Ether lipid content and fatty acid distribution in rabbit polymorphonuclear neutrophil phospholipids

This study was undertaken to determine if rabbit neutrophils contain sufficient ether-linked precursor for the synthesis of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activatin factor) by a deacylation-reacylation pathway. The phospholipids from rabbit peritoneal polymorphonuclear neutrophils were purified and quantitated, and the choline-containing and ethanolamine-containing phosphoglycerides were analyzed for ether lipid content. Choline-containing phosphoglycerides (37%), ethanolamine-containing phosphoglycerides (30%), and sphingomyelin (28%) were the predominant phospholipid classes, with smaller amounts of phosphatidylserine (5%) and phosphatidylinositol (<1%). The choline-linked fraction contained high amounts of 1-O-alkyl-2-acyl-(46%) and 1,2-diacyl-sn-glycero-3-phosphocholine (54%), with a trace of the 1-O-alk-1′-enyl-2-acyl species. The ethanolamine-linked fraction contained high amounts of 1-O-alk-1′-enyl-2-acyl-(63%) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (34%), and a low quantity of the 1-O-alkyl-2-acyl species (3%). The predominant 1-O-alkyl ether chains found in thesn-1 position of the choline-linked fraction were 16∶0 (35%), 18∶0 (14%), 18∶1 (26%), 20∶0 (16%), and 22∶0 (9%). The major 1-O-alk-1′-enyl ether chains found in thesn-1 position of the ethanolamine-linked fraction were 14∶0 (13%), 16∶0 (44%), 18∶0 (27%), 18∶1 (12%) and 18∶2 (3%). The major acyl groups in thesn-1 position of 1,2-diacyl-sn-glycero-3-phosphocholine and 1,2-diacyl-sn-glycero-3-phosphoethanolamine were 16∶0, 18∶0 and 18∶1. The most abundant acyl group in thesn-2 position of all classes of choline- and ethanolamine-linked phosphoglycerides was 18⩺2. Although this work does not define the biosynthetic pathway for platelet activating factor, it does show that there is ample precursor present to support its synthesis by a deacylation-reacylation pathway.     

1-Acyl-2-acetyl-sn-glycero-3-phosphocholine from stimulated human polymorphonuclear leukocytes

1-Acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl GPC) was found in the fraction of platelet-activating factor obtained from stimulated human polymorphonuclear leukocytes (PMN). The amount of 1-acyl-2-acetyl GPC obtained from 1×10⁷ PMN stimulated with ionophore A23187 at 37 C for 15 min ranged from 8 to 56 pmol (32±10 pmol, mean±standard error; n=4). The main species was 16∶0 palmitoyl (17±5 pmol), followed by 18∶0 stearoyl (8±3 pmol) and 18∶1 oleoyl (7±3 pmol). Although the physiological significance is unknown, 1-acyl-2-acetyl GPC was always detected when 1-alkyl-2-acetyl GPC was detected.     

1-O-alkyl-linked phosphoglycerides of human platelets: Distribution of arachidonate and other acyl residues in the ether-linked and diacyl species

