Antloxidant vitamins in the prevention of cardiovascular disease

Transcription

1 European Heart Journal (1997) 18, Clinical Perspective Antloxidant vitamins in the prevention of cardiovascular disease The epidemiological evidence Introduction Recent evidence suggests that in vivo oxidation, primarily by oxygen free radicals, may be involved in atherogenesis. In particular, the atherogenicity of low density lipoprotein appears to be increased by oxidative modification 1 ' 1. Oxidation of polyunsaturated fatty acids in low density lipoprotein is believed to occur locally within atherosclerotic lesions. Following oxidation, the low density lipoprotein is taken up by macrophages more rapidly than normal to form cholesterol-laden foam cells 121 (Fig. 1). Antioxidant molecules may influence atherogenesis by interfering with this oxidation process. Vitamin E is the major lipid-soluble antioxidant preventing the formation of lipid hydroperoxides from polyunsaturated fatty acids. It occurs in low density lipoprotein where its concentration increases by 2-4-fold when vitamin E is taken by mouth' 21. There is biochemical evidence that vitamin C can scavenge free radicals in the cytoplasm and may also participate in the regeneration of vitamin E [2]. Vitamin A (retinol) and certain carotenoids (notably beta-carotene, a vitamin A precursor, and the non-vitamin A precursors lutein and lycopene) have substantial singlet oxygen scavenging ability 131 and betacarotene is contained within low density lipoprotein 141. The antioxidant vitamins required by humans are derived from fresh fruit and vegetables, and from vegetable oils and polyunsaturated fatty acid margerines in the case of vitamin E, but cannot be synthesized from simple precursors. Thus, concentrations of vitamins in plasma and body tissues are determined by dietary intake, absorption, metabolism and storage. Evidence from vitamin supplementation studies and comparison of food frequency questionnaires with circulating vitamin levels, indicates that plasma levels of beta-carotene, vitamin E and vitamin C generally correlate well with dietary intake' 5 " 8 ', although dietary beta-carotene may not determine plasma levels as accurately in smokers Revision submitted 18 April 1996, and accepted 25 April Correspondence: Dr J. F. Price, Wolfson Unit for Prevention of Peripheral Vascular Diseases, University of Edinburgh, Teviot Place, Edinburgh EG8 9AG, U.K X/97/ $18.00/0 who have lower circulating levels of the pro-vitamin than non- smokers' 7 ' 81. However, increases in dietary vitamin A do not result in long-term increases in the level of circulating retinol as surplus vitamin is stored in the liver' 31. The influence of antioxidant vitamin levels in the diet on plasma concentrations, together with their biochemical properties, raises the possibility that increased consumption of these vitamins could prevent or decelerate the atherogenic process. We review the epidemiological evidence for such an effect in the context of coronary heart disease and subsequently, in peripheral arterial disease. Vitamin E Dietary intake There is fairly good evidence from two large-scale, prospective studies that a high intake of vitamin E is associated with a reduced risk of coronary artery disease' 9 ' 101. In the Nurses Health Survey, a 31% reduction in non-fatal myocardial infarction and cardiovascular death was evident in women taking at least 100 mg daily of supplementary vitamin E for over 2 years 191. This effect persisted after adjustment for age, smoking, obesity, exercise, blood pressure, cholesterol and use of postmenopausal oestrogen replacement, aspirin, vitamin C and beta-carotene. Men in the Health Professionals Follow-up Study had a 40% reduction in non-fatal myocardial infarction, cardiovascular death and coronary revascularization with increased dietary vitamin E intake, even after adjustment for age, coronary risk factors and intake of vitamin C and beta-carotene' 101. The main effect was again due to supplement use. An inverse relationship between dietary vitamin E and subsequent coronary mortality in 5000 Finnish men and women was not altered by adjustment for vitamin supplement use, although coronary mortality was again lower among supplement users (3% of the population) than among non-users'" 1. In a case-control study, the risk of newly diagnosed coronary heart disease was 1997 The European Society of Cardiology

