© 1997 American Heart Association, Inc.
Articles |
Interactions Between Dietary Fat, Fish, and Fish Oils and Their Effects on Platelet Function in Men at Risk of Cardiovascular Disease
the Department of Medicine, University of Western Australia, and the Western Australian Heart Research Institute, Royal Perth Hospital, Western Australia.
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Abstract |
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Recent studies have suggested that
3-fats of marine origin may have a protective role in heart disease. This study aimed to compare the effects of fish or fish oil, in the setting of a high- or low-fat diet, on platelet aggregation and platelet thromboxane in men with increased risk of cardiovascular disease. One hundred twenty men who were nonsmokers, 30 to 60 years old, with mildly elevated blood pressure and cholesterol were randomly allocated to one of five high-fat (40% of daily energy) or two low-fat (30%) groups for 12 weeks. The five high-fat groups took either 6 or 12 fish oil capsules daily; fish; a combination of fish and fish oil; or placebo capsules. The two low-fat groups took either fish or placebo capsules. Fish meals provided 1.3 g of eicosapentaenoic acid daily, equivalent to 6 fish oil capsules, and contained an average of 3.65 g/d of 3-fatty acids. Multiple regression analysis of the combined groups showed that all groups taking 3-fatty acids reduced platelet aggregation to both collagen (P<.0001) and platelet-activating factor (PAF) (P<.05) and platelet thromboxane B2 responses (P<.05) to collagen-induced aggregation. The low-fat diet alone had no effect on PAF-induced platelet aggregation and only a small effect on platelet responses to collagen (P<.05). Platelet aggregation responses to PAF were reduced more by fish oil than fish in a high-fat diet, whereas fish had a greater effect when part of a low-fat rather than a high-fat diet. There was no significant difference in collagen-induced platelet aggregation or platelet thromboxane between fish and fish oils on a high or low fat intake. In conjunction with our previous findings of improvements in lipoproteins, blood pressure, and heart rate in this population, these results on platelet function suggest that dietary 3-fatty acids incorporated into a low- rather than a high-fat diet have a wider spectrum of more favorable effects on cardiovascular risk factors.
Key Words: fish • fish oils • platelet aggregation • platelet thromboxane • cardiovascular disease
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Introduction |
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Numerous studies have demonstrated a multiplicity of functional effects of
3-fatty acids in human physiology, human diseases, and animal models.1 2 3 4 5 These include effects on plasma lipids and lipoproteins,6 eicosanoid metabolism, platelet–vessel wall interactions, blood viscosity, arterial blood pressure, coagulation, cytokines, and growth factors.1 2 3 4 5 Most of these changes are of potential benefit for the prevention of cardiovascular disease.
Over the past two decades, many studies have reported on the effects of 3-fatty acids on platelet lipid composition and platelet function (reviewed in Reference 7). Dietary 3-fatty acids have been shown to inhibit both platelet aggregation and platelet thromboxane release in response to collagen and ADP.7 8 9 Other studies have demonstrated an inhibition of thrombin-induced10 and adrenalin-induced11 platelet aggregation. Platelet response has also been inhibited after the addition of PAF.12 However, AA-induced13 and the PGH2 analogue U46619-induced14 platelet aggregations have not been affected by dietary 3-fatty acids.
Most studies that have examined the effects of 3-fatty acids on platelet aggregation have involved large amounts of fish oil or large numbers of capsules containing fish oil extracts.7 However, few studies have been reported in which fish was the source of dietary 3-fatty acids.15 16 17 Furthermore, most studies of 3-fatty acids have not adequately controlled other dietary constituents, and the question as to whether the effects of 3-fatty acids differ depending on whether they are taken as fish or fish oil has not been adequately addressed under carefully controlled dietary conditions. The importance of this is that in contrast to fish oil, the composition of fish, and in particular the relative amounts of EPA and DHA, is variable. Unlike purified fish oil, fish may also contain constituents other than the 3-fatty acids that could affect platelet function. Moreover, to date, no one has examined whether the effects of 3-fatty acid consumption on platelet function are influenced by the background dietary fat intake.
