Chronologic studies of the effects of a hypoproteinic diet followed by an equilibrated diet on delta-6 and delta-5 desaturations of linoleic acid in liver microsomes in the rat

A low protein diet affects amounts of linoleic and arachidonic acids in hepatic microsomal phospholipids of growing rats. Are the changes related to modifications in microsomal delta 6- and delta 5- linoleic acid desaturase activities? Two groups of Wistar rats weighing 80 +/- 5 g at the beginning of the experiment were used: Control group (T) was fed on a 16% gluten + 4% casein diet for 53 days; Experimental group (E) was fed on a 4% gluten + 1% casein diet for 26 days (MP) then Control diet for 27 days (RE). After 2, 14 and 26 days of MP and 2, 15 and 27 days of RE, rats of each group were sacrificed. Protein and water contents of liver, quantitative fatty acid, composition of total lipids in liver and hepatic microsomes were determined. delta 6- and delta 5- linoleic acid desaturase activities were estimated from incubation of liver microsomes with [1-14C] C 18: 2 n-6 or [2(14)C] C 20: 3 n-6 respectively. The low protein diet stops practically ponderal growth. The fatty-acid compositions of microsomal total lipids of E rats were affected in comparison with values of T rats. These modifications persist after 27 days of RE. The C 20: 4 n-6/C 18: 2 n-6 ratio in microsomal total lipids was slightly different between T and E rats but increased strongly during refeeding. Same modifications take place in the fatty-acid composition of hepatic total lipids. After two days of MP, delta 6- and delta 5- desaturase activities were depressed, phenomenon that not persist in the course of MP. These enzyme activities increase to higher values than those of the T after two days of RE.     

In vitro study of delta 6- and delta 5-desaturations of linoleic and dihomo-gamma-linolenic acids during the development of arterial hypertension as a function of age in spontaneously hypertensive rats (SHR) as compared to normotensive rats (WKY)

delta 6- and delta 5-desaturations of linoleic [1(14)C] or dihomo-gamma-linolenic [2(14)C] acids are partially inhibited, in vitro, in liver microsomes of spontaneously hypertensive rats (SHR) comparatively to normotensive rats (WKY). The inhibition is more light when the animals are young and the blood hypertension not well established. These results are particularly important, for these desaturations are fundamental steps in the biosynthesis of prostaglandins precursors, prostaglandins and their original fatty acids being involved in blood pressure regulation.     

Linoleate and linolenate desaturation by rat hepatoma cells

Morris 7777 rat hepatoma cells in culture possess high delta 6 and delta 5 desaturase activities over linolenic acid added to the medium as albumin or alpha-fetoprotein complexes. After 2 hours incubation with [1-14C] linolenic acid (7 microM), around 40% of the radioactivity was recovered in other polyene fatty acids, mainly pentaenes. After 24 hours incubation with this substrate the polyene derivatives raised to more than 60%. However, [1-14C] linoleic acid was poorly converted to other polyene fatty acids. Linoleic acid up to 58 microM concentration in the medium do not inhibited linolenic acid desaturation. Long-term supplementation with 50 microM linoleic or linolenic acid, which modified the fatty acid profile of hepatoma lipids, enhanced the desaturase activities against linoleic acid. Desaturase activities were not affected by the fatty acid protein carrier, alpha-fetoprotein or albumin.     

Comparative in vivo and in vitro study of the influence of experimental diabetes on rat liver linoleic acid delta 6- and delta 5-desaturation

Rats with experimental diabetes were administered in vivo a tracer dose of either [1-14C]-linoleic, [1-14C]-gamma-linolenic or [2-14C]-dihomo-gamma-linolenic acid and sacrificed 48 h later. With all three radioactive precursors, the radioactivity incorporated into arachidonic acid was lower in experimental diabetes, compared to nondiabetic rats similarly treated, while the weights of hepatic arachidonic acid were not significantly affected by the diabetic state. Streptozotocin-treated rats were administered moderate or excessive quantities of protamine-zinc-insulin. Streptozotocin diabetes inhibits rat liver homogenate [2-14C]-dihomo-gamma-linolenic acid delta 5-desaturation; only moderate injections of protamine-zinc-insulin restore the in vitro delta 5-desaturation. These results suggest that experimental diabetes, a reported inhibitor of delta 6-desaturation, also causes partial inhibition of delta 5-desaturation in rat liver; this suggests that dihomo-gamma-linolenic acid desaturation, a secondary regulatory step in linoleic acid metabolism, may be restored through an optimum insulin therapy.     

