Effects of long-term feeding of marine oils with different positional distribution of eicosapentaenoic and docosahexaenoic acids on lipid metabolism, eicosanoid production, and platelet aggregation in hypercholesterolemic rats

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were distributed mainly in the sn-1,3 positions of seal oil triglyceride and in the sn-2 position of squid oil triglyceride. Seal oil-rich or squid oil-rich fats having constant saturated/monounsaturated/polyunsaturated fatty acid (PUFA) and n−6/n−3 PUFA ratios were fed to exogenously hypercholesterolemic rats for 160 d. The control fat contained linoleic acid as the sole PUFA. Before starting the experimental diets, rats were orally treated with high doses of vitamin D for 4 d to accelerate atherogenesis. The percentage of arachidonic acid in phosphatidylcholine and phosphatidylethanolamine of liver, platelets, and aorta was lower in the marine oil groups than in the control group, seal oil being more effective than squid oil. Maximal platelet aggregation induced by collagen was significantly lower both marine oil groups. Platelet thromboxane (TX) A₂ production induced by collagen or thrombin was markedly reduced by feeding seal or squid oils, the reduction being more pronounced in the seal oil than in the squid oil group. Aortic prostacyclin (PGI₂) production was the same among the three groups. The ratio of the productions of aortic PGI₂ and platelet TXA₂ was significantly higher in the seal oil than in the control group. Although there was no difference in intimal thickness among the three groups, the aortic cholesterol content was significantly lower in the marine oil groups than in the control group. These results showed that the main effects in rats of the different intramolecular distributions of EPA and DHA in dietary fats were on arachidonic acid content in tissue phospholipids and on platelet TXA₂ production.     

Platelet and aorta arachidonic and eicosapentaenoic acid levels andin vitro eicosanoid production in rats fed high-fat diets

There is a significant interest in the interrelationship between long-chain n-3, and n-6 fatty acids due to their ability to modulate eicosanoid production. In general, the intake of arachidonic acid (AA) results in enhanced eicosanoid production, whereas n-3 polyunsaturated fatty acids (PUFA) decrease the production of eicosanoids from AA. The purpose of this study was to investigate whether the effects of dietary AA on eicosanoid production in the rat were correlated with the AA and EPA levels in platelets and aorta (eicosanoid-producing tissues). Four groups of male Sprague-Dawley rats were fed a highfat diet enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (approximately 100 mg/day of EPA+DHA) for 24 d. During the last 10 d, the four groups were orally supplemented with 0,30,60, and 90 mg/day of ethyl arachidonate. A further group of rats was fed a control diet (without longchain n-3 PUFA) for 24 d.In vitro aorta prostacyclin (PGI₂) production, serum thromboxane A₂ (TxA₂) production and plasma, and platelet and aorta phospholipid (PL) fatty acids were measured. Enriching the diet with n-3 PUFA resulted in significant reductions in tissue AA levels and an increase in the n-3 PUFA, particularly EPA. On this diet, the AA to EPA ratio was 1:1 in platelet PL, and it was 2:1 in the aorta PL. There were significant decreases in thein vitro PGI₂ and TxA₂ production compared with the control animals. The inclusion of AA in the diet resulted in marked increases in AA levels in the platelet and aorta PL with corresponding decreases in EPA. The lowest dose of AA (30 mg/rat) reversed the effects of 100 mg/day of n-3 PUFA on AA levels in platelet and aortic PL and onin vitro aorta PGI₂ and serum TxA₂ production. The dietary AA caused a differential (twofold) increase in TxA₂ relative to PGI₂ for all three levels of AA supplementation. There were greater changes in the levels of AA and/or EPA in platelet PL compared with the aorta PL, which might have accounted for the differential effects of these PUFA on thromboxane production compared with PGI₂ production in this study.     

