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Asia Pacific J Clin Nutr (1996) 5(3): 149-156

Essential fatty acids and modern lifestyle: a reappraisal

CM Smuts¹ MSc, HY Tichelaar¹ MSc, PJ van Jaarsveld¹ PhD, J van Rooyen² PhD, AJS Benadé¹ DSc

1. National Research Programme for Nutritional Intervention of the Medical Research Council, Tygerberg, South Africa.
2. Department of Human and Animal Physiology, University of Stellenbosch, Stellenbosch, South Africa

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Controversy surrounds the effects of dietary fish oil supplementation on atherosclerosis. Three studies were undertaken, where Vervet monkeys were fed either a Western atherogenic diet (WAD) or a high carbohydrate diet (HCD). The first study indicated that enhanced atherosclerosis may be the result of an imbalance of fatty acids in plasma and tissue lipids as eicosapentaenoic acid (EPA; C20:5w 3) was increased with fish oil (FO) supplementation at the expense of arachidonic acid (AA; C20:4w 6). The second study investigated the effect of diet on the metabolism of EPA. Disappearance of EPA, after EPA loading, was delayed in Vervets on the WAD in comparison with those on the HCD. Results of this study indicate that diet is able to modulate EPA metabolism, and that the beneficial effects of a HCD on plasma lipoprotein concentrations can be augmented by EPA supplementation. The third study investigated the combined effect of a supplement that contained different ratios and dosages of gamma-linolenic acid (GLA; C18:3w 6) and EPA during ingestion of the WAD. Based on a favourable response to plasma lipoprotein cholesterol and a phosphatidylcholine fatty acid metabolism with increases in both EPA and dihomogamma-linolenic acid (DGLA; C20:3w 6), we conclude that a 4:1 w 6/w 3 fatty acid supplement at 200 mg/day would be the optimum supplement in our animal model. The long-term effects of this supplement on lipoprotein metabolism and atherosclerosis in the non-human primate model, is currently under investigation.

Key words: Essential fatty acids, atherosclerosis, African Green monkey

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Introduction

The effects of fish oil (FO) supplementation on atherosclerosis va 1000 ry, with some reporting regression^1,2, whilst others report no effect³ or progression^4,5. The effects on lipids in plasma and tissue are also inconsistent^1,5-12. Unrealistically high doses of FO have been used in studies with primates^1,12. These conflicting reports encouraged us to evaluate commercially available fish oils at a realistic dose. In the first study a supplement that supplied 2.5% of dietary energy was evaluated to determine its effect on established atherosclerosis in Vervet monkeys¹³.

Thrombotic disorders are currently treated and prevented by utilising pharmacological concentrations of eicosapentaenoic acid (EPA; C20:5w 3)¹⁴. Controversy and disagreement about the beneficial effects of this essential fatty acid are legion. EPA has been shown to lower^12,15-18, elevate^19,20 or have no effect on cholesterol metabolism¹³. Supplementation of EPA to different diets may be the reason for the inconsistency of effects⁶. The purpose of the second study was therefore to investigate the metabolism of EPA (55% as free fatty acid) in Vervet monkeys as a supplement to a Western atherogenic diet (WAD) or a high carbohydrate diet (HCD).

Supplementation with w 3 fatty acids may enhance atherosclerosis¹⁹, probably due to a lower w 6/w 3 fatty acid ratio. According to the modified essential fatty acid hypothesis of coronary heart disease (CHD), this could be caused by an inadequate or imbalanced supply of w 6 fatty acids¹⁵. Increased cholesterol concentration, especially low density lipoprotein cholesterol (LDL-C), which may occur with increased w 3 fatty acid intake, is not desirable. In contrast, w 6 essential fatty acids, especially gamma-linolenic acid (GLA; C18:3w 6) and dihomogamma-linolenic acid (DGLA; C20:3w 6), reduce LDL-C^21,22. Supplementation of a WAD with w 3 essential fatty acids (EFA), without complementary supplementation with w 6 essential fatty acids, inhibits D 6-desaturation of linoleic acid (LA; C18:2w 6) and thereby levels of their respective metabolites, DGLA and arachidonic acid (AA; C20:4w 6). In turn, prostaglandin synthesis may be related to atherosclerosis. Favourable levels of EPA and DGLA could prevent atherosclerosis and reduce mortality^14,21. The third study was therefore designed to investigate the combined effect of a supplement that contained different ratios and dosages of GLA and EPA in Vervet monkeys on a WAD.

