Serine Phosphatides Research Articles

Metabolism of Exogenous Palmitic and Oleic Acids by Preimplantation Mouse Embryos.

In the present study, the oxidation of palmitic acid to carbon dioxide and the difference of incorporation rate of the same and oleic acid into embryo lipids were examined. To test the exact role of fatty acids for energy production, the oxidative rates of exogenous palmitic were assessed in M16 medium with and without carbohydrate substrates. The oxidation of palmitic acid in the medium with carbohydrates was lower than that without carbohydrates in the blastocyst stage. Incorporation rates of both palmitic and oleic acids into embryo lipids increased significantly from the 2-cell to the blastocyst stage (P<0.05), whereas the incorporation rates of palmitic acid were significantly higher than those of oleic acid at most of the cell stages except for the blastocyst stage (P<0.05). In the neutral lipid fraction, 3H-palmitic acid was predominately distributed in triacylglycerol species and a little in the other glycerides (P<0.05). However, higher percentages of 14C-oleic acid were recovered in fatty alcohols, diacylglycerols and monoacylglycerols. In the polar lipid fraction, the percentages of 3H-palmitic acid in choline and ethanolamine phosphatides were significantly higher than those of oleic acid among all cell stages except the 2-cell stage (P<0.05), whereas the percentages of 14C-oleic acid in inositol and serine phosphatides were significantly higher than those of 3H-palmitic acid during preimplantation development (P<0.05). According to the comparison of incorporations into the embryo lipids and distributions in individual lipid classes between palmitic and oleic acids, it could be inferred that exogenous oleic acid is not a main energy substrate but a major intermediate for synthesis of various embryo lipids.

Reversed-phase liquid chromatographic method for the determination of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labelled lipid analogues

This paper reports the development of a dual column system for the simultaneous separation of fluorescent short-chain ceramide, 6-[(7-nitrobenz-2-oxa-1,3,-diazol-4-yl[NBD])amino]hexanoyl-sphingosine and its metabolites, C6-NBD-sphingomyelin and C6-NBD-glucosylceramide, as well as the fluorescent derivatives of choline and serine phosphatides. The method enables the separation of these lipids in a single run on the basis of the polarity of their headgroups and hydrophobicity of their acyl backbone. The fluorescent properties of the NBD-label make it possible to quantitate small amounts of NBD-lipid analogues. The sensitivity of the presented method thus permits the use of small sample volumes and the determination of NBD-lipid analogues secreted into mouse bile directly, without prior extraction or concentration steps.

Incorporation and Distribution of Both 3H-Palmitic and 14C-Oleic Acids in Lipids by Preimplantation Mouse Embryos.

Both 3H-palmitic and 14C-oleic acids were simultaneously incorporated into mouse embryo lipids from the 1-cell to the blastocyst stage. Rates of incorporation of the two fatty acids into the embryo lipids increased and differed from each other significantly after 8-cell stage (P<0.05). That of palmitic acid into the polar lipid fraction was significantly higher than that of oleic acid at all corresponding cell stages, but that of palmitic acid into the neutral lipids was significantly higher than that of oleic acid at the morula and blastocyst stages (P<0.05). In the neutral lipid fraction, the radioactivity of 3H-palmitic acid distributed in triacylglycerols, monoakyldiglycerols and diacylglycerols were significantly higher than that of 14C-oleic acid at most corresponding cell stages with the exception of high monoacyldiglycerol content in 14C-oleic acid at the 2-cell stage (P<0.05). But that of 3H-palmitic acid in fatty alcohols and monoacylglycerols was significantly lower than that of 14C-oleic acid at the most corresponding cell stages (P<0.05). In the polar lipid fraction, the amounts of 3H-palmitic acid distributed in choline phosphatides, ethanolamine phosphatides and sphingomyelins were significantly greater at most corresponding cell stages than that of 14C-oleic acid (P<0.05) but the radioactivity of 14C-oleic acid recovered in inositol or serine phosphatides, lysophosphatidylcholines, monoglycosylglycerides was significantly higher than that of 3H-palmitic acid at almost every corresponding cell stage (P<0.05). These results demonstrated that the patterns of incorporation and distribution of double fatty acids in various lipid species were significantly different from those of the same single fatty acids in our previous report.

