Mitochondrial and peroxisomal oxidation of arachidonic and eicosapentaenoic acid studied in isolated liver cells

The partitioning between peroxisomal and mitochondrial beta-oxidation of [1-14C]eicosapentaenoic acid (20:5(n-3] and [1-14C]arachidonic acid (20:4(n-6)) was studied. In hepatocytes from fasted rats approximately 70% of the fatty acid substrate was oxidized with oleic, linoleic, eicosapentaenoic and docosahexaenoic (22:6(n-3)) acid, even more with adrenic (22:4(n-6)) and less with arachidonic acid. When the mitochondrial oxidation was suppressed by fructose refeeding and by (+)-decanoylcarnitine, the fatty acid oxidation in per cent of that in cells from fasted rats was with 18:1(n-9) 7%, 18:2(n-6) 8%, 20:4(n-6) 12%, 20:5(n-3) 20%, 22:4(n-6) 57% and for 22:6(n-3) 29%. The fraction of 14C recovered in palmitate and other newly synthesized fatty acids after fructose refeeding decreased in the order 22:4(n-6) greater than 22:6(n-3) greater than 20:5(n-3) greater than 20:4(n-6) and was very small with 18:1(n-9) and 18:2(n-6). In cells from both fed and fructose-refed animals 20:5(n-3) was efficiently elongated to 22:5(n-3) and 22:6(n-3). 20:5(n-3) and 20:4(n-6) were not elongated after fasting. The phospholipid incorporation with [1-14C]20:5(n-3) decreased during prolonged incubations while it remained stable with [1-14C]arachidonic acid. The results suggest that peroxisomes contribute more to the oxidation of 20:5(n-3) than with 20:4(n-6) although both substrates are probably oxidized mainly in the mitochondria.     

Selective incorporation of various C-22 polyunsaturated fatty acids in Ehrlich ascites tumor cells

Three 14C-labeled 22-carbon polyunsaturated fatty acids, 7,10,13,16-[14C]docosatetraenoic acid (22:4(n-6)), 7,10,13,16,19-[14C]docosapentaenoic acid (22:5(n-3)), and 4,7,10,13,16,19-[14C]docosahexaenoic acid (22:6(n-3)), were compared with [3H]arachidonic acid (20:4(n-6] and [14C]linoleic acid (18:2(n-6)) to characterize their incorporation into the lipids of Ehrlich ascites cells. The relatively rapid incorporation of the labeled 22-carbon acids into phosphatidic acid indicated that substantial amounts of these acids may be incorporated through the de novo pathway of phospholipid synthesis. In marked contrast to 20:4(n-6), the 22-carbon acids were incorporated much less into choline glycerophospholipids (CGP) and inositol glycerophospholipids (IGP). No selective preference was apparent for the (n-3) or (n-6) type of fatty acids. The amounts of the acids incorporated into diacylglycerophosphoethanolamine were in the order of: 22:6(n-3) greater than 20:4(n-6) much greater than 22:5(n-3) greater than or equal to 22:4(n-6) greater than 18:2(n-6), whereas for alkylacylglycerophosphoethanolamine they were in the order of: 22:4(n-6) greater than 22:6(n-3) greater than 22:5(n-3) much greater than 20:4(n-6) greater than 18:2(n-6). Of the mechanisms possibly responsible for the selective entry of 22-carbon acids into ethanolamine glycerophospholipids, the most reasonable explanation was that the cytidine-mediated ethanolamine phosphotransferase may have a unique double selectivity: for hexaenoic species of diacylglycerol and for 22-carbon polyunsaturated fatty acid-containing species of alkylacylglycerol. The relative distribution of fatty acids between newly incorporated and already maintained lipid classes suggested that IGP may function in Ehrlich cells as an intermediate pool for the retention of polyunsaturated fatty acids in glycerolipids.     

