Composition of phospholipid molecular species from mammary tumors

The molecular phospholipid species of mammary tumors induced by 7,12-dimethylbenz[a]anthracene in rats that were fed diets containing 20 or 3% sunflower-seed oil and different levels of calcium were analyzed by high-pressure liquid chromatography. Twenty-seven molecular species of phospholipids were identified. Phosphatidylcholine was predominantly composed of palmitoyl-arachidonoyl (16:0-20:4) (17-21%), palmitoyl-oleoyl (16:0-18:1) (19-21%), stearoyl-arachidonoyl (18:0-20:4) (12-13%), and 1,2-dipalmitoyl (16:0-16:0) (10-14%) species. The major molecular species of phosphatidylethanolamine were 18:0-20:4 (37-39%) and 16:0-20:4 (10-11%). The composition of diacyl phosphatidylcholine and diacyl phosphatidylethanolamine molecular species from rat mammary tumors was not greatly affected by the different diets.     

Molecular Species of Diacylglycerols and Phosphoglycerides and the Postmortem Changes in the Molecular Species of Diacylglycerols in Rat Brains

The molecular species of 1,2-diacyl-sn-glycerol (DAG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) from brains of adult rats (weighing 150 g) were determined. The DAG, isolated from brain lipid extracts by TLC, was benzoylated, and the molecular species of the purified benzoylated derivatives were separated from each other by reverse-phase HPLC. The total amount and the concentration of each species were quantified by using 1,2-distearoyl-sn-glycerol (18:0-18:0) as an internal standard. About 30 different molecular species containing different fatty acids at the sn-1 and sn-2 positions of DAG were identified in rat brains (1 min postmortem), and the predominant ones were 18:0-20:4 (35%), 16:0-18:1 (15%), 16:0-16:0 (9%), and 16:0-20:4 (8%). The molecular species of PC, PE, PS, and PI were determined by hydrolyzing the lipids with phospholipase C to DAG, which was then benzoylated and subjected to reverse-phase HPLC. PIP and PIP2 were first dephosphorylated to PI with alkaline phosphatase before hydrolysis by phospholipase C. The molecular species composition of phosphoinositides showed predominantly the 18:0-20:4 species (50% in PI and approximately 65% in PIP and PIP2). PS contained mainly the 18:0-22:6 (42%) and 18:0-18:1 (24%) species. PE was mainly composed of the 18:0-20:4 (22%), 18:0-22:6 (18%), 16:0-18:1 (15%), and 18:0-18:1 (15%) species. In PC the main molecular species were 16:0-18:1 (36%), 16:0-16:0 (19%), and 18:0-18:1 (14%). Studies on postmortem brains (30 s to 30 min) showed a rapid increase in the total amount (from 40-50 nmol/g in 0 min to 210-290 nmol/g in 30 min) and in all the molecular species of DAG. Comparatively larger increases (seven- to 10-fold) were found for the 18:0-20:4 and 16:0-20:4 species. Comparison of DAG species with the molecular species of different glycerolipids indicated that the rapid postmortem increase in content of DAG was mainly due to the breakdown of phosphoinositides. However, a slow but continuous breakdown of PC to DAG was also observed.     

Individual molecular species of phosphatidylcholine and phosphatidylethanolamine in myelin turn over at different rates.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of the myelin membrane exhibit heterogeneity with respect to metabolic turnover rate (Miller, S. L., Benjamins, J. A., and Morell, P. (1977) J. Biol. Chem. 252, 4025-4037). To test the hypothesis that this is due to differential turnover of individual molecular species (which differ in acyl chain composition), we have examined the relative turnover of individual molecular species of myelin PC and PE. Phospholipids were labeled by injection of [2-3H]glycerol into the brains of young rats. Myelin was isolated at 1, 15, and 30 days post-injection, lipids were extracted, and phospholipid classes were separated by thin-layer chromatography. The PC and PE fractions were hydrolyzed with phospholipase C, and the resulting diacylglycerols were dinitrobenzoylated and fractionated by reverse-phase high performance liquid chromatography. The distribution of radioactivity among individual molecular species was determined. The labeled molecular species of myelin PC were 16:0-16:0, 16:0-18:0, 16:0-18:1, and 18:0-18:1, with most of the label present in 16:0-18:1 and 18:0-18:1. Changes in distribution of label with time after injection indicated that 16:0-18:1 turned over more rapidly than 18:0-18:1. The labeled molecular species of myelin PE were 18:0-20:4, 18:1-18:1, 16:0-18:1, 18:0-18:2, and 18:0-18:1. As with myelin PC, 16:0-18:1 (and 18:1-18:1) turned over more rapidly than 18:0-18:1. The relative turnover of individual molecular species of PC in the microsomal fraction from forebrain was also examined. The molecular species profile was different from myelin PC, but again, 16:0-18:1 turned over more rapidly than the other molecular species. Thus, within the same membrane, individual molecular species of a phospholipid class are metabolized at different rates. Comparison of our results with previous studies of turnover of molecular classes of phospholipids indicates that in addition to polar head group composition (Miller et al., 1977), fatty acid composition is very important in determining the metabolic fate of a phospholipid.     

