Addiction B iolog y (1997) 2, 269 ± 276
INVITE D R E VIEW
Alcoholic beverages and lipid peroxidation: relevance to cardiovascular disease IAN B. PU DDEY & KEVIN CROFT Departm ent of M edicine and Western Australian H eart Research Institute, University of Western Australia, Australia
Abstract Overall there is good evidence that alcohol consum ption induces oxidative stress, and leads to lipid peroxidation, e Œects which have been linked to alcohol-related toxicity and disease and m ay be relevant to alcoholatherosclerosis inter relationships. On the other hand, a protective e Œect of light to m oderate alcohol consum ption against cardiovascula r disease is well recognized, with the further hypoth esis that red wine o Œers extra cardiovascula r protection due to its rich content of antiox idant phenolic com pound s. Although this hypothesis is given some credence from in vitro data, controlled stud ies in hum ans have produced con¯ icting results. Clearly, the equally well described pro-oxidant e Œects of alcohol and its m etabolism have been insu ciently consid ered in the pursuit of what to many is an intuitiv ely attractive hypoth esis. Further stud ies are required to deter m ine if red wine phenolics are actua lly absorbed from the gut and wheth er they o Œer any overall antioxid ant protection in vivo. The hypothesis that red wine o Œers extra cardiovascula r protection com pared to oth er alcoholic beverages is not proven and m ust await the outcom e of studies in which the full spectr um of the pro-oxidant and antioxid ant e Œects of alcoholic beverages are duly consid ered. In the absence of such studies, there are no ground s at present for the promotion of the consum ption of alcoholic beverages on the basis of their putative ``antiox idant’ ’ properties.
Introdu ction In attempting to understand the possible protective eŒect of alcohol against atherosclerotic cardiovascular disease, several eŒects of alcohol on lipoproteins have been canvassed together with other potential mechanisms.1 ,2 M ore recently, an anti-oxidant eŒect of certain alcoholic beverages, particularly red wine, has been proposed 3 on the basis of the oxidative hypothesis of atherogenesis. This evolving hypothesis points to free radical generation with subsequent oxidat-
ive modi® cation of low density lipoprotein-cholesterol (LDL) as possibly playing a major role in the development of the atherosclerotic lesion.4 A free radical is an atom or molecule that contains one or more unpaired electrons. They are generally represented in chemical formula by a dot, e.g. HO ´. Free radicals are produced in the body as part of normal metabolism, for exam ple superoxide, O ´22 and nitric oxide, NO ´, which have important physiological functions. In general, free radicals are highly reactive and can
Correspondence to: Dr Kevin Croft, Department of Medicine and Western Australian Heart Research Institute, University of Western Australia, Royal Perth H ospital, GPO Box X2213, Perth, Western Australia, 6000, Australia. e-m ail: [email protected] Received for publication 17th June 1996. Accepted 5th October 1996. 1355 ± 6215/97/030269 ± 08 Society for the Study of Addiction to Alcohol and Other Drugs
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attack membrane lipids, for example generating a carbon radical which in turn reacts with oxygen to produce a peroxyl radical which may attack adjacent fatty acids to generate new carbon radicals. This process leads to a chain reaction producing lipid peroxidation products.5 By this means a single radical m ay damage m any molecules by initiating lipid peroxidation chain reactions. Because of the potential damaging nature of free radicals the body has a number of antioxidant defence m echanisms which include enzymes such as superoxide dismutase, catalase, copper and iron transport and storage proteins and both water soluble and lipid soluble molecular antioxidants. Oxidative stress m ay result when antioxidant defences are unable to cope with the production of free radicals, and may result from the action of certain toxins or by physiological 5 stress. The attraction of the oxidative model for atherosclerosis, in attempting to understand any cardioprotective eŒect of alcoholic beverages, relates to the observation that polyphenolic constituents of wines, especially red wine3 ,6 ,7 can, at least in vitro , inhibit oxidation of LDL . These in vitro observations have been paralleled by population studies that suggest a greater cardiovascular protective advantage from the ingestion of wine compared to other alcoholic beverages.8 ± 1 0 As a consequence, it has been hypothesized that these substances may be responsible for the so-called French paradox, whereby a lower incidence of ischaemic heart disease is seen among the French despite a relatively high saturated fat intake.1 1 A recent review of the literature has however found no convincing evidence that wine is more protective against coronary disease than other alcoholic beverages such as beer and spirits.1 2 There is a further resulting tension in that this hypothesis needs to also encompass the simultaneous persuasive evidence for a pro-oxidant eŒect of alcohol itself to induce lipid peroxidation, an eŒect which has been linked to alcohol-related toxicity and disease in several organ systems.
