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Home , Lauric acid, Myristic acid, Palmitic acid, Saturated fat, Trimyristin

European Journal of Clinical Nutrition (1998) 52, 697±702 ß 1998 Stockton Press. All rights reserved 0954±3007/98 $12.00 http://www.stockton-press.co.uk/ejcn

Individual saturated fatty acids and effects on whole blood aggregation in vitro

EHM Temme*, RP Mensink and G Hornstra

Department of Human Biology, Maastricht University, Maastricht, the Netherlands; *Present address: Leuven University, Capucijnenvoer 35, Leuven B-3000, Belgium

Objectives: In two studies we have compared the effects of four different saturated diets (medium chain fatty acids (MCFA), and lauric, myristic and palmitic acids) with those of a monounsaturated diet on in- vitro whole blood aggregation in healthy women and men. Design: Study 1 had a cross-over design with three diet periods of each six weeks, and studied the effects of diets enriched in lauric, palmitic or oleic acids. Study 2 had a parallel design. After a three week oleic acid run-in diet, three groups of subjects were formed which consumed either an MCFA, or oleic acid rich diet for six weeks. Subjects: Eighteen women and 14 men in Study 1 and 37 women and 23 men in Study 2. All subjects were healthy and were aged 20±60 y. Interventions: The experimental diets were the same in composition except for on average 8 En% (Study 1) or 10 En% (Study 2) which was provided by either MCFA, , myristic acid, or oleic acid. Blood samples were taken at the end of each dietary period. Whole blood platelet aggregation, anti- coagulated with recombinant hirudin was assessed after administration of collagen (®nal concentration (fc): 0.38 mg/mL) in Study 1 and collagen (fc: 0.22 mg/mL) or ADP (fc: 1.25 mmol/L) in Study 2. Collagen-induced formation of (Tx)A2, measured as thromboxane (Tx)B2, was evaluated in Study 1 only. Results: The aggregation velocity between the saturated diets and the monounsaturated fatty acid diet did not differ. TxB2 concentrations measured in collagen activated blood samples, which correlated signi®cantly with aggregation velocity, did not differ between the lauric or the palmitic compared with the oleic acid diet. A stepwise regression analysis indicated that collagen-induced aggregation was negatively correlated with the number of red blood cells. ADP-induced aggregation also correlated negatively with red blood cell count, and positively with platelet count. Conclusions: The exchange of 7±10 En% from oleic acid for MCFA, lauric, myristic or palmitic acid does not affect in-vitro whole blood aggregation induced by collagen. ADP-induced aggregation is not affected when 10 En% from oleic acid is exchanged for MCFA or myristic acid. Sponsorship: This study was supported by a grant from the Dutch Dairy Foundation on Nutrition and Health. Descriptors: individual saturated fatty acids; whole blood aggregation; healthy humans; thromboxane B2; dietary intervention

Introduction Different types of dietary fatty acids have been demon- strated to change the reactivity of platelets. Renaud and Blood platelets play an important role in the etiology of coworkers (1986) found decreased thrombin-induced and coronary heart disease. Activated platelets provide tissue increased ADP-induced platelet rich plasma (PRP) aggre- factor, membrane surface and other regula- gation when farmers switched from high diets tory compounds, that are necessary for the coagulation to high polyunsaturated fat diets for one year. Mutanen and process. Increased platelets reactivity for ADP was demon- coworkers (1992) demonstrated lower ADP- and collagen- strated in middle aged men at risk for coronary heart induced PRP aggregation on a dairy fat diet compared with disease (Thaulow et al, 1991) and in patients with coronary diets rich in unsaturated fatty acids from rapeseed or heart disease (Elwood et al, 1990). Also, the protective sun¯ower . Ng et al (1992), in addition, found increased effect of aspirin against suggests a thromboxane B (TxB ) in collagen-activated whole blood major role of platelets in the development of coronary heart 2 2 comparing diets rich in with . This disease (Antiplatelet Trialists' Collaboration, 1988; Steer- observation (Ng et al, 1992) suggested that the various ing Committee of the Physicians' Health Study Research saturated fatty acids have different effects on platelet Group, 1989), as it inhibits platelet synthesis of thrombox- activity and that thromboxane dependent aggregation ane A (TxA ). 2 2 increases with diets rich in fatty acids from (for example, MCFA, lauric and myristic acid). However, effects of speci®c saturated fatty acids on platelet aggrega- Correspondence: Dr RP Mensink, Department of Human Biology, tion have hardly been investigated systematically. Maastricht University, Universiteitssingel 50, PO Box 616, 6200 MD Maastricht, the Netherlands. Platelet aggregation is frequently measured with the Received 16 July 1997; revised 10 April 1998; accepted 15 May 1998 optical density technique using citrated PRP and activators Saturated fatty acids and platelet aggregation EHM Temme et al 698 of aggregation such as ADP, collagen or thrombin. This postmenopausal and two women used oral contraceptives. technique might not always be preferable, since it does not Study 2 included sixty subjects, thirty-seven women and take into account the possible role of short-lived plasmatic twenty three men, aged between 22 and 60 y (mean, 41 y components and of red and white blood cells. In this old). Women had body mass indexes between 20 and present study, therefore, the effects of diets enriched in 30 kg/m2 (mean 24 kg/m2) and the men between 17 and MCFA, lauric, myristic and palmitic acid on aggregation in 29 kg/m2 (mean 25 kg/m2). Nine women were postmeno- whole blood in vitro were investigated and compared with pausal and 10 women used oral contraceptives, six women those of oleic acid. and ®ve men smoked.

