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Moderate alcohol consumption and platelet aggregation in healthy middle-aged men
Alcohol, Vol. 7, pp. 547-549. ©Pergamon Press plc, 1990. Printed in the U.S.A.
0741-8329/90 $3.00 + .00
BRIEF COMMUNICATION
Moderate Alcohol Consumption and Platelet Aggregation in Healthy Middle-Aged Men I J A N V E E N S T R A , H E T T I E VAN DE P O L AND GERTJAN SCHAAFSMA
Department of Human Nutrition, TNO-CIVO Toxicology and Nutrition Institute P.O. Box 360, 3700 A J, Zeist, Netherlands R e c e i v e d 21 N o v e m b e r 1989; A c c e p t e d 23 April 1990
VEENSTRA, J., H. VAN OE POL AND G. SCHAAFSMA. Moderate alcohol consumption and platelet aggregation in healthy middle-aged men. ALCOHOL 7(6) 547-549, 1990.--Changes in platelet aggregation have often been proposed as an explanation for the protective effect of moderate alcohol consumption on the development of coronary heart disease, observed in epidemiological studies. To test the tenability of this assumption, the acute effect of moderate alcohol consumption on platelet aggregation was studied in eight healthy middle-aged men in a controlled study. The intake of alcohol consisted of two glasses of red wine at dinner and two glasses of distilled liquor (Hollands gin), combined with a snack, before bedtime. No acute effects of moderate alcohol consumption on platelet aggregation were observed. Alcohol consumption
Platelet aggregation
Human healthy volunteers
Coronary heart disease
of the present study was to investigate the acute effects of alcohol on platelet aggregation under carefully controlled conditions mimicking normal life as closely as possible.
IN many epidemiological studies a protective effect of moderate alcohol consumption on coronary heart disease (CHD) has been observed (9, 12, 15, 16). One of the proposed explanations is that moderate alcohol consumption reduces platelet aggregation. In man, the acute effects of alcohol on platelet aggregation have been investigated in a number of studies (2-8, 11, 13). The results, however, are conflicting. No effects (2, 5, 8, 13), inhibitory effects (3, 4, 11) as well as stimulative effects (6,7) of alcohol consumption on platelet aggregation have been reported. This discrepancy may be explained by the large diversity in study protocols. In three studies (4, 6, 7) the volunteers consumed 100 g of alcohol or more (1 drink contains approximately 10 g), and in one study (2) alcohol was taken ad lib for four hours. In the other studies the amount of alcohol consumed varied broadly between 15 and 90 g. Furthermore, the studies varied considerably in type of alcoholic beverage (e.g., whiskey, white wine and alcohol in fruit juice). In all studies mentioned, the alcohol was consumed on an empty stomach, mostly in the morning. Until now, no data are available on the acute effects on platelet aggregation of a moderate dose of alcohol, consumed at a customary time of the day and under habitual conditions. The aim
METHOD
Subjects Eight apparently healthy, nonsmoking volunteers, between 45 and 55 years of age, participated in this study. Their average habitual consumption of alcohol varied between 10 and 40 g per day. They all had a normal liver function, had no familial history of alcoholism and were used to a normal, Western lifestyle, including dietary habits. No drugs were used during the experiment. The study protocol was approved by the Institute's external Medical-Ethical Committee and the participants signed an informed consent form.
Study Design The total experimental period lasted four weeks in which the subjects received four treatments, each of which lasted three days.
~Supported by the Central Commodity Board for Arable Products, The Hague, Netherlands and the Commodity Board for Spirits, Schiedam, Netherlands.