Alcoh ol as a pro-oxidan tÐ the evidence The possibility that ethanol or ethanol metabolism may lead to lipid peroxidation was ® rst raised by Di Luzio, who reported prevention of ethanolinduced fatty liver in rats who were simultaneously administered anti-oxidants1 3 and later observed increased formation of thiobarbituric
acid reactive substances when ethanol was added to normal rat liver homogenates.1 4 A growing body of evidence has since pointed to a possible primary role of ethanol-induced peroxidation in the aetiology of alcoholic liver disease. 1 5 ,1 6 Corroboration of such a m echanism has been provided by the ® nding of increased chemiluminescence, malondialdehyde production and diene formation in livers from rats acutely treated with alcohol.1 7 In primary rat hepatocyte cultures the metabolism of ethanol has also been shown to induce lipid peroxidation as evidenced by an 18 increase in free m alondialdehyde. Similarly, studies in rats treated for 15 months with ethanol demonstrated enhanced hepatic microsomal 19 malondialdehyde formation and there is evidence of increased urinary m alondialdehyde excretion in rats after both acute and chronic 20 alcohol administration. However, an increase in lipid peroxidation during chronic ethanol ingestion has not always been reported 2 1 and in both the acute and chronic rat studies, no eŒect on diene conjugates has been seen.1 8 ,1 9 Whether the administration of alcohol in vivo can also result in lipid peroxidation has been the 22 subject of continuing review and debate. More sophisticated approaches, with direct m easurement of free radical production through the assay of oxidation of chemical scavengers, are consistent with the thesis that the hepatic metabolism of alcohol leads to local free radical generation.2 3 ,2 4 The possibility that ethanol or its major metabolic product, acetaldehyde, can lead to the production of free radical species in other sites, where ethanol may not necessarily be m etabolised, has been recently reviewed 2 5 ,2 6 with the conclusion that a pro-oxidant eŒect of alcohol may also be responsible for alcohol-related toxicity and injury in other tissues. Studies in alcoholics are also consistent with the presence of systemic pro-oxidant stress with elevation of another marker of increased free radical activity, the m olar proportion of linoleic acid that is diene conjugated, which falls during subsequent abstinence from alcohol.2 7 ,2 8 Utilizing intragastric feeding of alcohol to rats, a systemic pro-oxidant eŒect is also suggested by the ® nding of higher plasma isoprostane (a non-cyclo-oxygenase prostanoid) levels, a unique in vivo marker of lipid peroxidation.2 9 In other rat studies increased ethane exhalation has been put forward as a m arker of alcohol-induced lipid peroxidation,3 0 while in man, ethane exhalation in alcohol abusers has
Alcoholic beverages and lipid peroxidation
been only weakly correlated with the level of daily ethanol intake reported before hospital admission.3 1 There is good evidence to implicate pro-oxidant stress in the pathogenesis of ethanol-related neurotoxicity3 2 and cerebellar degeneration, 2 5 33 ethanol-induced damage to gastric mucosa, mem brane injury to erythrocytes,3 4 alcoholic cardiomyopathy 3 5 and the prom otion by ethanol or oral carcinogenesis3 6 and oesophageal tumour growth.3 7 ,3 8 W ith such a broad range of sites where it may induce oxidative stress, a role for alcohol-induced lipid peroxidation in the promotion rather than prevention of atherosclerosis remains a possibility, especially with heavy alcohol intake and long-term alcohol abuse. In this setting, the recognized protective eŒect of light alcohol consumption may disappear, and increased carotid atherosclerosis with heavy alcohol consumption has been reported in population studies in man.3 9 ± 4 1 However, in alcohol-fed rats, despite the exhalation of greater quantities of ethane and an increase in aortic concentrations of cholesterol and phospholipids, no detectable change in the levels of aortic wall lipid peroxides 30 was seen.
M echan ism of the pro-oxidant stress indu ced by ethano l The pro-oxidant stress induced by ethanol m ay be a direct result of the production of free radical species during several stages of the metabolism of alcohol.2 6 ,4 2 Ethanol is oxidized to acetaldehyde by alcohol dehydrogenase, the microsomal ethanol oxidizing system and by catalase. The ® rst two pathways represent possible points at which excess free radical generation m ay occur. The induction of hepatic m icrosomal cytochrome P450 2E1 in habitual abusers of alcohol can 43 result in the production of free radical species, with m icrosomes producing both superoxide and H 2 O 2 . 3 8 ,4 4 Acetaldehyde production may cause 45 lipid peroxidation, the metabolism of acetaldehyde via xanthine oxidase or aldehyde oxidase is capable of generating free radicals. More recently, a vicious cycle has been proposed4 6 with alcohol dehydrogenase reducing NAD + to NADH which in turn is oxidized by aldehyde oxidase generating reactive oxidative species plus NAD + which is then available again for reduction by alcohol dehydrogenase. Ethanol or acetaldehyde, although not neces-
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sarily actively oxidised at other sites in the body, may still induce oxidative stress by several other postulated or dem onstrated m echanisms. Acetal4 5 ,4 7 dehyde can bind with cysteine or glutathione, both potentially important pathways for scavenging free radicals and prevention of lipid peroxidation. Ethanol appears to alter the activities of the anti-oxidant enzymes, copper± zinc superoxide dismutase and glutathione peroxidase,1 8 ,4 8 an action which may be direct or m ediated by eŒect of ethanol to decrease tissue concentrations of zinc and copper. In animal studies however, similar eŒects of alcohol have not been shown in cardiac tissue where Cu,Zn-superoxide dismutase and glutathione-S-transferase were higher when compared to controls while M n-superoxide dismutase, catalase and glutathione peroxidase activities were unaltered.3 5 These paradoxical changes could be understood possibly as adaptive responses to increased free radical production. In this context, rats chronically fed alcohol have demonstrated increases in both plasma alphatocopherol levels and hepatic glutathione despite increases in hepatic malonaldehyde.4 9 In cell culture, direct evidence of an eŒect of ethanol to interfere with anti-oxidant defence mechanisms has been provided, with alterations in cell phospholipid structure associated with a fall in alpha-tocopherol levels.5 0 Ethanol feeding markedly decreased both alpha and gammatocopherol in livers of normal and vitamin E de® cient rats but plasma levels fell only in normal 51 rats. Similarly, lower hepatic vitamin E content and lower serum vitamin E levels have been reported in alcoholics in some,5 2 ± 5 4 but not all studies.5 5 Beta carotene levels have been found to be lower in alcoholics and heavy drinkers 5 5 ,5 6 and even in healthy men changes in serum concentration of beta carotene have been negatively associated with changes in alcohol intake.5 7 This ® nding has not been consistent, however, with a positive correlation between beta carotene levels and alcohol intake in one study,5 6 and no correlation in another after allowing for the eŒects of smoking and diet.5 8 In a recent controlled study in premenopausal women alcohol increased plasma carotene but signi® cantly lowered lutein /zeaxanthin.5 9 These diŒerences may also re¯ ect direct eŒects of alcohol on beta-carotene metabolism, with delays in the clearance of beta carotene demonstrated in studies in primates.6 0 Levels of the anti-oxidant trace elem ent, selenium, have been consistently shown to be lower in
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serum and tissues of alcohol abusers and may also contribute to pro-oxidant stress. An eŒect of alcohol to mobilise and increase the cellular pool of redox-active iron is another potential pathway for alcohol-related pro-oxidant stress, with iron being an important metal catalyst 1 8 ,6 3 ,64 in initiating lipid peroxidation.