In this study, the 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine content of human platelets was determined. The distribution of arachidonate among the 1,2-diacyl, 1-O-alkyl-2-acyl, and 1-O-alk-l′-enyl-2-acyl classes of choline- and ethanolamine-containing phosphoglycerides was also assessed. The major platelet phospholipids were choline-containing phosphoglycerides (38%), ethanolamine-containing phosphoglycerides (25%) and sphingomyelin (18%), with smaller amounts of phosphatidylserine (11%) and phosphatidylinositol (4%). In addition to the diacyl class, the choline-linked fraction was found to contain both 1-O-alkyl-2-acyl (10%) and 1-O-alk-l′-enyl-2-acyl (9%) species. The ethanolamine-linked fraction, on the other hand, had an elevated level of the 1-O-alk-l′-enyl-2-acyl (60%) species and a small amount of the 1-O-alkyl-2-acyl component (4%). The major fatty acyl residues found in all classes of the choline and ethanolamine phospholipids were 16∶0, 18∶0, (Δ⁹), 18∶2(n−6) and 20∶4(n−6). The 1-O-alk-l and 1-O-alk-l′-enyl fraction of the ethanolamine-linked phospholipids also contained substantial amounts of 22∶4(n−6), 22∶5(n−3) and 22∶6(n−3) acyl chains. Arachidonate comprised 44% of the acyl residues in thesn-2 position of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine. Corresponding values for the diacyl and 1-O-alk-l′-enyl-2-acyl species were 23% and 25%, respectively, based on all 20∶4(n−6) being linked to thesn-2 position of all classes. In the ethanolamine-linked phosphoglycerides, arachidonate constituted 60%, 20% and 68% of the acyl groups in thesn-2 position of the 1,2-diacyl, 1-O-alkyl-2-acyl and 1-O-alk-l′-enyl-2-acyl classes, respectively. The content of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine appears sufficient to support the synthesis of platelet activating factor by a deacylation-reacylation pathway in platelets. Our findings also demonstrate that human platelets contain a significant amount of 1-O-alkyl-2-arachidonyl-sn-glycero-3-phosphocholine that could possibly serve as a precursor of both platelet activating factor and bioactive arachidonate metabolites.     

1-O-alk-1′-enyl-2-acyl and 1-O-alkyl-2-acyl glycerophospholipids in white muscle of bonitoEuthynnus pelamis (Linnaeus)

The existence of ether-linked phospholipids, including 1-O-alk-1′-enyl-2-acyl and 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholines and ethanolamines in bonitoEuthynnus pelamis (Linnaeus) white muscle, was investigated by gas chromatography and gas chromatography-mass spectrometry. Chemical ionization (iso-butane) mass spectrometry of trimethylsilyl ethers derived from the corresponding ether-linked glycerophospholipids proved effective not only for determining molecular weights but also for structural identification based on the ions [M−R]⁺, [M−RO]⁺ and [M+1]⁺. 1-O-Alk-1′-enyl-2-acyl-sn-glycero-3-phosphocholine and ethanolamine accounted for 3.0–6.0% and 3.6–7.6% of the total glycerophospholipids, respectively. 1-O-Alkyl-2-acyl-sn-glycero-3-phosphocholine and ethanolamine were also determined for one fish and accounted for 1.4% and 0.6% of the total glycerophospholipids, respectively. The predominant long chains in thesn-1 position of the glycerol moieties were 16∶0, 18∶0 and 18∶1 in the case of the alkenylacyl and alkylacyl components. Fatty acid distribution of individual glycerophospholipids was also determined.     

Molecular species of 1-O-alk-1′-enyl-2-acyl-, 1-O-alkyl-2-acyl- and 1,2-diacyl glycerophospholipids in Japanese oysterCrassostrea gigas (Thunberg)

Molecular species of 1-O-alk-1′-enyl-2-acyl-, 1-O-alkyl-2-acyl-, and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (EPL) andsn-glycero-3-phosphocholine (CPL) of Japanese oysterCrassostrea gigas were analyzed by selectedion monitoring gas chromatography/mass spectrometry using electron impact ionization. The characteristic fragment ions, [RCH=CH+56]⁺ due to the alkenyl residue in thesn-1 position and [RCO+74]⁺ due to the acyl residue in thesn-2 position of alkenylacylglycerols, [R+130]⁺ due to the alkyl residue in thesn-1 position and [RCO+74]⁺ due to the acyl residue in thesn-2 position of alkylacylglycerols, [RCO+74]⁺ due to the acyl residues in thesn-1 and/orsn-2 positions of diacylglycerols, and [M−57]⁺ being indicative of the corresponding molecular weight, were used for structural assignments. For alkenylacyl EPL and CPL, 19 and 16 molecular species were determined, respectively. Two molecular species, 18∶0alkenyl-22∶6n−3 and 18∶0-alkenyl-22∶2-non-methylene interrupted diene (NMID), amounted to 53.2% and 47.9%, respectively. The alkylacyl EPL and CPL consisted of 16 and 20 molecular species, respectively, and the prominent components were 18∶0alkyl-22∶2NMID, 20∶1alkyl-20∶1n−11 (27.4%) and 20∶1alkyl-20∶2NMID (16.3%) in the former, and 16∶0alkyl-20∶5n−3 (23.0%) and 16∶0alkyl-22∶6n−3 (21.6%) in the latter. For the diacyl EPL and CPL, 14 and 51 molecular species were determined, respectively. The major molecular species were 18∶0–20∶5n−3 (37.4%), 16∶0–20∶5n−3 (14.2%) and 18∶1n−7–22∶2NMID (13.2%) in the former, and 16∶0–20∶5n−3 (33.4%) and 16∶0–22∶6n−3 (22.3%) in the latter. It was found that there were significant differences in the molecular species between the alkylacyl and diacyl EPL and the alkylacyl and diacyl CPL; the number of molecular species was larger in CPL than in EPL, while the number of total carbons and double bonds of the major molecular species were larger in the EPL than in the CPL. Alkenylacyl EPL were similar to alkenylacyl CPL in molecular species composition.     

