The prostacyclin/thromboxane balance is favourably shifted in Greenland Eskimos

The rare incidence of cardiovascular disease in Eskimos has been ascribed to their diet rich in eicosapentaenoic acid (EPA, C20:5n-3) and hence a possible formation of trienoic prostanoids. In this study we compare endogenous formation of prostacyclin (PGI), which is formed by the endothelial cell, and thromboxane (TXA), which is formed by platelets in 20 Eskimos and 20 age and sex matched Danish controls by measurement of the main urinary metabolites. Considerable formation of bioactive PGI3 from dietary EPA was shown in Eskimos, which was barely detectable in the controls. Furthermore synthesis of PGI2 was significantly higher in Eskimos in spite of a markedly lower arachidonate content in membrane lipids. In contrast formation of TXA2,3 was lower in Eskimos as compared to the Danish controls. We conclude, that the balance between PGI and TXA, which may regulate the interaction of platelet and vessel wall, is favourably shifted in Greenland Eskimos to an antithrombotic state.     

Dietary docosahexaenoic acid is retroconverted in man to eicosapentaenoic acid, which can be quickly transformed to prostaglandin I3

In a 24 h kinetic study docosahexaenoic acid (DCHA, C22:6n-3) or eicosapentaenoic acid (EPA, C20:5n-3) were given in a single dose to healthy male volunteers. PGI₃-M, the main urinary metabolite of prostaglandin I₃ was below the detection limit in the control periods, but was excreted already in the first 4 h after ingestion of DCHA or EPA and decreased thereafter. Excretion of PGI₂-M did not change significantly. In a second dietary trial DCHA and EPA were given cross-over to 7 healthy male volunteers for 6 days. PGI₃-M was formed after DCHA and EPA in amounts of 35 and 20 % of PGI₂-M and showed a considerable interindividual variation. The structure of PGI₃-M was verified by independant biochemical synthesis. Our data indicate that dietary DCHA is retroconverted to EPA in man, which is quickly transformed - like dietary EPA itself - to prostaglandin I₃. DCHA may therefore serve as a precursor fatty acid for EPA and its cyclooxygenated and lipoxygenated products.     

Human umbilical blood vessel converts all cis-5, 8, 11, 14, 17 eicosapentaenoic acid to prostaglandin I3

All cis-5, 8, 11, 14, 17 eicosapentaenoic acid (EPA) is presented being evaluated for dietary prophylactic use in thrombo-embolic disorders. EPA inhibits the production of TXA₂ and platelet aggregation. We here present results demonstrating that human umbilical arteries convert ¹⁴C- EPA to a substance that in aqueous solutions decomposes to 14C-δ¹⁷-6-keto-PGF_1α. The conversion rate in rat aortic tissue was found substantially lower. These results in combination with earlier data indicating that EPA does not influence the conversion of arachidonic acid (AA) into PGI₂ in human vascular tissue, encourage further research along the lines initiated by the findings of high EPA/AA ratio and low incidence of myocardial infarction in Greenland Eskimos.     

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 Wishar 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 Λ¹⁷-6-keto-PGF_1α, 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.     

Effects of all CIS-5, 8,11,14,17 eicosapentaenoic acid and PGH3 on platelet aggregation

