Tetraenoic Species Research Articles

Separation of milk fat triacylglycerols by argentation thin‐layer chromatography

AbstractArgentation thin‐layer chromatography was investigated as a method of obtaining detailed compositional information about milk fat. A modified argentation thin‐layer chromatography procedure, developed to optimize the separation of the complex mixture of total milk fat triacylglycerols, provided nine different groups of triacylglycerols, based on both the degree of unsaturation and the total length of fatty acid groups. Fatty acid methyl ester (FAME) analysis was performed to determine the composition of each band. Separation on the basis of chainlength was most pronounced among the fully saturated triacylglycerol groups, as evidenced by the high level of C4:0 and C6:0 in bands 7 and 8, respectively. For the cis‐monoenoic triacylglycerols, the separation of C4:0 and C6:0 was less distinct. The cis,cis dienes and other dienoic, trienoic, or tetraenoic species were principally observed in two bands of retention factor <0.08 on the chromatography plate. Minimal cross‐contamination of bands was observed, with the exception of the lowest of the trisaturate bands, band 7, in which trans‐monoenes were found to be present. Three samples from different points of the New Zealand dairy season were separated by argentation thin‐layer chromatography, and their FAME distributions were measured. In addition to differences in the masses of band extracts from these samples, levels of C10:0 and C12:0 in all bands, and levels of monounsaturates in the dienoic and trienoic bands, were found to differ. These changes were generally consistent with a pattern of decreasing fat hardness over the November to March period of a typical dairy season.

Agonist-induced endocytosis of muscarinic cholinergic receptors: relationship to stimulated phosphoinositide turnover.

The ability of muscarinic cholinergic receptors to activate phosphoinositide turnover following agonist-induced internalization has been investigated. Incubation of SH-SY5Y neuroblastoma cells with oxotremorine-M resulted in a time-dependent endocytosis of both muscarinic receptors and alpha subunits of G0 and G11, but not of isoforms of phosphoinositide-specific phospholipase C, into a subfraction of smooth endoplasmic reticulum (V1). Agonist-induced increases in diacylglycerol mass and in 32P-phosphatidate labeling, much of which was of the tetraenoic species, were also observed in the V1 fraction, but these increases persisted when the agonist-induced translocation of receptors into the V1 fraction was blocked. All enzymes of the phosphoinositide cycle were detectable in the V1 fraction. However, with the exception of phosphatidylinositol 4-kinase, none was enriched when compared with cell lysates. Both 32P-labeling studies and enzyme assays point to a very limited capacity of this fraction to synthesize phosphatidylinositol 4,5-bisphosphate, whereas the synthesis of phosphatidylinositol 4-phosphate is robust. These results indicate that endocytosed receptors do not appear to retain their ability to activate phosphoinositide turnover. The availability of the substrate for phospholipase C, phosphatidylinositol 4,5-bisphosphate, may be one factor that limits the activity of muscarinic receptors in this subcellular compartment.

Arachidonic acid modulates [14C]stearic acid incorporation into phosphatidylinositol, in human neuroblastoma cells.

In the human neuroblastoma cell line SK-N-BE(2), arachidonic acid (AA), supplied in the medium at micromolar concentrations, markedly enhanced [14C]stearic acid (SA) (but not [14C]palmitic acid or [14C]oleic acid) incorporation into phosphatidylinositol (PtdIns). AA failed to stimulate [14C]SA incorporation into PtdIns precursors, namely phosphatidic acid and cytidinediphosphodiacylglycerol: furthermore, enhanced [14C]SA incorporation, brought about by exogenously administered AA, was not restricted to PtdIns tetraenoic species. When cells were pulsed for 1 h with [14C]SA (either in the presence or absence of AA) and then reincubated in AA- and [14C]SA-free medium, a marked loss of radioactivity from PtdIns was observed, that however was restricted to molecular species other than tetraenoic. These results are discussed in the light of possible mechanisms through which PtdIns achieves the 1-stearoyl-2-arachidonoyl configuration.

Inositol phospholipid arachidonic acid metabolism in GH3 pituitary cells.