Alcoh olic beverag es and L D L oxidationÐ in vitro studies The regular light to moderate consumption of alcohol may protect against atherosclerotic cardiovascular disease, an eŒect that is negated or reversed at higher levels of intake.1 ,2 It has been argued that this protective eŒect of m oderate alcohol consumption compared to abstainers is the result of migration of drinkers to the 65 abstainer group after they become ill. Others have argued that there is still a protective eŒect of moderate alcohol consumption even after this possible problem has been allowed for. 6 6 Given the recent convincing evidence that lipid peroxidation, in particular oxidative damage to LDL, may play a critical role in the development of 67 atherosclerotic lesions it is not surprising that there has been a focus by several investigators on the in vitro eŒects of alcoholic beverages on LDL oxidation. Studies by Frankel et al. 3 were among the ® rst to show that diluted red wine (with the ethanol rem oved) inhibited copper-induced LDL oxidations measured by hexanal form ation. It was suggested that the antioxidant eŒects were due to phenolic compounds which are particularly rich in red wine.6 8 The antioxidant eŒects were found to be more substantial than vitamin E; however, since vitamin E added externally to isolated LDL is poorly incorporated, it may exert only weak antioxidant activity compared to its eŒect when incorporated from the diet. It has been suggested that this may have led to an overstatement of the antioxidant eŒect of red wine.6 9 Several other studies have since con® rmed that, in vitro , red 7 0 ,7 1 wine is a potent inhibitor of LDL oxidation and can increase the antioxidant capacity of serum.7 2 In one of these studies polyphenolics were shown to be the primary antioxidants because when they were stripped from the red wine or grape juice all antioxidant activity was lost.7 1 The major ¯ avonoid constituents of red wine have been analysed and their in vitro antioxidant activity in inhibiting LDL oxidation has been determined. 7 Some cinnamic acid derivat-
ives have also been identi® ed in red wine and these also act as potent inhibitors of LDL oxidation.7 1 ,7 3 These polyphenolic compounds are not limited to red wine and are found in m any fruits and vegetables 7 4 and are particularly rich in tea.7 5 All in vitro studies are consistent with polyphenolic compounds derived from grapes and other plants as providing antioxidant protection against LDL oxidation. What is less clear is which polyphenolic compounds are absorbed and whether they are able to oŒer antioxidant protection in vivo .
Alcoholic beverage s and L DL oxidationÐ in vivo studies There is relatively little information on the eŒect of alcohol in vivo on LDL oxidation. One might predict that since alcohol (as outlined in the preceding discussion) can induce oxidative stress and reduce levels of natural antioxidants in the circulation, then LDL is likely to be m ore sensitive to oxidative damage following alcohol ingestion. In a recent controlled cross-over study conducted by us during which regular beer drinkers consumed a high versus a low alcohol beer, an increased susceptibility of LDL to oxidation was observed during the high alcohol phase.7 6 However another recent study using brandy as the source of alcohol found no eŒect of alcohol (0.5 g /kg /day over 4 weeks) on LDL oxidation,7 7 although b -carotene levels in plasma and LDL decreased signi® cantly with alcohol intake indicating a predominant pro-oxidant stress. The eŒect of an alcoholic beverage on lipoprotein oxidation in vivo may ultimately depend on the balance between the pro-oxidant eŒects of alcohol and the level of antioxidant polyphenolic compounds in the beverage. Several controlled studies in humans have now been reported looking at the eŒect of red wine on lipid and LDL oxidation. Fuhrman et al.7 8 compared red and white wine (containing 11% alcohol) consumed with meals (400 m l /day) for a period of 2 weeks, on the level of lipid peroxides in plasma and the susceptibility of LDL to oxidation. This study reported a substantial prolongation of the lag phase to initiation of LDL oxidation for subjects consuming red wine, while LDL isolated from subjects consuming white wine was m ore susceptible to oxidation. Two other studies, however, have found no such antioxidant eŒect of red wine. Sharpe et al. 7 9 studied 20 volunteers who were
Alcoholic beverages and lipid peroxidation
given either red or white wine (200 ml /day) for 10 days but with no eŒect on the susceptibility of LDL to oxidation. The lack of any antioxidant eŒect may have been due to the lower dose of red wine and the slightly shorter treatment period. However, this was not the case in another study in which subjects received 550 ml /day of red wine over 4 weeks with no eŒect at all on the oxidizability of LDL or level of antioxidants in plasma.8 0 In the latter study the alcohol content of the wines was reduced to 3.5% in order to remove possible interfering eŒects of alcohol, and slightly diŒerent methods were used to estimate LDL oxidation than Fuhrman et al. . 7 8 The extent to which these diŒerences can explain the discrepancy in results is di cult to estimate. None of the studies measured the absorption of any polyphenolic substance, although Fuhrman et al. 7 8 reported a substantial increase in LDL associated total polyphenols using a crude spectrophotometric method. From limited animal studies it would appear that polyphenolics such as catechins after oral administration reach maximal plasma concentrations after 1 hour then decrease quickly.8 1 Therefore, in red wine studies the time of blood sampling after the last drink m ay be a critical factor. Another approach to the study of the possible antioxidant eŒects of red wine has been to measure changes in total antioxidant capacity of serum. Using this method Whitehead et al.7 2 observed an 18% increase in serum antioxidant capacity 1 hour after drinking 300 ml of red wine. No signi® cant increase was seen in subjects drinking white wine. Similar results were observed by M axwell et al.8 2 who saw an increase in antioxidant activity in serum which was maximal at 90 minutes following consumption of red wine. In both of the above studies no measurement was made of plasma polyphenolics and it is uncertain whether the observed changes in antioxidant capacity can be accounted for by the polyphenolics in red wine. Day & Stansbie8 3 have suggested that the acute increase in total antioxidant capacity of serum after port wine can be accounted for by increased levels of urate. Urate is a signi® cant contributor to the total plasma peroxyl radical scavenging capacity.8 4 Polyphenolic com poun ds in alcoho lic beverage sÐ other con sideration s Inhibition of lipoprotein oxidation may not be the only mechanism by which plant (or red wine)
