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Enhancement of thrombin-induced degradation of phosphatidylcholine in reserpinized rabbit platelets.

When 1-(14)C-arachidonic acid-labeled, washed platelets from the reserpinized rabbits were exposed to thrombin, the decrease in radioactivity of phospholipids was significantly stimulated as compared with the control platelets. The increase in radioactivity of fatty acids and their oxygenated metabolites was also stimulated. TLC analysis revealed that the stimulation of the decrease in radioactivity of phospholipids was almost exclusively attributed to that of phosphatidylcholine (PC), and that thrombin induced a slight but significant increase in radioactivity of phosphatidylethanolamine (PE) in the reserpinized platelets. The results suggest that thrombin-induced degradation of PC is enhanced in the reserpinized platelets, and the overproduced fatty acids would be metabolized to the larger amount of oxygenated products which result in the activation of the platelets. Thrombin-induced mobilization of PC to PE in the reserpinized platelet phospholipids was also suggested.

Animals↗

Isolation and partial characterization of a cerulenin-sensitive mutant of Pseudomonas aeruginosa.

Sensitivity of Pseudomonas aeruginosa to cerulenin was first tested. The result indicated that this bacterium is resistant to cerulenin. Cerulenin-sensitive mutants were isolated from P. aeruginosa PML 1552 by 1-methyl-3-nitro-1-nitrosoguanidine treatment and following carbenicillin plus D-cycloserine screening. Isolated mutants were designated CSM-1 to CSM-19, and some characters of CSM-19, which showed rapid growth almost as well as parent strain in the medium without cerulenin, were examined. The cell growth of CSM-19 was greatly inhibited by 50 micrograms/ml of cerulenin, but when the mixture of cellular fatty acids or both cis-vaccenic acid and palmitic acid were added to the medium, the growth was partially recovered. Incorporation of radioactivity into fatty acids from [1-14C]acetate was lowered by cerulenin. Those results mean that the fatty acid synthesis of CSM-19 was decreased by cerulenin. Although cellular fatty acid composition and amount were not notably different between CSM-19 and PML 1552, CSM-19 had less phosphatidylethanolamine, and more phosphatidylglycerol and cardiolipin than PML 1552. CSM-19 was also supersensitive to several other antibiotics, especially to carbenicillin and tetracycline, when compared with PML 1552, although both strains showed identical sensitivity to D-cycloserine, polymyxin B, and chloramphenicol.

Anti-Bacterial Agents↗

Regulation of fatty acid biosynthesis in Ehrlich cells by ascites tumor plasma lipoproteins.

Fatty acid biosynthesis in Ehrlich cells in vitro was reduced when very low density lipoproteins (VLDL) isolated from the ascites tumor plasma were added to the incubation medium. The degree of inhibition was dependent on the VLDL concentration. At the VLDL concentrations usually present in the ascites plasma, there was a 30% decrease in biosynthesis as measured by (3)H(2)O incorporation into fatty acids. Analysis of the labeled fatty acids by gas liquid chromatography indicated that this decrease was due to a reduction in fatty acid de novo biosynthesis and that chain elongation actually was increased when VLDL were present. Although ascites plasma low- and high density lipoproteins also produced a concentration-dependent inhibition of fatty acid biosynthesis, their effects were much smaller than those of the VLDL. Studies employing VLDL and radioactive free fatty acids indicated that the cells took up utlilzed fatty acids derived from these lipoproteins. When VLDL were present, labeled free fatty acid incorporation into cell phospholipids, cholesteryl esters, and CO(2) decreased, whereas its incorporation into the cell free fatty acid pool increased. By contrast, the cells incorporated only very small amounts of fatty acid from either low- or high density lipoproteins. This suggests that the VLDL exert their inhibitory effect on fatty acid synthesis by supplying exogenous fatty acids to the cells.

Animals↗

Positional specificity of trans fatty acids in fetal lecithin.