The present study was designed to examine and compare the effects of dietary fish and fish oil, in the setting of a high-fat (40% of daily energy) or low-fat (30% of daily energy) diet, on blood pressure, blood lipids, and platelet function in otherwise healthy men whose blood pressure and blood lipids exposed them to an increased risk of cardiovascular disease. We have previously reported the changes in blood pressure18 19 and lipids19 20 ; here, we describe the changes in platelet aggregation and platelet thromboxane.
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Methods |
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Subject Recruitment
Healthy, nonsmoking men 30 to 60 years years old were recruited from the general community by media publicity. The aim was to select men having body mass index, blood pressure, and serum cholesterol toward the higher end of the "normal" range. After a preliminary telephone screening, respondents were invited to attend the Research Clinic on 3 separate days for screening measurements to determine suitability according to the following entry criteria: body mass index <33 kg/m2, systolic blood pressure 130 to 159 mm Hg, diastolic blood pressure 80 to 99 mm Hg (Dinamap Oscillometric 1846 SX/P Monitor, Critikon Inc; the average of five readings in the supine position recorded on two separate occasions), and serum cholesterol 5.2 to 6.9 mmol/L. They were excluded if they were taking any regular medication or if they had evidence of uncontrolled congestive cardiac failure or unstable angina, clinical or biochemical evidence of renal or liver disease, hypercholesterolemia, or asthma or suffered from any major allergies. Entry was also restricted to those who usually ate one or less than one fish meal per week and drank <210 mL ethanol per week (three standard drinks per day). Of 1248 men attending for screening, 138 met the trial entry criteria. Approval for the study was obtained from the hospital's Human Rights Committee, and all participants were informed of the purpose of the study and gave written consent.
Dietary Education and Intervention
Details of the dietary education and intervention have been described previously.18 19 20 Briefly, the screening of each subject took place over a 3-week period. Individual energy intake and average intake of nutrients was determined from three 24-hour weighed food records. Subjects were then randomly allocated to one of seven groups before entering a 1-week baseline measurement period before dietary intervention, during which time maintenance of usual dietary habits was encouraged (Fig 1
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During the baseline period, volunteers received detailed dietary counseling on three separate occasions about the diet plan and appropriate energy level to be followed for the duration of the study. A family counseling session was held during which the men and their spouses were advised on how to achieve their dietary goals in a family environment. A dietitian ensured that subjects also received individual counseling on how to achieve and maintain the planned diet. To assist in this, each subject was provided with a variety of recipes, as well as detailed information on preferred food items to be eaten and those to be avoided. To ensure an accurate assessment, the dietitian advised the men and their spouses on the use of digital food scales and the recording of all foods, beverages, minerals, and vitamins on a standardized food record form. During the subsequent 12-week intervention period, seven 24-hour weighed food records were completed on randomly selected days and manually checked at each visit to ensure compliance with the dietary instructions.
All groups reduced sodium intake to <90 mmol/d during the 12-week diet intervention phase. At 2-week visits and at the end of the intervention, weight was recorded and a health questionnaire was completed. Individual diet counseling was continued, and compliance was assessed by the dietitian throughout the intervention. During the 7-day baseline period, fasting blood samples were collected twice for platelet aggregation studies and the determination of platelet TXB2, twice for serum lipids,19 20 and once for platelet phospholipid fatty acids,18 20 and a lifestyle questionnaire was completed. Blood pressure and heart rate were measured automatically with the same Dinamap 1846 SX/P monitor for each subject.18 19 Measurements were taken twice during each of the screening and baseline periods, then every 4 weeks of the intervention, including twice during the final week. Fasting blood samples for platelet aggregation and platelet TXB2 studies, serum lipids, and platelet phospholipid fatty acids were also collected at the end of the intervention.