Methods

Experimental animals

The non-human primate is a well defined and acceptable model that can be used to study dietary treatments to improve blood lipid profiles. Availability and a well controlled environment²³ made it an excellent model for us to use. In the studies under discussion, African Green Vervet monkeys (Cercophithecus aethiops) were used as they were used successfully by us^24,25 and other workers^1,26 t 1000 o determine the effect of modulation by diets on lipids, lipoproteins and atherosclerosis. Vervets are also phylogenetically related to humans²⁶ and when fed an atherogenic diet have lipoprotein concentrations, distributions and compositions similar to human beings that are at risk of having coronary heart disease (CHD)¹⁴.

Experimental dietary treatment

Dietary manipulation was achieved in the studies under discussion by using either a WAD or a HCD. The WAD provided 40% and the HCD 20% energy from fat respectively. The dietary treatments have been described in previous work¹⁹. The diets used were formulated entirely from cooked foods normally consumed by people, with no extra synthetic cholesterol added and prepared as described^25,27. The food was prepared by the same individual in the same kitchen, and the diets were analysed during the studies to monitor the consistency of the lipid composition. Fatty acid supplements were injected as required at multiple sites per food patty, and subsequently fed to the animals without delay, one patty each morning and afternoon¹⁹.

Blood and tissue samples

Blood samples were collected into K₂-EDTA (1mg/mL blood) after an overnight fast, by femoral venipuncture without venous stasis, under ketamine-HCl anaesthesia (10mg/kg body weight i.m.) and atropine (0.05mg/kg). In the first study, monkeys were terminated and arteries collected as described¹⁹.

Ethical approval

All three studies were approved by the Ethics Committee of the South African Medical Research Council.

Biochemical analyses

Lipoproteins

Lipoproteins were isolated by sequential ultracentrifugation at 10°C in a Beckman L8-80M ultracentrifuge with a 40.3 rotor. Plasma density was adjusted by addition of NaBr/NaCl solutions and LDL was isolated within a density range of 1.019-1.063g/mL²⁸. High density lipoprotein (HDL) was isolated by precipitation of the apo B containing lipoproteins with heparin-manganese chloride²⁹.

Analytical methods

Total plasma cholesterol and high density lipoprotein cholesterol (HDL-C) were measured enzymatically (Boehringer Mannheim, CHOD-PAP, Cat. No. 237574). Cholesterol in the isolated lipoprotein fractions were determined by an enzymatic iodide method^30,31 as described¹³. Total protein was analysed by a modified Lowry procedure³².

Tissue samples

Erythrocyte membranes (EMB) were prepared by haemolysing erythrocytes with different phosphate buffers^33,34.

Fatty acids

Lipids from studies 1 and 3 were extracted from plasma and

EMB with chloroform/methanol (2:1; v/v), separated by thin layer chromatography and analysed for fatty acid composition of plasma triacylglycerol (TAG), cholesterol ester (CE) and phosphatidylcholine (PC), and EMB-PC by gas-liquid chromatography^35-37. In study two, neutral lipids were extracted from plasma by the Dole method³⁸.

Study 1: Effect of fish oil on atherosclerosis in Vervets fed Western atherogenic (WAD) and high carbohydrate (HCD) diets.

Aim

To determine the effect of fish oil (FO) supplementation to a WAD and HCD on plasma lipoproteins, arterial lipids and fatty acids in Vervet monkeys.

Experimental design This study was described previously13. Briefly the experimental design can be summarised as follows: The experimental Vervets were divided into four comparable groups, two groups were retained on the WAD, one of which was supplemented with fish oil (WAD/FO; n=9), whilst the other received a sunflower oil (WAD/SO; n=9) supplement. The remaining two groups were changed from the WAD to a HCD. One group was supplemented with the same FO supplement (HCD/FO; n=9) and the other group received the sunflower oil (HCD/SO; n= 10) supplement. Nine female Vervets that were never exposed to the WAD, served as a reference group. Blood was drawn prior to starting with the respective dietary regimens and oil supplementations, and again at 3, 8, 13 and 20 months. The Vervets were terminated after the experimental period of 20 months. Statistical analysis Statistical analysis was done as described13 and as groups consisted of male and female Vervets, diet/sex interactions were tested and the results interpreted accordingly. Results Results were described previously13 and can be summarised as follows:

Table 1. The effect of fish oil on lipoprotein and arterial total cholesterol levels13. 1000   WAD/SO WAD/FO HCD/SO HCD/FO HCD   n=9 n=9 n=10 n=9 n=9 Plasma 332.5 344.9 145.7 1000 144.3 180.7 (mg/dL) (125.2) (121.0) (23.1) (20.6) (24.2) LDL 300.9 265.9 49.5 49.7 86.9 (mg/dL) (158.9) 134.2) (21.2) (13.6) (26.6) Intima 32.5 89.2** 44.2 83.7* 10.5 (m g/mg protein) (26.6) (78.3) (70.9) (125.2) (4.9) WAD: Western atherogenic diet; SO: Sunflower oil; FO: Fish oil; HCD: High carbohydrate diet Significant difference between WAD/FO and WAD/SO or HCD/FO and HCD/SO: *p < 0.01; **p < 0.00l

Plasma lipoprotein and tissue cholesterol response FO supplementation did not change the cholesterol concentrations of plasma, HDL and LDL significantly (Table 1). Vervets of the WAD/FO group had an increased (2.7 times; p<0.001) content of total cholesterol in their aorta intima compared to the WAD/SO group. The same trend was also evident after FO was supplemented to the HCD. Fatty acid response EPA was increased 7.5- and 6.5-fold respectively (both p<0.001) in plasma and aortic intima PC (Table 2). DGLA (p<0.01) AA (p<0.001) levels were reduced in the plasma PC after FO supplementation of the WAD, and similar effects were seen after supplementing the HCD with FO. In the aorta intima the AA was also reduced (P<0.001) on the WAD/FO. Docosahexaenoic acid (DHA; C22:6w 3) was also increased after the FO supplementation. Correlations In the plasma and aorta intima PC, EPA and AA respectively demonstrated the strongest negative and positive correlations with the intimal CE and FC contents (Table 3). Study 2: The effect of diet on the metabolism of eicosapentaenoic acid. Aim We studied the effect of diet on the metabolism of EPA in our Vervet monkey model receiving either a WAD or HCD. Controversy surrounds the beneficial effects of EPA on lipoprotein metabolism because some researchers showed that EPA does lower plasma cholesterol concentrations12,15-18, while others suggested a cholesterol elevating effect19,20. Although many factors could possibly explain these divergent results obtained with EPA, Harris6 speculated that the type of diet which EPA is supplemented with, could possibly be the reason for these contradicting results Experimental design Ten healthy female Vervet monkeys were randomly assigned to two groups, namely a HCD group and a WAD group. Experimental procedures were carried out in three phases. Phase 1 During this phase of six weeks, animals received their respective WAD or HCD without any EPA supplementation. This phase was used to stabilise animals on their respective diets. Phase 2 EPA (Callanish Pharmaceuticals, 50% free acid) was used as a supplement to the respective diets and were pr 1000 ogressively increased every six weeks from 300mg/day to 2400 mg/day over a period of 24 weeks. The incorporation of EPA in plasma CE, plasma TAG, EMB-PC and in EMB phosphatidylethanolamine (PE) was investigated at the end of this phase.

