Vitamin E and heart disease:

Free Radical Biology & Medicine, Vol. 28, No. 1, pp. 141–164, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter

PII S0891-5849(99)00224-5

Review Article VITAMIN E AND HEART DISEASE: BASIC SCIENCE TO CLINICAL INTERVENTION TRIALS WILLIAM A. PRYOR The Biodynamics Institute, Louisiana State University, Baton Rouge, LA

Abstract—A review is presented of studies on the effects of vitamin E on heart disease, studies encompassing basic science, animal studies, epidemiological and observational studies, and four intervention trials. The in vitro, cellular, and animal studies, which are impressive both in quantity and quality, leave no doubt that vitamin E, the most important fat-soluble antioxidant, protects animals against a variety of types of oxidative stress. The hypothesis that links vitamin E to the prevention of cardiovascular disease (CVD) postulates that the oxidation of unsaturated lipids in the low-density lipoprotein (LDL) particle initiates a complex sequence of events that leads to the development of atherosclerotic plaque. This hypothesis is supported by numerous studies in vitro, in animals, and in humans. There is some evidence that the ex vivo oxidizability of a subject’s LDL is predictive of future heart events. This background in basic science and observational studies, coupled with the safety of vitamin E, led to the initiation of clinical intervention trials. The three trials that have been reported in detail are, on balance, supportive of the proposal that supplemental vitamin E can reduce the risk for heart disease, and the fourth trial, which has just been reported, showed small, but not statistically significant, benefits. Subgroup analyses of cohorts from the older three trials, as well as evidence from smaller trials, indicate that vitamin E provides protection against a number of medical conditions, including some that are indicative of atherosclerosis (such as intermittent claudication). Vitamin E supplementation also produces an improvement in the immune system and protection against diseases other than cardiovascular disease (such as prostate cancer). Vitamin E at the supplemental levels being used in the current trials, 100 to 800 IU/d, is safe, and there is little likelihood that increased risk will be found for those taking supplements. About one half of American cardiologists take supplemental vitamin E, about the same number as take aspirin. In fact, one study suggests that aspirin plus vitamin E is more effective than aspirin alone. There are a substantial number of trials involving vitamin E that are in progress. However, it is possible, or even likely, that each condition for which vitamin E provides benefit will have a unique dose-effect curve. Furthermore, different antioxidants appear to act synergistically, so supplementation with vitamin E might be more effective if combined with other micronutrients. It will be extremely difficult to do trials that adequately probe the dose-effect curve for vitamin E for each condition that it might affect, or to do studies of all the possible combinations of other micronutrients that might act with vitamin E to improve its effectiveness. Therefore, the scientific community must recognize that there never will be a time when the science is “complete.” At some point, the weight of the scientific evidence must be judged adequate; although some may regard it as early to that judgement now, clearly we are very close. In view of the very low risk of reasonable supplementation with vitamin E, and the difficulty in obtaining more than about 30 IU/day from a balanced diet, some supplementation appears prudent now. © 2000 Elsevier Science Inc. Keywords—Vitamin E, Tocopherol, Heart disease, Atherosclerosis, Oxidative stress, Free radical, Human, Rabbit, Review, Intervention trial, LDL oxidation, Myocardial infarction, Stroke

Address correspondence to: William A. Pryor, Biodynamics Institute, 711 Choppin, Louisiana State University, Baton Rouge, LA 70803, USA; Tel: (225) 388-2063; Fax: (225) 388-4936; E-Mail: [email protected]. William A. Pryor is the Thomas & David Boyd Professor and Director of the Biodynamics Institute at LSU. He holds a “Chicago-Plan” (i.e., no high school) Ph.B. and a B.S. in chemistry from the University of Chicago and a Ph.D. from the University of California at Berkeley. He has published over 700 articles and notes, and is author or editor of more than 25 books. He has received many honors including five medals from the American Chemical Society, three from the National Institutes of Health (including a MERIT award), a Guggenheim Fellowship, and several nutrition awards. He is the past president of the Oxygen Society and is Co-Editor-in-Chief of the journal Free Radical Biology & Medicine. His hobbies include reading modern European history, biking, and being the host of a show, Classic Jazz, on Public Radio that features jazz recorded from the 1930s up to today. 141

142

W. A. PRYOR INTRODUCTION

The chemistry and biology of vitamin E has been the subject of intensive study for more than 50 years [1– 6], and this enormous body of literature demonstrates conclusively that the principal role of vitamin E is to protect tissue against unwanted, destructive oxidation [7,8]. Although additional functions of vitamin E have been studied by various investigators [9 –12], the antioxidant function of vitamin E remains the most well established; in fact, vitamin E is the most effective lipid-soluble antioxidant present in our cells [7]. Because vitamin E is an antioxidant, and because it has long been known that oxidation plays an important role in carcinogenesis [13–15], it was presumed that vitamin E would be protective against cancer. In fact, in animal studies, many antioxidants do protect against chemically induced cancers in various organs [13,16 – 19]. However, perhaps because of the long induction times before overt tumors are observed and the long period over which supplementation must occur to provide protection, evidence for an anticancer effect for vitamin E in humans has developed more slowly than has evidence for the role of vitamin E in preventing heart disease. The hypothesis that links vitamin E and cardiovascular diseases (CVD) posits that the oxidation of unsaturated lipids in the LDL particle, as well as the complex sequelae that flow from this oxidation, play a crucial role in the pathogenesis of atherosclerosis. This theory, proposed by Morel et al. [21,22], Steinberg and his associates [22,23], and Mitchinson et al. [24], is now generally accepted, and this theory provides a strong biological plausibility for the role of vitamin E in preventing CVD. Recently, data from epidemiological studies, from prospective observational studies, and from clinical intervention trials have become available. The extensive data supporting a role for vitamin E in preventing heart disease, therefore, are impressive both in their quality and depth. These facts have become increasingly familiar to the American public. A substantial fraction of all Americans, including about half of the physicians in the field [25], now take vitamin E supplements; in fact, as many physicians take vitamin E as take aspirin. THE BIOLOGICAL EFFECTS OF OXIDATION

The random oxidation of biopolymers was suggested by Denham Harman [26,27] in the 1950s to be a factor responsible for aging. In recent years, this “free radical theory of aging” has been modified to state that the oxidation of specific biotarget molecules increases the susceptibility of animals and humans to specific diseases [28], and particularly the chronic, life-shortening dis-

eases such as cancer, heart disease, and some of the destructive neurological diseases [29 –36]. Oxidations can occur by one-electron pathways involving radicals such as the hydroxyl radical, or by two-electron paths that involve nonradical oxidants such as hydrogen peroxide, ozone, or peroxynitrite; however, even these nonradical oxidants can, under some circumstances, oxidize biomolecules by radical pathways [37, 38]. Therefore, a majority of the random, pathological oxidations of biopolymers occur by one-electron mechanisms involving free radicals. Because vitamin E is nature’s best lipid-soluble antioxidant, it would be expected to inhibit these pathologic oxidations.

THE OXIDATION OF THE LDL PARTICLE

The most developed theory for the etiology of atherosclerosis and CVD proposes that the oxidation of the LDL particle plays a key role [20 –24,39 – 42]. The typical LDL particle contains 2700 fatty acid molecules incorporated into several lipid classes; about half of these fatty acids are PUFA [41,42], which are very oxidation sensitive. The most prevalent antioxidant in LDL is vitamin E: each LDL contains five to nine vitamin E molecules along with smaller amounts of several other antioxidants (such as ␥-tocopherol and ␤-carotene) that are present at less than one molecule in the average LDL particle [41,43]. The PUFA in LDL can be caused to undergo autoxidation in vitro by various free radical initiators, by metals such as copper ions, and by radicals released by cells such as the endothelial cells that line the vascular wall [41]; the initiating species in vivo are not known, but it is thought that endothelial cells are involved in the initiation process. The kinetics of lipid autoxidation suggest that vitamin E should retard or stop the propagation steps involved in lipid autoxidation (and, thus, the rate of buildup of oxidized lipids), but it would not be expected to influence the rate of initiation. The oxidation of the LDL particle triggers a number of events that enhance the likelihood of atherosclerotic plaque development [41,44]. For example, oxidized LDL particles are marked for uptake by macrophages leading to foam cell formation [22,45]. Oxidized LDL also is chemotactic toward monocytes and inhibits macrophage exodus from the artery [46,47], induces endothelial cell damage [20,21], and stimulates cytokine and growth factor release from cells in the artery wall [48,49]. The 1994 review by Keaney and Frei [41] tabulates six studies in which vitamin E was given to humans and the oxidizability of their LDL measured ex vivo; in all six studies, supplementation with vitamin E was found to reduce the oxidizability of the LDL. One study measured LDL uptake by macrophages, which also is required for

Vitamin E and heart disease

143

Table 1. Effects of Vitamin E on the Atherosclerotic Process Target LDL Endothelial and other cells Endothelium Endothelium Smooth muscle cell Platelet Neutrophils Blood cells Monocytes

Function Antioxidant Potentiates arachidonate release and affects prostane production and activity Inhibits cell adhesion Protects nitric oxide and endothelial cell dependent vasoactivity Inhibits proliferation Inhibits adhesion and aggregation Reduces leukotriene synthesis Reduces leukotriene urinary excretion Reduced adhesion, decreased production of oxidants, and altered cytokine expression

foam cell formation [50], and this index too was reduced after vitamin E supplementation [51]. Vitamin E also may limit the progression of atherosclerosis, perhaps by stabilizing plaque and preventing its rupture and subsequent clot formation [52]. This could be an important contributor to the prevention of ischemic heart disease, because plaque types that are the most subject to rupture present the greatest threat [52]. Vitamin E supplementation also leads to a decrease in platelet aggregability; neither vitamin C nor ␤-carotene have this effect [53,54]. This antiplatelet aggregation activity of vitamin E also is seen in patients with diabetes or heart transplants [55,56]. A study that involved supplementation with a mixture of vitamins E and C, ␤-carotene, and selenium found reduced platelet aggregation in men on a high fat diet [57]. Vitamin E also can reduce the expression of adhesion molecules that can cause neutrophils to stick to the endothelial cells lining the artery [58]. The benefits of vitamin E may be particularly pronounced in patients with existing CVD and low dietary antioxidant intake [53]. And, finally, patients with transient ischemic attacks (TIA, a minor stroke resulting from blood clots) had a greater reduction in platelet adhesion on a combination of vitamin E and aspirin than on aspirin alone [59]. Parks et al. [60] measured the oxidizability of LDL from 25 patients with established CVD who were taking part in an atherosclerosis treatment program that involved intensive exercise, stress management, and a very low fat diet with no animal products. The treatment program significantly reduced total cholesterol. Although vitamin E supplements were not provided, the LDL content of ␣-tocopherol and ␤-carotene was increased 27 and 17%, respectively, and the ratio of LDL cholesterol to ␣-tocopherol and ␤-carotene decreased at 3 months. The oxidizability of the LDL also was decreased by the treatment program: the lag phase of LDL oxidation increased by 24% and the maximum rate of oxidation slowed by 29% (p ⫽ .01). The principal determinants of LDL oxidation were the concentrations of ␣-tocopherol and ␤-carotene in the LDL particle [60]. The authors of an important clinical study discussed

Reference [22,39,41,68–72] [73–75] [76–79] [66,80–83] [64,84–87] [54,88,89] [90] [91] [58,92–94]

below, the CHAOS study [24,61], comment that the reduction in risk of CVD may be due to reductions in platelet adhesion and aggregation, inhibition of vitamin K– dependent clotting factors by the vitamin E quinone, stimulation of endothelin production, and/or stimulation of nitric oxide production. In addition, a recent editorial by a member of this group suggests that the patients of CHAOS had a 3.5-fold increased frequency for a polymorphism in the gene for endothelial nitric oxide synthase, a gene that is associated with reduced endothelial function, and vitamin E may protect nitric oxide from destruction, conferring benefits independent of its role in protecting LDL from oxidation [62]. Other studies also have suggested that vitamin E may effect gene transcription and gene products. For example, natural vitamin E, RRR-␣-tocopherol, can affect protein kinase C, leading to changes in gene transcription and the inhibition of cell proliferation [63,64]. A high-fat meal containing triglyceride-rich lipoproteins produces a transient, endothelial cell-dependent decrease in vasoactivity [65]. In an observer-blinded randomized trial, 20 subjects with normal blood lipoproteins were given a high-fat meal, a low-fat meal, or a high-fat meal combined with 800 IU vitamin E and 1000 mg vitamin C [66]. Using ultrasound, flow-mediated vasoactivity was measured hourly for 6 h after the meal. After the high-fat meal, vasoactivity fell significantly (p ⫽ .001). However, no significant changes occurred after the low-fat meal or the high-fat meal with vitamins E and C. Thus, these antioxidant vitamins block the vasoconstrictive effect of the fat [66], suggesting that oxidation products of lipids may be responsible for the effect. Table 1, modified and expanded from the review by Chan [67], lists some of the diverse effects of vitamin E on the atherosclerotic process. Oxidized LDL as a biomarker for CVD If oxidation of LDL is a common theme in CVD, oxidized products from LDL might be a useful biomarker of risk from CVD. In a comparison of 23 men with proven CVD and 23 controls, the CVD patients had LDL

144

W. A. PRYOR

that contained significantly more of the cholesterol oxidation product 4-cholesten-3-one [95]. Also, TBARS levels were higher in the LDL of the patients than the controls. The authors conclude that “. . . LDL from patients with CVD have an elevated oxidized cholesterol content and are more susceptible to peroxidative modification [than are controls]” [95]. In addition, Chisolm et al. [96] have shown that 7-␤-hydroperoxycholest-5-en3-␤-ol is present in plaque and is a cytotoxin in oxidatively modified LDL. Twenty subjects with rapid progression and 20 controls with no progression in their atherosclerosis over 3 years were selected from the more than 400 participants in the Kuopio atherosclerosis prevention study in Finland [97]. Progression of carotid stenosis was assessed by high resolution B-mode ultrasonography. Eight cholesterol oxidation products and TBARS values were determined, along with the oxidizability of LDL. High concentrations of 7-␤-hydroxycholesterol (p ⫽ .0005), cigarette smoking (p ⫽ .0167), and the TBARS values of LDL (p ⫽ .1114) were the strongest predictors of a 3 year increase in carotid wall thickness of the more than 30 variables tested [97]. The authors comment: “These findings . . . provide further evidence to support an association of lipid oxidation and atherogenesis. 7-␤hydroxycholesterol could be a . . . useful marker in assessing this hypothesis” [97]. This group had previously developed an antibody against oxidized LDL and found the antibody titer of MDA-LDL to be an independent predictor of the progression of carotid atherosclerosis [98]. Kim et al. [99] have shown that antibodies raised against oxidatively modified proteins can distinguish occluded arteries from controls. These workers have developed antibodies to human glycodelin-derived peptide that has been oxidized by lipid hydroperoxides. The levels of lipid and lipid-protein–autoxidation products in occluded carotid arteries were compared to the levels found in normal arteries in a double-blind study by Mallat et al. [100]. Higher levels of some acyclic products of lipid peroxidation (e.g., hydroxyeicosatetraenoic acid, HETE’s) were detected in occluded arteries compared with normal arteries, although isoprostanes and some of the other oxy-derivatives were not. Interactions among antioxidants—the “cocktail” approach Because oxidative stress places a burden on the entire cell or organ, it is not surprising that oxidative stress in one cell can affect the redox balance in other cells, or that stress on one organ can affect the oxidative status of the entire animal. Antioxidants reflect this interaction as well. For example, in model experiments done in vitro,

vitamin C can reduce the vitamin E radical (the tocopheryloxyl radical) back to vitamin E, and thus extend the effectiveness of vitamin E [41,101–103]. In the guinea pig, higher dietary vitamin C increased the vitamin E content of the lung at all levels of supplementation with vitamin E [104]. The concept that antioxidants “talk to each other” and interlock in their protective effects, and, therefore, that the levels of all the antioxidants in the network are important, rather than just a single antioxidant, raises the possible importance of supplementing with a group of antioxidants (a “cocktail”) rather than a single antioxidant [41]. Several studies have investigated the effects of combinations of antioxidants on LDL oxidation. Two studies used vitamin E, ␤-carotene, and vitamin C, taken separately or together, and both found that the resistance of LDL to oxidation mainly derived from the vitamin E [44,105]. In contrast, a study by Esterbauer et al. [42] of the sequence in which antioxidants are used up during the oxidation of LDL found that vitamin E is used first, but that significant additional protection for LDL was provided by ␤-carotene. It is possible that the effectiveness of vitamin E is modulated by other dietary antioxidants in the diet that are not generally measured. After a review of dietary and epidemiological data, Gey has commented (p. 591 in ref. [106]): “. . . synergistic antioxidant [interactions] between common plant phenols, such as salicyclic acid, and specific phenols, such as the . . . bioflavonoids, as well as the nonphenolic vitamin C and . . . vitamin E await further exploration . . .” If synergistic interactions among antioxidants are important, then the total diet of the subjects being studied, as well as the vitamin E intake, would be important. The possibility that even greater benefits for vitamin E may be observed if vitamin E is taken with other micronutrients, although very real, may be very difficult to establish using intervention trials. Thus, human clinical intervention trials may lead to an underestimation of the effect of optimum protection of an individual against oxidative stress by the totality of dietary and supplemental antioxidants that they might have ingested. The “correct” dose of vitamin E: primary versus secondary prevention Intakes of vitamin E that are used in intervention studies, 100 to 800 IU/d, are virtually impossible to obtain from food alone. The principal source of vitamin E is plant oils that have high contents of fats, food components that are recommended to be only a small part of a well-designed diet. A recent analysis [107] of the data base from the Third National Health and Nutrition Examination Survey (1988 –1994) found that

