Dietary Antioxidants and Cardiovascular Disease J. MICHAEL GAZIANO,” JOANN E. MANSON,b JULIE E. B U R I N G , ~AND CHARLES H. HENNEKENS~J The Channing Laboratory and “The Division of Cardiology in the Department of Medicine and bThe Department of Preventive Medicine Brigham and Women’s Hospital and Harvard Medical School Boston. Massachusetts 02215-1204 For several years, it has been suggested that oxidation is involved in the pathogenesis of many diseases, including cancer, arthritis, and reperfusion injury during myocardial infarction (MI). Recently, studies of lipid metabolism have contributed to the formulation of the hypothesis that oxidative damage accelerates the process of atherogenesis.’ Despite a postulated role in atherogenesis, the clinical relevance of antioxidants to the prevention and treatment of cardiovascular disease (CVD) is unclear. In this report we review the hypothesis that oxidative damage, in particular to LDL, plays a role in atherogenesis. Second, we present recent evidence, including findings from three different populations by our research group, regarding the role of dietary antioxidants in reducing risks of CVD. Finally, we outline possible avenues of future research.
OXIDATION IN ATHEROSCLEROSIS Natural Defense against Free-Radical Oxidation
Oxidative metabolism clearly has many advantages, but free radicals generated through this process are highly reactive and can also react with and damage innocent bystander molecules, such as DNA, proteins, lipids, and carbohydrates.* The unpaired electron in the outer shell of free radicals is in a high energy state and is therefore very reactive. Free radicals of oxygen found in biological systems include the superoxide radical, the hydroxyl radical, and the hydroperoxyl radical. Hydrogen peroxide is not a free radical, but can easily form oxygen free radicals. Singlet oxygen is another highly reactive form that has been transformed into a free radical by the absorption of a single photon. These oxygen free radicals will attack and oxidize any nearby molecule, such as a fatty acid, in an attempt to shed the high energy electron. Free radicals initiate lipid peroxidation, a chain reaction resulting in the formation of a new fatty acid free radical. This fatty acid free radical will, in turn, attack another fatty acid
Address for correspondence: Charles H. Hennekens, MD, Brigham and Women’s Hospital, 900 Commonwealth Avenue East, Boston, MA 02215-1204. 249
250
ANNALS NEW YORK ACADEMY OF SCIENCES
and, in the process, be transformed into a lipid hydroperoxide. This propagation reaction, initiated by a single free radical, results in damage to thousands of fatty acid molecules. Free radical reactions terminate when two free radicals meet and combine in a way that the unpaired electrons bind. As humans evolved to use oxidative metabolism, many mechanisms developed to control this process and minimize random free radical oxidation. First, oxidative metabolism is compartmentalized, for example, in the mitochondria. Molecular oxygen and its reactive free radical species are tightly bound to enzymes, as is the case in the cytochrome system during oxidative phosphorylation. In order to prevent free radical formation, transition metals, such as copper and iron, which can catalyze free radical formation, are tightly bound to transport and storage proteins. Several enzymes exist within the cell to neutralize free radicals. Superoxide dismutase catalyzes the transformation of superoxide radicals to hydrogen peroxide. Catalase and glutathione dismutase neutralize hydrogen peroxide and fatty acid radicals. Damage caused by oxidation can be repaired by specific enzymes. Several nonenzymatic dietary antioxidants neutralize radicals outside the cell. Vitamin E, found in liver, egg yolks, cereal grains, nuts, and several vegetable oils; and beta-carotene, found in carrots, green leafy vegetables, squash, melons, and tomatoes are fat-soluble antioxidants that reside in circulating lipoproteins and lipid membranes. Vitamin C, found in many fruits and vegetables, is a watersoluble antioxidant. These three vitamins can quench free radicals and terminate chain reactions. Oxidation in Atherosclerosis
Elevated LDL increases risks of cardiovascular disease, but the possible mechanisms are only now becoming understood. In vitro and in vivo studies suggest that atherogenicity of LDL is greatly enhanced by oxidative damage. Oxidative modification of LDL (Ox-LDL) results in the formation of fatty acid hydroperoxides and subsequent fragmentation of the fatty acids, which leads to formation of short chain aldehydes, followed by fragmentation of apo B.j Oxidized LDL has been implicated in many steps of atherogenesis. Ox-LDL is toxic to cultured endothelial cells, altering both structure and function. An early step in atherogenesis is the adhesion of circulating monocytes to the endothelium. Once monocytes This adhesion appears to be aided by the presence of OX-LDL.~ adhere to the endothelium, the next step in atheroma formation is the migration of these cells into the subendothelium. Ox-LDL also stimulates the secretion of chemoattractants that may aid in this process.' Ox-LDL is not recognized by the native LDL receptor and therefore cannot be cleared by this pathway. Further, the uptake of LDL by foam cells is enhanced by oxidative modification through an alternative pathway (scavenger receptor).6 Once taken up by the foam cell, however, Ox-LDL degradation may be impaired, which leads to further accumulation within the c e L 7 Autoantibodies to the OxLDL may be involved in the later stages of atheroma formation.8 There is growing evidence that these processes also occur in vivo. Trace amounts of Ox-LDL have been detected in human and animal plasma and have been reported to be elevated in diabetics, smokers, patients with coronary artery stenoses, and in the elderly.'.'" Injection of lipid hydroperoxides into animals results in dramatic changes in the endothelial lining." Finally, antibodies to OxLDL have been detected in plasma of patients with carotid artery disease and in
GAZIANO et al.: DIETARY ANTIOXIDANTS
251
atherosclerotic plaque.’ Despite the body’s attempts to limit oxidative damage, uncontrolled damage is constantly occurring. The fact that antioxidants prevent such damage has raised the possibility of their role in inhibiting atherogenesis and preventing various manifestations of occlusive vascular disease.
DIETARY ANTIOXIDANTS IN THE PREVENTION OF ATHEROSCLEROSIS Vitamin E (tocopherol and tocotrienols), beta-carotene, and vitamin C (ascorbic acid) are three important naturally occurring antioxidants. Evidence suggests that these dietary agents may play a role in the prevention and treatment of CVD.
Animal Studies Over a dozen animal studies have been conducted to test the hypothesis that antioxidants delay atherosclerosis. Three trials (one in rabbits, one in monkeys, and one in restricted ovulatory hens) have reported decreased atheromatous lesion formation in vitamin E-fed animals compared to controls. Restricted ovulatory hens develop hyperlipidemia and subsequent aortic intimal thickening. Compared to controls, those fed 1000 mg of vitamin E per kilogram of feed had reduced levels of plasma peroxides and less aortic intimal thickening.’* Wojcicki reported a statistically significant 25% reduction in aortic atherosclerotic lesions in hypercholesterolemic mongrel rabbits fed 10 milligrams per kilogram per day of vitamin E compared to controls,13 and Verlangieri reported a 54% lesion reduction in monkeys fed 108 IU of vitamin E per day compared to control^.'^ Beta-carotene has been less well studied in animal models. Vitamin C deficiency has been shown to cause atherosclerotic lesions in guinea pigs.I5 Subsequent studies in deficient guinea pigs revealed regression of early atherosclerotic lesions after the administration of vitamin C.I6 Altman reported delayed progression and, in some cases, regression of atherosclerotic lesions among hypercholesterolemic rats supplemented with vitamin C.” Several animal trials have tested probucol in the development of atherosclerotic lesions. Probucol is both a cholesterol-lowering agent and a powerful antioxidant. Kita reported reduced LDL oxidation among rabbits administered probucol compared with those given placebo.I* Carew controlled for probucol’s cholesterollowering effect by using lovastatin in one of two control groups. Compared to lovastatin-treated rabbits, probucol further reduced the rate of formation of fatty streaks in Watanabe heritable hyperlipidemic rabbits, suggesting that the benefit of this agent may be at least in part due to its antioxidant effects.” Administration of BHT, another antioxidant, and selenium, an antioxidant enzyme cofactor, also results in reduced atherosclerotic lesions in hypercholesterolemic rabbit^.^.^^ Descriptive Studies Descriptive studies are extremely useful for hypothesis formulation, but they cannot be used to test hypotheses. Cross-cultural surveys have provided equivocal, but suggestive, results that dietary antioxidants are associated with reduced isch-
252
ANNALS NEW YORK ACADEMY OF SCIENCES
emic heart disease. Two studies in the United Kingdom reported a significant inverse relationship between consumption of fresh fruits and vegetables and rates of atherosclerotic disease.21.22 In the United States, the steady decline in cardiovascular mortality has generally been attributed to improvements in treatment, or secondary prevention, as well as primary prevention due to decreased cigarette smoking, better control of hypertension, and cholesterol lowering. Descriptive studies have also shown, however, that the early decline in CVD was preceded by significant changes in the intake of antioxidant-rich foods. Verlangieri has raised the hypothesis that part of the secular decline in CVD may be due to increased fresh fruit and vegetable intake resulting from greater year round availability of these foods.23Ginter reported an inverse relationship between vitamin C production and coronary disease mortality rates in the United States over the last twenty years, although, clearly, changes in other risk factors may hopelessly confound these results.24 Three studies by Gey and colleagues reported inverse associations between plasma vitamin E levels standardized to plasma cholesterol level and rates of CVD mortality in several European populations. The findings for vitamin A and selenium were less c~nsistent.~’-~’ Riemersma reported that plasma levels of vitamins A, C, and E were not consistently correlated with heart disease mortality rates among four European populations, although a trend for vitamin E was apparent.z8Finally, in a cross-sectional survey of 1,132 Finnish men, Salonen found no association between plasma vitamin C or E and prevalent ischemic heart disease but was, of course. unable to control for confounding variables or changes in diet subsequent to diagnosis of disease.29 Case-Control Studies Several case-control studies lend support to the possible role of antioxidants in the reduction of CVD risks. Riemersa et al. recently reported an inverse relationship between plasma levels of vitamins E and C and beta-carotene and angina pectoris assessed by a self-administered chest pain q ~ e s t i o n n a i r e .The ~ ~ .relation ~~ persisted for vitamin E after controlling for smoking and other cardiovascular risk factors, despite the fact that misclassification of angina from a chest pain questionnaire may have underestimated the true effect of each antioxidant. Rameriz et 01. reported decreased leukocyte ascorbic acid levels in cases of angiographically proven coronary artery disease (CAD) compared to controls without CAD referred for cardiac c a t h e t e r i ~ a t i o n Three . ~ ~ nested case-control studies have reported conflicting results. Because blood samples for antioxidant determination were obtained before disease occurrence, these studies are termed prospective blood-based investigations. Although a recent nested case-control study demonstrated an inverse association between prediagnostic serum beta-carotene level and subsequent risk of MI (relative risk (RR) = 0.42; 95% confidence interval (CI) = 0.19-0.89; p trend = 0.03),33two others reported no consistent association between serum antioxidants and subsequent CVD. Kok reported no association between serum vitamins A and E and selenium levels in frozen serum samples and CVD mortality in a nested case-control study of residents in Western Netherlands.74 A potential limitation of this study may be the instability of vitamin antioxidants in serum samples after nine years of storage. In a nested case-control study of Finnish men, Salonen found no consistent association between serum vitamins A, E, or selenium and death from CAD.35Samples were stored for seven years before analysis, so vitamin instability may also have affected these results.
