Takashi Terano

Effect of oral administration of highly purified eicosapentaenoic acid on platelet function, blood viscosity and red cell deformability in healthy human subjects

Eicosapentaenoic acid (EPA), which is abundant in seafood, has been reported to be a potent antagonist of platelet aggregation and also to reduce the incidene of cardiovascular disorders. We recently reported that EPA also reduces whole blood viscosity. A highly purified EPA, in a soft capsule (75% ethylester form of EPA; EPA-E), manufactured from sardine oil was administered to 8 healthy male subjects for 4 weeks. No side effects were observed. Platelet aggregation and platelet retention significantly decreased. The EPA content in platelet phospholipids markedly increased but docosahexaenoic acid (DHA) and arachidonic acid (AA) contents did not change. A reduction in whole blood viscosity and an increase in erythrocyte deformability were also observed after 4 weeks ingestion of EPA-E. The EPA content in erythrocyte membrane phospholipids markedly increased after 4 weeks, and was positively correlated with erythrocyte deformability. Reduction of platelet aggregation and improvement of the rheological properties of the erythrocyte might be explained by an increase in the EPA content in platelet and erythrocyte phospholipids.

Biosynthesis and biological activity of leukotriene B5

Several studies indicate that increased intake of eicosapentaenoic acid (EPA) in the diet may lead to decreased incidence of thrombotic events. Most investigators agree that this is achieved by competitively inhibiting the conversion of arachidonic acid (AA) to thromboxane A2 in the platelets. The effect of high EPA-intake on the formation of prostacyclin is less clear. However, EPA is a good substrate for lipoxygenase enzymes which results in formation of hydroperoxy- and hydroxy-acids, and, in some cases, leukotrienes. The biological activities of the leukotrienes derived from arachidonic acid suggest that they mediate or modulate some symptoms associated with inflammatory and hypersensitivity reactions. In order to clarify the possible effect of dietary manipulation on inflammatory processes, leukotriene B5 (LTB5) was prepared and its biological activities assessed. LTB5 was biosynthesized by incubation EPA with glycogen-elicited polymorphonuclear neutrophils (PMN) from rabbits in the presence of the divalent cation ionophore, A23187. The LTB5 was extracted from the incubate using mini-reverse phase extraction columns (Sep-pak) and purified by reverse-phase high pressure liquid chromatography (RP-HPLC). The purity of the product assessed by repeat RP-HPLC and straight phase (SP) HPLC was greater than 95%. Ultra-violet spectrophotometry of the product confirmed its purity and also provided assessment of the yield. The biological activity of LTB5 was assessed and compared with that of LTB4 in the following tests: aggregation of rat neutrophils, chemokinesis of human PMN, lysosomal enzyme release from human PMN and potentiation of bradykinin-induced plasma exudation. In all these tests, LTB5 was considerably less active (at least 30 times) than LTB4.

The effects of the oral administration of fish oil concentrate on the release and the metabolism of [14C]arachidonic acid and [14C]eicosapentaenoic acid by human platelets

It has been suggested by several investigators that eicosapentaenoic acid (C20:5 omega 3, EPA) might have anti-thrombotic effects. In this experiment, the effect of the oral administration of EPA rich fish oil concentrate on platelet aggregation and the release and the metabolism of [1-14C]arachidonic acid and [(U)-14C]eicosapentaenoic acid by human platelets was studied. Eight healthy male subjects ingested 18 capsules of fish oil concentrate (EPA 1.4 g) per day for 4 weeks. Plasma and platelet concentrations of EPA markedly increased, while those of arachidonic acid (C20:4 omega 6, AA) and docosahexaenoic acid (C22:6 omega 3, DHA) did not change. Platelet aggregation induced by collagen and ADP was reduced. Collagen induced [14C]thromboxane B2 (TXB2) formation from [14C]AA prelabeled platelets decreased. There was no detectable formation of [14C]TXB3 from [14C]EPA prelabeled platelets, and the conversion of exogenous [14C]EPA to [14C]TXB3 was lower than that of [14C]AA to [14C]TXB2. The release of [14C]AA from [14C]AA prelabeled platelets by collagen was significantly decreased. These observations raise the possibility that the release of arachidonic acid from platelet lipids might be affected by the alteration of EPA content in platelets.

Effects of orally administered ethyl ester of eicosapentaenoic acid (EPA; C20:5, ω-3) on PGI2-like substance production by rat aorta☆

A highly purified ethyl ester of EPA (EPAEE) (74%) was manufactured from sardine oil. Sixty mg/kg/day of EPAEE was given orally to male Wistar rats for 8 weeks. No side effect or toxicity from the administration of EPAEE was observed. Plasma EPA concentration and the ratio of EPA to arachidonic acid were significantly increased, compared with control Wistar rats. An enhancement of PGI2-like substance production by aortas obtained from rats fed EPAEE was noted. Conversion of EPA to delta 17-6-keto-PGF1 alpha, a stable metabolite of PGI3, could not be detected by an incubation study of 14C-EPA and aortas either from rats fed EPAEE or from control rats. Therefore, PGI2-like substance produced by rat aorta is most likely to be PGI2 itself and not PGI3.