Preparation and characterization of glucosylated aminoglycerophospholipids.

Natural aminophospholipids were isolated from egg yolk and from human red blood cells. Glucosylated ethanolamine and serine phosphatides were prepared by exposing synthetic and natural aminophospholipids to glucose for 3-18 h at pH 7.4. The glucosylation products were resolved from parent phospholipids by normal-phase high-performance liquid chromatography and were identified by on-line mass spectrometry with an electrospray interface. The soft ionization method allowed us to detect the glucosylation products as molecular ions of the Schiff bases. The Schiff bases could be stabilized by sodium cyanoborohydride reduction. The molecular species of the ethanolamine and serine phosphatides reacted in proportion to their molar concentration in the mixtures. The yields of the glucosylation products varied with time of reaction and the concentration of glucose in the medium. At 50 mM glucose and 0.6 mg/mL phosphatidylethanolamine, 20% of the aminophospholipid was glycated in 18 h at 37 degrees C.

Molecular species of glycerolipids of Ehrlich ascites cells and of their fat granules.

Ehrlich ascites cells were grown in mice and were isolated by centrifugation of the ascites fluid. The cells were lysed with distilled water, and the floating fat particles were collected by centrifugation. The particles contained about 90% neutral and 10% polar lipid. The neutral lipid was made up of about 50% triacylglycerol, 30% alkyldiacylglycerol, 3% cholesteryl esters, 3% free cholesterol and 4% free diacylglycerols. The phospholipid fraction was comprised of about 50% phosphatidylcholine, 35% phosphatidylethanolamine, 10% sphingomyelin and small amounts (less than 5% total) of serine and/or inositol phosphatides. The triacylglycerol and alkyldiacylglycerol fractions possessed total carbon number and fatty acid compositions closely similar to those reported in the literature for whole ascites cells and for a cell membrane preparation. Likewise, the fatty acid composition of phospholipids from the granules in general was similar to that reported for Ehrlich ascites cells. On the basis of the polar and neutral lipid ratio, the lipid granules of the ascites cells were calculated to possess lipid core diameters of 30-50 nm, which were 40-70 times smaller than those (up to 2 mu) measured for the lipid granules of the intact cells by electron microscopy. The characterization of the lipid composition of the Ehrlich ascites lipid granules was completed by determining the molecular species composition of the diacyl, alkylacyl and alkenylacyl phosphatidylethanolamines and of the diacyl and alkylacyl phosphatidylcholines of the ascites cells. It is concluded that the alkyldiacylglycerols of the Ehrlich ascites cells occur largely in the cytoplasmic lipid granules, which appear to consist of many particles of the size and structure of very low density lipoproteins enclosed in membranous sacs.

The metabolism of exogenous fatty acids by preimplantation mouse embryos developingin vitro

The utilization of fatty acids from the culture medium has been examined in preimplantation mouse embryos developing in vitro. Incorporation of exogenous fatty acid into embryo lipids was examined by culturing 8-cell mouse embryos for 2h in a medium containing 0.1 mM [9, 10-3H]palmitic acid (900 mCi/mmol). Lipids were extracted from the embryos, and the total lipid extract was fractionated into various neutral lipid and polar lipid classes by thin-layer chromatography. Most of the radioactivity, over 93%, was recovered in neutral glycerides (mono- di-, and triacylglycerols). About 2% of the total radioactivity was recovered in other neutral lipid species including fatty acids, fatty alcohols, and sterol esters. The remainder of the radioactivity was recovered in polar lipids. Seventy-four per cent of the polar lipid radioactivity was present in the amine phosphatides, inositol and/or serine phosphatides, sphingomyelin, choline lysophosphatides, sulfatides, cerebrosides, and monoglycosylglycerides. Chemical degradation studies of labelled embryo lipids indicated that the tritium lable was entering into embryo lipids as the fatty acid and not via metabolic recycling. The oxidation of exogenous fatty acids by mouse embryos was assessed by incubating variously staged embryos for 4h in medium containing 0.1 mM [U-14C]palmitic acid (50 mCi/mmol) and quantitating the production of 14CO2. The rate of fatty acid oxidation was found to be relatively constant from the unfertilized egg up to the 8-cell stage and then increase significantly between the 8-cell and late blastocyst stages. The results suggest that preimplantation mouse embryos developing in vitro can utilize fatty acids from the medium both for incorporation into embryo lipids and for energy production via oxidation.

