Metabolism of arachidonate and stearate injected simultaneously into the mouse brain

The metabolism of a polyunsaturated and a saturated fatty acid in brain membrane phosphoglycerides was examined by injecting simultaneously a mixture of¹⁴C-arachidonate and³H-stearate into the mouse brain and isolating the microsomal and synaptosomal fractions at 1–40 min after injections. Both types of labeled fatty acids were utilized more readily in the microsomal than the synaptosomal fractions in brain. However, labeled arachidonate was incorporated more rapidly into membrane phosphoglycerides than was stearate. In both subcellular fractions, the relative specific radioactivity (³H and¹⁴C) of diacyl-glycerophosphorylinositol (diacyl-GPI) was higher than other types of phosphoglycerides such as diacyl-glycerophosphorylcholine (diacyl-GPC) and diacyl-glycerophosphorylethanolamine (diacyl-GPE). Furthermore, the apparent rates of incorporation of radioactivity into diacyl-GPI was more rapid for the¹⁴C-arachidonate than for the³H-stearate. Results of the experiment have demonstrated obvious differences in metabolism between stearate and arachidonate in brain. The more rapid transfer of arachidonate to diacyl-GPI is probably due to the presence of an acyl transferase system specially active for the transfer of arachidonyl groups to diacyl-GPI.     

Stereospecific analysis of triacylglycerols and major phosphoglycerides fromLipomyces lipoferus

The stereospecific distribution of fatty acids in triacylglycerols, phosphatidylcholines and phosphatidylethanolamines ofLipomyces lipoferus was determined. Position sn-1 of the triacylglycerols had a predominance of unsaturated fatty acids of which C_18∶1 (61%) was the major component. Position sn-2 of the triacylglycerols contained 88% C_18∶1 and was more unsaturated than position sn-1 by 24.6%. Position sn-3 had equal proportions of saturated and unsaturated fatty acids. Phosphatidylcholine had a quantitatively distinctive fatty acid distribution in that position sn-2 was 26.7% more unsaturated than position sn-1. In phosphatidylethanolamine, position sn-2 was 10.8% more unsaturated than position sn-1. Positions sn-1 and sn-2 of these phosphoglycerides had a different fatty acid profile from positions sn-1 and sn-2 of the triacylglycerols. These results suggest a nonrandom distribution of fatty acids in the triacylglycerols and phosphoglycerides. Because triacylglycerols and phosphoglycerides are both derived from 1,2-diacylglycerols, these data suggest two possibilities: some selectivity in utilization of species of diacylglycerols to form triacylglycerols and phosphoglycerides or modification of the triacylglycerols and phosphoglycerides after they are formed or both. This study is the first of its kind in a yeast. Presented in part at the AOCS Fall Meeting, Ottawa, Canada, September 1972.     

Relative incorporation of acetate and glucose into glycerides ofGlomerella cingulata

Cultures of the fungusGlomerella cingulata were pulse-labeled for 30 min with 1-¹⁴C-acetate at ages from 2 to 12 days old. The greatest incorporation into triglycerides and phosphoglycerides occurred in the youngest cultures, but more of the radioactive acetate was diverted to the phosphoglycerides than to the triglycerides in these cultures. In another series of experiments the incorporation of 1-¹⁴C-acetate or 2-¹⁴C-glucose into the triglycerides and phosphoglycerides of the fungus was investigated at 15-minute intervals for 1 hr. Hydrolysis of the two classes of glycerides revealed some labeling of glycerol from acetate; but, not surprisingly, acetate was incorporated into the fatty acid moieties to a greater extent than into the glycerol moieties, and there was relatively greater incorporation of 2-¹⁴C-glucose into glycerol than into fatty acids. Some relationships of these results to the growth and development ofG. cingulata are suggested, and implications relative to control mechanisms are pointed out.     