Diets Methods The diets of both studies consisted of products in which the Design and statistical analysis normal fat was replaced by experimental . Total fat The project consisted of two separate studies also designed energy was on average 40% of energy (En%). The experi- to investigate the effects of separate saturated fatty acids on mental fats supplied 70% of the total fat energy in the ®rst lipoproteins, coagulation and ®brinolysis as reported else- and 63% in the second study. The nutrient composition of where (Temme et al, 1996, 1997b). The protocols of both the calculated diets was similar except that approximately studies had been approved by the ethical committee of 8 En% (Study 1) or 10 En% (Study 2) of oleic acid was Maastricht University. All subjects had given their written exchanged for lauric acid or palmitic acid (Study 1) and for informed consent. MCFA or myristic acid (Study 2). These differences were In the ®rst study (Study 1) the effects of lauric acid, achieved by the use of special experimental fats. The lauric palmitic acid and oleic acid were investigated using a acid fat was a mixture of (75%) (Wesco; cross-over study design. The three different diets were Karlshamns, Zaandijk, the Netherlands) and a high oleic consumed by 32 healthy subjects, 18 women and 14 men, acid sun¯ower oil (25%) (Trisun; SVO enterprises, Wick- for six weeks separated with wash out periods from two or liffe, USA). The palmitic acid fat consisted of dairy fat three weeks. The second study (Study 2) examined the (55%) (FDF, Leeuwarden, The Netherlands), palmstearin effects of MCFA and myristic acid versus oleic acid with a (36%) (Aarhus oil, Aarhus, Denmark) and sun¯ower oil parallel study design. After a run-in period of three weeks, (9%) and the fat high in oleic acid of dairy fat (37%) and in which 60 volunteers, 37 women and 23 men, consumed high oleic acid sun¯ower oil (63%). was added an oleic acid diet, the subjects were divided into three to the fats high in lauric and oleic acid, so that all fats groups; the ®rst group received an MCFA diet (n ˆ 21), the contained 145 mg of cholesterol per 100 g of fat. The fat second group a myristic acid diet (n ˆ 20) and the last high in MCFA was made by interesteri®cation of 34.0% group continued with the oleic acid diet (n ˆ 19). palm , 17.6% high oleic acid sun¯ower oil, 8.4% Data were analysed using the General Linear Models sun¯ower oil, and 40% medium chain (MCT) (GLM) procedure of the Statistical Analyses System. In oil. The composition of the MCT oil was 33.3% C6:0, Study 1, the aggregation parameters at the end of each 25.3% C8:0, and 41.5% C10:0. The myristic acid fat was dietary period were analysed with diet, period and diet-and- made by interesteri®cation of a blend of 34% , sex interaction as independent variables. Data of TxB2 were 17% high oleic acid sun¯ower oil, 9% sun¯ower oil and logarithmic transformed before analysis, because they were 40% . The oleic acid fat consisted of a blend of not normally distributed. In Study 2, changes in aggrega- 30% palm stearin and 70% high oleic sun¯ower oil tion parameters from the end of the run-in to the end of the (GruÈnau GMBH, Illertissen, Germany). were experimental period were analysed with diet, sex and diet- made from the fats and consisted of 83% fat and 17% and-sex interaction as independent variables. Pearson cor- water. The experimental margarines were given as such and relation coef®cients were calculated between aggregation were incorporated in bread, biscuits, pies, cakes (Study 1 velocity and hematological parameters. Correlations were and Study 2), and custards (Study 1 only). calculated when subjects consumed the oleic acid diet The study diets were formulated at different levels of during the experimental period (Study 1) or the run-in energy, so that each subject received a diet that met his or period (Study 2). To determine the relationship between her energy needs. According to these energy levels lists hematological parameters and aggregation in whole blood, were computed which stated the amount of experimental a stepwise regression procedure (forward selection) was products the subject had to eat each day or each week. performed with whole blood aggregation velocity as depen- Products were handed out on an individual basis and were dent and the main hematological parametersÐplatelet free of charge. 30% (Study 1) and 37% (Study 2) of the count, red blood cell count and white blood cell countÐ total fat intake was free of choice. The subject could choose as independent variables. The signi®cance level for entry in these `free' products from a list of fat containing products. the model was set at 0.05. Subjects came at least once a week to a special room in the In both studies differences in responses between women University to get a new supply of products and to be and men were evaluated with unpaired t-tests. weighted. They were also asked to maintain the same activity level, smoking and drinking habits throughout the Subjects study. Any changes were written down in a special diary as Details of both studies have been published previously. well as medication used, illnesses, daily use and Brie¯y, Study 1 included thirty-two subjects, eighteen menstrual cycle. women and fourteen men, aged between 20 and 60 y (mean, 41 y old). The women had body mass indexes Blood sampling and measurements from 19±30 kg/m2 (mean 25 kg/m2), the men had body Blood was sampled after an overnight fast and after