Inositol phospholipids in cultured GH3 cells, a prolactin secreting, thyrotropin-releasing hormone (TRH) sensitive rat pituitary cell line, exhibit a preferential selectivity for incorporating arachidonic acid. Fatty acid composition data show that all inositol phospholipids are enriched in stearic and arachidonic acids to a much greater degree than other cellular phospholipids. Incubation of GH3 cells with radioactive stearate, oleate, arachidonate, eicosapentaenoate or docosahexaenoate also showed that much more stearate and arachidonate were incorporated into inositol phospholipids. In short term incubations with tracer amounts of radioactive arachidonate, incorporation was initially into phosphatidylinositol (PtdIns), with phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] being labelled at later times. During longer incubations, all of the inositol phospholipids reach equilibrium at about 10 h, and the resulting specific activities of the three fractions were similar. These findings suggest that arachidonate is incorporated initially into PtdIns and that PtdIns is then phosphorylated. There was no release of either arachidonate or eicosanoid products when GH3 cells were incubated with TRH. However, TRH stimulation of 32P-labelled GH3 cells resulted in rapid breakdown of PtdIns(4,5)P2 and PtdIns4P, with concomitant increases in [32P]phosphatidic acid and [32P]PtdIns. When the [32P]PtdIns was further analysed by argentation chromatography to separate PtdIns molecular species, it was found that tetraenoic (stearate/arachidonate) species accounted for 80% of the stimulated labelling. The selectivity for arachidonate incorporation into inositol phospholipids coupled with turnover of the arachidonate-containing molecular species suggests that inositol phospholipids containing arachidonic acid or the diacylglycerol resulting therefrom may play a vital cellular role in GH3 cells. This role may involve the operation of the PtdIns cycle itself rather than a stimulated release of arachidonate for eicosanoid formation.

The biosynthesis of phosphatidylserine and phosphatidylethanolamine from l-[3- 14C]serine in isolated rat hepatocytes

Incorporation of l-[3- 14C]serine into phosphatidylserine (PS) and phosphatidylethanolamine (PE) has been studied in isolated rat hepatocytes. Ethanolamine inhibited the incorporation, indicating competition with serine in the base-exchange reaction. Choline, monomethylethanolamine, dimethylethanolamine and dimethyl-3-aminopropan-1-ol had no such effect. The observed rate of PS biosynthesis corresponded to 7–17 nmol/min per liver at 0.55 mM l-serine. The results indicate that only a small fraction ( 1 25 to 1 70 ) of the PS pool equilibrates with the base-exchange enzyme, and that decarboxylation to PE occurs preferentially from this pool. The rate of PS synthesis and decarboxylation can therefore not be calculated by methods which assume random, homogeneous labelling of the total PS pool. The apparent rate of PS decarboxylation increased approx. 4-fold when l-serine increased from 0.5 to 2.25 mM, suggesting that decarboxylation of PS to PE might be regulated by the concentration of l-serine or by the amount of PS present in the hepatocyte cell membranes. Lauric, palmitic, stearic, oleic and linoleic acid decreased the rate of PS synthesis. At 0.5 mM, lauric and palmitic acid were most inhibitory. At 1.0 mM, linoleic acid was the least inhibitory fatty acid. The saturated hexaenoic and saturated tetraenoic species of PS contained 51 and 29%, respectively, of the incorporated l-[3- 14C]serine. The combined monoene dienoic/diene dienoic fraction had the highest rate of synthesis judged by its relative specific activity. At 0.9 mM concentration, linoleic acid doubled the relative specific activity of the combined monoene dienoic/diene dienoic fraction of PS. Incorporation of l-[3- 14C]serine into molecular species of PE resembled that into PS, both in the absence and presence of linoleic acid, suggesting that the phosphatidylserine decarboxylase (EC 4.1.1.65) has a low specificity towards the fatty acid composition of PS. The results indicate that biosynthesis of PS from l-serine occurs mainly by the base-exchange with only negligible contribution from direct incorporation of phosphatidic acid or diacylglycerol. Furthermore, the deacylation-reacylation pathway seem to contribute only little to the determination of the fatty acid composition of hepatocyte PS. Active PS turnover seems to be confined to a small fraction of the PS pool.

Tetraenoic species are conserved in muscarinically enhanced inositide turnover.

Carbamylcholine enhances the labeling of phosphatidate and phosphatidylinositol from 32Pi in nerve endings. Approximately 74% of labeled phosphatidate and 85% of labeled phosphatidylinositol produced on muscarinic stimulation are accounted for by tetraenoic species, as detected by argentation TLC. Incubation of membranes derived from nerve endings with [gamma-32P]ATP under conditions of phosphodiesteratic degradation of endogenous polyphosphoinositides resulted in increased labeling of phosphatidate. Approximately 78% of the newly formed phosphatidate was in a tetraenoic fraction. It is concluded that in muscarinically stimulated nerve endings, the diacylglycerol moiety is conserved following diacylglycerol release from polyphosphoinositides through its resynthesis to inositol lipid via phosphatidate.