The effect of dietary arachidonic acid on platelet function, platelet fatty acid composition, and blood coagulation in humans

Arachidonic acid (AA) is the precursor of thromboxane and prostacyclin, two of the most active compounds related to platelet function. The effect of dietary AA on platelet function in humans is not understood although a previous study suggested dietary AA might have adverse physiological consequences on platelet function. Here normal healthy male volunteers (n=10) were fed diets containing 1.7 g/d of AA for 50 d. The control diet contained 210 mg/d of AA. Platelet aggregation in the platelet-rich plasma was determined using ADP, collagen, and AA. No statistical differences could be detected between the aggregation before and after consuming the high-AA diet. The prothrombin time, partial thromboplastin time, and the antithrombin III levels in the subjects were determined also. There were no statistically significant differences in these three parameters when the values were compared before and after they consumed the high-AA diet. The in vivo bleeding times also did not show a significant difference before and after the subjects consumed the high-AA diet. Platelets exhibited only small changes in their AA content during the AA feeding period. The results from this study on blood clotting parameters and in vitro platelet aggregation suggest that adding 1.5 g/d of dietary AA for 50 d to a typical Western diet containing about 200 mg of AA produces no observable physiological changes in blood coagulation and thrombotic tendencies in healthy, adult males compared to the unsupplemented diet. Thus, moderate intakes of foods high in AA have few effects on blood coagulation, platelet function, or platelet fatty acid composition.     

Effects of dietary fats on prostanoid production and aortic and plasma fatty acid composition in rats

Male Sprague-Dawley rats were fed diets with 10%, 30%, or 50% of energy derived from fat for two weeks. The fats used were beef tallow, olive oil, peanut oil and butter. Aortic prostacyclin (PGI₂) production, platelet aggregation and thromboxane A₂ (TXA₂) production and plasma and aortic phospholipid (PL) content were measured. Butter- and beef tallow-feeding reduced aortic PGI₂ production and collagen-induced TXA₂ production in a dosedependent manner as the level of fat in the diet increased. Neither olive oil nor peanut oil had any effect on aortic PGI₂ production or collagen-induced TXA₂ production. Butter-feeding also resulted in a decrease in collageninduced platelet aggregation; however, none of the other fats had any effect on collagen-induced platelet aggregation. The observed decreases in aortic PGI₂ and collagen-induced TXA₂ production were paralleled by similar decreases in aortic and plasma PL arachidonic acid content and an increase in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Only the most saturated fats, butter and beef tallow, had significant inhibitory effects on prostanoid production and platelet aggregation.     

Effects of dietary palm oil on arterial thrombosis,platelet responses and platelet membrane fluidity in rats

Wistar rats were fed a control diet containing 5 energy % (en %) sunflowerseed oil or diets containing 50 en % of either palm oil, rich in saturated fatty acids, or sunflowerseed oil, high in linoleic acid, for at least eight weeks. Arterial thrombosis tendency, measured by the aorta loop technique, tended to be lowered by the palm oil diet and was lowered significantly by the sunflowerseed oil diet, compared with the control. Aggregation of platelets in whole blood activated with collagen was not altered by palm oil feeding, but was enhanced in the sunflowerseed oil group, compared with the control. The concomitant formation of thromboxane A₂ was decreased by palm oil feeding, although formation of prostacyclin did not change; the ratio of thromboxane/prostacyclin formed was decreased significantly in the palm oil group. Compared with the control diet, platelet membrane fluidity, measured by fluorescence polarization, was not altered in the palm oil group and was significantly increased only by sunflowerseed-oil feeding. Thus, although palm oil contains about 50% saturated fatty acids, it did not increase arterial thrombosis tendency and tended to decrease platelet aggregation, as compared with highly polyunsaturated sunflowerseed oil.     

The effect of a salmon diet on blood clotting,platelet aggregation and fatty acids in normal adult men