Effect of dietary fats on arachidonic acid and eicosapentaenoic acid biosynthesis and conversion to C22 fatty acids in isolated rat liver cells

The desaturation, chain elongation and esterification of [1-14C]eicosapentaenoic acid, [1-14C]arachidonic acid, [1-14C]eicosatrienoic acid, [1-14C]linolenic acid and [1-14C]linoleic acid were studied in isolated liver cells. Rats fed diets with either 15% hydrogenated coconut oil or 15% partially hydrogenated marine oil, both deficient in essential fatty acids, 15% soybean oil or standard pellet diet with 6% fat, were used. The delta 4-desaturation of 22:5(n - 3) and 22:4(n - 6) as well as the delta 6-desaturase activity was distinctly higher in cells from animals fed coconut or marine oil than with soybean oil or standard pellet. The rate of delta 5-desaturation of 20:3(n - 6) and 20:4(n - 3) was nearly the same in cells from rats fed coconut, marine and soybean oils and higher than with standard pellet. The chain elongation of 20:5(n - 3) to 22:5(n - 3) was distinctly more pronounced than the elongation of 20:4(n - 6) with all four diets. 20:5(n - 3) was mainly esterified in the phospholipids with marine and coconut oils, and mainly in triacylglycerol with standard pellet and soybean oils. The proportion of [1-14C]20:4(n - 6) in the phospholipids to that in triacylglycerol decreased in the order marine oil greater than coconut oil greater than standard pellet greater than soybean oil. The different endogenous arachidonic acid content in the phospholipids induced by the different diets increased in the same order. 20:5(n - 3) was rapidly esterified in triacylglycerol and phospholipids, then liberated especially from the triacylglycerol fraction, chain elongated to 22:5(n - 3) and reesterified.     

Regional differences in lipid composition and incorporation of saturated and unsaturated fatty acids into microsomal membranes of rat small intestine

Incorporation of [1-14C]palmitic (16:0) and [1-14C]linoleic (18:2 omega 6) acids into microsomal membranes of proximal (jejunum) and distal (ileum) regions of rat small intestine was investigated, and the lipid composition, including fatty acid profiles of membrane phospholipids, was determined. Jejunal microsomes contained significantly higher amounts of total phospholipids, phosphatidylcholine, and phosphatidylinositol, and lower amounts of cholesterol and sphingomyelin when compared with ileal microsomes. Jejunal microsomal phospholipids contained higher levels of stearic (18:0), 18:2 omega 6, and eicosapentaenoic (20:5 omega 3) acids followed by reduced levels of oleic (18:1 omega 9), arachidonic (20:4 omega 6), and docosahexaenoic (22:6 omega 3) acids when compared with those from the ileum, except for phosphatidylinositol where no significant difference between 20:4 omega 6 content of each site was observed. In both jejunal and ileal microsomes, incorporation of [1-14C]18:2 omega 6 was significantly higher than that of [1-14C]16:0. Incorporation of both [1-14C]16:0 and [1-14C]18:2 omega 6 was significantly higher in jejunal microsomal lipid fractions (phospholipids, diacylglycerols, triacylglycerols) when compared with the ileal microsomal fraction. These data suggest that (1) jejunal and ileal microsomal membranes differ from each other in terms of lipid composition and lipid synthesis, (2) site variations in the specificity of acyltransferases for different fatty acids exist, and (3) higher delta 9-, delta 6-, delta 5-, and delta 4-desaturase activities exist in ileal compared with jejunal enterocytes.     

Fatty acid metabolism in human lymphocytes. II. Activation of fatty acid desaturase-elongase systems during blastic transformation

