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.     

Incorporation of polyunsaturated fatty acids into CT-26, a transplantable murine colonic adenocarcinoma

Previous studies in our laboratory have shown that marine oils, with high levels of eicosapentaenoic (EPA, 20∶5n−3) and docosahexaenoic acids (DHA, 22∶6n−3), inhibit the growth of CT-26, a murine colon carcinoma cell line, when implanted into the colons of male BALB/c mice. Anin vitro model was developed to study the incorporation of polyunsaturated fatty acids (PUFA) into CT-26 cells in culture. PUFA-induced changes in the phospholipid fatty acid composition and the affinity with which different fatty acids enter the various phospholipid species and subspecies were examined. We found that supplementation of cultured CT-26 cells with either 50 μM linoleic acid (LIN, 18∶2n−6), arachidonic acid (AA, 20∶4n−6), EPA, or DHA significantly alters the fatty acid composition of CT-26 cells. Incorporation of these fatty acids resulted in decreased levels of monounsaturated fatty acids, while EPA and DHA also resulted in lower levels of AA. While significant elongation of both AA and EPA occurred, LIN remained relatively unmodified. Incorporation of radiolabeled fatty acids into different phospholipid species varied significantly. LIN was incorporated predominantly into phosphatidylcholine and had a much lower affinity for the ethanolamine phospholipids. DHA had a higher affinity for plasmenylethanolamine (1-O-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) than the other fatty acids, while EPA had the highest affinity for phosphatidylethanol-amine (1,2-diacyl-sn-glycero-3-phosphoethanolamine). These results demonstrate that,in vitro, significant differences are seen between the various PUFA in CT-26 cells with respect to metabolism and distribution, and these may help to explain differences observed with respect to their effects on tumor growth and metastasis in the transplantable model.     

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%).     

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.     

Lyso platelet activating factor (LysoPAF) and its enantiomer. Total synthesis and carbon-13 NMR spectroscopy

Described is a reaction sequence for the total synthesis of lyso platelet activating factor (lysoPAF; 1-O-alkyl-sn-glycero-3-phosphocholine) and its enantiomer. The procedure is versatile and yields optically pure isomers of defined chain length. The synthesis is equally suited for the preparation of lysoPAF analogues and its enantiomers with unsaturation in the long aliphatic chain. First,rac-1(3)-O-alkylglycerol is prepared by alkylation ofrac-isopropylideneglycerol with alkyl methanesulfonate followed by acid-catalyzed removal of the ketal group. The primary hydroxy group of alkylglycerol is then protected by tritylation, the secondary hydroxy group is acylated, and the protective trityl group is removed under mild acidic conditions with boric acid on silicic acid, essentially without acyl migration. Condensation of the diradylglycerol with bromoethyl dichlorophosphate in diethyl ether, hydrolysis of the resulting chloride, and nucleophilic displacement of the bromine with trimethylamine givesrac-1-O-alkyl-2-acylglycero-3-phosphocholine in good overall yield. The racemic alkylacylglycerophosphocholine is finally treated with snake venom phospholipase A₂ (Ophiophagus hannah) which affords 1-O-alkyl-sn-glycero-3-phosphocholine (lysoPAF) of natural configuration in optically pure form. The “unnatural” 3-O-alkyl-2-O-acyl-sn-glycerol-1-phosphocholine enantiomer, which is not susceptible to phospholipase A₂ cleavage, gives 3-O-alkyl-sn-glycero-1-phosphocholine upon deacylation with methanolic sodium hydroxide. Homogeneity and structure of the intermediates and final products were ascertained by carbon-13 nuclear magnetic resonance spectroscopy on monomeric solutions.     

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.     

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.     

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.     

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.     

Platelet-activating factor modulates phospholipid acylation in human neutrophils

The present study showed that platelet-activating factor (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, PAF), but not lysoPAF (1-O-hexadecyl-sn-glycero-3-phosphocholine) rapidly (within 15 sec) stimulated the incorporation of both [1-¹⁴C]arachidonate and [1-¹⁴C]docosahexaenoate into phosphatidylinositol (PI) and phosphatidylcholine (PC) in human neutrophils. Concomitantly, it inhibited the formation of labeled phosphatidic acid from both fatty acids. The magnitude of stimulation (percentage of control) was greater in PI than in PC for the incorporation of arachidonate and vice versa for the incorporation of docosahexaenoate. It reached a maximum at 10⁻⁷ M and started to decline at 10⁻⁶ M. Extracellular Ca²⁺ was not essential for the action of PAF on phospholipid acylation. The distribution of labeled arachidonate in the molecular species of PC was not altered by PAF after 1 min incubation, suggesting that the increased formation of arachidonyl-PC during the early stage of neutrophil-PAF interaction was not originated from the added PAF. No measurable changes in the mass of each phospholipid were detected in neutrophils challenged by PAF from 15 sec to 2 min. The data suggest that the increased incorporated of extracellular fatty acids into PI and PC elicited by PAF may be secondary to increased deacylation of these phospholipids, and the magnitude of stimulation reflects the specificity of acyltransferase catalyzing the acylation of lysoPI and lysoPC by fatty acyl-CoA.     