This study was designed to measure the effect of dietary n−3 fatty acids (FA) on platelets and blood lipids. Healthy men (n=9), ages 31 to 65, were fed diets in which salmon was the source of n−3 fatty acids. They were confined in a nutrition suite at this Center for 100 days. Food intake and exercise levels were rigidly controlled. Initially they were placed on a stabilization diet for 20 days, then six men were fed the salmon diet for 40 days. The others remained on the stabilization diet. The two groups switched diets for the last 40 days of the study. Both diets were isocaloric [16% protein, 54% carbohydrate, and 30% fat by energy-% (En%)]. The salmon diet contained 7.5% of calories from n−6 FA and 2% from n−3 FA, primarily eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in a 40∶60 ratio, while the stabilization diet contained 7.5% of calories from n−6 FA and less than 0.3% n−3 FA, mainly 18∶3n−3. The bleeding time was unaffected by the diets in this study. The prothrombin time was shortened (11.6 sec.vs. 12.6 sec., p<0.01) for the subjects consuming the salmon diet as compared to that measured after 20 days of the stabilization diet. Mean platelet volume increased significantly during the period in which the volunteers consumed the salmon diet compared to the baseline diet (p<0.01), while the mean platelet levels decreased. Platelet aggregation (PA) was measured in platelet rich plasma before, during, and after the salmon diet using collagen, ADP, arachidonic acid (AA), and thrombin agonists. The PA threshold for ADP was significantly increased for the subjects on the salmon diet (p<0.05). No change in the PA threshold was detected for collagen or thrombin. The PA threshold for AA was unchanged also, but the platelets in subjects consuming the salmon diet had a prolonged time to maximum aggregation (p<0.01) with this reagent compared to platelets from men on the stabilization diet. Plasma, red cell, and platelet total FA composition was determined by capillary GLC. While the men consumed the salmon diets, there were marked increases (3 to 10-fold) in the EPA and DHA levels in all blood components with concomitant decreases in linoleic acid and AA levels. Thus, a salmon diet, high in n−3 FA, did not influence the bleeding times, but it decreased the sensitivity of platelets to ADP and AA, increased the mean platelet size, decreased the platelet count, and changed the FA composition of the plasma, RBC and platelet membrane lipids.     

The effect of linoleic,arachidonic and eicosapentaenoic acid supplementation on prostacyclin production in rats

We examined the effect of dietary supplementation of linoleic acid (LA), arachidonic acid (AA) or eicosapentaenoic acid (EPA) to rats fed a diet low in linoleic acid onin vitro andin vivo production of prostacyclin. Male Sprague Dawley rats were fed a high-fat diet (50% energy as fat, 1.5% linoleic acid) for two weeks. Three of the groups were then supplemented orally with either 90 mg/d of LA, AA or EPA, all as the ethyl esters, for a further two weeks while remaining on the high-fat diet. Forty-eight hour urine samples were collected at the end of the second and fourth weeks.In vivo prostacyclin production was determined by a stable isotope dilution, gas chromatography/mass spectrometry assay for the major urinary metabolite of prostacyclins (2,3-dinor-6-keto-PGF_1α or PGI₂-M and Δ¹⁷-2-3-dinor-6-keto-PGF_1α or PGI₃-M).In vitro prostacyclin production was determined by radioimmunoassay of the stable metabolite (6-keto-PGF_1α) following incubation of arterial tissue. Oral supplementation with AA resulted in a rise in plasma and aorta 20∶4n−6, and increasedin vitro prostacyclin and urinary PGI₂-M production. EPA supplementation resulted in a rise in plasma and aorta 20∶5n−3 and 22∶5n−3, and a decline in plasma 20∶4n−6, but not in the aorta. In the EPA-supplemented group, thein vitro prostacyclin and the urinary PGI₃-M increased, but urinary PGI₂-M decreased. The increase inin vitro prostacyclin production in the EPA-supplemented rats was unexpected and without obvious explanation. Supplementation with LA had minimal effect on fatty acid composition of plasma or aorta and caused no change in prostacyclin production with either method. Thein vivo measure of prostacyclin production was positively correlated with aorta AA levels, and negatively correlated with aorta levels of EPA. There was a significant positive correlation between thein vitro production of prostacyclin and thein vivo production (as measured by the urinary prostacyclin metabolite level), despite the differences observed in the EPA-fed group. There was a high inter-animal variability in prostacyclin production using either method. These results indicate that dietary AA stimulates and dietary EPA reducesin vivo PGI₂ production in the rat. An equivalent amount of dietary LA was without effect.     

Low concentration of oxidized low density lipoprotein suppresses platelet reactivity <Emphasis Type="Italic">in vitro</Emphasis>: An intracellular study

The intracellular mechanisms underlying oxidized low density lipoprotein (oxLDL)-signaling pathways in platelets remain obscure and findings have been controversial. Therefore, we examined the influence of oxLDL in washed human platelets. In this study oxLDL concentration-dependently (20–100 μg/mL) inhibited platelet aggregation in human platelets stimulated by collagen (1 μg/mL) and arachidonic acid (60 μM), but not by thrombin (0.02 U/mL). The activity of oxLDL was greater at 24 h in inhibiting platelet aggregation than at 12 h. At 24 h, oxLDL concentration-dependently inhibited intracellular Ca²⁺ mobilization and thromboxane B₂ formation in human platelets stimulated by collagen. In addition, at 24 h oxLDL (40 and 80 μg/mL) significantly increased the formation of cyclic AMP, but not cyclic GMP or nitrate. In an ESR study, 24 h-oxLDL (40 μg/mL) markedly reduced the ESR signal intensity of hydroxyl radicals (OH⁻) in both collagen (2 μg/mL)-activated platelets and Fenton reaction (H₂O₂+Fe²⁺). The inhibitory effect of oxLDL may induce radical-radical termination reactions by oxLDL-derived lipid radical interactions with free radicals (such as hydroxyl radicals) released from activated platelets, with a resultant lowering of intracellular Ca²⁺ mobilization followed by inhibition of thromboxane A₂ formation, thereby leading to increased cyclic AMP formation and finally inhibited platelet aggregation. This study provides new insights concerning the effect of oxLDL in platelet aggregation.     

