Metabolism of saturated fatty acids by Paramecium tetraurelia

Paramecium requires oleate for growth. The phospholipids of the ciliate contain high concentrations of palmitate and 18- and 20-carbon unsaturated fatty acids. We previously showed that radiolabeled oleate is desaturated and elongated to provide these 18- and 20-carbon unsaturated acids. We now report on saturated fatty acid (SFA) metabolism in Paramecium. Radiolabeled palmitate and stearate were incorporated directly into cellular phospholipids with little or no desaturation and/or elongation. Radiolabeled acetate, malonate, pyruvate, citrate, or glucose added to cultures were not incorporated into cellular phospholipid fatty acids indicating that these exogenously supplied putative precursors were not utilized for fatty acid synthesis by Paramecium. Radiolabel from octanoate or hexanoate appeared in fatty acyl groups of phospholipids, possibly by partial beta-oxidation and reincorporation of the label. Under oleate-free conditions in which cultures do not grow, radiolabel from these shorter chain SFA were beta-oxidized and preferentially used for the formation of arachidonate, the major end-product of fatty acid synthesis in Paramecium. Cerulenin inhibited culture growth apparently by inhibiting de novo fatty acid synthesis. Cerulenin-treated cells did not incorporate radioactivity from [1-14C]octanoate into esterified palmitate. However, total saponifiable phospholipid fatty acids, including SFA, per cell increased under these conditions.     

Metabolism of polyunsaturated fatty acids by rabbit reticulocytes

Rabbit reticulocytes obtained by repeated bleeding metabolize exogenous [1-14C]linoleic acid and [1-14C]arachidonic acid by three different pathways. 1. Incorporation into cellular lipids: 50% of the fatty acids metabolized are incorporated into phospholipids, mainly phosphatidylcholine (32.8%) but also into phosphatidylethanolamine (12%), whereas about 10% of the radioactivity was found in the neutral lipids (mono- di- and triacylglycerols, but not cholesterol esters). 2. Formation of lipoxygenase products: 30% of the fatty acids metabolized are converted via the lipoxygenase pathway mainly to hydroxy fatty acids. Their formation is strongly inhibited by lipoxygenase inhibitors such as 5,8,11,14-eicosatetraynoic acid or nordihydroguaiaretic acid. Inhibition of the lipoxygenase pathway results in an increase of the incorporation of the fatty acids into cellular lipids. 15-Hydroxy-5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid and 13-hydroxy-9,11(Z,E)-octadecadienoic acid are incorporated by reticulocytes into cellular lipids and also are metabolized via beta-oxidation. The metabolism of arachidonic acid and linoleic acid is very similar except for a higher incorporation of linoleic acid into neutral lipids. 3. beta-Oxidation of the exogenous fatty acids: about 10% of the polyenoic fatty acids are metabolized via beta-oxidation to 14CO2. Addition of 5,8,11,14-eicosatetraynoic acid strongly increased the 14CO2 formation from the polyenoic fatty acids whereas antimycin A completely abolished beta-oxidation. Erythrocytes show very little incorporation of unsaturated fatty acids into phospholipids and neutral lipids. Without addition of calcium and ionophore A23187 lipoxygenase metabolites could not be detected.     

Metabolism of phospholipids and the characterization of fatty acids in bovine corpus luteum

1. Phosphatidylcholine was the predominant phospholipid in bovine corpora lutea; it accounted for about 50% of the total phospholipid phosphorus. Phosphatidylethanolamine (13%) and ethanolamine plasmalogen (8-9%) were the next two major components. 2. After incubation of the tissue with [(32)P]orthophosphate the total radioactivity and specific radioactivity of phosphatidylinositol were higher than those of any other lipid. 3. Luteinizing hormone failed to increase significantly the incorporation of [(32)P]orthophosphate into total phospholipids from luteal tissue slices, but did stimulate progesterone synthesis and lactate production. 4. The proportion of oleate (18:1) in the neutral lipids and phospholipids was higher than that of any other fatty acid. 5. The proportion of unsaturated fatty acid in the tissue lipids exceeded 60%, and almost half of this was polyunsaturated. Arachidonate (20:4), docosatetraenoate (22:4) and docosapentaenoate (22:5) were the principal polyunsaturated fatty acids. 6. After incubation of luteal tissue with [1-(14)C]acetate, the greatest proportion of radioactivity in the fatty acids isolated from the total lipid fraction was in palmitate (16:0) and docosatetraenoate (22:4). Polyunsaturated fatty acids accounted for almost 50% of the (14)C radioactivity incorporated and this pattern was observed in phospholipids, triglycerides and free fatty acids.     