Platelet-activating factor regulates phospholipid metabolism in human neutrophils

This study extended the earlier finding that platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) promotes arachidonic acid incorporation into neutrophil phosphatidylinositol (PI) and phosphatidylcholine (PC). In the present study the effect of PAF on fatty acid uptake by human neutrophils and the incorporation of extracellular linoleic acid and palmitic acid into phospholipids were investigated. Incubation of 10⁻⁷ M PAF with neutrophils and radiolabeled arachidonic acid or linoleic acid or palmitic acid for 1–10 min resulted in an increased rate of loss of label from the incubation medium. PAF stimulated the incorporation of linoleic acid and palmitic acid most significantly into PI and PC. The magnitude of stimulation was greater in PI than in PC for the incorporation of linoleic acid, and vice versa for the incorporation of palmitic acid. The positional distribution of linoleic acid and palmitic acid in PI and PC and the mass of these phospholipids were not altered in PAF-stimulated neutrophils. An increased incorporation of all three fatty acids into both diacyl and alkylacyl species of PC was demonstrated after a two minute incubation of cells with PAF. While more radioactivity was recovered in the diacyl species, the magnitude of increase of radioactivity in the alkylacyl species was more pronounced than that in the diacyl species of PC. These results suggest that both increased fatty acid uptake and increased available lysophospholipids may be contributory to the increased phospholipid acylation induced by PAF.     

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.     

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.     

The effect of two synthetic phospholipids on cell proliferation and phosphatidylcholine biosynthesis in Madin-Darby canine kidney cells

The concentration-dependent effects of two different synthetic phospholipids on cell proliferation and phosphatidylcholine biosynthesis were compared in Madin-Darby canine kidney (MDCK) cells. The alkyllysophospholipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine and the alkylphosphocholine, hexadecylphosphocholine, inhibited cell proliferation with half-inhibitory concentrations (IC₅₀) of 75 and 135 μmol/L, respectively. The agents also inhibited phosphatidylcholine biosynthesis in confluent and proliferating MDCK cells. The IC₅₀ of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine was 40 μmol/L in confluent cells and 20 μmol/L in proliferating cells, whereas the IC₅₀ of hexadecylphosphocholine was higher in both experimental systems (67 μmol/L in confluent cells and 40 μmol/L in proliferating cells). Further experiments revealed that the effect of both agents on phosphatidylcholine biosynthesis was reversible and that the inhibition was mediated by translocation of the rate-limiting enzyme of this pathway, CTP: phosphocholine cytidylyltransferase (EC 2.7.7.15), from membranes to the cytosol, where it is inactive. The present findings suggest that the inhibition of phosphatidylcholine biosynthesis by both synthetic phospholipids might be related, in part, to their antiproliferative effects.     

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.     

Leukotriene B4 promotes phospholipid acylation in human neutrophils

The present study was undertaken to test the hypothesis that leukotriene B₄ (LTB₄) may promote extracellular fatty acid incorporation into neutrophil choline glycerophospholipids (PC) to replenish phospholipids after deacylation. Incubation of human neutrophils with LTB₄ (1.5 to 150 nM) for 1 for 5 min resulted in increased fatty acid incorporation into phosphatidylinositol (PI), diacyl-sn-glycero-3-phosphocholine (diacyl-GPC) and alkylacyl-GPC. The magnitude of stimulation (percentage of control) of fatty acid incorporation appears to reflect increased activity of the acyltransferases catalyzing acylation of the respective lysophospholipids. LTB₄ stimulation of arachidonic acid incorporation into PI was greater than into PC, whereas the stimulation of palmitic acid but not by arachidonic acid. LTB₄ and 1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (cPAF) exhibited a similar stimulatory effect on fatty acid incorporation into the PC fraction. Phosphate analysis could not detect changes in the mass of PI or of PC in neutrophils exposed to LTB₄ or cPAF. The results suggest that increased fatty acid incorporation into phospholipids in LTB₄-activated neutrophils reflects activation of phospholipase A₂ and acyltransferases as well as ofde novo phospholipid synthesis.     