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polyphenolic compounds can give possible protection against cardiovascular disease. Two red wine phenolics, resveratrol and quercetin, have been shown to block human platelet aggregation in vitro . 8 5 This eŒect is possibly due to inhibition of platelet thromboxane synthesis. In vivo studies in experimental animals suggest that platelet activation can be inhibited with red wine or grape juice.8 6 ,8 7 Both red wine and grape juice, but not white wine, inhibited thrombosis in stenosed canine coronary arteries8 6 while red wine or grape seed extracts were able to reduce the platelet 87 rebound eŒect seen after alcohol withdrawal. The suppression of the platelet rebound eŒect was related to the inhibition of alcohol-induced lipid peroxidation. Cer tain wines, grape juices and grape skin extracts have been shown to induce endothelium-dependent vasorelaxation which was mediated by the nitric oxide± cGMP pathway.8 8 If such responses were to occur in vivo they could conceivably also contribute to the protective eŒect of alcoholic beverages against atherosclerotic vascular disease, but would need to outweigh the vaso-constrictive eŒects of alcohol reported for several vascular beds, especially 89 the cerebrovasculature. Not all plant phenolics necessarily inhibit lipoprotein modi® cation, with reports that some ¯ avonoids increase LDL modi® cation.9 0 M any plant phenolics bind and reduce iron which can have oxidative eŒects on molecules other than lipids such as proteins and DNA. 9 1 ,9 2 Human atherosclerotic lesions contain available iron and copper ions 9 3 and the net in vivo eŒect of polyphenolics on atherosclerosis may not be easy to predict. One recent study has suggested that resveratrol increases atherosclerosis in cholesterol-fed rabbits. 9 4
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C, Testolin G. EŒect of alcohol intake on lipids and fat-soluble vitamins in blood. Miner va M ed 1993;84:4 47 ± 52. D’ Antonio JA, Laporte R E, Dai W S, H om DL, Wozniczak M , Kuller LH. Lipoprotein cholesterol, vitamin A, and vitamin E in an alcoholic population. Cancer 1986;57:17 98 ± 802. Ahm ed S, Leo M A, Lieber CS. Interactions between alcohol and beta-carotene in patients w ith alcoholic liver disease. Am J Clin Nutr 1994;60: 430 ± 6. Suzuki S, Sasaki R, Ito Y et al. Changes in serum concentrations of beta-carotene and changes in the dietary intake frequency of green-yellow vegetables among healthy male inhabitants of Japan. Jpn J Cancer Res 1990;81:4 63 ± 9. Rim m E, Colditz G . Sm oking, alcohol, and plasma levels of carotenes and vitamin E. Ann NY Acad Sci 1993;686: 323 ± 34. Forman M R, Beecher GR, Lanza E et al. EŒect of alcohol consumption on plasma carotenoid concentrations in premenopausal women: a controlled dietary study. Am J Clin Nutr 1995;62:1 31 ± 5. Leo MA, Kim C, Lowe N, Lieber CS. Interaction of ethanol w ith beta-carotene: delayed blood clearance and enhanced hepatotoxicty. Hepatology 1992;15: 883 ± 91. Ringstad J, Knutsen SF, Nilssen OR, Thom assen Y. A comparative study of serum selenium and vitamin E levels in a population of m ale risk drinkers and abstainers. A population-based m atched-pair study. B iol Trace Elem R es 1993;36: 65 ± 71. Lloyd B, Lloyd R, Clayton B. EŒect of sm oking, alcohol, and other factors on the selenium status of a healthy population. J Epidem iol Commun Health 1983;37:2 13 ± 7. Sergent O, M orel I, Cogrel P et al. Increase in cellular pool of low-molecular-weight iron during ethanol m etabolism in rat hepatocyte cultures. Relationship with lipid peroxidation. B iol Trace Elem Res 1995;47:18 5 ± 92. Shaw S. Lipid peroxidation, iron mobilization and radical generation induced by alcohol. Free R adic B iol Med 1989;7:541 ± 7. Shaper AG, Wannamethee G, Walker M . Alcohol and mortality in British m en: explaining the U shaped curve. Lancet 1988;2:126 7 ± 73. Jackson R, Scragg R, Beaglehole R. Alcohol consum ption and risk of coronary heart disease. B r J M ed 1991;303: 211 ± 16. W itztum JL. The oxidative hypothesis of atherosclerosis. Lancet 1994;344:7 93 ± 8. Kinsella JE, Frankel E, G erman B, Kanner J. Possible m echanisms for the protective role of antioxidants in wine and plant foods. Food Tech 1993; April:85 ± 9. Halliwell B. Antioxidants in wine (letter). Lancet 1993;341: 1538. Kanner J, Frankel E, Granit R, G erm an B, Kinsella JE. Natural antioxidants in grapes and wines. J Ag ric Food Chem 1994;42:64 ± 9. Abu-Amsh a R, Croft KD, Puddey IB, Proudfoot J, Beilin LJ. The phenolic content of various beverages
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determines the extent of serum and low denisty lipoprotein oxidation: identi® cation and m echanism of action of some cinnamic acid derivatives from red wine. Clin Sci 1996;91:4 49 ± 58. W hitehead TP, Robinson D, Allaway S, Syms J, Hale A. EŒect of red wine ingestion on the antioxidant capacity of serum. Clin Chem 1995;41:32 ± 5. Nardini M , D’ Aquino M, Tom assi G, G entili V, Felice M D, Scaccini C. Inhibition of hum an LDL oxidation by caŒeic acid and other hydroxycinnamic acid derivatives. Free Rad B iol M ed 1995;19: 541 ± 52. Halliwell B, Aeschbach R, Loliger J, Aruoma OI. The characterization of antioxidants. Food Chem Toxicol 1995;33:60 1 ± 17. Vinson JA, Dabbagh YA, Serry M M, Jang J. Plant ¯ avonoids, especially tea ¯ avonoids, are powerful antioxidants using an in vitro m odel for heart disease. J Agric Food Chem 1995;43:28 00 ± 2. Croft KD, Puddey IB, Rakic V, Abu-Am sha R, Dimm itt SB, Beilin LJ. Oxidative susceptibility of low density lipoproteins± in¯ uence of regular alcohol use. A lcohol Clin Exp Res 1996;20:98 0 ± 84. Suzukawa M , Ishikawa T, Yoshida H et al. EŒects of alcohol consumption on antioxidant content and susceptibility of low-density lipoprotein to oxidative modi® cation. J Am Coll Nutr 1994;13:23 7 ± 42. Fuhr man B, Lavy A, Aviram M. Consumption of red wine with meals reduces the susceptibility of hum an plasma and low-density lipoprotein to lipid peroxidation. Am J Clin Nutr 1995;61:5 49 ± 54. Sharpe PC, McGrath LT, M cClean E, Young IS, Archbold G P. EŒect of red wine consumption on lipoprotein (a) and other risk factors for atherosclerosis. Q JM 1995;88:1 01 ± 8. De Rijke YB, Demacker PNM, Assen NA et al. Red wine consumption does not eŒect oxidizability of low density lipoproteins in volunteers. Am J Clin Nutr 1996;63:32 9 ± 34. U nno T, Takeo T. Absorption of epigallocatechin gallate into the circulation system of rats. B iosci B iotech B iochem 1995;59:1 558 ± 9. Maxwell S, Cruickshank A, Thorpe G. Red wine and antioxidant acitivity in serum. Lancet 1994; 344:193 ± 4. Day A, Stansbie D. Cardioprotective eŒect of red wine m ay be mediated by urate. Clin Chem 1995; 41:1319 ± 20.