In vivo incorporation of exogenous stearic acid in synaptosomes: High occurrence of non-esterified fatty acids

Subcutaneously injected stearic acid is taken up by the brain through the ‘blood-brain barrier’ and is incorporated into synaptosomal lipids (as well as into other brain compartments). Phospholipids are potent acceptors (choline phosphatides, ethanolamine phosphatides, serine phosphatides). Moreover a high level of radioactivity was found in non-esterified fatty acids.

Distribution of newly formed fatty acids among glycerolipids of isolated perfused rat liver.

Livers of chow fed rats were perfused 1-3 h with buffer, glucose, albumin, and red blood cells, made up in 100 percent D(2)O. Glycerolipids were isolated and the deuterated fatty acids determined by gas chromatography with mass spectrometry on Silar 5 CP. Percentage of replacement by deuterated acids ranged from 1 to 14, of which palmitate was 87 percent. Differences were found in total lipid class and in subcellular distribution of the newly synthesized acids. Microsomes had 37 percent more deuterated acids than the total or floating fat. At 3 h the highest replacement was found in diacylglycerols (17 percent) and free fatty acids (11 percent). Of the palmitate in hepatic choline and ethanolamine phosphatides, 6.9 percent and 4.7 percent, respectively, contained dueterium. The serine and inositol phosphatides had a higher proportion of deuterated palmitate (7.7 percent) than other phosphatides. The data support the hypothesis that palmitate is incorporated into glycerolipids largely via de novo synthesis while stearate enters them by deacylation-acyl transfer replacement.

Activation of fibrinolysis by certain phospholipids

The fibrinolytic activity of plasmin was found to be significantly increased by phosphatidyl serine and inositol phosphatide. Only these two of a wide variety of lipid-related and micelle-forming agents were effective in enhancing fibrinolysis, though calcium, even at low concentrations, prevented the enhancement effect. Plasmin fibrinolytic activity was assessed by decreased turbidity in a fibrin powder suspension. The effect was not due to stabilization of plasmin by phospholipid under the conditions used. At temperatures near 48°C plasmin itself was destroyed by inositol phosphatide more rapidly than normal. This was taken to indicate some direct interaction between the enzyme protein and the phospholipid, rather than between the fibrin substrate and the phospholipid. The fibrin powder suspension assay was easily manipulated to give a range of substrate concentrations. Rates of fibrinolysis determined at several fibrin levels were expressed as a reciprocal plot assuming a normal three-step enzyme mechanism. Kinetic analysis suggested that the phosphatidyl inositol did not affect the first step of the mechanism, i.e. that involving binding of plasmin to fibrin, but increased the rate of one of the subsequent steps.

Lipid and fatty acid composition of myelin purified from normal and MS brains.

The concentrations of phospholipids and galactolipids and their fatty acid compositions were determined in purified myelin from apparently normal white matter of 11 multiple sclerosis (MS) and 11 control brains. Myelin from MS brains showed a significant decrease in serine phosphatide and a slight decrease in the ethanolamine phosphatide fraction when compared with normal myelin; increases were found in the lysocholine and lysoethanolamine phosphoglycerides. In the galactolipids, lower figures were obtained for sulfatides, resulting in a higher cerebroside/sulfatide ratio for MS myelin. Analysis of the fatty acid pattern of phospholipids in MS brains revealed lower values for 18:1 and, except for the lyso compounds, slightly higher amounts for 20:4 and 22:6. Lower levels for the sum of 18:3 and 20:1 fatty acids were found for the EPG and IPG fractions.