Enzymic regulation of arachidonate metabolism in brain membrane phosphoglycerides

The metabolism of arachidonate in brain membrane phosphoglycerides was investigated in vivo by intracerebral injection of labeled arachidonate and by in vitro assay of enzymic systems associated with the metabolism. After intracerebral injection, labeled arachidonate was incorporated rapidly into brain phosphoglycerides with radioactivity distributed mainly in diacyl-sn-glycero-3-phosphoinositols (GPI) and diacyl-sn-glycero-3-phosphocholines (GPC). Some evidence of a metabolic relationship between diacyl-sn-glycerophosphoinositols (diacyl-GPI) and diacylglycerols was observed. Among the phosphoglycerides labeled with [¹⁴C] arachidonoyl groups, diacyl-GPI were most rapidly metabolized in brain microsomal and synaptosomal fractions. The decay of diacyl-GPI in brain synaptosomes may be represented by two pools with half-lives of 5 hr and 5 days. Three types of enzymic systems related to metabolism of the polyunsaturated fatty acids in brain were investigated. The first system involves the cyclic events relating the ATP-dependent activation of polyunsaturated fatty acids (PUFA) to their acylCoA by the acylCoA ligase and subsequent hydrolysis of acylCoA to free fatty acids by the acylCoA hydrolase. It is apparent that fatty acid activation and hydrolysis is under strigent control in order to maitain suitable levels of free fatty acids and acylCoA in the brain tissue for various metabolic use. Factors involved in the regulation may include the level of ATP, divalent cations and the nature of substrates. The second enzymic system pertains to deacylation via phospholipase A₂ and reacylation via the acyltransferase of membrane phosphoglycerides. In brain tissue, activity of the acyl transferase is generally higher than that of the phospholipase A₂. Factors known to affect specificity of the acyltransferase include substrate concentration and the nature of the acyl groups and lysophosphoglycerides. The acyltranferase(s) in brain preferentially transfers arachidonate to 1-acyl-GPI. Activity of the acyltransferase can be inhited by a number of lypophilic compounds including local anesthetics and cell surface agents. Activity of the phospholipase A₂ in brain may depend on the physical form of the substrates, i.e., whether the substrates are in monomeric or micellar form. The third process is associated with the degradation of diacyl-GPI by enzymes present in brain subcellular membranes. Incubation of brain subcellular membranes with 1-acyl-2-[¹⁴C] arachidonoyl-GPI yielded labeled diacylglycerols and arachidonate. The phospholipase C action is specific for hydrolysis of diacyl-GPI. The arachidonate released from incubation of labeled diacyl-GPI may be the result of phospholipase A₂ action which is not specific for diacyl-GPI or the hydrolysis by lipase acting on the diacylglycerols formed from the phospholipase C activity. Enzymic hydrolysis of diacyl-GPI is most active in the microsomal fraction, but uoon disruption of synaptosomes, enzyme in synaptic plasma membranes is also active in degradating this glycerophospholipid. In general, the results of in vitro studies are in good agreement with those observed in vivo and the information yielded has contributed towards understanding the metabolism of polyunsaturated fatty acids in brain subcellular membranes.     

Differential trypsin effect upon (1-14C) acetate incorporation into choline and ethanolamine glycerophosphatides of rat brain and liver

Preincubation of rat brain and liver slices in a medium (5% glucose, 5% fructose, 1% albumin, 1% trypsin, 10 mM phosphate buffer pH 6.0) used to pretreat brain tissue for the separation of cell types was found to uncouple the incorporation of (1-¹⁴C) acetate into ethanolamine phosphoglycerides from that of the choline phosphoglycerides. Incorporation into ethanolamine phosphoglycerides was stimulated in both brain (330%) and liver (780%) slices, while the incorporation of (1-¹⁴C) acetate into choline phosphoglycerides was reduced for both brain (71%) and liver (63%) slices, compared to control values from nonpreincubated material. With (1-¹⁴C) linolenic acid as a precursor, no significant differences were found in incorporation into ethanolamine phosphoglycerides and choline phosphoglycerides.     

Acyltransferases in subcellular fractions of developing seeds of rape (Brassica napus L.)

