The role of antioxidant vitamins in the primary

Transcription

1 SPRING 2003 PREVENTIVE CARDIOLOGY 85 Treatment of Atherosclerosis in the New Millennium: Is There a Role for Vitamin E? Emma A. Meagher, MD From the Center for Experimental Therapeutics and Department of Medicine, University of Pennsylvania, Philadelphia, PA Address for correspondence: Emma A. Meagher, MD, Center for Experimental Therapeutics, 9055 West Gates Pavilion, 3400 Spruce Street, Philadelphia, PA Manuscript received January 25, 2001; accepted April 23, 2002 Oxidative stress appears to be of fundamental relevance to diseases as diverse as atherosclerosis, cancer, and Alzheimer s disease. Observational data in humans have suggested that antioxidant vitamin intake is associated with reduced cardiovascular disease. Animal studies are largely consistent with the concept that dietary supplementation with antioxidant vitamins reduces the progression of atherosclerosis. However, recent prospective, controlled clinical trials of vitamin E, including the Cardiovascular Disease, Hypertension and Hyperlipidemia, Adult- Onset Diabetes, Obesity, and Stroke (CHAOS) study, the Heart Outcomes Prevention Evaluation (HOPE) trial, Gruppo Italiano per lo Studio della Sopravvivvenza nell Infarto Miocardico (GISSI)- Prevenzione trial, the Secondary Prevention with Antioxidants of Cardiovascular Disease in End Stage Renal Disease (SPACE) trial, and the Heart Protection Study (HPS) present a confused picture. The various possibilities that have been advanced to explain this discrepancy are discussed in this review. A striking feature of these and other trials of antioxidants is the absence of a biochemical basis for patient inclusion or, indeed, dose selection. Patients with high levels of oxidant stress or depletion of natural antioxidant defense systems may be the most likely to benefit from antioxidant therapy. If this is the case, then reliable, quantitative indices of in vivo oxidant stress such as urinary isoprostane levels should be considered as an inclusion criterion for patient selection. Future trials of antioxidant therapy in cardiovascular disease should then be targeted toward such patients with high levels of oxidant stress or patients with depletion of natural antioxidant defense systems. Furthermore, the dose of antioxidant should be chosen based on a surrogate readout that is a reliable, reproducible, and easily obtainable in vivo measure of oxidant stress. In the interim, although the safety of vitamin E up to doses of 800 IU/day has been determined, the conflicting nature of the results published to date encourages us to avoid making premature recommendations with respect to vitamin E supplementation in the prevention and treatment of cardiovascular disease. (Prev Cardiol. 2003;6:85 90) 2003 CHF, Inc. The role of antioxidant vitamins in the primary and secondary prevention of cardiovascular disease (CVD) has been a topic of increasing interest over the last decade. This review will discuss the evidence that supports a role of vitamin E in this process. THE RATIONALE Free radical species derived from molecular oxygen are highly charged. They can react with all biologic molecules such as lipids and nucleic acids. 1 This interaction results in peroxidation. 1,2 The importance of lipid peroxidation in atherogenesis is now appreciated. Oxidative modification of the LDL molecule by a free radical facilitates macrophage uptake in vitro. 3 5 These oxidized moieties have been demonstrated in the atherosclerotic lesion. 6 Additionally, oxidized LDL has been shown to have important biologic properties such as induction of monocyte chemotaxis and expression of intercellular and vascular adhesion-1 molecules on endothelial cells. 3,7,8 Vitamin E (α-tocopherol) is a lipid-soluble molecule that exists in the lipid bilayer of cell membranes and lipoproteins. It has several important properties as they pertain to CVD. Vitamin E is considered to be nature s most effective lipid-soluble, chain-breaking antioxidant, protecting cell membranes from peroxidative damage Several studies have shown that dietary supplements of vitamin E inhibit the oxidative modification of LDL in vitro In addition it has been demonstrated