Inactivation of platelet activating factor by rabbit platelets. Lyso-platelet activating factor as a key intermediate with phosphatidylcholine as the source of arachidonic acid in its conversion to a tetraenoic acylated product.

[3H]PAF (platelet activating factor or 1-alkyl-2-acetyl-GPC) is converted to 1-alkyl-2-lyso-GPC and 1-alkyl-2-acyl-GPC by rabbit platelets (GPC is sn-glycero-3-phosphocholine). The deacetylation reaction does not involve the transfer of the acetate of PAF to any other lipid class and added exogenous lyso-PAF readily mixes with the cellular pool of the [3H]lyso-PAF intermediate formed from [3H]PAF. [3H]1-Alkyl-2-acyl-GPC produced during the inactivation of [3H]PAF contained primarily the tetraenoic acyl species (approximately 80% of the 3H in this fraction). The source of the arachidonic acid used for the reacylation of the lyso-PAF intermediate is the diacyl species, phosphatidylcholine.

Enhanced turnover of arachidonic acid-containing species of phosphatidylinositol and phosphatidic acid of concanavalin astimulated lymphocytes

The incorporation of [ 32P]orthophosphate into phosphatidylinositol (PI) of pig lymphocytes was markedly increased by stimulation with concanavalin A. The labeling of PI with [ 3H]glycerol was also enhanced significantly, indicating that both de novo synthesis and recircular system (PI response) of PI were accelerated. This rapid labeling of PI might be related to the rapid breakdown of phosphatidylinositol 4,5-bisphosphate which was observed in various stimulated tissues. Concanavalin A also accelerated the labeling of phosphatidic acid with 32P and [ 3H]glycerol. To determine the dependence of this phenomenon on the fatty acid composition of both phospholipids, we separated PI and phosphatidic acid into individual molecular species. The predominant molecular species in PI was tetraene (81.6%) and those in phosphatidic acid were monoene (53.0%), diene (15.8%) and tetraene (19.2%), respectively. Interestingly, the incorporation of 32P into arachidonic acid-containing species (tetraene) was most rapidly elevated. On the other hand, the increment of 32P into saturated + monoene, diene and triene was relatively smaller and resembled that of [ 3H]glycerol. Similarly, the incorporation of 32P into tetraene of phosphatidic acid was preferentially accelerated. This is the first report concerning the metabolism of molecular species of phosphatidie acid in stimulated cells. These results indicate that the PI recirculating system is virtually dependent on tetraenoic species and that the participation of other molecular species is small. The increased de novo synthesis mainly depends upon molecular species other than tetraene. Arachidonic acid-containing species which turn over rapidly via the PI cycle may have an important role in the mitogenic triggering.

The incorporation of [3H]glycerol and 1-[14C]acyl-sn-glycero-3-phosphocholine into different molecular species of phosphatidylcholine in human platelets.

The molecular species of phosphatidylcholine which are formed by de novo synthesis and by the acylation of 1-acyl-sn-glycero-3-phosphocholine were characterized and compared in human platelets. For this purpose, intact human platelets were incubated in the presence of [3H]glycerol or 1-[14C]palmitoyl-sn-glycero-3-phosphocholine and the newly formed radioactive phosphatidylcholine was isolated by thin-layer chromatography. The labelled phosphatidylcholines were converted to their 1,2-diacylglycerol acetate derivatives and fractionated into their various chemical classes (saturates, monoenes, dienes, trienes, tetraenes, and greater than tetraenes) by argentation thin-layer chromatography. Regardless of the precursor used, the radioactivity distributions among the various classes did not correspond closely to that of the mass. The highest percentage of the radioactivity incorporated from [3H]glycerol was found in the saturates (25% of total), followed closely by the tetraenes (21%) and monoenes (18%), with lesser amounts in the dienes (15%), pentaenes plus hexaenes (14%), and trienes (7%). These results indicate that the de novo pathway is capable of substantial synthesis of tetraenoic (1-acyl 2-arachidonoyl) phosphatidylcholine in human platelets in contrast to previous observations in rat liver. In close agreement with work in rat liver, 59% of the radioactivity in the [14C]phosphatidylcholine derived from 1-[14C]palmitoyl-sn-glycero-3-phosphocholine was found in the tetraenoic species. The present results, together with the existence of phospholipase A2 and acyltransferase activity in platelets, support the potential importance of a deacylation-acylation cycle enrichment of human platelet phosphatidylcholine in arachidonoyl species.