Table 2. The effect of fish oil on the fatty acid composition of plasma and intima phosphatidylcholine fatty acids13. 1000   WAD/SO WAD/FO HCD/SO HCD/FO HCD   n=9 n=9 n=10 n=9 n=9 Plasma           C18:2w 6 25.6 18.1** 33.3 23.6a* 31.5   (2.2) (2.1) (2.9) (1.6) (1.5) C20:3w 6 1.5 1.2* 2.6 1.1a* 3.9   (0.1) (0.1) (1.4) (0.3) (1.2) C20:4w 6 12.1 9.4** 8.0 5.9** 8.8   (1.0) (0.5) (1.0) (0.7) (0.8) C20:5w 3 0.8 6.0** 0.4 < 1000 font size="1">5.3a* 0.3   (0.1) (0.7) (0.1) (1.1) (0.1) C22:6w 3 5.5 8.2** 3.0 7.6** 3.0   (1.0) (1.5) (0.4) (0.8) (0.5) Intima           C18:2w 6 5.7 7.4* 6.3 7.0 5.1   (0. 1000 8) (1.3) (1.0) (1.7) (0.8) C20:3w 6 1.0 1.3 1.1 1.1 1.0   (0.3) (0.2) (0.4) (0.3) (0.2) C20:4w 6 19.6 15.0** 18.0 15.5 20.4   (1.9) (1.8) (2.8) (2.5) (1.9) C20:5w 3 0.2 1.3** 0.1 0.6a* ND   (0.1) (0.4) (0.1) (0.1)   C22:6w 3 3.1 3.7 2.1 3.0* 2.3   (0.7) (0.9) (0.5) (0.7) (0.5) WAD: Western atherogenic diet; HCD: High carbohydrate diet; SO: Sunflower oil; FO: Fish oil; a: Males only; ND: Not detected. Significant difference between WAD/FO and WAD/SO or HCD/FO and HCD/SO: *p<0.01; **p<0.001

Phase 3: Washout period At the beginning of the third phase, EPA supplementation was withdrawn. Animals then continued on their respective diets for a further 12 weeks. Blood samples Blood was sampled from each animal at the end of Phase 2. During Phase 3, blood samples were collected after weeks 1, 2, 4, 6, 8 and 12. Statistical analysis Each animal served as its own control. The baseline value was taken before supplementation of EPA was started. This value was compared with the blood sample value obtained at maximal level of supplementation and at one, two, four, six, eight and 12 weeks after cessation of EPA supplementation. In order to minimise the possible effects of diet, differences in fatty acid intake and differences between individual animals on the amount of fatty acid accumulated, the following relationship was used: FAt/FAtot where FAt is equivalent to the percentage of EPA in plasma or EMB after a given time during the washout period and FAtot is equivalent to the percentage of EPA at the beginning of the washout period (end of supplementation period).

Table 3. Correlation coefficients (r) and p-values between the cholesterol ester (CE) and free cholesterol (FC) content of the aorta intima and plasma and intimal phosphatidylcholine (PC) fatty acids13.   Intima-CE Intima-FC PC Fatty acid r p r p Plasma         C20:4w 6 -0.66 0.0029 -0.53 0.0245 C20:5w 3 0.75 0.0004 0.57 0.0126 Intimal         C20:4w 6 -0.73 0.0007 -0.72 0.0005 C20:5w 3 0.78 0.0001 0.59 0.0095

Results

In the WAD group the EPA content in the EMB-PC increased from 0.34%± 0.18% to 11.32± 3.06% (33-fold) during the supplementation period. In the HCD group the EPA content in the EMB-PC increased from 0.08± 0.1% to 8.68± 4.59 (109-fold) over the same period.

In the EMB-PE fraction, EPA content in the group consuming a WAD increased from 1.03± 0.24% to 19.51± 6.47% (19-fold) during the supplementation period. In the HCD group EPA content increased from 0.25± 0.05% to 11.19± 5.20% (45-fold) during the corresponding period.

EPA content in plasma

In plasma CE, EPA increased from 0.35± 0.11% to 19.46± 4.93% (56-fold) in the WAD group, while in the HCD group it increased from 0.05± 0.04% to 20.04± 10.94% (400-fold).

In the WAD group EPA content in plasma TAG increased from 0.16± 0.13% to 19.31± 7.98% (120-fold) during supplementation. In the HCD group EPA content increased from 0.03± 0.03% to 18.02± 11.78% (600-fold) during supplementation.

The disappearance role of EPA EPA content in EMB-PC gradually decreased during the washout phase. The estimation of the half-life (t½) of EPA is given by the median of the t½ which is 34.3 and 22.8 days for the WAD and the HCD groups respectively (Table 4). The t½ of EPA for the EMB-PE is estimated by the median of the individual median t½ which is 43.5 and 31.3 days for the WAD and HCD groups respectively (Table 4). The t½ of EPA for plasma CE was found to be 23.5 days for the WAD and 14.1 days for the HCD (Table 4). Different rates of disappearance of EPA from plasma TAG of the WAD and HCD animals were also observed. The WAD had a t½ of 17.4 days whereas the t½ for HCD was 9.4 days.