Fatty acid composition of total lipids and phospholipids of membrane preparations of transport ATPases

The fatty acid composition of total lipids and phospholipids of duck salt gland Na,K-ATPase (outer plasma membrane) and of rabbit skeletal muscle Ca-ATPase (intracellular membrane) was investigated. The bulk of Na,K-ATPase fatty acids is represented by palmitic (16:0), oleic (18:1), stearic (18:0) and arachidonic (20:4) acids. The duck salt gland is characterized by rather a high content of unsaturated fatty acids, especially of arachidonic acid. The unsaturation index of total-lipid fatty acids increases during purification of these preparations in the following order: homogenate greater than microsomal fraction greater than purified enzyme. The fatty acid composition of Na,K-ATPase total lipids and phospholipids reveals certain differences. Phospholipids contain more stearic and liholeic (18:2) acids than total lipids, but the level of arachidonic acid in them is twice as low. Besides, phospholipids were found to contain polyunsaturated docosohexaenic (22:6) acid. The bulk of fatty acids of rabbit skeletal muscle Ca-ATPase total lipids and phospholipids is represented by 16:0, 18:0, 18:1 and 18:2 acids. The content of polyunsaturated fatty acids in this preparation is much lower than in duck salt gland Na,K-ATPase. The fatty acid composition of total lipids and phospholipids in rabbit skeletal muscle Ca-ATPase differ insignificantly. The differences in the fatty acid composition of membrane preparations under study is conditioned mainly by the fractional composition of their lipids.     

Evidence for peroxisomal retroconversion of adrenic acid (22:4(n-6)) and docosahexaenoic acids (22:6(n-3)) in isolated liver cells

The intracellular localization of the oxidation of [2-14C]adrenic acid (22:4(n-6)) and [1-14C]docosahexaenoic acid (22:6(n-3)) was studied in isolated liver cells. The oxidation of 22:4(n-6) was 2-3-times more rapid than the oxidation of 22:6(n-3), [1-14C]arachidonic acid (20:4(n-6)) or [1-14C]oleic acid (18:1). (+)-Decanoylcarnitine and lactate, both known to inhibit mitochondrial beta-oxidation, reduced the oxidation of 18:1 distinctly more efficiently than with 22:4(n-6) and 22:6(n-3). In liver cells from rats fed a diet containing partially hydrogenated fish oil, the oxidation of 22:6(n-6) and 22:6(n-3) was increased by 30-40% compared with cells from rats fed a standard pellet diet. With 18:1 as substrate, the amount of fatty acid oxidized was very similar in cells from animals fed standard pellets or partially hydrogenated fish oil. Shortened fatty acids were not produced from [5,6,8,9,11,12,14,15-3H]arachidonic acid. In hepatocytes from rats starved and refed 20% fructose, a large fraction of 14C from 22:4 was recovered in 14C-labelled C14-C18 fatty acids. Oxidation of 22:4 thus caused a high specific activity of the extramitochondrial pool of acetyl-CoA. The results suggest that 22:4(n-6) and to some extent 22:6(n-3) are oxidized by peroxisomal beta-oxidation and by this are retroconverted to arachidonic acid and eicosapentaenoic acid.     

Sex differences in n-3 and n-6 fatty acid metabolism in EFA-depleted rats

We studied the effect of sex on the distribution of long-chain n-3 and n-6 fatty acids in essential fatty acid-deficient rats fed gamma-linolenate (GLA) concentrate and/or eicosapentaenoate and docosahexaenoate-rich fish oil (FO). Male and female weanling rats were rendered essential fatty acid deficient by maintaining them on a fat-free semisynthetic diet for 8 weeks. Thereafter, animals of each sex were separated into three groups (n = 6) and given, for 2 consecutive days by gastric intubation, 4 g/kg body wt per day of GLA concentrate (containing 84% 18:2n-6), n-3 fatty acid-rich FO (containing 18% 20:5n-3 and 52% 22:6n-3), or an equal mixture of the two oil preparations (GLA + FO). The fatty acid distributions in plasma and liver lipids were then examined. GLA treatment increased the levels of C-20 and C-22 n-6 fatty acids in all lipid fractions indicating that GLA was rapidly metabolized. However, the increases in 20:3n-6 were less in females than those in males, while those in 20:4n-6 were greater, suggesting that the conversion of 20:3n-6 to 20:4n-6 was more active in female than in male rats. FO treatment increased the levels of 20:5n-3 and 22:6n-3 and reduced those of 20:4n-6. The increase in n-3 fatty acids was greater in females than that in males and the reduction in 20:4n-6 was smaller. Consequently, the sum of total long-chain EFAs incorporated was greater in females than that in males. The administration of n-3 fatty acids also reduced the ratio of 20:4n-6 to 20:3n-6 in GLA + FO-treated rats indicating that n-3 fatty acids inhibited the activity of delta-5-desaturase. However, this effect was not affected by the sex difference.     