Acyltransferase activities responsible for the transfer of oleoyl moieties from oleoyl-CoA to various lipids have been examined in subcellular fractions of developing seeds of rape,Brassica napus L. In the absence of exogenous acyl acceptors, the microsomal and oil body fractions transferred oleoyl moieties mostly to phosphatidylcholines and phosphatidic acids, although there was substantial incorporation of the oleoyl moieties into monoacylglycerols, diacylglycerols and triacylglycerols. The soluble (150,000-g supernatant) fraction incorporated oleoyl moieties mainly into the neutral lipids (monoacylglycerols and diacylglycerols) and also exhibited a relatively high acyl-CoA hydrolase activity. In the presence of the exogenous acyl acceptors, lysophosphatidylcholine and lysophosphatidic acid, both microsomal and oil body fractions transferred most of the oleoyl moieties to phosphatidylcholines and phosphatidic acids, respectively. Other lysophospholipids, such as lysophosphatidylethanolamine and lysophosphatidylonositol, were not very effective as acyl acceptors, nor were glycerol-3-phosphate, monoacylglycerols or diacylglycerols. In contrast, the soluble fraction showed little or no stimulation of acyltransfer in the presence of exogenous lysophospholipids but was able to utilize, to some extent, exogenous monoacylglycerols and diacylglycerols as acyl acceptors.     

Chain length specificity in the utilization of long chain alcohols for ether lipid biosynthesis in rat brain

A mixture ofcis-9-[1-¹⁴C] octadecenol and [1-¹⁴C] docosanol was injected into the brains of 19-day-old rats, and incorporation of radioactivity into brain lipids was determined after 3, 12, and 24 hr. Both alcohols were metabolized by the brain but at different rates; each was oxidized to the corresponding fatty acid, but oleic acid was more radily incorporated into polar lipids. Substantial amounts of radioactivity were incorporated into 18∶1 alkyl and alk-1-enyl moieties of the ethanolamine phosphoglycerides and into 18∶1 alkyl moieties of the choline phosphoglycerides. Even after the disappearance of the 18∶1 alcohol from the substrate mixture (12 hr), the 22∶0 alcohol was not used to any measurable extent for alkyl and alk-1-enyl glycerol formation.     

Lipid metabolism of brain tissue in culture

Tissue explants from frontal lobes of rat brain were used for the study of cerebral fatty acid metabolism. After tissues had been maintained in serum-supplemented medium, a lipid free medium was substituted and metabolic studies were carried out. Under these conditions explants continued to take up lipid precursors for at least 48 hr. Stearic acid 1-C¹⁴, palmitic acid 1-C¹⁴ and lignoceric acid 1-C¹⁴ were bound to cells as the free fatty acids or incorporafed into neutral lipids (particularly triglycerides), glycolipids and phospholipids. In the galactolipid fraction, cerebrosides were the principal radioactive lipids. Choline phosphoglycerides, ethanolamine phosphoglycerides, inositol phosphoglycerides and serine phosphoglycerides were the principal radioactive phospholipids. Fatty acids were incorporated into cellular lipids either unchanged or after desaturation, chain elongation, or both. In a patient with a demyelinating disease, precursor uptake was reduced and chain elongation and desaturation of the fatty acid was diminished. In a patient with generalized G_M2 gangliosidosis, glycolipids other than cerebrosides were labeled to a greater extent than normal. These studies exemplify the usefulness of tissue explants for prolonged metabolic studies in normal and pathological specimens of brain. One of 13 papers presented at the symposium “Lipid Metabolism in Cells in Culture,” AOCS Meeting, Houston, May, 1971.     