GAZIANO el al.: DIETARY ANTIOXIDANTS
253
Prospective Cohort Studies Prospective cohort data, although limited, tend to support a protective role of dietary antioxidants in CVD. In 1980, the Nurses’ Health Study, a prospective cohort study among 120,700 female nurses from the United States, aged 30-55 at entry in 1976, began collecting detailed dietary intake data using a semiquantitative food frequency questionnaire. Based on the responses to the diet form, which asked women to quantitate their intake of various foods and vitamin supplements, intake of several micronutrients, including pro-vitamin A carotenoids and vitamin E, was estimated. There was a decreased risk of coronary heart disease among women in the highest quintile of vitamin E consumption compared to the lowest (RR = 0.66; 95% CI = 0.50-0.87; p trend > 0.001) after adjustment for age, sex, smoking, and other cardiovascular risk factors.j6 Similar results were obtained for carotene. Among those in the highest quintile of beta-carotene consumption, relative risk of a CVD event was 0.78 (95% CI = 0.59-1.03; p trend = 0.02) compared to the lowest, after adjustment for age, smoking, and other cardiovascular risk factors. There was also an apparent trend in risk reduction for vitamin C.37 In 1976, we collected data on 1,299 elderly Massachusetts residents identified from a statewide area probability sample. Dietary information was obtained using a food frequency questionnaire, and beta-carotene scores were derived by summing the total number of servings of items rich in this nutrient. An inverse relation was observed between consumption of fruits and vegetables high in beta-carotene and subsequent CVD death. Among those in the highest quartile of beta-carotene consumption, the relative risk of CVD mortality was 0.55 (95% CI = 0.34-0.87) compared to those in the lowest quartile after adjustment for age, sex, smoking, and alcohol intake.3s In a prospective cohort of Swedish women, estimates of vitamin C intake from a 24-hour recall dietary history were inversely correlated with CVD event rates; however, these findings did not persist after controlling for age.39 Whereas the data from both prospective blood-based and dietary intake studies are compatible with a possible benefit of antioxidants, the available observational data are sparse and not all consistent. Additional observational data would certainly be a valuable contribution to the totality of evidence concerning antioxidants and CVD. Regardless of the number or sample size of such studies, however, or the consistency of their findings, observational investigations are limited in their ability to provide reliable data on the most plausible small to moderate benefits of antioxidants. It may be, for example, that greater dietary intake of antioxidants, measured by blood levels or a diet assessment questionnaire, is only a marker for some other dietary practice or even non-dietary life-style variable that is truly protective. It is, in fact, plausible that intake of antioxidant-rich foods is indeed protective, but that the benefit results not from their antioxidant properties, but from some other component these foods have in common. In addition, the intake of individual dietary antioxidants is often highly correlated, making it difficult to determine the specific benefit of any one. Observational studies can control for the effects of known potential confounding variables, but they cannot take into account unknown or unmeasured confounding factors. In searching for small to moderate effects, the number of uncontrolled confounding variables in observational studies may be as large as the likely risk reduction. For these reasons, reliable data can only emerge from large-scale randomized trials, in which investigators allocate subjects at random
254
ANNALS N E W YORK ACADEMY OF SCIENCES
to either active treatment or placebo. If the trials are large enough, the randomization process will evenly distribute, among treatment groups, known as well as unknown confounding variables.
Randomized Trials To date, no large-scale randomized trials have been conducted of antioxidants in either the treatment or prevention of CVD. The limited data presently available derive from five trials with small samples. Three trials of vitamin E in the treatment of claudication revealed a clinical benefit over periods of 1 to 3 y e a r ~ , ~ Owhereas -~* a fourth failed to show any benefit of vitamin E in the treatment of angina pectoris after six months of follow-up.43 Finally, the role of beta-carotene in the treatment of CVD events was examined among a subgroup of men with angina in the Physicians' Health Study, a randomized, double-blind, placebo-controlled trial among 22,071 male physicians from the United States, aged 40-84 at baseline. This trial tested aspirin in primary prevention of CVD and beta-carotene in primary prevention of cancer among men with no prior history of MI, stroke, transient ischemic attack (TIA), or cancer. After 60.2 months of follow-up, compliance with pill-taking was 85% and morbidity and mortality data were available on 99.7% and 100% of participants, respectively. The aspirin component, which tested 325 mg on alternate days, was terminated in 1988, based primarily on the emergence of a statistically extreme 44% reduction in risk of a first MI. The beta-carotene component, which is testing 50 mg on alternate days, has proceeded uninterrupted and is scheduled to continue treatment and follow-up until 1995. This trial should provide importantly relevant information on the possible role of beta-carotene in the primary prevention of CVD. Based on the emerging hypotheses concerning antioxidants and CVD, the availability of the data base from the completed aspirin component of the trial afforded an opportunity to conduct an analysis of beta-carotene and cardiovascular events in a small subgroup of participants. Although physicians with prior MI, stroke, or TIA were ineligible to enroll, 333 doctors did enter the trial with chronic stable angina or a prior coronary revascularization-coronary artery bypass graft (CABG) or percutaneous transluminal coronary angioplasty (PTCA). In analyses among this subgroup of 333 high-risk participants, those assigned beta-carotene had a significant reduction in a combined end point of subsequent vascular events (nonfatal myocardial infarction, nonfatal stroke, coronary revascularization, and cardiovascular death) compared to those receiving placebo (RR = 0.46; 95% = CI 0.24-0.85).44 Because the postulated benefit of antioxidants is to delay the progression of atherosclerosis, the effects may not be evident during the initial period of follow-up. In these analyses, no effect of beta-carotene was seen until the second year of follow-up. Of 27 incident MIS, 10 occurred in the carotene group and 17 in the group receiving carotene placebo. With respect to aspirin, there were 7 and 20 in the active and placebo groups, respectively. For those assigned both carotene and aspirin, there were no incident MIS. Other mechanisms of action of dietary antioxidants may alter cardiac risk factors in ways that may or may not be related to their ability to prevent LDL oxidation. Specifically, vitamins C and E may improve the lipid p r ~ f i l e ~ ~ and -~' may inhibit platelet a g g r e g a t i ~ n ,though ~ ~ . ~ ~studies are not conclusive. Vitamin C has also been reported to reduce blood pressure among persons with hyperten~ion.~~.~'