mass indexes from 22±31 kg/m2 (mean 25 kg/m2). Four abstinence from drinking the preceding day and smoking women and ®ve men were smokers. Six women were on the morning before blood sampling. Blood for platelet Saturated fatty acids and platelet aggregation EHM Temme et al 699 aggregation was sampled at the end (week 6) of each period 41.8 En% with the oleic acid diet. As determined from in Study 1 and at the end of the run-in (week 3) and the end the duplicate portions, the difference in lauric acid was of the experimental period (week 9) in Study 2. Blood was 7.3 En% between the lauric and the oleic acid diet, while sampled with a minimum of stasis and using a 1.2 mm the palmitic acid diet was 6.1 En% higher in palmitic than needle (Strauû Kanule, Luer, WaÈchtersbach, Germany) the oleic acid diet. The lauric acid diet contained also some with the subject in recumbent position. All venipunctures (average 1.8 En%) more myristic acid than the other two were performed by the same person, at the same location, diets. The diets did not differ with respect to or and for each subject generally at the same time of the same a-linolenic acid intakes (Temme et al, 1997a). day of the week. In Study 2, the average intake of fat was 39 En%. The The ®rst 3±4 mL blood was collected in an EDTA tube average intake of oleic acid was 15.1 En% during the run-in to count the number of platelets (Hematology system, period and decreased in the test period to 6.6 En% with the Technicon Instruments Corporation, Tarrytown, NY, MCFA and to 6.3 En% with the myristic acid diet. It was USA) in Study 1 and to measure complete hematological exchanged for extra MCFA or myristic acid. Oleic acid parameters in Study 2 (Coulter microdiff 18, Coulter intake during the oleic acid diet did not change from the Corporation, Miami, FL, USA). Then, 1.5 mL of blood run-in to the test period. Changes of other , except was collected in a tube containing 67 mg/mL recombinant for En% saturated and monounsaturated fatty acids, did not desulphatohirudin variant 1 (TMRevasc; a gift from Novar- signi®cantly differ between the diets. tis Pharma AG, Horsham, Great Britain). Whole blood Relative to the oleic acid diet, fatty acid composition of aggregation was immediately measured after blood sam- pooled serum samples in both studies (Temme et al, 1997a, pling using a whole blood aggregometer (Chrono-log whole 1997b) did not indicate signi®cant changes in the propor- blood aggro-meters, model 500 (Study 1 and Study 2) and tions of linoleic acid, a-linolenic acid, or model 540 (Study 2)) (Chrono-log Corporation, Haverton, total polyunsaturated fatty acids. PA, USA). 1 mL of blood was transferred to a pre-warmed plastic cuvet and the electrodes were inserted in the cuv- Platelet aggregation and TxB2 ettes. The sample was stirred at 1000 rpm, kept at 37C and Table 1 shows that collagen-induced aggregation velocity allowed to reach equilibrium. After calibrating the instru- and TxB2 production after six weeks consumption of diets ment, 10 mL of the aggregation inducer (collagen or ADP) enriched in lauric, palmitic or oleic acids did not differ was added. Aggregation curves were registered for 15 min signi®cantly between the diets. Also, diets rich in MCFA and the velocity was expressed as the steepest slope of the and myristic acid (Study 2) did not affect collagen- and aggregation curve (O/min). When aggregation curves of ADP-induced aggregation velocity differently as compared ADP-induced aggregation consisted of two curves the with a diet rich in oleic acid (Table 2). steepest slope of the ®rst curve was determined. In Study 1, logarithmic transformed values of TxB2 In Study 1, platelet aggregation was induced by addition concentrations correlated with aggregation velocity of collagen (Collagen Horm, Hormon Chemie, Munich, (r ˆ 0.42 (P ˆ 0.0165), r ˆ 0.48 (P ˆ 0.0055), r ˆ 0.39 Germany) at a ®nal concentration of 0.38 mg/mL. In Study (P ˆ 0.0274), for subjects on the lauric, palmitic or oleic 2, collagen- and ADP- (Sigma Chemical Co, St Louis, MO, acid diet, respectively). USA) induced aggregation were measured. Final concen- In Study 2, collagen-induced aggregation velocity was trations were 0.22 mg/mL for collagen and 1.25 mmol/L for slightly higher in subjects that smoked compared with the ADP. Samples of the subjects were measured at the same nonsmokers (5.31Æ 2.93 O/min in smokers versus channel of the aggregometer along the study. Due to an 5.05Æ 1.72 O/min in nonsmokers; P ˆ 0.0221), but did occasional technical problem with the aggregometers, not differ between them in Study 1. Also, ADP-induced aggregation values of some subjects are missing, which aggregation in Study 2 did not signi®cantly differ between has been emphasized in the tables. smokers and non-smokers. Collagen-induced aggregation After 15 min of activation, collagen-activated aggrega- tion samples from Study 1 were centrifuged in an eppen- dorf centrifuge for 3 min, to measure TxB2 production. The plasma samples were then immediately snap-frozen and stored at 7 80C. The TxB concentration was determined 2 Table 1 Aggregation velocity (O/min) induced by collagen (fc: 0.38 mg/ with an Enzyme Immunoassay (Cayman Chemical Com- mL) and plasma thromboxane B2 concentrations of collagen activated blood pany, Ann Harbor, MI, USA). Coef®cient of variation (Study 1)a within runs was 6.1%. All samples from the same subject were analysed within one assay run. Lauric acid Palmitic acid Oleic acid diet diet diet