The fatty acid desaturation-elongation ability of human T-lymphocytes during blastic transformation was determined both by gas-liquid chromatography and incubation with radiolabeled precursors. Human peripheral blood mononuclear cells (PBMC) were activated with phytohemagglutinin (PHA) and cultured in media supplemented with different fatty acids (18:0, 18:1(n - 9), 18:2(n - 6), 18:3(n - 3) and 20:4(n - 6)) at a final concentration of 30 microM. All the fatty acids added were elongated by activated PBMC and the maximal activity was observed on 20:4(n - 6) (a 25% of conversion to 22:4(n - 6)). Supplementation with stearic acid increased the proportion of oleic (from 21.4% to 23.7%) and eicosaenoic (from 3.1% to 5.7%) acids in cellular lipids, indicating the existence of a delta 9-desaturase activity. Supplementation with linoleic and linoleic acids increased slightly the cell content in their more unsaturated derivatives. Direct measurement of desaturase activities was performed by incubating quiescent and activated PBMC with [1-14C]stearic, [1-14C]linoleic and [1-14C]linolenic acids. Quiescent cells exhibited a very low delta 9-desaturase and no sign of delta 6-desaturase activity. A moderate and progressive activation of delta 9-, delta 6- and delta 5-desaturases was observed during blastic transformation of human PBMC. Up to 8% of 18:0 was converted to monoenes, 4% and 1.5% of 18:2(n - 6) was converted to trienes and tetraenes, respectively, and 14.5% of 18:3(n - 3) was converted to pentaenes. The maximal relative activities were found after 48 h of PHA-stimulation for delta 9-desaturase (around 90 pmol of 18:0 converted per 10(6) cells in the last 24 h) and at 72 h for delta 6- and delta 5-desaturases (around 75 and 140 pmol of 18:2 and 18:3, respectively, converted per 10(7) cells in the last 24 h). Although these activities are not enough to explain all the changes in fatty acid composition of human PBMC during blastic transformation, they may contribute to a more controlled cell phospholipid composition.     

Sex-related differences in desaturation and chain elongation of essential fatty acids studied in isolated rat hepatocytes

When [14C]linoleic acid (18:2(n-6)) or [14C]dihomogammalinolenic acid (20:3(n-6)) was incubated with isolated liver cells from rats fed an essential fatty acid deficient diet, delta 6- and delta 5-desaturation, chain elongation and synthesis of 14C-labelled C14-C18 fatty acids (from [14C]acetate) were enhanced in female cells compared with male ones. No sex difference in total secretion of very low density lipoproteins (VLDL) was observed. However, VLDL secreted from female cells contained significantly more C16-C18 fatty acids than male cells. It is suggested that the observed sex differences, at least in part, may be related to the different content of fatty acid binding proteins in female cells compared with males.     

Microsomal fatty acid desaturation and elongation in a human lung carcinoma grown in nude mice

Since tumor cells show abnormal fatty acid composition, it is likely that their desaturase systems were affected to some extent. Although desaturase activities in experimental tumors have been evaluated, to our knowledge, fatty acid desaturases in human neoplasms and particularly in human tumors grown in nude mice have not been assessed yet. We have therefore, chosen a rapidly growing human lung mucoepidermoid carcinoma (HLMC) grown in nude mice to study microsomal fatty acid desaturation and chain elongation activities. Tumor microsomal proteins were incubated with unlabeled malonyl-CoA and one of the following fatty acids: [1-14C]palmitic (16:0), [1-14C]linoleic (18:2), alpha-[1-14C]linolenic (alpha-18:3), and unlabeled gamma-linolenic (gamma-18:3) plus [2-14C]malonyl-CoA. Data show that HLMC microsomes were capable to desaturate 16:0, alpha-18:3, and dihomogammalinolenic acids (20:3) by delta 9, delta 6 and delta 5 desaturase, respectively; however, delta 6 desaturase activity on [14C]18:2 was not detected. The microsomal elongation system was active in all fatty acid series tested except for 18:2. These findings show that the undetectable activity for 18:2 desaturation is not exclusively found in experimental tumors.     

Incorporation into liver microsomal lipids of linoleic and stearic acids and of their respective products of delta 6 and delta 9 desaturation, gamma-linolenic and oleic acids: effect of age and of blackcurrant seed oil.

The incorporation of [1-14C]linoleic and [1-14C]stearic acid and of their delta 6 and delta 9 desaturation products (gamma-linolenic and oleic acids, respectively) into different classes of lipids was studied in liver microsomes of rats in function of the diet (blackcurrant seed oil diet, containing gamma-linolenic acid, versus control diet) and in function of age (3, 6 and 9 months). After delta 6 desaturation, total radioactivity was distributed between phospholipids, especially phosphatidylcholine, and neutral lipids. The desaturation product, gamma-linolenic acid, was totally recovered in the phospholipid fraction. Blackcurrant seed oil, which decreased the rate of delta 6 desaturation in 6- and 9-month-old rats, also decreased the incorporation of radioactivity in total phospholipids, especially in phosphatidylcholine. At 6 months of age, after delta 9 desaturation, the majority of radioactivity was recovered in neutral lipids principally as oleic acid, the desaturation product. The precursor, stearic acid, was highly incorporated into phospholipids, especially in rats on a diet of blackcurrant seed oil.     