2 720 J. F. Price and F. G. R. Fowkes LDL Monocytes Endothelium Free radicals Oxidised LDL Macrophage Foam cell Internal elastic lamina Figure 1 Model for the formation of foam cells. Low density lipoprotein (LDL) in atherosclerotic lesions is oxidized by free radicals and causes endothelial cells to release a chemotactic factor for monocytes. Attracted macrophages subsequently endocytoze the oxidized low density lipoprotein via scavenger receptors and are converted into cholesterol-laden foam cells. lower in the highest quintiles of dietary vitamin E for men but not for women' 121. Although the opposite effect was observed for men who were already aware of their condition, this may have been due to changes in diet following diagnosis. Whilst dietary studies are highly suggestive of an association between vitamin E and coronary heart disease, in some cases, extra vitamin E in the form of supplements appears to be required to produce protective in vivo levels. Alternatively, individuals who choose to take vitamin supplements may have other characteristics which make them less prone to developing heart disease. Furthermore, it cannot be excluded that nutrients) other than vitamin E in vitamin E-rich foods could be responsible for many of the effects observed in studies relying on dietary questionnaires. This problem is partially addressed by measuring vitamin levels in plasma and other body fluids directly. Plasma levels In a recent cross-cultural survey, plasma levels of vitamin E in men aged 40^49 years correlated strongly, and inversely, with the age-specific mortality from ischaemic heart disease in 16 European regions' 131, although plasma levels did not reflect coronary mortality rates among a smaller group of four European populations' 141. There was no association between plasma vitamin E and prevalence of ischaemic heart disease in a cross-sectional survey of 1132 Finnish men, but dietary changes secondary to the diagnosis of heart disease were not evaluated 1 ' 51. Similarly, most case-control studies found no relationship between plasma vitamin E levels and subsequent coronary mortality 1 ' 6 ' 171, or risk of myocardial infarction' Adipose tissue concentrations were also found not to influence the risk of heart attack On the other hand, plasma vitamin E was found to be independently and inversely related to the risk of newly diagnosed angina pectoris after adjustment for age, smoking, blood pressure, lipids and weight' 2 ' 1. All but this latter study analysed vitamin levels from frozen blood samples which had been stored for several years. This raises the possibility that degeneration of vitamin E in storage was responsible for the negative results, although a recent review indicated that concentrations of vitamin E were fairly stable for at least 15 years if stored at 70 C or colder' 221. A single prospective study on 2975 healthy males, followed-up for 7 years, failed to show a relationship between plasma vitamin E and mortality from ischaemic heart disease' 231. This lack of association has been blamed on exceptionally high vitamin E levels in the population under study; all vitamin quintiles may have been above a presumed critical level for improving coronary artery disease risk' 241. However, the study also lacked power because the mortality rate was less than 3%. In summary, prospective data suggest that high dietary and/or supplemental intake of vitamin E is associated with a reduced risk of coronary heart disease, but support from blood- and tissue-based case control studies is scanty. Reasons for these disparate results are unknown but may include changes in diet following disease diagnosis, poor classification of controls and lack of variability in plasma levels within

3 Clinical Perspective 721 populations not using supplements. Ultimately, definitive conclusions must be sought from clinical trials. Clinical trials (Table 1) In an early clinical trial, 52 patients with angina pectoris demonstrated no symptomatic benefit following 6 months of vitamin E treatment compared with placebo' 25 '. However, both patient numbers and the period of follow-up in this study were limited. In the Alpha-Tocopherol and Beta-Carotene (ATBC) Study, vitamin E supplementation for 5-8 years had no effect on coronary mortality in male smokers' 261 and reduced the incidence of new angina pectoris by only a minor amount in men free of coronary artery disease at baseline (relative risk 0-91, P=0-04) [27]. However, the dose of vitamin E used was low (50 mg per day) and the synthetic preparation of alpha-tocopherol used has a low bioavailability so that mean blood levels were increased by only 50%, less than that produced by many dietary supplements of vitamin E. In the recent Cambridge Heart Antioxidant Study (CHAOS), higher doses of vitamin E (400 and 800 mg per day) were tested in 2002 patients with angiographically confirmed coronary artery disease. After an average of 17 months follow-up, patients taking vitamin E had 75% fewer non-fatal myocardial infarctions and 50% fewer nonfatal myocardial infarctions and cardiovascular deaths combined that those given placebo. Although