In human platelet-rich plasma (PRP) eicosapentaenoic acid (EPA) inhibited platelet aggregation induced by a stable analogue of PGH₂ (U46619), arachidonic acid, collagen or ADP. EPA was more potent than oleic, linoleic, α-linolenic or γ-linolenic acids. In aspirin-treated platelets, aggregation induced by U46619 was inhibited to a similar extent by arachidonic acid or by EPA over a range of concentrations of 0.05–0.3 mM. EPA incubated with PRP did not induce the generation of a thromboxane (TXA)-like activity; indeed it prevented the formation of TXA₂ induced by arachidonic acid or by collagen. The anti-aggregatory activity of EPA was not influenced by inhibitors of cyclo-oxygenase and lipoxygenase. The anti-aggregatory action of EPA may be caused by a rapid occupancy by EPA of TXA₂/PGH₂ “receptors” on platelet membrane as well as by a slower displacement of arachidonic acid from platelet phospholipids by chemically unchanged molecules of EPA.Not all samples of PRP were irreversibly aggregated by PGH₂, but in those that were, PGH₃ also induced an immediate dose-dependent but reversible aggregation. After a 4 min incubation of non-aggregating doses of PGH₂ or PGH₃ (100–300 nM) with PRP a stable anti-aggregatory compound was detected. The inhibitory activity produced from PGH₃ was apparently more potent (ca 10 times) than that obtained from PGH₂. The anti-aggregating compounds were identified by TLC and GLC-MS as PGD₂ and PGD₃. The apparent difference of potency between PGD₂ and PGD₃ was attributed to the concurrent production of PGE₂ and PGE₃. PGE₂ prevented the inhibitory effect of PGD₂ whereas PGE₃ did not affect the activity of PGD₃.It is concluded that one of the reasons for the low incidence of myocardial infarction in Eskimos could be that the pro-aggregatory arachidonic acid is replaced in their phospholipids by the anti-aggregatory EPA.     

Thromboxane A2 fails to induce proliferation of smooth muscle cells enriched with eicosapentaenoic acid and docosahexaenoic acid.

Thromboxane A2 (TXA2) released from aggregating platelets and injured vessel wall stimulates smooth muscle cell proliferation, which may contribute to the development of vascular lesion formation after percutaneous transluminal coronary angioplasty. Polyunsaturated fatty acids (n-3) present in the fish oils have been shown to have anti-atherosclerotic effects. In view of this, we examined the effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the active ingredients of fish oils on TXA2 induced smooth muscle cell proliferation. To find out the specificity of these fatty acids we used gamma-linolenic acid (n-6) and oleic acid (n-9) as controls. It was found that TXA2 failed to stimulate proliferation of smooth muscle cells preloaded with EPA or DHA but not with gamma-linolenic acid or oleic acid. Further, when smooth muscle cells were preloaded with both EPA and DHA, they acted together in preventing the TXA2 induced smooth muscle cell proliferation. These results demonstrate that one of the mechanisms by which fish oils may prevent neointima formation is by making smooth muscle cells less responsive to TXA2 induced proliferation of smooth muscle cells.     

Human umbilical blood vessel converts all cis-5, 8, 11, 14, 17 eicosapentaenoic acid to prostaglandin I3

All cis-5, 8, 11, 14, 17 eicosapentaenoic acid (EPA) is presently being evaluated for dietary prophylactic use in thrombo-embolic disorders. EPA inhibits the production of TXA2 and platelet aggregation. We here present results demonstrating that human umbilical arteries convert 14c-EPA to a substance that in aqueous solutions decomposes to 14C-delta 17-6-keto-PGF1 alpha . The conversion rate in rat aortic tissue was found substantially lower. These results in combination with earlier data indicating that EPA does not influence the conversion of arachidonic acid (AA) into PGI2 in human vascular tissue, encourage further research along the lines initiated by the findings of high EPA/AA ratio and low incidence of myocardial infarction in Greenland Eskimos.     

Eicosapentaenoic acid metabolism in human and rabbit anterior uvea

Eicosapentaenoic acid (EPA) metabolism into 3 series cyclooxygenase and 5 series lipoxygenase products was assessed in human and rabbit anterior uvea. Both tissues synthesized 3 series cyclooxygenase products such as delta¹⁷ 6-keto-PGF₁ (PGI₃ metabolite), PGF₃, PGE₃, PGD₃ and TxB₃ (a stable product of TxA₃) and lipoxygenase products 12-hydroxyeicosapentaenoic acid (HEPE), 5-HEPE and 5,12-diHEPE from ¹⁴C-EPA. EPA-derived cyclooxygenase product synthesis was considerably greater than the formation of lipoxygenase products from EPA in both tissues.     