Vitamin E and heart disease

“. . . important proportions of US adults have a low serum ␣-tocopherol concentration, which may increase their risk for chronic diseases. . .” It is important to ask, “What is the optimum dose of vitamin E?” However, it is likely that protection against different conditions will have different dose-response curves. If so, then, as has already been pointed out [108], different human clinical trials with different endpoints, may find different daily intakes of vitamin E are enough. In fact, a study in rats has shown that different physiological responses have very different dose-response curves [109]. In particular, normal weight and growth rates are obtained when the rats are fed 7.5 mg/kg vitamin E; a level of 15 mg/kg is adequate to prevent myopathy; 50 mg/kg is necessary for the prevention of red blood hemolysis; however, lymphocyte response to mitogens, a measure of the immune system response, continues to increase at higher intakes of vitamin E and the optimum level of vitamin E is ⬎ 50 mg/kg. In fact, the immune system response correlates with plasma vitamin E levels over the enormous range of 0.04 to 18.0 ␮g/mL [109]. Thus, if different responses have different dose-response curves in humans as well as in rats, one cannot simply ask, “How much vitamin E is enough?” Instead, one must ask, “How much vitamin E is enough to provide maximum protection against a particular health endpoint?” That is, it is possible that optimum protection against one condition, such as a second myocardial infarction (MI) in those who have already proven heart disease, might require a different dose of vitamin E than another condition, such as cancer prevention in a population with normal health, or protection against Alzheimer’s disease [110]. Thus, each trial, with different populations and different end points, may find different intakes of vitamin E are required to provide optimum protection. The problem of varying dose-response curves raises a number of interesting questions, among which is this: “Will the amount of vitamin E required for the secondary prevention of events in patients with already-proven heart disease differ from the amount necessary for the primary prevention of heart disease in the young and healthy?” Because atherosclerosis begins in the very young [111,112], it is tempting to speculate about the possible benefits of prolonged vitamin E supplementation, starting in the young, as a tool in primary prevention of CVD. However, it seems doubtful that this potential benefit could ever be tested in an intervention trial of sufficient length. Several ex vivo LDL oxidation studies have studied the response of LDL oxidation to different dietary intakes of supplemental vitamin E. Esterbauer’s group [113] has reported supplementation of 12 subjects with

145

150, 225, 800, or 1200 IU of RRR-␣-tocopherol (natural source vitamin E). Their results demonstrate that the vitamin E was taken up by LDL in a dose-response manner, and there was a correlation (r2 ⫽ 0.51) between the concentration of vitamin E in the LDL and the lag time for ex vivo, copper-initiated LDL oxidation, demonstrating a dose response [113]. Supplementation with ␣-tocopherol did not change cholesterol plasma concentrations, but it did appear to cause a reduction in ␥-tocopherol [113]. Princen et al. [114] also have reported that in men and women, the vitamin E content of LDL was proportional to the resistance of the LDL to undergoing autoxidation. Jialal et al. [115] gave doses of 60, 200, 400, 800, and 1200 IU/d to groups of humans with eight subjects in each group. Plasma and lipid-standardized vitamin E levels increased with supplementation in a dose-dependent fashion, and vitamin E in LDL appeared to follow the same trend. After 8 weeks of supplementation, there was no difference in LDL oxidation in groups that received placebo or vitamin E at levels of 60 or 200 IU/d, but levels above 400 IU/d did significantly decrease the susceptibility of LDL to undergo oxidation [115]. By some indices of oxidation, supplementation with 800 or 1200 was found to be slightly more effective than 400 IU/d, but by others there was little further improvement in antioxidant protection. (The authors studied the lag phase before oxidation ensues as well as the rate of oxidation, using either TBARS or the formation of conjugated diene-containing lipid hydroperoxides.) The authors [115] conclude that “. . . the minimum dose of vitamin E needed to significantly decrease the susceptibility of LDL to oxidation is 400 IU/d.” SAFETY OF VITAMIN E

There is very substantial consensus among experts that vitamin E is safe at levels up to 800 IU/d and probably safe at levels at least twice that [116 –119]. In a thorough appraisal, Kappus and Diplock [117] review tolerance, toxicological considerations, and the safety of vitamin E. They conclude that there are no side effects up to 800 ␣-tocopherol equivalents (␣-TE), equivalent to about 1200 IU. The therapeutic range is given as 200 to 1600 ␣-TE [117]. Kappus and Diplock state that side effects are only expected to begin at doses of 1000 to 3000 ␣-TE/d (that is, to begin at about 1500 IU/d), and to consist of: “. . . gastrointestinal complaints, creatinuria and impairment of blood coagulation, which are, however, generally not severe and which subside rapidly on reducing the dosage or on discontinuing the administration of vitamin E. . . . Thus, the entire range from the minimal requirement up to a dose of approxi-

146

W. A. PRYOR

mately 3000 mg [of RRR-␣-tocopherol] can be considered as a safe range. There is a risk of adverse effects above intakes of 3000 mg vitamin E per day.”

Thus, the levels of vitamin E consumed as a result of supplementation with 400 or even 800 IU/d is well within the safe limits. Vitamin E does decrease platelet adhesion and at levels of supplementation above 400 IU/d may increase clotting times [120]. Therefore, it may be prudent for those individuals who take anticoagulants to have periodic monitoring of their clotting times. ANIMAL DATA1

In rabbits fed a high fat atherogenic diet, 10 mg/kg natural vitamin E acetate and/or selenium, both vitamin E and selenium separately protected against the fatinduced elevation of plasma TBARS values, and both also protected against the fat-induced formation of atherosclerotic plaque in the aortic intima, although vitamin E and selenium together protected more than either alone [121]. This study supports the wisdom of testing antioxidant cocktails as well as single antioxidants. In a study in Watanabe hyperlipidemic rabbits, 0.5% w/w synthetic vitamin E added to the feed reduced the extent of the ex vivo oxidation of LDL and caused a significant 32% inhibition of surface area atherosclerotic lesion involvement in the arch region as determined by image analysis [122]. In another study [123], New Zealand White rabbits were fed a 1% cholesterol diet and either 1000 or 10,000 IU/kg ␣-tocopheryl acetate (source not specified). After 28 d, endothelial function was significantly impaired in the cholesterol group but preserved in the low-dose vitamin E group [123]. Surprisingly, however, the high-dose vitamin E group demonstrated significantly more intima atherosclerotic proliferation than other groups [123], indicating the wisdom of obtaining dose-response curves. In normal vessels, vitamin E had no effect on endothelial function [123]. The LDL from both vitamin E groups was significantly more resistant to oxidation than was the LDL from controls [123]. Ovulator chickens that are restricted in their movement, and consequently develop hyperlipidemia, were given 1000 IU/kg vitamin E (unspecified source) [124]. The vitamin E–fed chickens maintained their hyperlipidemia, but plasma TBARS values were reduced to those of laying hens; the authors suggest that in these hens 1

When the authors specify the source of the vitamin E used, it is reported here. Unfortunately, some (particularly older) studies do not report whether natural-source (RRR-␣-tocopherol) or synthetic (allrac-␣-tocopherol) was used. Nomenclature generally is reported as used by the original authors, even if it is older and no longer recommended; e.g., natural-source vitamin E is given the older name d-␣tocopherol, synthetic vitamin E is called dl-␣-tocopherol, and units are given as IU rather than the newer ␣-tocopherol equivalents, alpha-TE.

hyperlipidemia without lipid autoxidation does not promote atherogenesis [124]. The aorta intimal layer, measured by microscopy, was significantly thicker in restricted hens, but those fed vitamin E were protected and had intima thickness equal to those of laying hens [124]. In a study by Verlangieri and Bush [125], male monkeys were given 108 IU/d natural vitamin E (about 8-fold their required level), and the animals were monitored by ultrasound for 3 years. Some animals were given the vitamin at the beginning of the experiment, and some were given vitamin E only when ultrasound examination indicated the development of atherosclerosis. Vitamin E was found to significantly inhibit the progression of the disease when given from the beginning of the experiment, and the plasma concentration of vitamin E was proportional to the resistance of the carotid artery to stenosis [125]. This study points out the individual variation in blood levels achieved by the same dose of vitamin E, and therefore points up the need and value of results that include a dose-response study. Interestingly, animals given the vitamin after the development of arterial narrowing showed no further progression of the disease, and then a decrease in the degree of narrowing, much like animals given vitamin E from the beginning of the study [125]. Hypercholesterolemia can lead to enhanced LDL concentrations, increased oxidative stress, lowered control by endogenous nitric oxide, and lowered endothelial cell function. Several studies have shown that vitamin E can reverse some of these events and improve endothelial function. For example, in cholesterol-fed rabbits there is a decrease in aorta relaxation in response to acetyl choline [126]. Giving 50 IU/d synthetic vitamin E by gavage for 2 or 4 d reduced plasma LDL and vessel wall oxidation as measured by TBARS values [126]. After 6 d, an improvement in vessel wall endothelial function was observed [126]. OBSERVATIONAL STUDIES

Descriptive epidemiological studies Before reviewing the data, some caveats need be noted. First, the older epidemiological studies reflect largely dietary, rather than supplementary, vitamin E. The typical vitamin E intake from the diet alone is about 10 IU/d, and some groups ingest less than the USRDA [127]; even a “Mediterranean”-type diet would not provide more than 20 –30 IU/d [1]. Because most of the clinical trials have used supplements of from 200 to 800 IU/d, these two approaches measure very different ends of the dose-response curve. Second, the measurement of micronutrient intakes through the use of dietary recall questionnaires can be imprecise [128]. And finally, ob-

Vitamin E and heart disease

servational correlations provide interesting hypotheses, but they can not probe mechanisms and they can have inherent problems [129]. In the 33 populations in the WHO/MONICA study, the approximately 7-fold difference in heart disease mortality is correlated to only about 20% with classical risk factors (cholesterol, blood pressure, and smoking), and other factors must be involved [130]. Of the 16 populations that had been analyzed, both the blood levels of RRR-␣-tocopherol and the tocopherol/cholesterol ratio inversely correlate with mortality rates, but vitamin C and carotene were not found to be protective [130 –133]. This MONICA study provided some of the first supportive evidence for the antioxidant hypothesis for CVD [130]. Gey, a research worker who contributed many of the earliest epidemiological studies of micronutrients, has commented [134] that the MONICA cross-cultural studies (139 centers in 26 countries), the Edinburgh CaseControl study (6000 males), and the Basel Prospective Study (3000 males) all “. . . consistently reveal an increased risk of ischemic heart disease and stroke for low plasma levels of antioxidants, with the rank order: lipidstandardized vitamin E Ⰷ carotene ⬇ vitamin C ⬎ vitamin A, independent of classical risk factors . . .” In a later review (p. 597 in ref. [106]), Gey again states “. . . all currently available data reconfirm the predominant role of vitamin E . . . CVD mortality in the currently available European study populations is by far more strongly correlated to vitamin E than to the classical risk factors total plasma cholesterol and blood pressure. . .” Case-control studies Gey [106] has reviewed a number of case control studies that used plasma levels of “antioxidants” including ␤-carotene and vitamins A, C, and E. In two studies, angina patients had lower lipid-standardized vitamin E levels than did controls [135,136]. Vitamin C and carotene were also found to be protective, but these relations were decreased when an adjustment for cigarette smoking was made. In a large European case control study, the EURAMIC study, adipose levels of vitamin E and ␤-carotene were measured in samples collected from 1991 to 1992 from 683 subjects with acute MI and 727 controls [137]. The mean difference in ␤-carotene levels was 0.07 ␮g/g lower in cases than controls (95% CI ⫽ 0.04 to 0.10). No protective effect was found for ␣-tocopherol, which the authors suggest is due to the fact that most of the ␣-tocopherol was obtained from the diet and levels were not high enough. However, vitamin E seemed to modify the effect of ␤-carotene: the inverse association of ␤-carotene with MI was the strongest at the highest ␣-tocopherol concentrations [137].

147

Two studies report blood levels of vitamin E are lower in angina patients than controls [138,139]. Kostner et al. [139] studied lipid peroxidation products and vitamin E levels in 50 consecutive patients presenting at a Vienna clinic with stable angina, 50 consecutive patients with unstable angina, and 100 matched controls of clinically healthy individuals. As a measure of oxidative stress in these patients, blood levels of the following parameters were obtained: (i) TBARS, (ii) lipid hydroperoxides, (iii) blood lipids, and (iv) vitamin E. The patients had higher cholesterol and LDL cholesterol and lower HDL cholesterol than controls. Both peroxides and TBARS were higher in the group with unstable angina compared with those with stable angina or controls. Total plasma ␣-tocopherol levels were comparable in all three groups, but the ␣-tocopherol content in LDL was lowest in patients with unstable angina, and next lowest in patients with stable angina, relative to controls. The authors conclude that: “. . . lipid peroxidation products are increased in patients with unstable angina, and . . . these patients show a relative lack of ␣-tocopherol in their LDL. Therefore it might be useful to add antioxidants such as ␣-tocopherol to the drugs that are currently applied in the treatment of unstable angina.”

Prospective cohort studies Prospective cohort studies use dietary questionnaires and self-recalled intake levels of vitamin E, but collect these data before the development of disease, and, therefore, are thought to be less subject to confounding by recall bias and subject selection than are retrospective studies [140]. Two large studies of this type are ongoing at the Harvard School of Public Health [140,141]: the Nurses’ Health Study was started in 1980 and enrolled female nurses free of diagnosed cancer or heart disease [142]; the Health Professionals Study, started in 1986, involved more than 40,000 male health professionals who were free of heart disease and diabetes [143]. The Nurses’ Health Study, the largest study of this type reported to date, involved more than 120,000 nurses, 30 to 35 years old at the beginning of the study in 1976. A cohort of more than 80,000 of these nurses, free of CVD at entry, gave dietary information using a semiquantitative food-frequency questionnaire, and were followed for 8 years [141,142,144 –146]. The analysis was based on 552 cases of nonfatal MI and fatal heart disease events; of these 552 events, 503 (91%) occurred in nonusers of vitamin E supplements and just 49 occurred in users of vitamin E [142]. Women in the highest versus the lowest quintile of vitamin E intake had a relative risk of major coronary disease of 0.66 (95% CI ⫽ 0.50 to 0.87) after adjustment for age, smoking, and other related

148

W. A. PRYOR

factors [142]. Women who took vitamin E supplements for a short time showed little benefit, but those who took them for longer times had a relative risk of major coronary disease of 0.59 (95% CI ⫽ 0.38 to 0.91) after adjustment for age, smoking status, and the use of other vitamin supplements [142]. Those women in the Nurses’ Health Study who obtained their vitamin E from diet alone had a small and statistically nonsignificant reduction in the relative risk for developing CVD [145]. Women in the highest quintile of vitamin E intake from supplement use, and who had used supplements for at least 2 years, had a relative risk of nonfatal MI or death from coronary disease of 0.54 (95% CI ⫽ 0.36 to 0.82) [145]. A protective effect was not found for vitamin C. The relative risk for nonfatal MI or death from CVD for the highest versus the lowest quintile in vitamin C intake was 0.80 (95% CI ⫽ 0.58 to l.l0) [145], but vitamin E and vitamin C intake were highly correlated, and after controlling for vitamin E and multivitamin intake, the correlation for vitamin C became nonsignificant [142]. The second of these related studies, the Health Professionals Follow-Up Study, enrolled 39,910 men 40- to 75-years old at entry, from the total cohort of 51,529, who were free of heart disease, diabetes, and had normal cholesterol levels at entry; these men were followed for 4 years [143]. Again, dietary intakes of vitamin E were found to be strongly, but not significantly correlated with reduced risk for coronary heart disease or death [145]. Men in the highest quintile of vitamin E intake from supplements had a relative risk of nonfatal MI or fatal heart disease events of 0.68, and the effect was largely confined to those who took at least 100 IU/d supplemental vitamin E for at least 2 years [143]. For men taking more than 250 IU/d vitamin E versus nonusers of supplements, the relative risk for nonfatal MI, coronary revascularization, or heart disease death was reduced by 30% (95% CI ⫽ 0.55 to 0.89) [145]. The relative risk for CVD for the highest quintile of vitamin C intake was 1.29; therefore, this study, like the Nurses’ Study described above, did not find that vitamin C was protective. The relative risk for ␤-carotene supplement users was 0.75. The similarity of the relative risk for both women (0.66) and men (0.68) for the supplement users in these two independently run studies with quite different populations leads to increased confidence in the results [142]. Furthermore, the differences between the results for vitamin E and C suggests that self-selection of particularly healthy individuals to use vitamin supplements was not a confounding variable; if self-selection were a confounding variable, those healthy individuals that took vitamin E would have been equally likely to take vitamin C. However, no reduced risk for CVD was

found for persons taking vitamin C supplements. Persons who take vitamins do maintain a healthier lifestyle than do persons who do not take vitamins [147– 149]. However, the protective effects provided by vitamin E supplementation in the two Harvard prospective studies appear to be due to vitamin E itself rather than to a confounding variable [142,143]. The First National Health and Nutrition Examination Survey Longitudinal Study (NHANES-1) followed 11,349 subjects for a median of 10 years [150]. Evaluation of data from this study found a protective effect for vitamin C, but did not attempt to correlate vitamin C and vitamin E or multivitamin supplement usage, factors found important in the two Harvard studies described above [140,146]. Losonczy et al. [151] at the National Institute of Aging used a population from the Established Populations for Epidemiologic Studies of the Elderly (EPESE), which involves four communities in the eastern part of the U.S. In this study, 11,178 persons 67 to 105 years old were interviewed on two occasions 3 years apart and then followed for about 6 years [151]. Persons were defined as users of vitamin E and/or C if they reported using the individual supplements, not part of a multivitamin tablet. End points were all-cause death and death from heart disease. During the follow-up period there were 3490 deaths. The use of vitamin E supplements was associated with a significantly reduced risk for all-cause mortality, with the relative risk being 0.66 (95% CI ⫽ 0.53 to 0.83). The relative risk for death from heart disease for those taking vitamin E was 0.53 (95% CI ⫽ 0.34 to 0.84). The risk was reduced even more for persons who reported taking vitamin E at both of the interviews, suggesting long-term use of vitamin E. Persons reporting the simultaneous use of both vitamin E and C had a relative risk of all-cause mortality of 0.58 (95% CI ⫽ 0.42 to 0.79). The risk for cancer mortality was reduced, but not significantly (relative risk 0.41, 95% CI ⫽ 0.15 to 1.08). Adjustment for other risk factors did not substantially alter these data [151]. In a study by Meyer et al. in Quebec [152], a cohort of 2313 men provided data on vitamin use and CVD risk factors in 1985 and were followed for 5 years. The use of vitamin supplements was consistently associated with a lower incidence of CVD. The adjusted rate ratios and 95% CI were 0.31 (0.09 to 0.99) for death from ischemic heart disease, 0.53 (0.24 to 1.11) for MI, 0.76 (0.44 to 1.65) for angina, and 0.73 (0.44 to 1.22) for a first ischemic heart disease event. The authors review possible confounding variables and specifically exclude “healthy lifestyle” as well as several others as explaining their data. They comment: “The inverse association with ischemic heart disease was more consistent for vitamin E than for any other vitamin” [152].