Relative degradation of different molecular species of phosphatidylcholine in thrombin-stimulated human platelets.

The relative degradation of the various molecular species of [3H]phosphatidylcholine in response to thrombin was studied in human platelets following prelabeling with [3H]glycerol and compared to results obtained following labeling with [14C]oleic, [14C]linoleic, or [14C]arachidonic acids. This was of interest since previous work using radioactive fatty acids had led to the conclusion that the 1-acyl-2-arachidonoyl species of phosphatidylcholine is exclusively hydrolyzed in thrombin-stimulated platelets. Within 90 s, the thrombin-dependent release of [14C]arachidonic acid from phosphatidylcholine amounted to 25% but only 3 and 6% for oleic and linoleic acids, respectively, in general agreement with previous work. However, for [3H]glycerol-labeled phosphatidylcholine, all molecular species (saturates, monoenes, dienes, trienes, tetraenes, and greater than tetraenes) were subject to significant hydrolysis in the presence of thrombin within 90 s, ranging from 12-24% across the various classes. Furthermore, the degradation of the tetraenoic species (1-acyl-2-arachidonoyl) of [3H]phosphatidylcholine was found to be only 1.5 and 1.4 times that for the monoenoic (predominantly 1-acyl-2-oleoyl) and dienoic (predominantly 1-acyl-2-linoleoyl) species, respectively. A much heavier proportional labeling of plasma membrane relative to whole platelet phosphatidylcholine was observed with [3H]glycerol as compared to [14C] oleate or [14C]arachidonate. These results indicate that the 1-acyl-2-arachidonoyl species of phosphatidylcholine are not exclusively degraded by phospholipase A2 activity in thrombin-stimulated platelets and suggest that the differential compartmentation of molecular species of phosphatidylcholine according to their metabolic origins can influence their apparent susceptibility to hydrolysis.

Molecular species of phosphatidylcholine, -ethanolamine, -serine, and -inositol in microsomal and photoreceptor membranes of bovine retina.

The molecular species compositions of phosphatidylcholine, -ethanolamine, -serine, and -inositol in microsomes and rod outer segments from bovine retina were studied by means of argentation thin-layer chromatography (TLC) after conversion of the phospholipids to labeled acetyldiacylglycerols. A highly unsaturated group of molecular species, called "supraenes" to indicate the presence of more than six double bonds per molecule, was present in all four glycerophospholipids. Docosahexaenoic acid was the major component of these species, in combination with other unsaturated fatty acids, predominantly polyunsaturates. The presence of species containing docosahexaenoate in both positions of the glycerol backbone was suggested by the high percentage (95%) of these fatty acids in the most polar fraction of phosphatidylcholine. In rod outer segments, supraenes were the major component of phosphatidylserine (51% of the molecular species), followed by phosphatidylcholine (31%), phosphatidylethanolamine (21%), and phosphatidylinositol (9%). Hexaenes, which contained docosahexaenoate and saturated fatty acids, predominated in phosphatidylethanolamine (67%), followed by phosphatidylserine and phosphatidylcholine (about 36%), and phosphatidylinositol (12%). Tetraenes made up half the phosphatidylinositols, but amounted to less than 5% of the other glycerophospholipids. Disaturated and monoenoic species made up 14 and 6%, respectively, of phosphatidylcholine. In microsomes, there was less of the docosahexaenoate-containing species and more of the saturated to tetraenoic species. Monoenes predominated in phosphatidylcholine (35%), tetraenes in phosphatidylinositol (71%), and hexaenes in phosphatidylethanolamine and phosphatidylserine (50 and 42%). Supraenes amounted to less than 15% of each of the four glycerophospholipids in microsomes.-Aveldaño, M. I., and N. G. Bazán. Molecular species of phosphatidylcholine, -ethanolamine, -serine, and -inositol in microsomal and photoreceptor membranes of bovine retina.

Metabolic changes in scaly lesions of rat skin produced by essential fatty acid deficiency.