Adaptation of biological membranes to temperature: molecular species compositions of phosphatidylcholine and phosphatidylethanolamine in mitochondrial and microsomal membranes of liver from thermally-acclimated rainbow trout

Summary Molecular species profiles were determined for both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of mitochondrial and microsomal membrane fractions from liver tissue of thermally-acclimated rainbow trout,Salmo gairdneri. The predominant molecular species of PC were 16:0/22:6, 16:0/18:1, 16:0/20:3 and 16:0/22:5, whereas predominant molecular species of PE were 18:1/20:4, 14:0/16:0, 18:0/22:6 and 18:1/22:6. PE possessed short chain saturates (primarily 14:0/16:0) and monoenes (primarily 14:0/16:1) not present in PC and larger proportions of polyunsaturated (18:0/22:6, 18:0/22:5 and 18:1/22:6. and diunsaturated molecular species than PC. Differences between membrane fractions were most evident in warm (20°C)-acclimated trout. Mitochondria contained higher proportions of long-chain, polyunsaturated molecular species of PE, but less of the corresponding species of PC than other membrane fractions. Rankings based on unsaturation index were accordingly: mitochondria heavy microsomes>light microsomes for PE, but heavy microsomes>light microsomes>-mitochondria for PC. Mitochondria were notable for high proportions of diunsaturated molecular species of both phosphatides. Growth at cold temperatures (5°C) was generally associated with a replacement of shorter chain mono- and dienoic molecular species (16:0/18:1, 16:1/18:1, 14:0/16:2 and 18:1/18:1 in the case of PC and 14:0/16:1, 14:0/16:2 and 16:1/18:1 for PE), and occasionally saturates, with long-chain, polyunsaturated molecular species (for PC, C_36–38: 16:0/22:6, 16:1/22:6, 16:0/20:3 and 16:0/20:5; for PE, C_38–40: 18:1/20:4, 16:1/22:6, 18:0/20:5, 18:2/20:4, 18:0/22:5 and 18:0/22:6). However, compositions of mitochondrial PE and PC from heavy microsomes were not significantly influenced by acclimation temperature. The role of phospholipase A₂, in addition to other metabolic processes, in mediating these changes is discussed.Abbreviations ACL average chain length - UI unsaturation index     

Molecular species profiles of acidic phospholipids in lung fractions of adult and perinatal rabbits

The molecular species of phosphatidylglycerol (PG) and phosphatidylinositol (PI) in pulmonary surfactant and membrane fractions of adult and perinatal rabbit lungs were analyzed by high-performance liquid chromatography of the dinitrobenzoyl derivatives of the diacylglycerols (DGs), derived from the two acidic phospholipids. The PG in both surfactant and membrane fractions of adult lungs consisted mainly of the 16:0/16:0 species, followed in order by 16:0/18:1 and 16:0/18:2 species. In contrast, the prominent molecular species of PI in the membrane fractions were 18:0/20:4 and 16:0/18:1, while surfactant PI consisted mainly of 16:0/18:1 and 16:0/18:2, containing only 3% of 16:0/16:0. In the perinatal rabbit lungs, a compositional change between surfactant PG and PI was found, i.e., an increase in PG and a decrease in PI. The molecular species compositions of PG and PI in the perinatal lungs were generally the same as those observed in the adult lungs. It should, therefore, be noted that the species profiles of surfactant PG and PI, particularly in the content of 16:0/16:0 and 18:0/20:4, are not similar, but distinctly different from each other in both adult and developing lungs. Therefore, the present results strongly suggest two possibilities; (1) both acidic phospholipids which appear in pulmonary surfactant may originate from different pools of CDP-DGs having different molecular species; and/or (2) surfactant PG and PI may be synthesized by individual enzymes having different substrate specificities for different CDP-DGs in alveolar type II cells.     