Differences in the positional incorporation of 9-trans[1-(14)C] octadecenoic (elaidic) and 9-trans,12-trans[1-(14)C] octadecadienoic (linoelaidic) acids in fetal lecithin of rats were demonstrated. On the 20th day of gestation, a 14C-labeled albumin complex of elaidic or linoelaidic acid was injected into the jugular vein of pregnant rats. For comparative purposes, 9-cis[1-(14)C] octadecenoic (oleic) or 9-cis,12-cis[1-(14)C] octadecadienoic (linoleic acid) was injected into the maternal circulation of rats. Animals were killed 6 hr later. Distribution of label in total lipids and phospholipids (PL) of fetal issue was measured by TLC. Irrespective of the label, the highest percentage of total radioactivity was associated with PL-59 to 67%. Within PL, the major portion of radioactivity was found in choline phosphoglycerides (CPG)-53 to 67%, and in ethanolamine phosphoglycerides (EPG)-18 to 33%. While linoelaidic acid was predominantly esterified in the 2-position of CPG, elaidic acid was nearly equally distributed between positions 1 and 2 of lecithin. Distribution of radioactivity within fatty acid methyl esters (FAME) of CPG measured by radio-GLC suggested that oleic and possibly linoleic acids may be converted to nervonic and arachidonic acid, respectively, in the rat by the 20th day of gestation. Following injection of elaidate, radioacivity of FAME was distributed between palmitate and elaidic acid indicating that rat fetal tissue may metabolize elaidic acid via beta-oxidation. In contrast, following injection of linoelaidate, radioactivity of FAME was primarily associated with tt-18:2, suggesting little biotransformation to other fatty acids by fetal tissues.

Animals↗

Biologically active ether lipids: incorporation of long-chain precursors into 1(3),2-diacylglycero-3(1)-O-4'-(N,N,N-trimethyl)homoserines and other lipids of Chlorella fusca.

The lipids of Chlorella fusca are composed of the ester lipids typical of photosynthetically active cells. In addition, there occurs a class of less common ether lipids, the biologically active 1(3),2-diacylglycero-3(1)-O-4'-(N,N,N- trimethyl)homoserines, at a level of about 1.3% of total lipids. The acyl moieties of the total lipids include saturated as well as mono-, di- and tri-unsaturated species with chain lengths of 16 and 18 carbon atoms, the major constituents being palmitic and oleic acids. In both the diacylglycerophosphocholines, i.e., the major class of ester phospholipids, and the diacylglycero-4'-O-(N,N,N-trimethyl)homoserines palmitic acid is located predominantly at position 1 of the glycerol backbone, whereas oleic acid is almost equally distributed between positions 1 and 2; palmitoleic and polyunsaturated fatty acids are esterified preferentially at position 2. Incubation of C. fusca cultures with 14C-labeled fatty acids leads to their rapid incorporation into various lipid classes. Oleic and palmitic acids are incorporated at a faster rate than stearic acid (18:1 greater than 16:0 much greater than 18:0). 1,2- and 1,3-Diacylglycerols are the most prominent intermediates of early metabolism of the exogenous fatty acids. In the course of time, a steady decrease of radioactive 1,2-diacylglycerols is observed that is accompanied by an increase in labeled triacylglycerols, diacylglycerophosphocholines, and diacylglycero-O-(N,N,N-trimethyl)homoserines. The stereospecific distribution of acyl moieties in the diacylglycero-O-(N,N,N-trimethyl)homoserines in C. fusca indicates that these ether lipids are derived from 1,2-diacylglycerol intermediates. This notion is supported by the finding that during incubation with radioactively labeled fatty acids the formation of diacylglycero-O-(N,N,N-trimethyl)homoserines parallels the biosynthesis of both diacylglycerophosphocholines and diacylglycerophosphoethanolamines, two classes of phospholipids which are known to be derived from 1,2-diacylglycerols. The mechanism of the formation of the ether bond, however, is as yet unknown. Incubation of C. fusca cultures with 14C-labeled fatty acids or alcohols leads to the formation of fair proportions of wax esters that are labeled in both the acyl and the alkyl moieties, indicating that in these algae fatty acids and alcohols are interconverted. 14C-Labeled long-chain alcohols are not incorporated into the alkyl moieties of ether lipids, whereas labeled 1-O-alkylglycerols are used, though to a very small extent, as precursors of ether phospholipids.

Chlorella↗

Placental transport of trans fatty acids in the rat.