Glyceryl ethers in insects: Biosynthesis of ethanolamine phosphoglycerides containing alkyl and alk-1-enyl glyceryl ether linkages

When¹⁴C-labeled acetate, fatty acids or fatty alcohols were injected into or fed to the tobacco budworm, acyl, alkyl and alk-1-enyl moieties of the phospholipids incorporated radioactivity. Fatty acids were the principal precursor in acyl bond formation and fatty alcohols in the synthesis of alkyl and alk-1-enyl glyceryl ethers. Detailed analysis of the etherlinked phosphoglycerides revealed that most of the radioactivity was in the ethanolamine phosphoglycerides, and very little¹⁴C was found in the choline phosphoglycerides. In experiments of a short duration, the alkyl glyceryl ethers incorporated more radioactivity than the alk-1-enyl glyceryl ethers. The reverse was found with long term experiments, when the alk-1-enyl ethers had higher radioactivity. In addition to demonstrating the synthesis of ether-linked ethanolamine phosphoglycerides, the data suggested that fatty alcohols and acids were interconverted by insects and that the alk-1-enyl ethers were derived from the alkyl ethers. Presented at the AOCS Meeting, Houston, May 1971. The following abbreviations and terminology will be used: PE, PC, PI and PS for the generic terms ethanolamine, choline, inositol and serine phosphoglycerides, respectfully. Alkyl glyceryl ether for 1-alkyl-2-acyl-sn-glycerol-3-phosphoryl-, and alk-1-enyl glyceryl ether for 1-alk-1′-enyl-2-acyl-sn-glycerol-3-phosphoryl-(commonly called plasmalogen). These are adapted from the tentative rules published inJ. Lipid Res. 8:522–528 (1967).     

Lipids of cultured hepatoma cells: III. Triglyceride and phosphoglyceride biosynthesis in minimal deviation hepatoma 7288C

Minimal deviation hepatoma 7288 C cells were cultured on media containing 25% serum to the confluent stage. The growth media was replaced with serumfree media containing 1-¹⁴C-palmitate, and incubations were continued for 0.75, 1.5, 3, 6, 12, and 24 hr. The distribution of radioactivity among the major neutral lipids and phosphoglycerides was determined for cells and culture media. Radioactivity in individual fatty acids of cellular triglyceride, phosphatidylcholine, and phosphatidylethanolamine also was determined. After 24 hr, more than 95% of the administered radioactivity was recovered in neutral and phosphoglycerides, indicating that only a small amount of the fatty acid was oxidized. At any time period examined, over 80% of the incorporated radioactivity was found in triglyceride, phosphatidylcholine, and phosphatidylethanolamine. Incorporation of the label into cellular triglyceride and phosphatidylcholine plateaued at 12 hr, whereas incorporation of radioactivity into phosphatidylethanolamine still was increasing at 24 hr. In contrast, during the entire incubation period the relative distribution of¹⁴C among esterified lipid classes in the culture media remained constant. Elongation of palmitic acid to stearic acid and its subsequent desaturation to oleic acid suggests that these cells possess an active elongation and monoenoic desaturation system. Labeled glycerol ether diesters were not detected in the cells or culture media. Positional distribution of the¹⁴C label in the triglyceride and phosphatidylcholine suggests that minimal deviation hepatoma cells do not exhibit diglyceride selectivity in the biosynthesis of these two lipid classes.     

Effects of omega-3 fatty acids on vascular smooth muscle cells: Reduction in arachidonic acid incorporation into inositol phospholipids

A rapid increase in arachidonic acid incorporation into phosphatidylinositol (PI) occurred following exposure of cultured porcine pulmonary artery smooth muscle cells to calcium ionophore A23187. This response was specific for PI and phosphatidic acid; none of the other phosphoglycerides showed any increase in arachidonic acid incorporation. The incorporation of [³H]inositol also was increased, indicating that complete synthesis of PI rather than only fatty acylation occurred in response to the ionophore. The presence of omega-3 fatty acids, especially eicosapentaenoic acid (EPA), reduced arachidonic acid but not inositol incorporation into PI. Stimulated incorporation of EPA also occurred under these conditions, suggesting that EPA replaces arachidonic acid in the newly synthesized pool of PI. Although much less arachidonic acid was incorporated into the polyphosphoinositides following exposure to the ionophore, arachidonic acid incorporation into these phosphorylated derivatives also decreased when EPA was present. These findings suggest that when omega-3 fatty acids are available, less arachidonic acid is channeled into the inositol phospholipids of activated smooth muscle cells because of replacement by EPA. This may represent a mechanism whereby omega-3 fatty acids, especially EPA, can accumulate in the metabolically active pools of inositol phospholipids and thereby possibly influence the properties or responsiveness of vascular smooth muscle.     