Effect of orally administered eicosapentaenoic acid (EPA) on the formation of leukotriene B4 and leukotriene B5 by rat leukocytes

Eicosapentaenoic acid (EPA) is a poor substrate for the fatty acid cyclo-oxygenase but is a good substrate for lipoxygenase enzymes which catalyse the biosynthesis of hydroperoxy-acids, hydroxy-acids and leukotrienes. Recently, we reported that leukotriene B5 (LTB5) was at least 30 times less potent than LTB4 in causing aggregation, chemokinesis and degranulation of polymorphonuclear leukocytes in vitro. In this paper, the effect of oral administration of EPA on LTB4 and LTB5 production by rat leukocytes stimulated with the calcium ionophore, A23187, was assessed. The concentration of LTB was determined by radioimmunoassay and also by reverse-phase high pressure liquid chromatography using PGB3 as internal standard. Supplementation of a normal rat diet with EPA (240 mg/kg per day) for 4 weeks caused a significant increase in the formation of LTB5 and a decrease in the synthesis of LTB4 by stimulated leukocytes. The EPA-rich diet significantly increased the EPA content of leukocyte phospholipids without altering the content of arachidonic acid (AA) or linoleic acid. The ratio of EPA/AA in leukocytes correlated (r = 0.795, P less than 0.001) with the LTB5/LTB4 ratio produced after stimulation of leukocytes. If LTB4 has a chemotactic role during inflammation, the present data suggest that an EPA rich diet could decrease the accumulation of leukocytes at sites of inflammation.

Docosahexaenoic acid supplementation improves the moderately severe dementia from thrombotic cerebrovascular diseases

T. Terano a,*, S. Fujishiro b, T. Ban c, K. Yamamoto d, T. Tanaka e, Y. Noguchi e, Y. Tamura ~, K. Yazawa g and T. Hirayama h aDepartment of Internal Medicine, Chiba Municipal Hospital Chiba 260, bKyoundou Hospital, Tokyo, ~Kuniyoshi Hospital, Chiba, dKimitsu Central Hospital, Chiba, e2nd Department Internal Medicine, Chiba University, Chiba, ~Kyoundou Hiratsuka Hospital, Kanagawa, gSagami Chemical Research Center, Kanagawa, and hHirayama Hospital, Chiba, Japan

Eicosapentaenoic acid and docosahexaenoic acid suppress the proliferation of vascular smooth muscle cells.

Eicosapentaenoic acid, which is one of the n-3 polyunsaturated fatty acids (PUFA), is reported to exert its antithrombotic and anti-atherogenic effect partly through the modulation of vascular cell functions. Vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of atherosclerosis. We reported the differential effect of various PUFA on VSMC proliferation. First we established a method for preparing PUFA rich cells in culture to mimic the in vivo situation using PUFA triacylglycerol emulsion. Using these fatty acid rich cells, we found that only EPA and docosahexaenoic acid, although less potent than EPA, inhibited the proliferation of VSMC among the fatty acids tested. This effect of EPA was reversed by the addition of anti-oxidants. It is suggested that production of the oxidized species at a low concentration from EPA inhibited the proliferation of VSMC. This anti-proliferative effect of EPA and DHA on VSMC could partly explain the anti-atherosclerotic effect of marine lipids.

Eicosapentaenoic acid and adult diseases in Japan: epidemiological and clinical aspects

Abstract. Residents of a coastal fishing village in Japan consume larger amounts of fresh fish rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) than those in inland farming villages. A higher content of EPA and DHA in the plasma and reduced platelet aggregability was observed in the residents of the fishing village than in farmers. Incidence of thrombotic cardiovascular disorders was lower in the fishing area than in the farming area. Oral ingestion of highly purified EPA or DHA reduced platelet aggregability and improved serum lipid profile in healthy subjects and in hyperlipidaemic patients, though DHA seems to be much less potent. In clinical studies with highly purified EPA, improvement of clinical features was noted in patients with thrombotic cardiovascular disorders. It is also shown that EPA and DHA have an anti‐inflammatory effect in man although EPA is far more active than DHA.

Eicosapentaenoic Acid Suppressed the Proliferation of Vascular Smooth Muscle Cells Through Modulation of Various Steps of Growth Signals

Among fatty acids, only n-3 polyunsaturated fatty acids such as eicosapentanenoic acid (EPA) and docosahexaenoic acid (DHA) inhibited the proliferation of vascular smooth muscle cells. DHA was less effective than EPA. To clarify the anti-proliferative effect of n-3 polyunsaturated fatty acid, we have explored the effect of EPA on the signal transduction pathway of platelet derived growth factor (PDGF). EPA inhibited PDGF binding on its receptor and activation of protein kinase C. EPA also suppressed c-fos mRNA expression, one of immediate early genes, through partly inhibiting c-fos transcription. These data revealed that EPA could inhibit vascular smooth muscle cell proliferation through modulating various steps of the signal transduction by PDGF.

Studies on the mechanism of antiaggregatory effect of moutan cortex

The effects of Moutan Cortex and one of its major components, paeonol, on platelet aggregation and arachidonic acid (AA) metabolism in human platelets were studied. One week oral administration of water extract of Moutan Cortex [Moutan Cortex (w), 3 g/day] significantly reduced platelet aggregation and thromboxane B2 (TXB2) formation induced by collagen, epinephrine and ADP. Paeonol dose-dependently inhibited ADP and collagen induced platelet aggregation in vitro. Moutan Cortex (w) and paeonol dose-dependently inhibited the conversion of exogenous [14C]AA to [14C]heptadecatetraenoic acid [( 14C]HHT) and [14C]TXB2 by washed human platelets, while both of them increased its conversion to [14C]12-hydroxy eicosatetraenoic acid [( 14C]12-HETE). High dose of Moutan Cortex (w) inhibited the release of [14C]AA from prelabeled platelets in vitro, while paeonol did not. These results suggest that a reduction in platelet aggregation by the oral administration of Moutan Cortex might be ascribed to a decrease in thromboxane synthesis and that paeonol might play an important role in the antiaggregatory effect of Moutan Cortex because of its potent inhibitory effect on platelet aggregation and thromboxane formation.