Table 4. Summary of the comparison of the estimated half-life (t½) (median of the individual median measurement in days) of eicosapentaenoic acid (EPA). Compartment WAD HCD EMB-PE 43.5 31.3 EMB-PC 34.3 22.6 Plasma CE 23.5 14.1 Plasma TAG 17.4 9.4 EMB: Erythrocyte membrane; PC: Phosphatidylcholine; PE: Phospha-tidylethanolamine; CE: Cholesterol ester; TAG: Triacylglycerol; WAD: Western atherogenic diet; HCD: High carbohydrate diet.

Plasma total cholesterol and HDL-C

In the EPA treated groups plasma total cholesterol levels increased by 1 1000 7.1% in the WAD and decreased by 20.8% in the HCD. EPA supplementation reduced HDL-C by 36% in the WAD and 21.7% in the HCD.

Study 3: Establishment of an optimal w 6/w 3 essential fatty acid ratio and dosage for the prevention of atherosclerosis in nonhuman primates fed a Western atherogenic diet.

Aim

To determine the optimal w 6/w 3 fatty acid ratio and dosage of a fatty acid concentrate which will have the most favourable plasma lipid profile as well as the most favourable plasma and tissue fatty acid composition in a nonhuman primate model fed a WAD.

Experimental design Twenty adult male Vervets were stabilised on a high carbohydrate diet for three months prior to experimental intervention at which time the diet was changed to a WAD for 20 weeks. Vervets were then randomly allocated to receive one of four possible fatty acid supplements for a period of 12 weeks according to their LDL-C response to the WAD. The major fatty acid compositions of the fatty acid concentrate mixture are given in Table 5. Group A (n=5) received 200mg/day of a fatty acid supplement with an w 6/w 3 fatty acid ratio of 9:1 and group B (n=5) received 800 mg/day of the same fatty acid supplement. Group C (n=5) received 200mg/day of a fatty acid supplement with an w 6/w 3 fatty acid ratio of 1:1 and Group D (n=5) received 800mg/day of the same fatty acid supplement. The supplementation period was followed by a 20 week washout period. The washout period was followed by a second supplementation period during which the Vervet groups received a different fatty acid supplement albeit at the same dosage. Group A (n=5) received 200mg/day of a fatty acid supplement with an w 6/w 3 fatty acid ratio of 4:1 and group B (n=5) received 800 mg/day of the same fatty acid supplement. Group C (n=5) received 200mg/day of a fatty acid supplement with an w 6/w 3 fatty acid ratio of 2:1 and Group D (n=5) received 800 mg/day of the same fatty acid supplement.

Table 5. Major fatty acid compositions of the GLA (80) and EPA (50) concentrate mixtures. 1000   Ratios Fatty acid 9:1 4:1 2:1 1:1 C18:2w 6 13.6 11.8 9.8 7.2 C18:3w 6 70.6 60.2 47.8 31.9 C20:5w 3 7.0 14.4 23.3 34.7 C22:6w 3 1.1 2.2 3.6 5.4 S w 6 84.4 72.3 58.1 40.0 1000 S w 3 9.0 18.3 29.4 43.6 S w 6/S w 3 9.34 3.96 1.98 0.92 GLA: Gamma-linolenic acid; EPA: Eicosapentaenoic acid.

Blood and tissue samples

Blood samples were collected before and after each supplementation period and during the washout period.

Statistical Analysis The analysis of variance model39 used, assessed the effects of the experimental factors that included ratio of the w 6/w 3 fatty acid supplement (9:1, 4:1, 2:1 and 1:1), dosage of supplement (200 and 800mg/day), baseline measurements, as well as the interactions between these factors, so that the model could asses change that was observed in the experimental design. The change from baseline was accordingly investigated for significant effects of the different supplements. Results Figure 1 illustrates the cholesterol response to the w 6/w 3 fatty acid supplement. A beneficial effect on cholesterol concentration was achieved only with the 4:1 w 6/w 3 fatty acid ratio at 200mg/day and with the 2:1 w 6/w 3 fatty acid ratio at 800mg/day. The 4:1 w 6/w 3 fatty acid supplement at 200mg/day lowered the LDL-C concentration with 19.8%, while the 2:1 w 6/w 3 fatty acid supplement at 800mg/day lowered the LDL-C concentration with 28.9%. The experimental factors that contributed significantly towards the LDL-C response, were the baseline value and interactions between the dose and ratio used. HDL-C concentrations were more increased with the 4:1 than the 2:1 w 6/w 3 fatty acid supplement (19% versus 6.1% respectively). All the supplements at different ratios and dosages increased DGLA, EPA and DHA, with different responses (Figures 2 and 3). Based on the favourable plasma lipoprotein responses, the 4:1 w 6/w 3 fatty acid ratio at 200mg/day supplement was found to have a non-significant effect on plasma PC AA composition. EPA was only slightly increased by this supplement (Figure 3), favouring the accumulation of DGLA. The experimental factors that significantly contributed towards this accumulation, were the ratio, baseline value and interactions between the dose and ratio, and between baseline value and ratio.

