Effects of antioxidants on vascular health

Effects of antioxidants on vascular health Garry G Duthie and Mary C Bellizzi Rowett Research Institute, Aberdeen, UK Substantial in vitro and animal model evidence implicates the free radicalmediated oxidation of low density lipoprotein and its subsequent preferential uptake by macrophages in the arterial intima as an important factor in the development of vascular disease. In addition, antioxidants which prevent the oxidation of LDL in vitro also reduce the severity of vascular disease in animal models. Although some epidemiological studies also suggest that inadequate antioxidant status is related to the development of vascular disease, particularly cardiovascular disease, results from intervention trials have been contradictory. Whereas vitamin E may have a role in reducing the incidence of vascular disease, evidence is less strong for vitamin C, flavonoids and (J-carotene. Additionally, supplementation with some antioxidants such as p-carotene may increase the incidence of cancer in high risk groups. Although increasing antioxidant intake is generally beneficial for health, this should perhaps be achieved by an increased dietary intake of antioxidant-rich foods rather than by use of supplements. Correspondence to: Dr Garry G Duthie, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB25 95B, UK There is strong evidence that diets rich in fruit and vegetables protect against the development of vascular disease 1. This has been attributed, in part, to the antioxidants in such foods inhibiting cellular damage by free radicals. These are potentially reactive molecules with an unpaired electron which can confer considerable chemical reactivity on the molecule 2. Reactive oxygen species are formed endogenously during the stepwise reduction of oxygen to water and by secondary reactions with protons and transition metals such as copper and iron. For example, the superoxide anion is produced in many cell redox systems such as those involving xanthine oxidase, aldehyde oxidase, membrane-associated NADPH oxidases and the cytochrome P450 system. In addition, cells are exposed to radicals arising from cigarette smoke, pollution, UV light, ionising radiation, ultra-sound and mechanical shearing forces. Many of these free radicals have the potential to interact with biological molecules such as proteins, lipids and DNA and there is now substantial evidence that they are involved in the aetiology of many diseases including vascular disease 3. Antioxidants which prevent free radical reactions may, therefore, play an important role in modifying the development of such diseases. Living British Medical Bulletin 1999, 55 (No. 3): 568-577 The British Council 1999

Antioxidants and vascular disease organisms have evolved complex antioxidant defence systems to minimise the damaging effects of excessive exposure to endogenously and exogenously produced free radicals. Biological antioxidants are, therefore, defined 4 as 'compounds that protect biological systems against the potentially harmful effects of processes or reactions that can cause excessive oxidations' and are generally characterised by being readily absorbed, transported to the relevant site within the cell for optimum function and relatively non-toxic at nutritionally-relevant intakes. They inhibit the oxidation of cellular components by directly scavenging reactive oxygen and nitrogen species, by metabolising lipid peroxides to non-radical products and by the chelation of metal ions to prevent generation of oxidising species. The main antioxidant micronutrients of dietary origin are vitamins E and C, the carotenoids and polyphenols. Vitamin E (in particular d-atocopherol) is a phenolic that is probably the main lipid-soluble antioxidant in human tissues. It is an example of a chain-breaking type antioxidant. Vitamin C is water-soluble and efficiently scavenges a range of reactive oxygen species such as the superoxide anion, hydroxyl radicals, peroxyl radicals and singlet oxygen. It can also chelate trace elements such as Fe and Cu thus inhibiting the catalytic decomposition of hydroperoxides to potentially damaging products. Carotenoids are polyisoprenoid structures with an extensive conjugated double bond system. Although many carotenoids may have antioxidant function, most attention has been directed towards [3-carotene which is particularly effective at scavenging singlet oxygen. It is also an effective antioxidant at low partial pressures of oxygen under conditions where singlet oxygen is not formed and, therefore, may be important at the physiological oxygen tensions in tissues. Polyphenolic products of the plant phenylpropanoid biosynthetic pathway such as the flavonoids were originally regarded as being nutritionally inert, but there is now increasing interest in their potential antioxidant properties. In addition, a number of enzymes with antioxidant function require trace elements from the diet as cofactors. Cytoplasmic and mitochondrial superoxide dismutases (EC 1.15.1.1) require copper, zinc and manganese to catalyse the removal of superoxide radicals. Hydrogen peroxide in the cell is removed by catalase (EC 1.16.1.6) which requires iron and also by cytosolic glutathione peroxidase (EC 1.11.1.9). Several varieties of this latter enzyme also remove potentially toxic lipid hydroperoxides from the cell and are dependent on selenium for optimum function. There are also many other natural and synthetic products which have potential antioxidant activity. Detailed reviews of the structures, functions and mechanisms of antioxidants are readily available 5. If the antioxidant defence system is compromised, damage to biological molecules may occur and lead to the initiation and propagation of the British Medical Bulletin 1999;5S (No. 3) 569