Placental transport of 9-trans [1-14C] octadecenoic (elaidic) and 9-trans,12-trans [1-14C] octadecadienoic (linoelaidic) acids was demonstrated in rats. On the 18th day of gestation, a 14C-labeled albumin complex of elaidic or linoelaidic acid was injected into the jugular vein of pregnant rats. For comparison, 9-cis [1-14C] octadecenoic (oleic) or 9-cis,12-cis [1-14C] octadecadienoic (linoleic) acid also was injected into the maternal circulation of rats. All animals were sacrificed 1 hr following injection. Lipid composition and distribution of label were determined in maternal plasma, placental and fetal tissues. Differences in specific activities of plasma, placental and fetal total lipids indicated a decreasing concentration gradient for both cis and trans isomers of octadecenoic and octadecadienoic acids. Distribution of radioactivity in various lipid components was determined by thin layer chromatography. Irrespective of the label, the highest percentage of total radioactivity was carried by triglycerides (TG) in maternal plasma (approximately 60-80%), and was incorporated mainly in phospholipids (PL) of fetal tissue (approximately 50-60%). A nearly equal distribution of the label was found between PL and TG of placental lipids (approximately 40%). Radioactivity of fatty acid methyl esters (FAME) determined by radio-gas liquid chromatography indicated that after injection of linoelaidate, radioactivity of maternal plasma, placental and fetal tissue FAME was associated only with t,t-18:2. Following injection of elaidate, all the radioactivity in placental FAME was associated with t-18:1; however, in fetal tissues, the label was distributed between 16:0 and t-18:1. These findings suggest that, in contrast to linoelaidic acid, rat fetal tissues can metabolize elaidic acid via beta oxidation to form acetyl CoA and palmitic acid.

Animals↗

Investigation of [14C] linoleic acid conversion into [14C] arachidonic acid and placental transfer of linoleic and palmitic acids across the perfused human placenta.

In five separate experiments in which single placental lobes were perfused, [14C] antipyrine, [3H] dextran and a fatty acid mixture containing [14C] linoleic and [3H] palmitic acids were added to the maternal circuit. Samples of fetal and maternal perfusate, taken at intervals, were analysed for radioactivity and fatty acid content. The relative placental transport rates of antipyrine, linoleic and palmitic acids and the changes in maternal and fetal circuit content of linoleic, palmitic and arachidonic acids were measured. Mean transfer rates of 150, 0.023 and 0.034 mumol min-1 were obtained for [14C] antipyrine, [3H] palmitic and [14C] linoleic acids, respectively. The transfer of [3H] dextran was negligible. The transfer rates of linoleic and palmitic acids did not differ significantly from one another. The maternal circuit content of palmitic and arachidonic acids increased, whereas linoleic acid content decreased. The fetal content of all three acid increased but in the case of arachidonic and palmitic acids the increase was not as great as that seen in the maternal circuit. In no experiment was radioactivity detected in maternal or fetal perfusate arachidonic acid. It is concluded that linoleic and palmitic acids cross the placenta from the maternal to the fetal side. This transfer appears to be non-selective. The large amounts of arachidonic acid that are released into the perfusate probably originate from placental lipids other than free linoleic acid.

Antipyrine↗

Demonstration of the deposition and modification of dietary fatty acids in pinniped blubber using radiolabelled precursors.

Radioisotopes are commonly used to study the in vivo metabolism and deposition of dietary fatty acids in adipose tissue. The application of this approach to pinnipeds is problematic because of their large mass and blubber fat content. We have developed a method where labelled lipids can be fed to seals at financially feasible levels, with the radioactivity in individual fatty acids isolated from blubber detected with standard laboratory equipment. A combination of techniques including argentation thin layer chromatography, high performance liquid chromatography with ultraviolet detection, and independent liquid scintillation counting were employed. Juvenile gray seals (Halichoerus grypus) were fed either 0.5 mCi (3)H-labelled triolein (18:1n-9, n=2) or palmitic acid (16:0, n=2). Blubber samples were taken 12 h later, and the radioactivity in individual fatty acids was determined. Radioactivity was detected in only 18:1 from the animals fed (3)H-labelled triolein, indicating direct deposition without modification. Both animals fed (3)H-labelled palmitic acid showed clear peaks of radioactivity in 16:0; however, there was also significant activity (23%-29%) found in the desaturation product 16:1. Our results demonstrate that this method is sufficiently sensitive to track the deposition of labelled dietary lipids as well as modification products of ingested fatty acids and will be important in the application of fatty acid signatures to study predator diets.

Adipose Tissue↗

Phospholipid metabolism of stimulated lymphocytes. Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A.

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [3H]arachidonate and [14C]oleate or [3H]arachidonate and [14C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3-7-fold) and phosphatidylethanolamine (2-3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.

Acyl Coenzyme A↗

Fatty-acid-binding proteins. Occurrence of two fatty-acid-binding proteins in bovine liver cytosol and their binding of fatty acids, cholesterol, and other lipophilic ligands.