The molecular species composition of diacyl-, alkylacyl- and alkenylacylglycerophospholipids in rabbit alveolar macrophages. High amounts of 1-O-hexadecyl-2-arachidonyl molecular species in alkylacylglycerophosphocholine

The relative composition of molecular species of diacyl-, alkylacyl- and alkenylacylglycerophospholipids in rabbit alveolar macrophages was determined with reverse-phase high-performance liquid chromatography (HPLC). Diacylglycerophosphocholine (GPC) (22.3% of the total glycerophospholipids), alkylacylGPC (11.3%) and alkenylacylglycerophosphoethanolamine (GPE) (15.8%) were the predominant glycerophospholipids in rabbit alveolar macrophages. DiacylGPE (6.9%), diacylGPI (5.5%) and diacylGPS (3.6%) also occurred. 1,2-Diradyl-3-acetylglycerol derived from glycerophospholipids were each resolved into 19 separate peaks with reverse-phase HPLC. By gas-liquid chromatographic quantitation of each peak, 19-29 different molecular species were identified. DiacylGPC, GPE and GPS were mainly composed of saturate, monoene and diene species, such as the 16:0-16:0, 16:0-18:1, 18:0-18:1, and 18:0-18:2 species. The predominant molecular species composing diacylGPI was the 18:0-20:4 species, which represented 40% of this glycerophospholipid. Distinct differences were found in the distributions of arachidonyl molecular species between diacyl- and ether-containing GPC and GPE. Although diacylGPC and GPE included a small amount of arachidonyl molecular species, the 16:0-20:4 species was by far the most prevalent one which composed alkylacylGPC (39% of the total) and alkenylacylGPE (49% of the total). The 16:0-20:4 species of alkylacylGPC and alkenylacylGPE together comprised 60% of the total arachidonyl molecular species of glycerophospholipids. The high amounts of the 16:0-20:4 species in alkylacylGPC may serve as a good source of both the potent platelet-activating factor and the products of arachidonic cascade in the stimulated alveolar macrophages.     

Studies on Composition of Molecular Species of Phosphatidylglycerol in Relation to Cold-resistance of Poplar

The composition of molecular species and the positional distribution in fatty acids of phosphatidylglycerol (PG) isolated from poplar ( Populus deltoides cv. Lux 1-69/55 and Poeuramericarla cv.I- 45/51 ) leaves were analyzed by high-performance liquid chromatography (HPLC), enzym hydrolysis and gas phase chromatography (C,C), and the different cold-resistant poplars were compared with respect to the compositions of molecular species of PG isolated from their leaves. The results showed that the fatty acid compositions ( sn- 1/sn-2) of the major molecular species in PCs from poplar leaves were as follows: 18:3/18:2(18:2/18:3), 18:3/16: 1(3t); 18:3/16:0; 18:2/ 16:1 (3t); 16:0/18:2,18:2/16:0; 18: 1/16: l(3t); 16:0/16: l(3t); 18: 1/18: 1,16:0/18: 1( 18: 1/16:0); 16:0/16:0o The positional distribution of fatty acids in lPG from poplar leaves was found that 16:1(30 was exclusively occupied the sn-2 position, whereas 16:0 was present in both the sn1 position and the sn-2 position. The C18 acids were principally localized at the sn-2 position. The relative contents of the unsaturated molecular species of leaf PCs were more than 70% in both coldresistant poplar and cold-sensitive poplar. The ratio of the unsaturated/saturated molecular species of PG isolated from the cold-resistant Ⅰ -45 poplar was 3.10, which was higher than that of the PG from the cold-sensitive cottonwood, which was 2.38. The sum of the relative contents of the disaturated molecular species of the PG from poplar leaves was closely associated with the cold-resistance of plants. The ∑[ 16:0/16:0+ 16:0/16: l(3t) ] of the PG from cottonwood was higher than that of the PG from cold-resistant I -45 poplar. The differences in the compositions of molecular species and the phase transition temperatures of PCs between cold-resistant and cold-sensitive plants were discussed in terms of the pathways and the activities of selective acyhransferases involved in the PG biosynthesis in chloroplast.     

Relationships between phosphatidylglycerol molecular species of thylakoid membrane lipids and sensitivities to chilling-induced photoinhibition in rice