Metabolism of exogenous long-chain fatty acids by spinach leaves

When applied in liquid paraffin to the upper surface of expanding spinach leaves, [1-14C]palmitic acid was efficiently and exclusively incorporated into the sn-1 position of cellular glycerolipids, principally phosphatidylcholine and triacylglycerol. A slow transfer of fatty acids from phosphatidylcholine to chloroplast glycolipids subsequently occurred with the positional specificity of the label remaining intact. Labeled palmitate at the sn-1 position of monogalactosyldiacylglycerol was desaturated to hexadecatrienoate so that 1-[14C]hexadecatrienoyl-2-linolenoyl-3-galactosoylglycerol became the major labeled species of the lipid between 8 and 24 h. There was no evidence of deacylation/reacylation reactions modifying the acyl compositions of spinach leaf glycerolipids for at least 48 h after labeling with [1-14C]palmitic acid; even the partially prokaryotic glycerolipids remained firmly labeled at the sn-1 position. Exogenous [1-14C]stearic acid was also incorporated into the sn-1 position of MGD, presumably by the same mechanism, and was there desaturated to [14C]linolenate. Exogenous [1-14C]oleic acid was initially incorporated equally into both sn-1 and sn-2 positions of phosphatidylcholine, and was desaturated to linoleate at both positions before the label was rapidly transferred to monogalactosyldiacylglycerol. There, desaturation of linoleate to linolenate took place. Galactolipids remained equally labeled at both positions throughout the 6 days of the experiment, but label was concentrated in the 1-saturated-2-[14C]linolenoyl molecular species of phosphatidylcholine as those species with two [14C]linoleoyl residues were drawn off for monogalactolipid synthesis. Glycerolipids synthesised from exogenous [1-14C]acetate by spinach leaves were labeled equally at both the sn-1 and the sn-2 positions. These results are interpreted as providing strong support for the two-pathway scheme of glycerolipid synthesis in plants.     

The phospholipid-bound fatty acids of fowl and turkey spermatozoa

The composition of the phospholipid-bound fatty acids in the spermatozoa of the turkey, Meleagris gallopavo, and fowl, Gallus domesticus, was studied. The types of fatty acids were similar in the two birds. The ratio of polyunsaturated : saturated fatty acids was generally low but slightly higher in the turkey than in the fowl. The significance of the findings in relation to the origin of the semen collected in these gallinaceous birds and the greater difficulty of freezing turkey spermatozoa was discussed.     

Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa

Intact human sperm incorporated radiolabelled fatty acids into membrane phospholipids when incubated in medium containing bovine serum albumin as a fatty acid carrier. The polyunsturated fatty acids were preferentially incorporated into the plasmalogen fraction of phospholipid. Uptake was linear with time over 2 hr; at this time sufficient label was available to determine the loss of fatty acids under conditions of spontaneous lipid peroxidation. Loss of the various phospholipid types, the loss of the various fatty acids from these phospholipids, and the overall loss of fatty acids were all first order. The loss of saturated fatty acids was slow with first order rate constant k₁ = 0.003 hr^?1; for the polyunsaturated fatty acids, arachidonic and docosahexaenoic acids, k₁ = 0.145 and 0.162 hr^?1, respectively. The rate of loss of fatty acids from the various phospholipid types was dependent on the type, with loss from phosphatidylethanolamine being the most rapid. Among the phospholipid types, phosphatidylethanolamine was lost at the greatest rate. Analysis of fatty acid loss through oxidation products was determined for radiolabelled arachidonic acid. Under conditions of spontaneous lipid peroxidation at 37°C under air in the absence of albumin, free arachidonic acid was found in the medium, along with minor amounts of hydroxylated derivative. All the hydroperoxy fatty acid remained in the cells. In the presence of albumin, all the hydroperoxy fatty acid was found in the supernatant bound to albumin; none could be detected in the cells. Albumin is known as a very potent inhibitor of lipid peroxidation in sperm; its action may be explained, based on these results, as binding the damaging hydroperoxy fatty acids. These results also indicate that a phospholipase A₂ may act in peroxidative defense by excising a hydroperoxy acyl group from phospholipid and providing the hydroperoxy fatty acid product as substrate to glutathione peroxidase. This formulation targets hydroperoxy fatty acid as a key intermediate in peroxidative degradation. © 1995 wiley-Liss, Inc.     

