Host plasma low density lipoprotein particles as an essential source of lipids for the bloodstream forms of Trypanosoma brucei

In contrast to mammalian cells, bloodstream forms of Trypanosoma brucei show no activity for fatty acid and sterol synthesis and critically depend on plasma low density lipoprotein (LDL) particles for their rapid growth. We report here that these parasites acquire such lipids by receptor-mediated endocytosis of LDL, subsequent lysosomal degradation of apoprotein B-LDL, and utilization of these lipids. Uptake of LDL-associated [3H]sphingomyelin and of LDL-associated [3H]cholesteryl oleate paralleled each other, and that of 125I-apoprotein B-LDL showed saturation and could be inhibited by unlabeled LDL or by anti-LDL receptor antibodies. Metabolism of lipids carried by LDL was abolished by chloroquine and by the thiol protease inhibitor, leupeptin. Sphingomyelin was cleaved by an acid sphingomyelinase to yield ceramide, which was itself split up into sphingosine and fatty acids. The latter were further incorporated into phosphatidylcholine, triacylglycerols, or cholesteryl esters. Similarly, cholesteryl oleate was hydrolyzed by an acid lipase to yield free cholesterol, which was reesterified with fatty acids, presumably in the cytosol. Like free cholesterol, LDL provided substrate for cholesterol esterification. In the culture-adapted procyclic form of T. brucei, which is capable of sterol synthesis, exogenous LDL-cholesterol rather than endogenously synthesized sterol was utilized for sterol esterification. Interference with exogenous supply of lipids via receptor-mediated endocytosis of LDL should be explored to fight against trypanosomiasis.     

Increased cholesterol content and esterification in rabbit aortic medial cells cultured in hyperlipidemic serum

Cells of early atherosclerotic lesions have an increased content of cholesteryl esters and enhanced rate of cholesterol esterification. The present study was carried out to elucidate whether these changes could be reproduced in a purely in vitro system. Cultures of rabbit aortic medial cells were incubated in a medium containing 10% of either normal rabbit serum (control cells) or serum from rabbits with cholesterol-induced hyperlipidemia (hyperlipidemic cells). The incorporation rate of [1-14C]oleate into cellular cholesteryl esters increased two- to threefold after four hours' incubation with hyperlipidemic serum and remained elevated during the rest of the eight days' study. After seven days' incubation the hyperlipidemic cells incorporated significantly more (+13%) oleate into triglycerides and significantly less (-15%) oleate into phospholipids than did the control cells. Cholesteryl ester content of the hyperlipidemic cells rose steeply for about two days, with a slower rise thereafter. In hyperlipidemic cells the esterified cholesterol content was significantly higher (315 ng/mug DNA) than in the control cells (79 ng/mug DNA). Hyperlipidemic cells showed a slow but significant rise of free cholesterol as well (from 1.15 mug/mug DNA in control to 1.50 mug/mug DNA). The results indicate that lipid changes resembling those in early atherosclerotic lesions can be brought about in vitro 0y treatment of aortic cells with hyperlipidemic serum.     

Plasma insulin and growth hormone concentrations in pregnant sheep II: post-absorptive levels in mid- and late pregnancy

1. The incorporation of L-[U-14C]leucine, L[U-14C]histidine and L-[U-14C]phenylalanine into casein secreted during perfusion of isolated guinea-pig mammary glands was demonstrated. 2. The extent of incorporation of label into casein residues was consistent with their being derived from free amino acids of the perfusate plasma. 3. The mean transit time of the amino acids from perfusate into secreted casein was approx. 100 min. 4. Whereas radioactive histidine and phenylalanine were incorporated solely into milk protein, radioactivity from [U-14C]valine was also transferred to CO2 and to an unidentified plasma component, and from [U-14C]leucine to plasma glutamic acid. 5. Evidence from experiments with [U-14C]phenylalanine suggests that, as in rats, but in contrast with ruminant species, guinea-pig mammary tissue does not possess phenyl alanine hydroxylase activity. 6. The results are discussed in relation to the possible role of essential amino acid catabolism in the control of milk-protein synthesis.     