Atherosclerosis decreased prostacyclin formation in rabbit lungs and kidneys

Metabolism of arachidonic acid (AA) was studied in perfused lungs and kidneys of normal and atherosclerotic rabbits by determination of PGE₂, PGF_2α and the stable metabolites of PGI₂ (6-keto-PGF_1α) and TXA₂ (TXB₂). PGI₂ was the main AA metabolite formed by normal lungs and kidneys. Atherosclerosis reduced the formation of PGI₂ by about 50 % in both organs. TXA₂ formation was similarily decreased in lungs. In kidneys, the decrease in PGI₂ formation was accompanied by an increase in PGE₂ formation.     

Essential fatty acid metabolism in patients with essential hypertension, diabetes mellitus and coronary heart disease

Mortality and morbidity from coronary heart disease (CHD), diabetes mellitus (DM) and essential hypertension (HTN) are higher in people of South Asian descent than in other groups. There is evidence to believe that essential fatty acids (EFAs) and their metabolites may have a role in the pathobiology of CHD, DM and HTN. Fatty acid analysis of the plasma phospholipid fraction revealed that in CHD the levels of gamma-linolenic acid (GLA), arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are low, in patients with HTN linoleic acid (LA) and AA are low, and in patients with non-insulin dependent diabetes mellitus (NIDDM) and diabetic nephropathy the levels of dihomo-gamma-linolenic acid (DGLA), AA, alapha-linolenic acid (ALA) and DHA are low, all compared to normal controls. These results are interesting since DGLA, AA and EPA form precursors to prostaglandin E₁, (PGE₁), prostacyclin (PGI₂), and PGI₃, which are potent platelet anti-aggregators and vasodilators and can prevent thrombosis and atherosclerosis. Further, the levels of lipid peroxides were found to be high in patients with CHD, HTN, NIDDM and diabetic nephropathy. These results suggest that increased formation of lipid peroxides and an alteration in the metabolism of EFAs are closely associated with CHD, HTN and NIDDM in Indians. Since insulin resistance and hyperinsulinemia and features of obesity, NIDDM, HTN and CHD, diseases that are common in Indians, and as decreased insulin sensitivity is associated with decreased concentrations of polyunsaturated fatty acids (PUFAs) in skeletal muscle phospholipids and, possibly, in the plasma, the possibility is raised that changes in the metabolism of EFAs may have a fundamental role in the pathobiology of these conditions. If this is true, this suggests that supplementation of GLA, DGLA, AA, EPA and/or DHA may be indicated to prevent CHD, HTN and NIDDM in Indians.     

Fish oil administration as a supplement to a corn oil containing diet affects arterial prostacyclin production more than platelet thromboxane formation in the rat

The administration to male rats of 5 en % fish oil (FO) as supplement to a diet containing 5 en % corn oil (CO), selectively and markedly decreased arterial parameters (6-keto-PGF_1α formation and platelet antiaggregatory activity) assessed in isolated aortic segments perfused with autologous platelet rich plasma (PRP). Platelet parameters (ADP-induced aggregation, TxB₂ formation in thrombin-stimulated PRP and sensitivity to exogenous PGI₂) were instead minimally affected. Eicosapentaenoic acid (EPA, 20:5 n-3) did not accumulate in plasma, platelet and aorta lipids and arachidonic acid (AA, 20:4 n-6) levels declined markedly only in the plasma compartment. When FO was given alone at the same 5 en % level, both arterial and platelet parameters were similarly affected. EPA accumulated in plasma cholesterol esters and was present in appreciable concentrations also in platelets and aortic walls. AA levels declined markedly in plasma lipids and appreciably also in platelet and aorta lipids. It is concluded that a) arterial and platelet parameters are differentially affected by FO administration depending upon the presence of n-6 polyunsaturated fatty acids in the diet, b) 6-keto-PGF_1α production by arterial tissues does not seem to be related to changes of PG precursor fatty acid levels in the phospholipid fraction.     