In vivo conversion of 18- and 20-C essential fatty acids in rats using the multiple simultaneous stable isotope method

An important question for mammalian nutrition is the relative efficiency of C18 versus C20 essential fatty acids (EFAs) for supporting the tissue composition of n-3 and n-6 pathway end products. One specific question is whether C22 EFAs are made available to tissues more effectively by dietary alpha-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) or by dietary eicosapentaenoic acid (20:5n-3) and dihomo-gamma-linolenic acid (20:3n-6). To address this question in a direct manner, four stable isotope compounds were given simultaneously in a novel paradigm. A single oral dose of a mixture of 2H5-18:3n-3, 13C-U-20:5n-3, 13C-U-18:2n-6, and 2H5-20:3n-6 was administered to rats given a defined diet. There was a preferential in vivo conversion of arachidonic acid (20:4n-6) to docosatetraenoic acid (22:4n-6) and of 22:4n-6 to n-6 docosapentaenoic acid (22:5n-6) when the substrates originated from the C18 precursors. However, when the end products docosahexaenoic acid (22:6n-3) or 22:5n-6 were expressed as the total amount in the plasma compartment divided by the dosage, this parameter was 11-fold greater for 20:5n-3 than for 18:3n-3 and 14-fold greater for 20:3n-6 than for 18:2n-6. Thus, on a per dosage basis, the total amounts of n-3 and n-6 end products accreted in plasma were considerably greater for C20 EFA precursors relative to C18.     

Polyunsaturated fatty acid biosynthesis from [1-14C]20:3 n-6 acid in rat cultured Sertoli cells. Linoleic acid effect

Primary culture is a suitable system to study lipid metabolism and polyunsaturated fatty acid biosynthesis. Sertoli cell-enriched preparations were used to determine the fatty acid composition after 5 and 7 days in culture (serum free) as well as the uptake and metabolism of [1-14C]eicosa-8,11,14-trienoic acid. The addition of unlabeled linoleic acid (0.2 and 2.0 microg/ml) was also evaluated. Fatty acid methyl esters derived from cellular lipids were analyzed by gas liquid chromatography and radiochromatography. After 5 days in culture, cells had significantly less 18:2, 20:4, 22:5 and 24:5 and more 18:3, 20:3, 22:4 and 24:4 n-6 fatty acids than non-cultured cells. On day 7, an additional increment in 22:4 n-6 and a decrease in linoleic, gamma-linoleic and 24:4 n-6 fatty acids were observed. The presence of linoleic acid (low dose) produced a significant decrease in saturated and monounsaturated acids and an increase in 18:2, 20:4 and 22:5 n-6 fatty acids. At a high concentration almost all fatty acids belonging to 18:2 n-6 increased significantly. The drop in 20:4 n-6/20:3 n-6 ratio was considered as an indirect evidence of a Delta 5 desaturase activity depression. This assumption was corroborated by studying the transformation of [1-14C]eicosa-8,11,14-trienoic acid into 20:4, 22:4, 22:5, 24:4 and 24:5 n-6 fatty acids. We conclude that Sertoli cells after 7 days in culture evidenced changes in the fatty acid profile similar to those described under fat deprivation. The addition of linoleic acid reverted this pattern and indicated that the Delta 5 desaturase activity is a limiting step in the polyunsaturated fatty acid biosynthesis.     