Albumin-bound docosahexaenoic acid and collagen-induced human platelet reactivity

Anin vitro system designed to mimic the effect of various plasma nonesterified (polyunsaturated) fatty acids on platelet function and metabolism was employed. Human platelet aggregation induced by submaximal (1.8 μg/ml) collagen stimulation was significantly inhibited by 2 min preincubation with 20 μM albumin-bound docosahexaenoic acid (22∶6n−3) (DHA), but not by the other fatty acids tested. [³H]Phosphatidic acid (PA) formation, an indicator of phospholipase C activation following platelet stimulation, was moderately inhibited by eicosapentaenoic acid (20∶5n−3), 11,14,17-eicosatrienoic acid (20∶3n−3), dihomo-γ-linolenic acid (20∶3n−6), as well as DHA, but not by arachidonic acid (20∶4n−6); this inhibition of phospholipase C activation could not explain the differential effect of DHA on platelet aggregation. The decreased production of thromboxane A₂ (TxA₂), as assessed by [³H]12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) formation, may account for the inhibition of collagen-induced aggregation by 20 μM DHA. Surprisingly, preincubation with 40 μM albumin-bound DHA, even though resulting in greater inhibition of collagen-induced aggregation, had less impact on HHT formation. A small but significant increase in [³H]prostaglandin D₂ (PGD₂) levels following 3-min collagen stimulation may have contributed to the greater antiaggregatory effect of 40 μM DHA. It is concluded that increased plasma nonesterified DHA may contribute to the dampened platelet activation and altered metabolism following fish oil supplementation of the diet.     

Study of the two pathways for arachidonate oxygenation in blood platelets

During collagen-induced blood platelet aggregation, arachidonic acid is set free from membrane phospholipids and subsequently converted into 12-hydroxyeicosatetraenoic acid by arachidonate lipoxygenase and into thromboxane A₂, 12-hydroxyheptadecatrienoic acid (HETE) and malondialdehyde by cyclooxygenase and thromboxane synthase. Lipoxygenase and cyclooxygenase have optimal activity at neutral to basic pH, while the thromboxane synthase is pH-independent between 5 and 9. These enzymes are membrane-bound. The cyclooxygenase is rapidly inactivated upon membrane disruption by nonionic detergents or phospholipid degradation with phospholipase A₂. It was found that platelet phospholipase A₂ preferentially splits off fatty acid with four double bonds. Eicosatetraynoic acid was used to investigate the physiological function of the arachidonate lipoxygenase during collagen-induced aggregation of rat blood platelets. This fatty acid is a more efficient inhibitor of lipoxygenase than of cyclooxygenase. At an inhibitor concentration of 0.6 μg/ml, platelet aggreation, 12-hydroxyeicosatetraenoic acid production as well as 15-hydroxytryptamine release are completely inhibited, while there is an apparent stimulation of the cyclooxygenase. These results indicate that arachidonate lipoxygenase is essential for irreversible blood platelet aggregation.     

Decreased production of inflammatory mediators in human osteoarthritic chondrocytes by conjugated linoleic acids