Interactions between n-3 and n-6 essential fatty acids (EFAs) in the regulation of cardiovascular disorders and inflammation.

Much attention has recently been paid to the possible benefits of increasing the intake of eicosapentaenoic acid (EPA) by consuming fish oil. However, this can have adverse effects such as raising cholesterol levels in patients with hyperlipidaemia and causing a deterioration in glucose tolerance. High doses of EPA given to Westerners also lower levels of dihomogammalinolenic acid (DGLA), a substance with a wide range of desirable cardiovascular and antiinflammatory actions. This lowering of DGLA does not occur in Eskimos who consume large amounts of EPA, indicating that there may be differences in essential fatty acid metabolism between Westerners and Eskimos. Therapeutic strategies are required which raise both EPA and DGLA and which do not raise EPA at the cost of lowering DGLA.     

Dietary administration of eicosapentaenoic and linolenic acid increases arterial blood pressure and suppresses vascular prostacyclin synthesis in the rat

The role of the ‘prostacyclin-thromboxane system’ in the regulation of arterial blood pressure was investigated in rats receiving diets which contained different amounts of eixosapentaenoic (EPA) and linolenic acid (LNA). Forty rats were divided into five groups of 8 animals, each group receiving 25 energy (en) % as fat. All diets contained equal amounts of linoleic acid (5 en%) and oleic acid (5 en%). In the control group I, the remaining 15 en% of fat were given as saturated fat. Two groups of animals received cod liver oil as a source for EPA in amounts of 2.5 (group II)_and 5 en% (group III) while the two remaining groups were given diets supplemented with linseed oil as a source for LNA in amounts of 2.5 (group IV) and 5 en% (group V), respectively. After six weeks of feeding period the animals were sacrificed and portions of their isolated aorta incubated in Tris buffer (pH 9.3) for determination of prostacyclin (PGI₂)-like activity. Arterial blood pressure was uncharged in group I animals, but significantly increased in all rats receiving dietary EPA or LNA supplements. This rise is arterial blood pressure was associated with a marked suppression of the appearance of PGI₂-like activity in the incubation buffer while platelet thromboxane release during blood clotting was unchanged. Our results show that dietary adminis- tration of EPA and LNA increases arterial blood pressure in the rat and that this effect is associated with a suppressed generation of vasodilator prostacyclin by vascular tissue.     

12-Hydroxyeicosatetraenoic acid reduces prostacyclin production by endothelial cells

12-Hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase product released by activated platelets and macrophages, reduced prostacyclin (PGI₂) formation in bovine aortic endothelial cultures by as much as 70%. Maximal inhibition required 1 to 2 h to occur and after 2 hr, a concentration of 1 μM 12-HETE produced 80% of the maximum inhibitory effect. 5-HETE and 15-HETE also inhibited PGI₂ formation. The inhibition was not specific for PGI₂; 12-HETE reduced the formation of all of the radioactive eicosanoids synthesized from [1-¹⁴C]arachidonic acid by human umbilical vein endothelial cultures. Inhibition occurred in the human cultures when PGI₂ formation was elicited with arachidonic acid, ionophore A23187 or thrombin. These findings suggest that prolonged exposure to HETEs may compromise the antithrombotic and vasodilator properties of the endothelium by reducing its capacity to produce eicosanoids, including PGI₂.     

Major urinary metabolites of 6-keto-prostaglandin F2α in mice

Western diets are enriched in omega-6 vs. omega-3 fatty acids, and a shift in this balance toward omega-3 fatty acids may have health benefits. There is limited information about the catabolism of 3-series prostaglandins (PG) formed from eicosapentaenoic acid (EPA), a fish oil omega-3 fatty acid that becomes elevated in tissues following fish oil consumption. Quantification of appropriate urinary 3-series PG metabolites could be used for noninvasive measurement of omega-3 fatty acid tone. Here we describe the preparation of tritium- and deuterium-labeled 6-keto-PGF_2α and their use in identifying urinary metabolites in mice using LC-MS/MS. The major 6-keto-PGF_2α urinary metabolites included dinor-6-keto-PGF_2α (∼10%) and dinor-13,14-dihydro-6,15-diketo-PGF_1α (∼10%). These metabolites can arise only from the enzymatic conversion of EPA to the 3-series PGH endoperoxide by cyclooxygenases, then PGI₃ by prostacyclin synthase and, finally, nonenzymatic hydrolysis to 6-keto-PGF_2α. The 6-keto-PGF derivatives are not formed by free radical mechanisms that generate isoprostanes, and thus, these metabolites provide an unbiased marker for utilization of EPA by cyclooxygenases.     