Characterization and separation of the arachidonic acid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase (arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes

The cytosolic fraction of human polymorphonuclear leukocytes precipitated with 60% ammonium sulfate produced 5-lipoxygenase products from [14C]arachidonic acid and omega-6 lipoxygenase products from both [14C]linoleic acid and, to a lesser extent, [14C]- and [3H]arachidonic acid. The arachidonyl 5-lipoxygenase products 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) derived from [14C]arachidonic acid, and the omega-6 lipoxygenase products 13-hydroperoxy-9,11-octadecadienoic acid (13-OOH linoleic acid) and 13-hydroxy-9,11-octadecadienoic acid (13-OH linoleic acid) derived from [14C]linoleic acid and 15-hydroxyperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) derived from [14C]- and [3H]arachidonic acid were identified by TLC-autoradiography and by reverse-phase high-performance liquid chromatography (RP-HPLC). Products were quantitated by counting samples that had been scraped from replicate TLC plates and by determination of the integrated optical density during RP-HPLC. The arachidonyl 5-lipoxygenase had a pH optimum of 7.5 and was 50% maximally active at a Ca2+ concentration of 0.05 mM; the Km for production of 5-HPETE/5-HETE from arachidonic acid was 12.2 +/- 4.5 microM (mean +/- S.D., n = 3), and the Vmax was 2.8 +/- 0.9 nmol/min X mg protein (mean +/- S.D., n = 3). The omega-6 linoleic lipoxygenase had a pH optimum of 6.5 and was 50% maximally active at a Ca2+ concentration of 0.1 mM in the presence of 5 mM EGTA. When the arachidonyl 5-lipoxygenase and the omega-6 lipoxygenase were separated by DEAE-Sephadex ion exchange chromatography, the omega-6 lipoxygenase exhibited a Km of 77.2 microM and a Vmax of 9.5 nmol/min X mg protein (mean, n = 2) for conversion of linoleic acid to 13-OOH/13-OH linoleic acid and a Km of 63.1 microM and a Vmax of 5.3 nmol/min X mg protein (mean, n = 2) for formation of 15-HPETE/15-HETE from arachidonic acid.     

Neither delta- nor lambda-tris(phenanthroline)ruthenium(II) binds to DNA by classical intercalation.

Equilibrium binding studies and viscosity experiments are described that characterize the interaction of delta- and lambda-[Ru(o-phen)3]2+ with calf thymus DNA. The mode of binding of these compounds to DNA is a matter of controversy. Both isomers of [Ru(o-phen)3]2+ were found to bind but weakly to DNA, with binding constants of 4.9 (+/- 0.3) x 10(4) M-1 and 2.8 (+/- 0.2) x 10(4) M-1 determined for the delta and lambda isomers, respectively, at 20 degrees C in a solution containing 5 mM Tris-HCl (pH 7.1) and 10 mM NaCl. We determined that the quantity delta log K/delta log [Na+] equals 1.37 and 1.24 for the delta and lambda isomers, respectively. Application of polyelectrolyte theory allows us to use these values to show quantitatively that both the delta and lambda isomers are essentially electrostatically bound to DNA. Viscosity experiments show that binding the lambda isomer does not alter the relative viscosity of DNA to any appreciable extent, while binding of the delta isomer decreases the relative viscosity of DNA. From these viscosity results, we conclude that neither isomer of [Ru(o-phen)3]2+ binds to DNA by classical intercalation.     