Osteoarthritic chondrocytes (OC) produce excessive prostaglandin E₂ (PGE₂) and nitric oxide (NO), which function as inflammation mediators in the pathogenesis of osteoarthritis (OA). This study examined the effect of CLA alone and in combination with other PUFA on the FA composition and the production of PGE₂ and NO in OC cultures isolated from OA patients. Human OC were grown in monolayer and treated with one of the following PUFA treatments: CLA, CLA+arachidonic acid (CLA/AA), CLA_EPA (CLA/EPA), linoleic acid (LA), LA+AA (LA/AA), LA+EPA (LA/EPA), and ethanol (as a vehicle control) at 10 and 20 μM for 6 d. Supplementation of PUFA at 10 μM for 6 d did not introduce any cytotoxic effects or morphological changes in OC, whereas 20 μM resulted in apoptosis. Cultures of OC treated with CLA, CLA/AA, and CLA/EPA had higher concentrations of CLA isomers, and these isomers were not detected in other treatments. Supplementation of CLA or LA alone to the OC led to a lower PGE₂ production compared to the control. Combination of CLA/EPA resulted in the lowest PGE₂ production in cultured OC. OC cultures treated with CLA were lower in NO production than the control, whereas the LA/AA treatment demonstrated the lowest NO production. The fact that CLA alone or in combination with other PUFA modulated PGE₂ and NO production in human OC cultures suggests that these 18∶2 isomers may have the potential to influence OA pathogenesis.     

5c, 11c, 14c-Eicosatrienoic acid and 5c, 11c, 14c, 17c-eicosatetraenoic acid ofBiota orientalis seed oil affect lipid metabolism in the rat

The effects of 5c, 11c, 14c-eicosatrienoic acid (20∶3BSO) and 5c, 11c, 14c, 17c-eicosatetraenoic acid (20∶4BSO), polyunsaturated fatty acids (PUFA) contained inBiota orientalis seed oil (BSO), on lipid metabolism in rats were compared to the effects of fats rich in linoleic acid (LA) or α-linolenic acid (ALA) under similar conditions. The potential effect of ethyl 20∶4BSO as an essential fatty acid also was examined in comparison with the ethyl esters of LA. ALA and γ-linolenic acid (GLA). BSO- and ALA-rich fat decreased the concentration of plasma total cholesterol, high density lipoprotein cholesterol, triglyceride and phospholipid as compared to LA-rich fat. BSO was more effective in reducing plasma cholesterol concentrations than was the ALA-rich fat. Dietary BSO markedly decreased the hepatic triglyceride concentration as compared to the LA-rich or ALA-rich fats. Aortic production of prostaglandin I₂ tended to decrease in rats fed BSO or ALA-rich fat compared to those fed the LA-rich fat. Adenosine diphosphate-induced platelet aggregation was similar in the three groups. The proportion of arachidonic acid (AA) in liver phosphatidylcholine (PC) of rats fed BSO was lowest compared to that of rats fed ALA-rich or LA-rich fats. Administration of 20∶4BSO, ALA or GLA to essential fatty acid-deficient rats decreased the ratio of 20∶3n−9 to AA in liver PC to the same extent; administration of LA was more effective. The results indicate that the effects of specific PUFA contained in BSO on lipid metabolism are different from those of LA and ALA. It is also suggested that 20∶4BSO may exhibit some essential fatty acid effects.     

Butter-enriched diets reduce arterial prostacyclin production in rats

Rats were fed diets containing 10%, 30% or 50% energy as fat derived predominantly from butter or lard. The protein content of the diets was maintained at 20%. After three weeks on the diets, the rats were killed and the following parameters measured: prostacyclin production in vitro from abdominal aorta and mesenteric artery; platelet aggregation to ADP and thrombin; fatty acid composition of the phospholipids in plasma, thoracic aorta and liver; smooth muscle reactivity and release of endothelial derived relaxing factor (EDRF) from aortic endothelium stimulated by acetylcholine. There was no significant effect of increasing fat content of the diets (neither lard nor butter) on platelet aggregation. In contrast, prostacyclin production in both the mesenteric artery and the abdominal aorta fell in a concentration-dependent manner in the butter-supplemented rats. However, no effect on prostacyclin production was detected in arteries from the lard-supplemented animals. The effects of the diets on prostacyclin (PGI₂) production correlated very well with the changes in plasma, aortic and liver phospholipid arachidonic acid (AA) and eicosapentaenoic acid (EPA) contents. AA decreased in a concentration-dependent manner in the rats fed the butter-enriched diets but did not change in those fed the lard-enriched diets, whereas EPA rose in a concentration-dependent manner in the butter-fed rats and was unchanged in the lard-fed animals. The clear-cut effects of the butter-enriched diets on aortic phospholipid fatty acid composition and aortic PGI₂ production were accompanied by a significant reduction in smooth muscle relaxation to EDRF. These results indicate that in the rat, enrichment of the diet with butter can reduce the concentration of AA and increase that of EPA in plasma and tissue phospholipids with a parallel reduction in arterial PGI₂ production and EDRF.     