547
548
VEENSTRA, VAN OE POL AND SCHAAFSMA
During these days, the subjects were housed in the metabolic ward of the department and were not allowed to consume any food or drinks but the meals and drinks supplied. The four treatments comprised two different standard diets, one rich in saturated fatty acids and the other rich in polyunsaturated fatty acids, each at two different alcohol regimes, one with and one without alcohol (factorial design). For each subject the experimental days were always on the same days of the week. The first two days, in which no alcohol was consumed, served as a run-in period. On the third day 40 g of alcohol was consumed, two glasses of red wine during dinner and two glasses of Hollands gin just before bedtime combined with a snack, 5 hours after dinner. If no alcohol was given, exactly the same volumes of mineral water were consumed. Blood samples were taken at four moments; 1) three hours before dinner, 2) one hour after dinner, 3) one hour after the snack and 4) the next morning. Compliance was tested by measuring the blood alcohol concentration. All subjects were subjected to all four treatments in a randomized order, according to a Latin square design. Analyses Blood was taken from a vena mediana cubiti by means of a catheter. For platelet aggregation analyses 20 ml blood was collected by gentle suction into 200 ixl of 320 g/1 sodium citrate pH 7.4. Aggregation experiments were carried out in platelet-rich plasma (PRP) containing 220 × 106 platelets/ml in a Lumi Dual aggrometer (Chrono-Log Corp., Havertown, PA). After 3 minutes of preincubation at 37°C of 450 Ixl PRP, 50 txl aliquots of collagen or ADP suspensions were added to induce aggregation. Aggregation curves were recorded for 5 minutes. For collagen (Hormon Chemie, Miinchen, FRG) the final concentrations used were 0.2, 0.4 and 0.7 mg/1 and for ADP (Boehringer-Mannheim, Mannheim, FRG) 0.5, 1.0 and 2.0 IxM. From the aggregation curves the percentage of maximum aggregation and the velocity of aggregation were obtained. For each sample separately, the percentage of maximum aggregation or velocity of aggregation was plotted against the collagen or ADP concentrations. The areas under the curves thus obtained were used as derived variables. The use of these derived variables reduces the number of variables obtained from aggregation studies and also increases the precision of the aggregation parameters. Platelets were counted on a Sysmex CC-180 Microcellcounter (TOA Medical Electronics Co. Ltd., Carson, CA). Blood alcohol concentrations were determined enzymatically by the method of Beutler and Michal (1), using Boehringer biochemicals (Boehringer, Mannheim, FRG). Statistics Statistical evaluation comprised analysis of variance, according to the design, to test the significance of the main effects of alcohol and meal composition as well as their two-factor interactions. The measurements at moment l (before alcohol consumption) were used as a covariate. RESULTS
The mean blood alcohol concentration one hour after wine consumption was 0.12 g/1 (range 0.05 to 0.20 g/l) and one hour after the two glasses of Hollands gin 0.23 g/1 (range 0.16 to 0.32 g/l). After consumption of mineral water the blood alcohol concentration was always 0.00 g/1. In all subjects platelet counts were normal at the start of the
TABLE 1 THE ACUTE INFLUENCE OF MODERATE ALCOHOL CONSUMPTION ON PLATELET AGGREGATION IN HEALTHY MIDDLE-AGED VOLUNTEERS*
Parameter
Alcohol
Before Dinner
1 hr After Dinner
I hr After the Snack
The Next Morning
216 237 0.223
197 198 0.955
212 226 0.467
163 176 0.420
147 143 0.808
167 174 0.726
437 423 0.675
413 387 0.382
420 445 0.107
503 516 0.460
514 495 0.538
519 515 0.864
Collagen Percentage of aggregation p-value
+
Velocity aggregation p-value
+
221
169
ADP Percentage of aggregation p-value
+
Velocity of aggregation p-value
+
435
546
*Values refer to the area under the curve for percentage of aggregation and velocity of aggregation and are expressed in arbitrary units (see the Method section).
study (mean 195 109/1, range 154-297) and remained unchanged during the different treatments. The results of the platelet aggregation measurements are summarized in Table 1. There were no significant effects of alcohol or diet, or two-factor interactions between alcohol and diet on platelet aggregation. The percentage of maximum aggregation and the velocity of each collagen and ADP-induced aggregation was also statistically analyzed separately and in none of these analyses significant effects of alcohol were observed. DISCUSSION
Our study demonstrates that moderate alcohol consumption has no acute effects on platelet aggregation in healthy middle-aged men. Thus, the reported protective effect of alcohol against CHD is not likely to be attributable to such an acute effect. In a previous study (14), we have investigated the effects of 5 weeks of moderate alcohol consumption in a controlled study and observed an alcohol-induced decrease in platelet aggregability. Four days of moderate alcohol consumption, however, did not influence platelet aggregation (submitted for publication). Alcohol consumption and platelet aggregability has also been investigated in an epidemiological study (958 participants) by Meade et al. (10). They report a decrease in platelet aggregation with increasing dally alcohol consumption. We conclude that a moderate intake of alcohol has no acute effect on platelet aggregation in habitual moderate drinkers. Daily moderate alcohol consumption, on the other hand, causes a decreased platelet aggregability in the long term. This long-term effect of alcohol may, among other mechanisms, play a role in the proposed protection of moderate alcohol consumption against CHD. However, one should bear in mind that platelet aggregation is always measured ex vivo and that aggregability in vivo depends on many other factors.