Vitamin E and heart disease

There also have been studies of subjects that mainly relied on diet for vitamin E [145,153,154]. In a study of 19 European and 5 non-European populations, a strong inverse association was found between dietary vitamin E and CVD; an inverse association also was found for wine consumption but the association with vitamin E was stronger and vitamin E consumption, mainly from vegetable oils, was suggested as a possible explanation of the French paradox [155]. In a 14-year study in Finland, an inverse association was observed between dietary vitamin E and CVD in more than 5000 men and women from 30- to 69-years old; just 3% of this population took vitamin supplements [156]. In the men, those in the highest tertile of vitamin E usage versus the lowest had a relative risk of death from heart disease of 0.66 (p value for trend ⫽ .01); in the women, this relative risk was 0.35 (p value for trend ⬍ .01) [156]. Kushi et al. [157] have reported on the Iowa Women’s Health Study, a prospective study of 34,486 postmenopausal women, ages 55 to 69, who were recruited from the total cohort of 99,826 women and followed for 7 years. The end point of this study was death from heart disease. For this group, an inverse association was found between dietary vitamin E and death from heart disease; no association was found for vitamins A or C. Oddly, however, the vitamin E association was found for those women who obtained their vitamin E from food and did not report using vitamin E supplements [157]. The data show that 21,809 of the women obtained vitamin E from food only, and the remainder, 12,677, obtained the vitamin from both food and supplements. The nature of the supplements used by the women is not described, but the intake of vitamin E by quintiles varied from 0 –25 IU/d to ⬎ 250 IU/d, suggesting that some of the women who took supplements obtained more vitamin E than can be obtained from typical multivitamin capsules. However, no information was collected on the length of time the supplemental vitamins were used [157]. As described above, the Nurses’ Health Study and the Health Professionals Follow-up Study found benefit only for persons taking more than 100 IU/d vitamin E for at least 2 years. Conceivably, the Iowa women had used supplements for less than this period, which would explain the discordant results of this study. For women who obtained their vitamin E from food alone, the relative risk for death from heart disease for the quintile with the highest intake versus the lowest was 0.38 (the 95% CI is 0.18 to 0.80) [157]. No significant association was found between dietary vitamin E and CVD mortality in the 25-year follow-up of the 16 cohorts taken from the Seven Countries Study [158] and in the Nurses’ Health Study and the Allied Health Professionals studies discussed above [142,143]. A dietary study of 747 Massachusetts residents 60 years

149

old and older and 725 controls was conducted, and the subjects then followed for 9 to 12 years. Subjects with plasma vitamin C levels in the highest quintile had significantly reduced risk for total mortality and mortality from heart disease, even after adjustment for potential confounders [159]. Intake of vegetables also was inversely associated with overall mortality and mortality from heart disease. Decreased risk for the highest quintile was found for mortality from heart disease for carotenes and for vitamin E, but neither relation had statistical significance [159]. Studies that use dietary levels of vitamin E are forced to study a very narrow range of vitamin intakes and tissue levels, making correlations difficult [106,160]. In some countries, dietary vitamin E intakes are almost uniform across the cohort, and correlations with other factors are unlikely to be found [106,161]. In addition, plasma levels of vitamin E increase much more slowly than do intake levels, with relatively high intakes (which are not obtainable from diet alone) being required to change plasma levels substantially [1]. Despite these difficulties, Kushi [154], in a recent review of studies that use either the diet or supplements as the source of vitamin E, concludes that “. . . results from these studies are consistent with the theory that vitamin E intake from foods and supplements is associated with decreased risk of coronary heart disease.” Studies that utilize the levels of vitamin E in blood A study of 16 European populations showed a strong inverse correlation between blood levels of vitamin E and the risk of CVD death [106]. However, in a cohort from the MONICA study, a significant correlation was not found between serum vitamin E concentrations and the risk of MI [162]. In a Scottish study, blood levels of vitamin E were inversely related to the risk of angina [135]. In Japan, blood levels of vitamin E were lower in patients with angina than those with other forms of CVD or in healthy controls [163]. In a case-control study reported by Street, Comstock et al. [164], blood was collected in 1974 from a cohort of 25,802 persons. The group was followed for 16 years during which the blood was stored at ⫺70°C, which could have resulted in some loss of antioxidants [164]. At cholesterol levels ⬎ 240 mg/dl, low serum levels of d-␣-tocopherol were associated with increased risk of MI; this was not observed if serum levels of d-␣-tocopherol were above the median level [164]. Singh and colleagues [165] have published a study of an urban Indian population in which plasma levels of vitamins E, C, and ␤-carotene, as well as a number of measures of oxidative stress were measured and related

150

W. A. PRYOR

to the risk of CVD. In a sample of 72 elderly subjects taken from a cohort of 595 Indians 50 to 84 years old, vitamin E levels remained independently and inversely related to the risk of CVD after adjustment for age, smoking, diabetes, blood pressure, and other traditional risk factors; the adjusted relative risk between the lowest (⬍ 11.8 ␮M) and highest (⬎ 19.2 ␮M) quintiles of plasma vitamin E levels was 2.53 (95% CI ⫽ 1.11 to 5.31). This group also has published the results of a small, randomized, placebo-controlled intervention trial in which the effects of vitamin A (50,000 IU/d), vitamin C (1 g/d), vitamin E (400 mg/d), and ␤-carotene (25 mg/d) were given for 28 d to 63 patients with suspected MI and compared with 63 controls given placebo [166]. In the treated group, the mean infarct size was reduced as was serum glutamic-oxaloacetic transaminase. The QRS score in an electrocardiogram also was significantly less in the treated group. Serum TBARS values, a measure of generalized oxidative stress, also were decreased in the treated group [166]. Serum levels of ␣-tocopherol and ␤-carotene were studied in a group of 119 Koreans angiographically diagnosed within the preceding month with CVD and 249 healthy controls [167]. Although ␤-carotene levels were significantly lower in cases than controls, there was no significant difference between the cases and control group for serum ␣-tocopherol levels for either men or women [167]. In this study, both ␣-tocopherol and ␤-carotene derived from the diet [167]. A very thorough review by Gey [106] concludes that in 12 of 16 European study populations the cholesterol levels, blood pressure, and similar classical factors were almost invariant, and, therefore, the 6-fold differences in CVD mortality must arise from other factors. In these populations, in persons with low CVD mortality, the vitamin E plasma levels were between 27 and 30 ␮M and the vitamin E/cholesterol ratio (in ␮M/mM) was almost 5. In contrast, persons with high risk for CVD mortality had vitamin E levels around 20 ␮M and vitamin E/cholesterol ratios of about 3.5. Gey, a prominent research worker in this field, comments that vitamin E was: “. . . clearly in the leading rank [of factors explaining the higher CVD, and is more important than] the moderately strongly correlated vitamin C and the marginally correlated vitamin A . . . In summary, in the major cluster of European populations whose classical risk factors for CVD were very similar, the several-fold differences in CVD mortality could . . . be explained at least to about 60% by differences in the plasma levels of vitamin E and up to 90% by the combination of vitamins E, A, and C” [106]. Gey [106] further concludes that: “. . . all currently available data reconfirm the predominant role of vitamin E . . . The CVD mortality in the currently available European study populations is by far more strongly correlated to vitamin E [levels] than to classical risk factors [such as] total

plasma cholesterol and blood pressure . . .” (pp. 597–599 in ref. [106]).

There are, however, several European population studies that do not find an association between blood levels of vitamin E and endpoints of CVD [106,162,168,169]. Also, a European study found that adipose levels of vitamin E did not correlate with the relative risk for MI [137]. It is very likely that these contradictory results are due to the narrow range of vitamin E intakes and serum and adipose levels available to those whose only source of the vitamin is the diet, as discussed above. Supplementation with ␣-tocopherol has been found to reduce the plasma levels of ␤- and ␥-tocopherol [170]. Gamma-tocopherol (but not ␣) has an unsubstituted position on the phenolic ring that can be attacked by electrophiles, and therefore may [171], or may not [172], provide additional protection against agents such as peroxynitrite. This theory, however, has not been shown to have any relevance to human health. ARTERIAL IMAGING STUDIES

Any biomarker that would be predictive of trial outcomes in shorter times would be extremely useful. For example, and as reviewed above, there has been some effort at establishing LDL oxidation as a biomarker of risk for CVD [173]. The technique of measuring the degree of occlusion of an artery by angiography or the less invasive ultrasound method is a promising approach to determining the protection that various interventions can provide against atherosclerosis [174 –177]. In a study of the role of antioxidants in heart disease risk, Kritchevsky et al. [178] used ultrasound to measure carotid artery intima– media wall thickness in a group of 6318 women and 4989 men from 45 to 64 years old who were enrolled in the Atherosclerosis Risk in Communities Study (ARIC), a program to reduce or reverse the effects of atherosclerosis by dietary and other means. At baseline, 19% of the women and 13% of the men reported taking a multivitamin, 11 and 9% a vitamin C tablet, and 8 and 5% a vitamin E capsule. The ARIC study [178] found an inverse relationship between wall thickness and ␣-tocopherol intake; however, the relationship was significant only in women and was most significant for those over 55. The study, however, was limited because only 5% of the subjects took vitamin E supplements; furthermore, the use of vitamin supplements was asked as part of the dietary questionnaire, but the duration and dose used was not ascertained [178]. A Swedish group [179] found that lipid-adjusted serum and LDL vitamin E concentrations were significantly lower in a cohort of 64 men with a first MI before

Vitamin E and heart disease

the age of 45 than in 35 healthy controls. In addition, a significant inverse correlation was found between the lipid-adjusted LDL vitamin E concentration and a coronary stenosis score (r ⫽ ⫺0.48, p ⬍ .001). The stenosis score was obtained by angiography on the percutaneous transfemoral artery and stenoses graded by means of separate classification systems using 15 segments. Trials involving the direct visualization of arterial status while subjects are on antioxidant supplementation are relatively new; the Cholesterol Lowering Atherosclerosis Study (CLAS) was the first randomized, doubleblind study to follow the progression of atherosclerosis [177]. The intervention involved self-selected vitamin E supplementation in connection with administration of the cholesterol-lowering drugs colestipol and niacin [177, 180 –184]. The authors have studied subjects given or not given the drug combination and who were getting vitamin E only from their diet versus those getting additional vitamin E from supplements. The results have been reported for subjects with occlusions classed as mild-tomoderate lesions or all lesions [177,181,182]. Hodis [177], the lead investigator, summarizes the findings as follows: subjects with vitamin E intakes ⱖ 100 IU/d demonstrated less coronary artery lesion progression (judged as the per-subject change in percent of diameter) than did subjects with ⱕ 100 IU/d, with improvement observed both for the groups taking and not taking the colestipol-niacin drug combination [177,181,182]. Dietary intake of vitamin E (as opposed to vitamin E supplements) was inversely but not significantly associated with progression of CVD [177]. High-resolution ultrasound measurements of the arterial wall intima-media thickness (IMT) can measure the extent of atherosclerosis at early, subclinical stages [174, 177,185–187]. One trial [188] found little relation between antioxidant status and the degree of carotid wall thickening. The EVA trial, a 4-year longitudinal study on cognitive and vascular aging, involves 59 to 71 year-old subjects recruited from the city of Nantes, France [189]. A total of 1389 subjects were recruited and high-resolution ultrasound examination of their carotid arteries was done in 1384 [189]. The levels of vitamin E in the subjects’ red blood cells was significantly associated with less thickening of the arterial wall, after adjustment for normal CVD risk factors. Plasma carotenoid levels, however, were not associated with benefit. In the Kupio Ischemic Heart Disease Study, the relation between vitamin E and ␤-carotene plasma levels and the status of carotid IMT was examined over 12 months in 216 men with high LDL cholesterol levels [190]. After adjustment for other variables, there was a very significant inverse correlation between the progression of carotid artery narrowing and the vitamin E plasma levels as well as those of ␤-carotene [177,190]. The adjusted

151

mean 12-month carotid IMT increase was 78% greater in the lowest vs. the highest quartile of plasma levels of vitamin E [190]. The Multivitamins and Probucol (MVP) trial studied the effects of four treatments on preventing restenosis in 317 patients after coronary artery angioplasty [191]. The four treatments consisted of probucol, a mixture of 700 IU vitamin E, 500 mg vitamin C, and 30,000 IU ␤-carotene, or both probucol and the vitamin mixture, or a placebo. Probucol was found to be effective in reducing the percentage of restenosis, but the vitamin mixture was not [191]. Probucol has been thought to exert its action, at least in part, by being an antioxidant.

CONTROLLED INTERVENTION TRIALS

Vitamin E, vitamin C, and ␤-carotene: all antioxidants? In reviewing studies of clinical trials, many authors group vitamin E, vitamin C, and ␤-carotene together as “antioxidants” [140,177,192]. This may imply for some readers that all three of these micronutrients are similar, and if one “antioxidant” fails to protect us against diseases, then there is less hope for others. In fact, when the two large human trials of the effects of ␤-carotene on lung cancer in smokers (ATBC and CARET) failed to show protection, the authors of some commentaries concluded that the “antioxidant miracle” had been shown to be a false hope and all “antioxidants” were suspect (the ATBC and CARET trials are discussed below). Are vitamin E, vitamin C, and ␤-carotene all relatively similar radical-scavenging antioxidants? As discussed above, vitamin E is the most important lipidsoluble peroxyl radical scavenger in our cells. Acting as an antioxidant clearly is one of the principal roles of vitamin E, and quite possibly the most important. Although vitamin E is an excellent hydrogen atom donor toward free radicals, and particularly toward peroxyl radicals, it does not function as a generalized biological reducing agent, although the phenolate ion may reduce some transition metals in some circumstances. In contrast, vitamin C is an essential cofactor and an important biological reducing agent. Vitamin C is a reducing agent for ferric ion, a critical step in iron-mediated Haber Weiss chemistry [193–196]. An important role for vitamin C is acting as a general reducing agent toward a variety of oxidants, including supplying reducing equivalents in enzymatic reactions. It is true that vitamin C also is one of the important water-soluble antioxidants in our cells [197,198]. However, vitamin C is not unique in this role: there are other water-soluble antioxidants, including uric acid [199 –202] and glutathione [203]. Thus, vitamin C, in contrast with vitamin E, has many other

152

W. A. PRYOR

well-established functions in addition to being an antioxidant and is not unique in its antioxidant role. Both vitamins E and C have an aromatic hydroxyl group that can act as an electron donor. In contrast, ␤-carotene is a hydrocarbon, and, therefore, would be a poor structural choice as a general reducing agent. At least some authors believe that some of the important functions of ␤-carotene probably are not related to its antioxidant properties [204 –208]. For these reasons it appears wiser to review the activities of vitamin E, vitamin C, and the carotenoids independently. There probably is more confusion than enlightenment to be gleaned from comparisons of their activities. Small intervention trials A group of 60 patients, 41 to 70 years old, with angiographically documented coronary spastic angina (CSA), but normal coronary arteries, and 60 controls were studied to determine if vitamin E would relieve CSA [209]. CSA is thought to play a role in variant angina and ischemic heart disease in general, including other forms of angina, acute MI, and sudden death [209]. Patients with CSA were randomized either to the vitamin E group (300 mg/d ␣-tocopherol, source not specified) or placebo. Before treatment, patients with CSA had impaired flow dependent vasodilation, lower plasma levels of ␣-tocopherol, and higher plasma TBARS values compared with matched controls. Treatment with vitamin E restored flow-dependent vasodilation (p ⬍ .001) , and this treatment was associated with reductions in angina attacks and plasma TBARS values [209]. The effects of vitamin E on preserving the vasoactivity of the endothelium was reviewed above and references to this phenomenon are given in Table 1. Four trials on intermittent claudication Three small trials performed in the 1950s to 1970s and the more recent ATBC trial studied the effect of vitamin E on improving poor circulation in the leg resulting from occlusive arterial disease and leading to intermittent claudication. Intermittent claudication, which, like angina, can be quite painful, results from calf muscle ischemia and is a recognized clinical manifestation of atherosclerosis in the lower extremities. The three early trials were secondary prevention trials; they all studied patients who already had intermittent claudication, and they found that vitamin E provided significant benefit [210 –212]. In 1958, Livingstone and Jones [210] reported on 40 nondiabetic males with symptoms for at least 5 years, and a control

group, in a placebo-controlled, double-blind trial in which the subjects were given three 200 mg vitamin E capsules daily for 40 weeks. The authors comment on the difficulty of studies of intermittent claudication, but then conclude: “. . . 13 of . . . 17 patients on vitamin E improved, whereas only 2 of a control group of 17 showed improvement . . . Apparently vitamin E therapy does confer benefit . . .” In 1971, Williams et al. [212] reported on the use of selection criteria to pick a group of 74 patients from a cohort of 138; the patients were followed for 10 years, from 1957 to 1967. The patients were randomized to receive 400 mg vitamin E four times a day, or a placebo, and were divided into three classes, A, B, and C, with class C having the most severe symptoms. Only the subjects in class C were found to improve on vitamin E; these patients at entry exhibited “. . . poor collateral circulation . . . and reduced blood flow to the foot at rest” [212]. Of the group C patients, 10 of the 30 total on vitamin E showed “good” improvement and 9 showed “fair” improvement, whereas for the 15 total patients on placebo, none showed good improvement and only 2 showed fair improvement [212]. In 1974, Haeger [211] followed 47 men with intermittent claudication for 2 to 5 years; the group was divided and one half was given 300 mg d-␣-tocopheryl acetate daily. Interestingly, improvement in arterial flow was delayed until 12–18 months of treatment. After treatment, 29 of 32 patients given vitamin E had significantly improved blood flow; only 3 of 14 control subjects demonstrated improved flow. The ATBC trial selected 26,289 men from the total cohort who were free of intermittent claudication at entry; the first development of symptoms of intermittent claudication during the 4-year follow-up period was used as the end point [213]. The incidence of improvement per 1000 person-years for those on 50 mg synthetic vitamin E daily, 20 mg of ␤-carotene daily, both, or neither was compared. No significant effect on the prevention of intermittent claudication was found for any of the three treatments compared with the placebo group [213]. Possible explanations for the negative results of the ATBC study in comparison with the three smaller trials include: (i) the ATBC trial examined the ability of vitamin E to avoid or delay the first occurrence of intermittent claudication, whereas the three smaller trials examined the effect of vitamin E in alleviating the condition in patients already demonstrating intermittent claudication; and (ii) the ATBC trial used a very much smaller dose of synthetic vitamin E than used in the three smaller trials. At least one, and probably all, of the smaller trials used natural vitamin E. In addition, all the participants in the ATBC study were heavy smokers and smoking is known to place special demands on the total reducing capacity