In an attempt to explore the relationships between phosphatidylglycerol （PG） molecular species of thylakoid membrane lipids and sensitivities to chilling-induced photoinhibition, PG molecular species, D1 protein, electron transport activities of thylakoid membrane and the potential quantum yield （FvlFm） in rice treated under middle and low photon flux density （PFD） at 11℃ were analyzed by high performance liquid chromatography, enzyme hydrolysis, gas phase chromatography （GC） and so on. Results showed that the major molecular species of PGs in rice thylakoid membrane were 18：3/16：0, 18：3/16：1（3t）, 18：2/16：0, 18：2/16：1（3t）, 18：1/16：0, 18：1/16：1（3t）, 16：0/16：0, 16：0/16：1（3t）. There were large differences in the contents of unsaturated PG molecular species such as 18：1-3/16：0-16：1（3t） and saturated PG molecular species like 16：0/16：0-16：1（3t） among japonica cv 9516 0-9516）, japonica-indica hybrid F1 j-9516/i-SY63 （ji-95SY） and indica cv Shanyou 63 （i-SY63）. J-9516 containing higher contents of unsaturated PG molecular species was manifest in stable D1 protein contents under chill and tolerant to chill-induced photoinhibition. In contrast to j-9516, i-SY63 with lower contents of unsaturated PG molecular species, exhibited unstable D1 protein contents under chill and was sensitive to chill-induced photoinhibition, ji-95SY containing middle contents of unsaturated PG molecular species between those of j-9516 and i-SY63, exhibited mid extent of sensitivity to chill-induced photoinhibition. The losses in D1 protein also account for the inhibition in electron transport activity of thylakoid membrane and the observed decline in FvlFm. The PG molecular species that is efficient in raising chilling-resistant capacity were those containing unsaturated fatty acids, namely, unsaturated PG molecular species. These results implied that the substrate selectivity of the glycerol-3-phosphate acyltransferase in chloroplasts towards 16：0 or 18：1 displayed greatly the difference between japonica and indica rice. Itwas possible to enhance the capacity of resistance to chilling-induced photoinhibition by improving or modifying the GPAT gene.     

Separation of dimethylphosphatidates of alkylacyl glycerophosphocholine and their molecular species by high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) method for separation of the alkylacyl and diacyl analogs of choline glycerophospholipids (CGP) of guinea pig polymorphonuclear leukocytes and their molecular species is described. CGP were hydrolyzed with phospholipase D and then methylated with diazomethane to convert them to dimethylphosphatidates. The dimethylphosphatidates were then separated into the alkylacyl and diacyl subclasses by HPLC on a silica gel column within 15 min. The alkylacyl and diacyl analogs were then separated into individual molecular species by reverse-phase HPLC. Dimethylphosphatidates were resolved into 15 separate peaks, and 11-16 different molecular species of alkylacyl and diacyl glycerophosphocholines were identified on gas-liquid chromatography. The present results indicate that the CGP of polymorphonuclear leukocytes are composed of 27 major molecular species. In the alkylacyl subclass, the most predominant species was the 16:0-18:2 species (32%), followed by the 18:1-18:2 (18%), 16:0-16:0 (16%), and 16:0-18:1 (15%) species. The diacyl type consisted mainly of species with 18:2 at the 2-position, such as the 16:0-18:2, 18:0-18:2, and 18:1-18:2 species, the total percentage of which was 57%.     

Plasmodium knowlesi induces alterations in phosphatidylcholine and phosphatidylethanolamine molecular species composition of parasitized monkey erythrocytes

Using high performance liquid chromatography and gas-liquid chromatography, we have characterized the phosphatidylcholine and phosphatidylethanolamine molecular species composition of trophozoite and schizont forms of Plasmodium knowlesi parasitized erythrocytes. Similarly, we determined these parameters in the erythrocyte membranes of trophozoite parasitized cells, unparasitized erythrocytes from infected monkeys before and after a chloroquine treatment and erythrocytes from monkeys that had never been infected. Plasma phosphatidylcholine molecular species composition was also studied. P. knowlesi parasitized erythrocytes presented higher amounts of 16:0/18:2-phosphatidylcholine than the various control cells, which appeared to be compensated for by a decrease in 18:0/20:4-, 16:0/20:3-, 16:0/18:1-, 18:0/18:2-, 18:0/20:3-, 16:0/16:0- and 16:0/18:0-phosphatidylcholines. In the case of phosphatidylethanolamine, the alterations were quantitatively of greater importance and consisted of an increase in, again, 16:0/18:2-phosphatidylethanolamine and a decrease in several species containing 20:4, namely 16:0/20:4-, 18:0/20:4- and 18:1/20:4-phosphatidylethanolamine; also the levels of alkoxy-phosphatidylethanolamines were markedly decreased. P. knowlesi development within monkey erythrocytes therefore appears to be associated with changes in phosphatidylcholine and phosphatidylethanolamine molecular species in the whole parasitized cell. These alterations are also exhibited by the host cell membrane, which provides the first experimental evidence that the parasite is able to manipulate the erythrocyte membrane lipid species composition. The consequences of these alterations on membrane physiology are discussed, as well as the implications that these data may have on the trafficking of phosphatidylcholine and phosphatidylethanolamine in the erythrocytes of P. knowlesi infected monkeys.     