Educating primary care providers about HIV disease: multidisciplinary interactive mechanisms

We have recently demonstrated that macrophage conditioned medium (MP medium) and beta VLDL enhance cholesterol esterification in cultured aortic smooth muscle cells by LDL receptor mediated and other pathways (Stein, O. et al. (1993) Arteroscl. Thromb. 13, 1350-1358). In view of the presence of extracellular non-lipoprotein cholesteryl ester (in the form of lipid droplets) in the atheroma, the effect of MP medium on the cellular uptake of liposomal cholesteryl linoleyl ether (CLE) or cholesteryl ester (CE) was studied. After 4 h incubation in MP medium, the uptake of liposomal [3H]CLE was up to 10-fold higher than in the presence of control medium of the same composition but not conditioned with macrophages (DV medium). Similar results were seen also with HSF derived from LDL receptor negative donors. The MP medium-stimulated uptake of liposomal [3H]CE resulted also in hydrolysis of 70-90% of the labeled compound, indicating that the [3H]CE was intracellular. While the MP medium effect on liposomal [3H]CLE uptake was evident after 4 h, its effect on [3H]cholesterol esterification by SMC in the presence of beta VLDL could be demonstrated only after 24 h. Addition of apoE to MP medium resulted in a small (30-40%) increase in the uptake of liposomal [3H]CLE; however, it was augmented more than 4-fold when apoE was added to DV medium. The MP medium effect on the uptake of liposomal [3H]CLE was interfered with by heparin, anti-LPL antibody or heparinase, while these treatments did not affect [3H]cholesterol esterification in the presence of beta VLDL. These results suggest that the interaction between SMC and two potential sources of lipids in atheroma, i.e., lipoproteins and non-lipoprotein lipid droplets, could be governed by different components of the MP medium. In the case of the lipid droplets, as modeled here in the form of liposomes, macrophage-derived lipoprotein lipase could play a major role in cholesteryl ester transfer into SMC.     

Implication of the midgut of the centipede Lithobius forficatus in the heavy metal detoxification process

The ability of two rat liver fatty acid binding protein (L-FABP) isoforms to influence microsomal phosphatidic acid biosynthesis, a key intermediate in glycerolipid formation, and phospholipid fatty acid remodeling was examined in vitro. Isoform I enhanced microsomal incorporation of [1-14C]-oleoyl-CoA into phosphatidic acid 7-fold while isoform II had no effect relative to basal. In contrast, isoform II enhanced microsomal incorporation of [1-14C]-palmitoyl-CoA into phosphatidic acid 4-fold while isoform I had no effect. These results suggest that each L-FABP isoform selectively utilized different acyl-CoAs for glycerol-3-phosphate esterification. Both isoforms stimulated phosphatidic acid formation by increasing glycerol-3-phosphate acyltransferase activity, not by increasing lysophosphatidic acid acyltransferase activity. Furthermore, the effects of L-FABP on phosphatidic acid biosynthesis could not be correlated with protection from acyl-CoA hydrolysis. L-FABP isoforms also influenced phospholipid fatty acid remodeling in a phospholipid-dependent manner. Isoform I preferentially enhanced oleate and palmitate esterification into phosphatidylethanol-amine, while isoform II stimulated esterification into phosphatidylcholine, phosphatidylserine and sphingomyelin. Taken together, these data demonstrated a unique role of each L-FABP isoform in modulating microsomally derived phospholipid fatty acid composition. (c) 1998 Elsevier Science B.V.     

Relationship between ATP synthesis and 201Tl uptake in transformed and non-transformed cell lines

The effects of vitamin E on cholesteryl ester (CE) metabolism in J774 cells were examined. Pretreatment of J774 cells with vitamin E at concentrations above 50 microM significantly decreased acetylated low density lipoprotein (LDL)-induced incorporation of [14C]oleate into CE in cells in a dose-dependent manner. This was partly due to vitamin E also significantly inhibiting the uptake of [3H]CE-labeled acetylated LDL by J774 cells. A trend existed toward suppression of acyl-CoA:cholesterol acyltransferase (ACAT) activity in the cell lysate at high vitamin E concentration, but there was no effect on hydrolysis of CE. These data indicate that vitamin E reduces the uptake of modified LDL and suppresses ACAT activity, resulting in less cholesterol esterification in macrophages: a novel mechanism underlying the antiatherogenic properties of vitamin E.     

