly significant positive correlation (r2"0.55; P(0.001) between oxidative resistance or the lag-phase and the TOH content of 182 LDL samples, which were either supplemented or unsupplemented with TOH in vitro or in vivo. Abbey et al. (1993) supplemented ten men and twelve women with a daily dose of 18 mg b-carotene, 900 mg vitamin C and 200 mg TOH for 6 months and observed that the lag-time before the onset of oxidation was significantly lengthened after supplementation (P(0.01). Plasma TOH was significantly and independently correlated with lag-time (r"0.47, P(0.01). In another study, Princen et al. (1992) supplemented healthy volunteers with 1000 mg a-tocopheryl acetate for 7d and observed that the increase in TOH content of LDL and the increase in oxidative resistance were highly correlated (r"0.85, P"0.014). Studies in which b-carotene levels of LDL were increased have yielded less consistent results than those observed for TOH. Some human b-carotene supplementation studies failed to observe any significant effect on the susceptibility of LDL to oxidative modification (Princen et al., 1992; Reaven et al., 1993, 1994). For example, Reaven et al. (1993) failed to detect a protective

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effect of b-carotene on LDL oxidation despite a 20-fold increase in LDL b-carotene. Esterbauer et al. (1992) concluded that carotenoids may not be important determinants of the ability of LDL to withstand oxidative modification. Coronary heart disease (CHD) Several cross-sectional and prospective studies have reported an inverse relationship between biological levels of antioxidants and risk of CHD. Gey et al. (1991) compared antioxidant concentrations in plasma of middleaged men (40—59 yrs) representing sixteen European Study populations in its Vitamin Substudy of the WHO/MONICA Project. In 12 of the 16 populations, there was no evidence of an expected relationship between mortality and cholesterol. However, 63% of the differences in mortality were accounted for by plasma vitamin E (r2"0.63; P"0.002). Similar results were obtained for lipid-standardised plasma vitamin E (r2"0.62; P"0.0003). The relationship between risk of angina pectoris and plasma concentrations of vitamins A, C and E and bcarotene was examined in a population case-control study in Edinburgh of 110 patients with angina (Riemersma et al., 1991). Linear regression analysis of the data showed that only vitamin E remained independently and inversely related to risk of angina after adjustment for age, smoking, blood pressure, lipid and relative weight. The Basel prospective study (Gey et al., 1993a) showed that high vitamin E status was also associated with the lowest rate of CHD in Europe. Gey et al. (1993b) re evaluated the MONICA Project data and observed that the risk order for potential heart disease prevention in populations with low antioxidant concentrations is: lipid standardized vitamin E A carotene"vitamin C'vitamin A. These authors, using data from a number of studies, calculated that the following antioxidant plasma concentrations (mM L~1) are needed to reduce cardiovascular disease risk: '27.5—30 lipid-standardised vitamin E, 0.4—0.5 a-plus b-carotene, '40—50 vitamin C, and 2.2—2.8 lipid standardised vitamin A. Prospective studies of association between vitamin E intake (measured by dietary frequency questionnaire and a questionnaire on the use of multivitamin supplements) and risk of CHD showed significantly reduced risk in both women and men who used vitamin E supplements. In the Nurses’ Health Study (Stampfer et al., 1993), 87,245 female nurses (aged 34—59 yrs) were assessed over an 8-yr period. The risk of major coronary disease was about 40% lower in those who took vitamin E supplements (100 IU or more) for more than 2 years. The Health Professionals Follow-up Study (Rimm et al., 1993), where 39,910 males were followed for 4 years, showed that for men consuming at least 100 IU vitamin E/d the multivariate relative risk of CHD was 0.63 (95% confidence interval 0.47—0.84). Rimm et al. (1993) and Stampfer et al. (1993) concluded that supplemental vitamin E may reduce the risk of CHD in both men and women. However, they do not rule out the possibility that confounding factors may partly account for these results. In the Iowa Women’s Health Study, Kushi et al. (1996) studied 34,486 postmenopausal women and assessed (questionnaire) intake of vitamins A, E and C from food sources and supplements. Over a 7 year follow-up period,