GAZIANO et al.: DIETARY ANTIOXIDANTS
255
AVENUES OF FUTURE RESEARCH In summary, available epidemiologic evidence supports the possibility that antioxidants may have a protective effect in CVD, and basic research supports a plausible mechanism for the involvement of oxidative stress in atherogenesis. At present, antioxidants represent a promising but clearly unproven means to reduce risks of CVD. Several avenues should be pursued in future research on antioxidants and CVD. Further basic research and better animal models are needed to explore the mechanism of oxidation and the extent to which this occurs in viuo. Although additional descriptive and observational study findings may provide further support for a possible beneficial effect of dietary antioxidants, the only way to determine definitively whether antioxidants offer a benefit in both treatment or prevention of atherosclerotic disease is through randomized trials. A recent National Heart Lung and Blood Institute (NHLB1)-sponsored conference, titled “Antioxidants in the Prevention of Human Atherosclerosis,” addressed the issue of randomized trials of antioxidants. The consensus statement from this conference concluded that present data support the need for randomized trials of antioxidants in reducing risks of CVD.’? There are currently three large primary prevention trials testing dietary antioxidants. The beta-carotene component of the Physicians’ Health Study should provide reliable evidence about whether beta-carotene has a role in the primary prevention of CVD in over 20,000 apparently healthy men. The “CARET” study is testing beta-carotene among 18,000 individuals at high risk for lung cancer due to occupational asbestos exposure. Finally, our group is now beginning the Women’s Health Study, a randomized trial of beta-carotene, vitamin E, and lowdose aspirin in the primary prevention of CVD and cancer among over 40,000 healthy female nurses from the United States, aged 50 and older. Although these trials should provide reliable data on the role of antioxidants in primary prevention, there are currently no trials, ongoing or funded, of antioxidants in secondary prevention of CVD. Our group has proposed a secondary prevention trial of beta-carotene, vitamin C, and vitamin E using a factorial design in women. This trial would provide information on the role of these safe dietary agents, alone and in combination, in the treatment of atherosclerotic disease. Further, in the United Kingdom, a 2 x 2 factorial trial of cholesterol-lowering and vitamin E has been proposed. Results from secondary prevention trials, if funded, together with data that will become available over the next few years from ongoing trials in primary prevention, should provide reliable evidence on the role of antioxidants in both the treatment as well as primary prevention of cardiovascular disease. REFERENCES STEINBERG, D., S. PARTHASARKTHY, T. E. CAREW,J. C. KHOO& J. L. WITZTUM. 1989. Beyond cholesterol: Modifications of low-density lipoprotein that increase its atherogenicity. N. Engl. J. Med. 320(14): 915-924. P. A. & G. Powrs. 1988. Free radicals in medicine. I. Chemical nature and 2. SOUTHORN, biologic reactions. Mayo Clin. Proc. 63: 381-389. 3. FONG,L. G., S. PARTHASARATHY, J. L. WITZTUM& D. STEINBERG. 1987. Nonenzyrnatic oxidative cleavage of peptide bonds in apoprotein 8-100. J. Lipid Res. 28: 1466-1477. 4. BERLINER, J. A , , M. C. TERRITO,A. SEVANIAN, S. RAMIN,J . A. KIM,B. BAMSHAD, M. ESTERSON & A. M. FODLEMAN. 1990. J. Clin. Invest. 85: 1260-1266. I.
256
ANNALS NEW YORK ACADEMY OF SCIENCES
& D. STEINBERG. 1985. Endothelial cell-derived 5. QUINN,M. T., S. PARATHASARATHY chemotactic activity for mouse peritoneal macrophages and the effects of modified forms of low density lipoprotein. Proc. Natl. Acad. Sci. USA 82: 5949-5953. 6. FOGELMAN,A. M., I. SCHECHTER, M. HOKOM,J . S. CHILD& P. A. EDWARDS. 1980. Malondialdehyde alteration of low density lipoproteins leads to cholesterol ester accumulation in human monocyte macrophages. Proc. Natl. Acad. Sci. USA 77: 22 14-22 18. 1991. Oxidized LDL is resistent to cathepsins M. & U. P. STEINBRECHER. 7. L.OUGHEED. and accumulate in macrophages. J. Biol. Chem. 266: 14519-14525. R. YAMAMOTO, S. BUTLER,H. KORPELA,R. T . , S. YLA-HERTTUALA, 8. SALONEN, SALONEN, K. NYSSONEN, W. PALINSKI & J. L. WITZTUM.1992. Autoantibody against oxidized LDL and progression of carotid atherosclerosis. Lancet 339(8798): 883-887. B. C., P. WHITE,W. Tsou & D. FINKEL.1984. Peroxidation of plasma 9. BLACKMAN, and platelet lipids in chronic cigarette smokers and insulin-dependent diabetics. Ann. N. Y. Acad. Sci. 435: 385-387. T., T. KAMADA, H. ABE,S. KIKUCHI & K. YAGI.1977. Serumlipoperoxide 10. SUEMATSU, level in patients suffering from liver diseases. Clin. Chim. Acta 79: 267. N. ONISHI,M. YAMASHITA & T. NAKASHIMA. 1981. Lesion 11. YAGI, K., H. OHKAWA, of aortic intima caused by intravenous administration of linoleic acid hydroperoxide. J. Appl. Biochem. 3: 58-65. 1989. Effect of dietary vitamin E on plasma lipids 12. SMITH.T. L. & F. A. KUMMEROW. and atherogenesis in restricted ovulatory chickens. Atherosclerosis 75: 105-109. J., L. ROZEWICKA, B. BARCEW-WISZNIEWSKA, L. SAMOCHOWIEC, S. Juz13. WOJCICKI, W I A K , D. KADUBOWASKA, S. TUSTANOWSKI & Z. JUZSZYN. 1991. Effect of selenium and vitamin E on the development of experimental atherosclerosis in rabbits. Atherosclerosis 87: 9- 19. A. J. & M. BUSH.1992. Prevention and regression of atherosclerosis by 14. V ERLANGIERI, alpha-tocopherol. J. Am. Coll. Nutr. ll(2): 131-138. 15. WILLIS.G. C. 1953. An experimental study of the intimal ground substance in atherosclerosis. Can. Med. Assoc. J . 69: 17-22. 16. WILLIS,G. C. 1957. The reversibility of atherosclerosis, Can. Med. Assoc. J. 77: 106- 109. R. F. A , , G . M. V . SCHAEFFER, C. A. SALLES,A. S. RAMOSDE S o u z ~& 17. ALTMAN, P. M. T. COTIAS.1980. Phospholipids associated with vitamin C in experimental atherosclerosis. Drug. Res 30: 627-630. M. YOKODE,K. ISHI,N. KUME,A. OOSHIMA, H. YOSHIDA & 18. KITA,T., Y. NAGANO, C. KAWAI.1897. Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbit, and animal model for familial hypercholesterolemia. Proc. Natl. Acad. Sci. USA 84: 5928-5931. & D. STEINBERG. 1987. Antiatherogenic effect of probucol 19. CAREW,T., D. SCHWENKE unrelated to its hypercholesterolemic effect: evidence that antioxidants in uiuo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbits. Proc. Natl. Acad. Sci. USA 84: 7725-7729. 0. BREUER,U . DICZFALUSY, L. 20. BJOKHEM,I . , A. HENRIKKSSON-FREYSCHUSS, BERGLUND& P. HENRIKSSON. 1991. The antioxidant butylated hydroxytoluene protects against atherosclerosis. Arteriosclerosis Thrombosis 11: 15-22. 1983. Does consumption offruit and vegetables R. M. & D. R. R. WILLIAMS. 21. ACHESON, protect against stroke? Lancet 1: 1191-1 193. B. K., J. L. MANN,A. M. ADELSTEIN & F. ESKIN.1975. Commodity 22. ARMSTRONG, consumption and ischemic heart disease mortality, with special reference to dietary practices J . Chronic Dis. 36: 673-677. A. J., J. C. KAPEGHIAN, S. EL-DEAN& M. BUSH. 1985. Fruit and 23. VERLANGIERI, vegetable consumption and cardiovascular disease mortality. Med. Hypotheses 1 6 7-15. 24. GINTER,E. 1979. Decline of coronary mortality in the United States and vitamin C. Am. J. Clin. Nutr. 32: 51 1-512.