Fatty-acid-binding proteins (FABPs) are known as cytosolic binding sites for fatty acids and their CoA esters. Radioactively labeled and fluorescent fatty acids were used to locate and identify these proteins in bovine liver cytosol. The occurrence of two species of FABPs was demonstrated and these were designated pI6.0-FABP and pI7.0-FABP according to their isoelectric points in the delipidated state. Oleic acid/FABP binding ratios were 1 with pI6.0-FABP and 2 with pI7.0-FABP. Upon binding of oleic acid the isoelectric points of liganded FABPs shifted to pH 5.0-5.1 in each case. Both proteins were purified by removing nonbinding proteins by acid and heat denaturation and subsequent gel filtration. By making use of the pI shifts observed upon lipidation and delipidation of the binding proteins with ligand fatty acids, final purification was achieved in two fractionations by isoelectric focusing. The binding proteins (Mr 11 800 +/- 1 000) had similar amino-acid compositions (no Trp) and were not covalently modified by carbohydrate and fatty acid. Fatty acids and their CoA esters were complexed by either FABP, cholesterol only by pI-7.0-FABP, though non-stoichiometrically. 16-(9-Anthroyloxy)palmitic acid was bound by pI-7.0-FABP in a 1:1 ratio and precluded the additional binding of a straight-chain fatty acid. Electrophoretic titration curves indicated dissociation of the oleic acid/pI7.0-FABP complex below pH 5.0. It appears that fatty acids and their CoA esters are the foremost binding partners of FABPs in vivo. The results are discussed in terms of a single binding site for fatty acids per molecule FABP.

Amino Acids↗

The pattern of fatty acid synthesis in lactating rabbit mammary gland studied in vivo.

The biosynthesis of fatty acids has been studied in lactating rabbits at 6h after intravenous injection of sodium [1-(14)C]acetate. The specific radioactivities of the individual fatty acids (C(6:0) to C(14:0)) and the proportions of these fatty acids synthesized were similar in mammary tissue and milk. Hexanoic acid had the highest specific radioactivity, and the C(8:0)-C(14:0) fatty acids had similar specific radioactivities, which were about five times those of C(16) and C(18) acids. No radioactivity was detected in fatty acids of chain length C(14) in these tissues were similar to those of the long-chain fatty acids in the milk and mammary gland. The results show that the C(4:0)-C(14:0) fatty acids are synthesized within the mammary gland rather than by fatty acid uptake from circulating blood or by oxidation of long-chain fatty acids within the gland. We conclude that de novo synthesis of esterified fatty acids in vivo by this tissue has a high degree of chain-length specificity.

Acetates↗

Differential diagnosis of maldigestion and malabsorption of fat. I. Comparison of the radioactivity of triglyceride and non-esterified fatty acid in fecal fat using purified 131I-triolein.

To differentiate maldigestion from malabsorption of fat, we compared the radioactivity of triglyceride with non-esterified fatty acid in fecal fat after ingestion of radiochemically purified 131I-triolein. A large amount of radioactivity was present in the triglyceride fraction from patients with pancreatic insufficiency, while such was found only in non-esterified fatty acid fraction in patients with short bowel syndrome, as determined by thin-layer chromatogram scanning. Thus, the "Digestion-Absorption Index" was calculated as a ratio of the radioactivity of triglyceride to that of non-esterified fatty acid. Mean Digestion-Absorption Index was 0.946 in the maldigestive group, and 0.045 in the malabsorptive group, and there was a statistical significance. However, an overlap in a few cases was confirmed in the area of 0.10 +/- 0.02, when this index was plotted on a table of semilogarithms. We assumed that this overlap was chiefly the result of maldigestion plus malasorption of fat.

Adult↗

Renewal of fatty acids in the membranes of visual cell outer segments.