there was a non-significant excess of cardiovascular deaths in the vitamin E group, the number of such events in each group was small (27 vs 23) [28]. Several other randomized controlled trials which include vitamin E are currently in progress. The Woman's Health Study (WHS) is a primary prevention trial investigating the effects of vitamin E, beta-carotene and aspirin on cardiovascular disease and cancer in women aged 50 years and over' 291. In France, the Supplementation Vitamins, Minerals, and Antioxidant (SU.VI.MAX) Trial is testing a combination of antioxidant vitamins including vitamin E, vitamin C, and beta-carotene in healthy men and women' 301. The Heart Protection Study in Oxford is investigating the effects of vitamin E, vitamin C, and beta-carotene in subjects with above average risk of future myocardial infarction' 3 ' 1 and a secondary prevention trial using the same three vitamins in 8000 women has been established in the U.S.A. (Women's Antioxidant Cardiovascular Disease Trial, WACDT)' 30 '. The Heart Outcomes Protection (HOPE) Study is also assessing vitamin E in 9000 persons with previous myocardial infarction, stroke or peripheral vascular disease and diabetic patients' 321. Vitamin A and beta-carotene Dietary intake In the prospective Nurses Health Survey, consumption of vitamins A and beta-carotene in food and supplements correlated weakly with the incidence of coronary heart disease' 331 ; Gaziano and Hennekens calculated a 22% risk reduction for women in the highest quintile of beta-carotene compared with those in the lowest' 341. Unfortunately, no adjustment for the potentially confounding effect of antioxidant vitamins other than vitamins A and beta-carotene in multivitamin preparations was made. However, a small prospective study on 1271 elderly people also demonstrated an inverse relationship between beta-carotene intake in fruit and vegetables and subsequent cardiovascular death' In the Health Professionals Follow-Up Study, high beta-carotene intake was associated with reduced coronary artery disease in current smokers and ex-smokers (70% risk reduction) but not never-smokers, after adjustment for cardiovascular risk factors and vitamin E and C intake' 101. In Finland, there was no association between daily intake of carotenoids and subsequent coronary mortality, although a nonsignificant reduction was seen for women in the highest tertile compared with those in the lowest tertile' 11 '. In a single case control study, the risk of newly-diagnosed coronary artery disease was reduced in the highest quintiles of beta-carotene intake for men. The same effect was not seen in women, or in men who were already aware of their diagnosis' 121. Plasma levels In Gey's cross-cultural survey, neither plasma vitamin A nor beta-carotene correlated consistently with ischaemic heart disease mortality in 16 European populations; neither was there an association with the major subset of 12 populations with normal cholesterol' 131. The only significant association was a weak inverse correlation between lipid-standardized levels of vitamin A and coronary mortality in all populations. Five case control studies found variable results (Table 2). In Finland, mean retinol levels were higher in female cases of coronary death, but the same effect was not found for men' 161. In the Netherlands, there was no association between vitamin A and coronary mortality in either sex' 171. An initial inverse relationship between angina and low plasma carotene in Scottish males disappeared after adjustment for smoking; no relationship was found for vitamin A' 2 ' 1. More recently, an increased risk of myocardial infarction was demonstrated for smokers with reduced levels of beta-carotene' 19 ' although the same effect was not found in non-smokers. Similarly, an increased risk of myocardial infarction in the lowest quintile of adipose tissue beta-carotene was confined mainly to smokers' 20 '. In the Basle prospective study, there was an increased risk of mortality from ischaemic heart disease at initially low plasma levels of beta-carotene, independent of vitamin E and other cardiovascular risk factors' 23 '. In 1899 middle-aged hyperlipidaemic men, carotenoid levels were independently and inversely correlated with the subsequent risk of myocardial infarction