Metabolism of branched-chain fatty acids in Tetrahymena pyriformis W

Cultures of Tetrahymena pyriformis W were supplemented with the branched short-chain acids, isobutyrate and α-methyl-n-butyrate. Growth inhibition occurred which was directly related to the concentration of the supplement. Growth of the cells with isobutyrate resulted in an enhancement in the levels of even iso and normal fatty acids, a decrease in odd iso fatty acids, and an elevation in fatty acids less than 18 carbons in chain length. The data indicate a reduction in overall cellular branched-chain synthesis. Polyunsaturated, long-chain iso acids were not detected.Addition of α-methyl-n-butyrate led to minimal incorporation (2%) of saturated long-chain anteiso fatty acids and to a marked increase in odd normal acids at the expense of odd iso and even normal acids in the glycerophospholipids and in neutral lipids. An elevation of odd normal α-hydroxy acids in the sphingolipids was observed. The metabolism of α-methyl-n-butyrate to propionyl-CoA which serves as a primer for odd normal acids would account for the observed changes. Unsaturated anteiso components were not detected. Enhancement of the amount of anteiso acids occurred when the cells were grown at low temperature with α-methyl-n-butyrate.Long-chain anteiso acids (C₁₅, C₁₇ and C₁₉) were incorporated extensively into the glycerophospholipids at the expense of odd iso, odd normal and even normal acids. Anteiso unsaturates were not detected. Elongation of 15 : 0(ai) and 17 : 0(ai) occurred. Retroconversion of 17 : 0(af) and 19 : 0(ai) was observed. Growth with 17 : 0(ai) and 19 : 0(ai) but not 15 : 0(ai) led to the appearance of 19 : 0(ai, α-OH) in the sphingolipid fraction. The fact that addition of these long-chain anteiso saturates led to an enhanced incorporation into the glycerophospholipids compared to supplementation with α-methyl-n-butyrate indicates that a defect in the synthesis of long-chain acids occurs and that acyltransferase activity is not limiting in anteiso acid incorporation.A proposal is made to account for the low levels of unsaturated even iso acids and the lack of unsaturated anteiso acids based on Δ⁹ desaturase specificity toward carbon chain length and the position of the methyl substituent in the fatty acid.     

Metabolism of polyunsaturated fatty acids by larvae of the waxmoth,Galleria mellonella

The metabolic fates of linoleic (18:2n6) and linolenic (18:3n3) acids injected into the hemocoel of fifth instar larvae of the waxmoth, Galleria mellonella, were examined by radio-high-pressure liquid chromatography and radio-gas-liquid chromatography. In addition to undergoing β-oxidation and incorporation into neutral and phospholipid fractions, a portion of both of these C18 fatty acids was elongated and desaturated to longer chain and more unsaturated polyenoics. Radioactivity from linoleic acid was recovered in components that coeluted with 18:3, 18:4, 20:3, and 20:4. Radioactivity from linolenic acid was recovered in an unidentified component and in components that coeluted with 18:4, 20:3, and 20:5. Labeled arachidonic and eicosapentaenoic acids injected into waxmoth larvae were converted to prostaglandins, suggesting that one aspect of the biological significance of the elongation/desaturation reactions is to generate precursors for prostaglandin biosynthesis.     

Binding of long-chain fatty acids to bovine serum albumin

We have studied the binding of long-chain free fatty acids (FFA) to crystalline bovine serum albumin (BSA) that had been extracted with charcoal to remove endogenous fatty acids. The data were analyzed in terms of a model consisting of six high-energy binding sites and a large number of weak binding sites. The high-energy sites were resolved into two distinct classes, each containing three sites. At 37 degrees C and pH 7.4, k'(1) (the apparent association constant of a class of binding sites) was about 10(6) m(-1) for binding to the three primary sites, and k'(2) was about 10(5) m(-1) for binding to the three secondary sites. The number of weak (tertiary) sites was estimated to be 63 with a k'(3) of 10(3) m(-1). In general, palmitate and palmitoleate were bound more tightly than oleate, linoleate, stearate, or myristate, and much more tightly than laurate. The association of palmitate with human and rabbit albumin also was analyzed in terms of this model. Palmitate was bound less firmly by human or rabbit albumin than by BSA. Palmitate binding to BSA was dependent upon the pH and temperature of the incubation medium. Long-chain hydrocarbons that did not contain a free carboxyl group (methyl palmitate, cetyl alcohol, and hexadecane) were bound to a limited extent and weakly. The presence of positively charged protein sites and native protein tertiary structure were required for maximal binding of palmitate to BSA. Of nine other proteins tested, only -lactoglobulin exhibited a significant capacity to bind palmitate.