Stimulation of deacylation in Madin-Darby canine kidney cells. Specificity of deacylation and prostaglandin production in 12-O-tetradecanoylphorbol-13-acetate-treated cells

Madin-Darby canine kidney cells deacylate arachidonic acid from cellular phospholipid in response to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and convert the free arachidonic acid to prostaglandins. We have used this system to characterize the acyl specificity of deacylation. Cells were labeled with either [14C]linoleic, [14C]eicosatrienoic (delta 8,11,14 or delta 5,8,11), or [14C]arachidonic acid and stimulated with 10 nM TPA. We found that TPA stimulated the deacylation of all four acids, primarily from phosphatidylethanolamine and phosphatidylcholine.l Only products from linoleic (presumably through chain elongation and desaturation), eicosatrienoic (delta 8,11,14), and arachidonic acids produced prostaglandins. Those produced from linoleic and eicosatrienoic acid (delta 8,11,14)-labeled cells were determined to be primarily of the 1-series, while arachidonic acid-labeled cells produced prostaglandins of the 2-series. Together these results indicate that the stimulated deacylation of phospholipids is not specific for arachidonic acid and that the membrane acyl composition controls the particular series of prostaglandin which is produced.     

Effect of glucocorticoids on the oxidative desaturation of fatty acids by rat liver microsomes.

The effect of glucocorticoids on the oxidative desaturation of fatty acids by liver microsomal preparations of rats has been studied. Hydrocortisone produced a significant decrease in the conversion of [1-14C]linoleic acid to gamma-linolenic acid and [1-14C]eicosa-8, 11, 14-trienoic acid to arachidonic acid. Triamcinolone and dexamethasaone were more active than hydrocortisone in depressing delta 6 and delta 5 fatty acid desaturating activity in liver microsomes. The glucocorticoids evoked a maximal response approximately 24 hr after admission. Palmitic acid conversion to palmitoleic acid showed no statistically significant changes by any of the glucocorticoids. The mechanism of action of glucocorticoids is apparently different from other hyperglycemic hormones that produce similar effects.     

Evidence of isozymes for delta6 fatty acid desaturase in rat hepatocytes

The expression of delta6 fatty acid desaturase, previously identified, was suppressed almost completely by hyper expression of the corresponding antisense gene in a transformant of the rat hepatic cell line BRL-3A. Conversion rates of [1-14C] linoleic acid, alpha-linolenic acid, and tetracosapentaenoic acid into the respective delta6 fatty acids were equivalent to those in control cells. This finding suggested that all of these reactions were catalyzed by at least two delta6 desaturase isozymes in rat hepatocytes.     

ACTH depresses delta 6 and delta 5 desaturation activity in rat adrenal gland and liver

The in vivo and in vitro effect of ACTH on the biosynthesis of polyunsaturated fatty acid of rat adrenal gland and liver was studied. The administration of ACTH to intact rats produced a significant decrease in the conversion of [1-14C]linoleic acid to gamma-linolenic acid, [1-14C]alpha-linolenic acid to octadeca-6,9,12,15-tetraenoic acid, and [1-14C]eicosa-8,11,14-trienoic acid to arachidonic acid in liver and adrenal microsomes. Isolated adrenocortical cells and hepatocytes obtained from animals treated with ACTH showed a decrease in the incorporation and desaturation of exogenous [1-14C]eicosa-8,11,14-trienoic acid. The addition of ACTH to the incubation medium of adrenocortical cells and hepatocytes isolated from untreated rats also caused a decrease in delta 5 desaturation activity. The effect of ACTH on adrenal and liver desaturases could be produced as a consequence of the release of glucocorticoids, already measured in the experiments. However, the in vitro experiments carried out with hepatocytes isolated from untreated rats, where corticosterone was absent, indicated that ACTH can depress delta 5 desaturation per se.     

Glucosylation of phosphorylpolyisoprenol and sterol at the plasma membrane of soya-bean (Glycine max) protoplasts.

The mechanism of isomerization of delta 5-3-ox steroids to delta 4-3-oxo steroids was examined by using the membrane-bound 3-oxo steroid delta 4-delta 5-isomerase (EC 5.3.3.1) and the 3 beta-hydroxy steroid dehydrogenase present in the microsomal fraction obtained from full-term human placenta. (1) Methods for the preparation of androst-5-ene-3 beta, 17 beta-diol specifically labelled at the 4 alpha-, 4 beta- or 6-positions are described. (2) Incubations with androst-5-ene-3 beta, 17 beta-diol stereospecifically 3H-labelled either in the 4 alpha- or 4 beta-position showed that the isomerization reaction occurs via a stereospecific elimination of the 4 beta hydrogen atom. In addition, the complete retention of 3H in the delta 4-3-oxo steroids obtained from [4 alpha-3H]androst-5-ene-3 beta, 17 beta-diol indicates that the non-enzymic contribution to these experiments was negligible. (3) To study the stereochemistry of the insertion of the incoming proton at C-6, the [6-3H]androst-4-ene-3, 17-dione obtained from the oxidation isomerization of [6-3H]androst-5-ene-3 beta, 17 beta-diol was enzymically hydroxylated in the 6 beta-position by the fungus Rhizopls stolonifer. Retention of 3H in the 6 alpha-position of the isolated 6 beta-hydroxyandrost-4-ene-3, 17-dione indicates that in the isomerase-catalysed migration of the C(5) = C(6) double bond, the incoming proton from the acidic group on the enzyme must enter C-6 from the beta-face, forcing the existing 3H into the 6 alpha-position.     