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only the intake of vitamin E from food was inversely associated with the risk of death from CHD. The intakes of vitamins A and C were not associated with lower risks of dying from CHD. Results from intervention studies have been largely inconclusive. The largest trial is of the Alpha-Tocopherol Beta-Carotene (ATBC) Lung Cancer Prevention Study in Finland (Heinomen and Albanes, 1994; Albanes et al., 1995). In 1985, 29,133 male smokers aged 50— 69 yrs were enrolled in a randomised double-blind trial of b-carotene (20 mg d~1) or TOH (50 mg d~1). There was no apparent benefit of either vitamin on cardiovascular outcome. In fact, those assigned to b-carotene experienced a small, but significant, 11% increase in ischaemic heart disease (IHD) mortality. Men assigned to vitamin E had only a small, nonsignificant reduction in risk to IHD mortality. However, those assigned vitamin E experienced a significant 50% increase in deaths due to cerebral haemorrhage. The Physicians’ Health Study in the United States (Hennekens et al., 1996) investigated the effect of b-carotene supplementation (11,036 male physicians, 40—84 yrs, consuming 50 mg b-carotene on alternate days, 11,036, receiving placebo) and showed that 12 yrs of supplementation with b-carotene produced neither benefit nor harm in terms of the incidence of cardiovascular disease, or death from all causes. The b-Carotene and retinol efficacy trial (CARET) randomized 18,134 men and women at high risk of lung cancer to daily treatment with a combined supplement containing b-carotene (30 mg) and vitamin A (25,000 IU retinyl palmitate) or placebo for 4 years (Omenn et al., 1996). For cardiovascular disease (CVD) mortality, there was a non-significant 26% increase among those assigned the supplement combination. This trial was terminated 21 months earlier than planned. The Cambridge Heart Antioxidant Study (CHAOS) was designed to test the hypothesis that treatment with a high dose of a-tocopherol (400 or 800 IU/d) would reduce the risk of myocardial infarction in patients with evidence of coronary atherosclerosis (Stephens et al., 1996). A significant benefit was associated with TOH in the primary trial endpoint of nonfatal myocardial infarction compared with the placebo group (14 vs 41). Several prospective studies have reported an inverse association between consumption of fruit and vegetables and mortality from coronary disease (Knekt et al., 1994; Gaziano et al., 1995; Key et al., 1996). Knekt et al. (1994) reported a study where they ascertained dietary intake of vitamin E and other nutrients in a cohort of 5,133 Finnish men and women aged 30—69 yrs and followed the cohart for 14 yrs. Rates of coronary disease were lowest in men and women who consumed the highest amounts of vitamin E in the diet; trends were significant for men and women. Key et al. (1996) examined dietary factors associated with mortality among 11,000 health conscious people in the UK followed for an average 17 yrs. Daily consumption of fresh fruit was associated with a 24% reduction in mortality from ischaemic heart disease, a 32% reduction in mortality from cerebrovascular disease, and a 21% reduction in all cause mortality compared with less frequent consumption. In summary, data support an effect of vitamin E on reducing the risk of CHD. Evidence from the Nurses’ Health Study (Stampfer et al., 1993), the Health Professionals, Follow-up Study (Rimm et al., 1993) and CHAOS (Stephens et al., 1996) suggest that the major

effect is mainly found with an intake 5100 IU/d. In the case of b-carotene, epidemiological evidence is consistent with a protective association between b-carotene, carotenoids as a group, or carotenoid-rich foods and cardiovascular disease. However, the findings from intervention studies cast doubt on the ability of b-carotene for highrisk populations. b-Carotene may only represent a marker of dietary behaviour conducive to lower risk of cardiovascular disease (Kohlmeier and Hastings, 1995). Carotenoids may show protective effects because they are good markers of fruit and vegetable intake. The active substance in fruit and vegetables may be other carotenoids (lutein, lycopene) or phenolic compounds (quercetin). Cancer The association of vitamin E and other serum antioxidants and the risk of cancer in humans was reviewed by Block (1992), Knekt, (1994), Ziegler et al. (1992), Byers and Guerrero (1995), van Poppel and Goldholm (1995), and Steinmetz and Potter (1991a,b; 1996). The Finnish Mobile Clinic Health Survey (Knekt et al., 1991) suggested a significant inverse association between serum vitamin E and cancer risk. The association was strongest for some gastrointestinal cancers and for the combined group of cancers unrelated to smoking. The prospective study of 89,495 women in the Nurses’ Health Study (Hunter et al., 1993) showed that large intakes of vitamin C or vitamin E (from food-frequency questionnaires) did not protect women (aged 34—59 yrs in 1989) against breast cancer. In contrast, the prospective Iowa Women’s Health Study (Bostick et al., 1993) of 35,215 women showed that most of the association of vitamin E with reduced risk of colon cancer was associated with high intakes of supplemental vitamin E in women under 65 yrs of age. Those women who developed colon cancer had significantly lower mean daily intakes of dietary, supplemental and total vitamin E, C. The Linxian intervention trial provided evidence that nutritional supplementation during adulthood may lower the risks of cancers (primarily of the oesophagus and gastric cardia in a high-risk population) (Blot et al., 1995). A modest, but significant reduction in cancer mortality was observed in a general population trial (n"29,584; aged 40—69 yrs) in those receiving daily (for 5.25 yrs) a combination of b-carotene (15 mg), vitamin E (30 mg) and selenium (50 kg). The efficacy of supplementation with TOH and b-carotene on the prevention of certain cancers in male smokers has been raised in a recent report on the Finnish ATBC (Albanes et al., 1995). In this study, 29,133 male cigarette smokers aged 50—69 yrs were randomly assigned to receive b-carotene (20 mg), TOH (50 mg), bcarotene and TOH, or placebo daily for 5—8 yrs. b-Carotene treatment did not result in a decrease in cancer at any of the major sites, but rather in an increase at several sites, most notably lung, prostate and stomach. Vitamin E showed no overall effect on lung cancer. Prostate cancer incidence was 34% lower in the vitamin E group and colorectal cancer was 16% lower, but more cancers of the stomach were observed. In the CARET study (Omenn et al., 1996), a statistically significant increase of 28% was observed in lung cancer in the group assigned the supplement (30 mg b-carotene, 25,000 IU retinyl palmitate) compared with those on placebo. There were no significant differences in