2 86 PREVENTIVE CARDIOLOGY SPRING 2003 in vitro that LDL oxidation will only occur after depletion of endogenous vitamin E. 15 The oxidizability of LDL ex vivo is enhanced in men at increased risk for developing atherosclerosis such as diabetics, smokers, and patients with coronary heart disease. 16,17 More recently, vitamin E has been shown to have additional properties that may be relevant to protection against CVD. These include inhibition of endothelial cell adhesion, inhibition of platelet aggregation, 21,22 inhibition of vascular smooth muscle proliferation, 23 and reduction of leukotriene synthesis by neutrophils. 24 Analysis of this data in isolation gives credence to a potential role for Vitamin E in atherosclerosis. ANIMAL STUDIES Refined animal models of atherosclerosis have been employed to confirm the hypothesis that administration of antioxidants would reduce the progression of atherosclerosis. These sophisticated models have provided substantial insights. LDL oxidation has been shown to be markedly inhibited in LDL-receptor deficient mice fed a high-fat, high-cholesterol diet supplemented with vitamin E. 25 In this study, the cross-sectional surface area of fatty streak lesions in the aortic sinus was reduced by 60% in the antioxidant group compared with controls. Aortic lesion area and isoprostane excretion (an independent marker of in vivo oxidant injury) were significantly reduced in apolipoprotein-e deficient mice fed a chow diet supplemented with vitamin E. 26 A recent report of a new mouse model confirms the importance of vitamin E in preventing atherosclerosis in mice. Vitamin E deficiency caused by disruption of the α-tocopherol transfer protein gene (Ttpa) was associated with increased levels of isoprostanes in aortic tissue and increased severity of atherosclerotic lesions in the proximal aortas of apolipoprotein-e deficient mice. 27 OBSERVATIONAL STUDIES Early observations of an association between diet and CVD were made in Since that time several observational studies have reported an inverse relationship between dietary antioxidant intake and the incidence of CVD Very few of these studies made any adjustment for concomitant changes in risk factors such as smoking, exercise, blood pressure, or cholesterol level. The earlier studies reflect largely dietary, rather that supplementary vitamin E. The typical dietary intake of vitamin E ranges from about 10 IU/day in the American diet 33 to about IU/day in the Mediterranean diet. 34 In contrast the intervention trials that will be discussed have employed doses in the IU/day range. Therefore these two types of studies address opposite ends of the dose-response curve. The Nurses Health Study 35 is a prospective cohort study that addressed the association between vitamin E intake and the incidence of major coronary events in 87,245 females aged years who were free of symptomatic CVD at study entry. The use of antioxidant vitamin supplements was recorded and dietary intake of nutrients was measured. A total of 552 major coronary events, 437 nonfatal myocardial infarctions (MIs) and 115 coronary deaths were documented over an 8-year follow-up period. Of these 552 events, 503 (91%) occurred in subjects not taking vitamin E supplements. Adjusting for age and smoking status, the risk of a coronary event was significantly lower in the highest quintile of vitamin E intake as compared to those in the lowest quintile (relative risk 0.66; 95% confidence interval [CI], ). The significant protective effect persisted after adjusting for obesity, exercise level, dietary intake, hypertension, hypercholesterolemia, diabetes, menopausal status, use of estrogen replacement therapy and aspirin, alcohol intake and the use of multivitamins, β-carotene and vitamin C. The apparent benefit was attributable mainly to the use of vitamin E supplements. High vitamin E intake from diet alone was not associated with significant risk reduction. Women who took vitamin E for a short time showed little benefit. However, the use of vitamin E supplements for two or more years was associated with a decrease in risk of 41%. The Health Professionals Follow-Up Study, 36 started in 1986, investigated 39,910 males, years of age with no history of CVD, diabetes or hypercholesterolemia. The use of supplements