Results Collagen-induced aggregation velocity (O/min) Dietary composition All 5.5Æ 1.4 5.2Æ 1.5 5.6Æ 1.7 The mean daily energy intake and the composition of the Women 5.8Æ 1.2 5.0Æ 1.8 5.6Æ 1.7 Men 5.1Æ 1.7 5.0Æ 1.8 5.5Æ 1.8 diets was determined from four days records in Study

1 and three days food records in Study 2. In addition, Thromboxane B2 (ng/mL) duplicate portions of food and drinks consumed have been All 17.3Æ 5.4 17.7Æ 5.8 16.5Æ 5.5 collected by the subjects for two days (Study 1). of Women 17.6Æ 6.2 16.9Æ 6.1 15.7Æ 6.1 these portions were extracted and fatty acid composition Men 16.9Æ 4.5 18.7Æ 5.5 17.5Æ 4.7 determined. a Values are given as meansÆ standard deviations. 32 healthy subjects (18 In Study 1, total fat intake was 41.5 En% with the lauric women and 14 men) consumed diets enriched in lauric acid, palmitic acid acid diet, 41.0 En% with the palmitic acid diet and or oleic acid. Saturated fatty acids and platelet aggregation EHM Temme et al 700 Table 2 Aggregation velocity (O/min) induced by collagen (FC: 0.22 mg/mL) or by ADP (FC: 1.25 mmol/L) on diets enriched in MCFA, myristic acid or oleic acid (Study 2)a