Lipid composition of subcellular fractions from rat testis

1. 1. Golgi apparatus, residual body and microsomal fractions were isolated from rat testes. Lipid vesicles were subsequently isolated from residual body fractions. The lipid distribution and fatty acid composition of total lipids and individual phospholipids were determined and compared to whole testis. 2. 2. The lipid/protein ratios of the fractions were: whole testis, 0.4; Golgi apparatus, 1.26; residual bodies, 0.60; microsomes, 0.63; and lipid vesicles, > 19. Phospholipids, as percent of total lipids, decreased in the order microsomes > whole testis > Golgi apparatus > residual bodies > lipid vesicles. Neutral lipids followed the inverse order. With the exception of lipid vesicles, which were not assayed, whole testis and all fractions contained nearly equal levels of a glycolipid tentatively identified as an aminoglycolipid containing galactose and hexosamine as major carbohydrates. 3. 3. The major phospholipids encountered in all fractions were the choline, ethanolamine, inositol and serine phosphatides and sphingomyelin. The distribution of these phospholipids was nearly the same comparing whole testis to the isolated fractions. In addition to these phospholipids, whole testis and residual bodies contained cardiolipin. Triglycerides, free fatty acids and cholesterol were present as major constituents in the neutral lipids from all fractions. 4. 4. Total lipids and all polar lipids except sphingolipids contained 18:1, 20:4, and 22:5 acids as the major unsaturated fatty acids and 16:0 and 18:0 as major saturated acids. Sphingolipids were the most highly saturated lipids encountered.

The fatty acid composition of porcine spermatozoa phospholipids.

The fatty acid composition of three phospholipids of boar spermatozoa from 32 ejaculates was determined; choline phosphatide, ethanolamine phosphatide, and serine phosphatide were the phospholipids in which 19 fatty acids and two aldehydes were identified. Thin-layer and gas-liquid chromatography were employed for separation of phospholipids and determination of fatty acids. Major saturated fatty acids present were palmitic (16:0), stearic (18:0), and myristic (14:0). Palmitic and stearic acid were present in highest concentrations, ranging from 8 to 19% and 4 to 36%, respectively, in the three phosphatides. Unsaturated fatty acids predominated in the choline and ethanolamine phosphatides, while saturated acids predominated in the serine phosphatides. Docosapentaenoic acid (22:5ω6) was the major unsaturated acid at a high of 38.6%. Docosahexaenoic acid (22:6) was somewhat less at 25.2%. Oleic acid (18:1ω9), linoleic acid (18:2ω6), and arachidonic acid (20:4ω6) were present in lesser quantities.

Quantitative analysis of porcine spermatozoa and seminal plasma phospholipids as affected by frequency of ejaculation.

Summary. Sixty-four ejaculates were collected from four crossbred litter-mate boars. Two frequencies of ejaculation were employed; once every 3 days and daily. Total semen volume (ml) and total spermatozoa per ejaculate (\m=x\109) for the two frequencies were 157, 37\m=.\4; and 131\m=.\9, 19\m=.\7,respectively. Spermatozoa and seminal plasma phospholipids were determined by thin-layer chromatography and phosphorus analysis. The phospholipid components of spermatozoa, in order of decreasing concentration, were: choline phosphatides, ethanolamine phosphatides, sphingomyelin, serine phosphatides, phosphatidic acid and/or polyglycerol phosphatides. Differences between collection frequencies for the phospholipid components were not significant. The seminal plasma phospholipid contained the same component phospholipids as the spermatozoa in addition to trace quantities of lysolecithin. Sphingomyelin was the major phospholipid present in the seminal plasma.

Distribution of Fatty Acids in Cod Flesh Lipids

Cod flesh lipids were separated by silicic acid chromatography into eight fractions, and the fatty acid distribution in five of these was examined by gas–liquid chromatography (GLC). As compared with the fatty acid composition of total flesh lipids, sterol esters contained less 16:0 but more 20:5ω3; ethanolamine phosphatides contained less 16:0, less 20:5ω3, but more 22:6ω3; serine phosphatides contained less 16:0 and less 20:5ω3 but more 18:0; and choline phosphatides had a fatty acid composition roughly similar to that of total flesh lipid but containing slightly more 16:0 and 20:5ω3 and less 22:6ω3. In fatty acid composition, the triglycerides more closely resembled liver lipid than any of the flesh lipid fractions.