Restructuring of plasma membrane phospholipids in isolated hepatocytes of rainbow trout during brief in vitro cold exposure

Adaptive changes in membrane physical properties in response to changing environmental temperature (e.g., inereased fluidity at low growth temperatures) are well known in poikilotherms; however, the timecourse of this response has received little attention. In this study the plasma membrane lipids of hepatocytes prepared from 20°C-acclimated trout were analyzed for phospholipid class and molecular species composition and metabolism after the cells were exposed to 5°C for 6 hours. Proportions of phosphatidylethanolamine and phosphatidylcholine were not altered by in vitro incubation at either 5 or 20°C. Molecular species analysis revealed that proportions of 18:1/20:5-phosphatidylcholine were significantly lower in plasma membranes of 5°C incubated cells, while decreases in 16:0/20:4-phosphatidylcholine, an unidentified phosphatidylcholine species, and 16:0/16:0-phosphatidylethanolamine as well as increases in 16:0/16:1-phosphatidylethanolamine as well as increases in 16:0/16:1-phosphatidylcholine bordered on significance. Exogenous radiolabeled molecular species of phosphatidylcholine (16:0/16:0-phosphatidylcholine and 16:0/18:1-phosphatidylcholine) were converted into other species at both temperatures, and the formation of some was influenced by incubation temperature. For example, cells exposed to 5°C convert significantly more 16:0/16:0-phosphatidylcholine into 16:0/20:4-phosphatidylcholine and 18:0/16:1-phosphatidylcholine and less into 18:1/18:1-phosphatidylcholine and 16:0/22:6-phosphatidylcholine than cells incubated at 20°C. In addition, cells at 5°C metabolized 16:0/18:1-phosphatidylcholine to a lesser extent than those at 20°C. The profile of conversion products indicates that deacylation/reacylation, elongation and desaturation reactions all participate in this early membrane restructuring. It is concluded that the plasma membrane of trout hepatocytes is a highly dynamic structure characterized by continuous lipid restructuring/turnover which can be rapidly altered upon acute cold exposure to adjust membrane phospholipid molecular species composition to the prevailing thermal environment.Abbreviations BHT butylated hydroxytoluene - BSA bovine serum albumin - HEPES N-(2-hydroxyethyl)piperazine-N-(2-ethanesnlphonic acid) - HELC high-performance liquid chromatography - HVA homeoviscous adaptation - MS molecular species - MS-222 2-aminobenzoic acid ethyl ester (methanesulphonate salt) - RRT relative reteption time - PC phosphatidylcholine - PE phosphatidylethanolamine - TLC thin-layer chromatography - TRIS tris(hydroxymethyl)aminoethane - T _a ambient temperature     

凡纳滨对虾血蓝蛋白与病原菌凝集作用靶标的鉴定

血蓝蛋白是一种具有多种非特异性免疫学活性的多功能蛋白,以前的研究发现,血蓝蛋白具有凝集活性.本研究采用凝集抑制实验和亲和蛋白质组学等方法探索凡纳滨对虾血蓝蛋白与病原菌的凝集作用靶标.结果显示,大肠杆菌K12和副溶血弧菌外膜蛋白可以抑制血蓝蛋白对7种细菌的凝集活性,其中大肠杆菌K12中2种分子质量分别为16 kD、18 kD （命名为 p16、p18）的外膜蛋白可以与血蓝蛋白发生特异性的结合,经MALDI-TOF/MS鉴定,p16、p18 分别与大肠杆菌外膜蛋白OmpC、OmpX具有高度同源性.尤其是与大肠杆菌K12野生菌株相比,血蓝蛋白对 ΔOmpX 的凝集特异性明显降低,后者仅为前者的25％.由此推测,OmpX 应为血蓝蛋白与病原菌的凝集作用靶标.     

Molecular organization of cholesterol in polyunsaturated phospholipid membranes: a solid state 2H NMR investigation.

We compared the molecular organization of equimolar [3alpha-2H1]cholesterol in 18:0-18:1PC (1-stearoyl-2-oleoylphosphatidylcholine), 18:0-22:6PC (1-stearoyl-2-docosahexaenoylphosphatidylcholine), 18:0-20:4PC (1-stearoyl-2-arachidonylphosphatidylcholine) and 20:4-20:4PC (1,2-diarachidonylphosphatidylcholine) bilayers by solid state 2H NMR. Essentially identical quadrupolar splittings (delta v(r) = 45 +/- 1 kHz) corresponding to the same molecular orientation characterized by tilt angle alpha0 = 16 +/- 1 degrees were measured in 18:0-18:1PC, 18:0-22:6PC and 18:0-20:4PC. A profound difference in molecular interaction with dipolyunsaturated 20:4-20:4PC, in contrast, is indicated for the sterol. Specifically, the tilt angle alpha0 = 22 +/- 1 degrees (derived from delta v(r) = 37 +/- 1 kHz) is greater and its membrane intercalation is only 15 mol%.     