ALCOHOL AND PLATELET AGGREGATION
549
REFERENCES 1. Beutler, H. O.; Michal, G. Neue methode zur enzymatischen Bestimmung von ~thanol in Lebensmitteln. Z. Anal. Chem. 284:113-117; 1977. 2. Dunn, E. L.; Cohen, R. G.; Moore, E. E.; Hamstra, R. D. Acute alcohol ingestion and platelet function. Arch. Surg. 116:1082-1083; 1981. 3. Elm6r, 0.; G6ransson, G.; Zoucas, E. Impairment of primary hemostasis and platelet function after alcohol ingestion in man. Haemostasis 14:223-228; 1984. 4. Fenn, C. G.; Littleton, J. M. Interactions between ethanol and dietary fat in determining human platelet function. Thromb. Haemost. 51: 50-53; 1984. 5. Galli, C.; Colli, S.; Gianfranceschi, G.; Madema, P.; Petroni, A.; Tremoli, E.; Marinovich, M.; Sirtori, C. R. Acute effects of ethanol, caffeine, or both on platelet aggregation, thromboxane formation, and plasma-free fatty acids in human subjects. Drug Nutr. Interact. 3:61--67; 1984. 6. Hillbom, M.; Kangasaho, M.; Kaste, M.; Numminen, H.; Vapaatalo, H. Acute ethanol ingestion increases platelet reactivity: Is there a relationship to stroke? Stroke 16:19-23; 1985. 7. Hillbom, M.; Kangasaho, M.; L6wbeer, C.; Kaste, M.; Muuronen, A.; Numminen, H. Effects of ethanol on platelet function. Alcohol 2:429--432; 1985. 8. Hillbom, M.; Muuronen, A.; Neiman, J.; Bj6rk, G.; Egberg, N.; Kangasaho, M. Effects of vitamin E therapy on ethanol-induced changes in platelet aggregation, thromboxane formation, factor VII/
levels and serum lipids. Eur. J. Clin. Invest. 17:68-74; 1987. 9. Marmot, M. G. Alcohol and coronary heart disease. Int. J. Epidemiol. 13:160-167; 1984. 10. Meade, T. W., Vickers, M. V.; Thompson, S. G.; Stifling, Y.; Haines, A. P.; Miller, G. J. Epidemiologicai characteristics of platelet aggregability. Br. Med. J. 290:428-432; 1985. 11. Mikhailidis, D. P.; Jeremy, J. Y.; Barradas, M. A.; Green, N.; Dandona, P. Effect of ethanol on vascular prostacyclin (prostaglandin I2) synthesis, platelet aggregation, and platelet thromboxane release. Br. Med. J. 287:1495-1498; 1983. 12. Moore, R. D.; Pearson, T. A. Moderate alcohol consumption and coronary artery disease. Medicine 65:242-267; 1986. 13. Neiman, J.; Jones, A. W.; Numminen, H.; Hillbom, M. Combined effect of a small dose of ethanol and 36 hr fasting on blood-glucose response, breath-acetone profiles and platelet function in healthy men. Alcohol Alcohol. 22:265-270; 1987. 14. Pikaar, N. A.; Wedel, M.; van der Beek, E.; van Dokkum, W.; Kempen, H. J. M.; Kluft, C.; Ockhuizen, T.; Hermus, R. J. J. Effects of moderate alcohol consumption on platelet aggregation, fibrinolysis, and blood lipids. Metabolism 36:538-547; 1987. 15. Rohan, T. E. Alcohol and ischemic heart disease, a review. Aust. N. Z. J. Med. 14:75-80; 1984. 16. Veenstra, J. Moderate alcohol use and coronary heart disease: A U-shaped curve? World Rev. Nutr. Diet. Accepted for publication; 1990.