The turnover of the lipid components of myelin

The rate of loss of radioactivity in the lipid components of rat myelin labeled with acetate-1-C¹⁴ was determined over a period of one year. Rats were injected with acetate-1-C¹⁴ at 15–16 days of age and purified myelin was prepared by differential ultracentrifugation from brain and spinal cord of this group at 1 day, 2 weeks, 1 month, 2 months, 3 months, 6 months and 1 year after injection. Total lipid was extracted from the myelin preparations and the lipids were separated into their components by thin-layer chromatography. Cholesterol, galactolipid, ethanolamine phosphatide, choline phosphatide, inositol phosphatide, serine phosphatide and sphingomyelin specific activities at each age were measured. Three of the myelin lipid components, serine phosphatide, inositol phosphatide, and choline phosphatide decreased in specific activity faster than cerebroside, cholesterol, sphingomyelin, and ethanolamine phosphatide. Acetate-1-C¹⁴ injected into adult animals, though incorporated into myelin to a very small extent, is taken up primarly in the choline phosphatide. These experiments suggests that myelin does not behave as a fixed entity but that certain constituents may be more actively metabolized than others.     

Effect of pentadecan-2-one on lipid metabolism in HeLa cells

HeLa cells exposed to trace amounts of pentadecan-2-one showed changes in metabolism of 1-¹⁴C-palmitate. These changes consisted of an increased incorporation of radioactivity into the triglycerides and free fatty acids and a decreased¹⁴C incorporation into the ether moiety of alk-1-enyl acyl phosphoglycerides. Chemical analysis of the several lipid fractions showed a threefold increase in triglyceride content but no change in the amount of alk-1-enyl acyl or diacyl phosphoglycerides in the treated cells. Pentadecan-2-one added to the culture medium apparently gains entrance to the cell since both pentadecan-2-one and pentadecan-2-ol were detected in the ketone-treated cells and their culture medium.     

Metabolism of long-chain fatty acids,alcohols and alkylglycerols in the fish parasiteParatenuisentis ambiguus (Acanthocephala)

Specific differences between the acyl composition of lipids on the helminthParatenuisentis ambiguus and its host eel, as shown previously, prompted us to study the lipid metabolism in this intestinal fish parasite. Adults and larvae ofP. ambiguus were fed various lipid precursors, e.g., fatty acids, long-chain alcohols and 1-O-alkylglycerols, which may occur as common nutrients of intestinal parasites. Incorporation of [1-¹⁴C]palmitic acid into neutral and polar lipids was found to be similar under aerobic and near-anaerobic conditions. In adult parasites maintained in culture medium supplemented with glucose, [1-¹⁴C]palmitic acid was incorporated mainly into triacylglycerols and phosphatidylcholines, whereas [1-¹⁴C]oleic acid was incorporated preferentially into triacylglycerols. In fasted adults, as well as in larvae, [1-¹⁴C]oleic acid was mainly transferred to phosphatidylcholines. Lipolytic activity was detected in adult parasites that had been incubated with radioactive trioleoylglycerol. [1-¹⁴C]Hexadecan-1-ol was oxidized inP. ambiguus at a high rate to labeled palmitic acid, which was incorporated into various lipid classes ofP. ambiguus. Small but significant proportions of radioactivity from hexadecan-1-ol were incorporated into ether glycerolipids of the parasite. A more direct precursor in ether glycerolipid metabolism, i.e.,rac-1-O-[1′-¹⁴C] hexadecylglycerol, was incorporated into alkyl and 1′-alkenyl moieties of choline and etha-nolamine etherglycerophospholipids ofP. ambiguus in high yield. High proportions of labeled diacylglycerols, triacylglycerols and steryl esters were detected in surface lipids as well as lipid extracts of the culture media after incubation ofP. ambiguus with [1-¹⁴C]palmitic or [1-¹⁴C]oleic acids. The results suggest that palmitic acid and oleic acid are incorporated into neutral and polar lipids ofP. ambiguus maintained in glucose medium quite differently with oleic acid showing a strong preference for triacylglycerols. However, the incorporation of palmitic acid in glucose-fed parasites was similar to that of oleic acid in fasted parasites, as well as in larvae. This may be explained by partial fatty acid depletion in fasted worms and rapid cell division in larvae, respectively.     