Antioxidants, health and disease

the risk of other types of cancer. The authors concluded that after an average of four years of supplementation, the combination of b-carotene and vitamin A had no benefit and may have had an adverse effect on the incidence of lung cancer and the risk of death from lung cancer, cardiovascular disease, and any cause in smokers and workers exposed to asbestos. A rapidly increasing body of epidemiological evidence links dietary antioxidants and their rich food sources, especially vegetables and fruit, with reduced risk of cancer. Block (1992) reviewed about 200 epidemiological studies that examined the relationship between fruit and vegetable intake and cancer risks. The majority of these studies found statistically significant protective effects for a wide range of cancers. Zeigler et al. (1992) also reviewed epidemiological studies and concluded that increased intakes of vegetables, fruit and b-carotene and elevated blood levels of b-carotene are consistently associated with reduced risk of lung cancer. The scientific literature on the relationship between vegetable and fruit consumption and risk of cancer has been extensively reviewed (Steinmetz and Potter, 1991a, b, 1996). The evidence for a protective effect of greater vegetable and fruit consumption is consistent for cancers of the stomach, esophagus, lung, oral cavity and pharynx, pancreas and colon. These authors conclude that while the protective effect may be associated with specific nutrients (carotenoids, vitamin C, vitamin E, selenium), the possibilities of other constituents cannot be ignored. Plant components include: dithiothiones, glucosinolates and indoles, isothiocyanates, flavonoids, phenols, protease inhibitors, plant sterols, allium compounds and limonene. These agents have both complementary and overlapping mechanisms of action, including the induction of detoxification enzymes, inhibition of nitrosamine formation, dilution and binding of carcinogens in the digestive tract, alteration of hormone balance and antioxidant effect (Steinmetz and Potter, 1991a, b, 1996). Thus, consumption of fruit and vegetables exposes the body to a cocktail of ‘essential nutrients’ and ‘antinutrients’ which may block the development of certain cancers. Antioxidants and other disease situations Increased oxidative stress due to lower consumption of antioxidant nutrients, a decreased concentration of antioxidants, or both contribute to the decline of T-cellmediated function in aged persons (Meydani et al., 1995). Vitamin E supplementation resulted in a significant improvement in several clinical indicators of immune function (Meydani et al., 1990). Vitamin E is thought to exert its immuno enhancing effects by decreasing H O and 2 2 PGE production, both of which have been shown to 2 depress lymphocyte proliferation (Meydani et al., 1995). Data from human studies regarding the effect of b-carotene supplementation on immune response have yielded inconsistent results. Epidemiological evidence suggests an association between cataract incidence and antioxidant status (Jacques et al., 1994). Knekt et al. (1992) showed that low serum levels of vitamin E and b-carotene were predictors of increased risk of senile cataract in humans. A recent report (Sano et al., 1997) showed that in patients with moderately severe impairment from Alzheimer’s disease, treatment with TOH (2000 IU/d for 2 yrs) slowed the progression of the disease.

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CONCLUSIONS Oxidative processes are normal cellular events, but uncontrolled oxidation, particularly of membrane lipids and lipoproteins, has been implicated in a variety of degenerative diseases. The susceptibility of the body to peroxidative damage is related to the balance between the prooxidant load and the adequacy of antioxidant defences. This balance can be adversely modulated by suboptimal diets and nutrient intakes. Optimal protection against ageing and associated diseases depends not only on adequate intakes of vitamins C, E and carotenoids, but also on achieving and maintaining the correct balance of fatty acids, vitamin A, B vitamins (B , B , 6 12 folic acid), trace elements (Zn, Cu, Mn and Se) and non-essential nutrients. Defining the optimal intakes of these nutrients is one of the greatest challenges facing nutrition experts today. In the meantime, moderation, variety and balance and increasing intakes of fruit, vegetables and cereals remain key concepts in providing dietary advice to the general public.

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