Consequences of long-term selenium-deficient diet on the prostacyclin and thromboxane release from rat aorta

It is known that peroxides, which are increased during Se deficiency because of reduced glutathione peroxidase (GSH-Px) activity, can influence the prostacyclin I₂/thromboxane A₂ (PGI₂/TXA₂) ratio. In this study we analyzed the PGI₂ and TXA₂ formation of aortas of long-term Se-deficient rats. Despite low GSH-Px activity in the Se-deficient group, the basal PGI₂ and TXA₂ formation was not different versus control animals (PGI₂: 2295 ± 1134 pg/mg vs 2940 ± 1134 pg/mg; TXA₂: 3.83 ± 1.06 pg/mg vs 5.67 ± 2.99 pg/mg). However, we checked the capacity of the aortas of Se-deficient rats to compensate for a suddenly increased peroxide concentration. After peroxide stimulation, the PGI₂ release was significantly lower in the Se-deficient group compared to the control group (PGI₂: 3507 ± 1829 pg/mg vs 7986 ± 2636 pg/mg). Again, the TXA₂ release did not show any differences. The release ratio of PGI₂/TXA₂ decreased under peroxide stress in Se-deficient animals. Although long-term Se deficiency showed a relatively well-balanced metabolism under resting conditions, sudden stress, accompanied by an excessive radical production, cannot be compensated.     

Enhanced formation of PGI2, a potent hypotensive substance, by aortic rings and homogenates of the spontaneously hypertensive rat

Intact rings and homogenates of aorta from spontaneously hypertensive rats (SHR) contain enhanced capacity over normal rats (NR) to convert arachidonic acid into PGI₂. The PGI₂ synthetic system in SHR is stimulated to a greater extent than NR by norepinephrine. Indomethacin blocks this stimulation. PGE₂ and PGF_2α were detected in much smaller amounts in homogenates (undetected in rings) but their formation was not enhanced by the hypertensive tissue. The identity of PGI₂ was based on 1) direct pharmacological assay on the rat blood pressure. In this system identical vasodepressor responses to PGI₂ are observed after intracarotid and intrajugular administration 2) indirectly as 6-keto PGF_1α isolated after incubation of aortic homogenates with tritiated arachidonic acid and 3) indirectly by GC-MS assay of PGE₂, PGF_2α and 6-keto PGF_1α formed during incubation of aortic homogenates with excess unlabeled arachidonic acid. These results provide additional support to our recent hypothesis that PGI₂, of aortic origin, might actively participate in the regulation of systemic blood pressure. Its enhanced formation by intact hypertensive vascular tissue reflects an increase in the number of enzyme molecules immediately available to the substrate. This could probably be an adaptive response to the elevated levels of catecholamines in the circulation.     

Increased production of prostacyclin (PGI2) by vascular intimal smooth muscle cells from atherosclerotic rabbit aorta