Effect of epinephrine on the oxidative desaturation of fatty acids in the rat.

The effect of epinephrine on the oxidative desaturation of fatty acids by liver microsomal preparations of rats has been studied. Administration of epinephrine (1 mg/kg body weight) produced a significant decrease in desaturation of [l-14C]=linoleic acid to gamma-linolenic acid and of [L-14C]alpha-linolenic acid to actadeca-6,9,12,15-tetraenoic acid 12 hr after the infection. Lower doses produced a lesser effect on the delta6-desaturation activity. Epinephrine administration modified the V max of linoleic acid desaturation but not the K m. There was also a slight increase in palmityl desaturation activity. The effect of epinephrine on delta6-desaturation activity was postulated to be mediated through an enhancement of the intracellular cyclic AMP levels that lead to an increase of a glucose metabolite. This metabolite would inhibit delta6-desaturation activity.     

Astrocytes, Not Neurons, Produce Docosahexaenoic Acid (22:6ω-3) and Arachidonic Acid (20:4ω-6)

Elongated, highly polyunsaturated derivatives of linoleic acid (18:2 omega-6) and linolenic acid (18:3 omega-3) accumulate in brain, but their sites of synthesis are not fully characterized. To investigate whether neurons themselves are capable of essential fatty acid elongation and desaturation or are dependent upon the support of other brain cells, primary cultures of rat neurons and astrocytes were incubated with [1-14C] 18:2 omega-6, [1-14C]20:4 omega-6, [1-14C]18:3 omega-3, or [1-14C]20:5 omega-3 and their elongation/desaturation products determined. Neuronal cultures were routinely incapable of producing significant amounts of delta 4-desaturase products. They desaturated fatty acids very poorly at every step of the pathway, producing primarily elongation products of the 18- and 20-carbon precursors. In contrast, astrocytes actively elongated and desaturated the 18- and 20-carbon precursors. The major metabolite of 18:2 omega-6 was 20:4 omega-6, whereas the primary products from 18:3 omega-3 were 20:5 omega-3, 22:5 omega-3, and 22:6 omega-3. The majority of the long-chain fatty acids formed by astrocyte cultures, particularly 20:4 omega-6 and 22:6 omega-3, was released into the extracellular fluid. Although incapable of producing 20:4 omega-6 and 22:6 omega-3 from precursor fatty acids, neuronal cultures readily took up these fatty acids from the medium. These findings suggest that astrocytes play an important supportive role in the brain by elongating and desaturating omega-6 and omega-3 essential fatty acid precursors to 20:4 omega-6 and 22:6 omega-3, then releasing the long-chain polyunsaturated fatty acids for uptake by neurons.     

Synthesis of hydroxy fatty acids from linoleic acid by human blood platelets

The metabolism of linoleic acid by washed human platelets was investigated. [1.14C] linoleic acid was converted to [1.14C] hydroxy octadecadienoic acids (HODEs) at about the same rate with which [1.14C] 12-HETE was produced from [1.14C] arachidonic acid. The total radioactivity in HODEs was distributed among two isomers: 13-HODE (85%) and 9-HODE (15%) as defined by CG-MS. The production of HODEs by intact washed platelets was inhibited by indomethacin (IC50:5 x 10(-7) M) which suggest that hydroxy fatty acids were produced by PGH-synthase. By contrast, the production of HODEs by platelet cytosolic fractions was not modified under indomethacin treatment but completely abolished by NDGA (10(-3) M) and inhibited by the platelet lipoxygenase inhibitors 15-HETE (2.10(-5) M) and baicalein (10(-5) M). Platelets thus contain two different active systems which may convert linoleic acid to hydroxy fatty acids. Since these compounds remained essentially associated with the platelets, their presence may significantly participate in the mechanisms of platelet activation.