MCFA diet Myristic acid diet Oleic acid diet

Run-in Change Run-in Change Run-in Change

Collagen-induced aggregation velocity (O/min) Allb 5.0Æ 2.2 0.4Æ 2.7 5.4Æ 1.8 0.3Æ 1.8 4.8Æ 1.9 0.1Æ 2.6 Womenc 6.1Æ 1.7 7 0.5Æ 2.1 6.0Æ 1.9 0.3Æ 1.7 4.9Æ 2.4 0.1Æ 3.0 Men 2.9Æ 1.4 2.2Æ 2.9 4.5Æ 1.1 0.3Æ 2.0 4.6Æ 0.8 0.2Æ 1.9

ADP-induced aggregation velocity (O/min) All 5.6Æ 1.8 0.1Æ 2.0 5.2Æ 1.4 7 0.2Æ 1.5 5.9Æ 1.3 7 0.2Æ 1.5 Womenc 6.0Æ 2.0 7 0.2Æ 1.7 6.0Æ 1.4 7 0.4Æ 1.6 6.3Æ 1.4 0.0Æ 1.3 Men 5.0Æ 1.1 0.5Æ 2.6 4.1Æ 0.5 0.1Æ 1.3 5.3Æ 0.9 7 0.6Æ 1.8

a Values are given as meansÆ standard deviations. 18 subjects (12 women and 6 men) consumed a diet rich in MCFA, 20 subjects (12 women and 8 men) a diet rich in myristic acid and 19 subjects (12 women and 7 men) a diet rich in oleic acid. b Values of one woman and one man are missing in the myristic acid group and one woman and one man in the oleic acid group. c Values of women after the run-in period (all subjects combined) are signi®cantly different from those of men, P < 0.01.