Eicosanoid synthesis in 7,12-dimethylbenz(a)anthracene-induced mammary carcinomas in Sprague-Dawley rats fed primrose oil,menhaden oil or corn oil diet

The comparative effects of high-fat diets (20%, w/w) on eicosanoid synthesis during mammary tumor promotion in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rats were studied using diets containing 20% primrose oil (PO), 20% menhaden oil (MO) or 20% corn oil (CO). Sprague-Dawley rats fed the PO or MO diet had 21% or 24% fewer adenocarcinomas, respectively, than rats fed the CO diet. Histologically (i.e., mitotic figures, inflammatory cell infiltration and necrosis), the CO-fed rats exhibited the highest frequency of changes within tumors. Plasma fatty acid composition was significantly altered by diet, reflecting the composition of the oils which were being fed. Only the plasma of PO-fed rats contained detectable levels of gamma-linolenic acid (GLA). Arachidonic acid (AA) levels were significantly higher (p<0.05) in PO-fed than in CO- or MO-fed rats. MO-fed rats had significantly higher levels of plasma palmitic acid, while palmitoleic, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids were detected only in MO-fed rats. As expected, linoleic acid (LA) and AA levels were lower (p<0.05) in the MO-fed rats than in PO- or CO-fed groups. The plasma of the CO-fed rats contained significantly higher levels of oleic acid. Eicosanoid synthesis in mammary carcinomas of rats fed the 20%-fat diets was 2–10 times higher than in mammary fat pads of control rats. The synthesis of PGE₁ and LTB₄ was significantly (p<0.05) higher in PO-fed rats than in CO-fed or MO-fed rats, although PGE values were significantly (p<0.05) higher in CO-fed rats than in Mo or PO groups. The synthesis of eicosanoids in both mammary fat pads and mammary carcinomas of MO-fed rats was lower (p<0.05) than in tissues of rats fed either CO or PO diets due to less AA precursor being fed and/or to competition between n−6 and n−3 fatty acids for cyclooxygenase and lipoxygenase. The ratios of monoenoic to dienoic eicosanoids in both mammary fat pads and mammary carcinomas were higher in the PO group than in the MO or CO groups. These results suggest that inclusion of GLA (PO feeding) or EPA and DHA (MO feeding) in the diet may decrease malignancy by altering eicosanoid profiles.     

Characteristics of the formation of the platelet lipoxygenase product from endogenous arachidonic acid

The concentration of 12-hydroxyeicosatetraenoic acid (12-HETE) formed in rat platelets aggregated by collagen suspension increased continuously during a 115-min incubation period, whereas the concentration of TXB₂ or PGF_2α reached the maximum within 3 min and stayed at the plateau for the remaining incubation period. These data indicate that platelet lipoxygenase is not completely inactivated as is cyclooxygenase by the oxidizing agent. Platelets of essential fatty acid deficient (EFAD) rats resuspended in plasma of control rats produced more 12-HETE than platelet-rich plasma (PRP) of EFAD rats, whereas platelets of control rats resuspended in plasma of EFAD rats formed less 12-HETE than PRP of control rats. However, the concentration of TXB₂ or PGF_2α produced was not changed in both cases implying that platelet cyclooxygenase preferentially utilizes arachidonic acid (AA) derived from platelet lipids. Radioactivity of phosphatidylcholine (2-arachidonyl-1-¹⁴C) suspended in the plasma of PRP was incorporated into 12-HETE but not to TXB₂, indicating again that only lipoxygenase can utilize AA derived from plasma phospholipids. The significance of this observation is that the effects of platelet lipoxygenase products, although their physiological roles are not known, would be much more persistent than cyclooxygenase products after platelets are stimulated or aggregated in vivo.     