Studies of differential turnover of palmitoyl and stearoyl species of glycerophosphatides using labeled unsaturated acids

Normal and bile fistula rats were injected with 1-¹⁴C-linoleate and arachidonate as albumin complex and the glycerides and glycerophosphatides of the liver and bile were isolated at various time intervals. The distribution of radioactivity among the individual molecular species was determined by thin layer and radio gas chromatography and specific enzymic hydrolyses. At 30 min after administration of linoleate 30% of the radioactivity in liver was in lecithins and 8% in cephalins, while at 120 min 48% was in lecithins and 16% in cephalins. After arachidonate, 58% and 64% of the counts were in lecithins and 12% to 13% in the cephalins at the above periods of sampling. The specific activity of the palmitoyl linoleoyl lecithins and cephalins was two to three times higher than that of the corresponding stearoyl linoleoyl species, which was of the same order but much lower magnitude than found previously for lecithins using labeled phosphate and choline. The palmitoyl and stearoyl species of arachidonoyl lecithins possessed equal specific activities, in sharp contrast to previous findings with radioactive phosphate, which showed a 12 times higher specific activity for the palmitoyl arachidonate. The palmitoyl arachidonoyl cephalins had two to three times greater specific activity than the corresponding stearoyl species in agreement with previous work using labeled phosphate. The distribution of radioactivity suggests that the arachidonate was incorporated into the lecithins largely via acyl transfer, while the linoleate contributed to both acyl transfer and de novo synthesis. Interpretation of the mechanism of uptake of these acids into the cephalins awaits further studies. Presented in part at the Federation of American Societies for Experimental Biology Meeting, Atlantic City, April 1970.     

Incorporation of 1,2-14C-ethanolamine into subfractions of rat liver phosphatidylethanolamines and phosphatidylcholines

The incorporation of 1,2-¹⁴C-ethanolamine into the liver phosphatidylethanolamines (PE) and phosphatidylcholines (PC) in female rats was studied. These phosphatides were fractionated according to their degrees of unsaturation and the specific activities of monoenoic, dienoic, tetraenoic and hexaenoic fractions were measured at intervals of 1, 20, 60 and 300 min after injection of the labeled precursor. Hexaenoic and dienoic PE incorporated and lost the label rapidly. Although the labeled precursor was incorporated into tetraene PE at a similar rate, this fraction attained a relatively low specific activity that remained essentially constant between 10 and 300 min after injection of the label. Hexaenoic PC had the highest specific activity among the PC fractions at all time periods. Estimations of the rate of loss of radioactivity in the hexaenoic PE fraction and its appearance in hexaenoic PC indicate that the docosahexaenoic acid is conserved, possibly by being reincorporated into PE after becoming a part of the hexaenoic PC species. The high rate of turnover of the hexaenoic PE also suggests that this fraction might have some special role in endogenous choline synthesis.     