PGI₂ synthesis by aortic strips obtained from thoracic aorta of rabbits fed a high cholesterol diet was examined and compared with that of control rabbits fed a normal diet. In this report, the amounts of PGI₂ produced were shown as 6-keto-PGF_1α per μg of aortic tissue DNA instead of per mg wet weight. We also investigated PGI₂ synthesis by cultured smooth muscle cells (SMC) obtained from atherosclerotic intima.Basal PGI₂ production by aortic strips from atherosclerotic rabbit aorta was significantly augmented compared with that of controls. Arachidonic acid (AA)-induced PGI₂ production by atherosclerotic aorta was also significantly higher than that of controls. PGI₂ producing capacities of intimal and medial layers, separated from atherosclerotic aorta, were examined and the intimal layer was found to elicit a significantly greater PGI₂ production than the medial layer.Furthermore, cultured intimal SMC obtained from atherosclerotic rabbit aorta produced a greater amount of PGI₂ than medial SMC from normal rabbit aorta at various cultured conditions. These results suggest that the possibility of enhanced PGI₂ production by atherosclerotic aorta may well be considered as a defence mechanism of the vessel wall against damaging stimuli.     

Myocardial prostacyclin and thromboxane A2 synthetase activities during ischemia and reperfusion in isolated rabbit heart

The aim of the study was to determine the prostacyclin (PGI₂) and thromboxane A₂ (TXA₂) synthetase activities of myocardial tissue and their variation during ischemia and reperfusion. Regional ischemia was induced by 10 min occlusion of the left anterior descending coronary artery in isolated Langendorff rabbit hearts. Biosynthesis of PGI₂ and TXA₂ were carried out by using arachidonic acid as substrate and left ventricle microsomes (LVM) from ischemic and non-ischemic areas as sources of PGI₂ and TXA₂ synthetase. 6-keto-PGF_1α and TXB₂, stable metabolites of PGI₂ and TXA₂ respectively, were determined by radioimmunoassay. Experiments carried out under the adopted conditions showed that LVM were able to synthetise PGI₂ as well as TXA₂ from arachidonic acid. On the other hand, ischemia depressed both PGI₂ and TXA₂ synthetase activities of cardiac tissue: the depression was more pronounced on TXA₂ synthetase than on PGI₂ synthetase with no significant difference between ischemic and non-ischemic regions. Moreover, ischemia increased the ratio indicating therefore that it can facilitate the formation of PGI₂. The post ischemic reperfusion of the heart counteracted the decrease in PGI₂ synthetase induced by ischemia which returned to the normal level: reperfusion also slightly reversed the decrease in TXA₂ synthetase. However, the diminution in TXA₂ synthetase of non-ischemic myocardium was attenuated but it remained lower than the normal level. These results suggested that the whole left ventricle is affected by regional ischemia. Furthermore it appears that myocardial TXA₂ synthetase is more vulnerable than PGI₂ synthetase to a lack of oxygen and nutrients.     

6-Keto-prostaglandin E1 is not equipotent to prostacyclin (PGI2) as an antiaggregatory agent

A direct comparison of the relative potencies of the two anti-aggregatory prostaglandins PGI₂ and 6-keto-PGE₁ showed PGI₂ was at least 20 times more potent than 6-keto-PGE₁ when tested against ADP-induced human platelet aggregation. This marked difference in potency was even more evident when the ability of PGI₂ and 6-keto-PGE₁ to stimulate platelet cyclic AMP levels was determined. When cyclic AMP levels were measured direct comparisons were difficult because the respective dose response curves were not parallel, but 10 ng of PGI₂ was equivalent to 300 ng of 6-keto-PGE₁.PGI₂ was also more potent (10–20 times) than 6-keto-PGE₁ as a disaggregatory agent, and the disaggregatory activity of both prostaglandins was enhanced by the phosphodiesterase inhibitor 1-methyl-3-isobutylmethylxanthine.PGI₂ was also more active than 6-keto-PGE₁ as an inhibitor of thrombus formation in dog coronary arteries in vivo. In vivo, 6-keto-PGE₁ was at least 10 times less potent than PGI₂, the exact difference could not be determined because 6-keto-PGE₁ caused significant falls in blood pressure before anti-platelet activity could be detected.PGI₂ is an intrinsically more potent anti-aggregatory molecule than 6-keto-PGE₁, but these data do not rule out the possibility that some of the activities attributed to PGI₂ could be the result of the conversion of PGI₂ and/or 6-keto-PGF_1α to 6-keto-PGE₁.