The turnover of myelin in the adult rat

The rate of loss of radioactivity of lipids of brain subcellular fractions was followed for 56 days after injection of adult rats with uniformly labeled [ 14C]glucose. Myelin did not reach its maximum specific activity until 5 days after injection, then showed a slow rate of loss of radioactivity. When the turnover of the lipid components of purified myelin was measured, inositol phosphatide and lecithin were found to have a more active metabolism than the other myelin lipids which include serine phosphatide, sphingomyelin, ethanolamine phosphatide, cerebroside, cerebroside sulfate, and cholesterol. These results which agree with our previous findings indicate that the metabolism of myelin on the outer layers is identical to that laid down early in the life of the animal. Proteolipid protein showed a half-life of about 35 days, thus was one of the more active myelin components. The pattern of metabolism, though on a longer time scale in myelin, is similar to that of the most active brain membrane fraction. These results are discussed in relation to the possible structure of myelin membranes.

Serum Complement and the Enzymatic Degradation of Erythrocyte Phospholipid

Summary Small but significant and reproducible alterations in the phospholipid and titratable acid contents of reaction mixtures containing sensitized sheep erythrocytes and guinea pig serum occurred during and following the process of lysis. In experiments employing erythrocytes sensitized with anti-Forssman antibody, there was a fall in the total serine phosphatide content of the mixtures. In experiments using cells sensitized with hyperimmune antibody there was a fall in the total choline phosphatide content of the mixtures. In so far as studied, the lipid alterations had the characteristics of an enzyme reaction which is dependent for its activation on the presence of both sensitized erythrocytes and guinea pig serum, and which was influenced in terms of its substrate by the nature of the sensitizing antibody. Although the exact nature of the enzymatic process is unclear, the results suggest that the titratable acid changes are related to the action of C′8 or C′9 and are not the result of nonspecific activation of membrane, or of intracellular or serum enzymes following hemolysis of erythrocytes. Whereas it is likely that the lipid changes seen in these experiments are related to the action of serum complement, certain other data in this work indicate that the nature of this relationship is not clear.

On the possibility of lysophosphatide formation during Wallerian degeneration.

Abstract—During an extensive decomposition of phospholipids, at the end of the second week of Wallerian degeneration, the decomposition of glycerophosphatides were studied in detail. In a degenerative process lasting for 2 weeks about one‐third of the choline phosphatides, two‐thirds of the ethanolamine phosphatides, one‐third of the serine phosphatides and one‐quarter of the inositol phosphatides, were destroyed.The amount of lysophosphatidylcholine decreased proportionally to the destruction of choline phosphatide. On the other hand, the amount of lysophosphatides formed from‘kephalin'‐containing fatty aldehyde, during the marked destruction of these phospholipids, remained constant or increased to a small extent and its percentage distribution increased 2 or 3 times compared with other phospholipids. Ethanolamine phosphatides having a high fatty aldehyde content can be regarded as mainly responsible for the relative accumulation of lysophosphatides.

The biosynthesis of glycerides and glycerophosphatides by rabbit reticulocytes

1. 1. When rabbit reticulocytes were incubated with [1- 14C]glycerol, radioactivity was incorporated into glycerides and glycerophosphatides but no radioactivity was present in their fatty acid moieties or in cholesterol. 2. 2. Lecithin and ethanolamine phosphatide together contained about 70% and the neutral lipids about 20% of the total radioactivity. The triglyceride, which comprised less than 2% of the lipids, had the highest specific activity of all the lipids. 3. 3. Experiments with other radioactive precursors of the phosphatides gave results which indicate that the minor phosphatides are metabolically much less active than lecithin and that the formation of lecithin from serine phosphatide or ethanol-amine phosphatide is quantitatively unimportant in these cells. 4. 4. Incubation of rabbit reticulocytes with [2- 3H]inositol resulted in the formation of radioactive phosphoinositides. Most of this radioactivity was present in a monophosphoinositide.

The turnover of the lipid components of myelin

AbstractThe 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舑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.

Anticoagulant activity of phosphatidylserine free of lysophosphatidylserine.

SummarySerine phosphatide fractions free of lysophosphatidylserine have been prepared from brain tissue. The strong anticoagulant activity of these fractions is ascribed to a naturally occurring serine phosphatide in the diester form which has the capacity to block formation of plasma thromboplastin as well as neutralize its effect after it is formed.