Marked changes in the structure and metabolism of lipids were observed at scaly lesions induced in the skin of rats by the feeding for four months of a diet deficient in essential fatty acids. The concentration of neutral lipids increased in the experimental skin specimens with the exception of free cholesterol. Analysis of fatty acids in the skin lipids of these essential fatty acid deficient rats showed a marked increase in monoenoic and eicosatrienoic acids, with a concomitant decrease in linoleic and arachidonic acids. Incubation of skin specimens from the essential fatty acid deficient rats with [2-3H]glycerol and [1-14C]acetate showed a marked increase in the incorporation of both precursors into all lipid classes. Particularly, their incorporations into phosphatidylcholine were predominant compared to other lipid classes. With regard to the molecular species of phosphatidylcholine, saturated-monoenoic and monoenoic-monoenoic species were highly synthesized in the skin from essential fatty acid deficient animals, and the synthesis of saturated-dienoic and tetraenoic species was very low with either precursor. Significant decreases in these labelings were also noted in saturated-saturated species. Addition of prostaglandin E2 to the incubation medium did not significantly affect the metabolism of any of the lipid classes in the skin from the essential fatty acid deficient rats. The present results, therefore, suggest that phosphatidylcholine containing linoleic acid may be a key lipid in the epidermal barrier function.

The biosynthesis of phosphatidylserines by acylation of 1-acyl- sn-glycero-3-phosphoserine in rat liver

The conversion of 1-[ 14 C]acyl-sn- glycero-3- phosphoserine into molecular species of [ 14C] phosphatidylserine was studied using rat liver homogenate and microsomal preparations in the absence of added fatty acyl moieties. In liver homogenates, 81% of the newly-formed phosphatidylserines were tetraenoic (arachidonoyl) species while saturated, monoenoic, dienoic, trienoic, pentaenoic, and hexaenoic (docosahexaenoyl) species each represented 2–5% of the total. A similar pattern of molecular species was produced in liver microsomes. The selectivity of the microsomal acyl-CoA: 1-acyl- sn-glycero-3-phosphoserine acyltransferase towards different acyl-CoA derivatives was also investigated. The relative suitability of the various acyl-CoA esters as substrates was found to be of the following order: 20:4 = 18:2 > 18:1 > 16:0 = 18:0. These results with endogenous acyl donors suggest that the acylation of 1-acyl- sn-glycero-3-phosphoserine may partly account for the enrichment of liver phosphatidylserine in arachidonic acid but does not appear to be primarily responsible for the preponderance of docosahexaenoic acid in this phospholipid. The fatty acid specificity of the acyl-CoA: 1-acyl- sn-glycero-3-phosphoserine acyltransferase may contribute to the preferential formation of arachidonoyl phosphatidylserine.

Synthesis of phosphatidylcholine and phosphatidylethanolamine at different ages in the rat brain in vitro.

The de novo synthesis of choline and ethanolamine phosphoglycerides in brain microsomes from 18 month-old male rats was investigated in vitro by using labeled cytidine-5'-diphosphate choline and cytidine-5'-diphosphate ethanolamine as lipid precursors. The rate of synthesis of the two phospholipid classes was found to be noticeably decreased, as compared to that of adult animals. The addition of exogenous diacyl glycerols to microsomes from ageing rat brain brings the rate of synthesis nearly to the adult levels. The synthesis of choline and ethanolamine phosphoglycerides is not affected in the liver microsomes of ageing rats. The molar distribution of fatty acids in brain microsomal diacyl glycerols of ageing rats is noticeably different from that of adult animals. The content of monoenoic and dienoic species is increased, whereas that of the tetraenoic species is decreased. Base exchange reaction for choline and ethanolamine incorporation into respective phospholipids is not affected in the brain microsomes of the aged rats.