was recorded and a diet history was obtained. Dietary intakes of vitamin E were found to be strongly, but not significantly correlated with reduced risk for coronary heart disease or death. The age-adjusted risk for coronary heart disease was significantly lower in the highest quintile for vitamin E intake from supplements. The maximal reduction in risk was seen among men consuming at least 100 IU/day vitamin E for two or more years. The relative risk for nonfatal MI, coronary revascularization, or heart disease death was reduced by 30% (95% CI, ) for men taking more than 250 IU/day vitamin E versus nonusers of supplements. The Iowa Women s Health Study 37 is a prospective study of 34,486 postmenopausal women aged years who were free of CVD at the time of study entry. They were followed for a period of seven years. The end point for this study was death from CVD. The relative risk of death from heart disease for the quintile with the highest intake of vitamin E from food sources versus the lowest was 0.38 (95% CI, ). No associations were found for either vitamin C or β-carotene. 21,809 women obtained vitamin E from food only and the remainder 12,677 obtained the vitamin from both food and supplements. No information was collected about the duration of supplement use. The nature of the supplements is also not described, but the intake of vitamin E varied from 0 25 IU/day in

3 SPRING 2003 PREVENTIVE CARDIOLOGY 87 the lowest to >250 IU/day in the highest quintile for vitamin E intake. The Basel Study 38 is a large cohort study that enrolled 2975 healthy male pharmaceutical employees. Baseline antioxidant status was measured and subjects were monitored for coronary death for a 7-year follow-up period. No association between plasma vitamin E levels and coronary death was seen. Risk of death from coronary heart disease was increased in those in the lowest quartile of vitamin C and carotene level. In addition, low plasma concentrations of both of these vitamins were associated with an increased risk of death from stroke. This is a representative sample of the observational studies that have been published. There are of course inherent limitations with observational studies. Many confounders exist. For example, vitamin intake may reflect a healthier lifestyle, including general dietary habits, exercise and smoking patterns rather than reflect a direct beneficial role for vitamin supplementation alone. These types of studies prove association as opposed to causality. They do not address the mechanisms involved but are very valuable in hypothesis generation for future mechanistic studies and clinical trials. CLINICAL TRIALS OF VITAMIN E IN CVD A review of the experimental and epidemiologic evidence in 1991 led to the recommendation that clinical trials with antioxidants should proceed. 39 It is important to remember that studies in animal models, for the most part, measure the extent of the earliest atherosclerotic lesions (fatty streaks). Whether or not antioxidants have inhibitory effects on advanced atherosclerotic disease, plaque rupture, or thrombosis remains to be determined. Early mechanistic studies demonstrated that short- 40 and long-term 41,42 administration of large doses of vitamin E to humans strongly protects LDL against oxidative modification ex vivo. For example, treatment with 1000 IU α-tocopherol/day for seven days resulted in increased resistance of LDL to oxidation. The increase in resistance time highly correlated with LDL vitamin E content. 40 Other studies using either 800 IU α-tocopherol/day or 1600 mg vitamin E/day for several months also reported an increased protection of LDL, whereas three months supplementation with 60 mg/day of β-carotene had no effect on LDL oxidizability. 41,42 Two small prospective randomized controlled trials of subjects with intermittent claudication showed that exercise capacity improved significantly with vitamin E therapy. 