Isolation and mass spectrometry characterization of molecular species of lactosylceramides using liquid chromatography-electrospray ion trap mass spectrometry

Reverse-phase liquid chromatography/electrospray ion trap mass spectrometry (LC-ESI-MSn) was established for identification of the molecular species of lactosylceramides. Lactosylceramides derived from porcine blood cells were separated on a CapcellPak C8 column using a mixture of methanol and 1 mM ammonium formate from the C16 to C26 fatty acyl chains based on the length of total carbon chains and the nature of sphingoid bases (w') and fatty acyl chains (Y0'-w') was identified by MS3 as their [M+H]+ ions. The same number of fatty acyl moieties appeared in the order of unsaturated, (2-)hydroxylated, and saturated components. The molecular species of lactosylceramides derived from porcine blood cells totaled more than 33 and included mainly C24:0-d18:1, Ch24:0-d18:1, Ch24:1-d18:1, C24:1-d18:1, and C22:0-d18:1 in addition to 28 minor species from C16:0 to C26:0 fatty acyl moieties. The molecular species of lactosylceramides in the membrane microdomain fraction of HL-60 cells (70% were differentiated into macrophage-lineage cells) were identified as C24:0-d18:1, C24:1-d18:1, C22:0-d18:1, C16:0-d18:1, and more than 21 other minor species. Our results suggest that reverse-phase LC-ESI-MSn is a useful and simple method for identification of lactosylceramide molecular species.     

Occurrence of glyceryl ethers in the phosphatidylcholine fraction of surfactant from dog lungs

The molecular species of ether-linked lipids in the phosphatidylcholine (PC) fraction of the pulmonary surfactant obtained from the lavage fluid of dog were characterized. A combination of base-catalyzed methanolysis, phospholipase C treatment, gas-liquid chromatography, and mass spectrometry procedures were applied. The phospholipid composition of the surfactant, obtained by phosphorus assay of lipids separated by silica gel G thin-layer chromatography (TLC), was: PC (75%), phosphatidylglycerol (10%), phosphatidylethanolamine (7%), plus small amounts of sphingomyelin, phosphatidylinositol, and phosphatidylserine. The major components of the PC were 1,2-diacylPC (95%), and 1-O-alkyl-2-acylPC (5%). No detectable amounts of 1-O-alkyl-1'-enyl-2-acylPC or di-alkyl-1-enylPC were observed. The acyl groups present in the diacylPC were 14:0 (5%), 16:0 (68%), 16:1 (12%), 18:0 (6%), 18:1 (7%) and 18:2 (2%). The predominant alkyl ether chains located at the carbon 1 position of the 1-O-alkyl-2-acylPC were 16:0 (84%), 18:0 (5%) and 18:1 (14%). At the carbon 2 position only a 16:0 fatty acyl residue was detected. In three out of seven animals platelet-activating factor-like activity, as determined by a platelet aggregation assay, was isolated by TLC. This aggregating activity was lost upon base-catalyzed methanolysis, but was restored by functional levels after acetylation.     

Changes of membrane phospholipid composition of human erythrocytes in hyperlipidemias. II. Increases in distinct molecular species of phosphatidylethanolamine and phosphatidylcholine containing arachidonic acid.

The molecular species composition of red blood cell diacyl-phosphatidylcholine (PC), diacyl-phosphatidylethanolamine (PE) and alkenylacyl-PE (plasmalogen PE) has been analyzed in normolipidemic and hyperlipidemic donors. In all three phospholipid subclasses the percentages of the species 16:0/20:4 were increased in hyperlipidemic patients. In diacyl-PE, 18:1/20:4 was also elevated. No changes were observed in the other quantitatively important molecular species containing arachidonic acid at sn-2, namely 18:0/20:4. The rise in 16:0/20:4 in diacyl-PC and diacyl-PE of hyperlipidemic donors was accompanied by a fall in molecular species with linoleic acid (18:2) at sn-2 (in particular 18:1/18:2). In alkenylacyl-PE the elevation of 16:0/20:4 was compensated by a decrease in species with docosatetraenoic acid (22:4) at sn-2 in particular by a fall in 16:0/22:4. Among all donors, the percentages of 16:0/20:4 in diacyl-PC and PE were positively associated with plasma total cholesterol levels. The changes in molecular species composition of PC and PE in hyperlipidemia are expected to alter the function of erythrocyte membrane transport proteins and--if present also in other cell types--to affect eicosanoid metabolism.     