0741-8329/90 $3.00 + .00
BRIEF COMMUNICATION
Moderate Alcohol Consumption and Platelet Aggregation in Healthy Middle-Aged Men I J A N V E E N S T R A , H E T T I E VAN DE P O L AND GERTJAN SCHAAFSMA
Department of Human Nutrition, TNO-CIVO Toxicology and Nutrition Institute P.O. Box 360, 3700 A J, Zeist, Netherlands R e c e i v e d 21 N o v e m b e r 1989; A c c e p t e d 23 April 1990
VEENSTRA, J., H. VAN OE POL AND G. SCHAAFSMA. Moderate alcohol consumption and platelet aggregation in healthy middle-aged men. ALCOHOL 7(6) 547-549, 1990.--Changes in platelet aggregation have often been proposed as an explanation for the protective effect of moderate alcohol consumption on the development of coronary heart disease, observed in epidemiological studies. To test the tenability of this assumption, the acute effect of moderate alcohol consumption on platelet aggregation was studied in eight healthy middle-aged men in a controlled study. The intake of alcohol consisted of two glasses of red wine at dinner and two glasses of distilled liquor (Hollands gin), combined with a snack, before bedtime. No acute effects of moderate alcohol consumption on platelet aggregation were observed. Alcohol consumption
Platelet aggregation
Human healthy volunteers
Coronary heart disease
of the present study was to investigate the acute effects of alcohol on platelet aggregation under carefully controlled conditions mimicking normal life as closely as possible.
IN many epidemiological studies a protective effect of moderate alcohol consumption on coronary heart disease (CHD) has been observed (9, 12, 15, 16). One of the proposed explanations is that moderate alcohol consumption reduces platelet aggregation. In man, the acute effects of alcohol on platelet aggregation have been investigated in a number of studies (2-8, 11, 13). The results, however, are conflicting. No effects (2, 5, 8, 13), inhibitory effects (3, 4, 11) as well as stimulative effects (6,7) of alcohol consumption on platelet aggregation have been reported. This discrepancy may be explained by the large diversity in study protocols. In three studies (4, 6, 7) the volunteers consumed 100 g of alcohol or more (1 drink contains approximately 10 g), and in one study (2) alcohol was taken ad lib for four hours. In the other studies the amount of alcohol consumed varied broadly between 15 and 90 g. Furthermore, the studies varied considerably in type of alcoholic beverage (e.g., whiskey, white wine and alcohol in fruit juice). In all studies mentioned, the alcohol was consumed on an empty stomach, mostly in the morning. Until now, no data are available on the acute effects on platelet aggregation of a moderate dose of alcohol, consumed at a customary time of the day and under habitual conditions. The aim
METHOD
Subjects Eight apparently healthy, nonsmoking volunteers, between 45 and 55 years of age, participated in this study. Their average habitual consumption of alcohol varied between 10 and 40 g per day. They all had a normal liver function, had no familial history of alcoholism and were used to a normal, Western lifestyle, including dietary habits. No drugs were used during the experiment. The study protocol was approved by the Institute's external Medical-Ethical Committee and the participants signed an informed consent form.
Study Design The total experimental period lasted four weeks in which the subjects received four treatments, each of which lasted three days.
~Supported by the Central Commodity Board for Arable Products, The Hague, Netherlands and the Commodity Board for Spirits, Schiedam, Netherlands.