Vitamin E and heart disease

in the cells and organs of the smokers, with the result that smokers require more vitamin E to achieve comparable blood levels as nonsmokers [214]. Possibly for this reason, at least one of the three small studies excluded smokers from the study. Four large clinical intervention trials examining the effect of vitamin E on CVD There are eight randomized, placebo-controlled, blinded intervention trials that have been reported and that examined the effects on CVD of one or more of the “antioxidant” micronutrients, vitamin E, vitamin C, and/or ␤-carotene. However, only four of these trials involved vitamin E [177,215]. These four are (i) the Alpha Tocopherol Beta Carotene (ATBC) trial that was designed to study cancer in Finnish male heavy smokers; (ii) the Linxian China trial that was designed to study cancers in a population susceptible to esophageal cancer; (iii) Cambridge Heart Antioxidant Study (CHAOS) in England; and (iv) Gruppo Italiano per lo Studio Della Sopravvivenza nell’Infarto Miocardico (GISSI) in Italy. Only the latter two trials, CHAOS and GISSI, had as their primary endpoint the study of the preventive effects of vitamin E on CVD, and both are secondary prevention trials. The CHAOS trial is a secondary prevention trial that enrolled subjects with already-established heart disease. The authors state that: “The primary outcome variables were a combined endpoint of cardiovascular death and nonfatal MI, and nonfatal MI alone” [61]. A total of 2002 subjects with angiographically proven CVD were randomized to receive vitamin E or placebo; the first 546 subjects in the tocopherol group were given 800 IU/d and the remainder were given 400 IU/d, but the two groups were combined for statistical analysis (the trial was not designed to determine a dose-response curve for vitamin E, and both 400 and 800 IU/d significantly increased serum levels above the nonsupplemented levels). After 510 d, those on vitamin E experienced a significant 47% reduction (95% CI ⫽ ⫺66 to ⫺17%; p ⫽ .005) in CVD death and nonfatal MI [61,216]. This effect was due to a very significant 77% reduction (95% CI ⫽ ⫺89 to ⫺53%; p ⫽ .005) in the risk for nonfatal MI. However, there was no effect on CVD death alone. (A nonsignificant 18% increase in CVD death was found in the test group; 95% ⫽ ⫺38 to ⫹127% [61,216].) The nonsignificant increase in deaths due to CVD in this trial has been subjected to a later analysis [216]. The original authors have now published an analysis of the deaths due to heart disease in the subjects that were compliant with the vitamin E regime and the noncompliant group [216]. This analysis shows that of the total of 72 deaths, just 6 were in the group that was compliant

153

with the vitamin E regimen, 21 were in the noncompliant group, and 32 were in the placebo group [216]. The authors comment: “These findings can not . . . lead to different conclusions from an intention-to-treat analysis, which however may have underestimated the true benefit of alpha tocopherol . . . patients currently taking alpha tocopherol (nearly a quarter of all CHAOS patients) accounted for only 6 of the 59 [total] deaths [that were due to] ischemic heart disease . . .” [216]

This subsequent analysis lessens concern about possible dangers of the vitamin E regimen in this group of patients with established CVD. The GISSI trial has just been completed. The GISSI trial is a secondary prevention trial that enrolled 11,324 Italian patients who had survived an MI within the 3-month period before enrollment [217]. The primary endpoints were death, nonfatal MI, and stroke [217]. There were four treatment groups; the four involved 300 mg/d of synthetic vitamin E, or 0.9 g/d of a mixture of n-3 PUFA consisting of a two-to-one ratio of docosahexaenoate (DHA) and eicosapentaenate (EPA) esters, or both vitamin E and the n-3 PUFA mixture, or neither. The study design dispensed with using a placebo, but rather used “open labeling,” and also dispensed with independent monitors at each center [62,217]. In addition, the hypothesis and trial design proposed a 20% benefit, but only a 10% benefit was obtained, so the study did not achieve statistical significance for some observations [62,217]. Even though only a single report had been published at the time this review was written, the published analysis is quite complex, with 55 different relative risks presented [217]. In summary, and for the comparison of each supplement against no treatment, the n-3 PUFA reduced risk for the primary endpoints by 15% and vitamin E by 11%, but only the former value achieved statistical significance (p ⫽ .023) [62,217]. Table 2 presents some of the analysis. The relative risks for most endpoints are similar for vitamin E and the n-3 PUFA, although the 95% CI are greater (and often extend past unity) for vitamin E. However, for stroke, which we will discuss in more detail below, vitamin E shows a (nonsignificant) protective effect whereas PUFA appear to increase risk or have no effect. The lack of agreement between GISSI and CHAOS is troublesome, and an editorial by Brown [62], one of the CHAOS authors, has commented on possible explanations. This editorial [62] notes that: (i) the GISSI subjects presumably ate a “Mediterranean” diet rich in antioxidants, whereas the CHAOS subjects ate an English diet that was poorer in fruits and vegetables; (ii) about 50% of the GISSI subjects were on CVD-preventive drugs, such as the statins. With respect to these facts, Brown comments:

154

W. A. PRYOR Table 2. Relative Risk (and 95% Confidence Intervals) for the GISSI Study [62,217]

Data for n-3 PUFA Death & nonfatal MI & stroke CVD death & nonfatal MI & stroke CVD death and nonfatal MI Fatal & nonfatal stroke Data for vitamin E Death & nonfatal MI & stroke CVD death & nonfatal MI & stroke CVD death and nonfatal MI Fatal & nonfatal stroke

“Whether patients who have an MI despite a lifetime of Mediterranean diet, and are subsequently treated with a statin would be expected to benefit from vitamin E is not clear, especially since many of the complications of MI depend more on the state of the myocardium than of the coronary arteries.” [62]

Brown additionally comments that the CHAOS subjects have been found to have a 3.5-fold increased frequency for a polymorphism in the gene for endothelial nitric oxide synthase, which is associated with reduced endothelial function, and, as reviewed above, vitamin E may provide independent protection toward the nitric oxide– dependent endothelial cell– dependent vascular responsiveness. An additional, perhaps important, difference distinguishes the CHAOS and GISSI trial participants: CHAOS used from 400 to 800 IU of natural vitamin E, whereas GISSI used just 300 mg of synthetic, equivalent [218,219] to about 150 mg of natural-source vitamin E. Thus, the smaller response observed in GISSI relative to CHAOS may be explicable by the pronounced differences in the two populations, East Anglia and Italian, differences in amounts of vitamin E used, different diet and drug profiles, or even possibly different gene pools. The Linxian China study is a primary prevention trial testing the effect of four combinations of micronutrients on overall mortality and cancer mortality [220]. The subjects were 29,584 persons 40 to 69 years old in the Linxian, China, area. The participants were randomized to receive placebo; retinol and zinc; riboflavin and niacin; vitamin C and molybdenum; or synthetic vitamin E (30 IU), ␤-carotene (15 mg), and selenium (50 ␮g). This area of China has one of the world’s highest rates of esophageal/gastric cancer and a low dietary intake of several micronutrients. After a 5.25-year follow-up period, there were a total of 2127 deaths among the trial participants with 32% of all deaths due to esophageal or stomach cancer and 25% due to cerebrovascular disease [220]. Of the four vitamin regimens tested, only the group on vitamin C, vitamin E, and selenium showed

By two-way analysis

By four-way analysis

0.90 (0.82–0.99) 0.89 (0.80–1.01) 0.87 (0.76–0.99) 1.21 (0.91–1.63)

0.85 (0.74–0.98) 0.80 (0.68–0.95) 0.75 (0.62–0.90) 1.30 (0.87–1.96)

0.95 (0.86–1.05) 0.98 (0.87–1.10) 1.00 (0.88–1.14) 0.87 (0.65–1.17)

0.89 (0.77–1.03) 0.88 (0.75–1.04) 0.87 (0.73–1.04) 0.95 (0.61–1.47)

benefits. In this group there was a significant 9% reduction in total mortality; the relative risk of all-cause mortality was 0.91 (95% CI ⫽ 0.84 to 0.99) for the group on the antioxidants. This reduction in total mortality was mainly due to a 13% reduction in cancer rates (relative risk 0.87; 95% CI ⫽ 0.75 to 1.00), especially stomach cancer, which was reduced 13% (95% CI ⫽ 0.64 to 0.99). The reduced risk began to arise about 1 to 2 years after the start of the supplementation with these micronutrients. Notice that a similar 2-year induction period was found before benefit from supplementation with vitamin E was found to be significant in the Nurses’ and the Health Professionals Studies discussed above. There is biological plausibility for an induction period before the benefits of vitamin E are fully evident, because vitamin E must build up in tissues and membranes in which lipids are present. There also was a reduced relative risk for cerebrovascular mortality that was not statistically significant; the relative risk for the group on vitamin E, ␤-carotene, and selenium was 0.90 (95% CI ⫽ 0.76 to 1.07) [220]. Notice that this trial used a small dose of synthetic vitamin E, 30 IU, much smaller than the amount that the Nurses’ Health Study, the Health Professionals Study, or CHAOS intervention trial found to be protective. The ATBC trial was designed to test whether supplementation with either vitamin E or ␤-carotene, or both micronutrients together would reduce the risk of lung and other cancers in heavy smokers [221]. The first analysis of the data was published in 1994 [222]. A group of 29,133 Finnish males who were smokers were given synthetic vitamin E (50 mg), ␤-carotene (20 mg), both, or a placebo. Surprisingly, the ATBC trial found an increased risk for lung cancer, the primary end point of the trial, for smokers who took ␤-carotene rather than a placebo [222]. In 1998, an analysis of prostate cancer risk was published, although prostate cancer was not a prespecified end point [223]. Those subjects on vitamin E experienced a 32% (95% CI ⫽ 47 to 12%) lower risk for

Vitamin E and heart disease

occurrence of prostate cancer and a 41% lower mortality from prostate cancer (95% CI ⫽ 65% to 1%) [223]. An analysis of heart disease in the ATBC trial also was published in 1998 [224]. In 27,271 men with no prior history of MI, and after a median follow-up of 6.1 years, primary major coronary events decreased 4% (95% CI ⫽ ⫺12 to ⫹4%) among recipients of vitamin E and increased 1% (95% CI ⫽ ⫺7 to ⫹10%) among recipients of ␤-carotene compared with nonrecipients. In addition, neither agent affected the incidence of nonfatal MI. Vitamin E decreased the incidence of fatal coronary heart disease by 8% (95% CI ⫽ ⫺19% to ⫹5%) while ␤-carotene had no effect on this end point. Thus, although vitamin E provided a slight protection against ischemic heart disease mortality, a statistically significant benefit was not found for either micronutrient on heart disease [224]. Notice that the ATBC trial, like the Linxain trial, used a small dose of synthetic vitamin E (50 mg). In 1998, an analysis of the prevention of recurrence of angina was published for subjects in the ATBC trial [225]. The relative risk for recurrence of angina in those who took vitamin E alone was 1.06 (95% CI ⫽ 0.85 to 1.33); thus, no significant protective effect for vitamin E was found. The results for ␤-carotene were almost identical to those for vitamin E [225]. In the original 1994 report on the ATBC trial [222], small increases were found for stroke fatalities for subjects on both micronutrients. For hemorrhagic stoke, the number of deaths were: 66 and 44 for those on or not on vitamin E, respectively, and 59 and 51 for those on or not on ␤-carotene [222]. For ischemic stroke, the number of deaths were: 56 and 67 for those on and not on vitamin E, and 68 and 55 for those on or not on ␤-carotene [222]. Thus, this early report found a 50% increase in hemorrhagic stroke mortality for those on vitamin E versus those not on vitamin E (66 vs. 44%) and a 16% increase for those on ␤-carotene (59 vs. 51%). For ischemic stroke there was a 20% protection by vitamin E (56 vs. 67%) but a 19% increase in risk for those on ␤-carotene (68 vs. 55%). Note that the numbers of subjects who suffered a stroke are small relative to the numbers of subjects that developed cancer, the primary end point; e.g., a total of 564 subjects either on or not on ␤-carotene developed lung cancer [222]. DOES VITAMIN E HAVE AN EFFECT ON THE RISK FOR STROKE?

The question of an increased risk of hemorrhagic stroke for persons on vitamin E requires some discussion because of the findings of the ATBC trial. It is doubtful that vitamin E increases the risk for hemorrhagic stroke, for the following reasons:

155

1) While it is frequently stated that vitamin E acts as an anticlotting agent, supplementation at modest levels (up to about 400 IU) of vitamin E has only a mild antiplatelet aggregation effect [226,227]. 2) The total number of stroke victims in the ATBC trial was small, and differences in the treatment groups could result from chance. 3) The CHAOS trial used an 8- to 16-fold higher dose of natural vitamin E compared with the lower dose of synthetic vitamin E (with lower biological activity) used in the ATBC trial. However, the CHAOS study reported no increased risk for hemorrhagic stroke [61,216]. 4) Neither the Nurses’ Health Study [142] nor the Health Professional Study [143] report the observation of a higher risk for hemorrhagic stroke in those participants taking vitamin E supplements. In fact, in the Nurses’ Health Study [142] vitamin E lessened the risk for ischemic stroke, although not statistically significantly; vitamin E supplement users had a relative risk for stroke of 0.71 (95% CI ⫽ 0.39 to 1.31). 5) A cohort of 100 patients with transient ischemic attacks (TIA), minor strokes, or residual ischemic neurologic deficits were enrolled in a 2-year study that compared aspirin alone vs. aspirin plus 400 IU vitamin E [59]. Preliminary results show a significant reduction in the incidence of ischemic events in patients taking vitamin E plus aspirin compared with patients taking aspirin alone, and there was no significant difference in the incidence of hemorrhagic stroke [59]. 6) In the nutritional studies done in Linxian China, there was a reduced relative risk for cerebrovascular mortality, which did not reach statistical significance, for the group given vitamin E, beta-carotene, and selenium [220]. (The relative risk for cerebrovascular mortality was 0.90 (95% CI ⫽ 0.76 to 1.07) [220].) 7) As reviewed above, the GISSI trial found a reduced risk for total stroke for those on vitamin E, although the data did not achieve significance. 8) In the Austrian Stroke Prevention Study [228], 1769 patients, 50 to 75 years of age, with no history of neuropsychiatric disease, were given the Mattis Dementia Rating test and had plasma levels of six carotenoids, retinol, ␣- and ␥-tocopherol, and vitamin C measured. The authors state: “Only ␣-tocopherol remained significantly associated with cognitive functioning when . . . adjust[ment was made] for possible confounders including age, sex, month of blood sampling, years of education, smoking, lipid status, and major risk factors for stroke (p ⫽ .019).” This study primarily involved patients who obtained antioxidants from food, and the

156

W. A. PRYOR

range of plasma concentrations was small. However, similar results have been reported in other studies quoted by these authors [228]. 9) The cohort studied in Linxian China trial also was utilized to ask if various combinations of nutritional supplements can be used to lower the risk of stroke or hypertension [229]. Participants were randomly assigned to a placebo group or one of seven groups that were given different combinations of micronutrients. All seven of the groups showed some lowering of stroke mortality, although for most the differences did not achieve statistical significance [229]. The reduction in risk for stroke was found in the groups at high risk (ages ⬎ 60 and with systolic blood pressure ⬎ 160 mm); high blood pressure is the best-established risk factor for stroke [229]. For most nutrients, the protection did not differ based on age or blood pressure, but the group given both “factors A and B” (vitamin E, zinc, riboflavin, and niacin) showed a relative risk of 0.47 (95% CI ⫽ 0.26 to 0.86) for high-risk subjects. This study, therefore, serves to lessen possible concern over an increase in hemorrhagic stroke for persons taking supplemental vitamin E. In fact, as the authors [229] state: “Only the lipid soluble antioxidants (called “factor D” and consisting of vitamin E, ␤-carotene, and selenium) had a measurable impact on stroke mortality (relative risk ⫽ 0.91, 95% CI ⫽ 0.84 – 0.99) as well as other causes of death (although) . . . factor C [vitamin C and molybdenum] had a small beneficial effect on . . . hypertension.”