Fatty chain compositon of ether and ester glycerophospholipids in the Japanese oysterCrassostrea gigas (Thunberg)

The fatty chain compositions of 1-O-alk-1′-enyl-2-acyl, 1-0-alkyl-2-acyl, and 1,2-diacyl glycerophospholipids of the Japanese oysterCrassostrea gigas (Thunberg) were investigated. Major fatty chains in thesn-1 position of 1-alk-1′-enyl-2-acyl ethanolamine phospholipids (EPL) were 18∶0 (64.7%) and 20∶1 (11.1%). Majorsn-1 chains of alkenylacyl choline phospholipids (CPL) were 18∶0 (63.3%) and 16∶0 (22.2%). In the case of 1-alkyl-2-acyl EPL, the predominant fatty chains in thesn-1 position were 18∶0 (51.5%), 16∶0 (16.0%) and 20∶1 (12.5%); in the case of 1-alkyl-2-acyl CPL, the majorsn-1 chains were 16∶0 (44.0%) and 14∶0 (23.4%). Saturated fatty chains were predominant in both EPL and CPL. Prominent fatty acids in thesn-2 position of the alkenylacyl EPL were 22∶6n−3 (29.0%), 20∶5n−3 (19.0%) and 22∶2 NMID (non-methylene interrupted dienes, 16.6%) contributing to about 65% of the total fatty acids, while alkenylacyl CPL was rich in the saturated acids 16∶0 (32.0%) and 18∶0 (9.2%). In the alkylacyl EPL, 16∶0, 18∶1n−9, 18∶0 and 16∶1n−7 were prominentsn-2 fatty acids and accounted for 30.6%, 10.0%, 9.8%, and 8.3%, respectively. Polyunsaturated fatty acids were detected, but were present at extremely low percentages. Majorsn-2 fatty acids in alkylacyl CPL were 16∶0 (25.4%), 22∶6n−3 (16.0%) and 20∶5n−3 (8.4%). The major fatty acids of diacyl EPL were 20∶5n−3 (22.3%), 16∶0 (17.9%), and 18∶0 (16.1%), and those of diacyl CPL were 16∶0 (30.4%), 20∶5n−3 (17.6%) and 18∶1n−7 (7.4%).     

Resolution of radiolabeled molecular species of phospholipid in human platelets: Effect of thrombin

Resolution of individual molecular species of human platelet 1,2-diradyl-sn-glycero-3-phosphocholines and 1,2-diradyl-sn-glycero-3-phosphoethanolamines by reverse phase high pressure liquid chromatography (HPLC) allowed a thorough analysis of those phospholipids labeled with [³H]arachidonic acid. Approximately 54% and 16% of the total incorporated radiolabel was found in choline glycerophospholipids and ethanolamine glycerophospholipids, respectively, with ca. 90% of this being found in the 1,2-diacyl molecular species. Eighty percent of [³H]-arachidonic acid incorporated into 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine in resting platelets was equally distributed between 1-palmitoyl-2-arachidonoyl and 2-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, while 70% of the radiolabel in 1-acyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine was found in 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine. Thrombin stimulation (5 U/ml for 5 min) resulted in deacylation of all 1-acyl-2-[³H]arachidonoyl molecular species of 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-acyl-2-arachidonoyl-sn-glycero-3-ethanolamine. There was also a slight increase in 1-O-alkyl-2-[³H]arachidonoyl-sn-glycero-3-phosphocholine and a significant increase in 1-O-alk-1′-enyl-2-[³H]arachidonoyl-sn-glycero-3-phosphoethanolamine molecular species of over 300%. Thus, HPLC methodology indicates that arachidonoyl-containing molecular species of phosphatidylcholine and phosphatidylethanolamine are the major source of arachidonic acid in thrombin-stimulated human platelets, while certain ether phospholipid molecular species become enriched in arachidonate.     