The effects of dietary α-linolenic acid compared with docosahexaenoic acid on brain, retina, liver, and heart in the guinea pig

The aim of this study was to compare two different strategies to elevate brain, retina, liver, and heart docosahexaenoic acid (DHA) levels in guinea pigs. Fist, we used an increasing dose of α-linolenic acid (AIA) relative to a constant linoleic acid (LA) intake, and second, we used two levels of dietary DHA provided in conjunction with dietary arachidonic acid (AA). The percentage DHA and AA of total phospholipids in retina, liver, and heart, and in the brain phosphotidylethanolamine and phosphatidylcholine was studied in female pigmented guinea pigs (3 wk old) fed one of five semisynthetic diets containing 10% (w/w) lipid for 12 wk. The LA content in the diets was constant (17% of total fatty acids), with the ALA content varying from 0.05% (diet SFO), to 1% (diet Mix), and to 7% (diet CNO). Two other diets LCP) and LCP3) had a constant LA/ALA ratio (17.5∶1) but varied in the levels of dietary AA and DHA supplementation. Diet LCP1 was structured to closely replicate the principal long chain polyunsaturated fatty acids (PUFA) found in human breast milk and contained 0.9% AA and 0.6% DHA (% of total fatty acids) whereas diet LCP3 contained 2.7% AA and 1.8% DHA. At the end of the study, animals were sacrificed and tissues taken for fatty acid analyses. We found no significant effects of diets on the growth of guinea pigs. Diets containing ALA has profoundly different effects on tissue fatty acid compositions compared with diets which contained the long chain PUFA (DHA and AA). In the retina and brain phospholipids, high-ALA diets or dietary DHA supplementation produced moderate relative increases in DHA levels. There was no change in retinal or brain AA proportions following dietary AA supplementation, even at the highest level. This was in contrast to liver and heart where tissue DHA proportions were low and AA predominated. In these latter tissues, dietary ALA had little effect on tissue DHA proportions although the proportion of AA was slightly depressed at the highest dietary ALA intake, but dietary DHA and AA supplements led to large increases (up to 10-fold) in the proportions of these PUFA. Tissue uptake of dietary AA and DHA appeared maximal for the LCP1 diet (replicate of breast milk) in the heart. There were no significant changes in the plasma levels of 11-dehydrothromboxane B₂ (a thromboxane A₂ metabolite), for any diet. The data confirm that dietary ALA is less effective than dietary DHA supplementation (on a gram/gram basis) in increasing tissue DHA levels and that tissues vary greatly in their response to exogenous AA and DHA, with the levels of these long chain metabolites being most resistant to change in the retina and brain compared with liver and heart. Dietary DHA markedly increased tissue DHA proportions in both liver and heart, whereas the major effect of dietary AA was in the liver. Future studies of the effects of dietary DHA and AA supplementation should examine a variety of tissues rather than focusing only on neural tissue.     

Interrelations of linoleic acid with medium-chain and long-chain saturated triglycerides

1. Effects of medium-chain (C₆–C₁₂) saturated triglycerides (MCT) and long-chain (C₁₄–C₁₈) saturated triglycerides (LCT) with and without linoleic acid (LA) supplementation were studied on rats fed purified diets
2. With 2% linoleic acid rats fed MCT and LCT grew somewhat better than those on a low-fat diet with the same supplement. Without linoleic acid those fed MCT grew better, and those fed LCT grew worse than those on the corresponding low-fat diet. MCT seemed to decrease, and LCT to increase linoleic acid requirements.
3. In survival studies 14 out of 18 rats fed 20% MCT were alive after 2 years; of their controls fed 20% lard, 10 out of 19 survived.
4. Reproduction studies in females gave equally poor results on unsupplemented low-fat, MCT, and LCT diets regarding implantation, birth weight, and survival rate. The weaning weights of the young on MCT were however the highest. With 2% LA weaning weights were equally high with LCT and MCT but lower with low-fat diet.
5. In animals fed low-fat diets not supplemented with LA, low serum cholesterol was associated with high liver cholesterol. With MCT, serum values were higher and liver values were significantly lower. With unsupplemented LCT, serum and liver values were high. When the three diets were supplemented with 2% LA, there were no longer any differences in the serum levels and in the liver levels. Whether ar not the presence of some oleate in the MCT and LCT influenced the cholesterol results is not certain.
6. The differences in the effects of MCT and LCT are discussed.