Synthesis and removal of different cholesterol esters by aortic smooth muscle cells in culture

The incorporation of 3H-labelled oleic acid and of 14C-labelled linoleic acid into phospholipid, triglyceride and cholesterol ester in smooth muscle cells grown in incubation medium supplemented with either 5% normal or 5% hyperlipemic serum has been studied. Both fatty acids were incorporated into cholesterol esters to a greater extent when cells grown in incubation medium containing hyperlipemic serum. Oleic acid was incorporated into cholesterol esters in preference to linoleic acid. The addition of hyperlipemic serum to the incubation medium did not increase the incorporation fo either 3H-labelled oleic acid or of 14C-labelled linoleic acid into phospholipid or triglyceride. The removal of labelled lipid fractions has also been followed for four days in cells pulse labelled for 24 hours with 3H-labelled oleic acid and 14C-labelled linoleic acid. Both 3H- and 14C-labelled cholesterol esters were removed more rapidly when the smooth muscle cells were grown in medium containing normal serum than in medium containing hyperlipemic serum. The removal of both phospholipid and triglyceride was similar in normal and hyperlipemic serum. Comparison of the 3H/14C ratio indicated that the cholesterol oleate and cholesterol linoleate were removed at similar rates.     

Metabolism of [1-(14)C] and [2-(14)C] leucine in cultured skin fibroblasts from patients with isovaleric acidemia. Characterization of metabolic defects

Leucine metabolism in cultured skin fibroblasts from patients with isovaleric acidemia was compared with that in normal fibroblasts and in cells from patients with maple syrup urine disease using [1-(14)C] and [2-(14)C] leucine as substrates. Inhibitory effects of methylenecyclopropylacetic acid on leucine metabolism in normal cells were also investigated. Production of 14CO2 from [2-(14)C] leucine was very reduced (96-99%) in both types of mutant cells. Radioactive isovaleric acid accumulated in assay media with isovaleric acidemia cells but not in those with maple syrup urine disease cells. Unexpectedly, 14CO2 production from [1-(14)C] leucine was partially depressed (80%) in isovaleric acidemia cells whereas in maple syrup urine disease cells it was strongly depressed (99%) as expected. These two mutant cells were clearly distinguished by detection of 14C-isovaleric acid accumulation after incubation with [2-(14)C] leucine. A pattern of inhibition of leucine oxidation similar to that seen in isovaleric acidemia cells was induced in normal cells by the addition of 0.7 mM methylenecyclopropylacetic acid to the assay medium. The partial inhibition of [1-(14)C] leucine oxidation seen in isovaleric acidemia cells and also in normal cells in the presence of the inhibitor appears to be, at least in part, due to an accumulation of isovalerate in the cells. Isovaleric acid (5-10) mM) inhibited [1-(14)C] leucine oxidation 32-68% when added to the assay medium with normal cells. Addition of flavin adenine dinucleoside to culture medium or assay medium or both did not restore oxidation of either leucine substrate in isovaleric acidemia cells.     

An HPLC-GC/MS reference method for serum total cholesterol with control for ester hydrolysis

Currently used isotope-dilution mass spectrometry methods for serum total cholesterol are performed without control in each sample for completeness of hydrolysis of cholesterol esters. In order to monitor this step in the analysis, we developed a method based on both high-pressure liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). 13C3-cholesterol and cholesteryl [1-14C] oleate were added to serum that was saponified and extracted into hexane. The extract was subjected to HPLC with collection of the fractions corresponding to cholesterol and cholesteryl oleate. The radio-activity of the latter was counted in order to estimate the nonhydrolysed fraction that on average amounted to less than 0.1% for commonly used ester hydrolysis procedures. The cholesterol fraction was subjected to GC/MS for quantitation using the traditional isotope-dilution principle. Evaluation of accuracy by assaying sera from the National Institute of Standards and Technology showed a deviation from the target value of < 1% with a coefficient of variation of < or = 1.0%. In conclusion, the present reference method for serum total cholesterol assures results based on complete hydrolysis of the cholesterol ester fraction.     

Comparison of the effects of cyclic AMP analogues on cholesterol metabolism in cultured rat and hamster hepatocytes

The effects of two cell-permeable cyclic AMP analogues, 8-chloro cyclic AMP (8-Cl cAMP) and 8-(4-chlorophenylthio) cyclic AMP (8-CPT cAMP), on cholesterol esterification, cholesteryl ester hydrolysis and bile acid synthesis were compared in cultured rat and hamster hepatocytes. Cholesterol esterification, as measured by the incorporation of [3H]oleate into cholesteryl ester, was increased by 58-88% by the analogues in rat hepatocytes and by 33-43% in hamster cells. The response in rat hepatocytes, however, was observed after a relatively short incubation time (28% increase after 1 hr), whereas that in hamster cells required a longer period (36% after 12 hr) to become apparent. The activity of the cytosolic neutral cholesteryl ester hydrolase in rat hepatocytes was also stimulated by both cyclic AMP analogues (31-37%, but the microsomal activity was unaffected. In hamster hepatocytes, however, microsomal cholesteryl ester hydrolase activity was increased (47-80%) in the presence of 8-Cl cAMP or 8-CPT cAMP. Bile acid synthesis was increased by 8-CPT cyclic AMP in rat cells (approximately 25%) but was unchanged by both analogues in hamster hepatocytes. These results indicate significant differences in the way in which cholesterol metabolism responds to cyclic AMP in cultured rat and hamster hepatocytes.     