Biphasic action of platelet-activating factor on isolated guinea-pig ileum

1-0-Hexadecyl-2-0-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) at 10⁻¹⁰-10⁻⁹ M induced slow contraction of isolated guinea-pig ilcal muscles and the contraction persisted for a long time. At a higher concentration of 10⁻⁷ M, this phospholipid induced more rapid, but not greater, contraction. At higher concentrations (10⁻⁶-10⁻⁵ M), this phospholipid induced a biphasic response: rapid contraction followed by relaxation. At high concentrations, this compound inhibited acetylcholine-induced contractions. The stimulatory effect of this phospholipid was ca. 300 times that of 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphocholine, while its inhibitory potency on induced contraction was similar to those of 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphocholine and its lyso derivative. It was suggested that the differences in effects on contraction of different concentrations of 1-0-hexadecyl- and 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphocholine were due to the dual effects of these compounds on the ileum: a strong stimulatory effect and a moderate inhibitory effect on contraction.     

Distributions of hydroperoxide positional isomers generated by oxidation of 1-palmitoyl-2-arachidonoyl-<Emphasis Type="Italic">sn</Emphasis>-glycero-3-phosphocholine in liposomes and in methanol solution

The differences in distribution of geometric isomers of unsaturated PC hydroperoxides generated by free radical oxidation were compared, as corresponding hydroxy analogs, in heterogeneous liposomes and in a homogeneous methanol solution by using HPLC with UV detection due to the presence of conjugated dienes. Identification of fractionated peak components was carried out by GC-MS. When the oxidation of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine, PC(16∶0/18∶2), was initiated in liposomes by a hydrophilic azo radical initiator, and in a methanol solution by a hydrophobic azo radical initiator, there was no significant difference in the relative percentages of 1-palmitoyl-2-(9-hydroxy-trans-10,trans-12-octadecadienoyl)-sn-glycero-3-phosphocholine (9-t,t-OH PC) and 1-palmitoyl-2-(13-hydroxy-trans-9,trans-11-octadecadienoyl)-sn-glycero-3-phosphocholine (13-t,t-OH PC) between the PC oxidized in liposomes and in the methanol solution. For the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, PC(16∶0/20∶4), the relative percentage of 1-palmitoyl-2-(5-hydroxy-trans-6,cis-8,11,14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (5-OH PC) was significantly higher (P<0.01) than that of 1-palmitoyl-2-(15-hydroxy-cis-5,8,11,trans-13-eicosatetraenoyl)-sn-glycero-3-phosphocholine (15-OH PC) in liposomes. For the homogeneous methanol solution of PC(16∶0/20∶4), the relative percentage of 5-OH PC was close to that of 15-OH PC. For the PC(16∶0/20∶4) oxidized in bulk with added pentamethylchromanol, the individual amount of 15-OH PC, 1-palmitoyl-2-(11-hydroxy-cis-5,8trans-12,cis-14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (11-OH PC), 1-palmitoyl-2-(12-hydroxy-cis-5,8,trans-10,cis-14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (12-OH PC), 1-palmitoyl-2-(8-hydroxy-cis-5,trans-9,cis-11,14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (8-OH PC), 1-palmitoyl-2-(9-hydroxy-cis-5,trans-7,cis-11,14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (9-OH PC), and 5-OH PC were close to each other compared to the corresponding values in liposomes and in methanol solution. The results obtained by gel permeation chromatography of the PC liposomes containing hydrophilic 2,2′-azobis-2-amidinopropane) dihydrochloride (AAPH) suggest that the AAPH added to the liposomes of PC(16∶0/20∶4) was partitioned into the water phase and out of the hydrophobic region of the fatty acyl moieties of the PC. These results confirm that the distance that exists in the bis-allylic carbons of the unsaturated fatty acyl moieties of PC from the interface between the hydrophilic region of PC and the water phases played an important role in influencing hydrogen abstraction to form a symmetrical distribution of hydroperoxide isomers in both the heterogeneous liposomes and the homogeneous methanol solution.     