Fatty acid utilisation and metabolism in caecal enterocytes of rainbow trout (Oncorhynchus mykiss) fed dietary fish or copepod oil

A combined fatty acid metabolism assay was employed to determine fatty acid uptake and relative utilisation in enterocytes isolated from the pyloric caeca of rainbow trout. In addition, the effect of a diet high in long-chain monoenoic fatty alcohols present as wax esters in oil derived from Calanus finmarchicus, compared to a standard fish oil diet, on caecal enterocyte fatty acid metabolism was investigated. The diets were fed for 8 weeks before caecal enterocytes from each dietary group were isolated and incubated with [1-14C]fatty acids: 16:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:1n-9, 20:4n-6, 20:5n-3, and 22:6n-3. Uptake was measured over 2 h with relative utilisation of different [1-14C]fatty acids calculated as a percentage of uptake. Differences in uptake were observed, with 18:1n-9 and 18:2n-6 showing the highest rates. Esterification into cellular lipids was highest with 16:0 and C18 fatty acids, accounting for over one-third of total uptake, through predominant incorporation in triacylglycerol (TAG). The overall utilisation of fatty acids in phospholipid synthesis was low, but highest with 16:0, the most prevalent fatty acid recovered in intracellular phosphatidylcholine (PC) and phosphatidylinositol (PI), although exported PC exhibited higher proportions of C20/C22 polyunsaturated fatty acids (PUFA). Other than 16:0, incorporation into PC and PI was highest with C20/C22 PUFA and 20:4n-6 respectively. Recovery of labelled 18:1n-9 in exported TAG was 3-fold greater than any other fatty acid which could be due to multiple esterification on the glycerol 'backbone' and/or increased export. Approximately 20-40% of fatty acids taken up were beta-oxidised, and was highest with 20:4n-6. Oxidation of 20:5n-3 and 22:6n-3 was also surprisingly high, although 22:6n-3 oxidation was mainly attributed to retroconversion to 20:5n-3. Metabolic modification of fatty acids by elongation-desaturation was generally low at <10% of [1-14C]fatty acid uptake. Dietary copepod oil had generally little effect on fatty acid metabolism in enterocytes, although it stimulated the elongation and desaturation of 16:0 and elongation of 18:1n-9, with radioactivity recovered in longer n-9 monoenes. The monoenoic fatty acid, 20:1n-9, abundant in copepod oil as the homologous alcohol, was poorly utilised with 80% of uptake remaining unesterified in the enterocyte. However, the fatty acid composition of pyloric caeca was not influenced by dietary copepod oil.     

Sex differences in the metabolism of essential fatty acids studied in isolated rat liver cells

The triacylglycerol synthesis from exogenous linoleic acid (18:2(n-6], linolenic acid (18:3(n-3], dihomogammalinolenic acid (20:3(n-6], eicosapentaenoic acid (20:5(n-3] and oleic acid (18:1(n-9] was observed to be significantly increased in isolated liver cells from female rats compared with males. The rate of fatty acid oxidation and phospholipid biosynthesis was concomitantly more important in male cells. With the C22-polyenoic fatty acids, adrenic acid (22:4(n-6] and docosahexaenoic acid (22:6(n-3), only a minor sex-related difference in fatty acid metabolism was found.     