velocity did not differ between women and men in Study 1. women, whereas platelet count and mean platelet volume Collagen- and ADP-induced aggregation velocities after did not differ between the two sexes (see Table 3). Whereas the run-in period in Study 2, however, were signi®cantly collagen-induced aggregation was not correlated with pla- higher in women compared with men (P ˆ 0.0020 and telet count (r ˆ 0.20, P ˆ 0.1368), ADP-induced aggrega- P ˆ 0.0010, respectively). The data of collagen-induced tion velocity was (r ˆ 0.39, P ˆ 0.0026). Both collagen- aggregation velocity of the females consuming the oleic and ADP-induced aggregation velocity were inversely acid diet (oleic acid diet period in Study 1 and oleic acid correlated with the concentration of red blood cells run-in period in Study 2) were combined to determine (r ˆ 7 0.38, P ˆ 0.0035 for collagen-induced aggregation, differences between pre- and postmenopausal women. r ˆ 7 0.48, P < 0.0001 for ADP-induced aggregation). Collagen-induced aggregation velocity was (meansÆ stan- Independent hematologic determinants of whole blood dard deviation) 5.2Æ 1.8 O/min in premenopausal women platelet aggregation velocity as measured after the run-in not on contraceptives (n ˆ 27, one missing value), period in Study 2 were evaluated with a stepwise forward 6.6Æ 1.4 O/min in premenopausal women on contracep- regression analysis and are shown in Table 4. The red blood tives (n ˆ 11, one missing value) and 5.9Æ 2.2 O/min in cell count was negatively related to collagen- and ADP- postmenopausal women (n ˆ 15) and did not differ signi®- induced aggregations. ADP-induced aggregation was also, cantly between these groups of women. ADP-induced but to a lesser extent, positively associated with platelet aggregation velocity, which was only measured in Study count. 2, was 6.6Æ 1.9 O/min in premenopausal women not on contraceptives (n ˆ 17, one missing value), 5.6Æ 1.2 O/min in premenopausal women on contraceptives (n ˆ 10) and Discussion 5.7Æ 1.1 O/min in postmenopausal women (n ˆ 9) and did Compared with monounsaturated fatty acids (oleic acid), not differ in a signi®cant different way between these diets enriched in saturated fatty acids (MCFA, lauric acid, women. myristic acid or palmitic acid) did not change aggregation velocity in vitro in whole blood samples anticoagulated Hematologic parameters with hirudin. From measurements in PRP, Mutanen et al Platelet count in Study 1 did not change throughout the (1992) reported lower ADP- and collagen-induced aggre- study and did not differ between women and men (data not gations after consumption of diets rich in dairy fat com- shown). Platelet counts correlated signi®cantly with platelet pared with diets rich in rapeseed oil (oleic acid and a- aggregation velocity (r ˆ 0.40 (P ˆ 0.0217), r ˆ 0.47 linolenic acid) or sun¯ower oil (linoleic acid). It was (P ˆ 0.0069), r ˆ 0.48 (P ˆ 0.0049), on the lauric, palmitic suggested (Mutanen et al, 1992) that these effects were or oleic acid diet, respectively). due to differences in the linoleic acid and a-linolenic acid In Study 2 more hematologic parameters were measured ratio of the diets and not to the exchange of saturated for (Table 3). Although changes did not differ signi®cantly unsaturated fatty acids. In our study, the diets given did not between the three diets, a trend was observed for a differ with respect to linoleic acid (Study 1 and 2) or a- decreased mean platelet volume with the myristic acid linolenic acid (Study 1) content as determined from the (mean response of 7 0.2 fL; P ˆ 0.0006) compared with recorded food intakes (Study 2) or the duplicate portions the MCFA diet (difference of changes 7 0.3 fL; (Study 1) (Temme et al, 1997a). Therefore, the present P ˆ 0.0659, 95% Con®dence Interval 7 0.7±0.1 fL). study does not necessarily contradict this suggestion (Muta- After the run-in period in Study 2, red blood cell concen- nen et al, 1992), but can also not con®rm because collagen- trations were higher in men than in women and ADP-induced aggregation have now been measured in (4.9Æ 0.3 6 1012/L vs 4.4Æ 0.3 6 1012/L; P < 0.0001). whole blood samples. Earlier, Renaud and co-workers Other red blood cell related parametersÐhemoglobin and (1986) also reported increased ADP-induced aggregation hematocritÐwere also higher in men compared with when farmers replaced their saturated fat diets for one year, Saturated fatty acids and platelet aggregation EHM Temme et al 701 Table 3 Hematologic parameters on diets enriched in MCFA, myristic acid or oleic acid (Study 2)a