GAZIANO et al.: DIETARY ANTIOXIDANTS
257
& H. B. STAHELIN.1987. Plasma levels of antioxidant 25. GEY, K. F., G. B. BRUBACHER vitamins in relation to ischemic heart disease and cancer. Am. J. Clin. Nutr. 45: 1368-1 377. 26. GEY.K. F . & P. PUSKA.1989. Plasma vitamins E and A inversely correlated to mortality from ischemic heart disease in cross-cultural epidemiology.-Ann. N . Y . Acad. Sci. -. 570: 268-282. P. PUSKA& A. EVANS.1987. Relationship of plasma 27. GEY. K. F.. H. B. STAHELIN, level of vitamin C to mortality from ischemic heart disease. Ann. N . Y: Acid. Sci. 498: 110-123. R. A., M. OLIVER, R. A. ELTON,G. ALFTHAN,E. VARTIAINEN, M. SALO, 28. RIEMERSMA, P. RUBBA,M. MANCICI,H . GEORGI,J . VUILLEUMIER & K. F. GEY. 1990. Plasma antioxidants and coronary heart disease: vitamins C and E and selenium. Eur. J. Clin. Nutr. 44: 143-150. K. SEPPANEN,M. KANTOLA,M. PARVIAINEN, G. 29. SALONEN,J. T., R. SOLONEN, ALFTHAN,P. H . MAENPAA,E. TASKINEN & R. RAURAMAA. 1988. Relationship of serum selenium and antioxidants to plasma lipoproteins, platelet aggregability and prevalent ischaemic heart disease in eastern Finnish men. Atherosclerosis 7 0 155- 160. R. A., D. A. WOOD,C . C. H . MACINTYRE, R. A. ELTON,K. F. GEY& 30. RIEMERSMA, M. F . OLIVER.1991. Risk of angina pectoris and plasma concentrations of vitamins A, C, E , and carotene. Lancet 337(8732): 1-5. R. A., D. A. WOOD, C. C. A. MACINTYRE, R. ELTON,K. F . GEY & 31. RIEMERSMA, M. F. OLIVER.1989. Low plasma vitamins E and C: Increased risk of angina in Scottish men. Ann. N. Y. Acad. Sci. 570: 291-295. J. & N . C. FLOWERS. 1980. Leukocyte ascorbic acid and its relationship to 32. RAMIREZ, coronary heart disease in man. Am. J. Clin. Nutr. 33: 2079-2087. 33. STREET,D. A., G. W. COMSTOCK, R. M. SALKELD,W. SCHUEP& M. KLAG.1991. A population based case-control study of serum antioxidants and myocardial infarction. Am. J . Epidemiol. 134: 719-720. A. HOFMAN,A. VANLAAR,M. 34. KOK, F. J., A. M. DE BRUIJN,R. VERMEEREN, 1987. Serum selenium, vitamin DEBRUIN, R. J. T. HERMUS& H. A. VALKENBERG. antioxidants and cardiovascular mortality: a 9 year follow-up study in the Netherlands. Am. J. Clin. Nutr. 45: 462-468. J. T., R. SALONEN, I. PENTTILA,J . HERRANEN, M. JAUHIAINEN, M. KAN35. SALONEN, TOLA, R. LAPPETELAINEN, P. MAENPAA, G. ALFTHAN,& P. PUSKA.1985. Serum fatty acids, apolipoproteins, selenium and vitamin antioxidants and risk of death from coronary artery disease. Am. J. Cardiol. 56: 226-231. W. C. WILLET,G. A. COLDITZ,B. ROSNER,F. E. 36. MANSON,J. E., M. J. STAMPFER, SPEIZER& C. H . HENNEKENS.1991. A prospective study of antioxidant vitamins and incidence of coronary heart disease in women. Circulation (Abstract)84(4) Suppl 11: 2168. W. C. WILLETT,G. A. COLDITZ,B. ROSNER,F . E. 37. MANSON,J. E., M. J. STAMPFER, SPEIZER & C. H . HENNEKENS. 1992. A prospective study of vitamin C and incidence of coronary heart disease in women. Circulation. (Abstract) 85(3): 865. J. M., J . E. MANSON,L. G. BRANCH,F. LAMOTT,G. A. COLDITZ,J. E. 38. GAZIANO, BURINC& C. H. HENNEKENS. 1992. Dietary beta carotene and decreased cardiovascular mortality in an elderly cohort. J. Am. Coll. Cardiol. (abstract) 19(3 Suppl. A): 371A. C. BENCTSON& 1. BOSCEUS.1986. Dietary habits in 39. LAPIDUS,L., H. ANDERSON, relation to incidence of cardiovascular disease and death in women; a 12 year followup of participants in the study of women in Gothenberg, Sweden. Am. J. Clin. Nutr. 44: 444-448. 40. LIVINGSTON,P. D. & JONESC. 1958. Treatment of intermittent claudication with vitamin E. Lancet 2: 602-604. & R. A. MACBETH.1971. Alpha-tocopherol in the 41. WILLIAMS,H. T. G., D. FENNA treatment of intermittent claudication. Surg. Gynecol. Obstet. 132(4): 662-666. 42. HAEGER,K . 1974. Long-time treatment of intermittent claudication with vitamin E. Am. J. Clin. Nutr. 27: 1179-1181.
258
ANNALS NEW YORK ACADEMY OF SCIENCES
& J. R. WARBASSE. 1977. Quantitative evaluation of 43. GiLLILAN, R. E., B. MANDELL vitamin E in the treatment of angina pectoris. Am. Heart J. 93(4): 444-449. J. M., J . E. MANSON, P. M. RIDKER, J. E. BURING& C. H. HENNEKENS. 44. GAZIANO, 1990. Beta carotene therapy for chronic stable angina. Circulation 82(4, Supplement 111): 111-202. A. L. 1958. Influence of some factors on the development of experimental 45. MYASNIKOV, cholesterol atherosclerosis. Circulation 17: 99-1 13. C. R. 1971. Atherosclerosis and vitamin C. Lancet 2 1280-1281. 46. SPITTLE, V . BALAZ,F. HRUBA,V. ROCH& E. SASKO. 47. GINTER,E., 0. CERNA,J . BUDLOVSKY, 1977. Effect of ascorbic acid on plasma cholesterol in humans in a long term experiment. Int. J. Vitam. Nutr. Res. 47: 123-34. C., A. MUSCA,F. VIOLA,A. PERRONE & C. ALESSANDRI. 1982. Influence 48. CORDOVA, of ascorbic acid on platelet aggregation in vifroand in uiuo. Atherosclerosis 41: 15-19. 49. BORDIA.A. & S . K. VERMA.1985. Effects of vitamin C on blood b i d s . fibrinolvtic activity and platelet aggregation in coronary artery disease patients.'Clin. Cardioi. 8: 552-554. 50. YOSHIOKA,M., T. MATSUSHITA & Y. CHUMAN. 1984. Inverse association of serum ascorbic acid level and blood pressure or rate of hypertension in male adults aged 30-39 years. Int. J. Vitam. Nutr. Res. 54:343-347. 51. KOH, E. T. 1984. Effect of vitamin C on blood parameters of hypertensive subjects. J. Oklahoma State Med. Assoc. 77: 177-182. 52. STEINBERG, D. & WORKSHOP PARTICIPANTS. 1992. Antioxidants in the prevention of human atherosclerosis: Summary of the proceedings of a National Heart, Lung, and Blood Institute Workshop: September 5-6, 1991, Bethesda, Maryland. Circulation 85(6): 2337-2344.
DISCUSSION
H. SIES(University ofDusseldorf, Diisseldorf, Germany):Hermann Esterbauer calculated that the LDL particle contains less than one molecule of beta-carotene and indeed any other carotenoid. In other words, there might be subsets of LDL populations that are more prone to oxidation. If so, in these studies, is the intent to saturate to at least one beta-carotene molecule per LDL? C. H. HENNEKENS (Harvard Medical School, Boston, MA): The oxidation of LDL and its possible retardation by these antioxidants is attractive, but it may represent only one of a number of very plausible mechanisms by which these agents are operative. We performed dosing studies ten years ago to decide on the dose of beta-carotene, based on the goal that it would be sufficient to put people in the highest 3 or 4% of the distribution of intake in the United States and yet not give any orange discoloration of the skin, because this was a primary prevention trial. With vitamin E the dose is titrated to what I will call the vegetable epidemiology studies, that is, comparing the top fifth to the lowest fifth of the distribution of intake seems to demonstrate a benefit. We're basically comparing the top few percent of the distribution of intake to the median intake, and we're hoping that this will be a large enough differential to find a benefit if one truly exists. I think that the findings in the 333 men with the chronic stable angina or coronary revascularization procedure are suggestive but certainly by no means conclusive. If we find data like that in the 22,000, then I think we would have really conclusive data on the question. UNiDEN-rrFIED SPEAKER: Please comment about the future potential use of
GAZIANO
et af.:
DIETARY ANTIOXIDANTS
259
other antioxidants not mentioned in the previous two studies, such as selenium and glutathione, and their role in countering oxidized LDL. Second, would you comment on certain populations that have relatively high cholesterol but relatively low incidence of cardiovascular disease, such as the Masai, and how they fit into the oxidized LDL hypothesis? HENNEKENS: We have proposed trials of selenium in the past and submitted them to the NIH. We were told that it was the feeling of the reviewers, at least, that in certain areas of the country these trials could pose a hazard to people; in addition, they felt that the wealth of evidence was not sufficient to justify randomized trials. I also think it is a promising hypothesis, however, that could and should be tested. Regarding your second question, we can certainly get clues from such unique populations. I do feel, however, that the totality of evidence is that total cholesterol is the leading avoidable cause of cardiovascular mortality in most developed countries. I think that about a 1% lowering of blood cholesterol across these usual levels will correspond to a 2-3% decline in vascular mortality. My colleague, Richard Peto, has found that the absolute risk of cardiovascular disease in China is about 4% that of the United States. The average cholesterol level in China is 130 compared to 210 in the United States. He found that there was a 20-30% lower risk of cardiovascular disease in those that had cholesterol of 117 compared to those that had a value of 130, which is the mean for the area. This is a fairly strong argument, in my view, against the idea of a threshold for cholesterol. A. BENDICH(Hoffmunn-LaRoche I n c . , Nutley, N J ) : With the nurses' study, could you tell us what those quintiles mean for dietary intake? I think the average intake of beta-carotene in the nurses was HENNEKENS: about 2 mg or so, and I think the highest quintile was up to about 7 or 8 milligrams.