4 Randomized controlled trials of antioxidant vitamins in coronary artery disease Participants Duration Vitamins (dose) Outcome l [2S] Men with angina Health Study 1990' 39 ' Men with stable angina or coronary surgery y ) Male smokers years udy 1994' 4 ' 1 udy 1996' 281 Health Study 13 " 1 1 X Trial 1301 ealth Study 1291 ction Study 13 ' 1 y 1321 Men and women Heavy smokers and asbestos workers Men and women with coronary atheroma Healthy males years Healthy men and women Healthy females years Men and women at increased risk of future MI Women with coronary artery disease Men and women at increased risk of future MI 52 6 months years years years Vitamin E (1600 mg. day ~') Beta-carotene (50 mg. altday ~ ') Vitamin E (50 mg. day" ') Beta-carotene (20 mg. day ~ ') Vitamin A ( IU. day " ') Beta-carotene (30 mg. day" ') days (median 510) Vitamin E (400 mg or 800 mg. day" ') years Ongoing Ongoing (Begun 1992) Ongoing (Begun 1994) 8000 Ongoing (Begun 1995) 9000 Ongoing (Begun 1995) Beta-carotene (50 mg. altday Vitamin E Beta-carotene Vitamin C Vitamin E (600 mg. altday"') Beta-carotene (50 mg. altday " ') Vitamin E (600 mg. day " ') Beta-carotene (20 mg. day "') Vitamin C (250 mg. day " ') Vitamin E (400 mg. day " ') Beta-carotene (20mg. day " ') Vitamin C (1 g.day"') Vitamin E ') No symptomatic improvement 44% reduction in major corona events No effect on coronary mortality (increased incidence of lung can with beta-carotene) Terminated 1996, cardiovascula results awaited, 28% increase in cancer, 17% increase in deaths 75% reduction in non-fatal MI, significant increase in cardiovas death Cancer and cardiovascular resu awaited Cardiovascular endpoints Cancer and cardiovascular end (Beta-carotene terminated 1996 Incidence of coronary and all-c mortality Cardiovascular endpoints Cardiovascular endpoints

5 Case control studies on plasma and tissue levels of vitamin A and B-carotene in coronary artery disease Definition of disease Vitamin Sex Cases Mean vitamin levels** Controls Notes al. 1985" 6 ' Death from coronary artery disease Vitamin A M 653 g.l" g.r 1 ns F 647 g.p g.i"' < ' 71 Death from coronary artery disease Vitamin A M + F 56-5ug.dr'* 59-Oug.dl"'* ns 1991 et al [2I) Angina pectoris Beta-carotene M 0-30umol.r' 0-49umol.l~' <0-001 ns after adjustment for sm Vitamin A M 2-29umol.r 2-32umol.r' ns 1993 et al ' Acute myocardial infarction Beta-carotene M 0-35 ug. g ' fatty acid 0-42 ug. g ' fatty acid <005 Effect confined mainly to s. 1994" 91 First diagnosis of myocardial infarction Beta-carotene M + F 17-2ug.dl" 20-6ug. dl" 0-03 Effect confined to smokers ed for level of serum cholesterol. concentrations in plasma except for Kardinaal et al. where concentration measured in adipose tissue. nificant; M = male, F = female.

6 724 J. F. Price and F. G. R. Fowkes or coronary death' 371. Mean carotenoid levels were lower in the events group in both smokers and nonsmokers, although the difference was greater in nonsmokers' 37 '. Thus, there is some indication that increased dietary intake of beta-carotene is associated with a reduced risk of coronary artery disease, although the evidence is less convincing than that for vitamin E. Results of three case control and two prospective studies also suggest that plasma beta-carotene levels are associated with a reduced risk of disease. Several studies indicate that dietary and circulating levels of the provitamin affect smokers more than non-smokers' 10 ' 19 ' 201, although the opposite effect was observed in hyperlipidaemic men' 371. It may be that a high dietary intake of beta-carotene is especially important in smokers who have both an increased demand for antioxidants (to combat smoking-induced free radicals) and a correspondingly lower circulating level of the pro-vitamin for a given dietary intake' 381. Studies on vitamin A are scarce but in general do not indicate an independent role for this vitamin in coronary artery disease, perhaps unsurprisingly in view of the resistance of plasma vitamin A levels to changes in dietary intake. Definitive conclusions on the effects of these vitamins on the risk of coronary artery disease await the final analysis of ongoing randomized controlled trials. Clinical trials (Table 1) In the ATBC Study, beta-carotene had no significant effect on coronary mortality' 26 ' or on the incidence of new angina pectoris' 27 ' in male smokers, but did result in an 8% increase in total mortality and an 18% increase in lung cancer. A second large randomized controlled trial of beta-carotene in the primary prevention of cancer and cardiovascular disease, the Harvard Physicians Health Study' 341, ended in December Early results from a subset of 333 men with chronic stable angina or coronary revascularization at the time of randomization, indicated a 44% reduction in all major coronary events in those receiving beta-carotene' 39 '. Detailed results from the full trial on healthy men are awaited, although a preliminary report suggested that betacarotene had no effect on disease or death' 40 '. The Beta-Carotene and Retinol Efficacy Trial (CARET), a trial of beta-carotene and vitamin A against lung cancer in smokers and asbestos workers' 41 ' was terminated almost 2 years early due to a 28% increase in lung cancer and a 