84. Wayner DDM, Burton G W, Ingold KU, Barclay LRC, Locke SJ. The relative contributions of vitam in E, urate, ascorbate and proteins to the total peroxyl radical trapping antioxidant activity of hum an plasma. B iochim B iophys A cta 1987;924: 408 ± 19. 85. Pace-Asciak CR, H ahn S, Diamandis EP, Soleas G , Goldberg DM . The red wine phenolics transresveratrol and quercetin block hum an platelet aggregation and eicosanoid synthesis: implications for protection against coronary heart disease. Clin Chim Acta 1995;235: 207 ± 19. 86. Demrow HS, Slane PR, Folts JD. Adm inistration of w ine and grape juice inhibits in vivo platelet activity and thrombosis in stenosed canine coronary arteries. Circulation 1995;91:11 82 ± 8. 87. Ruf JC, Berger JL , Renaud S. Platelet rebound eŒect of alcohol withdrawal and wine drinking in ratsÐ relation to tannins and lipid peroxidation. Arter ioscler Thromb Vasc B iol 1995;15:14 0 ± 4. 88. Fitzpatrick DF, Hirsch® eld SL, CoŒey RG. Endothelium-dependent vasorelaxing activity of wine and other grape products. Am J Physiol 1993;265: H 774 ± 8. 89. Mayhan W G, Didion SP. Acute eŒects of ethanol on responses of cerebral arterioles. Stroke 1995;26: 2097 ± 101. 90. Rankin SM , Dewhalley CV, Hoult JR et al. T he m odi® cation of low density lipoprotein by the ¯ avonoids m yricetin and gossypetin. B iochem Phar macol 1993;45:6 7 ± 75. 91. Laughton MJ, Halliwell B, Evans PJ, Hoult JR. Antioxidant and pro-oxidant actions of the plant phenolics quercetin, gossipol and m yricetin. B iochem Phar macol 1989;38:28 59 ± 65. 92. Ahm ed MS, Ainley K. Parish JH, H adi SM. Free radical-induced fragmentation of proteins by quercetin. Carcinogenesis 1994;15:1 627 ± 30. 93. Sm ith C, M itchinson J, Ar uoma OI, Halliwell B. Stim ulation of lipid peroxidation and hydroxylradical generation by the contents of hum an atherosclerotic lesions. B iochem J 1992;286:9 01 ± 5. 94. W ilson T, Knight T J, Beitz DC, Lewis DS, Engen DC. Resveratrol promotes atherosclerosis in hypercholesterolemic rabbits. Life Sci 1996;59:P L15 ± 21.
INVITE D R E VIEW
Alcoholic beverages and lipid peroxidation: relevance to cardiovascular disease IAN B. PU DDEY & KEVIN CROFT Departm ent of M edicine and Western Australian H eart Research Institute, University of Western Australia, Australia
Abstract Overall there is good evidence that alcohol consum ption induces oxidative stress, and leads to lipid peroxidation, e Œects which have been linked to alcohol-related toxicity and disease and m ay be relevant to alcoholatherosclerosis inter relationships. On the other hand, a protective e Œect of light to m oderate alcohol consum ption against cardiovascula r disease is well recognized, with the further hypoth esis that red wine o Œers extra cardiovascula r protection due to its rich content of antiox idant phenolic com pound s. Although this hypothesis is given some credence from in vitro data, controlled stud ies in hum ans have produced con¯ icting results. Clearly, the equally well described pro-oxidant e Œects of alcohol and its m etabolism have been insu ciently consid ered in the pursuit of what to many is an intuitiv ely attractive hypoth esis. Further stud ies are required to deter m ine if red wine phenolics are actua lly absorbed from the gut and wheth er they o Œer any overall antioxid ant protection in vivo. The hypothesis that red wine o Œers extra cardiovascula r protection com pared to oth er alcoholic beverages is not proven and m ust await the outcom e of studies in which the full spectr um of the pro-oxidant and antioxid ant e Œects of alcoholic beverages are duly consid ered. In the absence of such studies, there are no ground s at present for the promotion of the consum ption of alcoholic beverages on the basis of their putative ``antiox idant’ ’ properties.