OXIDATION IN ATHEROSCLEROSIS Natural Defense against Free-Radical Oxidation
Oxidative metabolism clearly has many advantages, but free radicals generated through this process are highly reactive and can also react with and damage innocent bystander molecules, such as DNA, proteins, lipids, and carbohydrates.* The unpaired electron in the outer shell of free radicals is in a high energy state and is therefore very reactive. Free radicals of oxygen found in biological systems include the superoxide radical, the hydroxyl radical, and the hydroperoxyl radical. Hydrogen peroxide is not a free radical, but can easily form oxygen free radicals. Singlet oxygen is another highly reactive form that has been transformed into a free radical by the absorption of a single photon. These oxygen free radicals will attack and oxidize any nearby molecule, such as a fatty acid, in an attempt to shed the high energy electron. Free radicals initiate lipid peroxidation, a chain reaction resulting in the formation of a new fatty acid free radical. This fatty acid free radical will, in turn, attack another fatty acid
Address for correspondence: Charles H. Hennekens, MD, Brigham and Women’s Hospital, 900 Commonwealth Avenue East, Boston, MA 02215-1204. 249
250
ANNALS NEW YORK ACADEMY OF SCIENCES
and, in the process, be transformed into a lipid hydroperoxide. This propagation reaction, initiated by a single free radical, results in damage to thousands of fatty acid molecules. Free radical reactions terminate when two free radicals meet and combine in a way that the unpaired electrons bind. As humans evolved to use oxidative metabolism, many mechanisms developed to control this process and minimize random free radical oxidation. First, oxidative metabolism is compartmentalized, for example, in the mitochondria. Molecular oxygen and its reactive free radical species are tightly bound to enzymes, as is the case in the cytochrome system during oxidative phosphorylation. In order to prevent free radical formation, transition metals, such as copper and iron, which can catalyze free radical formation, are tightly bound to transport and storage proteins. Several enzymes exist within the cell to neutralize free radicals. Superoxide dismutase catalyzes the transformation of superoxide radicals to hydrogen peroxide. Catalase and glutathione dismutase neutralize hydrogen peroxide and fatty acid radicals. Damage caused by oxidation can be repaired by specific enzymes. Several nonenzymatic dietary antioxidants neutralize radicals outside the cell. Vitamin E, found in liver, egg yolks, cereal grains, nuts, and several vegetable oils; and beta-carotene, found in carrots, green leafy vegetables, squash, melons, and tomatoes are fat-soluble antioxidants that reside in circulating lipoproteins and lipid membranes. Vitamin C, found in many fruits and vegetables, is a watersoluble antioxidant. These three vitamins can quench free radicals and terminate chain reactions. Oxidation in Atherosclerosis
Elevated LDL increases risks of cardiovascular disease, but the possible mechanisms are only now becoming understood. In vitro and in vivo studies suggest that atherogenicity of LDL is greatly enhanced by oxidative damage. Oxidative modification of LDL (Ox-LDL) results in the formation of fatty acid hydroperoxides and subsequent fragmentation of the fatty acids, which leads to formation of short chain aldehydes, followed by fragmentation of apo B.j Oxidized LDL has been implicated in many steps of atherogenesis. Ox-LDL is toxic to cultured endothelial cells, altering both structure and function. An early step in atherogenesis is the adhesion of circulating monocytes to the endothelium. Once monocytes This adhesion appears to be aided by the presence of OX-LDL.~ adhere to the endothelium, the next step in atheroma formation is the migration of these cells into the subendothelium. Ox-LDL also stimulates the secretion of chemoattractants that may aid in this process.' Ox-LDL is not recognized by the native LDL receptor and therefore cannot be cleared by this pathway. Further, the uptake of LDL by foam cells is enhanced by oxidative modification through an alternative pathway (scavenger receptor).6 Once taken up by the foam cell, however, Ox-LDL degradation may be impaired, which leads to further accumulation within the c e L 7 Autoantibodies to the OxLDL may be involved in the later stages of atheroma formation.8 There is growing evidence that these processes also occur in vivo. Trace amounts of Ox-LDL have been detected in human and animal plasma and have been reported to be elevated in diabetics, smokers, patients with coronary artery stenoses, and in the elderly.'.'" Injection of lipid hydroperoxides into animals results in dramatic changes in the endothelial lining." Finally, antibodies to OxLDL have been detected in plasma of patients with carotid artery disease and in
GAZIANO et al.: DIETARY ANTIOXIDANTS
251
atherosclerotic plaque.’ Despite the body’s attempts to limit oxidative damage, uncontrolled damage is constantly occurring. The fact that antioxidants prevent such damage has raised the possibility of their role in inhibiting atherogenesis and preventing various manifestations of occlusive vascular disease.
DIETARY ANTIOXIDANTS IN THE PREVENTION OF ATHEROSCLEROSIS Vitamin E (tocopherol and tocotrienols), beta-carotene, and vitamin C (ascorbic acid) are three important naturally occurring antioxidants. Evidence suggests that these dietary agents may play a role in the prevention and treatment of CVD.
Animal Studies Over a dozen animal studies have been conducted to test the hypothesis that antioxidants delay atherosclerosis. Three trials (one in rabbits, one in monkeys, and one in restricted ovulatory hens) have reported decreased atheromatous lesion formation in vitamin E-fed animals compared to controls. Restricted ovulatory hens develop hyperlipidemia and subsequent aortic intimal thickening. Compared to controls, those fed 1000 mg of vitamin E per kilogram of feed had reduced levels of plasma peroxides and less aortic intimal thickening.’* Wojcicki reported a statistically significant 25% reduction in aortic atherosclerotic lesions in hypercholesterolemic mongrel rabbits fed 10 milligrams per kilogram per day of vitamin E compared to controls,13 and Verlangieri reported a 54% lesion reduction in monkeys fed 108 IU of vitamin E per day compared to control^.'^ Beta-carotene has been less well studied in animal models. Vitamin C deficiency has been shown to cause atherosclerotic lesions in guinea pigs.I5 Subsequent studies in deficient guinea pigs revealed regression of early atherosclerotic lesions after the administration of vitamin C.I6 Altman reported delayed progression and, in some cases, regression of atherosclerotic lesions among hypercholesterolemic rats supplemented with vitamin C.” Several animal trials have tested probucol in the development of atherosclerotic lesions. Probucol is both a cholesterol-lowering agent and a powerful antioxidant. Kita reported reduced LDL oxidation among rabbits administered probucol compared with those given placebo.I* Carew controlled for probucol’s cholesterollowering effect by using lovastatin in one of two control groups. Compared to lovastatin-treated rabbits, probucol further reduced the rate of formation of fatty streaks in Watanabe heritable hyperlipidemic rabbits, suggesting that the benefit of this agent may be at least in part due to its antioxidant effects.” Administration of BHT, another antioxidant, and selenium, an antioxidant enzyme cofactor, also results in reduced atherosclerotic lesions in hypercholesterolemic rabbit^.^.^^ Descriptive Studies Descriptive studies are extremely useful for hypothesis formulation, but they cannot be used to test hypotheses. Cross-cultural surveys have provided equivocal, but suggestive, results that dietary antioxidants are associated with reduced isch-
252
ANNALS NEW YORK ACADEMY OF SCIENCES
emic heart disease. Two studies in the United Kingdom reported a significant inverse relationship between consumption of fresh fruits and vegetables and rates of atherosclerotic disease.21.22 In the United States, the steady decline in cardiovascular mortality has generally been attributed to improvements in treatment, or secondary prevention, as well as primary prevention due to decreased cigarette smoking, better control of hypertension, and cholesterol lowering. Descriptive studies have also shown, however, that the early decline in CVD was preceded by significant changes in the intake of antioxidant-rich foods. Verlangieri has raised the hypothesis that part of the secular decline in CVD may be due to increased fresh fruit and vegetable intake resulting from greater year round availability of these foods.23Ginter reported an inverse relationship between vitamin C production and coronary disease mortality rates in the United States over the last twenty years, although, clearly, changes in other risk factors may hopelessly confound these results.24 Three studies by Gey and colleagues reported inverse associations between plasma vitamin E levels standardized to plasma cholesterol level and rates of CVD mortality in several European populations. The findings for vitamin A and selenium were less c~nsistent.~’-~’ Riemersma reported that plasma levels of vitamins A, C, and E were not consistently correlated with heart disease mortality rates among four European populations, although a trend for vitamin E was apparent.z8Finally, in a cross-sectional survey of 1,132 Finnish men, Salonen found no association between plasma vitamin C or E and prevalent ischemic heart disease but was, of course. unable to control for confounding variables or changes in diet subsequent to diagnosis of disease.29 Case-Control Studies Several case-control studies lend support to the possible role of antioxidants in the reduction of CVD risks. Riemersa et al. recently reported an inverse relationship between plasma levels of vitamins E and C and beta-carotene and angina pectoris assessed by a self-administered chest pain q ~ e s t i o n n a i r e .The ~ ~ .relation ~~ persisted for vitamin E after controlling for smoking and other cardiovascular risk factors, despite the fact that misclassification of angina from a chest pain questionnaire may have underestimated the true effect of each antioxidant. Rameriz et 01. reported decreased leukocyte ascorbic acid levels in cases of angiographically proven coronary artery disease (CAD) compared to controls without CAD referred for cardiac c a t h e t e r i ~ a t i o n Three . ~ ~ nested case-control studies have reported conflicting results. Because blood samples for antioxidant determination were obtained before disease occurrence, these studies are termed prospective blood-based investigations. Although a recent nested case-control study demonstrated an inverse association between prediagnostic serum beta-carotene level and subsequent risk of MI (relative risk (RR) = 0.42; 95% confidence interval (CI) = 0.19-0.89; p trend = 0.03),33two others reported no consistent association between serum antioxidants and subsequent CVD. Kok reported no association between serum vitamins A and E and selenium levels in frozen serum samples and CVD mortality in a nested case-control study of residents in Western Netherlands.74 A potential limitation of this study may be the instability of vitamin antioxidants in serum samples after nine years of storage. In a nested case-control study of Finnish men, Salonen found no consistent association between serum vitamins A, E, or selenium and death from CAD.35Samples were stored for seven years before analysis, so vitamin instability may also have affected these results.