17% increase in deaths' 40 '. Results for the effect of treatment on cardiovascular disease are awaited. In view of the adverse findings of the ATBC and CARET studies, the Woman's Health Study, which begun in 1992' 29 ', has also terminated the beta-carotene arm of its trial, but results to date have not been reported' 40 '. The three other trials which involve beta-carotene, factored with vitamins E and C are continuing' 3031 '. Vitamin C Dietary intake In the Harvard Physicians Follow-Up Study, a high intake of vitamin C was not associated with a lower risk of coronary heart disease in men' 101, whilst in women from the Nurses Health Survey, an initial effect was attenuated after adjustment for multivitamin use' 42 '. There was no association between vitamin C intake and subsequent cardiovascular events in 1462 Swedish women followed up for 5 years, but a total of only 23 myocardial infarctions was recorded' 43 '. Vitamin C intake was associated with a reduced risk of coronary mortality among 2385 women in Finland, but not among a similarly sized group of men and no adjustment was made for intake of vitamin E and beta-carotene'"'. Indeed, only one prospective study involving adults demonstrated an inverse relationship between vitamin C intake and cardiovascular mortality' 441. This effect was due largely to the use of vitamin C in supplements and again may have been due to antioxidant vitamins other than vitamin C in multivitamins, the supplement most widely used by study participants. In a single case-control study, men with previously undiagnosed ischaemic heart disease had lower vitamin C intake than healthy controls' 121. No association between vitamin C levels and risk of coronary artery disease was found in women, or in men who were already aware of their diagnosis. Plasma and leucocyte levels In Gey's cross-cultural study, plasma vitamin C correlated weakly with coronary mortality in the major subgroup of 12 populations with normal cholesterol levels, but this association disappeared on inclusion of all 16 populations' 131. Plasma levels were not correlated with coronary mortality rates among four European populations' 141, or with prevalent coronary disease in Finland' 15 '. Decreased leucocyte ascorbic acid levels were found in cases of angiographically proven coronary artery disease compared with controls referred for cardiac catheterization' 451. However, an initial relationship between newly diagnosed angina and low vitamin C was non-significant after controlling for cigarette smoking In the Basle prospective study, low levels of vitamin C alone did not increase the risk of ischaemic heart disease, although the risk of disease at low levels of both vitamin C and beta-carotene was greater than that for beta-carotene alone' 23 '. In conclusion, there is very little evidence in support of a role for vitamin C in the aetiology of coronary artery disease. Further information should be forthcoming from the two ongoing secondary prevention trials' 30 ' 31 ' and one primary prevention trial' 301 in which vitamin C is factored with vitamin E and betacarotene (Table 1).

7 Clinical Perspective 725 Table 3 Randomized controlled trials of vitamin E in the treatment of peripheral arterial disease Study No. of subjects Definition of disease Dose and duration of vitamin E Outcome Hamilton el al. 1953' 56 ' 41 Livingston and Jones Williams et al t54 > 33 Boyd and Marks 1963 (51) 33 Haeger 1968 [52 > 227 Williams et al ' Haeger 1974 [53 > Positive arteriogram >75% stenosis at angiography Positive arteriogram Femoropopliteal occlusion <450 mg. day ~' 12 weeks 600 mg. day" 1 40 weeks l-6g.day~' 3-43 months 400 mg. day ~ ' 13 weeks 300 mg. day" 1 ** 2-3 years 1-6 g. day" ' 9 months 300 mg. day" 1 ** 3-4 months No change in symptoms No change in exercise tolerance Improvement in exercise tolerance Improvement in walking distance* Improvement in walking distance Improvement in symptoms Improvement in walking distance Reduction in amputation rate Improvement in walking distance* Improvement in walking distance Improvement in muscle blood flow months improvement only for patients with femoropopliteal occlusion and poor distal arterial bed. "Vitamin E compared to alternative treatment regimes (vasodilators, anticoagulants or multivitamins without vitamin E). Peripheral arterial disease In comparison with coronary artery disease, relatively few studies have investigated the relationship between antioxidant vitamins and peripheral arterial disease. In a recent case control study, dietary vitamin C intake was significantly lower in cases of lower limb atherosclerosis than controls, but there were no significant differences in the intake of vitamins A and E' 46 '. Reduced levels of vitamin E were found in the skeletal muscle of