Introdu ction In attempting to understand the possible protective eŒect of alcohol against atherosclerotic cardiovascular disease, several eŒects of alcohol on lipoproteins have been canvassed together with other potential mechanisms.1 ,2 M ore recently, an anti-oxidant eŒect of certain alcoholic beverages, particularly red wine, has been proposed 3 on the basis of the oxidative hypothesis of atherogenesis. This evolving hypothesis points to free radical generation with subsequent oxidat-
ive modi® cation of low density lipoprotein-cholesterol (LDL) as possibly playing a major role in the development of the atherosclerotic lesion.4 A free radical is an atom or molecule that contains one or more unpaired electrons. They are generally represented in chemical formula by a dot, e.g. HO ´. Free radicals are produced in the body as part of normal metabolism, for exam ple superoxide, O ´22 and nitric oxide, NO ´, which have important physiological functions. In general, free radicals are highly reactive and can
Correspondence to: Dr Kevin Croft, Department of Medicine and Western Australian Heart Research Institute, University of Western Australia, Royal Perth H ospital, GPO Box X2213, Perth, Western Australia, 6000, Australia. e-m ail: [email protected] Received for publication 17th June 1996. Accepted 5th October 1996. 1355 ± 6215/97/030269 ± 08 Society for the Study of Addiction to Alcohol and Other Drugs
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attack membrane lipids, for example generating a carbon radical which in turn reacts with oxygen to produce a peroxyl radical which may attack adjacent fatty acids to generate new carbon radicals. This process leads to a chain reaction producing lipid peroxidation products.5 By this means a single radical m ay damage m any molecules by initiating lipid peroxidation chain reactions. Because of the potential damaging nature of free radicals the body has a number of antioxidant defence m echanisms which include enzymes such as superoxide dismutase, catalase, copper and iron transport and storage proteins and both water soluble and lipid soluble molecular antioxidants. Oxidative stress m ay result when antioxidant defences are unable to cope with the production of free radicals, and may result from the action of certain toxins or by physiological 5 stress. The attraction of the oxidative model for atherosclerosis, in attempting to understand any cardioprotective eŒect of alcoholic beverages, relates to the observation that polyphenolic constituents of wines, especially red wine3 ,6 ,7 can, at least in vitro , inhibit oxidation of LDL . These in vitro observations have been paralleled by population studies that suggest a greater cardiovascular protective advantage from the ingestion of wine compared to other alcoholic beverages.8 ± 1 0 As a consequence, it has been hypothesized that these substances may be responsible for the so-called French paradox, whereby a lower incidence of ischaemic heart disease is seen among the French despite a relatively high saturated fat intake.1 1 A recent review of the literature has however found no convincing evidence that wine is more protective against coronary disease than other alcoholic beverages such as beer and spirits.1 2 There is a further resulting tension in that this hypothesis needs to also encompass the simultaneous persuasive evidence for a pro-oxidant eŒect of alcohol itself to induce lipid peroxidation, an eŒect which has been linked to alcohol-related toxicity and disease in several organ systems.
Alcoh ol as a pro-oxidan tÐ the evidence The possibility that ethanol or ethanol metabolism may lead to lipid peroxidation was ® rst raised by Di Luzio, who reported prevention of ethanolinduced fatty liver in rats who were simultaneously administered anti-oxidants1 3 and later observed increased formation of thiobarbituric
acid reactive substances when ethanol was added to normal rat liver homogenates.1 4 A growing body of evidence has since pointed to a possible primary role of ethanol-induced peroxidation in the aetiology of alcoholic liver disease. 1 5 ,1 6 Corroboration of such a m echanism has been provided by the ® nding of increased chemiluminescence, malondialdehyde production and diene formation in livers from rats acutely treated with alcohol.1 7 In primary rat hepatocyte cultures the metabolism of ethanol has also been shown to induce lipid peroxidation as evidenced by an 18 increase in free m alondialdehyde. Similarly, studies in rats treated for 15 months with ethanol demonstrated enhanced hepatic microsomal 19 malondialdehyde formation and there is evidence of increased urinary m alondialdehyde excretion in rats after both acute and chronic 20 alcohol administration. However, an increase in lipid peroxidation during chronic ethanol ingestion has not always been reported 2 1 and in both the acute and chronic rat studies, no eŒect on diene conjugates has been seen.1 8 ,1 9 Whether the administration of alcohol in vivo can also result in lipid peroxidation has been the 22 subject of continuing review and debate. More sophisticated approaches, with direct m easurement of free radical production through the assay of oxidation of chemical scavengers, are consistent with the thesis that the hepatic metabolism of alcohol leads to local free radical generation.2 3 ,2 4 The possibility that ethanol or its major metabolic product, acetaldehyde, can lead to the production of free radical species in other sites, where ethanol may not necessarily be m etabolised, has been recently reviewed 2 5 ,2 6 with the conclusion that a pro-oxidant eŒect of alcohol may also be responsible for alcohol-related toxicity and injury in other tissues. Studies in alcoholics are also consistent with the presence of systemic pro-oxidant stress with elevation of another marker of increased free radical activity, the m olar proportion of linoleic acid that is diene conjugated, which falls during subsequent abstinence from alcohol.2 7 ,2 8 Utilizing intragastric feeding of alcohol to rats, a systemic pro-oxidant eŒect is also suggested by the ® nding of higher plasma isoprostane (a non-cyclo-oxygenase prostanoid) levels, a unique in vivo marker of lipid peroxidation.2 9 In other rat studies increased ethane exhalation has been put forward as a m arker of alcohol-induced lipid peroxidation,3 0 while in man, ethane exhalation in alcohol abusers has
Alcoholic beverages and lipid peroxidation
been only weakly correlated with the level of daily ethanol intake reported before hospital admission.3 1 There is good evidence to implicate pro-oxidant stress in the pathogenesis of ethanol-related neurotoxicity3 2 and cerebellar degeneration, 2 5 33 ethanol-induced damage to gastric mucosa, mem brane injury to erythrocytes,3 4 alcoholic cardiomyopathy 3 5 and the prom otion by ethanol or oral carcinogenesis3 6 and oesophageal tumour growth.3 7 ,3 8 W ith such a broad range of sites where it may induce oxidative stress, a role for alcohol-induced lipid peroxidation in the promotion rather than prevention of atherosclerosis remains a possibility, especially with heavy alcohol intake and long-term alcohol abuse. In this setting, the recognized protective eŒect of light alcohol consumption may disappear, and increased carotid atherosclerosis with heavy alcohol consumption has been reported in population studies in man.3 9 ± 4 1 However, in alcohol-fed rats, despite the exhalation of greater quantities of ethane and an increase in aortic concentrations of cholesterol and phospholipids, no detectable change in the levels of aortic wall lipid peroxides 30 was seen.