The reduced risk from ischemic and/or hemorrhagic stroke from vitamin E must be confirmed by a larger, longer-term study, preferably one that can provide doseresponse data. It also should be recognized that hemorrhagic stroke is very much less common than ischemic heart disease [230]. Thus, populations (such as those with proven atherosclerosis) might derive an overall benefit from vitamin E supplementation if hemorrhagic stroke rates were only very slightly increased but significant reductions in ischemic heart disease, including ischemic stroke, occurred. FURTHER THOUGHTS AND CONCLUSIONS

We have reviewed the enormous number of studies on the effects of vitamin E on heart disease, studies encompassing basic science, animal studies, epidemiologicalal data, and human intervention trials. The mass of the data leaves little doubt that vitamin E provides significant protection both to those without diagnosed heart conditions and to those with proven heart disease. Both the lay public and much of the medical community has already

reached this conclusion. About one half of American cardiologists take supplemental vitamin E, about the same number as take aspirin [25]. A substantial fraction of the lay public also takes vitamin supplements [150]. Before reaching conclusions, however, several comments should be made. An important limitation of the clinical trials we have reviewed is that just a single dose is tested and a particular population is used. Thus, none of the clinical intervention trials have yet provided doseresponse data, and, because of the expense involved, we may never have such data. Also, trials have not yet fully distinguished between persons who are “responders”and those who are “nonresponders,” where responders derive a greater benefit from supplemental vitamin E than nonresponders for a given condition. The recent editorial by Brown [62], which points out that some of the subjects in the CHAOS trial had a 3.5-fold increased frequency for a polymorphism in the gene for endothelial nitric oxide synthase, is the most recent reminder that individuals may differ in their responses to vitamin E, and that particularly susceptible subgroups may exist. Traber and coworkers [231,232] find that some subjects discriminate less well than normal controls between the natural isomer, RRR-␣-tocopherol, and synthetic vitamin E. Subgroup analyses may be very important. Aspirin appears to be particularly helpful in reducing the risk of CVD in men who have higher vascular inflammation, as predicted by higher plasma levels of high-sensitivity–Creactive protein [233]. Inflammatory processes produce a high flux of the superoxide radical, which leads to a cascade of potent oxidants such as the hydroxyl radical, peroxynitrite, and hypochlorite. Therefore, vitamin E might be found to be particularly effective against an inflammatory component of atherosclerotic plaque development, or perhaps more effective in those subjects with higher levels of vascular inflammation. Interestingly, aspirin and vitamin E appear to work synergistically against the risk of CVD [59,234]. In the future, it may be possible to identify particular classes of individuals who require vitamin E supplementation more immediately and can be expected to derive greater-than-usual benefit. However, at present, there is little reason not to supplement the general population, because the side reactions and toxicity of vitamin E, at levels used in these trials, is known to be very low. Some have argued [140,145,235–238] that it would be wise to wait for the conclusion of the intervention trials now in progress before allowing a public recommendation for supplementation with vitamin E. It is true that there are a large number of trials of various types in progress. Table 3, which is modified from a version prepared by Dr. Howard Hodis, lists a number of such trials. However, despite the trials in progress, there are ar-

Vitamin E and heart disease

157

Table 3. Randomized, Placebo-controlled, Double-blind, Clinical Trials Now Underway that Involve Vitamin E Name of trial Primary prevention trials Physicians’ Health Study II (PHS II)

Cohort

Agents used

Primary end point(s)

15,000 US physicians recruited to date (recruiting is ongoing)

on alternate days: vitamin E: 400 IU ␤-carotene; 50 mg daily: vitamin C: 500 mg/d RDA-style multivitamin

MI, stroke, and CVD death

Women;s Health Study (WHS)

40,000 US women

on alternate days: vitamin E: 600 IU aspirin: 100 mg

MI, stroke, and CVD death

Supplementation en Vitamines et Mineraux Antioxidants trial (SU.VI.M.AX)

15,000 French men and women

vitamin E: 15 IU/d ␤-carotene: 6 mg/d vitamin C: 120 mg/d selenium: 0.1 mg/d zinc: 20 mg/d

MI, stroke, and CVD death

20,000 U.K. men and women with CVD or diabetes

Vitamin E: 600 IU/d ␤-carotene: 20 mg/d vitamin C: 250 mg/d simvastatin

MI, stroke, and CVD death

Heart Outcomes Prevention Evaluation (HOPE) (Part of this study has been completed, but the results have not yet published; see the Note added in proof.

9,500 Canadian men and women with CVD or diabetes

Vitamin E: 400 IU/d ramipril: 10 mg

MI, stroke, and CVD death

Women’s Antioxidant Cardiovascular Study (WACS)

8,000 US women with CVD or ⱖ3 risk factors

On alternate days: vitamin E: 600 IU ␤-carotene: 50 mg Given every day: vitamin C: 500 mg/d folic acid: 2.5 mg/d vitamin B6: 50 mg/d vitamin B12: 1 mg

MI, stroke, revascularization, CVD death

353 healthy men and women

Vitamin E: 400 IU/d

Progression of carotid artery intima-media thickness followed by ultrasound

Study to Evaluate Carotid Ultrasound Changes in Patients Treated with Ramipril and Vitamin E (SECURE)

700 men and women with CVD

Vitamin E: 400 IU/d ramipril

Progression of carotid artery lesions followed by ultrasound

Women’s Antioxidant Vitamin Estrogen Trial (WAVE)

400 postmenopausal women with CVD

Twice a day: vitamin E: 400 IU vitamin C: 500 mg Premarin in women with hysterectomy and Prempro in women without

Progression of CVD by quantitative coronary angiography

HDL Atherosclerosis Treatment Study (HATS)

160 men and women with CVD and low HDL

Twice a day: vitamin E: 400 IU vitamin C: 500 mg selenium: 50 ␮g nicotinic acid

Progression of CVD by quantitative coronary angiography

St. Michael’s Atherosclerosis Regression Trial with Fenofibrate and Vitamin E in Diabetics (SMARTFED)

150 men and women with diabetes and dyslipidemia

Vitamin E: 400 IU/d

Progression of CVD by quantitative coronary angiography

Munich Coronary Bypass Intervention Trial (MCBIT)

600 men and women who have had bypass surgery

Vitamin E: 800 IU/d lovastatin

Progression of CVD by quantitative coronary angiography

Secondary prevention trials Heart Protection Study (HPS)

Arterial imaging studies Vitamin E Atherosclerosis Prevention Study (VEAPS)

158

W. A. PRYOR

guments for beginning supplementation with vitamin E now. First, vitamin E at the supplemental levels being used in the current trials, 100 to 800 IU/d, is established as safe, and there is little likelihood that increased risk will be found for those taking supplements. Second, the data already in hand are quite compelling, and postponing the benefits of supplementation seems unnecessarily cautious. And last, as has been argued here, it is likely that each condition for which vitamin E provides benefit will have a unique dose-effect curve. Furthermore, different antioxidants appear to act synergistically, so supplementation with vitamin E might be more effective if combined with other micronutrients. It will be extremely difficult, and probably impossible, to do trials that adequately probe the dose-effect curve for vitamin E for each condition that it might affect, or to do studies of all the possible combinations of other micronutrients that might act with vitamin E to improve its effectiveness. For these reasons there never will be a time when the science is “complete.” At some point, the weight of the scientific evidence will have to be judged adequate. It is this reviewer’s opinion, in view of the very low risk of supplementation with vitamin E and the difficulty in obtaining more than about 15–30 IU/d from a balanced diet, that there now is sufficient evidence to recommend modest vitamin E supplementation (100 to 400 IU/d) as part of a general program of heart-healthy behavior that includes a fruit- and vegetable-rich diet and regular exercise. Acknowledgements — Work in my laboratory on vitamin E has been supported in part by grants from the National Institutes of Health and by the Vitamin E Research and Information Service (VERIS). I thank Dr. Howard Hodis for allowing me to read a preprint of his review of human trial data on antioxidants, and Drs. Julie E. Buring and Howard Hodis for correspondence on the use of nomenclature. I also thank the following persons who read an earlier draft of this paper and offered valuable comments: Dr. Jeffery Blumberg, Dr. James P. Clark, Dr. Balz Frei, Dr. Howard Hodis, Dr. Meir Stampfer, Dr. Daniel Steinberg, Dr. Walter Willett, and two insightful, anonymous referees. Note added to the galley proofs — The results from the GISSI trial, as discussed above, are complex, with 55 relative risks presented. After this article went to press, seven letters were published back-to-back in Lancet that offer further analysis of the GISSI trial. Jialal et al. [239] comment: “. . . the investigators’ conclusion that ␣-tocopherol was without benefit is inappropriate and misleading. . . . a careful analysis of their findings reveals that ␣-tocopherol supplementation resulted in the following significant effects when the . . . appropriate four-way analysis was undertaken: 20% reduction in cardiovascular deaths, 23% reduction in cardiac death, 25% reduction in coronary death, 35% reduction in sudden death . . .” These authors conclude: “Thus, the GISSI results are in accord with the published work on the potential beneficial effects of ␣-tocopherol therapy in the secondary prevention of CVD.” Salen et al. [240] comment in particular on sudden vs. nonsudden cardiac deaths. They conclude: “Cardiovascular mortality was significantly reduced by vitamin E in GISSI and the effect on overall survival showed a very favorable trend . . .” Ng et al. [241] similarly comment:

“There is a trend towards a benefit with vitamin E in 3.5 years’ follow-up.” The other letters, including that by Hopper et al. [242] and the reply by the GISSI authors [243] also should be read. The complexities revealed by these letters suggest that fuller discussions of some of the points raised can be expected in the literature over the coming years. The HOPE trial, which is listed in Table 3 as ongoing, still has not been published. However, some of the results were presented at the European Society of Cardiology in Barcelona in August 1999 and the American Heart Association meeting in Atlanta in November 1999, and some results have been posted electronically on the Internet, which also announces that formal publication in the New England Journal of Medicine will occur early in 2000. This trial, as described in Table 3, is a secondary trial involving 9297 high-risk Canadian men and women, 55 years and older, who were given 400 IU/d vitamin E and/or 10 mg/d ramipril, an angiotensin-converting enzyme (ACE) inhibitor. The results indicate that ramipril appears to be strongly protective against the primary end points (MI, stroke, or CVD death). The results for vitamin E did not achieve statistical significance in the 4 years of the study, and the vitamin E portion of this study is to be continued for a longer period. The complexities described both in the text and in this Note regarding the GISSI trial make it clear that analysis of the HOPE trial on the basis of the electronically-presented data would be premature. The work of Stocker and his collaborators on tocopherol-mediated peroxidation (TMP) of LDL in vitro was not reviewed here, because it is complex and a number of authors believe TMP does not occur in vivo. However, readers may wish to examine a recent publication from that group [244] that presents the theory as well as a review of the animal data on LDL oxidation.

REFERENCES [1] Machlin, L. J., ed. Vitamin E: a comprehensive treatise. New York: Marcel Dekker; 1980. [2] Lubin, B.; Machlin, L. J., eds. Vitamin E: biochemical, hematological, and clinical aspects. New York: New York Academy of Sciences; 1982. [3] Diplock, A. T.; Machlin, L. J.; Packer, L.; Pryor, W. A., eds. Vitamin E: biochemistry and health implications. New York, NY: New York Academy of Sciences; 1989. [4] Sauberlich, H. E.; Machlin, L. J., eds. Beyond deficiency: new views on the function and health effects of vitamins. New York, NY: New York Academy of Sciences; 1992. [5] Packer, L.; Fuchs, J., eds. Vitamin E in health and disease. New York, NY: Marcel Decker, Inc.; 1993. [6] Frei, B., ed. Natural antioxidants in human health and disease. San Diego, CA: Academic Press; 1994. [7] Burton, G. W.; Joyce, A.; Ingold, K. U. Is vitamin E the only lipid-soluble, chain-breaking antioxidant in human blood plasma and erythrocyte membranes? Arch. Biochem. Biophys. 221:281– 290; 1983. [8] Pryor, W. A. Free radicals in autoxidation and in aging. Part I. Kinetics of the autoxidation of linoleic acid in SDS micelles: calculations of radical concentrations, kinetic chain lengths, and the effects of vitamin E. Part II. The role of radicals in chronic human diseases and in aging. In: Armstrong, D.; Sohal, R. S.; Cutler, R. G.;Slater, T. F., eds. Free radicals in molecular biology, aging and disease. New York, NY: Raven Press; 1984: 13– 41. [9] Tasinato, A.; Boscoboinik, D.; Bartoli, G.-M.; Maroni, P.; Azzi, A. ␣-Tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations, correlates with protein kinase C inhibition, and is independent of its antioxidant properties. Proc. Natl. Acad. Sci. USA 92:12190 –12194; 1995. [10] Ricciarelli, R.; Tasinato, A.; Clement, S.; Ozer, N. K.; Boscoboinik, D.; Azzi, A. Alpha-tocopherol specifically inactivates cellular protein kinase C alpha by changing its phosphorylation state. Biochem. J. 334:243–249; 1998. [11] Cachia, O.; El Benna, J.; Pedruzzi, E.; Descomps, B.; GougerotPocidalo, M.-A.; Leger, C.-L. Alpha-tocopherol inhibits the re-

Vitamin E and heart disease

[12]

[13] [14]

[15]

[16]

[17] [18] [19] [20]

[21]

[22]

[23] [24] [25] [26] [27]

[28]

[29] [30]

[31]

[32]

[33] [34]

[35]

spiratory burst in human monocytes. Attenuation of Pruphox membrane translocation and phosphorylation. J. Biol. Chem. 273:32801–32805; 1998. Infante, J. P. A function for the vitamin E metabolite ␣-tocopherol quinone as an essential enzyme cofactor for the mitochondrial fatty acid desaturases. FEBS Lett. 446:1–5; 1999. Prasad, K. N.; Cole, W. C., eds. Cancer and nutrition. Amsterdam: IOS Press; 1998. Pryor, W. A. Cancer and free radicals. In: Shankel, D.; Hartman, P.; Kada, T.; Hollaender, A., eds. Antimutagenesis and anticarcinogenesis mechanisms. New York, NY: Plenum Press; 1986: 45–59. Pryor, W. A. Cigarette smoke and the involvement of free radical reactions in chemical carcinogenesis. Br. J. Cancer 55(Suppl. VIII):19 –23; 1987. Weisburger, J. H. Nutritional approach to cancer prevention with emphasis on vitamins, antioxidants, and carotenoids. Am. J. Clin. Nutr. 53(Suppl.):226S–237S; 1991. Shamberger, R. J. Antioxidants and malonaldehyde in cancer. ACS Symposium Series 277:111–118; 1985. Wattenberg, L. W. Chemoprevention of cancer. Cancer Res. 45:1– 8; 1985. Ames, B. N. Micronutrients prevent cancer and delay aging. Toxicol. Lett. 102–103:5–18; 1998. Morel, D. W.; DiCorleto, P. E.; Chisolm, G. M. Endothelial and smooth muscle cells alter low density lipoprotein in vitro by free radical oxidation. Arteriosclerosis 4:357–364; 1984. Cathcart, M. K.; Morel, D. W.; Chisolm, G. M. Monocytes and neutrophils oxidize low– density lipoproteins making it cytotoxic. J. Leukoc. Biol. 38:341–350; 1985. Steinbrecher, U. P.; Parthasarthy, S.; Leake, D. S.; Witztum, J. L.; Steinberg, D. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids. Proc. Natl. Acad. Sci. USA 81:3883–3887; 1984. Steinberg, D.; Witztum, J. L. Lipoproteins and atherogenesis: current concepts. J. Am. Med. Assoc. 264:3047–3052; 1990. Mitchinson, M. J. The new face of atherosclerosis. Br. J. Clin. Pharmacol. 48:149 –151; 1994. Mehta, J. Intake of antioxidants among American cardiologists. Am. J. Cardiol. 79:1558 –1560; 1997. Harman, D. Atherosclerosis: a hypothesis concerning the initiating steps in pathogenesis. J. Gerontol. 12:199 –202; 1957. Harman, D. The free-radical theory of aging. In: Pryor, W. A., eds. Free radicals in biology, Vol. V. New York, NY: Academic Press; 1982:255–275. Pryor, W. A. The free-radical theory of aging revisited: a critique and a suggested disease-specific theory. In: Warner, H. R.; Butler, R. N.;Sprott, R. L., eds. Modern biological theories of aging. New York, NY: Raven Press; 1987:89 –112. Sokol, R. J. Vitamin E deficiency and neurologic disease. Ann. Rev. Nutr. 8:351–373; 1988. Sohal, R. S.; Allen, R. G. Relationship between metabolic rate, free radicals, differentiation and aging: a unified theory. In: Woodhead, A. D.; Blackett, A. D.;Hollaender, A., eds. Molecular biology of aging. New York: Plenum Publishing Corporation; 1985:75–104. Sohal, R. S.; Sohal, B. H.; Orr, W. C. Mitochondrial superoxide and hydrogen peroxide generation, protein oxidative damage, and longevity in different species of flies. Free Radic. Biol. Med. 19:499 –504; 1995. Agarwal, S.; Sohal, R. S. DNA oxidative damage and life expectancy in houseflies. Proc. Natl. Acad. Sci. USA 91:12332– 12334; 1994. Dawson, V. L.; Dawson, T. M. Free radicals and neuronal cell death. Cell Death Differ. 3:71–78; 1996. Lyras, L.; Evans, P. J.; Shaw, P. J.; Ince, P. G.; Halliwell, B. Oxidative damage and motor neuron disease difficulties in the measurement of protein carbonyls in human brain tissue. Free Radic. Res. 24:397– 406; 1996. Lohr, J. B.; Cadet, J. L.; Lohr, M. A.; Larson, L.; Wasli, E.;