Micronutrients in health and disease underlying processes involved in the pathogenesis of many diseases. The aim of this article is to discuss the hypothesis that the free radicalmediated oxidation of low density lipoprotein (LDL) is a key event in the development of vascular disease and the therapeutic benefits of intervention with antioxidant nutrients. Oxidation of low density lipoprotein Considerable in vitro evidence implicates the oxidation of low density lipoprotein (LDL) in atherogenesis. In brief, LDL is a heterogeneous structure containing phospholipids, free and esterified cholesterol, triglycerides, and amino acids which form apolipoprotein B (apob). The proteins and the polyunsaturated fatty acid components of the LDL are susceptible to free radical-mediated oxidation, particularly if the antioxidant content of the LDL is low. When LDL is oxidised in vitro, there is a loss of polyunsaturated fatty acids to yield a range of fragments of 3-9 carbon lengths including hydroperoxides, aldehydes and ketones which conjugate with other LDL-bound lipids and the apob 6. In cell cultures, this 'minimally-modified' LDL has a number of properties that could increase its atherogenicity. It is recognised by at least three types of scavenger receptors in macrophages which rapidly internalise the oxidised LDL. The macrophages are transformed into 'foam-like cells' which, in vivo, are regarded as precursors to the development of the occlusive plaque 7. In addition, oxidised LDL stimulates the release of macrophage colony stimulating factor and monocyte chemoattractant protein 1 from cells 8. Such observations have lead to the proposal that, in vivo, LDL in arterial endothehal cells may be oxidised by cellular enzymes such as NADPH oxidase, myeloperoxidase, lipoxygenase or by the leakage of free radicals from the mitochondrial electron transport chain. The presence of minimally-modified LDL induces the surrounding vascular cells to produce the chemoattractants and stimulating factors that cause monocytic accumulation and their subsequent differentiation to macrophages. On transformation of monocytes to macrophages, the oxidised LDL limits further macrophage mobility and decreases their ability to migrate away from the artery wall. The enhanced rate of uptake of oxidised LDL by the macrophages via the scavenger receptor pathways may then convert them into foam cells. In addition, since the macrophage can oxidatively modify native LDL via the respiratory burst, autocatalytic progression may lead to their continuous growth to form the plaque that begins to occlude the artery. These proposed events have not as yet been demonstrated in vivo. However, LDL extracted from human atherosclerotic lesions, but not normal arteries, appears to contain products of 570 British Medical Bulletin 1999,55 (No. 3)