Diets
The seven dietary groups are shown in Fig 1
. Five of the groups were assigned to diets supplying 40% and two to diets supplying 30% of total dietary energy from fat. The five groups on the 40%-fat diet were given either placebo capsules alone (group 1), fish (group 2), fish oil capsules (group 3), fish plus fish oil capsules (group 4), or twice the dosage of fish oil capsules (group 5). One of the two 30%-fat groups was a control group, and the other was given fish (groups 6 and 7, respectively). Placebo capsules were given to groups 1, 2, 6, and 7 as shown in Fig 1. Diets were designed such that subjects consuming fish and placebo capsules, fish oil capsules alone, or a combination of fish and fish oil capsules had the same total fat intake as their respective control group.
Diets for each of the seven groups were at six energy levels, from 6270 kJ (1500 kcal) to 11 495 kJ (2750 kcal) in 1045-kJ (250-kcal) increments, to cater to individual requirements. The 40%-fat diets recommended a ratio of polyunsaturated to saturated fats of <0.3 and included 16% to 17% protein, 43% to 44% carbohydrate, and 10 g/4180 kJ (1000 kcal) fiber. Cholesterol was >300 mg/d. The 30%-fat diets had a ratio of polyunsaturated to saturated fats of >1, 16% to 17% protein, 53% to 54% carbohydrate, fiber >20 g/4180 kJ, and cholesterol <230 mg/d. The recommended saturated-to-monounsaturated-to-polyunsaturated fatty acid compositions of the 40% and 30% diets were 20%:16%:4% and 10%:10%:10%, respectively.
Subjects in groups 2, 4, and 7 were instructed to eat one fish meal per day every day of the week, prepared according to a variety of recipes. Fish was provided free of cost and included Greenland turbot fillets (
160 g/d), canned sardines (95 g/d), tuna (90 g/d), and salmon (90 g/d). This quantity of fish provided 1.5, 1.7, 1.3, and 2.4 g/d, respectively, of DHA and 3.5, 4.1, 3.2, and 3.8 g/d, respectively, of total 3-fatty acids.18 20 Menus were designed to include all the types of fish, and thus the average daily intake of 3-fats was likely to be similar between the fish-eating groups. Fish were derived from the same batch to avoid seasonal variation in fatty acid composition. The amount of fish was calculated to provide 1.32 g/d of EPA, or about the same quantity (6 g) as six fish oil capsules (Lipitac; Reckitt & Colman Pharmaceuticals and Jahres Fabrikker). The capsules also contained 0.8 g/d DHA and a total of 2.6 g/d in total 3-fatty acids.18 20 Placebo capsules (Reckitt & Colman Pharmaceuticals and Jahres Fabrikker) contained olive, palm, and safflower oils in the ratio 1:4.5:4.5 and provided approximately the same ratio of saturated to monounsaturated to polyunsaturated fatty acids as in the fish oil capsules.18 20 The complete fatty acid, vitamin, and electrolyte composition of the fish, fish oil, and placebo capsules has been presented elsewhere.18 20
Platelet Phospholipid Fatty Acids
The platelet phospholipid fraction was isolated and fatty acid methyl esters were prepared and analyzed as described previously.20
Platelet Aggregation Studies
Whole blood (50 mL) was collected into 3.8% (wt/vol) trisodium citrate (1/10), and PRP was prepared by centrifugation at 190g for 10 minutes. After removal of PRP, blood was further centrifuged at 2000g for 10 minutes to provide PPP. The platelet preparation was counted on a Coulter model 5 (Coulter Electronics), and the PRP was diluted with PPP to give a platelet count of 2.5x108/mL. Platelet aggregation was measured in a model 560 Chronolog Aggregometer (Chrono-log Corp) by the optical density method in response to collagen and PAF. Aggregation was carried out in 500-µL aliquots of PRP, and after equilibration at 37°C for 5 minutes, the aggregant was added to the bottom of the cuvette and mixed efficiently with a stirrer. Five concentrations of each aggregant were used at final concentrations of 0.125, 0.25, 0.5, 1.0, and 2.0 µg/mL collagen (Chronolog 385) and 0.05, 0.10, 0.25, 0.50, and 1.0 µmol/L PAF (Swiss Bioproducts). Aggregation was measured as percentage light transmittance at 1, 3, and 5 minutes after addition of the aggregant. For the purpose of statistical comparison, areas under the aggregation plots constructed from measurements taken at 0, 1, 3, and 5 minutes were calculated by the method of Matthews et al.21
Platelet TXB2 was measured only from aggregations with the four highest doses of collagen (final concentrations of 0.25, 0.5, 1.0, and 2.0 µg/mL), during which aliquots of PRP were removed 5 minutes after the addition of the aggregant and collected into PGE1 and aspirin (final concentrations of 2.6 µmol/L and 9.2 mmol/L, respectively). Samples were centrifuged at 12 000g for 2 minutes and stored at -70°C until assayed for TXB2 (the degradation product of TXA2) as previously described.22