[36]

[37]

[38]

[39]

[40] [41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

159

Wade, L.; Hylton, R.; Vidoni, C.; Jeste, D. V.; Wyatt, R. J. Vitamin E in the treatment of Tardive dyskinesia: the possible involvement of free radical mechanisms. Schizophr. Bull. 14: 291–296; 1988. Meydani, M.; Macauley, J. B.; Blumberg, J. B. Effect of dietary vitamin E and selenium on susceptibility of brain regions to lipid peroxidation. Lipids 23:405– 409; 1988. Pryor, W. A. Mechanisms of radical formation from reactions of ozone with target molecules in the lung. Free Radic. Biol. Med. 5:451– 465; 1994. Squadrito, G. L.; Pryor, W. A. Oxidative chemistry of nitric oxide: the roles of superoxide, peroxynitrite, and carbon dioxide. Free Radic. Biol. Med. 25:392– 403; 1998. Steinberg, D.; Parthasarthy, S.; Carew, T. E.; Khoo, J. C.; Witztum, J. L. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N. Engl. J. Med. 320:915–924; 1989. Steinberg, D. Oxidative modification of LDL and atherogenesis. Circulation 95:1062–1071; 1997. Keaney, J. F., Jr.; Frei, B. Antioxidant protection of low density lipoprotein and its role in the prevention of atherosclerotic vascular disease. In: Frei, B., ed. Natural antioxidants in human health and disease. San Diego, CA: Academic Press; 1994:303– 351. Esterbauer, H.; Dieber-Rothender, M.; Striegl, G.; Waeg, G. Role of vitamin E in preventing the oxidation of low– density lipoprotein. Am. J. Clin. Nutr. 53(Suppl.):314S–321S; 1991. Esterbauer, H.; Gebicki, J.; Puhl, H.; Jurgens, G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic. Biol. Med. 13:341–390; 1992. Reaven, P. D.; Khouw, A.; Beltz, W. F.; Parthasarthy, S.; Witztum, J. L. Effect of dietary antioxidant combinations in humans: protection of LDL by vitamin E but not by ␤-carotene. Arterioscler. Thromb. 13:590 – 600; 1993. Henriksen, T.; Mahoney, E. M.; Steinberg, D. Enhanced macrophage degradation of low density lipoprotein previously incubated with cultured endothelial cells: Recognition by the receptor for acetylated low density lipoproteins. Proc. Natl. Acad. Sci. USA 78:6499 – 6503; 1981. Quinn, M. T.; Parthasarathy, S.; Steinberg, D. Endothelial cell derived chemotactic activity for mouse peritoneal macrophages and the effects of modified forms of low density lipoprotein. Proc. Natl. Acad. Sci. USA 82:5949 –5953; 1985. Quinn, M. T.; Parthasarthy, S.; Fong, L. G.; Steinberg, D. Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocyte/macrophages during atherogenesis. Proc. Natl. Acad. Sci. USA 84:2995–2998; 1987. Liao, F.; Berliner, J. A.; Mehrabian, A. J.; Navab, M.; Demer, L. L.; Lusis, A. J.; Fogelman, A. M. Minimally modified lowdensity lipoprotein is biologically active in vivo in mice. J. Clin. Invest. 87:2253–2257; 1991. Cushing, S. D.; Berliner, J. A.; Valente, A. J.; Territo, M. C.; Navab, M.; Parhami, F.; Gerrity, R.; Schwartz, C. J.; Fogelman, A. M. Minimally modified low-density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells. Proc. Natl. Acad. Sci. USA 87:5134 –5138; 1990. Sparrow, C. P.; Parthasarthy, S.; Steinberg, D. A macrophage receptor that recognizes oxidized low density lipoprotein but not acetylated low density lipoprotein. J. Biol. Chem. 264:2599 – 2604; 1989. Harats, D.; Ben-Naim, M.; Dabach, Y.; Hollander, G.; Havivi, E.; Stein, O. Effect of vitamin C and E supplementation on susceptibility of plasma lipoproteins to peroxidation induced by acute smoking. Atherosclerosis 85:47–54; 1990. Diaz, M. N.; Frei, B.; Vita, J. A.; Keaney, J. F., Jr. Antioxidants and atheroslcerotic heart disease. N. Engl. J. Med. 337:408 – 416; 1997. Calzada, C.; Bruckdorfer, K. R.; Rice-Evans, C. A. The influence of antioxidant nutrients on platelet function in healthy volunteers. Atherosclerosis 128:97–105; 1997.

160

W. A. PRYOR

[54] Mower, R.; Steiner, M. Synthetic byproducts of tocopherol oxidation as inhibitors of platelet function. Prostaglandins 24: 137–147; 1982. [55] Colette, C.; Pares-Herbute, N.; Monnier, L. H.; Cartry, E. Platelet function in type I diabetes: effects of supplementation with large doses of vitamin E. Am. J. Clin. Nutr. 47:256 –261; 1988. [56] De Lorgeril, M.; Boissonnat, P.; Salen, P.; Monjaud, I.; Monnez, C.; Guidollet, J.; Ferrera, R.; Dureau, G.; Ninet, J.; Renaud, S. The beneficial effect of dietary antioxidant supplementation on platelet aggregation and cyclosporine treatment in heart transplant recipients. Transplantation 58:193–194; 1994. [57] Salonen, J. T.; Salonen, R.; Seppanen, K.; Rinta-Kikka, S.; Kuukka, M.; Korpela, H.; Alfthan, G.; Kantola, M.; Schalch, W. Effects of antioxidant supplementation on platelet function: a randomized pair-matched, placebo-controlled, double-blind trial in men with low antioxidant status. Am. J. Clin. Nutr. 53:1222– 1229; 1991. [58] Yoshikawa, T.; Yoshida, N.; Manabe, H.; Terasawa, Y.; Takemura, T.; Kondo, M. alpha-Tocopherol protects against expression of adhesion molecules on neutrophils and endothelial cells. BioFactors 7:15–19; 1998. [59] Steiner, M.; Glantz, M.; Lekos, A. Vitamin E plus aspirin compared with aspirin alone in patients with transient ischemic attacks. Am. J. Clin. Nutr. 62(Suppl.):1381S–1384S; 1995. [60] Parks, E. J.; German, J. B.; Davis, P. A.; Frankel, E. N.; Kappagoda, C. T.; Rutledge, J. C.; Hyson, D. A.; Schneeman, B. Reduced oxidative susceptibility of LDL from patients participating in an intensive atherosclerosis treatment program. Am. J. Clin. Nutr. 68:778 –785; 1998. [61] Stephens, N. G.; Parsons, A.; Schofield, P. M.; Kelly, F.; Cheeseman, K.; Mitchinson, M. J.; Brown, M. J. Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge heart antioxidant study (CHAOS). Lancet 347:781– 786; 1996. [62] Brown, M. Do vitamin E and fish oil protect against ischaemic heart disease? Lancet 354:441– 442; 1999. [63] Azzi, A. M.; Bartoli, G.; Boscoboinik, D.; Hensey, C.; Szewczyk, A. ␣-Tocopherol and protein kinase C regulation of intracellular signaling. In: Packer, L.; Fuchs, J., eds. Vitamin E in health and disease. New York: Marcel Dekker, Inc.; 1993: 371–383. ¨ zer, N. K.; [64] Azzi, A.; Aratri, E.; Boscoboinik, D.; Cle´ment, S.; O Ricciarelli, R.; Spycher, S. Molecular basis of alpha-tocopherol control of smooth muscle cell proliferation. BioFactors 7:3–14; 1998. [65] Vogel, R. A.; Corretti, M. C.; Plotnick, G. D. Effect of a single high-fat meal on endothelial function in healthy subjects. Am. J. Cardiol. 79:350 –354; 1997. [66] Plotnick, G. D.; Corretii, M. C.; Vogel, R. A. Effect of antioxidant vitamins on the transient impairment of endotheliumdependent brachial artery vasoactivity following a single highfat meal. J. Am. Med. Assoc. 278:1682–1686; 1997. [67] Chan, A. C. Vitamin E and atherosclerosis. J. Nutr. 128:1593– 1596; 1998. [68] Mitchinson, M. J. Macrophages, oxidised lipids and atherosclerosis. Med. Hypotheses 12:171–178; 1983. [69] Hessler, J. R.; Morel, D. W.; James, L. J.; Chisolm, G. M. Lipoprotein oxidation and lipoprotein-induced cytotoxicity. Arteriosclerosis 3:215–222; 1983. [70] Morel, D. W.; Catheart, M. K.; Chisolm, G. M. Cytotoxicity of low density lipoprotein oxidized by cell generated free radicals. J. Cell Biol. 97:427A; abstr. 1983 [71] Ju¨rgens, G.; Hoff, H. F.; Chisolm, G. M., III; Esterbauer, H. Modification of human serum low density lipoprotein by oxidation— characterization and pathophysiological implications. Chem. Phys. Lipids 45:315–336; 1987. [72] Cohn, W.; Loechleiter, F.; Weber, F. ␣-Tocopherol is secreted from rat liver in very low density lipoproteins. J. Lipid Res. 29:1359 –1366; 1988. [73] Tran, K.; Chan, A. C. R,R,R-␣-tocopherol potentiates prostacyclin release in human endothelial cells. Evidence for structural

[74]

[75]

[76]

[77]

[78] [79]

[80]

[81]

[82] [83] [84]

[85]

[86]

[87]

[88]

[89] [90]

[91]

[92]

specificity of the tocopherol molecule. Biochim. Biophys. Acta 1043:189 –197; 1990. Tran, K.; Wong, J. T.; Lee, E.; Chan, A. C.; Choy, P. C. Vitamin E potentiates arachidonate release and phospholipase A2 activity in rat heart. Biochem. J. 319:385–391; 1996. Thorin, E.; Hamilton, C. A.; Dominiczak, M. H.; Reid, J. L. Chronic exposure of cultured bovine endothelial cells to oxidized LDL abolishes prostacyclin release. Arterioscler. Thromb. 14:453– 459; 1994. Faruqi, R.; de la Motte, C.; DiCorleto, P. E. ␣-Tocopherol inhibits agonist-induced monocytic cell adhesion to cultured human endothelial cells. J. Clin. Invest. 94:592– 600; 1994. Cominacini, L.; Garbin, U.; Pasini, A. F.; Davoli, A.; Campagnola, M.; Contessi, G. B.; Pastorino, A. M.; Cascio, V. L. Antioxidants inhibit the expression of intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 induced by oxidized LDL on human umbilical vein endothelial cells. Free Radic. Biol. Med. 22:117–127; 1997. Ross, R. The pathogenesis of atherosclerosis. Nature 362:801– 809; 1993. Islam, K. N.; Devaraj, S.; Jialal, I. ␣-Tocopherol enrichment of monocytes decreases agonist-induced adhesion to human endothelial cells. Circulation 98:2255–2261; 1998. Bo¨ger, R. H.; Bode-Bo¨ger, S. M.; Phivthong-ngam, L.; Brandes, R. P.; Schwedhelm, E.; Mu¨gge, A.; Bo¨hme, M.; Tsikas, D.; Fro¨lich, J. C. Dietary L-arginine and ␣-tocopherol reduce vascular oxidative stress and preserve endothelial function in hypercholesterolemic rabbits via different mechanisms. Atherosclerosis 141:31– 43; 1998. Schwarzacher, S. P.; Hutchison, S.; Chou, T. M.; Sun, Y.-P.; Zhu, B.-Q.; Chatterjee, K.; Glantz, S. A.; Deedwania, P. C.; Parmley, W. W.; Sudhir, K. Antioxidant diet preserves endothelium-dependent vasodilatation in resistance arteries of hypercholesterolemic rabbits exposed to environmental tobacco smoke. J. Cardiovasc. Pharmacol. 31:649 – 653; 1998. Shimokawa, H. Primary endothelial dysfunction: atherosclerosis. J. Mol. Cell. Cardiol. 31:23–37; 1999. Drexler, H.; Hornig, B. Endothelial dysfunction in human disease. J. Mol. Cell. Cardiol. 31:51– 60; 1999. ¨ zer, N. K.; Sta¨uble, Azzi, A.; Boscoboinik, D.; Marilley, D.; O B.; Tasinato, A. Vitamin E: a sensor and an information transducer of the cell oxidation state. Am. J. Clin. Nutr. 62:1337S– 1346S; 1995. ¨ zer, N. K.; Boscoboinik, E. D.; Azzi, A. New roles of low O density lipoproteins and vitamin in the pathogenesis of atherosclerosis. Biochem. Mol. Biol. Int. 35:117–124; 1995. Boscoboinik, D.; Szewczyk, A.; Hensey, C.; Azzi, A. Inhibition of cell proliferation by ␣-tocopherol. Role of protein kinase C. J. Biol. Chem. 266:6188 – 6194; 1991. Chatelain, E.; Boscoboinik, D. O.; Bartoli, G.-M.; Kagan, V. E.; Gey, F. K.; Packer, L.; Azzi, A. Inhibition of smooth muscle cell proliferation and protein kinase C activity by tocopherols and tocotrienols. Biochim. Biophys. Acta 1176:83– 89; 1993. Freedman, J. E.; Farhat, J. H.; Loscalzo, J.; Keaney, J. F., Jr. ␣-Tocopherol inhibits aggregation of human platelets by a protein kinase C-dependent mechanism. Circulation 94: 2434 – 2440; 1996. Jandak, J.; Steiner, M.; Richardson, P. D. ␣-Tocopherol, an effective inhibitor of platelet adhesion. Blood 73:141–149; 1989. Chan, A. C.; Tran, K.; Pyke, D. D.; Powell, W. S. Effects of dietary vitamin E on the biosynthesis of 5-lipoxygenase products by rat polymorphonuclear leukocytes (PMNL). Biochim. Biophys. Acta 1005:265–269; 1989. Denzlinger, C.; Kless, T.; Sagebiel-Kohler, S.; Lemmen, C.; Jacob, K.; Wilmanns, W.; Adam, O. Modulation of the endogenous leukotriene production by fish oil and vitamin E. J. Lipid Mediat. Cell Signal. 11:119 –132; 1995. Devaraj, S.; Li, D.; Jialal, I. The effects of alpha tocopherol supplementation on monocyte function. Decreased lipid oxidation, interleukin 1␤ secretion and monocyte adhesion to endothelium. J. Clin. Invest. 98:756 –763; 1996.

Vitamin E and heart disease [93] Martin, A.; Foxall, T.; Blumberg, J. B.; Meydani, M. Vitamin E inhibits low density lipoprotein induced adhesion of monocytes to human aortic endothelial cells. Arterioscler. Thromb. Vasc. Biol. 17:429 – 436; 1997. [94] Molenaar, R.; Visser, W. J.; Verkerk, A.; Koster, J. F.; Jongkind, J. F. Peroxidative stress and in vitro ageing of endothelial cells increases the monocyte-endothelial cell adherence in a human in vitro system. Atherosclerosis 76:193–202; 1989. [95] Liu, K.; Cuddy, T. E.; Pierce, G. N. Oxidative status of lipoproteins in coronary disease patients. Am. Heart J. 123:285–290; 1992. [96] Chisolm, G. M.; Ma, G.; Irwin, K. C.; Martin, L. L.; Gunderson, K. G.; Linberg, L. F.; Morel, D. W.; DiCorleto, P. E. 7-␤Hydroperoxycholest-5-en-3␤ol, a component of human atherosclerotic lesions, is the primary cytotoxin of oxidized human low density lipoprotein. Proc. Natl. Acad. Sci. USA 91: 11452– 11456; 1994. [97] Salonen, J. T.; Nyysso¨nen, K.; Salonen, R.; Porkkala-Sarataho, E.; Tuomainen, T.-P.; Diczfalusy, U.; Bjo¨rkhem, I. Lipoprotein oxidation and progression of carotid atherosclerosis. Circulation 95:840 – 845; 1997. [98] Salonen, J. T.; Yla¨-Herttuala, S.; Yamamoto, R.; Butler, S.; Korpela, H.; Salonen, R.; Nyyssonen, K.; Plainski, W.; Witztum, J. Autoantibody against oxidized LDL and progression of carotid atherosclerosis. Lancet 339:883– 887; 1992. [99] Kim, J. G.; Taylor, W. R.; Parthasarathy, S. Demonstration of the presence of lipid peroxide-modified proteins in human atherosclerotic lesions using a novel lipid peroxide-modified antipeptide antibody. Atherosclerosis 143:335–340; 1999. [100] Mallat, Z.; Nakamura, T.; Ohan, J.; Leseche, G.; Tedgui, A.; Maclouf, J.; Murphy, R. C. The relationship of hydoxyeicosatetraenoic acids and F2-isoprostanes to plaque instability in human carotid atherosclerosis. J. Clin. Invest. 103:421– 427; 1999. [101] May, J. M.; Qu, Z.-C.; Morrow, J. D. Interaction of ascorbate and ␣-tocopherol in resealed human erythrocyte ghosts. Transmembrane electron transfer and protection from lipid peroxidation. J. Biol. Chem. 271:10577–10582; 1996. [102] Packer, J. E.; Slater, T. F.; Wilson, R. W. Direct observation of a free radical interaction between vitamin E and vitamin C. Nature 278:737–738; 1979. [103] Frei, B. Ascorbic acid protects lipids in human plasma and low-density lipoprotein against oxidative damage. Am. J. Clin. Nutr. 54:1113S–1118S; 1991. [104] Bendich, A.; D’Apolito, P.; Gabriel, E.; Machlin, L. J. Interaction of dietary vitamin C and vitamin E on guinea pig immune responses to mitogens. J. Nutr. 114:1588 –1593; 1984. [105] Abbey, M.; Nestel, P. J.; Baghurst, P. A. Antioxidant vitamins and low-density-lipoprotein oxidation. Am. J. Clin. Nutr. 58: 525–532; 1993. [106] Gey, K. F. Vitamin E and other essential antioxidants regarding coronary heart disease: risk assessment studies. Epidemiological basis of the antioxidant hypothesis of cardiovascular disease. In: Packer, L.; Fuchs, J., eds. Vitamin E in health and disease. New York, NY: Marcel Dekker, Inc.; 1993:589 – 633. [107] Ford, E. S.; Sowell, A. Serum alpha-tocopherol status in the United States population: findings from the third national health and nutrition examination survey. Am. J. Epidemiol. 150:290 – 300; 1999. [108] Pryor, W. A. The antioxidant nutrients and disease prevention–– what do we know and what do we need to find out? Am. J. Clin. Nutr. 53(Suppl.):391S–393S; 1991. [109] Bendich, A.; Gabriel, E.; Machlin, L. J. Dietary vitamin E requirement for optimum immune responses in the rat. J. Nutr. 116:675– 681; 1986. [110] Morris, M. C.; Beckett, L. A.; Scherr, P. A.; Hebert, L. E.; Bennett, D. A.; Field, T. S.; Evans, D. A. Vitamin E and vitamin C supplement use and risk of incident alzheimer disease. Alzheimer Dis Assoc Disord 12:121–126; 1998. [111] Berenson, G. S.; Srinivasan, S. R.; Bao, W.; Newman, W. P., III; Tracy, R. E.; Wattigney, W. A. Association between multiple