GAZIANO el al.: DIETARY ANTIOXIDANTS
253
Prospective Cohort Studies Prospective cohort data, although limited, tend to support a protective role of dietary antioxidants in CVD. In 1980, the Nurses’ Health Study, a prospective cohort study among 120,700 female nurses from the United States, aged 30-55 at entry in 1976, began collecting detailed dietary intake data using a semiquantitative food frequency questionnaire. Based on the responses to the diet form, which asked women to quantitate their intake of various foods and vitamin supplements, intake of several micronutrients, including pro-vitamin A carotenoids and vitamin E, was estimated. There was a decreased risk of coronary heart disease among women in the highest quintile of vitamin E consumption compared to the lowest (RR = 0.66; 95% CI = 0.50-0.87; p trend > 0.001) after adjustment for age, sex, smoking, and other cardiovascular risk factors.j6 Similar results were obtained for carotene. Among those in the highest quintile of beta-carotene consumption, relative risk of a CVD event was 0.78 (95% CI = 0.59-1.03; p trend = 0.02) compared to the lowest, after adjustment for age, smoking, and other cardiovascular risk factors. There was also an apparent trend in risk reduction for vitamin C.37 In 1976, we collected data on 1,299 elderly Massachusetts residents identified from a statewide area probability sample. Dietary information was obtained using a food frequency questionnaire, and beta-carotene scores were derived by summing the total number of servings of items rich in this nutrient. An inverse relation was observed between consumption of fruits and vegetables high in beta-carotene and subsequent CVD death. Among those in the highest quartile of beta-carotene consumption, the relative risk of CVD mortality was 0.55 (95% CI = 0.34-0.87) compared to those in the lowest quartile after adjustment for age, sex, smoking, and alcohol intake.3s In a prospective cohort of Swedish women, estimates of vitamin C intake from a 24-hour recall dietary history were inversely correlated with CVD event rates; however, these findings did not persist after controlling for age.39 Whereas the data from both prospective blood-based and dietary intake studies are compatible with a possible benefit of antioxidants, the available observational data are sparse and not all consistent. Additional observational data would certainly be a valuable contribution to the totality of evidence concerning antioxidants and CVD. Regardless of the number or sample size of such studies, however, or the consistency of their findings, observational investigations are limited in their ability to provide reliable data on the most plausible small to moderate benefits of antioxidants. It may be, for example, that greater dietary intake of antioxidants, measured by blood levels or a diet assessment questionnaire, is only a marker for some other dietary practice or even non-dietary life-style variable that is truly protective. It is, in fact, plausible that intake of antioxidant-rich foods is indeed protective, but that the benefit results not from their antioxidant properties, but from some other component these foods have in common. In addition, the intake of individual dietary antioxidants is often highly correlated, making it difficult to determine the specific benefit of any one. Observational studies can control for the effects of known potential confounding variables, but they cannot take into account unknown or unmeasured confounding factors. In searching for small to moderate effects, the number of uncontrolled confounding variables in observational studies may be as large as the likely risk reduction. For these reasons, reliable data can only emerge from large-scale randomized trials, in which investigators allocate subjects at random
254
ANNALS N E W YORK ACADEMY OF SCIENCES
to either active treatment or placebo. If the trials are large enough, the randomization process will evenly distribute, among treatment groups, known as well as unknown confounding variables.
Randomized Trials To date, no large-scale randomized trials have been conducted of antioxidants in either the treatment or prevention of CVD. The limited data presently available derive from five trials with small samples. Three trials of vitamin E in the treatment of claudication revealed a clinical benefit over periods of 1 to 3 y e a r ~ , ~ Owhereas -~* a fourth failed to show any benefit of vitamin E in the treatment of angina pectoris after six months of follow-up.43 Finally, the role of beta-carotene in the treatment of CVD events was examined among a subgroup of men with angina in the Physicians' Health Study, a randomized, double-blind, placebo-controlled trial among 22,071 male physicians from the United States, aged 40-84 at baseline. This trial tested aspirin in primary prevention of CVD and beta-carotene in primary prevention of cancer among men with no prior history of MI, stroke, transient ischemic attack (TIA), or cancer. After 60.2 months of follow-up, compliance with pill-taking was 85% and morbidity and mortality data were available on 99.7% and 100% of participants, respectively. The aspirin component, which tested 325 mg on alternate days, was terminated in 1988, based primarily on the emergence of a statistically extreme 44% reduction in risk of a first MI. The beta-carotene component, which is testing 50 mg on alternate days, has proceeded uninterrupted and is scheduled to continue treatment and follow-up until 1995. This trial should provide importantly relevant information on the possible role of beta-carotene in the primary prevention of CVD. Based on the emerging hypotheses concerning antioxidants and CVD, the availability of the data base from the completed aspirin component of the trial afforded an opportunity to conduct an analysis of beta-carotene and cardiovascular events in a small subgroup of participants. Although physicians with prior MI, stroke, or TIA were ineligible to enroll, 333 doctors did enter the trial with chronic stable angina or a prior coronary revascularization-coronary artery bypass graft (CABG) or percutaneous transluminal coronary angioplasty (PTCA). In analyses among this subgroup of 333 high-risk participants, those assigned beta-carotene had a significant reduction in a combined end point of subsequent vascular events (nonfatal myocardial infarction, nonfatal stroke, coronary revascularization, and cardiovascular death) compared to those receiving placebo (RR = 0.46; 95% = CI 0.24-0.85).44 Because the postulated benefit of antioxidants is to delay the progression of atherosclerosis, the effects may not be evident during the initial period of follow-up. In these analyses, no effect of beta-carotene was seen until the second year of follow-up. Of 27 incident MIS, 10 occurred in the carotene group and 17 in the group receiving carotene placebo. With respect to aspirin, there were 7 and 20 in the active and placebo groups, respectively. For those assigned both carotene and aspirin, there were no incident MIS. Other mechanisms of action of dietary antioxidants may alter cardiac risk factors in ways that may or may not be related to their ability to prevent LDL oxidation. Specifically, vitamins C and E may improve the lipid p r ~ f i l e ~ ~ and -~' may inhibit platelet a g g r e g a t i ~ n ,though ~ ~ . ~ ~studies are not conclusive. Vitamin C has also been reported to reduce blood pressure among persons with hyperten~ion.~~.~'
GAZIANO et al.: DIETARY ANTIOXIDANTS
255
AVENUES OF FUTURE RESEARCH In summary, available epidemiologic evidence supports the possibility that antioxidants may have a protective effect in CVD, and basic research supports a plausible mechanism for the involvement of oxidative stress in atherogenesis. At present, antioxidants represent a promising but clearly unproven means to reduce risks of CVD. Several avenues should be pursued in future research on antioxidants and CVD. Further basic research and better animal models are needed to explore the mechanism of oxidation and the extent to which this occurs in viuo. Although additional descriptive and observational study findings may provide further support for a possible beneficial effect of dietary antioxidants, the only way to determine definitively whether antioxidants offer a benefit in both treatment or prevention of atherosclerotic disease is through randomized trials. A recent National Heart Lung and Blood Institute (NHLB1)-sponsored conference, titled “Antioxidants in the Prevention of Human Atherosclerosis,” addressed the issue of randomized trials of antioxidants. The consensus statement from this conference concluded that present data support the need for randomized trials of antioxidants in reducing risks of CVD.’? There are currently three large primary prevention trials testing dietary antioxidants. The beta-carotene component of the Physicians’ Health Study should provide reliable evidence about whether beta-carotene has a role in the primary prevention of CVD in over 20,000 apparently healthy men. The “CARET” study is testing beta-carotene among 18,000 individuals at high risk for lung cancer due to occupational asbestos exposure. Finally, our group is now beginning the Women’s Health Study, a randomized trial of beta-carotene, vitamin E, and lowdose aspirin in the primary prevention of CVD and cancer among over 40,000 healthy female nurses from the United States, aged 50 and older. Although these trials should provide reliable data on the role of antioxidants in primary prevention, there are currently no trials, ongoing or funded, of antioxidants in secondary prevention of CVD. Our group has proposed a secondary prevention trial of beta-carotene, vitamin C, and vitamin E using a factorial design in women. This trial would provide information on the role of these safe dietary agents, alone and in combination, in the treatment of atherosclerotic disease. Further, in the United Kingdom, a 2 x 2 factorial trial of cholesterol-lowering and vitamin E has been proposed. Results from secondary prevention trials, if funded, together with data that will become available over the next few years from ongoing trials in primary prevention, should provide reliable evidence on the role of antioxidants in both the treatment as well as primary prevention of cardiovascular disease. REFERENCES STEINBERG, D., S. PARTHASARKTHY, T. E. CAREW,J. C. KHOO& J. L. WITZTUM. 1989. Beyond cholesterol: Modifications of low-density lipoprotein that increase its atherogenicity. N. Engl. J. Med. 320(14): 915-924. P. A. & G. Powrs. 1988. Free radicals in medicine. I. Chemical nature and 2. SOUTHORN, biologic reactions. Mayo Clin. Proc. 63: 381-389. 3. FONG,L. G., S. PARTHASARATHY, J. L. WITZTUM& D. STEINBERG. 1987. Nonenzyrnatic oxidative cleavage of peptide bonds in apoprotein 8-100. J. Lipid Res. 28: 1466-1477. 4. BERLINER, J. A , , M. C. TERRITO,A. SEVANIAN, S. RAMIN,J . A. KIM,B. BAMSHAD, M. ESTERSON & A. M. FODLEMAN. 1990. J. Clin. Invest. 85: 1260-1266. I.