43,44 The first study published in 1958 administered 200 mg α-tocopheryl acetate/day for 40 weeks to 40 nondiabetic subjects with symptoms of intermittent claudication, and the second used 600 mg vitamin E/day for 10 months for a similar population. Both of these determined outcome based on symptomatic improvement in a walking test. In a double-blind, placebo-controlled trial, DeMaio et al. 45 investigated the effects of α-tocopherol on restenosis following successful percutaneous transluminal coronary angioplasty. The study randomized 115 subjects to receive 1200 IU per day α-tocopherol (n=52) or placebo (n=48). After four months of follow-up, subjects receiving vitamin E had a 34% incidence of restenosis compared with a 50% incidence in the placebo group. The 31% risk reduction did not reach statistical significance (p<0.06), but the small numbers and the short duration of treatment/follow-up may limit the study s power to detect a difference between the groups. More recently the Secondary Prevention With Antioxidants of Cardiovascular Disease in End Stage Renal Disease (SPACE) trial 46 investigated the effect of high-dose vitamin E supplementation on CVD outcomes in hemodialysis patients with preexisting heart disease. One hundred and ninety-six hemodialysis patients aged years were randomized to receive 800 IU once a day vitamin E or placebo in a double-blind design. Patients were followed for approximately 17 months. The primary end points included fatal and nonfatal MI, ischemic stroke, peripheral vascular disease and unstable angina. Secondary outcomes included cardiovascular mortality and total mortality. Results of this study demonstrated a highly significant reduction (46%) in the composite primary outcome measure that was primarily attributable to a 70% reduction in acute MI. No effect on total or cardiovascular mortality was seen. The Study to Evaluate Carotid Ultrasound Changes in Patients Treated With Ramipril and Vitamin E (SECURE), 47 a substudy of the Heart Outcomes Prevention Evaluation (HOPE) trial, randomized 732 patients with known CVD to receive ramipril, vitamin E or placebo in a 3 2 factorial design for 4.5 years. Vitamin E 400 IU per day had neutral effects on ultrasound measurements of atherosclerosis progression. Cambridge Heart Antioxidant Study (CHAOS) 48 is a secondary prevention study in which 2002 subjects with angiographically proven coronary artery disease were randomized to vitamin E (400 mg or 800 mg) or placebo and followed for an average of 510 days. Patients were prestratified by planned therapy (angioplasty, bypass grafting) and by risk factors for coronary disease and then randomly assigned to a placebo or treatment group. The primary end points were a combination of cardiovascular death plus nonfatal MI and nonfatal MI alone. The first 546 subjects in the α-tocopherol group received 800 IU/day and the remainder was given 400 IU/day, but the two groups were combined for statistical analysis. There was a 47% reduction (95% CI, 66% to 17%; p=0.005) in

4 88 PREVENTIVE CARDIOLOGY SPRING 2003 the primary end point of cardiovascular death and nonfatal MI compared with the placebo group. This effect was primarily attributable to a 77% reduction in the risk for nonfatal MI. There was no reduction in total mortality and an 18% nonsignificant increase (95% CI, 38% to 127%) in cardiovascular death in the vitamin E group. This increase in CVD death was subjected to a later analysis. Results of this have shown that of the total 72 deaths only six were in the group that was compliant with the vitamin E regimen, 21 were in the noncompliant group and 32 were in the placebo group. 49 The authors have suggested that this data could imply that the true benefit of the therapy may have been underestimated in the initial analysis. The Gruppo Italiano per lo Studio della Sopravvivvenza nell Infarto Miocardico (GISSI)- Prevenzione Trial 50 is another secondary prevention study that enrolled 11,324 Italian subjects with a history of a recent MI within three months of the enrollment period. Patients were randomized to receive 300 mg/day of synthetic vitamin E, 1 g/day n-3 polyunsaturated fatty acids, both or nothing in a 2 2 factorial design and followed for 3.5 years. There was no placebo group and the study was an open label design. The primary combined efficacy end point was death, nonfatal MI, and stroke. Treatment with n-3 PUFA but not vitamin E significantly lowered the risk of the primary end point. No additional benefit was noted with combination therapy. The results of this study are interesting and