Acylation of lyso platelet-activating factor by splenocytes of the rainbow trout,Oncorhyncus mykiss

In mammalian systems, platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, (PAF) is rapidly inactivated by a deacetylation/reacylation system that produces 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine which is highly enriched in arachidonic acid. There is some evidence that n−3 fatty acids may have an impact on this system in humans but the nature of this impact is unclear. In rainbow trout, n−3 fatty acids are known to be essential dietary components which are derived through the food chain. Substantial quantities of n−3 fatty acids are found in trout membrane phospholipids. We show here that in sharp contrast to mammalian cells, trout cells acylate lyso platelet-activating factor, alkyl-GPC, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine, (lyso-PAF) with a high degree of specificity for n−3 fatty acids. When [³H]lysoPAF was incubated with these cells, only three molecular species of alkylacylglycerophosphocholine were produced, and 92% contained n−3 fatty acids. Since isolated membranes yielded similar results, it appears that the acylation proceedsvia a coenzyme A-independent transacylase as found in mammalian systems.     

Lipid changes in HL-60 cells on differentiation into macrophages by treatment with a phorbol ester

We studied changes in lipid composition of human promyelocytic leukemia cells (HL-60) on differentiation to the macrophage/monocytic lineage by treatment with the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate. Differentiation was accompanied by: (i) a decrease in the level of phospholipids; (ii) a greater amount of triacylglycerols; (iii) an increase in 1-alk-1′-enyl-2-acyl- and 1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine and a decrease in 1-alkyl-2-acyl-sn-glycero-3-phosphocholine; and (iv) an increase in the level of arachidonic acid in ethanolamine phospholipids. The increased levels of ether-linked lipids and of arachidonic acid in ethanolamine phospholipids are consistent with an enhanced biosynthesis of platelet-activating factor and eicosanoids, which are particularly important in the macrophage function.     

Hydrocarbon chain distribution of ether phospholipids of the ascidianHalocynthia roretzi and the sea urchinStrongylocentrotus intermedius

The contents and compositions of the 1-O-alk-1′-enyl-2-acyl, 1-O-alkyl-2-acyl, and 1,2-diacyl glycerophospholipids in the muscle and viscera of the ascidianHalocynthia roretzi, and of the gonad of the sea urchinStrongylocentrotus intermedius, which are eaten to some extent in Alaska and in Asia, were analyzed by gas-liquid chromatography. 1-O-Alk-1′-enyl-2-acyl glycerophospholipids were found in all of the samples, accounting for 64.4–69.0% of the ethanolamine glycerophospholipid (EPL). By contrast, the levels of the 1-O-Alk-1′-enyl-2-acyl choline glycerophospholipids (CPL) were low (3.1–5.7%). CPL was rich in the 1-O-alkyl-2-acyl subclass amounting to 12.5–23.9% in the ascidian sample. The level of CPL in the sea urchin gonad was extremely high, amounting to 46.1%. The most prominent alkyl chains in thesn-1 position of CPL from the ascidian muscle were 16∶0 (44.6%), 18∶1 (26.5%), and 18∶0 (10.7%), and of CPL from the sea urchin gonad were 18∶0 (36.2%), 16∶0 (33.0%), and 18∶1 (17.8%). Unusually high levels of odd-numbered alkyl chains, e.g., 19∶0 andanteiso 17∶0, were detected in the CPL of all samples. The prominent alkenyl chains of EPL were 18∶0 (69.4%), 16∶0 (10.0%), and 18∶1 (8.54%) (not counting the vinyl double bond) for the sea urchin gonad. Relatively high levels of 20∶1 alkenyl chains were also present. The glycerolsn-2 positions contained high proportions of polyunsaturated fatty acids. Thus, 20∶5n-3 (43.6%) and 22∶6n-3 (20.1%) were most abundant in the alkylacyl CPL from the ascidian muscle and 20∶5n-3 (54.9%) and 20∶4n-6 (30.1%) in alkylacyl CPL from the sea urchin gonad. Despite a possible interconversion of the alkyl and alkenyl chains of each class of the ether phospholipids, they showed few features in common.     