Metabolism of eicosapentaenoic acid by aorta: formation of a novel 13-hydroxylated prostaglandin

We have investigated the metabolism by fetal calf aorta of eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6), two polyunsaturated fatty acids found in high concentrations in marine oils. The major product formed from 20:5 by particulate fractions from fetal calf aorta is delta 17-6-oxoprostaglandin F1 alpha. In addition, we detected a novel isomer of delta 17-6-oxoprostaglandin F1 alpha, in which a hydroxyl group is present in the 13-position instead of the 15-position. Eicosapentaenoic acid is also converted to 12-hydroxy-5,8,10,14-heptadecatetraenoic acid as well as to five monohydroxy isomers with hydroxyl groups present in the 11, 12, 14, 15, and 18 positions. Although 20:5 was metabolized at about one-third the rate of arachidonic acid (20:4), greater amounts of monohydroxy fatty acids, the major one being the 11-hydroxy metabolite, were formed from 20:5. Unlike 20:5, 22:6 was not metabolized to any detectable products by fetal calf aorta, but both of these polyunsaturated fatty acids inhibited the oxygenation of 20:4 by cyclooxygenase from aorta with IC50 values of 4.1 microM (22:6) and 15 microM (20:5). These results suggest that 20:5 has a high affinity for cyclooxygenase, but that the intermediate 11-oxygenated intermediate has a lower affinity than the corresponding intermediate from 20:4, resulting in a greater loss of substrate after a single oxygenation. The formation of oxygenation products from both 20:4 and 20:5 was inhibited by 13-hydroperoxy-9,11-octadecadienoic acid (13hp-18:2). The IC50 values for inhibition of cyclooxygenase products by 13hp-18:2 were about twice as high as those for inhibition of prostacyclin synthase products. Consequently, there was little diversion of prostaglandin endoperoxides to other prostaglandins in the presence of 13hp-18:2.     

Role of triglycerides in endothelial cell arachidonic acid metabolism

Arachidonic acid was incorporated into triglycerides by cultured bovine endothelial cells in a time- and concentration-dependent manner. At 75 microM or higher, more arachidonic acid was incorporated into triglycerides than into phospholipids. The triglyceride content of the cells increased as much as 5.5-fold, cytoplasmic inclusions appeared, and arachidonic acid comprised 22% of the triglyceride fatty acids. Triglyceride turnover occurred during subsequent maintenance culture; there was a 60% decrease in the radioactive arachidonic acid contained in triglycerides and a 40% decrease in triglyceride content in 6 hr. Most of the radioactivity was released into the medium as free fatty acid. The turnover of arachidonic acid, but not oleic acid in cellular triglycerides, decreased when supplemental fatty acid was added to the maintenance medium. Incorporation and turnover of radioactive arachidonic acid in triglycerides also was observed in human skin fibroblasts, 3T3-L1 cells, and MDCK cells. Other fatty acids were incorporated into triglycerides by the endothelial cells; the amounts after a 16-hr incubation with 50 microM fatty acid were 20:3 greater than 20:4 greater than 18:1 greater than 18:2 greater than 22:6 greater than 16:0 greater than 20:5. These findings indicate that triglyceride formation and turnover can play a role in the fatty acid metabolism of endothelial cells and that arachidonic acid can be stored in endothelial cell triglycerides.     

Positional Distribution of Acyl and Alk-1-enyl Groups in Grey and White Matter Ethanolamine and Choline Phosphoglycerides of a Marsupial, the Koala (Phascolarctos cinereus)

The major phosphoglycerides in grey and white matter from the brain of the koala have been separated and examined. The major polyunsaturated fatty acids present in both the diacyl- and alk-1-enyl acylglycerophosphorylethanolamines from grey matter were 22:6 omega 3, 20:4 omega 6, and 22:4 omega 6. In both grey and white matter, 22:6 omega 3 and 20:4 omega 6 were concentrated in the 2-position of diacylglycerophosphorylethanolamines and 22:4 omega 6 in the 2-position of alk-1-enylacylglycerophosphorylethanolamines; polyunsaturated fatty acid levels were higher in diacylglycerophosphorylethanolamines. Ethanolamine phosphoglyceride fractions from grey matter were enriched in polyunsaturated fatty acids compared with those from white matter. The acyl groups 18:0, 18:1, and 16:0 and their alk-1-enyl analogues were prominent in grey and white matter ethanolamine phosphoglycerides; 18:1 was dominant in white matter alk-1-enylacylglycerophosphorylethanolamines. The plasmalogen composition of ethanolamine phosphoglycerides was 55% in grey matter and 76% in white matter. Choline phosphoglycerides contained negligible plasmalogen and low polyunsaturated fatty acid levels. Diacylglycerophosphorylcholine was characterized by high levels of 16:0 and 18:1. Similar acyl group distributions were estimated in the 1-position in both grey and white matter, 16:0 being present at greater than 50%. The presence of the molecular species 18:0/22:6 omega 3 was indicated in grey matter diacylglycerophosphorylethanolamine, 18:1/18:1 in white matter alk-1-enylcylglycerophosphorylethanolamine, and 16:0/18:1 in white matter diacylglycerophosphorylcholine.     