547
548
VEENSTRA, VAN OE POL AND SCHAAFSMA
During these days, the subjects were housed in the metabolic ward of the department and were not allowed to consume any food or drinks but the meals and drinks supplied. The four treatments comprised two different standard diets, one rich in saturated fatty acids and the other rich in polyunsaturated fatty acids, each at two different alcohol regimes, one with and one without alcohol (factorial design). For each subject the experimental days were always on the same days of the week. The first two days, in which no alcohol was consumed, served as a run-in period. On the third day 40 g of alcohol was consumed, two glasses of red wine during dinner and two glasses of Hollands gin just before bedtime combined with a snack, 5 hours after dinner. If no alcohol was given, exactly the same volumes of mineral water were consumed. Blood samples were taken at four moments; 1) three hours before dinner, 2) one hour after dinner, 3) one hour after the snack and 4) the next morning. Compliance was tested by measuring the blood alcohol concentration. All subjects were subjected to all four treatments in a randomized order, according to a Latin square design. Analyses Blood was taken from a vena mediana cubiti by means of a catheter. For platelet aggregation analyses 20 ml blood was collected by gentle suction into 200 ixl of 320 g/1 sodium citrate pH 7.4. Aggregation experiments were carried out in platelet-rich plasma (PRP) containing 220 × 106 platelets/ml in a Lumi Dual aggrometer (Chrono-Log Corp., Havertown, PA). After 3 minutes of preincubation at 37°C of 450 Ixl PRP, 50 txl aliquots of collagen or ADP suspensions were added to induce aggregation. Aggregation curves were recorded for 5 minutes. For collagen (Hormon Chemie, Miinchen, FRG) the final concentrations used were 0.2, 0.4 and 0.7 mg/1 and for ADP (Boehringer-Mannheim, Mannheim, FRG) 0.5, 1.0 and 2.0 IxM. From the aggregation curves the percentage of maximum aggregation and the velocity of aggregation were obtained. For each sample separately, the percentage of maximum aggregation or velocity of aggregation was plotted against the collagen or ADP concentrations. The areas under the curves thus obtained were used as derived variables. The use of these derived variables reduces the number of variables obtained from aggregation studies and also increases the precision of the aggregation parameters. Platelets were counted on a Sysmex CC-180 Microcellcounter (TOA Medical Electronics Co. Ltd., Carson, CA). Blood alcohol concentrations were determined enzymatically by the method of Beutler and Michal (1), using Boehringer biochemicals (Boehringer, Mannheim, FRG). Statistics Statistical evaluation comprised analysis of variance, according to the design, to test the significance of the main effects of alcohol and meal composition as well as their two-factor interactions. The measurements at moment l (before alcohol consumption) were used as a covariate. RESULTS
The mean blood alcohol concentration one hour after wine consumption was 0.12 g/1 (range 0.05 to 0.20 g/l) and one hour after the two glasses of Hollands gin 0.23 g/1 (range 0.16 to 0.32 g/l). After consumption of mineral water the blood alcohol concentration was always 0.00 g/1. In all subjects platelet counts were normal at the start of the
TABLE 1 THE ACUTE INFLUENCE OF MODERATE ALCOHOL CONSUMPTION ON PLATELET AGGREGATION IN HEALTHY MIDDLE-AGED VOLUNTEERS*
Parameter
Alcohol
Before Dinner
1 hr After Dinner
I hr After the Snack
The Next Morning
216 237 0.223
197 198 0.955
212 226 0.467
163 176 0.420
147 143 0.808
167 174 0.726
437 423 0.675
413 387 0.382
420 445 0.107
503 516 0.460
514 495 0.538
519 515 0.864
Collagen Percentage of aggregation p-value
+
Velocity aggregation p-value
+
221
169
ADP Percentage of aggregation p-value
+
Velocity of aggregation p-value
+
435
546
*Values refer to the area under the curve for percentage of aggregation and velocity of aggregation and are expressed in arbitrary units (see the Method section).
study (mean 195 109/1, range 154-297) and remained unchanged during the different treatments. The results of the platelet aggregation measurements are summarized in Table 1. There were no significant effects of alcohol or diet, or two-factor interactions between alcohol and diet on platelet aggregation. The percentage of maximum aggregation and the velocity of each collagen and ADP-induced aggregation was also statistically analyzed separately and in none of these analyses significant effects of alcohol were observed. DISCUSSION
Our study demonstrates that moderate alcohol consumption has no acute effects on platelet aggregation in healthy middle-aged men. Thus, the reported protective effect of alcohol against CHD is not likely to be attributable to such an acute effect. In a previous study (14), we have investigated the effects of 5 weeks of moderate alcohol consumption in a controlled study and observed an alcohol-induced decrease in platelet aggregability. Four days of moderate alcohol consumption, however, did not influence platelet aggregation (submitted for publication). Alcohol consumption and platelet aggregability has also been investigated in an epidemiological study (958 participants) by Meade et al. (10). They report a decrease in platelet aggregation with increasing dally alcohol consumption. We conclude that a moderate intake of alcohol has no acute effect on platelet aggregation in habitual moderate drinkers. Daily moderate alcohol consumption, on the other hand, causes a decreased platelet aggregability in the long term. This long-term effect of alcohol may, among other mechanisms, play a role in the proposed protection of moderate alcohol consumption against CHD. However, one should bear in mind that platelet aggregation is always measured ex vivo and that aggregability in vivo depends on many other factors.