Biodegradation of phenols by the alga Ochromonas danica

The eukaryotic alga Ochromonas danica, a nutritionally versatile, mixotrophic chrysophyte, grew on phenol as the sole carbon source in axenic culture and removed the phenol carbon from the growth medium. Respirometric studies confirmed that the enzymes involved in phenol catabolism were inducible and that the alga oxidized phenol; the amount of oxygen consumed per mole of oxidized substrate was approximately 65% of the theoretical value. [U-14C]phenol was completely mineralized, with 65% of the 14C label appearing as 14CO2, approximately 15% remaining in the aqueous medium, and the rest accounted for in the biomass. Analysis of the biomass showed that 14C label had been incorporated into the protein, nucleic acid, and lipid fractions; phenol carbon is thus unequivocally assimilated by the alga. Phenol-grown cultures of O. danica converted phenols to the corresponding catechols, which were further metabolized by the meta-cleavage pathway. This surprising result was rigorously confirmed by taking the working stock culture through a variety of procedures to check that it was axenic and repeating the experiments with algal extracts. This is, as far as is known, the first definitive identification of the meta-cleavage pathway for aromatic ring degradation in a eukaryotic alga, though its incidence in other eukaryotes has been (infrequently) suggested.     

Synthesis and release of sulfolipid by Mycobacterium avium during growth andcell division

Mycobacterium avium exhibits a life cycle wherein small cells elongate to form filaments. The life cycle is unique in that elongated cells will undergo rapid division by fragmentation only if fatty acid is present. The utilization of [14C]palmitic acid and [3H]oleic acid by M. avium during the life cycle was assessed. Four glycolipids, identifiable by elution patterns from hydroxylapatite columns, were associated with postfission cells and contained isotope from the precursor fatty acid. The incorporation of 3H from oleic acid into the cellular glycolipids was maximal during cell division, but as much as 73% of the radioactivity was lost to the lipids from cells in the postfission status. Three of the glycolipids were sulfatides into which 36S was incorporated by M. avium. The [35]sulfatides were synthesized by cells undergoing fragmentation and were recovered from the medium at the termination of cell fission. These results demonstrated that the isotope was not lost to the cells because of turnover, but rather that the labeled compounds were released, intact, from the cells after fission. Because of the facile release of the sulfolipids, it was suggested that they were part of the cell envelope of M. avium cells during the division process.     

Amino acid metabolism in the piglet. 2. Influence of fasting on plasma free amino acid concentration and in vivo oxidation of methionine, isoleucine and threonine

1. The influence of a 24 h fast on the concentrations of free amino acids in the plasma, and upon the oxidation rates of methionine, isoleucine and threonine was studied (using early weaned, 4-week-old piglets which were receiving a semi-purified diet. 2. There was no change in the total concentration of the essential amino acids as a result of the 24 h fast: the concentration of the branched-chain amino acids increased, but the effect of this was offset by decreases in the concentrations of arginine, histidine, lysine, methionine and phenylalanine. There was a reduction in the concentration of the non-essential amino acids. 3. The piglets received infusions of L-[I-14C]methionine, L-[U-14C]isoleucine and L-[U-14C]-threonine, and the recovery of the label in carbon dioxide was determined. Less than 5% of the activity from methionine was recovered in the CO2 from the fed piglets, whereas 12% was recovered from the fasted piglets. The corresponding values with threonine were 11 and 19% but there was no effect of fasting on the recovery of the label from isoleucine in CO2. 4. The initial dilution of a single dose of a labelled amino acid infused into the bloodstream depends on the plasma concentration of the amino acid. Nutritional regimens may effect the free amino acid concentration in the plasma. Thus comparisons based upon direct determination of activity recovered in CO2 from the labelled dose of an amino acid with animals on different nutritional regimens could not misleading, unless the differences in the concentrations of the amino acid in the plasma are considered.     