Comparison of molecular structure of glycerolipids in rat lung

The major individual molecular species of glycerolipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and diacylglycerol of rat lung were determined quantitatively as diacylglycerol acetates. 1. 1. In three glycerolipids such as phosphatidylcholine, phosphatidylglycerol and diacylglycerol, the main molecular species was dipalmitoyl. The species was not detectable in phosphatidylethanolamine and was not of quantitative importance in phosphatidylinositol. 2. 2. The oligoenoic molecular species in phosphatidylethanolamine and phosphatidylinositol contained palmitoyl and stearoyl species in almost equal amounts, while those of phosphatidylcholine, phosphatidylglycerol and diacylglycerol were principally of the palmitoyl types. 3. 3. Tetraenoic species of phosphatidylinositol and diacyl types of phosphatidylethanolamine were predominantly of stearoyl types. Tetraenoic diacylglycerol also contained significant amounts of stearoyl-arachidonyl species. The value was about 3-fold that of palmitoyl-arachidonyl species. However, palmitoyl types were rather higher in phosphatidylcholine and phosphatidylglycerol. 4. 4. The alkenyl-acyl types of lung phosphatidylethanolamine consisted predominantly of polyenoic species, namely, tetraenoic, pentaenoic and hexaenoic species. The pentaenoic and hexaenoic species of all glycerolipids examined consisted mainly of palmitoyl types.

Metabolism of lecithin and phosphatidylglycerol in surfactant fraction of the lung.

The metabolisms of lecithin and phosphatidylglycerol in the surfactant and residual fractions of rat lung were compared using the in vitro system where tissue slices and radioactive precursors such as [14C]palmitic acid, [14C]palmitoyl lysolecithin and [14C]stearoly lysolecithin were inculbated. The incorporation of precursors into the residual lecithin proceeded linearly during the incubation time, while the time curve on the incorporation into the surfactant lecithin exhibited a lag period before the incorporation increased significantly. A similar tendency was also observed in the incorporation into phosphatidylglycerol. These findings suggested a precursor-product relationship between the corresponding lipids in both the fractions. The turnover time of lecithin was approx. 340-370 min and that of phosphatidylglycerol was 118 min in the intracellular surfactant fraction. The turnover rates of lecithin and phosphatidlyglycerol were approx. 0.6 and 2 nmoles/g of wet tissue, respectively. The transfer of lecithin from the residual fraction to the surfactant fraction was hardly influenced by the difference of fatty acid moiety at the 2-position of 1-palmitoly-lysolecithin. However, the turnover time of 1-palmitoyl lecithin (360 min) was distinctly shorter than that of 1-stearoly species (730 min). The turnover time of the saturated lecithin was also shown to be 328 min. On the other hand, polyenoic species showed the shorter turnover time; 182 min for the tetraenoic species.

Synthesis of molecular classes of cytidine diphosphate diglyceride by rat liver in vivo and in vitro.

Cytidine diphosphate (CDP) diglyceride isolated from pooled rat liver was shown like phosphatidyl inositol to contain stearate and arachidonate as the major fatty acids. On the other hand palmitate and oleate were the predominant fatty acids of total liver phosphatidic acid.Labelling of molecular classes of phosphatidic acid, CDP-diglyceride, and phosphatidyl inositol was examined in liver at various time intervals after either intraportal or intrajugular injections of [3H]glycerol. In general the major flux of radioactivity was through the oligoenoic classes [Δ1 and Δ2] of phosphatidic acid. In contrast the labelling of oligoenoic classes of CDP-diglyceride was less and of polyenoic classes [Formula: see text] significantly enhanced, raising the possibility that there may be some discrimination in selection of species of phosphatidic acid for liponucleotide synthesis. After intrajugular injections of isotope the monoenoic classes of phosphatidyl inositol were most highly labelled but between 5 and 240 min there was a progressive decrease of radioactivity in this class and increase in the tetraenoic species, presumably as a result of deacylation and reacylation reactions. The backflow of radioactivity from phosphatidyl inositol was deemed not a likely explanation for the observed labelling pattern of CDP-diglyceride.The conversion of sonicated dispersions of [3H]phosphatidic acid, labelled in the glycerol moiety, to CDP-diglyceride by rat liver microsomes was examined. There was slightly less label in the oligoenoic classes and slightly more in trienoic and polyenoic (> Δ4) classes of CDP-diglyceride as compared with phosphatidic acid. These differences were much smaller in vitro than those observed in vivo. The in vitro data provide no convincing evidence for a high enough selectivity to explain the differences in arachidonate content between phosphatidic acid and the liponucleotide.

