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The Association of Diet and Thrombotic Risk Factors in Healthy Male Vegetarians and Meat-Eaters

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The Association of Diet and Thrombotic Risk Factors in Healthy Male Vegetarians and Meat-Eaters European Journal of Clinical Nutrition (1999) 53, 612±619 ß 1999 Stockton Press. All rights reserved 0954±3007/99 $12.00 http://www.stockton-press.co.uk/ejcn The association of diet and thrombotic risk factors in healthy male vegetarians and meat-eaters DLi1, A Sinclair1*, N Mann1, A Turner2, M Ball3, F Kelly1, L Abedin1 and A Wilson1 1Departments of Food Science, RMIT University, Melbourne 3001, Australia; 2Medical Laboratory Science, RMIT University, Melbourne 3001, Australia; and 3School of Nutrition and Public Health, Deakin University, Malvern, VIC 3144, Australia Objective: The aim of this study was to assess thrombosis tendency in subjects who were habitual meat-eaters compared with those who were habitual vegetarians. Design: Cross-sectional comparision of habitual meat-eaters and habitual vegetarians. Setting: Free living subjects. Subjects: One hundred and thirty-nine healthy male subjects (vegans n 18, ovolacto vegetarians n 43, moderate-meat-eaters n 60 and high-meat-eaters n 18) aged 20 ± 55 y who were recruited in Melbourne. Outcome measures: Dietary intake was assessed using a semi-quantitative Food Frequency Questionnaire. The parameters of thrombosis were measured by standard methods. Results: Saturated fat and cholesterol intakes were signi®cantly higher and polyunsaturated fat (PUFA) was signi®cantly lower in the meat-eaters compared with vegetarians. In the meat-eaters, the platelet phospholipids AA levels were signi®cantly higher than in the vegetarians, but there was no increase in ex vivo platelet aggregation and plasma 11-dehydro thromboxane B2 levels. Vegetarians, especially the vegans, had a signi®cantly increased mean collagen and ADP stimulated ex vivo whole blood platelet aggregation compared with meat-eaters. The vegan group had a signi®cantly higher mean platelet volume than the other three dietary groups. However, meat-eaters had a signi®cantly higher cluster of cardiovascular risk factors compared with vegetarians, including increased body mass index, waist to hip ratio, plasma total cholesterol (TC), triacylgly- cerol and LDL-C levels, ratio of TC=HDL-C and LDL-C=HDL-C and plasma factor VII activity. Conclusions: Consumption of meat is not associated with an increased platelet aggregation compared with vegetarian subjects. Sponsorship: Meat Research Corporation, Australia. Descriptors: vegetarians; meat; hemostatic factors; platelet aggregation; lipoprotein lipids; fatty acids Introduction Arterial thrombosis is generally recognised to play a major role in the transition from stable to acute ischaemic The major cause of premature mortality in Western Society heart disease, manifested by unstable angina, acute myo- is cardiovascular disease. High fat, particularly saturated fat cardial infarction and sudden death. Beside local stimuli (SFA) intake has been implicated as one of the major such as disruption of plaque, systemic thrombogenic factors causes primarily because it raises blood low density lipo- (platelet hyperreactivity, increased ®brinogen (FIB) con- protein cholesterol (LDL-C) levels. High meat consump- centration and factor VII activity (VII), defective ®brino- tion has been linked with high plasma cholesterol levels, lysis and abnormalities of blood ¯ow) may contribute to the since most meats are sold with attached depot or visible fat occurrence, extent, and persistence of coronary thrombosis which is rich in SFA (Sinclair & O'Dea, 1990). Acute and its clinical sequelae. Therefore, it may be bene®cial to clinical disorders of the cardiovascular system are often detect thrombosis tendency in both patients and healthy caused by formation of thrombus and subsequent occlusion people by using appropriate laboratory tests. of a blood vessel. The traditional cardiovascular disease Platelet aggregation is an early event in the development risk factors of a positive family genetic history, cigarette of thrombosis. It is initiated by the production of throm- smoking, elevated blood LDL-C and blood pressure do not boxane A (TXA ) produced from arachidonic acid (AA, account for the majority of cases at risk for future throm- 2 2 20:4 n-6), a long chain n-6 polyunsaturated fatty acid botic events. (PUFA) in the platelet membrane. Since the Western diet contains an excess of n-6 PUFA, it is argued that this situation may lead to an overproduction of TXA2 and *Correspondence: Dr A Sinclair, Department of Food Science, RMIT therefore an increase in thrombosis tendency (Berner, University, GPO Box 2476V, Melbourne, VIC 3001, Australia. 