Molecular species composition of glycerophospholipids from white matter of human brain

The molecular species composition of the major glycerophospholipids from white matter of human brain were determined by high-performance liquid chromatography of the 3,5-dinitrobenzoyl derivatives of the corresponding diradylglycerols. In phosphatidylcholine (PC) and phosphatidylserine (PS), molecular species containing only saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) comprised 85.7 and 82.4% of the respective totals, with 18∶0/18∶1 predominant in PS and 16∶0/18∶1 in PC. These molecular species were also abundant in phosphatidylethanolamine (PE), but in this phospholipid species containing polyunsaturated fatty acids (PUFA), largely 18∶0/22∶6n−3 and 18∶0/20∶4n−6, accounted for over half the total; 18∶1/18∶1 was also abundant in PE. In contrast, 1-O-alk-1′-enyl-2-acylsn-glycero-3-phosphoethanolamine (GPE) had much more SFA- and MUFA-containing species, predominantly 16∶0a/18∶1, 18∶0a/18∶1 and 18∶1a/18∶1, with low amounts of species containing 20∶4n−6 and 22∶6n−3. In alkenylacyl GPE, 22∶4n−6 was the major PUFA and 16∶0a/22∶4n−6 and 18∶1a/22∶4n−6 the main PUFA-containing species. There was six times more 22∶6n−3, twice as much 20∶4n−6 and half the amount of 22∶4n−6 in PE as compared to alkenylacyl GPE. Molecular species are abbreviated as follows:e.g., 16∶0/18∶1 PE is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; the corresponding alkenylacyl species, 1-O-hexadec-1′-enyl-2-oleoyl-sn-glycero-3-phosphoethanolamine is 16∶0a/18∶1.     

Synthesis of trideuteratedO-alkyl platelet activating factor and lyso derivatives

Racemic heavy isotope analogs of 1-O-alkyl-sn-glycero-3-phosphocholine (lysoPAF) and 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (PAF) were prepared for use as internal standards to facilitate quantitative studies based on mass spectrometry. Starting from pentadencane-1,15-diol andrac-glycerol-1,2-acetonide, a convergent synthesis of 1-O-[16′-²H₃]hexadecyl and 1-O-[18′-²H₃]octadecylrac-glycero-3-phosphocholine and their acetyl derivatives is described. Three deuterium atoms were introduced at the terminal position of the 1-O-alkyl group by displacement of thep-toluensulfonyl group from 1-O-alkyl-15′-p-toluensulfonate and 1-O-alkyl-17′-p-toluensulfonate with [²H₃]-methylmagnesium iodide. The 1-O-alkyl-17′-p-toluensulfonate was obtained by reaction of the 1-O-alkyl-15′-p-toluensulfonate with allylmagnesium bromide, followed by reductive ozonolysis and treatment withp-toluenesulfonyl chloride. The hydroxyl group at C-2 was protected by a benzyl group and removed at a late stage in the synthesis. This provided the corresponding lysoderivatives or allowed preparation of racemic PAF by subsequent acetylation of the free hydroxy group. The phosphocholine moiety was introduced at glycerol C-3 by reaction with bromoethyldichlorophosphate and trimethylamine. The synthetic compounds were analyzed by FAB/MS and GC/NICIMS. They were shown to contain less than 0.6% protium impurity.     