have raised some questions about the trial design. Firstly, the hypothesis and trial design proposed a 20% benefit from intervention as clinically significant. Thus the results have shown that the n-3 reduced the risk of the primary end point by 15% and vitamin E by 11% but only the former attained statistical significance. A recent editorial by Brown 51 attempted to address the diverging results of CHAOS and GISSI. He proposes that they may be explained in part by the different diet consumed by the two groups. The subjects in the GISSI trial consumed a typical Mediterranean diet rich in fruits and vegetables whereas the CHAOS participants ate an English type diet higher in fat and lower in fruits and vegetables. In addition, it has been reported that approximately 50% of the GISSI participants were receiving CVD-preventive drugs such as statin therapy which have been suggested to inhibit LDL oxidation, an effect that is independent of LDL-cholesterol lowering. The dose of vitamin E is different. CHAOS employed either 400 IU/day or 800 IU/day of natural vitamin E and GISSI subjects received 300 mg/day of synthetic vitamin E which is equivalent to about 150 mg of the natural source vitamin E. 52 Finally, the authors of the CHAOS study have recently reported a 3.5-fold increased frequency of a polymorphism in the gene for endothelial nitric oxide (NO) synthase that is associated with impaired endothelial cell function. 49 As mentioned previously vitamin E may protect NO from destruction. This genetic polymorphism may therefore make the CHAOS study population more susceptible to the antioxidant effects of vitamin E administration. Results from the HOPE study 53 were reported late last year. This study enrolled 9541 subjects 55 years with both symptomatic and asymptomatic CVD. Subjects were randomized to receive naturally occurring vitamin E (400 IU per day) or placebo and followed for 4.5 years. This trial was a 2 2 factorial that also involved the angiotensin-converting enzyme inhibitor ramipril. Initial analysis has reported that no clinical benefit or adverse events were noted in the vitamin E-treated group. The HOPE study has been described as a primary prevention study. This is somewhat misleading. The patients enrolled had documented atherosclerosis and were considered high risk with respect to the development of clinical CVD but they did not have heart failure. One of the main objectives was to assess the role of angiotensin-converting enzyme inhibition in delaying the onset or progression of heart failure. There was no difference between the vitamin E and placebo groups with regard to cardiovascular events. These data fly in the face of the GISSI and CHAOS studies. In this study there does not appear to be a difference in the comorbidities or the additional CVD preventive strategies that could explain the negative results. The Heart Protection Study 54 tested the effect of an antioxidant cocktail (625 mg vitamin E, 250 mg vitamin C, and 20 mg β-carotene) in a 2 2 factorial design with a lipid-lowering agent for five years in 20,000 high-risk individuals without known CVD. Preliminary data show no benefit or harm in those who received the antioxidant cocktail. The Alpha-Tocopherol/Beta-Carotene Cancer (ATBC) prevention study 55 was a randomized double-blind, placebo-controlled primary prevention trial undertaken to determine whether supplementation with 50 mg/day of synthetic vitamin E, 20 mg/day β-carotene or both would reduce the incidence of lung cancer in male smokers. A secondary outcome of interest was secondary prevention of CVD. A total of 29,133 Finish male smokers aged were studied for a period of 5 8 years. The initial data reported in 1994 demonstrated an increased incidence of lung cancer in those receiving β-carotene after five years of supplementation. 55 A subsequent analysis of CVD risk was published in In the 22,271 men with no prior history of MI there was a 4% (95% CI, 12% to 4%) reduction in major coronary events and an 8% (95%CI, 19% to 5%) decreased incidence of fatal coronary heart disease in those subjects receiving vitamin E. These results did not reach statistical significance. Neither agent affected the incidence of nonfatal MI.