M echan ism of the pro-oxidant stress indu ced by ethano l The pro-oxidant stress induced by ethanol m ay be a direct result of the production of free radical species during several stages of the metabolism of alcohol.2 6 ,4 2 Ethanol is oxidized to acetaldehyde by alcohol dehydrogenase, the microsomal ethanol oxidizing system and by catalase. The ® rst two pathways represent possible points at which excess free radical generation m ay occur. The induction of hepatic m icrosomal cytochrome P450 2E1 in habitual abusers of alcohol can 43 result in the production of free radical species, with m icrosomes producing both superoxide and H 2 O 2 . 3 8 ,4 4 Acetaldehyde production may cause 45 lipid peroxidation, the metabolism of acetaldehyde via xanthine oxidase or aldehyde oxidase is capable of generating free radicals. More recently, a vicious cycle has been proposed4 6 with alcohol dehydrogenase reducing NAD + to NADH which in turn is oxidized by aldehyde oxidase generating reactive oxidative species plus NAD + which is then available again for reduction by alcohol dehydrogenase. Ethanol or acetaldehyde, although not neces-
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sarily actively oxidised at other sites in the body, may still induce oxidative stress by several other postulated or dem onstrated m echanisms. Acetal4 5 ,4 7 dehyde can bind with cysteine or glutathione, both potentially important pathways for scavenging free radicals and prevention of lipid peroxidation. Ethanol appears to alter the activities of the anti-oxidant enzymes, copper± zinc superoxide dismutase and glutathione peroxidase,1 8 ,4 8 an action which may be direct or m ediated by eŒect of ethanol to decrease tissue concentrations of zinc and copper. In animal studies however, similar eŒects of alcohol have not been shown in cardiac tissue where Cu,Zn-superoxide dismutase and glutathione-S-transferase were higher when compared to controls while M n-superoxide dismutase, catalase and glutathione peroxidase activities were unaltered.3 5 These paradoxical changes could be understood possibly as adaptive responses to increased free radical production. In this context, rats chronically fed alcohol have demonstrated increases in both plasma alphatocopherol levels and hepatic glutathione despite increases in hepatic malonaldehyde.4 9 In cell culture, direct evidence of an eŒect of ethanol to interfere with anti-oxidant defence mechanisms has been provided, with alterations in cell phospholipid structure associated with a fall in alpha-tocopherol levels.5 0 Ethanol feeding markedly decreased both alpha and gammatocopherol in livers of normal and vitamin E de® cient rats but plasma levels fell only in normal 51 rats. Similarly, lower hepatic vitamin E content and lower serum vitamin E levels have been reported in alcoholics in some,5 2 ± 5 4 but not all studies.5 5 Beta carotene levels have been found to be lower in alcoholics and heavy drinkers 5 5 ,5 6 and even in healthy men changes in serum concentration of beta carotene have been negatively associated with changes in alcohol intake.5 7 This ® nding has not been consistent, however, with a positive correlation between beta carotene levels and alcohol intake in one study,5 6 and no correlation in another after allowing for the eŒects of smoking and diet.5 8 In a recent controlled study in premenopausal women alcohol increased plasma carotene but signi® cantly lowered lutein /zeaxanthin.5 9 These diŒerences may also re¯ ect direct eŒects of alcohol on beta-carotene metabolism, with delays in the clearance of beta carotene demonstrated in studies in primates.6 0 Levels of the anti-oxidant trace elem ent, selenium, have been consistently shown to be lower in
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serum and tissues of alcohol abusers and may also contribute to pro-oxidant stress. An eŒect of alcohol to mobilise and increase the cellular pool of redox-active iron is another potential pathway for alcohol-related pro-oxidant stress, with iron being an important metal catalyst 1 8 ,6 3 ,64 in initiating lipid peroxidation.
Alcoh olic beverag es and L D L oxidationÐ in vitro studies The regular light to moderate consumption of alcohol may protect against atherosclerotic cardiovascular disease, an eŒect that is negated or reversed at higher levels of intake.1 ,2 It has been argued that this protective eŒect of m oderate alcohol consumption compared to abstainers is the result of migration of drinkers to the 65 abstainer group after they become ill. Others have argued that there is still a protective eŒect of moderate alcohol consumption even after this possible problem has been allowed for. 6 6 Given the recent convincing evidence that lipid peroxidation, in particular oxidative damage to LDL, may play a critical role in the development of 67 atherosclerotic lesions it is not surprising that there has been a focus by several investigators on the in vitro eŒects of alcoholic beverages on LDL oxidation. Studies by Frankel et al. 3 were among the ® rst to show that diluted red wine (with the ethanol rem oved) inhibited copper-induced LDL oxidations measured by hexanal form ation. It was suggested that the antioxidant eŒects were due to phenolic compounds which are particularly rich in red wine.6 8 The antioxidant eŒects were found to be more substantial than vitamin E; however, since vitamin E added externally to isolated LDL is poorly incorporated, it may exert only weak antioxidant activity compared to its eŒect when incorporated from the diet. It has been suggested that this may have led to an overstatement of the antioxidant eŒect of red wine.6 9 Several other studies have since con® rmed that, in vitro , red 7 0 ,7 1 wine is a potent inhibitor of LDL oxidation and can increase the antioxidant capacity of serum.7 2 In one of these studies polyphenolics were shown to be the primary antioxidants because when they were stripped from the red wine or grape juice all antioxidant activity was lost.7 1 The major ¯ avonoid constituents of red wine have been analysed and their in vitro antioxidant activity in inhibiting LDL oxidation has been determined. 7 Some cinnamic acid derivat-
ives have also been identi® ed in red wine and these also act as potent inhibitors of LDL oxidation.7 1 ,7 3 These polyphenolic compounds are not limited to red wine and are found in m any fruits and vegetables 7 4 and are particularly rich in tea.7 5 All in vitro studies are consistent with polyphenolic compounds derived from grapes and other plants as providing antioxidant protection against LDL oxidation. What is less clear is which polyphenolic compounds are absorbed and whether they are able to oŒer antioxidant protection in vivo .