Studies on molecular species of choline glycerophospholipids of developing rat brain.

The chronological changes in molecular species of choline glycerophospholipids were studied for cerebra of 17-, 19- and 21-day-old rat fetuses, and 3-, 6-, 12-, 24- and 90-day-old rats. The molecular species found by gas chromatography-mass spectrometry and selected ion retrieval technique were phosphatidylcholines of '30 : 0, 32 : 0, 32 : 1, 34 : 0, 34 : 1, 34 : 2, 36 : 0, 36 : 1, 36 : 2, 36 : 3, and 36 : 4' where the larger number indicates the sum of chain lengths on positions C-1 and C-2; the smaller number is the total number of double bonds. Of these molecular species, '32 : 0' (mainly 16 : 0/16 : 0, dipalmitoyl glycerophosphorylcholine), '34 : 1' (mainly 16 : 0/18 : 1, palmitoyloleoyl glycerophosphorylcholine), '34 : 0' (16 : 0/18 : 0, palmitoylstearoyl glycerophosphorylcholine), '32 : 1' (mainly 16 : 0/16 : 1, palmitoylpalmitoleoyl glycerophosphorylcholine and '30 : 0' (14 : 0/16 : 0, myristoylpalmitoyl glycerophosphorylcholine) were main species. The '32 : 0' species increased to about 44% at around the 10th day and thereafter remained nearly constant. '34 : 1' and '34 : 0' decreased to about 17 and 6% at that time and then increased to about 30 and 14%, respectively. '30 : 0' increased from last stage of gestation to the 6th day and then decreased. '32 : 1' was about 16% for 17-day-old fetus and decreased grandually. '36 : 1' (18 : 0/18 : 1, stearoyloleoyl glycerophosphorylcholine) increased at the latter part of development.     

Molecular species of phosphatidylcholine in abetalipoproteinemia: effect of lecithin:cholesterol acyltransferase and lysolecithin acyltransferase

In order to study the role of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) in determining the molecular species composition of phosphatidylcholine (PC) and the specificity of lecithin:cholesterol acyltransferase (LCAT) in human plasma, we studied the PC species composition in plasma from abetalipoproteinemic (ABL) and control subjects before and after incubation at 37 degrees C. The ABL plasma contained significantly higher percentages of sn-2-18:1 species (16:0-18:1, 18:0-18:1, and 18:1-18:1) and lower percentages of sn-2-18:2 species (16:0-18:2, 18:0-18:2, and 18:1-18:2) as well as sn-2-20:4 species (16:0-20:4, 18:0-20:4, and 18:1-20:4). Similar abnormalities were found in the PC of ABL erythrocytes, while the PE of the erythrocytes was less affected. The relative contribution of various PC species towards LCAT reaction in ABL plasma was significantly different from that found in normal plasma. Thus, while 16:0-18:2 and 16:0-18:1 contributed, respectively, 43.8% and 15.9% of the total acyl groups used for cholesterol esterification in normal plasma, they contributed, respectively, 21.5% and 37.9% in ABL plasma. The relative contribution of 16:0-20:4 was also significantly lower in ABL plasma (4.7% vs. 9.0% in normal), while that of 16:0-16:0 was higher (6.4% vs. 0.5%). However, the selectivity factors of various species (percent contribution/percent concentration) were not significantly different between ABL and normal plasma, indicating that the substrate specificity of LCAT is not altered in the absence of VLDL and LDL. Incubation of ABL plasma in the presence of normal VLDL or LDL resulted in normalization of its molecular species composition and in the stimulation of its LCAT activity. Addition of LDL, but not VLDL, also resulted in the activation of lysolecithin acyltransferase (LAT) activity. The incorporation of [1-14C]palmitoyl lysoPC into various PC species in the presence of LDL was similar to that observed in normal plasma, with the 16:0-16:0 species having the highest specific activity. These results indicate that the absence of apoB-containing lipoproteins significantly affects the molecular species composition of plasma PC as well as its metabolism by LCAT and LAT reactions.     