     

Biochemical and functional abnormalities in hypercholesterolemic rabbit platelets

This study was designed to elucidate changes in rabbit platelet lipids induced by a cholesterol rich diet and to explore the possible correlation of these lipid changes with platelet abnormalities. Pronounced biochemical alterations were observed when serum cholesterol levels of 700–1000 mg% were reached. Hypercholesterolemic (HC) platelets contained 37% more neutral lipids and 16% less phospholipids than the controls. Lysolecithin, cholesterol esters and phosphatidylinositol (PI) levels were increased in HC platelets, and the levels of phosphatidylcholine (PC) were decreased. The cholesterol/phospholipid molar ratio of lipidemic platelets increased from 0.55±0.011 to 0.89±0.016 (P<0.01) in eight weeks. HC platelets had 90% more arachidonic acid (AA) in the PI than normal platelets. No significant changes in AA of PC were observed. Platelet function was monitored by the uptake and release of [¹⁴C]serotonin in platelet rich plasma (PRP), using varying concentrations of collagen as an aggregating agent. The uptake of [¹⁴C]serotonin in HC and normal platelets ranged from 78–94%. The percent of [¹⁴C]serotonin released from normal and HC platelets was proportional to the concentration of collagen. However, lipidemic platelets were hyperreactive to low concentrations of collagen. Incorporation of 50 μM acetylsalicylic acid into the aggregating medium suppressed the release of [¹⁴C]serotonin in normal PRP by more than 90%, but had only a partial effect on lipidemic PRP.     

Modulation of phorbol ester-associated events in epidermal cells by linoleate and arachidonate

To elucidate the events elicited by the skin tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), which are modulated by linoleic acid (LA) and arachidonic acid (AA), the activity of these fatty acids in cultured mouse epidermal cells was compared. Approximately 94% of either exogenous radiolabelled fatty acid was incorporated into the total phospholipid pool over 15 h. The relative distribution among the phospholipid classes differed, however, such that approximately 70% of phospholipid-associated [¹⁴C]-LA was found in phosphatidylcholine, compared to approximately 30% for [¹⁴C]AA. Phosphatidylethanolamine and phosphatidylinositol/phosphatidylserine contained 17 and 13% of the phospholipid [¹⁴C]LA, and 34 and 30% of [¹⁴C]AA, respectively. Prostaglandin (PG) E₂ production was low but similar in unstimulated cultures prelabelled with either [¹⁴C]LA or [¹⁴C]AA. However, in cultures treated with TPA (1.6 μM), [¹⁴C]AA-prelabelling resulted in approximately three times the amount of [¹⁴C]PGE₂ compared with cultures prelabelled with [¹⁴C]LA. Cultured cells were found to contain significant δ6 desaturase activity, which may enable conversion of LA to AA, and thus may account for the observed PGE₂ production from [¹⁴C]LA treated cells. AA-Supplemented (1.6 μM) cultures supported approximately twice the induction of ornithine decarboxylase activity by TPA compared with cultures treated with 1.8 μM LA. Activation of partially purified protein kinase C was similar for either fatty acid tested over a 10–300 μM dose range. Overall, the results suggest that LA does not have the same biological activity as AA with regard to several TPA-associated events known to be important in skin tumor promotion. This reduced biological activity of LA may be partly responsible for the known inhibition of mouse skin tumor promotion by high dietary levels of LA [Leyton, J., Lee, M.L., Locniskar, M.F., Belury, M.A., Slaga, T.J., Bechtel, D., and Fischer, S.M. (1991)Cancer Res. 51, 907–915].     

Oral polyunsaturated phosphatidylcholine reduces platelet lipid and cholesterol contents in healthy volunteers

The effects of orally administered polyunsaturated phosphatidylcholine (PPC) on plasma lipids, lipoproteins and platelet function and composition were studied in seven healthy male volunteers. PPC (Nattermann & Cie, GmbH, Cologne, Federal Republic of Germany), 10 g/day, was given for a 6-week period after a 4-week wash out; laboratory tests were repeated after a further 4-week period after the end of treatment. PPC did not appear, during treatment, to modify the levels of plasma total cholesterol and triglycerides. High density lipoprotein (HDL) cholesterol levels were, however, increased after six weeks of PPC. The most dramatic changes occurred in platelet membrane composition: the total lipid/total protein and the cholesterol/protein ratios were reduced significantly, whereas increases of the phospholipid/total lipid ratio and of the linoleic acid membrane content were observed. Platelet function tests, both in whole blood and in platelet rich plasma, were not modified. Similarly, the thromboxane B₂ formation after standard stimuli and the sensitivity to exogenous prostaglandin I₂ also were unchanged. During the final wash out period following treatment, a reduction of plasma total and low density lipoprotein (LDL) cholesterol levels also was recorded. PPC appears to be capable of modulating lipid exchanges between cell membranes and the plasma compartment.