The metabolism of 1-acyl-PAF in rabbit platelets and its possible interaction with PAF

The metabolism of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-PAF), a naturally occurring analogue of platelet activating factor (PAF), was investigated in rabbit platelets. Our studies showed that 1-acyl-[³H]PAF (1-palmitoyl-2-acetyl-sn-glycero-3-phospho[N-methyl-³H]-choline) was converted by platelets into phosphatidyl-[³H]choline ([³H]PC) in a time-dependent fashion. The formation of [³H]PC occurred at a rate similar to that observed when lyso-[³H]PC (palmitoyl-sn-glycero-3-phospho[N-methyl-³H]choline) was used as substrate. In addition, a time-dependent increase in the level of water-soluble radioactivity was observed during the incubation of platelets with either 1-acyl-[³H]PAF or lyso-[³H]PC. This increase was parallel to the formation of [³H]PC and was not observed in the presence of [¹⁴C]PAF (1-octadecyl-2-acetyl-sn-glycerol-3-phospho[N methyl-¹⁴C]choline). Analysis by thin-layer chromatography showed that the soluble radioactivity was mainly associated with glycerophosphocholine (GPC). On the other hand, the preincubation of platelets with phenylmethylsulfonyl fluoride, an inhibitor of the acetylhydrolase, reduced the hydrolysis of 1-acyl-[³H]PAF to [³H]GPC with a concomitant accumulation of radioactivity in 1-acyl-PAF. These findings suggest that 1-acyl-PAF is converted into PC through deacetylation-reacylation with lysoPC as an obligatory intermediate. The findings also indicate that the lysoPC resulting from 1-acyl-PAF is either reacylated to phosphatidylcholine (PC) or hydrolyzed to GPC by lysophospholipase. Finally, we showed that the stimulation of platelets with PAF led to a time- and concentration-dependent increase in the conversion of 1-acyl-[³H]PAF to [³H]PC. The stimulatory effect of PAF was not observed when platelets were lysed before incubation, suggesting that PAF enhances the metabolism of 1-acyl-PAF, probably by accelerating its translocation through the plasma membrane.     

Molecular species analysis of phosphoglycerides from the ripe roes of cod (Gadus morhua)

Molecular species of the 3,5-dinitrobenzoyl derivatives of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) were quantitated by UV detection at 254 nm after reversed-phase HPLC using solvent systems modified from Takamuraet al. (Lipids 21, 356–361, 1986). Three isocratic solvent systems were used and a total of 39 different molecular species detected. Four species, 16∶0/20∶5, 18∶1/20∶5, 16∶0/22∶6 and 18∶1/22∶6 contributed 67.2% and 61.8% of PC and PE respectively but only 23.0% of PI. In PI the most important species was 18∶0/20∶4 at 36.7% but this species only constituted 0.7% in each of PC and PE. Small amounts of dipolyunsaturated species were also found in PC and PE. Molecular species are abbreviated as follows: e.g., 16∶0/20∶5 PC is 1-palmitoyl-2-eicosapentaenoyl-sn-glycero-3-phosphocholine.     

Separation of the enantiomers of 1-alkyl-2-acyl-rac-glycerol and of 1-alkyl-3-acyl-rac-glycerol by high performance liquid chromatography on a chiral column

High performance liquid chromatographic separations of two enantiomeric pairs of 1-alkyl-2-acyl-rac-glycerol (1-alkyl-2-acyl- and 3-alkyl-2-acyl-sn-glycerols) and 1-alkyl-3-acyl-rac-glycerol (1-alkyl-3-acyl- and 3-alkyl-1-acyl-sn-glycerols) as 3,5-dinitrophenylurethanes (3,5-DNPUs) were carried out on a chiral stationary phase, N-(R)-1-(α-naphthyl)ethylaminocarbonyl-(S)-valine chemically bonded to γ-aminopropyl silanized silica (Sumipax OA-4100). Good separation of the enantiomers of 1-hexadecyl-2-hexadecanoyl-rac-glycerol was easily achieved within 10 min using hexane/ethylene dichloride/ethanol (80∶20∶1, v/v/v) as a mobile phase. Separation of the enantiomers of 1-hexadecyl-3-hexadecanoyl-rac-glycerol was more difficult and required about 80 min to achieve satisfactory peak resolution (0.8) using hexane/ethylene dichloride/ethanol (250∶20∶1, v/v/v) as a mobile phase. Presented at the American Oil Chemists' Society 79th Annual Meeting, Phoenix, AZ, May, 1988.     