[112]

[113]

[114]

[115]

[116] [117]

[118]

[119]

[120] [121]

[122]

[123]

[124]

[125]

[126]

[127] [128]

[129] [130]

[131]

161

cardiovascular risk factors and atherosclerosis in children and young adults. N. Engl. J. Med. 338:1650 –1656; 1998. Strong, J. P.; Malcom, G. T.; McMahan, C. A.; Tracy, R. E.; Newman, W. P., III; Herderick, E. E.; Cornhill, J. F. Prevalence and extent of atherosclerosis in adolescents and young adults. J. Am. Med. Assoc. 281:727–735; 1999. Dieber-Rothender, M.; Puhl, H.; Waeg, G.; Striegl, G.; Esterbauer, H. Effect of oral supplementation with D-alpha-tocopherol on the vitamin E content of human low density lipoproteins and resistance to oxidation. J. Lipid Res. 32:1325–1332; 1991. Princen, H. M. G.; van Duyvenvoorde, W.; Buytenhek, R.; van der Laarse, A.; van Poppel, G.; Leuven, J. A. G.; van Hinsbergh, V. W. M. Supplementation with low doses of vitamin E protects LDL from lipid peroxidation in men and women. Arterioscler. Thromb. Vasc. Biol. 15:325–333; 1995. Jialal, I.; Fuller, C. J.; Huet, B. A. The effect of ␣-tocopherol supplementation on LDL oxidation. A dose-response study. Atheroscler. Thromb. Vasc. Biol. 15:190 –198; 1995. Bendich, A.; Machlin, L. J. Safety of oral intake of vitamin E. Am. J. Clin. Nutr. 48:612– 619; 1988. Kappus, H.; Diplock, A. T. Tolerance and safety on vitamin E: a toxicological position report. Free Radic. Biol. Med. 13:55–74; 1992. Meydani, S. N.; Meydani, M.; Blumberg, J. B.; Leka, L. S.; Pedrosa, M.; Diamond, R.; Schaefer, E. J. Assessment of the safety of supplementation with different amounts of vitamin E in healthy older adults. Am. J. Clin. Nutr. 68:311–318; 1998. Bendich, A.; Machlin, L. J. The safety of oral intake of vitamin E: Data from clinical studies from 1986 to 1991. In: Packer, L.; Fuchs, J., eds. Vitamin E in health and disease. New York: Marcel Dekker, Inc.; 1993:411– 416. Kim, J. M.; White, R. H. Effect of vitamin E on the anticoagulant response to warfarin. Am. J. Cardiol. 77:545–546; 1996. Wo´jcicki, J.; Ro´zewicka, L.; Barcew-Wiszniewska, B.; Samochowiec, L.; Juzwiak, S.; Kadlubowska, D.; Tustanowski, S.; Juzyszyyn, Z. Effect of selenium and vitamin E on the development of experimental atherosclerosis in rabbits. Atherosclerosis 87:9 –16; 1991. Williams, R. J.; Motteram, J. M.; Sharp, C. H.; Gallagher, P. J. Dietary vitamin E and the attenuation of early lesion development in modified Watanabe rabbits. Atherosclerosis 94:153– 159; 1992. Keaney, J. F., Jr.; Gaziano, J. M.; Xu, A. I.; Frei, B.; Currancelentano, J.; Shwaery, G. T.; Loscalzo, J.; Vita, J. A. Lowdose a-tocopherol improves and high-dose ␣-tocopherol worsens endothelial vasodilator function in cholesterol-fed rabbits. J. Clin. Invest. 93:844 – 851; 1994. Smith, T. L.; Kummerow, F. A. Effect of dietary vitamin E on plasma lipids and atherogenesis in restricted ovulator chickens. Atherosclerosis 75:105–109; 1989. Verlangieri, A. J.; Bush, M. J. Effects of d-␣-tocopherol supplementation on experimentally induced primate atherosclerosis. J. Am. Coll. Nutr. 11:131–138; 1992. Jorge, P. A. R.; Neyra, L. C.; Ozaki, R. M.; de Almeida, E. Improvement in the endothelium-dependent relaxation in hypercholesterolemic rabbits treated with vitamin E. Atherosclerosis 140:333–339; 1998. Rosenberg, I. H. Vitamin needs of older Americans. BackGrounder 7:1–11; 1999. Armstrong, B. K.; Mann, J. I.; Adelstein, A. M.; Eskin, F. Commodity consumption and ischemic heart disease mortality, with special reference to dietary practices. J. Chron. Dis. 28: 455– 469; 1975. Willett, W. Nutritional epidemiology. New York: Oxford University Press; 1998. Gey, K. F.; Puska, P.; Jordan, P.; Moser, U. K. Inverse correlation between plasma vitamin E and mortality from ischemic heart disease in cross-cultural epidemiology. Am. J. Clin. Nutr. 53:326S–334S; 1991. Grey, K. F.; Brubacher, G. B.; Sta¨helin, H. B. Plasma levels of

162

[132]

[133]

[134]

[135]

[136]

[137]

[138]

[139]

[140] [141] [142]

[143]

[144]

[145] [146] [147]

[148]

[149]

[150]

[151]

W. A. PRYOR antioxidant vitamins in relation to ischemic heart disease and cancer. Am. J. Clin. Nutr. 45:1368 –1377; 1987. Gey, K. F.; Puska, P. Plasma vitamins E and A inversely correlated to mortality from ischemic heart disease in crosscultural epidemiology. Ann. NY. Acad. Sci. 570:268 –282; 1989. Gey, K. F.; Stahelin, H. B.; Puska, P.; Evans, A. Relationship of plasma vitamin C to mortality from ischemic heart disease. Ann. N. Y. Acad. Sci. 498:110 –123; 1987. Gey, K. F.; Moser, U. K.; Jordan, P.; Sta¨helin, H. B.; Eichholzer, M.; Lu¨din, E. Increased risk of cardiovascular disease at suboptimal plasma concentrations of essential antioxidants: an epidemiologicalal update with special attention to carotene and vitamin C. Am. J. Clin. Nutr. 57(Suppl.):787S–797S; 1993. Riemersma, R. A.; Wood, D. A.; Macintyre, C. C. A.; Elton, R. A.; Gey, K. F.; Oliver, M. F. Risk of angina pectoris and plasma concentrations of vitamins A, C, and E and carotene. Lancet 337:1–5; 1991. Riemersma, R. A.; Wood, D. A.; Macintyre, C. C. A.; Elton, R.; Gey, K. F.; Oliver, M. F. Low plasma vitamins E and C: increased risk of angina in Scottish men. Ann. NY. Acad. Sci. 570:291–295; 1989. Kardinaal, A. F. M.; Kok, F. J.; Ringstad, J.; Gomez-Aracena, J.; Mazaev, V. P.; Kohlmeier, L.; Martin, B. C.; Aro, A.; Kark, J. D.; Delgado-Rodriguez, M.; Riemersma, R. A.; van’t Veer, P.; Huttunen, J. K.; Martin-Moreno, J. M. Antioxidants in adipose tissue and risk of myocardial infarction: the EURAMIC study. Lancet 342:1379 –1384; 1993. Sklodowska, M.; Wasowicz, W.; Gromadzinska, J.; Miroslaw, W.; Strzelczyk, M.; Malczyk, J.; Goch, J. H. Selenium and vitamin E concentrations in plasma and erythrocytes of angina pectoris patients. Trace Elem. Med. 8:113–117; 1991. Kostner, K.; Hornykewycz, S.; Yang, P.; Neunteufl, T.; Glogar, D.; Weidinger, F.; Maurer, G.; Huber, K. Is oxidative stress causally linked to unstable angina pectoris? A study in 100 CAD patients and matched controls. Cardiovasc. Res. 36:330 –336; 1997. Gaziano, J. M. Antioxidant vitamins and cardiovascular disease. Proc. Assoc. Am. Physicians 111:2–9; 1999. Rimm, E. B. Vitamin E and cardiovascular disease. VERIS 7:1–10; 1999. Stampfer, M. J.; Hennekens, C. H.; Manson, J. E.; Colditz, G. A.; Rosner, B.; Willett, W. C. Vitamin E consumption and the risk of coronary disease in women. N. Engl. J. Med. 328:1444 – 1449; 1993. Rimm, E. B.; Stampfer, M. J.; Ascherio, A.; Giovannucci, E.; Colditz, G. A.; Willett, W. C. Vitamin E consumption and the risk of coronary heart disease in men. N. Engl. J. Med. 328: 1450 –1456; 1993. Manson, J. E.; Stampfer, M. J.; Willett, W. C. A prospective study of vitamin C and incidence of coronary heart disease in women. Circulation 85:8651992. Rexrode, K. M.; Manson, J. E. Antioxidants and coronary heart disease: observational studies. J. Cardiovasc. Risk 3:363–367; 1996. Buring, J. E.; Hennekens, C. H. Antioxidant vitamins and cardiovascular disease. Nutr. Rev. 55:S53–S601997. Gartside, P. S.; Wang, P.; Glueck, C. J. Prospective assessment of coronary heart disease risk factors: the NHANES I Epidmiologic Follow-up Study (NHEFS) 16-year follow-up. J. Am. Coll. Nutr. 17:263–269; 1998. Houstion, D. K.; Johnson, M. A.; Daniel, T. D.; Poon, L. W. Health and dietary characteristics of supplement users in an elderly population. Int. J. Vit. Nutr. Res. 67:183–191; 1997. Slesinski, M. J.; Subar, A. F.; Kahle, L. L. Dietary intake of fat, fiber and other nutrients is related to the use of vitamin and mineral supplements in the United States: the 1992 National Health Interview Survey. J. Nutr. 126:3001–3008; 1996. Enstrom, J. E.; Kanim, L. E.; Klein, M. A. Vitamin C intake and mortality among a sample of the United States population. Epidemiology 3:194 –202; 1992. Losonczy, K. G.; Harris, T. B.; Havlik, R. J. Vitamin E and vitamin C supplement use and risk of all-cause and coronary heart disease mortality in older persons: the established popula-

[152]

[153]

[154] [155]

[156]

[157]

[158]

[159]

[160]

[161]

[162]

[163] [164]

[165]

[166]

[167]

[168]

[169]

tions for epidemiological studies of the elderly. Am. J. Clin. Nutr. 64:190 –196; 1996. Meyer, F.; Bairati, I.; Dagenais, G. R. Lower ischemic heart disease incidence and mortality among vitamin supplement users. Can. J. Cardiol. 12:930 –934; 1996. Moushumi, L.; Sumati, B. Studies on possible protective effect of plant derived phenols and the vitamin precursors— beta-carotene and alpha-tocopherol on 7,12-dimethylbenz(a)anthracene induced tumour initiation events. Phytother. Res. 4:237–240; 1994. Kushi, L. H. Vitamin E and heart disease: a case study. Am. J. Clin. Nutr. 69:1322S–1329S; 1999. Bellizzi, M. C.; Franklin, M. F.; Duthie, G. G.; James, W. P. T. Vitamin E and coronary heart disease: the European paradox. Eur. J. Clin. Nutr. 48:822– 831; 1994. Knekt, P.; Reunanen, A.; Ja¨rvinen, R.; Seppa¨nen, R.; Helio¨vaara, M.; Aromaa, A. Antioxidant vitamin intake and coronary mortality in a longitudinal population study. Am. J. Epidemiol. 139:1180 –1189; 1994. Kushi, L. H.; Folsom, A. R.; Prineas, R. J.; Mink, P. J.; Wu, Y.; Bostick, R. M. Dietary antioxidant vitamins and death from coronary heart disease in postmenopausal women. N. Engl. J. Med. 334:1156 –1162; 1996. Kromhout, D.; Bloemberg, B. P. M.; Feskens, E. J. M.; Hertog, M. G. L.; Menotti, A.; Blackburn, H. Alcohol, fish, fibre and antioxidant vitamins intake do not explain population differences in coronary heart disease mortality. Int. J. Epidemiol. 25:753–759; 1996. Sahyoun, N. R.; Jacques, P. F.; Russell, R. M. Carotenoids, vitamins C and E, and mortality in an elderly population. Am. J. Epidemiol. 144:501–511; 1996. Willett, W. C.; Stampfer, M. J.; Underwood, B. A.; Speizer, F. E.; Rosner, B.; Hennekens, C. H. Validation of a dietary questionnaire with plasma carotenoid and ␣-tocopherol levels. Am. J. Clin. Nutr. 38:631– 639; 1983. Gey, K. F.; Stahelin, H. B.; 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. 71:3– 6; 1993. Hense, H. W.; Stender, M.; Bors, W.; Keil, U. Lack of an association between serum vitamin E and myocardial infarction in a population with high vitamin E levels. Atherosclerosis 103:21–28; 1993. Miwa, K.; Miyagi, Y.; Igawa, A.; Nakagawa, K.; Inoue, H. Vitamin E deficiency in variant angina. Circulation 94:14 –18; 1996. Street, D. A.; Comstock, G. W.; Salkeld, R. M.; Schuep, W.; Klag, M. J. Serum antioxidants and myocardial infarction. Are low levels of carotenoids and alpha-tocopherol risk factors for myocardial infarction? Circulation 90:1154 –1161; 1994. Singh, R. B.; Ghosh, S.; Niaz, M. A.; Singh, R.; Beegum, R.; Chibo, H.; Shoumin, Z.; Postiglione, A. Dietary intake, plasma levels of antioxidant vitamins, and oxidative stress in relation to coronary artery disease in elderly subjects. Am. J. Cardiol. 76:1233–1238; 1995. Singh, R. B.; Niaz, M. A.; Rastogi, S. S.; Rostogi, S. Usefulness of antioxidant vitamins in suspected acute myocardial infarction (the Indian experiment of infarct survival-3). Am. J. Cardiol. 77:232–236; 1996. Kim, S. Y.; Lee-Kimm, Y. C.; Kim, M. K.; Suh, J. Y.; Chung, E. J.; Cho, S. Y.; Cho, B. K.; Suh, I. Serum levels of antioxidant vitamins in relation to coronary artery disease: a case control study of Koreans. Biomed. Environ. Sci. 9:229 –235; 1996. Kok, F. J.; de Bruijin, A. M.; Vermeeren, R.; Hofman, A.; van Laar, A.; de Bruin, M.; Hermus, R. J. J.; Valkenburg, H. A. Serum selenium, vitamin antioxidants, and cardiovascular mortality: a 9-year follow-up study in the Netherlands. Am. J. Clin. Nutr. 45:462– 468; 1987. Salonen, J. T.; Salonen, R.; Penttila¨, I.; Herranen, J.; Jauhiainen, M.; Kantola, M.; Lappetela¨inen, R.; Ma¨enpa¨a¨, P. H.; Alfthan, G.; Puska, P. Serum fatty acids, apolipoproteins, selenium and vitamin antioxidants and the risk of death from coronary artery disease. Am. J. Cardiol. 56:226 –231; 1985.