Regulation of selectivity of CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase in rat liver microsomes towards different molecular species of 1,2-diacyl-sn -glycerols

The suitability of monoenoic, dienoic, tetraenoic, and hexaenoic molecular species of 1,2-diacyl-sn-glycerols as substrates for the CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase (EC 2.7.8.2) was studied in rat liver microsomes. No statistically significant difference in the rates of phosphatidylcholine synthesis with the various diacylglycerols was found at 0.40 mM, although a moderate discrimination against hexaenoic species relative to monoenoic and dienoic species was observed at 0.25 mM. The addition of palmitoyl-CoA (7.5 micron) significantly enhanced cholinephosphotransferase activity when tetraenoic diacylglycerols were added at 0.25 or 0.40 mM. CDPethanolamine at 24.4 micron was found to inhibit the rates of phophatidylcholine biosynthesis by 54 and 39% with hexaenoic and monoenoic 1,2-diacyl-sn-glycerols, respectively, whereas no significant effects were observed in the case of dienoic and tetraenoic species. These latter findings may partially explain why 1-saturated 2-docosahexaenoyl diacylglycerols are used to a greater extent for phosphatidylethanolamine than for phosphatidylcholine synthesis in rat liver in vivo. The present results also suggest that the selectivity of the cholinephosphotransferase for certain molecular species of 1,2-diacyl-sn-glycerols is a function of diacylglycerol concentration and may be mediated under physiological conditions by substrates for enzymes which compete for common diacylglycerol precursors.

The metabolic turnover of molecular species of phosphatidylinositol and its precursor phosphatidic acid in guinea-pig cerebral hemispheres.

Abstract– The molecular species composition of phosphatidylinositol from guinea‐pig cerebral hemispheres was studied and found similar to that of phosphatidylinositol from ox cerebral hemispheres. In both cases the tetraenoic species was predominant. Phosphatidic acid from guinea‐pig cerebral hemispheres contained two major molecular species; the monoenoic and hexaenoic (33.4 and 24 mol/100 mol respectively).In order to study the metabolism of molecular species of phosphatidic acid and phosphatidylinositol in the cerebral hemispheres, guinea‐pigs were injected intracisternally with 32Pi and [U‐14C]glucose. After 5 min of isotopic exchange, the specific radioactivity of 32P in phosphatidylinositol was nearly equal to that in phosphatidic acid, whereas specific radioactivity of 14C in the glycerol was 1.4 times and in the fatty acids nearly 0.5 times that in the phosphatidic acid respectively, indicating metabolic heterogeneity of both phospholipids.The glycerol specific radioactivity was different in all the molecular species of phosphatidic acid being greatest in the monoenoic and least in the tetranenoic species. When the molecular species were arranged in this way, the order was representative of their relative rates of synthesis by acylation of glycerol‐3‐phosphate. An almost opposite order was obtained when the molecular species were arranged according to their phosphate/glycerol radioactivity ratios, indicating the relative contribution of the diacylglycerol kinase pathway to their formation.When the specific radioactivity values and ratios of phosphatidylinositol were similarly considered, the orders of the molecular species were, on the whole, similar to that of phosphatidic acid. This indicated that synthesis de novo (Paulus & Kennedy, 1960) was operative in the formation of most of its molecular species, but due to other considerations it was concluded that part of the tetraenoic, and probably the whole of saturated phosphatidylinositol may be formed by transacylation reactions.The results are discussed in terms of the experimental limitations of previous and present techniques for the analysis of phospholipid molecular species.

Specific formation of arachidonoyl phosphatidylinositol from 1‐acyl‐sn‐glycero‐3‐phosphorylinositol in rat liver

The conversion of 1-acyl-sn-glycero-3-phosphorylinositol-3H into phosphatidylinositol-3H was studied using rat liver microsomal and homogenate preparations. The nature of the molecular species of phosphatidyl inositol so formed in the absence of added acyl moieties was determined after fractionating the radioactive product by means of argentation thin layer chromatography. In other experiments, the possible specificity of the microsomal acyl-CoA:1-acyl-sn-glycero-3-phosphorylinositol acyltransferase towards different acyl-CoA derivatives was investigated. Maximum conversion of 1-acyl GPI to the diacyl analogue was dependent on the addition of adenosine triphosphate and CoA when exogensou acyl groups were omitted from the incubation medium. Under these latter conditions, the tetraenoic species comprised 56-74% of the total molecular species of newly-formed phosphatidylinositol. The microsomal acyl-CoA:1-acyl-sn-glycero-3-phosphorylinositol acyltransferase showed a marked preference for arachidonoyl-CoA. The present results suggest that the enrichment of rat liver phosphatidyl inositol in arachidonic acid may arise when 1-acyl-sn-glycero-3-phosphorylinositol is acylated to form phosphatidylinositol.