MCFA Diet Myristic Acid Diet Oleic Acid Diet

Run-in Change Run-in Change Run-in Change

White blood cells ( 6 109/L)b 5.9Æ 1.6 7 0.1Æ 1.8 6.0Æ 1.3 0.2Æ 1.4 5.7Æ 1.1 0Æ 1.2 Women 6.2Æ 1.7 0.6Æ 1.7 5.9Æ 1.3 0.4Æ 1.5 5.6Æ 1.2 7 0.5Æ 1.0 Men 5.6Æ 1.7 0.9Æ 1.8 6.1Æ 1.3 7 0.2Æ 1.1 5.9Æ 0.9 0.7Æ 1.1 Red blood cells ( 6 1012/L) 4.6Æ 0.5 7 0.1Æ 0.3 4.7Æ 0.3 7 0.1Æ 0.2 4.5Æ 0.4 0Æ 0.3 Women 4.4Æ 0.4c 7 0.2Æ 0.4 4.5Æ 0.3c 7 0.0Æ 0.2 4.4Æ 0.2c 0Æ 0.2 Men 5.0Æ 0.2 7 0.1Æ 0.2 5.0Æ 0.3 7 0.1Æ 0.2 4.8Æ 0.5 7 0.1Æ 0.4 Hemoglobin (mmol/L) 8.3Æ 0.8 7 0.2Æ 0.3 8.6Æ 0.8 7 0.1Æ 0.3 8.4Æ 0.7 0Æ 0.4 Women 7.9Æ 0.5c 7 0.2Æ 0.4 8.2Æ 0.4c 0Æ 0.4 8.1Æ 0.3c 0Æ 0.4 Men 9.1Æ 0.6 7 0.1Æ 0.3 9.4Æ 0.5 7 0.2Æ 0.2 9.1Æ 0.7 0Æ 0.4 Hematocrit (L/L) 0.40Æ 0.04 7 0.01Æ 0.02 0.42Æ 0.03 0.01Æ 0.02 0.41Æ 0.03 0Æ 0.02 Women 0.38Æ 0.03c 7 0.01Æ 0.02 0.40Æ 0.02c 7 0.01Æ 0.02 0.39Æ 0.01c 0Æ 0.02 Men 0.44Æ 0.02 7 0.01Æ 0.02 0.45Æ 0.02 7 0.01Æ 0.01 0.43Æ 0.04 0Æ 0.03 Platelets ( 6 109/L) 270Æ 67 3Æ 52 277Æ 43 3Æ 35 281Æ 62 1Æ 47 Women 280Æ 69 7 6Æ 53 273Æ 45 7Æ 34 286Æ 74 1Æ 46 Men 252Æ 64 19Æ 49 284Æ 43 7 2Æ 38 272Æ 33 2Æ 52 Mean platelet volume (fL) 8.5Æ 0.6 0.1Æ 0.8 8.3Æ 0.5 7 0.2Æ 0.2 8.3Æ 0.7 0Æ 0.3 Women 8.5Æ 0.7 0.2Æ 1.0 8.3Æ 0.6 7 0.2Æ 0.2 8.2Æ 0.9 0Æ 0.3 Men 8.5Æ 0.6 0.1Æ 0.6 8.2Æ 0.5 7 0.2Æ 0.3 8.3Æ 0.4 0Æ 0.3 a Values are given as meansÆ standard deviations. 21 subjects (13 women and 8 men) consumed a diet rich in MCFA, 20 subjects (12 women and 8 men) a diet rich in myristic acid and 19 subjects (12 women and 7 men) a diet rich in oleic acid. b Values of two subjects are missing in the MCFA group, and the value of one subject is missing in the myristic acid group. c Signi®cantly different from men, P < 0.01.