Statistical Analysis
Results are expressed as mean±SEM. The difference between the mean values of the two baseline and of the two postintervention measurements was calculated, and these differences were analyzed by one-way ANOVA between the seven diet groups by use of the Statistical Package for the Social Sciences (SPSS Inc). Between-group differences were determined by group contrast or Mann-Whitney U tests, with adjustment of the 
-level by the Bonferroni method. Data from platelet aggregations to collagen and PAF as well as platelet TXB2 were assessed by multiple linear regression analysis. Areas under the aggregation plots constructed from measurements taken at 0, 1, 3, and 5 minutes after addition of the aggregant were calculated.21 Platelet responses used regression models with pooled time series analyses and assessed the postintervention (week 16) response to each aggregant after adjustment for aggregation responses at baseline (week 4; calculated as areas under the aggregation plots) and dose of aggregant. Platelet TXB2 used pooled time series analyses and assessed the postintervention (week 16) response after adjustment for responses at baseline (week 4). Groups were compared by use of dummy variables constructed relative to the 40%-fat control group (group 1).
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Results |
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Of the 138 men who began the trial, 120 completed the 12-week dietary intervention and were included in the analysis. Withdrawals were primarily in the first 2 to 3 weeks of the intervention and for reasons unrelated to the dietary intervention. Subjects across the seven dietary groups were well matched for the selection variables, and there was no significant difference between the groups at baseline.18 20 For all the groups combined, the average age of the men was 45.7±0.6 years, with a body mass index of 27.5±0.2 kg/m2, blood pressure of 136.5±0.8 mm Hg systolic and 84.8±0.6 mm Hg diastolic, serum cholesterol of 6.09±0.06 mmol/L, triglycerides of 1.74±0.07 mmol/L, and HDL cholesterol of 1.24±0.02 mmol/L. Compliance with the dietary intervention was >95%, as assessed by analysis of diet records20 and confirmed by changes in platelet and plasma fatty acids. Analysis of lifestyle questionnaires indicated no major changes in physical activity during the study, and diet records confirmed that total energy intake, nutrient balance, and alcohol intake were not significantly altered. The mean body weight for all groups combined fell 0.65±0.13 kg, with the greatest fall in the 30%-fat groups (1.35 and 0.82 kg for groups 6 and 7, respectively).
Platelet Phospholipid Fatty Acids
The fatty acid composition of platelet phospholipids at baseline was similar across all groups and consisted of 26.82±0.13% 20:4 6, 0.52±0.03% 20:5 3, and 4.69±0.10% 22:6 3. The changes in total 3-fatty acids (ie, 20:5 3+22:5 3+22:63) and 6-fatty acids (ie, 20:3 6+20:4 6+22:4 6) are shown in Fig 2. These represent the difference between baseline (week 4) and the end of the intervention (week 16). Those groups consuming 3-fatty acids from fish or fish oil capsules showed a significant increase (P<.00001) in total 3-fatty acids, with a concomitant significant reduction (P<.00001) of comparable magnitude in total 6-fatty acids. The largest changes in fatty acids occurred in the composition of 20:4 6, which decreased markedly (P<.00001), and increases in 20:5 3 (P<.00001) and 22:6 3 (P<.00001).18 20
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Platelet Aggregation
Figs 3 and 4 illustrate platelet aggregation responses to collagen and PAF over the entire 5-minute period for individual dietary groups. Plots were constructed from the mean aggregation at 0, 1, 3, and 5 minutes after addition of each aggregant. As expected, platelet aggregation responses were dose dependent with collagen or PAF stimulation. Furthermore, Figs 3 and 4 demonstrate that although platelet aggregation in all groups taking 3-fatty acids was reduced relative to the high- and low-fat control groups (groups 1 and 6, respectively) to all doses of collagen and PAF by the end of the intervention period, the effect was more pronounced at the lower doses of each aggregant. For statistical analyses, however, multiple linear regression models (shown in Tables 1 and 2) used data of the postintervention responses, measured as areas under the aggregation plots shown in Figs 3 and 4, to assess and compare the effects of fish and fish oil.