Figure 1. Cholesterol response to w 6/w 3 ratio and dosage.

Discussion The most significant finding from Study 1 is that under the conditions of the study, neither plasma lipoprotein nor the arterial cholesterol concentrations improved as a result of supplementation of either the WAD or the HCD with FO. The lack of effect of dietary supplementation with FO on the plasma total cholesterol is in agreement with other studies that have compared FO with diets lacking a supplement, with monounsaturated fatty acids or with w 6 polyunsaturated fatty acid (PUFA) supplements2-5. In animal studies the effect of FO on LDL-C varied when diets were compared that lacked a supplement or with w 6 PUFA supplements and was either decreased, or had no effect or was increased within the same animal model2,7,8. Most animal studies compared the effect of FO with saturated fat diets that resulted in reduced LDL-C concentrations9,10,40. Our findings are consistent with studies in humans, especially normolipidaemic patients in which w 3 fatty acids had no effect on LDL-C6. Regardless of the mechanism, the final assessment of FO must be at the arterial wall. The aorta intima total cholesterol concentration was almost three times higher on the WAD/FO and two times higher on the HCD/FO when compared to the respective SO supplemented groups13. Our results clearly disagree with reports that demonstrated no effect11 of FO on 1000 the progressive accumulation of cholesterol in the aorta of rabbits, or decreases in the aorta of primates when compared to predominantly saturated fat diets12. On the contrary, not even platelet function was significantly inhibited by FO supplementation to either the WAD or the HCD41. None of the changes measured in terms of atherosclerosis after FO supplementation were favourable in the same individuals20.

Figure 2. Plasma phosphatidylcholine w 6 fatty acid response to w 6/w 3 ratio and dosage.

Study 1 strongly supported13 the hypothesis14 that a fatty acid imbalance of plasma and tissue lipids could be responsible for atherosclerosis, although others disagree with this hypothesis44. The increased EPA and decreased AA found in plasma and intimal PC after FO supplementation, supports the results of other animal43,44 and human studies45,46. The exact mechanism through which FO enhanced the accumulation of intimal cholesterol remains unclear from our results. The increased plasma and intimal EPA levels were nevertheless positively associated with intimal CE and FC contents13. This increase in EPA occurred at the expense of AA that was negatively associated with the intimal CE and FC contents13. The fatty acid results clearly indicated that the FO dose used, disturbed the fatty acid composition of plasma and arterial PC with dire consequences. This finding therefore supports the view that there may be an optimal anti-atherogenic ratio of w 6 and w 3 PUFAs in the diet13. Results from the disappearance rate studies (Study 2) that more EPA was incorporated in tissues of animals consuming a WAD than those consuming the HCD, was probably due to the fact that EPA displaced saturated and/or other fatty acids. Furthermore, the disappearance rate of EPA from EMB and plasma was slower in the animals consuming the WAD than in those consuming the HCD, suggesting a slower rate of metabolism of EPA in animals consuming the WAD. This difference in disappearance rate observed between the two diets, however, appears to be unrelated to the level of incorporation of EPA into the tissues of animals on the respective diets. Differences in disappearance rates were not only observed between the two diets, but also between the EMB-PC and -PE fractions and between plasma CE and TAG. The disappearance of EPA from EMB-PE took nine days longer than from EMB-PC, and five to six days longer in plasma CE than in plasma TAG, independent of the type of diet.

Figure 3. Plasma phosphatidylcholine w 3 fatty acid response to w 6/w 1000 3 ratio and dosage.