Effects of clofibrate feeding on essential fatty acid desaturation and oxidation in isolated rat liver cells.

The effects of clofibrate feeding on the metabolism of polyunsaturated fatty acids were studied in isolated rat hepatocytes. Administration of clofibrate stimulated the oxidation and particularly the peroxisomal beta-oxidation of all the fatty acids used. The increase in oxidation products was markedly higher when n-3 fatty acids were used as substrate, indicating that peroxisomes contribute more to the oxidation of n-3 than n-6 fatty acids. The whole increase in oxidation could be accounted for by a corresponding decrease in acylation in triacylglycerol while the esterification in phospholipids remained unchanged. A marked stimulation of the amounts of newly synthesized C16 and C18 fatty acids recovered, was observed when 18:2(n-6), 20:3(n-6), 18:3 (n-3) and 20:5(n-3), but not when 20:4(n-6) and 22:4(n-6) were used as substrate. This agrees with the view that extra-mitochondrial acetyl-CoA produced from peroxisomal beta-oxidation is more easily used for fatty acid new synthesis than acetyl-CoA from mitochondrial beta-oxidation. The delta 6 and delta 5 desaturase activities were distinctly higher in cells from clofibrate fed rats indicating a stimulating effect.     

Fatty acid composition of muscle and heart tissue of Nile perch,Lates niloticus,and Nile tilapia,Oreochromis niloticus,from various populations in Lakes Victoria and Kioga,Uganda

The fatty acid composition in the heart tissue and muscle tissue of the Nile perch, Lates niloticus, and Nile tilapia, Oreochromis niloticus populations from Lakes Kioga and Victoria was determined by methanolysis and gas chromatography of the resulting fatty acid methyl esters. The analytical data were treated by multivariate principal component analysis. The most abundant individual fatty acids were palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1n9), vaccenic acid (18:1n7), arachidonic acid (20:4n6) and docosahexaenoic acid (22:6n3). Due to high levels of both n6 and n3 fatty acids, the ratios of n3 to n6 were between 1 and 2, typical for freshwater fish species. Two Lake Victoria and one Lake Kioga populations of Nile tilapia and Nile perch were distinguished by the fatty acid profiles in their heart and muscle tissue. The heart tissue showed better separation than muscle tissue, due to dominance of polar phospholipids. It is rationalised that genetics are more important than diet in determining the fatty acid composition of the tissues.     

Linolenic acid desaturation and chain elongation and rapid turnover of phospholipid n - 3 fatty acids in isolated rat liver cells

The desaturation and chain elongation of [1-14C]linolenic acid was studied in isolated liver cells from rats fed a diet deficient in essential fatty acids. 14C-labelled 18:4, 20:3, 20:4, 20:5, 22:5 and 22:6, all n - 3 fatty acids, were formed. In the presence of lactate relatively large amounts of 20:5, 22:5 and 22:6 were formed. 20:5 was mainly present in phospholipids, 22:5 and 22:6 were present in both phospholipids and triacylglycerols. (+)-Decanoylcarnitine and (-)-hydroxycitrate decreased the formation of 20:5, 22:5 and 22:6 and increased the recovery of 18:4. The unchanged 18:3 substrate was also initially rapidly incorporated both in the phospholipids and in the triacylglycerol fraction. During long incubation periods, continued after nearly all the [14C]linolenic acid substrate had been metabolized either by esterification or by oxidation, the phospholipid content of labelled 18:3 and 18:4 decreased while the content of 20:5, 22:5 and 22:6 increased markedly, suggesting a remodeling of the phospholipid n - 3 fatty acid content by a series of deacylations-reacylations. The n - 3 fatty acid pattern in the triacylglycerol fraction changed little. 22:5 and 22:6 appeared in the VLDL fraction secreted by the isolated liver cells.     