Lipase catalyzed synthesis of neutral glycerides rich in micronutrients from rice bran oil fatty acid distillate

Neutral glycerides with micronutrients like sterols, tocopherols and squalene may be prepared from cheap raw material like rice bran oil fatty acid distillate (RBO FAD). RBO FAD is an important byproduct of vegetable oil refining industries in the physical refining process. Glycerides like triacylglycerols (TAG), diacylglycerols (DAG) and monoacylglycerols (MAG) containing significant amounts of unsaponifiable matter like sterols, tocopherols and hydrocarbons (mainly squalene) may certainly be considered as novel functional food ingredients. Fatty acids present in RBO FAD were esterified with glycerol of varying amount (1:0.33, 1:0.5, 1:1 and 1:1.5 of FAD : glycerol ratio) for 8 h using non-specific enzyme NS 40013 (Candida antartica). After esterification the product mixture containing mono, di- and triglycerides was purified by molecular distillation to remove excess free fatty acids and also other volatile undesirable components. The purified product containing sterols, tocopherols and squalene can be utilized in various food formulations.     

The incorporation of ethanolamine into ether-containing lipids in rat brain

After intracerebral injection of C¹⁴-ethanolamine into rats, the ethanolamine phosphoglycerides were isolated and hydrolyzed with mild alkali and acid. The specific radioactivity of the diacyl, acyl alkenyl, and acyl alkyl glyceryl-3-phosphorylethanolamine, the diacyl and acyl alkenyl glyceryl-3-phosphorylcholine, and sphingomyelin was determined at 0.5, 2, 24, and 48 hours. The specific radioactivity-time relationships show that the ethanolamine plasmalogen is not a precursor for the glyceryl ether form but suggest that acyl alkyl glyceryl-3-phosphorylethanolamine is desaturated to form some of the acyl alkenyl glyceryl-3-phosphorylethanolamine. The radioactivity in the choline portion of the choline phospholipids was very low. Presented at the AOCS Meeting, Cincinnati, October 1965.     

Accumulation of neutral lipids by human skin fibroblasts: Differential effects of saturated and unsaturated fatty acids

The accumulation of neutral lipids by human skin fibroblasts grown in medium supplemented with fatty acids has been investigated. GM-10 cells incorporated exogenous fatty acids into both phospholipids and neutral lipids. More [¹⁴C] oleate, linoleate, or linolenate was incorporated into triacylglycerol than was [¹⁴C] palmitate or stearate. Supplementation of medium containing delipidized serum with unsaturated fatty acids resulted in far more stimulation of [¹⁴C] glycerol incorporation into triacylglycerol than did supplementation with saturated fatty acids. Palmitate- and stearate-fed cells incorporated sizable amounts of [¹⁴C] fatty acids and [¹⁴C] glycerol into diacylglycerol as well as triacylglycerol, especially at higher fatty acid concentrations. Increased oleate supplementation from 10–300 μM resulted in increased triacylglycerol synthesis and accumulation of discrete cytoplasmic lipid droplets; palmitate concentrations above 70 μm were toxic. Micrographs of the palmitate-fed cells showed electron translucent slits, suggesting solid depositions of saturated fat, rather than the discrete osmiophilic droplets found in oleate-fed cells. Although GM-10 cells can synthesize fully saturated triacylglycerols, these data suggest that in cells fed saturated fatty acids, solid depositions of neutral lipids may sequester diacylglycerols and thus limit triacylglycerol synthesis.     

Positional specificity oftrans fatty acids in fetal lecithin

Differences in the positional incorporation of 9-trans[1-¹⁴C] octadecenoic (elaidic) and 9-trans,12-trans[1-¹⁴C] octadecadienoic (linoelaidic) acids in fetal lecithin of rats were demonstrated. On the 20th day of gestation, a¹⁴C-labeled albumin complex of elaidic or linoelaidic acid was injected into the jugular vein of pregnant rats. For comparative purposes, 9-cis[1-¹⁴C] octadecenoic (oleic) or 9-cis,12-cis[1-¹⁴C] 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 tissue 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, radioactivity of FAME was distributed between palmitate and elaidic acid indicating that rat fetal tissue may metabolize elaidic acid via β-oxidation. In contrast, following injection of linoelaidate, radioactivity of FAME was primarily associated withtt-18∶2, suggesting little biotransformation to other fatty acids by fetal tissues.