5 SPRING 2003 PREVENTIVE CARDIOLOGY 89 ONGOING STUDIES Randomized trials of sufficient power and duration are under way to establish the efficacy of high dose antioxidant supplementation in both primary and secondary prevention of CVD. The Women s Health Study 57 is a primary prevention trial designed to study the effects of vitamin E, β-carotene and low-dose aspirin on major cardiovascular end points in 40,000 healthy females aged 45 years and older. The β-carotene arm of the study was stopped because of the null findings in a similar study in men (the Physicians Health Study 58 ) and the possible harmful effects of β-carotene found in the Carotene And Retinol Efficacy Trial (CARET) 59 and ATBC study. 55 The vitamin E and aspirin components are ongoing. The Women s Antioxidant Cardiovascular Disease study is determining the effect of supplementation with 20 mg β-carotene, 400 IU of vitamin E, and 100 mg of vitamin C per day in 8000 nurses with established CVD. SUMMARY Oxidative stress appears to be of fundamental relevance to diseases as diverse as atherosclerosis, cancer and Alzheimer s disease. Observational data in humans have suggested that antioxidant vitamin intake is associated with reduced CVD. Animal studies are largely consistent with the concept that dietary supplementation with antioxidant vitamins reduces the progression of atherosclerosis. However, prospective, controlled clinical trials of antioxidants present a confused picture. For example, while vitamin E appeared to benefit patients with coronary disease in the CHAOS study, the HOPE, GISSI-Prevenzione and the Heart Protection studies have failed to detect such a benefit. Various possibilities have been advanced to explain this discrepancy. However, a striking feature of these and other trials of antioxidants is the absence of a biochemical basis for patient inclusion or, indeed, dose selection. In fact, the SPACE trial was performed in end-stage renal disease patients, who are known to have high levels of oxidant stress. Patients with high levels of oxidant stress or depletion of natural antioxidant defense systems may be the most likely to benefit from antioxidant therapy. If the latter were true then reliable, quantitative indices of in vivo oxidant stress 60 such as urinary isoprostane levels should be considered as an inclusion criterion for patient selection. We recently utilized this approach to investigate the dose-response relationships of vitamin E under double-blind, placebo-controlled conditions in healthy volunteers. In this population, with intact endogenous antioxidant defenses, a wide dose range of exogenous vitamin E had no effect on a reliable and reproducible index of lipid peroxidation. 61 Such observations further question the benefits to be derived by healthy individuals consuming vitamin E supplements. It is the author s belief that future trials of antioxidant therapy in CVD should be targeted toward patients with high levels of oxidant stress or patients with depletion of natural antioxidant defense systems. Furthermore, the dose of antioxidant should be chosen based on a rational surrogate that is a reliable, reproducible and easily obtainable in vivo measure of oxidant stress. In conclusion, although the safety of vitamin E up to doses of 800 IU/day has been determined, 62 the conflicting nature of the results published to date reinforces the need to avoid making premature recommendations with respect to vitamin E supplementation in the prevention and treatment of CVD. REFERENCES 1 Halliwell B. Free radicals and antioxidants. Nutr Rev. 1994; 52(8): Halliwell B, Chirico S. Lipid peroxidation: its mechanism, measurement, and significance. Am J Clin Nutr. 1993; 57(suppl):715S 725S. 3 Steinberg D, Parthasarathy S, Carwe TE, et al. Beyond cholesterol: modifications of low density lipoprotein (LDL) that increase its atherogenicity. N Engl J Med. 1989;320: Witztum JL. The oxidation hypothesis of atherosclerosis. Lancet. 1994;334: Witztum JL, Steinberg D. Role of oxidized LDL in atherogenesis. J Clin Invest. 1991;88: Palinski W, Rosenfeld ME, Yla-Herttuala S, et al. LDL undergoes oxidative modification in vivo. Proc Natl Acad Sci U S A.1989;86(4): Witztum JL, Berliner JA. Oxidized phospholipids and isoprostanes in atherosclerosis. Curr Opin Lipidol. 