Altered acyl chain compositions of alkylacyl, alkenylacyl, and diacyl subclasses of choline and ethanolamine glycerophospholipids in rat heart by dietary fish oil

The effects of dietary fish oil containing n - 3 polyunsaturated fatty acids on the fatty acid compositions of the alkylacyl and alkenylacyl species of choline glycerophospholipids (CGP) and ethanolamine glycerophospholipids (EGP) were studied in rat heart and compared with the corresponding diacylglycerophospholipids. After a 7 week feeding period, all phospholipid classes from the fish oil group exhibited much higher levels of the n - 3 polyunsaturated fatty acids including eicosapentaenoic acid (20:5(n - 30)), docosapentaenoic acid (22:5(n - 3)) and docosahexaenoic acid (22:6(n - 3)), as well as lower levels of the n - 6 series (18:2, 20:4, 22:4 and 22:5), relative to animals given sunflower seed oil-enriched in 18:2(n - 6). However, the docosahexaenoic acid rather than eicosapentaenoic acid provided a much greater contribution to the n - 3 accumulation (fish oil group) in the ether-containing CGP, as indicated by the 20:5(n - 3)/22:6(n - 3) molar ratios of 0.32, 0.26 and 0.56 in the alkylacyl, alkenylacyl and diacyl classes, respectively. In addition to accumulating very high levels of docosahexaenoic acid (e.g., 47.2 mol% of fatty acids in alkenylacylglycerophosphoethanolamine of fish oil group), both ether-linked classes of EGP exhibited significantly higher levels of docosapentaenoic acid than the diacylglycerophosphoethanolamine (GPE) and all classes of CGP. These findings may bear relevance to possible beneficial effects of dietary fish oil on pathophysiological states (including myocardial ischemia) in cardiac tissue and their mediation via platelet-activating factor, 1-alkyl-2-acetylglycerophosphocholine (PAF) and arachidonic acid (20:4(n - 6))-derived eicosanoids.     

Lipids of Leishmania promastigotes.

A chromatographic analysis of lipids of cultured promastigotes of Leishmania donovani, L. braziliensis, L. mexicana, L. tropica, L. enriettii, L. hertigi, L. adleri, and L. tarentolae showed that total lipids were 2--15% of dry wt, and neutral and polar lipids were 14--55% and 45--86% of total lipids. Major lipid classes were as follows: sterol ester, triacylglycerol, sterol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine. Predominant fatty acids were 18:2 (n - 6) greater than 18:3 (n - 3) greater than 18:1 (n - 9) greater than 18:0 greater than 22:6 (n - 3) greater than 22:5 (n - 6) greater than 16:0 greater than 14:0 greater than 18:4 (n - 3) greater than 20:3 (n - 3). Some remarkable distributions of fatty acids among the phospholipid fractions were observed, as follows: diphosphatidylglycerol 18--33% 22:6 (n - 3); phosphatidylinositol 31--68% 18:1 (n - 9); phosphatidylcholine 13--41% 18:3 (n - 3). Alk-l-enyldiacyl glycerols, and alk-l-enylacyl and alkylacyl forms of phosphatidylethanolamine and of phosphatidylinositol were found, and their glyceryl ethers and fatty adehydes analyzed. Notable in the phosphatidylethanolamine of some leishmanias was a cyclopropane fatty acid (4--11%), identified as cis-9,10-methylene octadecanoic acid by chromatographic, and by mass and proton resonance spectrometric analyses. The comparative biochemistry of the cyclopropane fatty acid, characteristic of many prokaryotes, and of alpha-linolenic acid, characteristic of photosynthetic plants, are commented upon.     