1993). Meats are the major source of dietary arachidonic Guarantor: A Sinclair Contributors: D Li, A Sinclair, N Mann, A Turner and M Ball initiated the acid and this arachidonic acid may contribute to the pool of study. D Li prepared the drafts of paper, did laboratory assays, collected tissue arachidonic acid and thereby to an increased risk of data, and did the statistical analysis. A Sinclair selected the study site, platelet aggregation and thrombosis (Mann et al, 1995). On supervised the project, and secured the funding. N Mann, F Kelly, L the other hand, lean meats and ®sh are also the major Abedin and A Wilson helped data collection and provided technical sources of eicosapentaenoic acid (EPA, 20:5 n-3) and assistance. All investigators contributed to the drafts of paper. Received 7 November 1998; revised 15 February 1999; accepted docosahexaenoic acid (DHA, 22:6 n-3) which may provide 23 February 1999 a protective effect against thrombin formation. Habitual diet and thrombotic risk factors in man DLiet al 613 In general, vegetarians have lower blood pressure, lower Blood specimen collections fat intake, lower body mass index, and higher antioxidant Subjects attended the RMIT Medical Centre in the morning intake than omnivores. These factors are known to reduce following an overnight fast. Subjects were allowed to sit the risk of cardiovascular disease (CVD). However, vege- relaxed for 10 min, and then venous blood was taken in tarians also have a relatively high intake of linoleic acid plain, EDTA, citrate and CTAD (citrate, theophylline, (18:2 n-6, a precursor of AA) and low EPA and DHA adenosine and dipyridamole) vacuum tubes with 21-gauge intakes (Sanders & Roshanai, 1992). needles. After blood collection, the subject's weight, Previous short term intervention studies from this group height, waist=hip ratio and blood pressure were measured. have indicated that low fat diets enriched in AA signi®- Whole blood platelet aggregation, full blood examination cantly increased plasma AA levels and TXA2 production, and platelet isolation were performed during the 3 h follow- whereas diets containing AA and long chain (LC) n-3 ing blood sampling. Plasma and serum samples were PUFA or diets rich in LC n-3 PUFA do not raise TXA2 prepared during the 2 h after blood was drawn, aliquoted levels (Sinclair & Mann, 1996; Mann et al, 1997). One of into separate tubes and stored at 720C until analysis. the dif®culties with previous studies is that they have been only conducted for short periods (2 ± 3 weeks), due to the Full blood examination dif®culty subjects had consuming higher amounts of meat A full blood cell count was performed on a Coulter STKR than normal. Therefore, it was decided to conduct a cross- analyser (Coulter Electronics Inc, Hialeah, USA). sectional study of thrombosis risk indicators in healthy male subjects who habitually consumed high quantities of Plasma lipid and lipoprotein lipids meat. In this study, we compared blood cell counts, Triacylglycerol (TAG) and total cholesterol (TC) concen- lipoprotein lipids, coagulation factors, plasma and platelet tration of fasting EDTA plasma were determined by stan- phospholipid fatty acid pro®les, plasma 11-dehydro throm- dard enzymatic methods on a centrifugal autoanalyser boxane B2 (11-dehydro TXB2) and ex vivo agonist induced (Hitachi Autoanalyser System 705, Japan), using commer- whole blood platelet aggregation in meat consumers and cially available kits (Boehringer Mannheim, Sydney, Aus- vegetarians. The latter group were chosen because they tralia) as published previously (Sravropoulous & Crouch, have a low intake of AA. We hypothesised that meat 1974). High density lipoprotein cholesterol (HDL-C) was consumption does increase the levels of blood indicators determined after precipitating all plasma lipoproteins of increased thrombosis potential risk. except HDL with polyethylene glycol 6000 (PEG 6000). LDL-C was calculated using the Friedwald equation as developed by DeLong et al (1986). Methods Platelet and plasma fatty acids Two CTAD tubes of whole blood were spun at 900 rpm for Subjects 10 min. Platelet rich plasma (PRP) was removed and This project was approved by the Human Research Ethics platelets were isolated from PRP using the method pub- Committee of RMIT University, and all subjects gave lished by Castaldi & Smith (1980). Remaining platelet poor written informed consent. One hundred and forty seven blood was further spun at 3000 rpm for 15 min, and platelet healthy male non-smokers aged between 20 ± 50 y were poor plasma (PPP) was removed and stored at 7 20C for recruited through advertisements in University newsletters later analysis of 11-dehydro TXB2. Total lipid of platelets and local newspapers. The exclusion criteria for this study and EDTA plasma was extracted with chloroform:methanol were: evidence of CVD, hypertension, renal disease, hyper- 1:1 (C:M, v=v) containing 10 mg=L of butylated hydroxy- lipaemia, hematological disorders, diabetes, family history toluene (Labco,
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