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Platelet Aggregation to Collagen
Collagen-stimulated platelet aggregation was reduced significantly to all doses of fish and fish oil on both the high- and low-fat diets (Fig 3). Stimulation of platelet aggregation was dose dependent, with 0.125 to 2.0 µg/mL final concentration of collagen. In multiple regression analysis, the postintervention platelet aggregation for all doses of collagen combined, after adjustment for baseline values (week 4), was significantly reduced in all groups taking fish or fish oil compared with the high-fat control group (group 1) (Table 1). In subjects receiving a high-fat diet, platelet aggregation was reduced significantly (P<.0001) after 3-fatty acids, with no difference between fish and fish oil, nor was there any difference in the response between the different 3-fatty acid doses (ie, groups 2 and 3 versus groups 4 and 5). Aggregation in groups receiving fish or fish oil (groups 2 through 5) was reduced by 7% compared with the high-fat control diet (group 1). Subjects on the low-fat control diet (group 6) had a small but significantly reduced response to collagen-induced platelet aggregation (P=.0478) compared with the high-fat control diet (group 1). The incorporation of fish into the low-fat diet (group 7) further reduced platelet aggregation to approximately the same extent (5.3%, P=.0003) as in those subjects consuming 3-fatty acids in a high-fat diet.
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Platelet Aggregation to PAF
Platelet aggregation in response to PAF was significantly reduced in all groups consuming 3-fatty acids in conjunction with both high- and low-fat diets (Fig 4). Stimulation of platelet aggregation was dose dependent, with PAF added at 0.05 to 1.0 µmol/L final concentration. Subjects in groups taking 3-fatty acids (groups 2 through 5 and 7) showed considerable reversible aggregation at PAF doses of 0.10 and 0.05 µmol/L final concentration. In multiple regression analysis, after adjustment for baseline values (week 4), the postintervention platelet aggregation for all doses of PAF combined was significantly reduced in all groups taking fish or fish oil in conjunction with a high-fat diet compared with the high-fat diet alone (group 1) (Table 2). 3-Fatty acids given as fish oil supplements, when part of a high-fat diet, were more potent than fish in reducing platelet aggregation. Subjects taking fish in groups 2 and 4 showed a 4.4% (P=.0544) and 4.6% (P=.0440) fall in platelet aggregation, respectively. Platelet aggregation in groups 3 and 5 taking fish oil capsules fell by 9.7% (P<.0001) and 8.5% (P=.0003), respectively. The low-fat diet alone (group 6) had no effect on platelet aggregation compared with the 40%-fat control diet (group 1), whereas the incorporation of fish to this diet (group 7) led to a significant (P<.0001) reduction (10.3%).
Platelet TXB2
Platelet-derived TXB2 from collagen-induced aggregation was significantly reduced in all groups taking 3-fatty acids (Fig 5 and Table 3). There was a significant correlation between the mean platelet aggregation measured as area under the aggregation curve and platelet TXB2 (r=.4119, P<.0001). Platelet aggregation measured as percentage light transmittance at 5 minutes was also significantly correlated with platelet TXB2 (r=.4694, P<.0001). Multiple regression analysis showed that after correction for baseline values (week 4), the postintervention platelet TXB2 from the four highest doses of collagen aggregation (final concentrations of 0.25, 0.5, 1.0, and 2.0 µg/mL) combined was significantly reduced in groups 2 (P=.0084), 3 (P=.0545), 4 (P=.0371), and 5 (P=.0004) on a high-fat diet and in group 7 (P=.0533) on a low-fat diet compared with the high-fat control diet (group 1). The low-fat diet alone (group 6) had no effect on platelet TXB2. The biggest fall occurred in group 5 subjects, who were taking 12 g of fish oil capsules per day.