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.     

Protein kinase C activation in mixed micelles. Mechanistic implications of phospholipid, diacylglycerol, and calcium interdependencies

The phospholipid, sn-1,2-diacylglycerol, and calcium dependencies of rat brain protein kinase C were investigated with a mixed micellar assay (Hannun, Y., Loomis, C., and Bell, R.M. (1985) J. Biol. Chem. 260, 10039-10043). Protein kinase C activity was independent of the number of Triton X-100, phosphatidylserine (PS), and sn-1,2-dioleoylglycerol (diC18:1) mixed micelles. Activation was strongly dependent on the mole per cent of PS and diC18:1. Activity of protein kinase C was dependent on PS, diC18:1, and calcium in mixed micelles prepared from detergents other than Triton X-100. This is consistent with the micelle providing an inert surface into which the lipid cofactors partition. Molecular sieve chromatography provided direct evidence for the homogeneity of Triton X-100, PS, and diC18:1 mixed micelles. Mixing studies and surface dilution studies indicated that PS and diC18:1 rapidly equilibrate among the mixed micelles. At saturating calcium, the diC18:1 dependence was strongly dependent on the mole per cent PS present. At 10 mol % PS, 0.25 mol % diC18:1 gave maximal activity whereas 6 mol % PS and 6 mol % diC18:1 did not give maximal activity. diC18:1 dependencies were hyperbolic at all PS levels tested. The data support the conclusion that a single molecule of diC18:1/micelle is sufficient to activate monomeric protein kinase C. The mole per cent PS required for maximal activation was reduced markedly as the mole per cent diC18:1 increased. Under all conditions tested, the PS dependence of protein kinase C activation lagged until greater than 3 mol % PS was present. Then activation occurred in a cooperative manner with Hill numbers near 4. These data indicate that 4 or more molecules of PS are required to activate monomeric protein kinase C. PS was the most effective of all the phospholipids tested in the mixed micelle assay. diC18:1 was found to modulate the amount of calcium required for maximal activity. As the level of Ca2+ increased, the mole per cent PS required reached a limiting value of 3 mol %. A number of sn-1,2-diacylglycerols containing short chain fatty acids activated protein kinase C in a saturable manner in mixed micelles. The data are discussed in relation to a model for protein kinase activation.     

The levels of essential unsaturated fatty acids in human milk on the 3rd, 4th, 5th, and 6th days after labour

The composition of fatty acids in human milk lipids was determined in 41 women on the 3rd, 4th, 5th and 6th days after labour by the method of gas chromatography. In these investigations no significant differences were demonstrated in the fatty acids in the lipid fractions between these consecutive days. The level of polyunsaturated fatty acids of the n-6 and n-3 groups was about 11.9-13.6%, including linoleic acid (18:2, n-6) about 7.7-9.8%, and alpha-linolenic acid (18:3, n-3) about 0.7-1%. In the analysis group of n-6 fatty acids the determined acids were: linoleic acid (18:2, n-6), gamma-linolenic acid (18:3, n-6), eicosadienoic acid (20:2, n-6), eicosatrienoic acid (20:3, n-6), arachidonic acid (20:4, n-6), docosahexaenoic acid (22:6, n-6). From the group of n-3 acids the identified ones were: alpha-linolenic acid (18:3, n-3), eicosapentaenoic acid (20:5, n-3), docosapentaenoic acid (22:5, n-3) and docosahexaenoic acid (22:6, n-3). The obtained quotients of fatty acids n-6 through n-3 on the consecutive days were: 7.2:1-7.8:1, indicating a too low level of the n-3 acids in the investigated milk. The acids prevailing in human milk lipids were: oleic (18:1, n-9) and palmitic (16:0) which accounted for 37-39% and 25-26% respectively. The polyunsaturated to saturated fatty acid ratio (P:S) ranged from 0.28 to 0.33.