The EMB-PC fraction is situated on the outside and -PE on the inside of the EMB⁴⁷. Phospholipid renewal on the outside of the EMB can occur via direct exchange of the phospholipid between EMB and plasma lipoprotein, whereas phospholipids on the inside are renewed via acylation of lysophospholipids⁴⁸ and could therefore take longer to be renewed and metabolised. Present results strongly suggests that these acylation and metabolic processes were delayed by the WAD.

Differences between the disappearance of EPA from plasma CE and TAG probably relates to differences in their respective turnover and metabolic rates. The effect of the WAD diet on the slower disappearance of EPA from the CE and TAG relative to the HCD, is in line with the reported effect of the WAD on LDL metabolism in these animals consuming a WAD²⁸. Results of Study 2 clearly showed that diet can modulate EPA metabolism and that the beneficial effects of a HCD on plasma lipoprotein levels can be augmented by EPA.

Although the LDL-C concentration was lowered more by the 2:1 w 6/w 3 fatty acid supplement at 800mg/day than the 4:1 w 6/w 3 fatty acid supplement at 200mg/day in Study 3, the HDL-C concentration was increased concomitantly more by the latter. The LDL-C lowering effect of the w 6/w 3 fatty acid supplements are promising indeed, as it has been shown that EPA can increase LDL-C levels in patients with coronary disease⁴⁹, hypercholesterolaemic men⁵⁰ and in hypertensive patients⁵¹, although the consumption of cod liver oil was unable to affect HDL-C in male patients with myocardial infarction⁵², probably as a result of its low EPA content⁵³. HDL-C can, however, be increased by weight reduction⁵⁴, alcohol consumption⁵⁵ and a regular consumption of w 3 PUFA that reduces cholesterol transport to cells by LDL and increases efflux of cholesterol from cells by HDL⁵⁶. The substantial HDL-C increase of 19% in this study by the 4:1 w 6/w 3 fatty acid supplement at 200mg/day indicates that a balanced w 6/w 3 fatty acid supplement is necessary as we were unable to change the HDL-C concentration in Vervets with established atherosclerosis on a fish oil supplement¹³.

The EPA increase from baseline due to the 4:1 w 6/w 3 fatty acid supplement at 200mg/day was three times less than the EPA increase due to the 2:1 w 6/w 3 fatty acid supplement at 800mg/day. The 4:1 w 6/w 3 fatty acid supplement at 200mg/day reduced AA by 3%, while the 2:1 w 6/w 3 fatty acid supplement at 800mg/day reduced AA by 13%. T 1000 he 2:1 w 6/w 3 fatty acid supplement at 800mg/day also increased DGLA nearly twice as much as the 4:1 w 6/w 3 fatty acid supplement at 200mg/day. As the w 6/w 3 PC fatty acid balance is easily disturbed by dietary imbalances^35,57,59, it seems that the w 6/w 3 fatty acid supplement that affects this delicate balance the least, would be the w 6/w 3 fatty acid supplement of choice.

Based on the favourable response to HDL-C and PC fatty acid metabolism, we therefore conclude that the 4:1 w 6/w 3 fatty acid supplement at 200 mg/day would be the optimum supplement to use in prospective intervention trials to test the hypothesis that w 3 fatty acids may regress atherosclerosis if the balance with w 6 fatty acids is not disturbed.

Conclusions

Supplementation of a WAD with w 3 fatty acids (FO; 32% w 3 fatty acids) did not regress atherosclerosis in Vervets on the WAD or prevent the progression of atherosclerosis on the HCD. The WAD delayed the metabolism of w 3 and w 6 (results not shown) fatty acids that could possibly explain the accumulation of lipids in arteries in Vervets on WAD studies as well as elevations of plasma lipoprotein. Recent experimental evidence in our model strongly suggest that we reappraise the role of w 3 and w 6 fatty acids in atherosclerosis. Results indicate that the impact of w 3 and w 6 fatty acids in the diet on atherosclerosis took place at very low levels, where not only amount but also the ratio appears to be critical.

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Essential fatty acids and modern lifestyle: a reappraisal

CM Smuts, HY Tichelaar, PJ van Jaarsveld, J van Rooyen, AJS Benadé

Asia Pacific Journal of Clinical Nutrition (1996) Volume 5, Number 3: 149-156

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