Vitamin E and heart disease [170] Melchert, H.-U.; Pabel, E. The tocopherol pattern in human serum is markedly influenced by intake of vitamin E drugs— results of the German national health surveys. J. Am. Oil Chem. Soc. 75:213–216; 1998. [171] Christen, S.; Woodall, A. A.; Shigenaga, M. K.; SouthwellKeely, P. T.; Duncan, M. W.; Ames, B. N. gamma-Tocopherol traps mutagenic electrophiles such as NOx and complements ␣-tocopherol: physiological implications. Proc. Natl. Acad. Sci. USA 94:3217–3222; 1997. [172] Goss, S. P. A.; Hogg, N.; Kalyanaraman, B. The effect of ␣-tocopherol on the nitration of ␥-tocopherol by peroxynitrite. Arch. Biochem. Biophys. 363:333–340; 1999. [173] Holvoet, P. Role of oxidatively modified low density lipoproteins and anti-oxidants in atherothrombosis. Expert Opin. Invest. Drugs 8:527–544; 1999. [174] Hodis, H. N.; Mack, W. J.; LaBree, L.; Selzer, R. H.; Liu, C.-R.; Liu, C.-H.; Azen, S. P. The role of carotid arterial intima-media thickness in predicting clinical coronary events. Ann. Intern. Med. 128:262–269; 1998. [175] Salonen, J. T.; Salonen, R. Ultrasound B-mode imaging in observational studies of atherosclerotic progression. Circulation 87 (Suppl II):II-56 –II-65; 1993. [176] Pignoli, P.; Tremoli, E.; Poli, A.; Oreste, P.; Paoletti, R. Intimal plus medial thickness of the arterial wall: a direct measurement with ultrasound imaging. Circulation 74:1399 –1406; 1986. [177] Hodis, H. N. Antioxidant vitamins and atherosclerosis. In press; 2000. [178] Kritchevsky, S. B.; Shimakawa, T.; Tell, G. S.; Dennis, B.; Carpenter, M.; Eckfeldt, J. H.; Peacher-Ryan, H.; Heiss, G. Dietary antioxidants and carotid artery wall thickness. The ARIC study. Circulation 92:2142–2150; 1995. [179] Rengstro¨m, J.; Nilsson, J.; Moldeus, P.; Stro¨m, K.; Båvenholm, P.; Tornvall, P.; Hamsten, A. Inverse relation between the concentration of low-density-lipoprotein vitamin E and severity of coronary artery disease. Am. J. Clin. Nutr. 63:377–385; 1996. [180] Blankenhorn, D. H.; Johnson, R. L.; Nessim, S. A.; Azen, S. P.; Sanmarco, M. E.; Selzer, R. H. The cholesterol lowering atherosclerosis study (CLAS): design, methods, and baseline results. Controlled Clin. Trials 8:354 –387; 1987. [181] Hodis, H. N.; Mack, W. J.; LaBree, L.; Cashin-Hemphill, L.; Sevanian, A.; Johnson, R.; Azen, S. P. Serial coronary angiographic evidence that antioxidant vitamin intake reduces progression of coronary artery atherosclerosis. J. Am. Med. Assoc. 273:1849 –1854; 1995. [182] Azen, S. P.; Qian, D.; Mack, W. J.; Sevanian, A.; Selzer, R. H.; Liu, C.-R.; Liu, C.-H.; Hodis, H. N. Effect of supplementary antioxidant vitamin intake on carotid arterial wall intima-media thickness in a controlled clinical trial of cholesterol lowering. Circulation 94:2369 –2372; 1996. [183] Blankenhorn, D. H.; Nessim, S. A.; Johnson, R. L.; Sanmarco, M. E.; Azen, S. P.; Cashin-Hemphill, L. Beneficial effects of combined colestipol-niacin therapy on coronary atherosclerosis and coronary venous bypass grafts. J. Am. Med. Assoc. 257: 3233–3240; 1987. [184] Blankenhorn, D. H.; Selzer, R. H.; Crawford, D. W.; Barth, J. D.; Liu, C. R.; Liu, C. H.; Mack, W. J.; Alaupovic, P. Beneficial effects of colestipol-niacin therapy on the common carotid artery: two- and four-year reduction of intima-media thickness measured by ultrasound. Circulation 88:20 –28; 1993. [185] Mack, W. J.; Hodis, H. N.; LaBree, L.; Liu, C. R.; Liu, C. H.; Selzer, R. H. Progression of carotid intima-media thickness correlated with angiographic progression of coronary disease. Circulation 94:I-122; 1996 (abstr.). [186] Cashin-Hemphill, L.; Mack, W. J.; Pogoda, J.; Sanmarco, M. E.; Azen, S. P.; Blankenhorn, D. H. Beneficial effects of colestipolniacin on coronary atherosclerosis: a 4-year follow-up. J. Am. Med. Assoc. 264:3013–3017; 1990. [187] Selzer, R. H.; Hodis, H. N.; Kwong–Fu, H.; Mack, W. J.; Lee, P. L.; Liu, C.-R.; Liu, C.-H. Evaluation of computerized edge tracking for quantifying intima-media thickness of the common carotid artery from B-mode ultrasound images. Atherosclerosis 111:1–11; 1994.

163

[188] Kritchevsky, S. B.; Shimakawa, T.; Dennis, B.; Eckfeldt, J.; Carpenter, M.; Heiss, G. Dietary antioxidants and carotid artery wall thickness: the ARIC study. Circulation 87:679; 1993. [189] Bonithon-Kopp, C.; Coudray, C.; Berr, C.; Touboul, P.-J.; Fe`ve, J. M.; Favier, A.; Ducimetie`re, P. Combined effects of lipid peroxidation and antioxidant status on carotid atherosclerosis in a population aged 59 –71 y: the EVA study. Am. J. Clin. Nutr. 65:121–127; 1997. [190] Salonen, J. T.; Nyysso¨nen, K.; Parviainen, M.; Kantola, M.; Korpela, H.; Salonen, R. Low plasma beta carotene, vitamin E and selenium levels associate with accelerated carotid atherogenesis in hypercholesterolemic eastern Finnish men. Circulation 87:678; 1993. [191] Tardif, J.-C.; Coˆte´, G.; Lespe´rance, J.; Bourassa, M.; Lambert, J.; Doucet, S.; Bilodeau, L.; Nattel, S.; de Guise, P.‘Probucol and multivitamins in the prevention of restenosis after coronary angioplasty. N. Engl. J. Med. 337:365–372; 1997. [192] Lee, I.-M. Antioxidant vitamins in the prevention of cancer. Proc. Assoc. Am. Physicians 111:10 –15; 1999. [193] Bucher, J. R.; Tien, M.; Morehouse, L. A.; Aust, S. D. Redox cycling and lipid peroxidation: the central role of iron chelates. Fund. Appl. Toxicol. 3:222–226; 1983. [194] Buettner, G. R. Ascorbate autoxidation in the presence of iron and copper chelates. Free Radic. Res. Comm. 1:349 –353; 1986. [195] Buettner, G. R. Ascorbate oxidation: UV absorbance of ascorbate and ESR spectroscopy of the ascorbyl radical as assays for iron. Free Radic. Res. Comm. 10:5–9; 1990. [196] Gerster, H. High-dose vitamin C: A risk for persons with high iron stores? Int. J. Vit. Nutr. Res. 69:67– 82; 1999. [197] Seib, P. A.; Tolbert, B. M., eds. Ascorbic acid: chemistry, metabolism, and uses. Washington, D.C.: American Chemical Society; 1982. [198] Packer, L.; Fuchs, J., eds. Vitamin C in health and disease. New York: Marcel Dekker, Inc.; 1997. [199] Ames, B. N.; Cathcart, R.; Schwiers, E.; Hochstein, P. Uric acid provides antioxidant defense in humans against oxidant–and radical caused aging and cancer: a hypothesis. Proc. Natl. Acad. Sci. USA 78:6858 – 6862; 1981. [200] Hochstein, P.; Hatch, L.; Sevanian, A. Uric acid: functions and determination. Meth. Enzymol. 105:162–166; 1984. [201] Niki, E.; Yamamoto, Y.; Kamiya, Y. Role of uric acid, cysteine, and glutathione as chain breaking antioxidant in aqueous phase. Chem. Lett. 1267–1270; 1985. [202] Peden, D. B.; Hohman, R.; Brown, M. E.; Mason, R. T.; Berkebile, C.; Fales, H. M.; Kaliner, M. A. Uric acid is a major antioxidant in human nasal airway secretions. Proc. Natl. Acad. Sci. USA 87:7638 –7642; 1990. [203] Halliwell, B.; Gutteridge, J. M. C., eds. Free radicals in biology and medicine, 3rd edition. Oxford: Oxford University Press; 1999. [204] Pryor, W. A.; Stahl, W.; Rock, C. L. Beta carotene: from biochemistry to clinical trials. Nutr. Rev. (In press); 2000. [205] Pryor, W. A.; Cornicelli, J. A.; Devall, L. J.; Tait, B.; Trivedi, B. K.; Witiak, D. T.; Wu, M. A rapid screening test to determine the antioxidant potencies of natural and synthetic antioxidants. J. Org. Chem. 58:3521–3532; 1993. [206] Krinsky, N. I. Cellular aspects of carotenoid actions. In: Cadenas, E.; Packer, L., eds. Handbook of antioxidants. New York: Marcel Dekker; 1996:315–336. [207] Krinsky, N. I.; Wang, X.-D.; Tang, G.; Russell, R. M. Cleavage of beta carotene to retinoids. In: Livrea, M. A.; Vidali, G., eds. Retinoids: from basic science to clinical applications. Basel, Switzerland: Birkhauser Verlag; 1994:21–28. [208] McCall, M. R.; Frei, B. Can antioxidant vitamins materially reduce oxidative damage in humans? Free Radic. Biol. Med. 26:1034 –1053; 1999. [209] Motoyama, T.; Kawano, H.; Kugiyama, K.; Hirashima, O.; Ohgushi, M.; Tsunoda, R.; Moriyama, Y.; Miyao, Y.; Yoshimura, m.; Ogawa, H.; Yasue, H. Vitamin E administration improves impairment of endothelium-dependent vasodilation in patients with coronary spastic angina. J. Am. Coll. Cardiol. 32:1672–1679; 1998.

164

W. A. PRYOR

[210] Livingstone, P. D.; Jones, C. Treatment of intermittent claudication with vitamin E. Lancet 2:602– 604; 1958. [211] Haeger, K. Long-time treatment of intermittent claudication with vitamin E. Am. J. Clin. Nutr. 27:1179 –1181; 1974. [212] Williams, H. T. G.; Fenna, D.; MacBeth, R. A. Alpha tocopherol in the treatment of intermittent claudication. Surg. Gynecol. Obstet. 132:662– 666; 1971. [213] To¨rnwall, M. E.; Virtamo, J.; Haukka, J. K.; Aro, A.; Albanes, D.; Edwards, B. K.; Huttunen, J. K. Effect of alpha tocopherol (vitamin E) and beta carotene supplementation on the incidence of intermittent claudication in male smokers. Arterioscler. Thromb. Vasc. Biol. 17:3475–3480; 1997. [214] Pryor, W. A. Cigarette smoke radicals and the role of free radicals in chemical carcinogenicity. Environ. Health Perspect. 105:875– 882; 1997. [215] Lonn, E. V.; Yusuf, S. Is there a role for antioxidant vitamins in the prevention of cardiovascular diseases? An update on epidemiologicalal and clinical trials data. Can. J. Cardiol. 13:957–965; 1997. [216] Mitchinson, M. J.; Stephens, N. G.; Parsons, A.; Bligh, E.; Schofield, P. M.; Brown, M. J. Mortality in the CHAOS trial. Lancet 353:381–382; 1999. [217] Marchioli, R.; GISSI-Prevenzione Investigators. Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Lancet 354:447– 455; 1999. [218] Burton, G. W.; Traber, M. G.; Acuff, R. V.; Walters, D. N.; Kayden, H.; Hughes, L.; Ingold, K. U. Human plasma and tissue alpha-tocopherol concentrations in response to supplementation with deuterated natural and synthetic vitamin E. Am. J. Clin. Nutr. 67:669 – 684; 1998. [219] Landvik, S. An overview of vitamin E efficacy. VERIS 1–36; November, 1998. [220] Blot, W. J.; Li, J.-Y.; Taylor, P. R.; Guo, W.; Dawsey, S.; Wang, G.-Q.; Yang, C. S.; Zheng, S.-F.; Gail, M.; Li, G.-Y.; Yu, Y.; Liu, B.-Q.; Tangrea, J.; Sun, Y.-H.; Liu, F.; Fraumeni, J. F., Jr.; Zhang, Y.-H.; Li, B. Nutrition intervention trials in Linxian, China: supplementation with specific vitamin/mineral combinations, cancer incidence, and disease-specific mortality in the general population. J. Natl. Cancer Inst. 85:1483–1492; 1993. [221] Albanes, D.; Heinonen, O. P.; Taylor, P. R.; Virtamo, J.; Edwards, B. K.; Rautalahti, M.; Hartman, A. M.; Palmgren, J.; Freedman, L. S.; Haapakoski, J.; Barrett, M. J.; Pietinen, P.; Malila, N.; Tala, E.; Liippo, K.; Salomaa, E.-R.; Tangrea, J. A.; Teppo, L.; Askin, F. B.; Taskinen, E.; Erozan, Y.; Greenwald, P.; Huttunen, J. K. ␣-Tocopherol and ␤-carotene supplements and lung cancer incidence in the alpha-tocopherol, beta-carotene cancer prevention study: effects of base-line characteristics and study compliance. J. Natl. Cancer Inst. 88:1560 –1570; 1996. [222] The Alpha-Tocopherol Beta Carotene Cancer Prevention Study Group. The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. N. Engl. J. Med. 330:1029 –1035; 1994. [223] Heinonen, O. P.; Albanes, D.; Virtamo, J.; Taylor, P. R.; Huttunen, J. K.; Hartman, A. M.; Haapakoski, J.; Malila, N.; Rautalahti, M.; Ripetti, S.; Maenpaa, H.; Teerenhovi, L.; Koss, L.; Virolainen, M.; Edwards, B. K. Prostate cancer and supplementation with ␣-tocopherol and ␤-carotene: incidence and mortality in a controlled trial. J. Natl. Cancer Inst. 90:440 – 446; 1998. [224] Virtamo, J.; Rapola, J. M.; Ripatti, S.; Heinonen, O. P.; Taylor, P. R.; Albanes, D.; Huttunen, J. K. Effect of vitamin E and beta carotene on the incidence of primary nonfatal myocardial infarction and fatal coronary heart disease. Arch. Intern. Med. 158:668–675; 1998. [225] Rapola, J. M.; Virtamo, J.; Ripatti, S.; Haukka, J. K.; Huttunen, J. K.; Albanes, D.; Taylor, P. R.; Heinonen, O. P. Effects of alpha tocopherol and beta carotene supplements on symptoms, progression, and prognosis of angina pectoris. Heart 79:454 – 458; 1998. [226] Steiner, M.; Anastasi, J. Vitamin E: an inhibitor of the platelet release reaction. J. Clin. Invest. 57:732–737; 1976. [227] Steiner, M. Effect of ␣-tocopherol administration on platelet function in man. Thromb. Haemost. 49:73–77; 1983. [228] Schmidt, R.; Hayn, M.; Reinhart, B.; Roob, G.; Schmidt, H.;

[229]

[230] [231]

[232] [233]

[234] [235] [236]

[237] [238] [239] [240] [241] [242] [243] [244]

Schumacher, M.; Watzinger, N.; Launer, L. J. Plasma antioxidants and cognitive performance in middle-aged and older adults: results of the austrian stroke prevention study. J. Am. Geriatr. Soc. 46:1407–1410; 1998. Mark, S. D.; Wang, W.; Fraumeni, J. F., Jr.; Li, J.-Y.; Taylor, P. R.; Wang, G.-Q.; Dawsey, S. M.; Li, B.; Blot, W. J. Do nutritional supplements lower the risk of stroke or hypertension? Epidemiology 9:9 –15; 1998. Landis, S. H.; Murray, T.; Bolden, S.; Wingo, P. A. Cancer statistics, 1999. Cancer J. Clin. 49:8 –31; 1999. Traber, M. G.; Sokol, R. J.; Kohlschu¨tter, A.; Yokota, T.; Muller, D. P. R.; Dufour, R.; Kayden, H. J. Impaired discrimination between stereoisomers of ␣-tocopherol in patients with familial isolated vitamin E deficiency. J. Lipid Res. 34:201–210; 1993. Traber, M. G.; Cohn, W.; Muller, D. P. R. Absorption, transport and delivery to tissues. In: Packer, L.; Fuchs, J., eds. Vitamin E in health and disease. New York: Marcel Dekker, Inc.; 1993:35–51. Ridker, P. M.; Cushman, M.; Stampfer, M. J.; Tracy, R. P.; Hennekens, C. H. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy man. N. Engl. J. Med. 336:973–979; 1997. Podhaisky, H.-P.; Abate, A.; Polte, T.; Oberle, S.; Schro¨der, H. Aspirin protects endothelial cells from oxidative stress–possible synergism with vitamin E. FEBS Lett. 417:349 –351; 1997. Mendis, S. Vitamins and coronary heart disease: where do we stand? Ceylon Med. J. 43:68 –73; 1998. Tribble, D. L. Antioxidant consumption and risk of coronary heart disease: emphasis on vitamin C, vitamin E, and beta carotene: a statement for healthcare professionals from the American Heart Association. Circulation 99:591–595; 1999. Gaziano, J. M. Antioxidants in cardiovascular disease: randomized trials. Nutr. Rev. 54:175–184; 1996. Tribble, D. L.; Frank, E. Dietary antioxidants, cancer, and atherosclerotic heart disease. West. J. Med. 161:605– 613; 1994. Jialal, I.; Devaraj, S.; Huet, B. A.; Traber, M. GISSI-prevenzione trial (letter to the editor). Lancet 354:1554; 1999. Salen, P.; De Lorgeril, M. GISSI-prevenzione trial (letter to the editor). Lancet 354:1555; 1999. Ng, W.; Tse, H.-F.; Lau, C.-P. GISSI-prevenzione trial (letter to the editor). Lancet 354:1555–1556; 1999. Hooper, L.; Ness, A.; Higgins, J. P. T.; Moore, T.; Ebrahim, S. GISSI-prevenzione trial (letter to the editor). Lancet 354:1557; 1999. Marchioli, R.; Tognoni, G.; Valagussa, F. GISSI-prevenzione trial (authors’ reply). Lancet 354:1556 –1557; 1999. Upston, J. M.; Terentis, A. C.; Stocker, R. Tocopherol-mediated peroxidation of lipoproteins: implications of vitamin E as a potent antiatherogenic supplement. FASEB J. 13:977–994; 1999. ABBREVIATIONS

CI— confidence interval CVD— cardiovascular disease CSA— coronary spastic angina DHA— docosahexaenoic acid EPA— eicosapentaenoic acid IMT—arterial wall intima media thickness IU—International Units LDL—low-density lipoprotein MDA—malonaldehyde (malondialdehyde) MI—myocardial infarction PUFA—polyunsaturated fatty acid(s) TBARS—thiobarbituric acid reactive substances TIA—transient ischemic attack ␣-TE—␣-tocopherol equivalents