ALCOHOL AND PLATELET AGGREGATION
549
REFERENCES 1. Beutler, H. O.; Michal, G. Neue methode zur enzymatischen Bestimmung von ~thanol in Lebensmitteln. Z. Anal. Chem. 284:113-117; 1977. 2. Dunn, E. L.; Cohen, R. G.; Moore, E. E.; Hamstra, R. D. Acute alcohol ingestion and platelet function. Arch. Surg. 116:1082-1083; 1981. 3. Elm6r, 0.; G6ransson, G.; Zoucas, E. Impairment of primary hemostasis and platelet function after alcohol ingestion in man. Haemostasis 14:223-228; 1984. 4. Fenn, C. G.; Littleton, J. M. Interactions between ethanol and dietary fat in determining human platelet function. Thromb. Haemost. 51: 50-53; 1984. 5. Galli, C.; Colli, S.; Gianfranceschi, G.; Madema, P.; Petroni, A.; Tremoli, E.; Marinovich, M.; Sirtori, C. R. Acute effects of ethanol, caffeine, or both on platelet aggregation, thromboxane formation, and plasma-free fatty acids in human subjects. Drug Nutr. Interact. 3:61--67; 1984. 6. Hillbom, M.; Kangasaho, M.; Kaste, M.; Numminen, H.; Vapaatalo, H. Acute ethanol ingestion increases platelet reactivity: Is there a relationship to stroke? Stroke 16:19-23; 1985. 7. Hillbom, M.; Kangasaho, M.; L6wbeer, C.; Kaste, M.; Muuronen, A.; Numminen, H. Effects of ethanol on platelet function. Alcohol 2:429--432; 1985. 8. Hillbom, M.; Muuronen, A.; Neiman, J.; Bj6rk, G.; Egberg, N.; Kangasaho, M. Effects of vitamin E therapy on ethanol-induced changes in platelet aggregation, thromboxane formation, factor VII/
levels and serum lipids. Eur. J. Clin. Invest. 17:68-74; 1987. 9. Marmot, M. G. Alcohol and coronary heart disease. Int. J. Epidemiol. 13:160-167; 1984. 10. Meade, T. W., Vickers, M. V.; Thompson, S. G.; Stifling, Y.; Haines, A. P.; Miller, G. J. Epidemiologicai characteristics of platelet aggregability. Br. Med. J. 290:428-432; 1985. 11. Mikhailidis, D. P.; Jeremy, J. Y.; Barradas, M. A.; Green, N.; Dandona, P. Effect of ethanol on vascular prostacyclin (prostaglandin I2) synthesis, platelet aggregation, and platelet thromboxane release. Br. Med. J. 287:1495-1498; 1983. 12. Moore, R. D.; Pearson, T. A. Moderate alcohol consumption and coronary artery disease. Medicine 65:242-267; 1986. 13. Neiman, J.; Jones, A. W.; Numminen, H.; Hillbom, M. Combined effect of a small dose of ethanol and 36 hr fasting on blood-glucose response, breath-acetone profiles and platelet function in healthy men. Alcohol Alcohol. 22:265-270; 1987. 14. Pikaar, N. A.; Wedel, M.; van der Beek, E.; van Dokkum, W.; Kempen, H. J. M.; Kluft, C.; Ockhuizen, T.; Hermus, R. J. J. Effects of moderate alcohol consumption on platelet aggregation, fibrinolysis, and blood lipids. Metabolism 36:538-547; 1987. 15. Rohan, T. E. Alcohol and ischemic heart disease, a review. Aust. N. Z. J. Med. 14:75-80; 1984. 16. Veenstra, J. Moderate alcohol use and coronary heart disease: A U-shaped curve? World Rev. Nutr. Diet. Accepted for publication; 1990.