256
ANNALS NEW YORK ACADEMY OF SCIENCES
& D. STEINBERG. 1985. Endothelial cell-derived 5. QUINN,M. T., S. PARATHASARATHY chemotactic activity for mouse peritoneal macrophages and the effects of modified forms of low density lipoprotein. Proc. Natl. Acad. Sci. USA 82: 5949-5953. 6. FOGELMAN,A. M., I. SCHECHTER, M. HOKOM,J . S. CHILD& P. A. EDWARDS. 1980. Malondialdehyde alteration of low density lipoproteins leads to cholesterol ester accumulation in human monocyte macrophages. Proc. Natl. Acad. Sci. USA 77: 22 14-22 18. 1991. Oxidized LDL is resistent to cathepsins M. & U. P. STEINBRECHER. 7. L.OUGHEED. and accumulate in macrophages. J. Biol. Chem. 266: 14519-14525. R. YAMAMOTO, S. BUTLER,H. KORPELA,R. T . , S. YLA-HERTTUALA, 8. SALONEN, SALONEN, K. NYSSONEN, W. PALINSKI & J. L. WITZTUM.1992. Autoantibody against oxidized LDL and progression of carotid atherosclerosis. Lancet 339(8798): 883-887. B. C., P. WHITE,W. Tsou & D. FINKEL.1984. Peroxidation of plasma 9. BLACKMAN, and platelet lipids in chronic cigarette smokers and insulin-dependent diabetics. Ann. N. Y. Acad. Sci. 435: 385-387. T., T. KAMADA, H. ABE,S. KIKUCHI & K. YAGI.1977. Serumlipoperoxide 10. SUEMATSU, level in patients suffering from liver diseases. Clin. Chim. Acta 79: 267. N. ONISHI,M. YAMASHITA & T. NAKASHIMA. 1981. Lesion 11. YAGI, K., H. OHKAWA, of aortic intima caused by intravenous administration of linoleic acid hydroperoxide. J. Appl. Biochem. 3: 58-65. 1989. Effect of dietary vitamin E on plasma lipids 12. SMITH.T. L. & F. A. KUMMEROW. and atherogenesis in restricted ovulatory chickens. Atherosclerosis 75: 105-109. J., L. ROZEWICKA, B. BARCEW-WISZNIEWSKA, L. SAMOCHOWIEC, S. Juz13. WOJCICKI, W I A K , D. KADUBOWASKA, S. TUSTANOWSKI & Z. JUZSZYN. 1991. Effect of selenium and vitamin E on the development of experimental atherosclerosis in rabbits. Atherosclerosis 87: 9- 19. A. J. & M. BUSH.1992. Prevention and regression of atherosclerosis by 14. V ERLANGIERI, alpha-tocopherol. J. Am. Coll. Nutr. ll(2): 131-138. 15. WILLIS.G. C. 1953. An experimental study of the intimal ground substance in atherosclerosis. Can. Med. Assoc. J . 69: 17-22. 16. WILLIS,G. C. 1957. The reversibility of atherosclerosis, Can. Med. Assoc. J. 77: 106- 109. R. F. A , , G . M. V . SCHAEFFER, C. A. SALLES,A. S. RAMOSDE S o u z ~& 17. ALTMAN, P. M. T. COTIAS.1980. Phospholipids associated with vitamin C in experimental atherosclerosis. Drug. Res 30: 627-630. M. YOKODE,K. ISHI,N. KUME,A. OOSHIMA, H. YOSHIDA & 18. KITA,T., Y. NAGANO, C. KAWAI.1897. Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbit, and animal model for familial hypercholesterolemia. Proc. Natl. Acad. Sci. USA 84: 5928-5931. & D. STEINBERG. 1987. Antiatherogenic effect of probucol 19. CAREW,T., D. SCHWENKE unrelated to its hypercholesterolemic effect: evidence that antioxidants in uiuo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbits. Proc. Natl. Acad. Sci. USA 84: 7725-7729. 0. BREUER,U . DICZFALUSY, L. 20. BJOKHEM,I . , A. HENRIKKSSON-FREYSCHUSS, BERGLUND& P. HENRIKSSON. 1991. The antioxidant butylated hydroxytoluene protects against atherosclerosis. Arteriosclerosis Thrombosis 11: 15-22. 1983. Does consumption offruit and vegetables R. M. & D. R. R. WILLIAMS. 21. ACHESON, protect against stroke? Lancet 1: 1191-1 193. B. K., J. L. MANN,A. M. ADELSTEIN & F. ESKIN.1975. Commodity 22. ARMSTRONG, consumption and ischemic heart disease mortality, with special reference to dietary practices J . Chronic Dis. 36: 673-677. A. J., J. C. KAPEGHIAN, S. EL-DEAN& M. BUSH. 1985. Fruit and 23. VERLANGIERI, vegetable consumption and cardiovascular disease mortality. Med. Hypotheses 1 6 7-15. 24. GINTER,E. 1979. Decline of coronary mortality in the United States and vitamin C. Am. J. Clin. Nutr. 32: 51 1-512.