Table 4 Independent hematologic determinants of whole blood after diets rich in lauric or palmitic acids compared with aggregation velocity in vitro as determined from a forward stepwise re- oleic acid, in collagen activated samples. Others, however, gression procedure showed that diets rich in coconut oil (lauric and myristic Partial acids) compared with palm oil (palmitic acid) or (oleic acid) increased the concentrations of TxB2, in col- Dependent variable Parameter estimate R2 P lagen activated whole blood samples, whereas the concen- tration of 6-ketoPGF (a PGI -metabolite) decreased Collagen-induced aggregation (FC: 0.22 mg/mL) 1a 2 Intercept 13.811 compared with palm oil (Ng et al, 1992). The experimental Red blood cell count 7 1.896 0.15 0.0032 periods in our and their (Ng et al, 1992) study were of the same duration. The exchange of lauric acid for palmitic ADP-induced aggregation (FC: 1.25 mmol/L) acid in our study, however, was smaller (7 En% instead of Intercept 10.404 Red blood cell count 7 1.631 0.21 0.0003 12 En% exchanged), which might have in¯uenced the Platelet count 0.009 0.13 0.0023 results. Aggregation was not measured in that study (Ng et al, 1992). Dependent variable ˆ Platelet count and Red blood cell count and White Our results seem in agreement with those of Mustad et blood cell count. al (1993), who did not indicate an altered excretion of metabolites (TxB2 and 6-ketoPGF1a) in urine after 26 d consumption of diets enriched in fat compared with or (4 En% with diets rich in linoleic acid. For the next three years, lauric plus myristic acid was exchanged for ). when they increased the intake of oleic and a-linolenic acid This lack of changes was observed despite an increase in at the expense of linoleic acid, ADP-induced PRP aggrega- the proportion of arachidonic acid in platelet phospholipid tion was similar to levels on the saturated fat diet. Both membranes with the butter fat compared with the cocoa replacements, however, reduced collagen- and thrombin- butter diet (Mustad et al, 1993). Whether urinary TxB2 induced aggregation compared with the saturated fat diet. really re¯ects platelet thromboxane biosynthesis, however, Therefore, it remains uncertain whether this ratio of dietary has been disputed (Catella et al, 1986). linoleic acid and a-linolenic acid might also be important The components of blood that in¯uenced whole blood for aggregations induced with other stimuli than ADP. aggregation most were red blood cell counts and platelet One of the mechanisms to alter platelet aggregation is counts, the latter for ADP-induced aggregation only. The via the production of TxA2, which is synthesized by observed inverse association between red blood cell count platelets and promotes platelet aggregation, and of PGI2, and whole blood aggregation was also present in the study which is produced by endothelial cells and inhibits platelet that demonstrated a marked association between ADP- aggregation. Both are derived from arachidonic induced whole blood aggregation and prevalent ischemic acid, an important fatty acid of membrane , heart disease (Elwood et al, 1990; Sharp et al, 1990) and a the concentrations of which can be altered by changes in cross sectional study in elderly subjects (Emery et al, dietary fatty acid composition. Our results did not indicate 1995). However, for collagen-induced whole blood aggre- changes in the TxB2 concentrations (a TxA2-metabolite) gation, this association was not observed (Emery et al, Saturated fatty acids and platelet aggregation EHM Temme et al 702 1995). This inverse association might explain the sex Emery JD, Leifer DW, Moura GL, Southern P, Morrissey JH & Lawrence differences in ADP-induced aggregation velocity, as JB (1995): Whole-blood platelet aggregation predicts in vitro and in vivo hemostatic function in the elderly. Arterioscler. Thromb. Vasc. women have lower red blood cell counts than men. Why Biol. 15, 748±753. the gender differences were less pronounced with the Mustad VA, Kris-Etherton PM, Derr J, Reddy CC & Pearson TA (1993): collagen-induced aggregation is not clear, as also for Comparison of the effects of diets rich in stearic acid versus myris-tic collagen-induced aggregation an inhibitory effect of red acid and lauric acid on platelet fatty acids and excretion of thromb- blood cells was demonstrated. oxane A2 and PGI2 metabolites in healthy young men. Metabolism 42, 463±469. Mutanen M, Freese R, Valsta L, Ahola I & AhlstroÈm A (1992): Rapeseed oil and sun¯ower oil diets enhance platlet in vitro aggregation and Conclusions thromboxane production in healthy men when compared with milk fat The combined action of the blood components on in-vitro or habitual diets. Thromb. Haemostas. 67, 352±356. Ng TKW, Hayes KC, DeWitt GF, Jegathesan M, Satgunasingam N, Ong whole blood aggregation induced by collagen, however, ASH & Tan D (1992): Dietary palmitic and oleic acids exert similar was not affected by the exchange of 7±10 En% from oleic effects on serum cholesterol and lipoprotein pro®les in normocholes- acid for MCFA, lauric, myristic or palmitic acid. Also, a teremic men and women. J. Am. Coll. Nutr. 11, 383±390. 10 En% exchange from oleic for MCFA or myristic acid Renaud S, Godsey F, Dumont E, Thevenon C, Ortchanian E & Martin JL did not affect ADP-induced aggregation. (1986): In¯uence of long-term diet modi®cation on platelet function and composition in Moselle farmers. Am. J. Clin. Nutr. 43, 136±150. Sharp DS, Beswick AD, O'Brien JR, Renaud S, Yarnell JWG & Elwood AcknowledgementsÐWe gratefully thank the volunteers for their coopera- PC (1990): The association of platelet and red cell count with platelet tion and interest. We acknowledge the technical assistance of Mrs TJFMB impedance changes in whole blood and light-scattering changes in Hermans, Mr FJJ Cox, Mr R Kalafusz ({) and Mr JW Wierts. We thank platelet rich plasma: evidence from the Caerphilly Collaborative heart Novartis Pharma AG (Horsham, Great Britain) for the provision of disease study. Thromb. Haemost. 64, 211±215. recombinant desulphatohirudin variant 1 (TMRevasc). Steering Committee of the Physicians' Health Study Research Group (1989): Final report on the aspirin component of the ongoing Physi- cians' Health Study. N. Engl. J. Med. 321, 129±135. Temme EHM, Mensink RP & Hornstra G (1996): Comparison of the effects of diets enriched in lauric, palmitic, or oleic acids on serum lipids and lipoproteins in healthy women and men. Am. J. Clin. Nutr. References 63, 897±903. 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