elderly claudicants 1471 and serum and leucocyte vitamin C levels were lower in seven patients with intermittent claudication than in 13 controls' 481. Finally, baseline plasma beta-carotene and vitamin E levels were inversely related to the 12 month progression of carotid artery wall thickness in hypercholesterolaemic men [49]. There have been at least six clinical trials of vitamin E in lower limb atherosclerosis (Table 3). Although these early trials have been small and of variable quality, all but one [56) indicated an improvement in peripheral arterial disease with vitamin E supplementation in the range of 300 mg to 1-6 g per day [5O ~ 551. Of all the studies, that finding no benefit was amongst the shortest performed and used a dose of wheat germ vitamin E which may have been much lower than the 400 mg per day estimated by the authors. A systematic review of vitamin E treatment in peripheral arterial disease is currently in progress (J. Kleijnen, personal communication). In addition, a randomized controlled trial of fatty acids and an antioxidant combination including vitamin C, beta-carotene, selenium, zinc and pyridoxine is currently underway in claudicants (G. Leng, personal communication). The results should be available in 1997, although it will not be possible to examine the effects of individual antioxidant vitamins in this combined preparation. Despite limited data, there is some indication that the effects of antioxidant vitamins in peripheral arterial disease may be similar to those in coronary artery disease. However, further prospective studies and clinical trials will be required before firm conclusions can be drawn. Conclusions Available epidemiological evidence is consistent with the possibility that antioxidant vitamins have a protective role in cardiovascular disease. Evidence with respect to vitamins C and A is weak, that for beta-carotene somewhat stronger and evidence for a role for vitamin E highly suggestive. Most investigators and reviewers have concentrated on the effect of antioxidant vitamins in coronary artery disease and, as yet, there is very little information on the role of antioxidant vitamins in peripheral arterial disease. However, clinical trials of vitamin E in claudicants have been encouraging and further work is required, especially in view of the possible link between beta-carotene and smoking (a particularly strong risk factor for peripheral arterial disease). Whilst there may be specific requirements for all the antioxidant vitamins, their proposed common effect on atherosclerotic disease via inhibition of low density lipoprotein oxidation raises the possibility of additive or even synergistic effects. Synergistic interactions between antioxidants have been described in many experimental studies and in the Basle prospective study there was an over-multiplicative increase in cardiovascular risk at low concentrations of vitamin C and beta-carotene' 57 '. Although plasma levels of vitamin E can be increased only approximately twofold by dietary supplementation,

8 726 J. F. Price and F. G. R. Fowkes there is evidence that vitamin C may regenerate and spare vitamin E [58), such that the protection afforded by the two given together should be additive. Although detailed epidemiological evidence on possible interactions between the antioxidant vitamins is lacking, it is generally felt that antioxidant vitamins in ischaemic heart disease prevention should be tested by an antioxidant mixture aimed at optimizing the total antioxidant effect' 591. Several ongoing large-scale trials are therefore using a factorial design in which it should be possible to test the effects of all the antioxidant vitamins combined as well as their individual effects' 30 ' 3 ' 1. It is tempting to suggest that as these antioxidant vitamins are potentially beneficial and do not appear to be harmful, then their consumption should be encouraged. However, current evidence does not support the routine use of antioxidant vitamins against cardiovascular disease, and dietary guidelines should await the outcome of clinical trials presently in progress. J. F. PRICE F. G. R. FOWKES Wolf son Unit for Prevention of Peripheral Vascular Diseases, University of Edinburgh, Edinburgh, U.K. References [1] Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 1989; 320: [2] Leake DS. Oxidised low density lipoproteins and atherogenesis. Br Heart J 1993; 69: [3] Diplock AT. Antioxidant nutrients and disease prevention: an overview. Am J Clin Nutr 1991; 53: 189S-93S. [4] Duthie GG, Wahle KJ. Smoking, antioxidants, essential fatty acids and coronary heart disease. Biochem Soc Transactions 1990; 18: 1051^4. [5] Willett WC, Stampfer MJ, Underwood BA, Taylor JO, Hennekens CH. 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