Biochemical characterization of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase in rat spleen microsomes

Acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF) ultrasonic disruption in the presence of 25% glycerol from rat spleen microsomes. About 26% of the enzymatic activity was recovered in the 225,000×g supernatant by this treatment, although the specific activity was slightly decreased compared with the original microsomes. The solubilized enzyme was remarkably susceptible to various kinds of metal ions. Sulfhydryl reagents such as p-chloromercuribenzoate and N-ethyl-maleimide significantly inhibited the enzyme reaction, suggesting that the enzyme is an SH enzyme. Based on the sedimentation pattern in sucrose density centrifugation, the isoelectric point, the kinetic characteristics and the sensitivity to tryptic digestion of microsomes, it appears that acetyl-CoA:lyso-PAF acetyltransferase does not differ from the acetyltransferase responsible for the transfer of acetate from acetyl-CoA to 1-acyl-2-lyso-sn-glycero-3-phosphocholine.     

1-O-alk-1′-enyl-2-acyl-glycerophosphoethanolamine content and molecular species composition in fish brain

Ethanolamine glycerophospholipids from the brains of both trout and cod comprised 36–38% of 1-O-alk-1′-enyl-2-acyl-glycerophosphoethanolamine (GPE) determined using two methods. In 1-O-alk-1′-enyl-2-acyl-GPE from trout brain, the main molecular species were 18∶1a/18∶1, 18∶0a/18∶1 and 16∶0a/18∶1, which totalled 63.3%, while polyunsaturated fatty acid (PUFA) containing species totalled only 18.2%. 1-O-Alk-1′-enyl-2-acyl-GPE from cod brain was much more unsaturated with PUFA containing species totalling 52.6%, of which 18∶0a/20∶5n−3, 18∶1a/20∶5n−3 and 18∶1a/22∶6n−3 were predominant. In cod 18∶1a/18∶1, 18∶0a/18∶1 and 16∶0a/18∶1 were the only other species present at over 5% each, totalling 31.8%. In both cod and trout, small amounts of species containing 22∶4n−6 were found. The results of this and earlier studies indicate that there is considerable specificity of composition at the level of molecular species between different lipid classes and subclasses. Molecular species of 1-O-alk-1′-enyl-2-acyl-GPE are abbreviated as follows:e.g., 16∶0a/18∶1 GPE is 1-O-hexadec-1′-enyl-2-oleoyl-sn-glycero-3-phosphoethanolamine. The corresponding diacyl species, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, is abbreviated as 16∶0/18∶1.     

Vitamin E. Deficiency increases the synthesis of platelet-activating factor (PAF) in rat polymorphonuclear leucocytes

Vitamin E deficiency was found to stimulate FMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine)-induced biosynthesis of PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in polymorphonuclear leucocytes (PMN) from rat peritoneum. In three separate experiments each, the amounts of PAF synthesized during 6min and 12 min incubation of PMN cells from control, vitamin E-supplemented, and vitamin E-deficient rats were 129–240, 131–227 and 248–354 pmol/10⁶ cells, respectively. The activity of the acetyl-transferase, which transfers the acetyl moiety of [³H]acetyl-CoA to 2-lysoPAF (1-O-alkyl-sn-glycero-3-phosphocholine) to form [³H]PAF, was higher in PMN homogenates from vitamin E-deficient rats (2.28±0.07 nmol/min/mg protein) than in those from E-supplemented rats (1.06±0.10 nmol/min/mg protein). However, there was no difference between the two groups in the activity of acetylhydrolase (4.26±0.71 and 4.26±0.06 nmol/min/mg protein, respectively), measured as degradation of [³H]PAF to [³H]lysoPAF.In vitro addition of α-tocopherol did not inhibit the increased activity of acetyl-transferase in vitamin E-deficient rats, in-dicating that the enzyme in vitamin E-supplemented rats was not directly inhibited by α-tocopherol. The acetyltransferases of the two groups showed similar Km values for acetyl-CoA, but different Vmax values (225 μM and 6.4 nmol/min/mg protein in vitamin E-deficient rats, and 216 μM and 3.6 nmol/min/mg protein in vitamin E-supplemented rats), suggesting that the enzyme was not activated but increased in amount in vitamin E deficiency.