Biosynthesis of vernoleate (cis-12-epoxyoctadeca-cis-9-enoate) in microsomal preparations from developing endosperm of Euphorbia lagascae

Epoxidated fatty acids are the major constituents of the triacylglycerols in a few plant species. We have investigated the biosynthesis of vernolic acid (cis-12-epoxyoctadeca-cis-9-enoic acid) in the seed oil of Euphorbia lagascae. Microsomes were isolated from developing endosperm. The membrane lipids were labeled in situ with [14C]oleate or [14C]linoleate, which mainly were recovered in phosphatidylcholine (PC), and the metabolization of the radioactive fatty acids was followed in incubations with or without NADPH. In the presence of NADPH, [14C]vernoleate was formed. After short incubations, most of the vernolic acid was found in PC, but with increasing incubation times, the free acid dominated. The synthesis of vernoleate was inhibited by carbon monoxide, but not by cyanide. The presence of anticytochrome b5 antibodies inhibited both the desaturation of [14C]oleate to [14C]linoleate and the epoxidation of [14C]linoleate to [14C]vernoleate. Free linoleic acid did not serve as substrate for epoxidation. The results indicate that, in the endosperm of E. lagascae, vernoleate is synthesized on PC from linoleate, and that the epoxidation is catalyzed by a cytochrome P450 and involves cytochrome b5.     

Ethanol inhibits phosphatidylcholine and phosphatidylethanolamine biosynthesis in human leukemic monocyte-like U937 cells

The effect of ethanol (ETOH) on the incorporation of [14C]oleic acid (18:1) into lipid in human monocyte-like U937 cells was investigated. With increasing time of exposure to ETOH, the percentage of the label distributed into neutral lipid (NL) declined from 35 per cent (3 h) to 10 per cent (24 h) accompanied by increased incorporation into phospholipid (PL). [14C] 18:1 was preferentially incorporated into triglyceride (TG) and phosphatidylcholine (PC), comprising over 65 per cent and 50 per cent of the label associated with NL and PL, respectively. Low concentrations of ETOH (< or = 1.0 per cent; v/v) had no effect. At concentrations greater than 1.5 per cent, there was enhanced incorporation into TG and diacylglycerol (DAG) in a 24-h incubation period, while at 16 h the label in phosphatidylethanolamine (PE) was decreased. The effect of ETOH on the CDP-choline or ethanolamine pathway was examined by monitoring the incorporation of [3H]choline or [14C]ethanolamine into PC or PE, respectively. At low concentrations ETOH had no effect on either choline uptake or the incorporation into PC. Higher concentrations (> or = 1.5 per cent) for 3 and 6 h resulted in a slightly decreased choline uptake, and the reduction (40-50 per cent) of incorporation into PC suggests that the CDP-choline pathway was inhibited. There was a similar inhibition of the incorporation of [14C]ethanolamine into PE. When the cells were incubated for 3 h in the presence of 2 per cent ETOH and with labelled 18:1 and PL-base, the ratios of incorporation (base/18:1) into PC and PE fractions decreased, indicating that the major inhibition lay in blockage of the availability of the base moiety for PL formation. Analysis of the distribution of the label into metabolites revealed that ETOH inhibited the conversion of [14C]ethanolamine into [14C]phosphorylethanolamine. The reduction in incorporation was not due to the enhanced breakdown of base-labelled PL. Our results indicate that ETOH has an inhibitory effect on the CDP-choline or ethanolamine pathway.     

Binocular processes in vernier acuity

The contribution of synthesis and dietary sequestration to the high arachidonate content of the lone star tick, Amblyomma americanum, salivary glands was investigated by assessing the salivary metabolites of various radiolabeled fatty acid substrates administered to partially fed females. A portion of each of the fatty acids studied was incorporated into the fatty acid moiety of the phospholipid fraction. [14C]acetate was metabolized only into myristic, palmitic, palmitoleic, steric, and oleic acids. [3H]oleic acid, [14C]linoleic acid, [14C]gamma-linolenic acid and [14C]eicosatrienoic acids were incorporated into salivary gland phospholipids but underwent little change including elongation and/or desaturation to arachidonate. Ingested [3H]arachidonic acid was readily taken up by the salivary gland and distributed among the lipid classes in a pattern markedly different from that of the other fatty acids tested. We conclude that ticks are unable to synthesize arachidonic acid for incorporation into the salivary glands, but rather sequester it from the host bloodmeal.     