Substrate specificity of human plasma lecithin-cholesterol acyltransferase towards molecular species of phosphatidylcholine in native plasma

The specificity of human plasma lecithin-cholesterol acyltransferase for molecular species of phosphatidylcholine (PC) was studied by determining the molecular species composition of whole plasma before and after incubation at 37 degrees C. Since the disappearance of PC under the conditions employed is entirely due to the activity of lecithin-cholesterol acyltransferase, its specificity can be determined from the decrease in the concentration of each species after the reaction. The selectivity factor for each species was calculated by dividing its observed contribution by its concentration at zero time. The major species contributing to cholesterol esterification in whole plasma were 16:0-18:2 (46%), 18:0-18:2 (16%), 16:0-18:1 (15%), 16:0-20:4 (10%), 18:0-20:4 (5%) and 18:1-18:2 (5%). The specificity, as determined from the selectivity factors for whole plasma, was in the order: 16:0-18:2 greater than 18:1-18:2 greater than 16:0-18:1 greater than 18:0-18:2 greater than 16:0-22:6 greater than 18:0-20:4 greater than 16:0-20:4. The high-density lipoproteins (HDL) contained a significantly higher percentage of 16:0-20:4 and 18:0-20:4 and a lower percentage of 16:0-18:1 and 18:0-18:1 compared to the very-low and low-density lipoproteins. These differences disappeared after incubation of the plasma for 24 h. Using selectivity factors for HDL PCs only, the specificity of the enzyme was found to be in the order: 16:0-18:2 greater than 18:1-18:2 greater than 18:1-18:1 greater than 16:0-22:6 greater than 18:0-18:2 greater than 16:0-18:1 greater than 16:0-20:4. These results indicate that in native plasma, lecithin-cholesterol acyltransferase prefers 16:0 greater than 18:1 greater than 18:0 at the 1-position and 18:2 greater than 18:1 greater than 22:6 greater than 20:4 at the 2-position of PC.     

Hormonal stimulation of diacylglycerol formation in hepatocytes. Evidence for phosphatidylcholine breakdown

The molecular species of 1,2-diacylglycerol in control and agonist-stimulated rat hepatocytes were analyzed by high performance liquid chromatography. Twelve species were identified which were increased nonuniformly by 100 nM vasopressin. Most species were increased 2-3-fold, but some (C16:0/C20:4 and C18:0/C20:4) were increased 3-6-fold. Selectively greater increases in the latter two species were also induced by ATP, angiotensin II, and A23187 ionophore, however, phorbol ester caused uniform increases. Calcium depletion of the cells with chelator resulted in a uniform 2-fold effect of vasopressin on 1,2-diacylglycerol species, with greater increases in C16:0/C20:4 and C18:0/C20:4 being restored by Ca2+ readdition. Comparison of the increases in 1,2-diacylglycerol species caused by the Ca2+-mediated agents with the molecular species present in rat hepatocyte phospholipids supports the concept that phosphatidylcholine is a major source of the 1,2-diacylglycerol that accumulates. In hepatocytes incubated for 5 min to 2 h with 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine, the label was incorporated mainly into phosphatidylcholine, and subsequent incubation with vasopressin, angiotensin II, ATP, epinephrine, A23187, and phorbol ester caused formation of [3H]alkyl-acylglycerol, but not [3H]alkyl-phosphatidic acid. The time course and concentration dependence of the vasopressin effect were similar to those reported previously for total 1,2-diacylglycerol (Bocckino, S. B., Blackmore, P. F., and Exton, J. H. (1985) J. Biol. Chem. 260, 14201-14207). Calcium depletion induced by chelator inhibited the effect of vasopressin, and readdition of Ca2+ largely restored the effect. In cells incubated with [14C]lyso-phosphatidylcholine, [3H]phosphatidylcholine, or [14C]phosphatidylethanolamine for 5 or 30 min to label hepatocyte phosphatidylcholine, vasopressin also induced the formation of labeled 1,2-diacylglycerol, but not phosphatidic acid. In contrast, in hepatocytes prepared from rats injected intraportally with [3H]alkyl-lyso-glycerophosphocholine 20 h previously, the hormone induced the rapid formation of both labeled 1,2-diacylglycerol and phosphatidic acid. In summary, these isotopic data indicate that a rapidly labeled pool of phosphatidylcholine is hydrolyzed to 1,2-diacylglycerol and a slowly labeled pool is broken down to both 1,2-diacylglycerol and phosphatidic acid in hepatocytes stimulated by Ca2+-mobilizing agents. It is concluded from both the analyses of molecular species of 1,2-diacylglycerol and the labeling experiments that phosphatidylcholine is a major source of the 1,2-diacylglycerol that accumulates in hepatocytes stimulated with Ca2+-mobilizing agonists and that the mechanisms responsible may involve both Ca2+ and protein kinase C.