Alcoholic beverage s and L DL oxidationÐ in vivo studies There is relatively little information on the eŒect of alcohol in vivo on LDL oxidation. One might predict that since alcohol (as outlined in the preceding discussion) can induce oxidative stress and reduce levels of natural antioxidants in the circulation, then LDL is likely to be m ore sensitive to oxidative damage following alcohol ingestion. In a recent controlled cross-over study conducted by us during which regular beer drinkers consumed a high versus a low alcohol beer, an increased susceptibility of LDL to oxidation was observed during the high alcohol phase.7 6 However another recent study using brandy as the source of alcohol found no eŒect of alcohol (0.5 g /kg /day over 4 weeks) on LDL oxidation,7 7 although b -carotene levels in plasma and LDL decreased signi® cantly with alcohol intake indicating a predominant pro-oxidant stress. The eŒect of an alcoholic beverage on lipoprotein oxidation in vivo may ultimately depend on the balance between the pro-oxidant eŒects of alcohol and the level of antioxidant polyphenolic compounds in the beverage. Several controlled studies in humans have now been reported looking at the eŒect of red wine on lipid and LDL oxidation. Fuhrman et al.7 8 compared red and white wine (containing 11% alcohol) consumed with meals (400 m l /day) for a period of 2 weeks, on the level of lipid peroxides in plasma and the susceptibility of LDL to oxidation. This study reported a substantial prolongation of the lag phase to initiation of LDL oxidation for subjects consuming red wine, while LDL isolated from subjects consuming white wine was m ore susceptible to oxidation. Two other studies, however, have found no such antioxidant eŒect of red wine. Sharpe et al. 7 9 studied 20 volunteers who were
Alcoholic beverages and lipid peroxidation
given either red or white wine (200 ml /day) for 10 days but with no eŒect on the susceptibility of LDL to oxidation. The lack of any antioxidant eŒect may have been due to the lower dose of red wine and the slightly shorter treatment period. However, this was not the case in another study in which subjects received 550 ml /day of red wine over 4 weeks with no eŒect at all on the oxidizability of LDL or level of antioxidants in plasma.8 0 In the latter study the alcohol content of the wines was reduced to 3.5% in order to remove possible interfering eŒects of alcohol, and slightly diŒerent methods were used to estimate LDL oxidation than Fuhrman et al. . 7 8 The extent to which these diŒerences can explain the discrepancy in results is di cult to estimate. None of the studies measured the absorption of any polyphenolic substance, although Fuhrman et al. 7 8 reported a substantial increase in LDL associated total polyphenols using a crude spectrophotometric method. From limited animal studies it would appear that polyphenolics such as catechins after oral administration reach maximal plasma concentrations after 1 hour then decrease quickly.8 1 Therefore, in red wine studies the time of blood sampling after the last drink m ay be a critical factor. Another approach to the study of the possible antioxidant eŒects of red wine has been to measure changes in total antioxidant capacity of serum. Using this method Whitehead et al.7 2 observed an 18% increase in serum antioxidant capacity 1 hour after drinking 300 ml of red wine. No signi® cant increase was seen in subjects drinking white wine. Similar results were observed by M axwell et al.8 2 who saw an increase in antioxidant activity in serum which was maximal at 90 minutes following consumption of red wine. In both of the above studies no measurement was made of plasma polyphenolics and it is uncertain whether the observed changes in antioxidant capacity can be accounted for by the polyphenolics in red wine. Day & Stansbie8 3 have suggested that the acute increase in total antioxidant capacity of serum after port wine can be accounted for by increased levels of urate. Urate is a signi® cant contributor to the total plasma peroxyl radical scavenging capacity.8 4 Polyphenolic com poun ds in alcoho lic beverage sÐ other con sideration s Inhibition of lipoprotein oxidation may not be the only mechanism by which plant (or red wine)
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polyphenolic compounds can give possible protection against cardiovascular disease. Two red wine phenolics, resveratrol and quercetin, have been shown to block human platelet aggregation in vitro . 8 5 This eŒect is possibly due to inhibition of platelet thromboxane synthesis. In vivo studies in experimental animals suggest that platelet activation can be inhibited with red wine or grape juice.8 6 ,8 7 Both red wine and grape juice, but not white wine, inhibited thrombosis in stenosed canine coronary arteries8 6 while red wine or grape seed extracts were able to reduce the platelet 87 rebound eŒect seen after alcohol withdrawal. The suppression of the platelet rebound eŒect was related to the inhibition of alcohol-induced lipid peroxidation. Cer tain wines, grape juices and grape skin extracts have been shown to induce endothelium-dependent vasorelaxation which was mediated by the nitric oxide± cGMP pathway.8 8 If such responses were to occur in vivo they could conceivably also contribute to the protective eŒect of alcoholic beverages against atherosclerotic vascular disease, but would need to outweigh the vaso-constrictive eŒects of alcohol reported for several vascular beds, especially 89 the cerebrovasculature. Not all plant phenolics necessarily inhibit lipoprotein modi® cation, with reports that some ¯ avonoids increase LDL modi® cation.9 0 M any plant phenolics bind and reduce iron which can have oxidative eŒects on molecules other than lipids such as proteins and DNA. 9 1 ,9 2 Human atherosclerotic lesions contain available iron and copper ions 9 3 and the net in vivo eŒect of polyphenolics on atherosclerosis may not be easy to predict. One recent study has suggested that resveratrol increases atherosclerosis in cholesterol-fed rabbits. 9 4
R eferen ces 1. Srivastava LM , Vasisht S, Agarwal DP, G oedde H W. Relation between alcohol intake, lipoproteins and coronary heart disease: the interest continues. Alcohol A lcohol 1994;29:11 ± 24. 2. G oldberg DM, H ahn SE, Parkes JG. Beyond alcohol: beverage consumption and cardiovascular m ortality. Clin Chim Acta 1995;237: 155 ± 87. 3. Frankel EN, Kanner J, German JB, Parks E, Kinsella, JE. Inhibition of oxidation of hum an lowdensity lipoprotein by phenolic substances in red wine. Lancet 1993;341:4 54 ± 7. 4. W itztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest 1991;88:1 785 ± 92.
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