1998;9: Berliner JA, Navab M, Fogelman AM, et al. Atherosclerosis: basic mechanisms. Oxidation, inflammation, and genetics. Circulation. 1995;91: Burton GW, Ingold KU. Vitamin E as an in vitro and in vivo antioxidant. Ann N Y Acad Sci. 1989;570: Gey KF. On the antioxidant hypothesis with regard to arteriosclerosis. Bibl Nutr Dieta. 1986;37: Esterbauer H, Striegl G, Phul H, et al. The role of vitamin E and carotenoids in preventing oxidation of LDLs. Ann N Y Acad Sci. 1989;570: Esterbauer H, Gebicki J, Jurgens G. The role of lipid peroxidation and antioxidants in oxidative modifications of LDL. Free Radic Biol Med. 1992;13: Reaven PD, Khouw A, Beltz WF, et al. Effect of dietary antioxidant combinations in humans: protection of LDL by vitamin E but not by β-carotene. Arterioscler Thromb. 1993;13: Jialal I, Grundy SM. Effect of dietary supplementation with α-tocopherol on the oxidative modification of LDL. J Lipid Res. 1992;33: Jialal I, Grundy SM. Influence of antioxidant vitamins on LDL oxidation. Ann N Y Acad Sci. 1992;669: Esterbauer H, Dieber-Rotheneder M, Striegel M, et al. Role of vitamin E in preventing the oxidation of LDL. Am J Clin Nutr. 1991;53:314S 321S. 17 Blackman C, White P, Tsou W, et al. Peroxidation of plasma and platelet lipids in chronic cigarette smokers and insulindependent diabetics. Ann N Y Acad Sci. 1984;435: Faruqi R, de la Motte C, DiCorleto PE. Alpha-tocopherol inhibits agonist-induced monocytic cell adhesion to cultured human endothelial cells. J Clin Invest.1994;94: Cominacini L, Garbin U, Pasini AF, et al. Antioxidants inhibit the expression of intercellular cell adhesion molecule-1 and vascular call adhesion molecule-1 induced by oxidized LDL on human umbilical vein endothelial cells. Free Radic Biol Med. 1997;22: Islam KN, Devaraj S, Jialal I. Alpha-tocopherol enrichment

6 90 PREVENTIVE CARDIOLOGY SPRING 2003 of monocytes decreases agonist-induced adhesion to human endothelial cells. Circulation. 1998;98: Salonen JT, Salonen R, Seppanen K, et al. Effects of antioxidant supplementation on platelet function: a randomized pairmatched, placebo-controlled, double-blind trial in men with low antioxidant status. Am J Clin Nutr. 1991;53: Colette C, Pares-Herbute N, Monnier LH, et al. Platelet function in type 1 diabetics: effects of supplementation with large doses of vitamin E. Am J Clin Nutr. 1988;47: Azzi A, Aratri E, Boscoboinik D, et al. Molecular basis of α- tocopherol control of smooth muscle cell proliferation. Bio Factors. 1998;7: Denzlinger C, Kless T, Sagebiel-Kohler S, et al. Modulation of the endogenous leukotriene production by fish oil and vitamin E. J Lipid Mediat Cell Signal. 1996;98: Crawford RS, Kirk EA, Rosenfeld ME, et al. Dietary antioxidants inhibit development of fatty streak lesions in the LDL receptor-deficient mouse. Arterioscler Thromb Vasc Biol. 1998;18: Pratico D, Tangirala RK, Rader DJ, et al. Vitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis in apolipoprotein-e deficient mice. Nat Med. 1998; 4: Jacobs SC, Stephenson JR, Wilkinson GW. High-level expression of the tick-borne encephalitis virus NS1 protein by using an adenovirus-based vector: protection elicited in a murine model. J Virol. 1992;66: Rouse IL, Armstrong BK, Beilin W, et al. Vegetarian diet, blood pressure and cardiovascular risk. Aust N Z J Med. 1934;14: Acheson RM, Williams DRR. Does consumption of fruit and vegetables protect against stroke? Lancet. 1983;1: Armstrong K, Mann JI, Adelstein AM, et al. Commodity consumption and ischemic heart disease mortality, with special reference to dietary practices. J Chronic Dis. 1975;28: Smith WCS, Tunstall-Pedoe H, Crombie IK, et al. Concomitants of excess coronary deaths: major risk factor and lifestyle findings from 10,359 men and women in the Scottish Heart Health Study. Scott Med J. 1989;34: Riemersma RA, Oliver M, Elton RA, et. al. Plasma antioxidants and coronary heart disease: vitamins C and E and selenium. Eur J Clin Nutr. 1990;44: Food and Nutrition Board National Research Council. Recommended Dietary Allowances (RDA). 