Antioxidants and vascular disease lipid peroxidation such as F2-isoprostanes and malonaldehyde 9. Moreover, antibodies raised against oxidised LDL react with such lesions 1011 and elevated amounts of oxidised LDL are present in blood of patients with atherosclerotic disease 12. Therefore, it is plausible that oxidised LDL is involved in the atherosclerotic process although such observations do not necessarily imply a causal relationship between oxidised LDL and vascular disease. Intervention with antioxidants Animal studies Many in vitro studies have shown that the oxidation of LDL by endothelial cells, macrophages and Cu 2+ can be inhibited by the addition of synthetic and natural antioxidants 13. This suggests that antioxidants may play a role in limiting the progression of vascular disease. However, there is an important distinction between the in vitro antioxidant effectiveness of a substance and its ability to prevent the oxidation of LDL in vivo. The effectiveness of biological antioxidants in vivo is also dependent on their need to be readily absorbed, and transported and incorporated within the LDL for optimum function. Nevertheless, several animal models do suggest that increasing dietary intakes of some antioxidants can inhibit the progression of vascular disease. For example, the feeding of antioxidants such as vitamin E, probucol, butylhydroxytoluene and BO-653 increases the resistance of LDL to oxidation and reduces the lesions in arteries of rabbits, mice and monkeys which are either genetically-susceptible to vascular disease or have been fed atheroscleroticpromoting diets 14 ' 15. Human studies A number of clinical studies indicate that supplementation with antioxidants such as probucol, vitamin E and vitamin C (but not P-carotene) increases the resistance of LDL to oxidation ex vivo 16, although it is no.t clear in such short-term studies whether they also reduce the severity of atherosclerosis. Moreover, the few randomised, double blind, placebocontrolled trials with human volunteers aimed at answering this question (Table 1) have given inconsistent results. For example, the CHAOS study 17 involving 2000 men and women with angiographically defined coronary artery disease resulted in a marked reduction in nonfatal myocardial infarction in subjects taking vitamin E compared with the placebo group. In contrast, the ATBC study 18 with 29,000 male British Medial Bulletin 1999;55 (No. 3) 571

Micronutrients in health and disease Table 1 Summary of intervention trials with antioxidant supplements and vascular disease Authors Acronym Treatment Comments Stephens et ap 1 CHAOS 400 or 800 IU vitamin E/day or placebo in 2002 patients with diagnosed atherosclerosis A 47% decrease in cardiovascular related mortality and non fatal Ml after 200 days Rapola et ap ATBC 1862 male smokers with previous Ml history received daily, either 50 mg vitamin E, 20 mg P-carotene, both, or placebo over 5 years No significant differences among the 4 groups in total number of coronary events. Risk of non fatal Ml slightly lower in vitamin E group but fatal heart disease significantly lower in placebo group and 43% higher in p-carotene group Omenn eta/" CARET 18,314 smokers, ex-smokers and workers exposed to asbestos supplemented with 30 mg p-carotene + 25,100 IU vitamin A or placebo over 4 years Relative risk of death from cardiovascular disease 26% higher in supplemented group compared with placebo. Trial terminated early because also increased incidence of lung cancer in treatment group Hennekens et ap Physicians 22,071 healthy males allocated either Health 50 mg p-carotene or placebo for 12 years No significant differences in cardiovascular disease deaths, or incidence of Ml between the groups Abbreviations: CHAOS, Cambridge Heart Antioxidant Study; ATBC Alpha-Tocopherol Beta-Carotene Cancer Prevention Study; CARET, p-carotene and Retinol Efficacy Trial; IU, international unit; Ml, myocardial infarction. smokers found no effect of vitamin E on coronary artery events. Although this study was primarily aimed at investigating the effects of antioxidant supplements on the incidence of lung cancer, a subpopulation of 1862 male smokers with a history of myocardial infarction received daily in a double blind design 50 mg vitamin E, or 20 mg p-carotene or both micronutrients or a placebo. After a 5 year follow-up, there were no statistically significant differences among the 4 groups in the total number of major coronary events. However, adverse effect were apparent as the incidence of fatal heart disease was significantly lower in the placebo group and significantly higher (by 43%) in those given P-carotene. In addition, in the overall study, there were 18% more lung cancers and 8% greater mortality amongst subjects taking P-carotene compared with those on placebo. Similarly the Beta-Carotene and Retinol Efficacy Trial (CARET) 19 involving 18,000 smokers, former smokers and those who had been exposed to asbestos indicated that daily supplementation with 30 mg p-carotene and 25,000 IU of vitamin A increased the incidence of cardiovascular disease by 26% compared with the placebo group. This trial was terminated 21 months prematurely as, after 4 years, there were also 28% more lung cancers and 17% more deaths in the supplemented subjects compared with the placebo group. Such differences in outcome between intervention studies may reflect the range of doses used, the 572 British Medical Bulletin 1999;55 (No. 3)

Antioxidants and vascular disease duration of supplementation and the health status of the volunteers at the start of the study. For example, many antioxidants can also act as pro-oxidants under a range of circumstances and it is possible that unforeseen metabolic disturbances may occur after prolonged use of highly bio-available pure compounds; such effects may not be apparent when antioxidants are obtained from foods. Epidemiology Several epidemiological studies have suggested that low antioxidant status, as determined by analysis of plasma and tissue and by dietary assessment, is an important risk factor for the development of vascular disease (Table 2). Some large cohort studies indicated that a high intake of vitamin E, usually by self-supplementation, was associated with a significant reduction in cardiovascular disease as measured by various fatal and non-fatal endpoints. For example, the US Nurses Health Study 21, involving 87,000 females in a 8 year follow-up, concluded that those women taking 100 mg or more vitamin E/day for at least 2 years had a statistically significant reduction of relative risk ratio by 31% for myocardial infarction and cardiovascular mortality compared with women who did not use supplements. However, significant risk reductions were not observed for subjects taking supplements of vitamin C and p- carotene. Similarly, the Health Professionals Study 22, which included 39,000 men, did not find any clear associations between risk and vitamin C and (3-carotene intake. However, men in the upper quintile of vitamin E intake had a 40% reduction in relative risk ratio than those in the lower quintile after adjustment for potentially confounding factors such as age, smoking, body mass, calorie intake, fibre intake, alcohol intake, hypertension, aspirin use, family history and exercise. Paradoxically, vitamin E supplementation was not associated with changes in cardiovascular risk in a 7 year follow-up of 35,000 postmenopausal women 23, but there was a significant inverse association between vitamin E intake from food and incidence of cardiovascular disease. Some ecological studies have also revealed associations between intakes of antioxidants and cardiovascular disease (Table 2). For example, vitamin E intakes (in particular a-tocopherol) calculated from food supply data bases across 22 countries were strongly and inversely associated with mortality from cardiovascular disease 24. Similar significant associations were found across 7 countries in relation to the intake of flavonoids". However, other studies have failed to reveal any relationship between supply of nutritional antioxidants and disease (Table 2). Whether this reflects inappropriate study design and methodology or reflects a real lack of association between antioxidants British Medical Bulletin 1999;55 (No. 3) 573

Micronutrients in health and disease Table 2 Summary of epidemiological studies relating antioxidants to vascular disease Authors Type of study Comment Bellizzi et ap* Cross sectional Comparison of food and nutrient supply with CHD mortality in men across 24 countries between 1962 and 1987. Dietary vitamin E and vitamin C but not p-carotene significantly and negatively correlated with CVD mortality Riemersma et ap* Cross sectional Plasma vitamin E, vitamin C and selenium in men from Scotland, Finland and Southern Italy did not reflect regional CVD mortality rates Hertog et ap 5 Cross sectional Estimated intakes of flavonoids by FFQ across 7 countries significantly and negatively associated with incidence of CVD Gey 27 Cross sectional Plasma vitamin E and 'cumulative antioxidant index' comprising vitamin E, vitamin C, (^-carotene and selenium significantly and negatively associated with CVD mortality Rimm et ap 2 Prospective cohort Asymptomatic males followed for 4 years. Those on highest intakes of vitamin E (median 300 mg/day) as assessed by FFQ at lowest risk for CVD. No significant risk reduction for vitamin C and p-carotene Stampler et a/ 21 Prospective cohort Healthy female nurses followed up for 8 years. Those on highest intakes of vitamin E (median 150 mg/day) as assessed by FFQ at lowest risk for CVD. Significant risk reductions not observed for vitamin C or (}-carotene Hertog et ap* Prospective cohort Dietary intake of flavonoids significantly and negatively associated with CVD in 5 year follow-up of 805 males Knekt et ap* Prospective cohort Flavonoid intake associated with reduced risk of CVD in a 20 year follow up of 5113 subjects Hertog et ap Prospective cohort No relationship between dietary flavonoid intake estimated by FFQ and CVD mortality in 14 year follow-up of 1900 men Rimm et ap 2 Prospective cohort Re-analyses of Health Professional Study with a 6 year follow-up 34,789 males showed no clear relationship between flavonoid intake and CVD incidence Knekt et a/ 31 Prospective cohort A 14 year follow-up of Finnish men and women showed an inverse association between dietary vitamin E established from interview and CVD mortality Kushi et ap 3 Prospective cohort Postmenopausal women followed up for 12 years with the highest dietary intakes of vitamin E had lowest risk of CVD Losonczy et ap 2 Prospective cohort A CVD protective effect of vitamin E intakes > 70 mg/day found in a 9 year follow-up of elderly subjects. No effect of vitamin C Meyer etar 1 Prospective cohort In 2313 men followed over 7 years, vitamin supplement use, in particular vitamin E, was associated with a lower incidence and death from ischaemic heart disease Daviglus et a/ Prospective cohort Modest decrease in risk of stroke in highest quartile of intakes of fj-carotene and vitamin C in 1843 middle-aged men from the Chicago Western Electric Study Evans et a/ 33 Nested case control No significant association between serum (n = 734) concentrations of vitamin E or carotenoids with CVD risk or in fatal Ml Hense et ap 4 Nested case control No difference in serum vitamin E between controls and subjects with fatal and non-fatal coronary events Salonen et a/ 35 Nested case control No difference in serum vitamin E or selenium between subjects who had died of CVD and matched controls Duthie et a/ 3 * Case control No difference in Scottish men with stable angina and matched controls in plasma vitamin C, vitamin A and cholesterol-adjusted vitamin E Kardinaal et a/ 37 Case control Men from 10 European countries with first acute Ml did not have significantly different adipose tissue vitamin E compared with healthy controls Sklodowska et ap* Case control Plasma and red cell vitamin E of patients with unstable angina lower than in normal subjects Riemersma et a/ Case control Males with untreated angina had lower plasma vitamin E compared with controls Abbreviations: CVD, cardiovascular disease; FFQ, food frequency questionnaire; Ml, myocardial infarction. 574 British Medical Bulletin 1999;55 (No. 3)

Antioxidants and vascular disease and disease is still unclear. Some randomised trials may not have been specifically designed to assess cardiovascular disease. Others, such as nested case control studies, may have used archived blood samples in which there had been loss of antioxidants during storage. Moreover, plasma antioxidant concentrations, which were used as a measure of antioxidant status in some studies, may not necessarily reflect concentrations in target tissues such as the arterial intima where plaque development occurs. Conclusion There is very strong biochemical and animal model evidence that the oxidation of LDL is an important event in the development of vascular disease and that antioxidants which inhibit LDL oxidation can prevent or inhibit disease progression. However, this has not always been corroborated and indeed has even been contradicted by the results from epidemiological studies. Some intervention studies appear to show marked benefits from supplementation with antioxidants, particularly with vitamin E, but there are also disconcerting reports that supplements can have adverse effects in certain population groups. This is particularly the case for P-carotene which has increased mortality rates in at least 2 studies. Consequently, the role of antioxidants in the prevention or cure of vascular disease is still problematical although there is now increasing awareness that there is a distinction between the amounts required to avoid overt deficiency and those needed to promote optimum health. For example, estimated dietary vitamin E intakes for the UK population range from 4.7-11.9 mg/person/day whereas they appear to be 18-25 mg/day in Spain, where the incidence of vascular disease is lower 24. This latter range may be an important benchmark for setting nutritional requirements for optimum health. Although larger amounts may be therapeutically appropriate in certain circumstances and for particular risk groups, very high doses may induce adverse effects through pro-oxidant mechanisms 40. Therefore, increasing consumption of foods rich in antioxidants such as fresh fruit and vegetable is possibly more suitable for the UK population. Having said that, one of the authors (GGD) having experienced a mild cardiovascular event in his early forties now takes 90 mg vitamin E every second day in addition to the prescribed 75 mg aspirin and 40 mg simvastatin each day. Acknowledgement The authors are grateful for financial support from the Scottish Office Agriculture, Environment and Fisheries Department (SOAEFD). British Medical Bulletin 1999;55 (No. 3) 575

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