The renewal of fatty acids in the visual cells and pigment epithelium of the frog retina was studied by autoradiographic analysis of animals injected with tritiated palmitic, stearic, or arachidonic acids. Most of the radioactive material could be extracted from the retina with chloroform-methanol, indicating that the fatty acids had been esterified in lipids. Analysis of the extracts, after injection of [(3)H]palmitic acid, revealed that the radioactivity was predominantly in phospholipid. Palmitic acid was initially concentrated in the pigment epithelium, particularly in oil droplets which are storage sites for vitamin A esterified with fatty acid. The cytoplasm, but not the nucleus of these cells, was also heavily labeled. Radioactive fatty acid was bound immediately to the visual cell outer segment membranes, including detached rod membranes which had been phagocytized by the pigment epithelium. This is believed to be due to fatty acid exchange in phospholipid molecules already situated in the membranes. Gradually, the concentration of radioactive material in the visual cell outer segment membranes increased, apparently as a result of the addition of new phospholipid molecules, possibly augmented by the transfer from the pigment epithelium of esterified vitamin A. Injected fatty acid became particularly concentrated in new membranes which are continually assembled at the base of rod outer segments. This localized concentration was short-lived, apparently due to the rapid renewal of fatty acid. The results support the conclusion that rods renew the lipids of their outer segments by membrane replacement, whereas both rods and cones renew the membrane lipids by molecular replacement, including fatty acid exchange and replacement of phospholipid molecules in existing membranes.

Animals↗

Metabolism of a n-paraffin, heptadecane, in rats.

14C-heptadecane incorporated in rat diet was largely absorbed, and a balance study showed extensive 14CO2 excretion (65%). There was no elimination of the hydrocarbon in the urine, and only minute quantities of labeled metabolites. Radioactivity in the feces was entirely in heptadecane. About 7% of the heptadecane absorbed was stored in the carcass, whereas the rest was omega-oxidized to heptadecanoic acid. This fatty acid was incorporated into neutral lipids and phospholipids, underwent the normal fatty acid degradation pathway, and contributed to the synthesis of lipids, including fatty acids, squalene and cholesterol, and nonlipids (7-10%). Heptadecanoic acid was desaturated to heptadecenoic acid. The even distribution of radioactivity in the fatty acids of the various phospholipid classes indicated that heptadecane did not interfere with the biochemical mechanisms of these functional lipids.

Adipose Tissue↗

Uptake of fatty acids by the yeasts, Saccharomyces uvarum and Saccharomycopsis lipolytica.

Yeast cells take up exogenous fatty acids with subsequent rapid incorporation into glycerolipids. beta-Oxidation does not occur in Saccharomyces uvarum and is observed in Saccharomycopsis lipolytica only 2-5 min after addition of radioactively labeled fatty acid. Rates of fatty acid uptake are linear up to 30 s with S. lipolytica and up to 2 min with S. uvarum. The uptake kinetics are consistent with a dual mode of transport, comprising a saturable component with KT values in the range 10(-5)-10(-6) M, and apparently simple diffusion that predominates at high substrate concentrations. Kinetics of fatty acid permeation are independent of metabolic energy and membrane potential. At least two fatty acid carrier systems exist in both S. lipolytica and S. uvarum, one being specific for fatty acids with 12 and 14 C atoms, respectively, the other for C16 and C18 saturated or unsaturated fatty acids. Octanoic acid and decanoic acid are not taken up by S. lipolytica. Internalization of lauric acid and oleic acid by S. lipolytica cells is preceded by a rapid (less than 5 s) initial uptake which most likely represents irreversible adsorption. This phenomenon was not observed with heat-inactivated S. lipolytica cells or with viable S. uvarum. In azide-poisoned cells of S. lipolytica an up to 20-fold accumulation of unesterified fatty acid was observed within 30 s after the addition of substrate.

Adsorption↗

High-performance liquid chromatographic study of the regulation of phospholipid metabolism in cultured adrenocortical cells.

A rapid high-performance liquid chromatographic (HPLC) method for the separation of phospholipids was developed for minute samples of total lipids (ca. 200 micrograms). The method was applied to the study of the phospholipid metabolism in adrenocortical cell cultures. A complete separation of the different cellular phospholipid classes was achieved in 40 min. Good resolution of the phospholipid peaks was obtained, which allowed the collection of each individual class of phospholipids for further analysis of radioactivity and fatty acid composition by gas chromatography. When cells were incubated with [U-14C]glycerol or [U-14C]palmitate the bulk of the radioactivity was found in cellular phosphatidylcholines. Exogenous phospholipids were incorporated into cellular lipids to a large extent, however without an increase in the cellular phospholipid content. 12-O-Tetradecanoyl-phorbol-13-acetate induced a 20% increase in the polyunsaturated fatty acid content of the cellular phosphatidylethanolamines, but no change was detected in the cellular phosphatidylcholines. The developed method is well-suited to the study of the phospholipid metabolism in adrenocortical cells where the phospholipid metabolism is closely linked to the specialized functions of the cells.

Adrenal Cortex↗