The effect of propionate on lipid synthesis in rat lymphocytes

1. The effect of propionate on lipid synthesis in lymphocytes cultured for 24 hr and incubated for 2 hr was investigated. 2. [1-14C]-propionate was incorporated mainly into phospholipids in both control and concanavalin A (Con A) stimulated cultured lymphocytes. 3. The content of free coenzyme A markedly decreased in 2 hr incubated lymphocytes when propionate was added to the medium at concentrations from 10 to 100 mmol/l. 4. Propionate at 40 mmol/l decreased the incorporation of [1-14C]-palmitate into phospholipids (86%), triacylglycerol (87%) and cholesterol ester (98%) and increased in cholesterol (133%) of cultured lymphocytes. 5. Addition of propionate into the culture medium at 2.5 and 5.0 mmol/l concentrations markedly increased the activity of hydrolases of various acylCoA derivatives. 6. The results suggest that propionate may reduce the content of acylCoA and so its esterification and this might be important for the regulation of lymphocytes proliferation.     

Inhibition of oxidation of low density lipoprotein by troglitazone

The effect of a new oral hypoglycemic agent troglitazone, (+/-)-5-[4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl-methoxy)benz yl]-2,4-thiazolidinedione as an antioxidant against the free radical-mediated oxidation of low density lipoprotein (LDL) was studied. The oxidation of LDL gives cholesteryl ester hydroperoxide and phosphatidylcholine hydroperoxide as major primary products. Troglitazone incorporated exogenously into LDL inhibited the oxidations of LDL induced by either aqueous or lipophilic peroxyl radicals and suppressed the formation of lipid hydroperoxides efficiently. Ascorbic acid added into the aqueous phase spared both endogenous alpha-tocopherol and troglitazone in LDL. It was also found by absorption spectroscopic and electron spin resonance (ESR) studies that troglitazone reacted rapidly with a galvinoxyl radical to give a chromanoxyl radical which gives the same ESR spectrum as alpha-tocopherol. This ESR spectrum disappeared rapidly when ascorbic acid was added into the system. These results show that troglitazone acts as a potent antioxidant and protects LDL from oxidative modification.     

Incorporation of exogenous fatty acids into molecular species of rat hepatocyte phosphatidylcholine

Freshly isolated rat hepatocytes were incubated for 20 and 60 min with [U-14C]glycerol and unlabeled palmitic (16:0), oleic (18:1), or arachidonic (20:4) acid, added as albumin complex in 10% ethanol. Each fatty acid increased glycerol incorporation into total lipids by a factor of 8-10 over control, whereas ethanol alone (final concentration 100 mM) yielded a threefold increase of glycerol uptake. Glycerol incorporation stopped after 20 min and cellular acyl turnover continued in the absence of useable labeled substrate. In each case, radioactivity recovered in hepatocyte lipids was present primarily in triacylglycerol (37-64%), phosphatidylcholine (22-37%), and phosphatidylethanolamine (10-22%). Separation by high-performance liquid chromatography of the diacylglycerol dinitrobenzoates derived from phosphatidylcholine showed that the molecular species had drastically different labeling patterns in the presence of the exogenous fatty acids, whereas the pattern obtained in the presence of ethanol alone was virtually the same as that for the control incubations. The labeling patterns indicated that exogenous fatty acids, including arachidonic acid, were incorporated into phosphatidylcholine primarily by the de novo pathway yielding highly labeled species with the exogenous fatty acid esterified at both the sn-1 and sn-2 positions of glycerol. After 20 min incubation with arachidonic acid, the 20:4-20:4 phosphatidylcholine contained about one-half of the [U-14C]glycerol label recovered in this lipid class. The data also showed that newly synthesized molecular species were extensively remodeled within 1 h.     

Elongation of (omega-14C)oleic acid and (omega-14C)nervonic acid

During feeding experiments with [omega-14C]oleic acid and [omega-14c]nervonic acid to adult rats, 14C-labelled C26, C28 and C30 fatty acids were recovered from the intestinal mucosa, liver, plasma, kidney and stools. The structures of these fatty acids were determined by g.l.c., radio-g.l.c. and mass spectrometry. The Schmidt and Ginger degradation methods indicated that most of the 14C found in these extra-long fatty acids remained in the omega position. These radioactive extra-long fatty acids were found mainly in the polar lipids of rats killed 3 or 15 h after being fed on labelled oleic acid or nervonic acid. Rats killed 63 h later yielded only traces of these extra-long fatty acids. When the rats were given antibiotics or received the same radioactive fatty acids by intravenous injection, the labelled extra-long fatty acids could not be detected in any of the tissues. We conclude that they were probably synthesized by elongation of oleic acid and nervonic acid by intestinal micro-organisms (probably yeasts) and then absorbed by the intestinal mucosa.