GAZIANO et al.: DIETARY ANTIOXIDANTS
257
& H. B. STAHELIN.1987. Plasma levels of antioxidant 25. GEY, K. F., G. B. BRUBACHER vitamins in relation to ischemic heart disease and cancer. Am. J. Clin. Nutr. 45: 1368-1 377. 26. GEY.K. F . & P. PUSKA.1989. Plasma vitamins E and A inversely correlated to mortality from ischemic heart disease in cross-cultural epidemiology.-Ann. N . Y . Acad. Sci. -. 570: 268-282. P. PUSKA& A. EVANS.1987. Relationship of plasma 27. GEY. K. F.. H. B. STAHELIN, level of vitamin C to mortality from ischemic heart disease. Ann. N . Y: Acid. Sci. 498: 110-123. R. A., M. OLIVER, R. A. ELTON,G. ALFTHAN,E. VARTIAINEN, M. SALO, 28. RIEMERSMA, P. RUBBA,M. MANCICI,H . GEORGI,J . VUILLEUMIER & K. F. GEY. 1990. Plasma antioxidants and coronary heart disease: vitamins C and E and selenium. Eur. J. Clin. Nutr. 44: 143-150. K. SEPPANEN,M. KANTOLA,M. PARVIAINEN, G. 29. SALONEN,J. T., R. SOLONEN, ALFTHAN,P. H . MAENPAA,E. TASKINEN & R. RAURAMAA. 1988. Relationship of serum selenium and antioxidants to plasma lipoproteins, platelet aggregability and prevalent ischaemic heart disease in eastern Finnish men. Atherosclerosis 7 0 155- 160. R. A., D. A. WOOD,C . C. H . MACINTYRE, R. A. ELTON,K. F. GEY& 30. RIEMERSMA, M. F . OLIVER.1991. Risk of angina pectoris and plasma concentrations of vitamins A, C, E , and carotene. Lancet 337(8732): 1-5. R. A., D. A. WOOD, C. C. A. MACINTYRE, R. ELTON,K. F . GEY & 31. RIEMERSMA, M. F. OLIVER.1989. Low plasma vitamins E and C: Increased risk of angina in Scottish men. Ann. N. Y. Acad. Sci. 570: 291-295. J. & N . C. FLOWERS. 1980. Leukocyte ascorbic acid and its relationship to 32. RAMIREZ, coronary heart disease in man. Am. J. Clin. Nutr. 33: 2079-2087. 33. STREET,D. A., G. W. COMSTOCK, R. M. SALKELD,W. SCHUEP& M. KLAG.1991. A population based case-control study of serum antioxidants and myocardial infarction. Am. J . Epidemiol. 134: 719-720. A. HOFMAN,A. VANLAAR,M. 34. KOK, F. J., A. M. DE BRUIJN,R. VERMEEREN, 1987. Serum selenium, vitamin DEBRUIN, R. J. T. HERMUS& H. A. VALKENBERG. antioxidants and cardiovascular mortality: a 9 year follow-up study in the Netherlands. Am. J. Clin. Nutr. 45: 462-468. J. T., R. SALONEN, I. PENTTILA,J . HERRANEN, M. JAUHIAINEN, M. KAN35. SALONEN, TOLA, R. LAPPETELAINEN, P. MAENPAA, G. ALFTHAN,& P. PUSKA.1985. Serum fatty acids, apolipoproteins, selenium and vitamin antioxidants and risk of death from coronary artery disease. Am. J. Cardiol. 56: 226-231. W. C. WILLET,G. A. COLDITZ,B. ROSNER,F. E. 36. MANSON,J. E., M. J. STAMPFER, SPEIZER& C. H . HENNEKENS.1991. A prospective study of antioxidant vitamins and incidence of coronary heart disease in women. Circulation (Abstract)84(4) Suppl 11: 2168. W. C. WILLETT,G. A. COLDITZ,B. ROSNER,F . E. 37. MANSON,J. E., M. J. STAMPFER, SPEIZER & C. H . HENNEKENS. 1992. A prospective study of vitamin C and incidence of coronary heart disease in women. Circulation. (Abstract) 85(3): 865. J. M., J . E. MANSON,L. G. BRANCH,F. LAMOTT,G. A. COLDITZ,J. E. 38. GAZIANO, BURINC& C. H. HENNEKENS. 1992. Dietary beta carotene and decreased cardiovascular mortality in an elderly cohort. J. Am. Coll. Cardiol. (abstract) 19(3 Suppl. A): 371A. C. BENCTSON& 1. BOSCEUS.1986. Dietary habits in 39. LAPIDUS,L., H. ANDERSON, relation to incidence of cardiovascular disease and death in women; a 12 year followup of participants in the study of women in Gothenberg, Sweden. Am. J. Clin. Nutr. 44: 444-448. 40. LIVINGSTON,P. D. & JONESC. 1958. Treatment of intermittent claudication with vitamin E. Lancet 2: 602-604. & R. A. MACBETH.1971. Alpha-tocopherol in the 41. WILLIAMS,H. T. G., D. FENNA treatment of intermittent claudication. Surg. Gynecol. Obstet. 132(4): 662-666. 42. HAEGER,K . 1974. Long-time treatment of intermittent claudication with vitamin E. Am. J. Clin. Nutr. 27: 1179-1181.
258
ANNALS NEW YORK ACADEMY OF SCIENCES
& J. R. WARBASSE. 1977. Quantitative evaluation of 43. GiLLILAN, R. E., B. MANDELL vitamin E in the treatment of angina pectoris. Am. Heart J. 93(4): 444-449. J. M., J . E. MANSON, P. M. RIDKER, J. E. BURING& C. H. HENNEKENS. 44. GAZIANO, 1990. Beta carotene therapy for chronic stable angina. Circulation 82(4, Supplement 111): 111-202. A. L. 1958. Influence of some factors on the development of experimental 45. MYASNIKOV, cholesterol atherosclerosis. Circulation 17: 99-1 13. C. R. 1971. Atherosclerosis and vitamin C. Lancet 2 1280-1281. 46. SPITTLE, V . BALAZ,F. HRUBA,V. ROCH& E. SASKO. 47. GINTER,E., 0. CERNA,J . BUDLOVSKY, 1977. Effect of ascorbic acid on plasma cholesterol in humans in a long term experiment. Int. J. Vitam. Nutr. Res. 47: 123-34. C., A. MUSCA,F. VIOLA,A. PERRONE & C. ALESSANDRI. 1982. Influence 48. CORDOVA, of ascorbic acid on platelet aggregation in vifroand in uiuo. Atherosclerosis 41: 15-19. 49. BORDIA.A. & S . K. VERMA.1985. Effects of vitamin C on blood b i d s . fibrinolvtic activity and platelet aggregation in coronary artery disease patients.'Clin. Cardioi. 8: 552-554. 50. YOSHIOKA,M., T. MATSUSHITA & Y. CHUMAN. 1984. Inverse association of serum ascorbic acid level and blood pressure or rate of hypertension in male adults aged 30-39 years. Int. J. Vitam. Nutr. Res. 54:343-347. 51. KOH, E. T. 1984. Effect of vitamin C on blood parameters of hypertensive subjects. J. Oklahoma State Med. Assoc. 77: 177-182. 52. STEINBERG, D. & WORKSHOP PARTICIPANTS. 1992. Antioxidants in the prevention of human atherosclerosis: Summary of the proceedings of a National Heart, Lung, and Blood Institute Workshop: September 5-6, 1991, Bethesda, Maryland. Circulation 85(6): 2337-2344.
DISCUSSION
H. SIES(University ofDusseldorf, Diisseldorf, Germany):Hermann Esterbauer calculated that the LDL particle contains less than one molecule of beta-carotene and indeed any other carotenoid. In other words, there might be subsets of LDL populations that are more prone to oxidation. If so, in these studies, is the intent to saturate to at least one beta-carotene molecule per LDL? C. H. HENNEKENS (Harvard Medical School, Boston, MA): The oxidation of LDL and its possible retardation by these antioxidants is attractive, but it may represent only one of a number of very plausible mechanisms by which these agents are operative. We performed dosing studies ten years ago to decide on the dose of beta-carotene, based on the goal that it would be sufficient to put people in the highest 3 or 4% of the distribution of intake in the United States and yet not give any orange discoloration of the skin, because this was a primary prevention trial. With vitamin E the dose is titrated to what I will call the vegetable epidemiology studies, that is, comparing the top fifth to the lowest fifth of the distribution of intake seems to demonstrate a benefit. We're basically comparing the top few percent of the distribution of intake to the median intake, and we're hoping that this will be a large enough differential to find a benefit if one truly exists. I think that the findings in the 333 men with the chronic stable angina or coronary revascularization procedure are suggestive but certainly by no means conclusive. If we find data like that in the 22,000, then I think we would have really conclusive data on the question. UNiDEN-rrFIED SPEAKER: Please comment about the future potential use of
GAZIANO
et af.:
DIETARY ANTIOXIDANTS
259
other antioxidants not mentioned in the previous two studies, such as selenium and glutathione, and their role in countering oxidized LDL. Second, would you comment on certain populations that have relatively high cholesterol but relatively low incidence of cardiovascular disease, such as the Masai, and how they fit into the oxidized LDL hypothesis? HENNEKENS: We have proposed trials of selenium in the past and submitted them to the NIH. We were told that it was the feeling of the reviewers, at least, that in certain areas of the country these trials could pose a hazard to people; in addition, they felt that the wealth of evidence was not sufficient to justify randomized trials. I also think it is a promising hypothesis, however, that could and should be tested. Regarding your second question, we can certainly get clues from such unique populations. I do feel, however, that the totality of evidence is that total cholesterol is the leading avoidable cause of cardiovascular mortality in most developed countries. I think that about a 1% lowering of blood cholesterol across these usual levels will correspond to a 2-3% decline in vascular mortality. My colleague, Richard Peto, has found that the absolute risk of cardiovascular disease in China is about 4% that of the United States. The average cholesterol level in China is 130 compared to 210 in the United States. He found that there was a 20-30% lower risk of cardiovascular disease in those that had cholesterol of 117 compared to those that had a value of 130, which is the mean for the area. This is a fairly strong argument, in my view, against the idea of a threshold for cholesterol. A. BENDICH(Hoffmunn-LaRoche I n c . , Nutley, N J ) : With the nurses' study, could you tell us what those quintiles mean for dietary intake? I think the average intake of beta-carotene in the nurses was HENNEKENS: about 2 mg or so, and I think the highest quintile was up to about 7 or 8 milligrams.