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Relationships Between Changes in Platelet Aggregation and TXB2 and in Platelet Phospholipid 3- and 6-Fatty Acids
In multiple regression analysis, the postintervention platelet TXB2 concentration was significantly associated with changes in platelet 3- and 6-fatty acid composition (Table 4). After adjustment for differences in baseline platelet TXB2 (week 4), the 3-fatty acids (20:5+22:5+22:6) were shown to be significantly (P=.0068) inversely associated with platelet TXB2 concentration (Table 4A). In addition, the 6-fatty acids (20:3+20:4+22:4) were significantly (P=.0013) positively associated with platelet TXB2 (Table 4B). That is, for an increase in platelet 3-fatty acids and a fall in the 6-fatty acids, there was a significant reduction in platelet TXB2. The change (fall) in platelet phospholipid 20:4 6 was the greatest predictor and showed a positive association with changes (reductions) in platelet TXB2 (P=.0098) (Table 4C). This may simply be due to the significantly higher concentration of 20:4 6 relative to 20:5 3 in platelet membranes.
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Discussion |
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This randomized controlled study in men at increased risk of cardiovascular disease examined and compared the effects of dietary fish and fish oil in the setting of different fat intakes on platelet aggregation and platelet thromboxane formation. The results show first, that
3-fatty acid effects on platelet aggregation are influenced by the background level of dietary fat and second, that after dietary 3-fatty acid intake, platelet aggregation responses are dependent on the source of the 3-fatty acids and the agonist used to induce platelet aggregation. 3-Fatty acids reduced platelet aggregation induced by collagen and PAF regardless of whether they were ingested as daily fish meals or fish oil capsules. Dietary 3-fatty acids also reduced platelet TXB2 release from collagen-aggregated platelets. Although platelet aggregation was reduced in response to all doses of collagen and PAF by the end of the fish and fish oil intervention period, the effect was more pronounced at the lower doses of each aggregant close to the threshold dose.
By multiple regression analysis, there were significant inverse relationships between platelet responses induced by both collagen and PAF and an increase in total dietary 3-fatty acid composition. However, platelet aggregation responses were differentially affected by the two agonists. Collagen and PAF were chosen as platelet agonists because they induce platelet aggregation by different mechanisms, with PAF being one of the most potent natural stimulators of platelet aggregation.23 In response to collagen, platelet aggregation and thromboxane were similarly reduced by 3-fats given as fish meals and fish oil capsules. The collagen response was also independent of the dose of 3-fatty acids and the dietary fat intake. Across all groups taking fish or fish oils, aggregation responses were reduced by 5% to 7% and platelet thromboxane by 6% to 11% at the end of the dietary period. Furthermore, the 30% low-fat diet alone (group 6) led to a significant, albeit small, reduction in platelet aggregation compared with the 40% high-fat diet (group 1). Of significance, however, the incorporation of one daily fish meal as part of a low-fat diet further reduced platelet aggregation to collagen.
In contrast, PAF-induced aggregation responses were dependent on the source of the 3-fatty acids. Fish oils were more potent in reducing platelet aggregation than fish meals (4.5% versus 9%, respectively) when given with a high-fat diet. However, whereas the low-fat diet alone had no effect on PAF-induced platelet aggregations, fish had a greater effect in reducing platelet aggregation to PAF stimulation on a low-fat rather than a high-fat diet. It is not clear why there were differences in platelet responses to collagen and PAF, although it may be explained in part by the different mechanisms by which the two aggregants induce platelet aggregation.23
Platelets play a critical role in atherogenesis and its complications through their interaction with the vascular endothelium and in the initial events leading to thrombus formation.24 25 Results with regard to measurements of platelet aggregation after dietary 3-fatty acids have yielded inconsistent findings, partly because of differences in study design, the source and quantity of 3-fatty acids given, and the choice of aggregant. However, previous short-term studies have demonstrated that dietary 3-fatty acids can reduce platelet responses to physiological stimuli, such as ADP and collagen,7 8 9 16 26 27 thrombin,7 10 and adrenalin.7 11 Our research group12 26 and others28 have also shown that fish oil supplements inhibit platelet aggregation by PAF. However, platelet aggregation does not appear to be affected in response to AA7 13 and the PGH2 analogue U46619.14 Such impaired platelet responses may be due in part to decreased platelet TXA2 production because of either a reduced availability of AA substrate or inhibition of platelet cyclooxygenase.28 29 It has been suggested that the replacement of AA by EPA, in particular, shifts the balance of the intravascular factors to an antiaggregatory, antithrombotic state. The production of TXA2 from platelets stimulated by a variety of agonists has been shown to be reduced by 60% to 80% after 3-fatty acid supplementation.7 30 31 At the same time, there is an increase in levels of platelet TXA3,32 which is derived from EPA and has significantly reduced biological activity.33
Despite these findings, the mechanism for the reduction of platelet–vessel wall interactions by diets rich in 3-fatty acids is certain to be more complex than just a reduction in the susceptibility of platelets to aggregation to an agonist. Moreover, the degree of inhibition of ex vivo platelet aggregation and platelet thromboxane formation by dietary 3-fatty acids is mild compared with the effects induced by aspirin. Thorngren et al,34 for example, noted that when aspirin was given to people supplementing their diets with fish, the inhibitory effect on platelet function was additive. In a subsequent report, it was shown that bleeding time was prolonged to a greater extent by a combination of aspirin and fish oil than by either alone, implying that inhibition of thromboxane production is not the only mode of action of 3-fatty acids.35
One of the aims of this study was to determine whether there are differences in the effects of 3-fatty acids provided as fish or fish oils on platelet function. Fish contain variable amounts of EPA and DHA, with proportionally more of the latter than is found in purified fish oil, such as the Lipitac used in this study. Fish meals may also provide constituents other than 3-fatty acids that could affect platelet function. In this study, the fish was analyzed for fatty acid composition and was stored in batches, with a mixture of fish types given to each subject. The amount of daily fish was calculated to provide 1.32 g of EPA, which equated to about the same quantity as 6 g of fish oil. Because of the variation in fatty acid composition of the fish, the intake of DHA in particular varied from 1.3 to 2.4 g/d and led to small differences in the total 3-fats provided. The ratio of EPA to DHA in the fish also varied considerably, being lowest (0.55) in salmon, which is rich in DHA, and highest (1.02) in tuna. In contrast, the ratio of EPA to DHA of the fish oil was 1.53. However, because the diets were designed to include all the types of fish, the average daily intakes of 3-fatty acids were likely to be similar. The study was not designed to determine whether EPA and DHA differ in their effect on platelet function, although there is evidence suggestive of biological differences between the two fatty acids. For example, an in vitro study showed that the incorporation of DHA into human platelets produced greater inhibition of platelet aggregation than either EPA alone or a combination of EPA and DHA.36 It was observed that the combination of EPA and DHA led to less inhibition of platelet aggregation than was seen for DHA alone. The authors concluded that EPA may alter thromboxane metabolism and DHA may act directly at the membrane level in a mechanism related to phospholipid modification.
A potentially important mechanism by which platelet function may be altered involves the cytochrome P450–dependent metabolism of fatty acids. This pathway leads to formation of epoxides or epoxyeicosatrienoic acid derivatives of AA, which have been shown to inhibit platelet aggregation.37 In addition, the 11,12-epoxyeicosatrienoic acid derived from AA inhibited platelet aggregation independently of thromboxane production.37 Recently, it was also demonstrated that the epoxides of DHA and EPA inhibited platelet aggregation at concentrations below those that affected thromboxane synthesis.38 The same study showed differences in the responsiveness of EPA and DHA in inhibiting platelet aggregation, whereby the epoxides of DHA were significantly more potent than the EPA and AA epoxides.38 These results provide more evidence for potential differences in platelet responsiveness to EPA and DHA and may possibly explain, in part, the differential effects observed herein between fish meals and the fish oil concentrate.
Effects of dietary 3-fatty acids on platelet function measured ex vivo may differ from platelet-vascular interactions in vivo. Patients who suffer from atherosclerotic vascular disease have been shown to have shortened platelet survival, which is presumably due to accelerated interactions of platelets with the damaged vasculature. The administration of 3-fatty acids has led to a significant lengthening of platelet survival in such patients, suggesting that dietary 3-fatty acids may reduce platelet adhesion to atherosclerotic plaques.39 40 We have previously shown that fish oil supplementation reduced platelet aggregation in patients with symptomatic and angiographically demonstrated peripheral vascular disease on their usual high fat intake.26 The results of the present study suggest that these effects would be greater with less dietary fat.
Although it is difficult to be certain of the clinical relevance of the observed changes in platelet aggregation to collagen and PAF in this study, given that the reduction in platelet aggregation and thromboxane production was on the order of 5% to 11%, the use of platelet aggregation in vitro has been reported to be a useful biological marker for the prediction of coronary events and mortality both in healthy men and in survivors of myocardial infarction.41 42 Thus, a reduction of platelet activity as shown here could be of clinical significance, particularly since our subjects were men with increased cardiovascular risk factors.
In summary, we have shown that in men at increased cardiovascular risk, platelet aggregation responses after dietary 3-fatty acids are dependent on the agonist, the source of 3-fatty acids, and the dietary fat intake. Platelet aggregation was reduced to both collagen and PAF stimulation after a 12-week intervention including one daily fish meal or fish oil capsules. Collagen-induced aggregation was independent of the source and dose of 3-fatty acids. In addition, platelet responses to collagen were reduced by the low-fat diet alone. In contrast, the low-fat diet alone did not affect PAF aggregation, and platelet responses to PAF were reduced more by fish oil than fish in the presence of a high-fat diet. The combination of a fish and low-fat diet had the greatest effect in reducing platelet aggregation to PAF stimulation.
From a nutritional and public health point of view, it would seem reasonable to recommend a diet reduced in total fat and including several fish meals a week. Eating fish regularly as a substitute for meat products with higher saturated fat will normally lead to a reduced intake of total and saturated fat. Moreover, the inclusion of fish rather than fish oil supplements will also ensure adequate provision of other dietary macronutrients, such as protein, calcium, and vitamin D, without increasing total fat. The reduction in platelet aggregation, in conjunction with our previous findings of improvements in blood pressure and heart rate18 20 and a substantial improvement in the lipoprotein profile,19 20 suggests that dietary 3-fatty acids have more consistent favorable effects on these cardiovascular risk factors when given as part of a low-fat rather than a high-fat diet.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by a programme grant from the National Health and Medical Research Council of Australia. The authors acknowledge the late Professor Robert Vandongen for his great contribution to and inspiration for this study. We acknowledge the excellent technical assistance of Lynette McCahon, Andrea Douglas, and Anne Tunney, and we thank Dr Anne Barden for the platelet thromboxane analyses. We would like to thank Kailis and France Pty Ltd, Perth, Western Australia, for their generous donation of frozen filleted fish and canned tuna and salmon. King Oscar Fine Foods Pty Ltd, Melbourne, Australia, subsidized the cost of canned sardines. Lipitac fish oil and placebo capsules were kindly donated by Reckitt & Colman Pharmaceuticals, Sydney, Australia, and Jahres Fabrikker, Sandefjord, Norway.
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Footnotes |
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Reprint requests to Dr Trevor A. Mori, University Department of Medicine, Medical Research Foundation Building, Box X2213 GPO, Perth, Western Australia 6001. E-mail tmori@cyllene.uwa.edu.au.
Received June 14, 1996;
revision received June 25, 1996;
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1. Lands WEM. Fish and Human Health. Orlando, Fla: Academic Press; 1986.
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