Positional distribution of 24∶6(n−3) in triacyl-sn-glycerols from flathead flounder liver and flesh

This paper presents the positional distribution of very long-chain fatty acids, 24∶6(n−3), in triacyl-sn-glycerols (TG) of flathead flounder (Hippoglossoides dubius). Each of the liver and flesh TGs was subjected to the stereospecific analysis. The liver TGs contained 24∶6(n−3) at concentrations of 1.5, 1.2 and 1.7 mole % in thesn-1,sn-2 andsn-3 positions, respectively, and the flesh TGs had 9.0, 7.8 and 7.1 mole % in thesn-1,sn-2 andsn-3 positions, respectively. This fatty acid was distributed almost evenly among the three positions of the TGs. No preference for thesn-2 position was observed in contrast to the general tendency for the distribution of longer-chain polyunsaturated fatty acids, such as 22∶6(n−3), 22∶5(n−3) and 20∶5(n−3). There was essentially no difference in the positional distributions of the liver and flesh TGs. The results obtained in this study give new fundamental information to the investigation of very long-chain fatty acids.     

Lipid composition of the pineal organ from rainbow trout (Oncorhynchus mykiss)

The lipid composition of the pineal organ from the rainbow trout (Oncorhynchus mykiss) was determined to establish whether the involvement of this organ in the control of circadian rhythms is reflected by specific adaptations of lipid composition. Lipid comprised 4.9% of the tissue wet weight and triacylglycerols were the major lipid class present (47% of total lipid). Phosphatidylcholine (PC) was the principal polar lipid, and smaller proportions of other phospholipids and cholesterol were also present. Plasmalogens contributed 11% of the ethanolamine glycerophospholipids (EGP). No cerebrosides were detected. The fatty acid composition of triacylglycerols was generally similar to that of total lipids in which saturated, monounsaturated and polyunsaturated fatty acids (PUFA) were present in almost equal proportions. Each of the polar lipid classes had a specific fatty acid composition. With the exception of phosphatidylinositol (PI), in which 20∶4n−6 comprised 27.4% of the total fatty acids, 22∶6n−3 was the principal PUFA in all lipid classes. The proportion of 20∶5n−3 never exceeded 6.0% of the fatty acids in any lipid class. The predominant molecular species of PC were 16∶0/22∶6n−3 and 16∶0/18∶1, which accounted for 33.2 and 28.5%, respectively, of the total molecular species of this phospholipid. Phosphatidylethanolamine (PE) contained the highest level of di-22∶6n−3 (13.0%) of any phospholipid. There was also 4.9% of this molecular species in phosphatidylserine (PS) and 4.1% in PC. In PE, the species 16∶0/22∶6, 18∶1/22∶6 and 18∶0/22∶6 totalled 45.1%, while in PS 18∶0/22∶6 accounted for 43.9% of the total molecular species. The most abundant molecular species of PI was 18∶0/20∶4n−6 (37.8%). The lipid composition of the pineal organ of trout, and particularly the molecular species composition of PI, is more similar to the composition of the retina than that of the brain. Molecular species are abbreviated as follows: e.g., 16∶0/22∶6 PC is 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine.     

Biotransformation of alkylglycerols in plant cell cultures: Production of platelet activating factor and other biologically active ether lipids

Plant cells in culture are capable of incorporating exogenous 1-O-alkyl-sn-glycerols into various neutral and ionic ether lipids. 1-O-Alkyl-2-acyl-sn-glycerol-3-phosphocholines, the major class of compounds thus formed, are used for the preparation of platelet activating factor (PAF) in high yields. Similarly, the prochiral 2-O-alkyl-sn-glycerols are transformed to chiral 2-O-alkyl glycerophospholipids from which compounds can be obtained that exhibit antiviral activity in plant and animal cells. Reaction of 1-O-alkyl-2-acyl-sn-glycerol-3-phosphocholines with phospholipase D in the presence of ethanolamine leads to 1-O-alkyl-2-acyl-sn-glycerol-3-phosphoethanolamines, which serve as starting material, for the preparation of 1-O-alkyl-2-acyl-sn-glycero-3-phospho-(N-acyl)ethanolamines, compounds known to have antitumor activity. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989. Dedicated to Professor Morris Kates, Ottawa, on the occasion of his retirement.