10th ed. Washington, DC: National Academy Press; Machlin LJ, ed. Vitamin E: A Comprehensive Treatise. New York, NY: Marcel Dekker; Stampfer MJ, Hennekens CH, Manson JE, et al. Vitamin E consumption and the risk of coronary disease in women. N Engl J Med. 1993;328: Rimm EB, Stampfer MJ, Ascherio A, et al. Vitamin E consumption and the risk of coronary artery disease in men. N Engl J Med. 1993;328: Kushi LH, Folsom AR, Prineas RJ. Dietary antioxidant vitamins and death from coronary heart disease in postmenopausal women. N Engl J Med. 1996;34: Gey KF, Stahelin HB, Eichholzer M. Poor plasma status of carotene and vitamin C is associated with higher mortality from ischemic heart disease and stroke: Basel Prospective Study. Clin Invest. 1993;71: Steinberg D, Workshop Participants. Antioxidants in the prevention of human atherosclerosis. Summary of the proceedings of a National Heart, Lung and Blood Institute Workshop. Circulation. 1992;85: Princen HMG, van Poppel G, Bogelzang C, et al. Supplementation with vitamin E but not β-carotene in vivo protects LDL from lipid peroxidation in vitro. Effect of cigarette smoking. Arterioscler Thromb. 1992;12: Reaven PD, Khouw A, Beltz WF, et al. Effect of dietary antioxidant combinations in humans. Arterioscler Thromb. 1993;13: Jialal I, Grundy SM. Effect of dietary supplementation with α-tocopherol on the oxidative modification of LDL. J Lipid Res. 1992;33: Livingstone PD, Jones C. Treatment of intermittent claudication with vitamin E. Lancet. 1958;12: Haeger K. Long-time treatment of intermittent claudication with vitamin E. Am J Clin Nutr. 1987;45: DeMaio SJ, King SB, Lembo NJ, et al. Vitamin E supplementation, plasma lipids and incidence of restenosis after percutaneous transluminal coronary angioplasty. J Am Coll Nutr. 1992;11: Boaz M, Smetana S, Weinstein T, et al. Secondary Prevention with Antioxidants of Cardiovascular disease in End-stage renal disease (SPACE): randomized placebo-controlled trial. Lancet. 2000;356: Lonn E, Yusuf S, Dzavik V, et al. Effects of ramipril and vitamin E on atherosclerosis: the Study to Evaluate Carotid Ultrasound changes in patients treated with Ramipril and vitamin E (SECURE). Circulation. 2001;20;103(7): Stephens NG, Parsons A, Schofield PM, et al. Randomized controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS). Lancet. 1996;347: Mitchinson MJ, Stephens NG, Parsons A, et al. Mortality in the CHAOS trial. Lancet. 1999;353: Anonymous. Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after MI: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della Sopravvivenza nell Infarto miocardico. Lancet. 1999;354: Brown M. Do vitamin E and fish oil protect against ischemic heat disease? Lancet. 1999;345: Burton GW, Traber MG, Acuff RV, et al. Human plasma and tissue α-tocopherol concentrations in response to supplementation with deuterated natural and synthetic vitamin E. Am J Clin Nutr. 1998;67: Yusuf S, Dagenais G, Pogue J, et al. Vitamin E supplementation and cardiovascular events in high-risk patients. The Heart Outcomes Prevention Evaluation Study Investigators. N Engl J Med. 2000;342: Collins R, Peto R, Armitage J. The MRC/BHF Heart Protection Study preliminary results. Int J Clin Pract. 2002;56(1): The α-tocopherol β-carotene Cancer Prevention Study Group. The effect of vitamin E and β-carotene on the incidence of lung cancer and other cancers in male smokers. N Engl J Med. 1994;330: Vitramo J, Rapola JM, Ripatti S, et al. Effect of vitamin E and β-carotene on the incidence of primary nonfatal MI and fatal coronary heart disease. Arch Intern Med. 1998;158: Rexrode KM, Lee IM, Cook NR, et al. Baseline characteristics of participants in the Women s Health Study. JWomens Health Gend Based Med. 2000;9(1): Hennekens CH, Buring JE, Manson JE, et al. Lack of effect of long-term supplementation with β-carotene on the incidence of malignant neoplasms and CVD. N Engl J Med. 1996;334: Omenn GS, Goodman GE, Thornquist MD, et al. Effects of a combination of β-carotene and vitamin A on lung cancer and CVD. N Engl J Med. 1996;334: Meagher EA, FitzGerald GA. Indices of lipid peroxidation in vivo: strengths and limitations. Free Rad Biol Med. 2000;28: Meagher EA, Barry OP, Lawson JA, et al. Effects of vitamin E on lipid peroxidation in healthy persons. JAMA. 2001; 285(9): Meydani SN, Meydani M, Blumberg JB, et al. Assessment of the safety of supplementation with different amounts of vitamin E in healthy older adults. Am J Clin Nutr. 1998;68: