© 1997 American Heart Association, Inc.
Articles |
Influence of n-6 versus n-3 Polyunsaturated Fatty Acids in Diets Low in Saturated Fatty Acids on Plasma Lipoproteins and Hemostatic Factors
From the Nutrition, Food & Health Research Centre, Kings College London (T.A.B.S., F.R.O.); the MRC Epidemiology and Medical Care Unit, Wolfson Institute of Preventive Medicine, St Bartholomew's, and the Royal London School of Medicine and Dentistry, London (G.J.M., K.A.M.); Wynn Division of Metabolic Medicine, Imperial College School of Medicine at the National Heart and Lung Institute, London (D.C.); and the National Heart & Lung Institute, Imperial College School of Medicine, London (M.F.O.).
Correspondence to Professor T.A.B. Sanders, Nutrition, Food & Health Research Centre, Kings College London, Campden Hill Rd, London, W8 7AW.
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Abstract |
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Abstract Modification of dietary fat composition may influence hemostatic variables, which are associated with increased risk of coronary heart disease (CHD). To address this question, we performed a controlled feeding study on 26 healthy male nonsmoking subjects with diets of differing fat composition. For the first 3 weeks, the subjects were given a diet calculated to supply 30% energy as total fat: 8% as monounsaturated, 4% as polyunsaturated, and 16% energy as saturated fatty acids, respectively (saturated diet). This was followed immediately by two diets taken in random order, each of 3-week duration and separated by an 8-week washout period on the subject's usual diet. Both diets were calculated to supply 30% of energy as fat: 14% monounsaturated, 6% as polyunsaturated, and 8% energy as saturated fatty acids. They both provided 5 g (approximately 1.7% energy) more of polyunsaturated fatty acids than the saturated fat diet; in one diet as long-chain n-3 fatty acids (n-3 diet) and in the other as linoleic acid (n-6 diet). Fasting plasma lipids, lipoproteins, and hemostatic factors were measured on the final 3 days of each dietary period. In a subset of 9 subjects the postprandial responses to a test meal were studied on the penultimate day of each period, each meal having the fat composition of its parent diet. On the n-3 diet compared with the n-6 diet, plasma triglyceride, HDL3 cholesterol, apoprotein AII, and fibrinogen concentrations were lower and HDL2 cholesterol concentration was higher (P=.0001, P=.003, P=.0001, P=.004 and P = .001, respectively). On both the n-3 and n-6 diets compared with the saturated diet, fasting plasma total and LDL cholesterol, apoprotein B, ß-thromboglobulin concentrations, and platelet counts were lower (P<.0001, P<.0001, P<.001, P<.01, and P<.05 respectively) and plasma Lp(a) and von Willebrand factor concentrations were higher (P=.02 and P<.01, respectively). Fasting factor VII coagulant activity (VIIc) was increased and apoprotein AI concentration reduced following the n-3 diet (P=.004 and P=.01, respectively) compared with the saturated diet. Plasma fibrinogen concentration was significantly greater following the n-6 diet than on the saturated diet (P=.02). Postprandially, plasma triglyceridemia was greater on the n-6 diet and lowest on the n-3 diet (P<.001) with the saturated diet being intermediate. Plasma VIIc was increased at 4 hours following the standardized test meals on the n-3 and n-6 diets (both P<.05) but not on the saturated diet. An increased intake of long chain n-3 fatty acids decreases fasting plasma triglyceride and apoprotein AII concentrations and increases HDL2 cholesterol concentrations and results in less postprandial lipemia but leads to an increase in VIIc. An increased intake of linoleic acid may raise plasma fibrinogen concentration. Decreasing the intake of saturated fatty acids reduces plasma LDL cholesterol and apoprotein B without affecting HDL cholesterol concentration independent of the type of polyunsaturated fatty acids in the diet. When advice is given to reduce saturated fat intake, it is important to ensure an appropriate ratio of n-3/n-6 fatty acids in the diet.
Key Words: lipids • lipoproteins • dietary fat • fibrinogen • coagulation
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Introduction |
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Dietary recommendations1 2 with respect to the intake of fat and the prevention of CHD specify that saturated fatty acids and total fat intakes should provide no more than 10% and 30% to 35% of dietary energy, respectively. Such advice is based on the direct and causal associations between saturated fat consumption and blood cholesterol levels3 and between cholesterol concentration and risk of CHD.4 Some recommendations suggest the partial replacement of saturated fatty acids with polyunsaturated or monounsaturated fatty acids, which appear to have a neutral influence on plasma cholesterol concentrations at the levels recommended.5 However, n-6 and n-3 polyunsaturated fatty acids can influence mechanisms concerned with hemostasis and inflammation,6 sometimes in opposing ways, so that an altered balance between the n-6 and n-3 series may influence risk of CHD.
Coronary thrombosis is a major cause of sudden cardiac death,7 acute myocardial infarction,8 unstable angina pectoris,9 and silent myocardial ischaemia.10 It has been argued that a hypercoagulable state not only predisposes to coronary thrombosis but is also associated with accelerated atherogenesis. Raised plasma fibrinogen concentrations and increased plasma factor VII coagulant activity (VIIc) are powerful predictors of risk of fatal CHD in middle-aged men11 12 13 even after adjustment for other known risk factors such as blood pressure and plasma cholesterol. Increased plasma concentrations of von Willebrand factor, which is synthesized and stored in endothelial cells and plays a role in platelet aggregation and adhesion, have been associated with a raised risk of fatal CHD14 and of thrombotic events in patients with atherosclerotic heart disease.13 15 Levels of plasminogen activator inhibitor type 1 (PAI-1) and tissue plasminogen activator (t-PA) are also elevated in patients with CHD16 and along with von Willebrand factor are thought to be markers of endothelial dysfunction.
An increase in VIIc occurs following the consumption of a high fat meal17 18 and raised levels of VIIc are associated with habitual high fat intakes.19 20 High plasma triglyceride concentrations are also associated with increased VIIc21 22 23 24 and PAI-1 activities.16 25 However, the influence of dietary fat composition on VIIc and PAI-1 has not been resolved. A post-hoc analysis of an atherosclerosis regression study found lower von Willebrand factor concentrations in subjects receiving a lipid lowering diet26 and this was attributed to an increased intake of polyunsaturated fatty acids. A cross-sectional study found inverse relationships between the dietary intake of n-3 polyunsaturated fatty acids and von Willebrand factor and fibrinogen concentrations.27 Although an increase in bleeding time and altered platelet reactivity has been reported following the consumption of long chain n-3 fatty acids, the influence of these and other polyunsaturated fatty acids on plasma fibrinogen, VIIc, PAI-1 and t-PA remains controversial.6 Moreover, few studies of n-3 fatty acids have attempted to standardize the intake of each fatty acid and in almost all the dietary fat has been provided in a single food.28 29 30 31 Nordoy et al32 investigated the effects of long-chain n-3 diets on plasma lipids and hemostatic function in 6 men in diets low or high in total and saturated fatty acids and concluded that the effects on plasma lipids of n-3 fatty acids were independent of those of saturated fatty acids. However, this study lacked the statistical power to answer these questions. Perhaps more importantly, the consequences for hemostatic activity of different ratios of n-6/n-3 fatty acids in diets high in monounsaturated fatty acids and low in saturated fatty acids are not known. This is relevant because an increased intake of oils rich in oleic acid (such as olive oil) is being advocated to replace saturated fatty acids in the diet.2 Recent dietary guidelines suggests that polyunsaturated fatty acids should provide approximately 6% of the energy intake, whereas the intake traditionally supplied by European and North American diets was 4% of the dietary energy.3 The aim of the present study was to compare the effects on plasma lipid concentrations and hemostatic factors of n-6 and n-3 fatty acids in diets providing 30% of the energy from fat in which saturated fatty acids provide less than 10% of energy and oleic supplies 14% of energy.
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Methods |
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Subjects and Setting
The study protocol was reviewed and approved by the local Human Experimentation Committee and all participants gave written informed consent. Twenty-seven healthy nonobese males aged 18 to 34 years were recruited from among staff and students of King's College, London. Fasting plasma lipoprotein lipid concentrations, body weight, whole blood cell count, plasma fibrinogen concentration and VIIc were confirmed to be within normal limits before to entry (Table 1
). Their habitual dietary intake was
then assessed by 3-day weighed dietary intake record, which was
converted to nutrient intake by computer from food tables based on
those of McCance and Widdowson (Royal Society of Chemistry, Cambridge).
Smoking was an exclusion criterion because of its effect on fibrinogen
concentration33 and nonsmoking status was
confirmed by analysis of random urine samples for
cotinine34 by gas
chromatography35 throughout the
study. One subject was found to be positive for urinary cotinine and
the results were excluded from the statistical analysis.
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Experimental Diets and Design
The subjects took three experimental diets standardized to an
energy intake of 13MJ/d (consistent with energy balance) and
designed to supply 30%, 55%, and 15% of energy as fat, carbohydrate,
and protein, respectively (Table 2). Each
experimental diet was taken for 21 days, and all meals were provided by
the metabolic unit at King's College on a 7-day rotating
menu. On each day the mid-day meal provided 60% of energy requirements
and 85% of the total fat intake. On all weekdays this meal was taken
in a dining facility, but those during weekends and all breakfasts and
evening meals were supplied prepacked for consumption at home. The
design of the study was such that all subjects were fed the saturated
fat diet for 3 weeks and then randomized to receive either the
n-3 or n-6 diet for 3 weeks when after an 8-week
washout period the treatment sequences were reversed.
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The first experimental diet (saturated) had a target composition of 16% of total energy supplied by saturated fatty acids, 8% by monounsaturated fatty acids, and 4% from polyunsaturated fatty acids (energy from glycerol bringing total energy from fat to 30%). The target saturated fatty acids intake in the n-6 and n-3 diets was reduced to 9% of total energy, the deficit of 8% energy being restored by increased monounsaturated and polyunsaturated fatty acids to 14% and approximately 6% of energy, respectively. In the n-3 diet, 1.5% of energy from polyunsaturated fatty acids was supplied as eicosapentaenoic (EPA; 20:5n-3) and docosahexaenoic (DHA; 22:6n-3) acid (approximately 5 g/day), whereas in the n-6 diet additional linoleic acid (approximately 5 g/day) was added in place of EPA and DHA. Thus both the n-6 and n-3 diets had similar contents of total polyunsaturated, monounsaturated, and saturated fatty acids.
All diets were formulated using ordinary foodstuffs. In the saturated
fat diet, the fat was provided mainly as butter. In the n-6
diet, the major fat was olive oil, while in the diet enriched with
n-3 fatty acids was comprised a mixture of olive oil and
fish oil (MaxEPA, Seven Seas). The fish oil was incorporated into bread
and foods. As the fish oil contained added vitamin E and 650mg
cholesterol/100g, vitamin E intakes were
standardized at 26mg RRR-
-tocopherol equivalents/d and
dietary cholesterol at 250mg/d across all diets, using
natural RRR--tocopherol added to an oil-based salad
dressing and dried egg powder in biscuits. Coffee intake was restricted
to not more than 4 cups/d. Alcohol up to two standard units (ie, about
20 mL) was permitted on each day, except the final 3 days of each
dietary period, when it was forbidden. Black tea, white tea (with milk
from the daily allowance), and low-calorie soft drinks were permitted
ad libitum. Vitamin preparations were not allowed. In addition subjects
were asked to avoid all aspirin-containing medication throughout the
study.
Test Meals
On the penultimate morning of each experimental regimen, 9
subjects were given a test meal in a clinical research facility in
place of their usual breakfast. The test meal provided 80 g fat, 55 g
protein, and 240 g carbohydrate. The fat composition of the meal was
similar to that of the background diet for that particular experimental
regimen.
Replicate Meal Analysis
Replicates of the daily diet and test meals were
analyzed chemically for total energy (bomb calorimetry),
protein (Kjeldahl), total fat (Soxhlet), and fatty acids
(gas-liquid chromatography) as described
previously.36 The contribution of individual
fatty acids to total energy intake was estimated using 9 kcal/g fatty
acid.
Blood Sampling
Venous blood samples were obtained after an overnight fast on
the last 3 days of each dietary period. Additional samples were
collected at 1 hour, 2 hours, 4 hours, and 6 hours after each test
meal. Citrated samples were kept at room temperature,
centrifuged, and the plasma separated with plastic Pasteur
pipettes. Aliquots of 0.25 mL were than snap-frozen in liquid nitrogen
for assays of PAI-1, fibrinogen, and factor VII. For tPA assays, 4.5 mL
of blood was collected into a chilled vacutainer containing 0.5 mL of
0.5 mol/L citrate buffer pH 4.0, the plasma separated and snap-frozen
in liquid nitrogen. Samples for the analysis of
ß-thromboglobulin were collected into special
vacutainers (Diatube H, Becton Dickinson) chilled to 4°C. The upper
layer of plasma was carefully collected after
centrifugation and snap frozen in liquid nitrogen. For
measurement of the prothrombin fragment F11+2
(F1.2) 4.5 mL of blood was collected into 0.5 mL
of an anticoagulant mixture of trasylol, EDTA and a thrombin
inhibitor (Byk-Sangtec) in a chilled tube,
centrifuged promptly, and the plasma snap-frozen. All samples
for hemostatic studies were taken into silicone-coated tubes and stored
in sealed polypropylene tubes (Nunc) at -70°C until assayed. Blood
counts were determined on a sample collected into EDTA. Samples for
lipid assays were collected into vacutainers (Becton Dickinson)
containing EDTA chilled to 4°C and centrifuged within 3
hours. The plasma was then held at 4°C pending analysis of
cholesterol, triglycerides, HDL subfractions
and apoproteins within 5 days.
Hemostatic Assays
All samples for one subject were assayed in duplicate in a
single batch. Fibrinogen concentration was measured by a
thrombin-clotting method.37 Plasma VIIc was
determined by a one-stage bioassay using a rabbit brain thromboplastin
(Diagen, Thame, Oxon) and a factor VII-deficient substrate plasma
described elsewhere.38 Factor VII antigen (VIIag)
was measured by ELISA (Novo Nordisk). Plasma tPA and PAI-1 activities
were determined by chromogenic assay (Kabi). Plasma
F1.2 concentration was measured by
radioimmunoassay in the laboratory of Professor RD Rosenberg and Dr KA
Bauer.39
Lipid Assays
Total cholesterol and triglyceride
concentrations were measured by full enzymatic procedures using a
discrete analyzer (Roche Diagnostics, Welwyn,
Herts). Plasma high-density lipoprotein (HDL) and
HDL3 were separated by sequential precipitations
with heparin and manganese ions40 and dextran
sulfate41 and their cholesterol
concentrations determined. Low-density lipoprotein (LDL)
cholesterol concentration was calculated using the
Friedewald formula.42 Serum concentrations of
apolipoproteins AI, AII, and B were measured by commercial immuno
turbidimetry43 and Lp(a) by enzyme-linked
immunosorbent assay (Biopool AB, Ume
, Sweden).
The remaining analyses were performed on one blood sample only at the end of each dietary period. Erythrocyte phosphoglyceride fatty acid composition28 and plasma fatty acid concentrations44 were determined by gas-liquid chromatography. Plasma vitamin E concentration was measured by high-pressure liquid chromatography.45 Blood count was determined on a Coulter counter. Plasma concentrations of ß-thromboglobulin (Stago) and von Willebrand factor antigen46 were estimated by ELISA.
Statistical Analysis
Twenty-six subjects completed all dietary treatments and 1
subject did not enter the third dietary period. Each individual's
results for the 3 fasting samples were averaged in each period. Tests
for differences between dietary periods were performed by
analysis of variance within subject. This approach assumes that
the effects of treatment and the effects of period operate
independently, and the model is invalid when significant interaction
exists. Order effects and interaction were tested using the method of
Pocock.47 When significant differences were found
between diets in the analysis of variance pairs of diets were
compared using paired t tests, with adjustment for order
effect when necessary. The study had greater than 80% power to detect
a 5% difference in VIIc between pairs of diets at the 5% level of
statistical significance. For Lp(a), in which the distribution was
highly skewed, period effects and interaction were sought on
untransformed values using the Mann-Whitney test. In the comparison of
3 diets and paired comparisons, the nonparametric Friedman
test and Sign test were employed, respectively, on untransformed data.
Plots of plasma total cholesterol,
HDL3 cholesterol, apolipoprotein B,
apolipoprotein AI, and apolipoprotein AII revealed a tendency for
concentrations to decrease for several hours postprandially. The
statistical significance of this curvature was tested by fitting
quadratic polynomial scores to the data48 and
comparing differences in these scores between diets by ANOVA.
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Results |
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Table 1
presents the baseline characteristics in the 26 men
whose results were used for statistical analysis. All were
normolipidemic and none was obese. Plasma VIIc (reported only on 17
subjects owing to accidental cold activation in one batch of samples
during transport) and fibrinogen concentrations were normal. The
subjects usual average energy intake was 13.5 MJ/d with fat providing
38% of dietary energy. Urinary cotinine levels were consistent
with nonsmoking status throughout the study.
Dietary Characteristics and Fatty Acid Composition of
Erythrocyte Phosphoglycerides
Table 2 summarizes the results of
replicate chemical analysis of the diets. Daily energy intake
differed between diets by 0.1 MJ/d at most. Total fat intake was
99 g/day on the saturated and n-3 diets, and 105
g/day on the n-6 diet. The distinctive features of the
saturated fat diet were its higher content of short and
medium chain saturated fatty acids (8.7 g/day as compared with 0.5
g/day or less in the other diets), and cholesterol raising
(12:0 + 14:0 + 16:0) fatty acids (26.5 g/day as compared with
18.8 g/day for the n-6 diet and 17.0 g/day for the
n-3 diet). Daily intakes of oleic (18:1n-9cis)
and -linolenic acid (18:3n-3) were similar on the
n-6 and n-3 diets. The n-3 diet
contained 5g of EPA + DHA together with 12.7 g/day as
linoleic (18:2 n-6), whereas the n-6 diet
contained EPA + DHA only in trace amounts with a compensatory increase
in 18:2 n-6 to 17.3 g/day. The intakes of trans fatty
acids were low in all diets but were slightly higher on the saturated
fat diet. The daily intakes of dietary cholesterol and
dietary fiber were 250mg and 12 g/1000kcal (4.2MJ), respectively
in all phases. Erythrocyte phosphoglyceride fatty acid composition was
measured to check compliance with the diets and to demonstrate the
bioavailability of the n-3 fatty acids from the diets (Fig 1 and 2). When the
n-6 diet was taken before the n-3 diet the
proportion of EPA and DHA increased on the n-3 diet (the
mean values (SD) for EPA expressed as wt % total fatty acids were as
follows: saturated fat diet 0.6 (0.31), n-6 diet 0.6 (0.36),
n-3 diet 3.0 (0.67); for DHA they were saturated fat 5.1
(1.13), n-6 diet 4.9 (0.73), n-3 diet 6.2
(0.60)). However, when the treatment order was reversed, some of the
increase in EPA and especially DHA persisted into the n-6
dietary period despite the intervening 8-week wash-out period (the mean
values with SD for EPA expressed as wt% total fatty acids were as
follows: saturated 0.7 (0.30), n-6 diet 1.1 (0.20),
n-3 diet 3.5 (0.7); and for DHA they were 4.9 (1.03),
n-3 diet 6.4 (0.72), n-6 diet 6.1 (0.68). As a
carry-over of DHA and EPA might weaken the comparison of differences in
blood lipids and hemostatic factors, we measured plasma concentrations
of EPA and DHA. These analyses showed no evidence of a
carryover effect of dietary EPA and DHA (see Figs 3 and 4).
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Dietary Characteristics and Plasma Lipids and Hemostatic Factors in
the Fasting State
Table 3 summarizes the fasting
lipoprotein lipid and apolipoprotein concentrations for the 3 dietary
periods. The saturated fat diet was associated with significantly
higher total cholesterol (P<.0001),
LDL cholesterol (P<.0001), and
apolipoprotein B (P<.001) concentrations than the
n-3 and n-6 diets, which did not differ
significantly in these respects. The n-3 fat diet was
distinguished by significantly lower HDL3
cholesterol (P<.003), apolipoprotein
AI (P<.005), apolipoprotein AII
(P<.0001) and total triglyceride concentrations
(P<.0001) and a higher HDL2
cholesterol (P=.001) concentration than the
other diets, which did not differ significantly in these respects.
Plasma total HDL cholesterol concentrations were not
significantly different between diets. Plasma Lp(a) concentration was
significantly lower on the saturated fat diet than on either
n-6 (P<.02) or n-3
diets (P<.01), in which the median levels
were similar.
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Table 4 presents the fasting values
of the hemostatic variables at the end of each dietary period.
Plasma VIIc did not differ between the saturated
and n-6 diets, but on completion of the n-3 diet
it was increased on average by 7% of standard in comparison with the
saturated fat diet (P=.004) and by 5% of standard in
comparison with the n-6 diet (not statistically significant
P=.19). In contrast, VIIag concentration did not differ
between dietary periods. Fibrinogen concentration was increased by
about 10% on the n-6 diet as compared with the
n-3 diet (P= .004) and the saturated fat
diet (P=.02). There were no statistically
significant differences between F1.2, PAI-1
or tPA levels between the 3 dietary periods. Plasma von
Willebrand factor antigen concentration was
significantly lower (P<.01) and
both plasma ß-thromboglobulin concentrations
(P<.01) and platelet count
(P < .05) were significantly higher on the
saturated fat diet than on the other diets, which themselves were
similar in these respects.
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Other Variables
Body weight, plasma tocopherol (µmol/mmol
cholesterol), and white cell count were similar at the end
of each dietary period.
Test Meals and Postprandial Responses
Chemical analysis of replicates showed that the test meals
contained 79 g of fat, 54 g of protein, and 238 g of
carbohydrate (energy value, 7.73MJ). Table 5 presents the fatty acid composition
of these meals, confirming their close similarity to their parent diets
in this respect. Saturated fatty acids accounted for 46% of total
fatty acids in the saturated fat test meal, as compared with about 21%
in the other test meals. EPA and DHA accounted for about 6% of fatty
acids in the n-3 test meal, but were found in only trace
quantities in the other test meals.
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Fig 5 shows a curvilinear response in
total cholesterol concentration following all test meals,
levels decreasing up to about 2 hours postprandially and tending to
recover thereafter, though the recovery after n-3 test meal
was retarded. Plasma apolipoprotein B concentration showed a similar
postprandial pattern to that of total cholesterol (Fig 6). This curvilinearity was statistically
significant as indicated by the quadratic effect, but for simplicity
the findings of the statistical analysis are shown as the %
reduction between baseline and 2 hours postprandially in Table 6. Plasma HDL cholesterol and
HDL3 cholesterol concentrations also
showed distinct and statistically significant curvilinearity, the
latter decreasing on average by 11% after the n-6 meal,
15% after the saturated fat meal, and 20% after the n-3
meal. The pattern of responsiveness for apolipoproteins AI and AII
concentration were of the same sign, although less marked than that for
HDL cholesterol concentration, but the results applied to
only 7 of the 9 subjects. The quadratic scores were statistically
significant for both apolipoproteins AI and AII concentration after the
saturated fat meal, for apolipoprotein AI only after the n-6
meal, and for apolipoprotein A-II only after the n-3
meal.
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=
saturated diet 
= n-3 diet 
= n-6 diet.
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The postprandial response of Lp(a) differed from that of the other lipoproteins. None of the quadratic scores for postprandial curvilinearity in concentration was statistically significant when the results were analyzed according to geometric mean levels. However, the geometric mean was significantly reduced from the baseline value by 2 hours following the saturated fatty acid meal, but was unchanged from the baseline value after the n-6 meal. A 5% reduction after the n-3 meal was not statistically significant on data for 6 subjects. When the data were described instead as median levels, the quadratic scores were then statistically significant for the saturated fat meal and the n-6 meal but not for the n-3 meal.
Plasma triglyceride concentration increased following
all test meals, the peak measured level being at 2 hours. However, the
area under the curve differed significantly between test meals
(P<.0001), being highest after the
n-6 meal and lowest after the n-3 test meal fat
meal (Fig 7). The postprandial responses of
VIIc to the 3 test meals were of the same general form, there being an
increase from baseline to a peak value at 4 hours (Fig 8). The peak increase (% above baseline) was
21.2 (P=.02) for the n-6 meal, 17.3
(P=.03) for the n-3 meal, but only 4.2 (not
statistically significant) for the saturated fat meal.
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Discussion |
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This study was primarily designed to compare the effects of n-6 and n-3 fatty acids in diets low in saturated fatty acids. We took a number of precautions to ensure factors such as aspirin use and smoking did not confound the results. In order to decrease regression dilution bias, triplicate estimates of the main variables of interest (plasma lipids, fibrinogen, VIIc, t-PA, and PAI-1) were made for each dietary period. Nonrandomization of the saturated diet make comparisons with the n-6 and n-3 diets less secure because the findings may have been biased by systematic drifts with time. Nevertheless, the design does not negate such comparisons provided that the potential for bias is taken into consideration. We chose to use a crossover design as the within-subject variation of both the lipid and hemostatic variables is much smaller than that for between subjects. A wash-out period of 8 weeks was introduced to minimize any effect of any carry-over particularly from the n-3 diet. Analysis of the erythrocyte lipids showed some carry-over effect in the subjects who received the n-3 fatty acids first. This was most marked for the proportion of docosahexaenoic acid and is consistent with previous studies.28 However, analysis of the plasma concentrations of EPA and DHA revealed no evidence of a carry-over effect. Furthermore, the statistical analysis of the plasma lipoprotein and hemostatic variable did not find any evidence of a carry-over effect.
Dietary Fat Composition and Blood Lipids
This study shows that in a group of healthy young men with an
average plasma cholesterol concentration of 4.2 mmol/l
(163 mg/dl) when consuming a diet in which fat supplies only about 30%
of energy requirements, removal of saturated fatty acid leads to a
reduction in their plasma cholesterol concentration. A
reduction in saturated fatty acid intake from 16% to close to 8% of
dietary energy, and substitution with unsaturated fatty acids, caused a
modest but significant average reduction in plasma
cholesterol of 0.3 mmol/l. This effect was independent
of the source of polyunsaturated fatty acid used for replacement. The
result is consistent with meta-analyses of dietary
trials.5
The exchange of about 8% of dietary energy as saturated fatty acid for monounsaturated fatty acid and n-6 polyunsaturated fatty acid was of no consequence for the plasma concentrations of HDL cholesterol, apolipoproteins AI and AII, or the major subclasses HDL2 and HDL3. These results accord with those of Mensink and Katan,49 who observed no significant change in HDL cholesterol concentration on exchange of 6.5% of total dietary energy as saturated fat for unsaturated fat. Only when the diet contains more than 14% of energy as n-6 polyunsaturated fatty acids are LDL and HDL cholesterol concentrations reduced.50 51
Exchanging 5 g of EPA + DHA for 5 g linoleic acid in the diet had no effect on total cholesterol or LDL cholesterol concentrations. Some studies29 52 have found that fish oil supplements as well as the diets rich in oily fish lead to a rise in LDL cholesterol concentration. A criticism of these studies has been that no allowance was made for either hypercholesterolemic fatty acids in fish oil, such as 14:0, 16:0, and 16:1, or dietary cholesterol (see 53 for a review). Two recent studies54 55 using purified esters of EPA and DHA did not find the increases in LDL cholesterol to be statistically significant. Nevertheless, LDL levels may rise in certain hypertriglyceridemic patients when given n-3 fatty acids or other triglyceride lowering compounds.56 57 The n-3 diet was accompanied by a nonsignificant 3% increase in HDL cholesterol concentration. Our results agree with the conclusions of Harris53 indicating that in normolipidemic subjects an intake of approximately 5 g of long-chain n-3 fatty acids would not influence LDL cholesterol and would increase HDL cholesterol only by about 3% to 4%. The effects of fish oil on HDL subclasses and apoproteins AI and AII are less clear. A review of studies53 using long-chain n-3 fatty acid supplements reported several instances of an increase in HDL2 cholesterol concentration as in the present study, consistent with a previous report45 from our group. In agreement with Nestel,58 we observed a significant reduction in plasma apolipoprotein AI concentration on the n-3 diet. Apolipoprotein A-II concentrations were also significantly reduced on the n-3 diet. The enrichment of the depleted HDL population with cholesterol (ie, increase in cholesterol/AI ratio) could be a result of the reduction in triglyceride-rich lipoproteins (plasma triglyceride decreased on fish oil), which would impair the transfer of cholesterol from HDL to VLDL particles by way of cholesterol ester transfer protein.59
Many studies have shown that n-3 fatty acids lower plasma triglyceride concentration by decreasing the rate of hepatic secretion of VLDL-triglyceride and several studies have reported a blunted postprandial lipemia after a test meal rich in fish oils in subjects consuming a diet rich in n-3 fatty acids,60 61 as in the present study. The mechanism is poorly understood. A decreased rate of absorption is an unlikely mechanism as the plasma triglyceride concentration was similar at 2 hours compared with the saturated fat diet. Although dietary n-3 fatty acids decrease the hepatic synthesis of VLDL,62 63 64 thereby raising the possibility of increased lipolysis of chylomicrons owing to reduced competition between substrates for LPL,65 Brunzell et al66 have shown that LPL does not approach saturation kinetics until the plasma concentration of triglyceride approaches 5.7 mmol/l, making this explanation unlikely. Moreover, postheparin plasma LPL and hepatic triglyceride lipase activities are uninfluenced by an n-3 diet,60 61 but this does not exclude the possibility of increased LPL activity due to an increased surface area of vascular endothelium exposed to blood in the postprandial period. Such an effect could be mediated via the synthesis of eicosanoids from the long chain n-3 fatty acids, which have net vasodilator actions relative to those synthesized by n-6 fatty acids.
Bergeron and Havel67 examined the effects of diets rich in saturated fatty acids and n-6 fatty acids on the postprandial responses of plasma triglyceride-rich lipoproteins. The peak percentage increase of triglyceride concentration above baseline was greater after the n-6 diet than after the saturated fat diet, but by 6 hours concentrations were similar on both diets, as in the present study. It was suggested that n-6 fatty acids may be absorbed more rapidly than saturated fatty acids. In the present study, butter was the main source of saturated fatty acids. Butter contains a substantial proportion of short and medium chain triglycerides, which are transported via the hepatic portal vein to the liver in the post-absorptive phase rather than being incorporated into chylomicrons.68 This coupled with its relatively high content of stearic acid, which is less well absorbed,69 may explain why the degree of postprandial lipemia was lower with the saturated fat diet compared with the n-6 diet. Meals rich in palmitic acid might produce a postprandial lipemia that is more similar to that given by the n-6 and n-3 test meals.
Differences between fasting and nonfasting levels of cholesterol are generally regarded as too small to be of clinical relevance, although Cohn et al70 showed that levels could increase significantly in some individuals and decrease significantly in others after a fatty meal. Our study found that plasma cholesterol decreased significantly by at least 5% and often 10% after a fatty meal, suggesting that the time relation between blood sampling and meals is an important source of variation in blood cholesterol concentration. Similar observations were made for the apolipoproteins. The reasons for these postprandial responses in lipoproteins are unclear but they may arise in part from a postprandial dilution effect,71 changes in their rates of synthesis and catabolism,70 and exchanges between HDL and other lipoprotein classes.72
The effects of dietary fatty acids on Lp(a) concentrations have not been fully elucidated and most attention has focused on the potentially deleterious effects of trans fatty acids on this lipoprotein.73 In the present study intakes of trans fatty acids were uniformly low. Thus the higher concentrations of Lp(a) on the n-6 and n-3 diets cannot be attributed to a higher intake of trans fatty acids. In agreement with others we could find no Lp(a) lowering effect of n-3 fatty acids.74 75 A review of the literature suggests that the Lp(a) elevating effects attributed to trans fatty acids may have been misinterpreted, and that elevations in Lp(a) concentration have been found only when the proportion of fatty acids greater or equal to C18 is increased, as in the present study. Hornstra et al76 found that replacing palm oil for fat in the average Dutch diet led to a fall in Lp(a) concentrations that the authors suggested were normally maintained by trans fatty acids in the Dutch diet. Nestel et al77 reported a high Lp(a) concentration on a diet rich in elaidic acid (18:1trans) compared with one rich in palm oil. However, the high elaidic acid diet did not differ significantly in its 18:1trans content from that of a high oleic acid diet, which was not associated with any increase in Lp(a). Mensink et al73 have reported that elaidic acid increased Lp(a) concentration when compared with a saturated fat diet rich in palm oil. Judd et al78 could find no effect of 18:1trans compared with 18:1cis on Lp(a). However, a recent study79 found that partially hydrogenated soy bean oil (PHSBO) containing predominantly C18 fatty acids, and partially hydrogenated fish oil (PHFO) containing C18 to 22 isomeric fatty acids increased median Lp(a) concentration from 91mg/dL on a butter rich diet to 103mg/dL on the PHSBO diet and 121mg/dL and on the PHFO diet. Mensink et al73 also reported an increase Lp(a) concentration with diets enriched in oleic and linoleic acids compared with a diet rich in palmitate. Tholstrup et al69 have recently described an increase in Lp(a) concentration with a diet enriched with stearate compared with a diet rich in palmitate or one rich in laurate and myristate. We therefore suggest that the observed increases in Lp(a) are related to fatty acid chain length rather than degree or geometry of unsaturation. The significance of these small changes in Lp(a) concentrations with regard to risk of CHD is uncertain.
Dietary Fat Composition and Hemostatic Factors
Platelet counts and plasma
ß-thromboglobulin concentrations were higher on
the saturated fat diet compared with both the n-6 and
n-3 diets. Plasma ß-thromboglobulin is
a platelet-specific protein released from -granules of
activated platelets.80 In agreement
with this study, Salomaa et al81 have reported an
inverse association between plasma
ß-thromboglobulin level and the proportion of
dietary energy supplied by n-6 fatty acids. Knapp et
al81 reported lower plasma
ß-thromboglobulin concentrations in subjects
receiving fish oil. The lower von Willebrand factor antigen
concentration on the saturated fat diet in the present is at
variance with a recent report of lower levels inpatients with
arterial disease following a lipid lowering
diet.26 These three findings require confirmation
in studies in which there has been full randomization of diets
containing differing amounts of saturated fatty acids.
A high plasma fibrinogen concentration is a powerful predictor of increased risk of CHD in middle-aged men.11 13 In the present study we were careful to exclude any influence of cigarette smoking, which is known to elevate plasma fibrinogen,33 and nonsmoking status was confirmed by measurement of urinary cotinine. In this study the within subject standard deviation for fibrinogen was approximately 5%. Consequently, the study had more than a 90% power to detect a 5% change in fibrinogen concentrations at P<.05. A reduced plasma fibrinogen concentration has been reported in a few studies with diets containing long-chain n-3 fatty acids83 84 85 but at least as many studies have reported no effect28 29 30 31 85 86 87 or even an increase.88 In the present study, plasma fibrinogen concentrations on the n-3 diet were almost identical to those on the saturated fat diet and to the baseline level on screening. The major difference was a 10% higher plasma fibrinogen concentration on the n-6 diet compared with n-3 and saturated fat diets. This potentially important observation requires confirmation.
There have been reports of both an increase29 89 90 and a reduction91 in PAI-1 activity following the consumption of diets containing fish or long-chain n-3 fatty acids. However, in the present study we could not find any influence on PAI-1 activity. This finding, along with the lack of effect on tPA activity is consistent with there being no influence of n-3 fatty acids on fibrinolytic activity.28
Although a reduction in the intake of total fat is associated with a fall in VIIc,19 92 the effect of dietary fat composition on this hemostatic factor is far from clear. The influence of different saturated fatty acids on VIIc was studied by Tholstrop et al93 who found that a particular type of stearic acid rich diet, in which stearate was taken in the form of shea butter, led to lower levels of factor VIIc compared with either a palmitate rich or laurate + myristate rich diets. On the other hand, Mitropoulos et al94 argued that a raised plasma concentration of stearic acid is associated with increased VIIc. In the present study, fasting VIIc did not differ significantly between the saturated fat and n-6 diets. Most previous studies have been unable to detect any significant effect of dietary fat unsaturation on factor VII19 95 96 but these studies have lacked the statistical power. Many studies using fish oil supplements have also resulted in an increase in total fat intake that can confound the interpretation of the results while others have used bioassays that are poorly sensitive to changes in the proportion of factor VII circulating in the activated form.38 However, Hendra et al87 reported an increase in VIIc in a relatively large controlled trial in noninsulin dependent diabetic patients given a fish oil supplement, using the same assay as in the present study. The within subject deviation for VIIc was 7% in the present study, which therefore had 80% power to detect a 5% change in VIIc at P<.05. The 7% increase in VIIc on the n-3 diet compared with the saturated fat diet was unexpected as it was accompanied by decreased postprandial lipemia. Because this change was not accompanied by any similar alteration in factor VII antigen, it presumably reflected activation of factor VII in the fasting sample. That such activation was probably real is supported by the responses to the test meals. However, the higher fasting level of VIIc was not accompanied by an increase in plasma F1.2, a marker of prothrombin's conversion to thrombin by factor Xa.39
Postprandial activation of factor VII after a fatty meal, but not after a low-fat isoenergetic meal, is now well recognized17 18 and appears to be due to an increase in fraction of factor VII circulating in the activated form.18 We are unaware of any previous studies of the effect of n-3 fatty acids on postprandial factor VIIc. In a more recent study we have found that a standardized amount of n-3 fatty acids given in a low-fat meal containing 15 g fish oil did not increase VIIc but when consumed with 75 g of olive oil it was followed by similar increase in VIIc to that obtained with 90 g of olive oil.97 The findings of the present study suggest that a relatively high intake of n-3 fatty acid over a longer period of time may promote activation of factor VII after fatty meals.
Both n-3 and n-6 polyunsaturated fatty acids are essential nutrients in human diets but the optimal levels of these fatty acids, both absolute and relative to each other, remain uncertain. Estimates of minimum requirements for linoleic acid and n-3 fatty acids are in the range of 1% to 2% energy and 0.2% to 0.5% energy, respectively, and it has been proposed that the ratio between the two series should be between 5:1 and 10:1.98 There is considerable commercial interest in fortifying foods with long-chain n-3 fatty acids and several food products are being marketed in Japan and Europe as "functional foods." The findings of this study are, therefore, particularly pertinent. While an increased intake of long-chain n-3 fatty acids showed some potentially beneficial effects on plasma lipids such as a reduced postprandial lipemia and an increase in HDL2 cholesterol, it was also associated with a potentially adverse increase in VIIc. There may be other disadvantages with high intakes of n-3 fatty acids such as an increased susceptibility of LDL to peroxidation.99 The amounts of n-3 fatty acids used in this study were relatively high. It remains to be established whether an increase in VIIc occurs at more moderate intakes in the order of 1g/day (an amount that would be supplied by 2 oil-rich fish meals/wk). A very moderately increased intake of linoleic acid from approximately 3.5% to 5% of dietary energy appeared to increase plasma fibrinogen concentrations. In addition, concern has been expressed about the potentially adverse effects that an increased intake of linoleic acid may have on the conversion of linolenic acid to docosahexaenoic acid, which has implications both for normal brain development98 and susceptibility to cardiac arrhythmias.100 It is clear, therefore, that both series of polyunsaturated fatty acids need to be balanced with respect to one another, and that the optimum ratio may differ with age and gender. It has been proposed that the ratio of n-6/n-3 fatty acids in the diet should be between 5:1 and 10:1.98 However, further research is needed to determine the optimum ratio.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This research was supported by a grant from the British Heart Foundation. We thank Professor R.D. Rosenberg and Dr I.C.A. Bauer for the determination of F1.2 concentration, David Howorth and Janet Martin for the other coagulation assays, and Jackie Cooper for assistance with the statistical analysis.
Received November 18, 1996; accepted May 12, 1997.
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