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.     

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.     

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.     

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.     

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.     

Intestinal absorption of ester and ether glycerophospholipids in guinea pig. Role of a phospholipase A2 from brush border membrane

In vivo intestinal perfusion was used to follow the absorption of three different choline glycerophospholipids (CGP) in guinea pig. These included 1-[³H]palmitoyl-2-acyl-sn-glycero-3-phosphocholine (diacyl-GPC), 1-[³H]-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) and 1,2-di-O-hexadecyl-sn-glycero-3-phospho-[³H]-choline (dialkyl-GPC). About 80% of diacyl-GPC was absorbed within 4 hr, compared to 60% of alkylacyl-GPC and 30% of dialkyl-GPC. The radioactivity disappearing from the perfusion fluid was recovered in intestinal lipids, mostly triacylglycerol, free fatty acid and CGP from diacyl-GPC, CGP from alkylacyl-GPC and dialkyl-GPC. These results indicated that the nonhydrolyzable substrate dialkyl-GPC was much less absorbed, whereas diacyl-GPC, which released over 80% of [³H]palmitic acid in the perfusion fluid, displayed the highest absorption rate. The intermediate picture observed for alkylacyl-GPC suggested the possible involvement of a phospholipase A₂, which was detected in the entire intestinal tract. This enzyme was further found to concentrate in villus cells, where it is localized in the brush border membrane, as shown using two different subcellular fractionation procedures. These data suggest a possible role of this new enzyme in the digestion of alimentary phospholipids.     

Molecular heterogeneity of platelet-activating factor (PAF) in rat glandular stomach determined by gas chromatography/mass spectrometry. PAF molecular species changes upon water-immersion stress

The molecular heterogeneity of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC) and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (acylacetyl-GPC) in normal rat glandular stomach was studied by gas chromatography/mass spectrometry (GC/MS) and tandem mass spectrometry. The percentage compositions of the molecular species of 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC in the antrum were, respectively. 1-alkyl [16∶0 (34%) and 18∶0 (66%)]-2-acetyl-GPC and 1-acyl [16∶0 (60%), 18∶0 (14%) and 18∶1 (26%)]-2-acetyl-GPC. The alkyl chain composition of 1-alkyl-2-acyl-GPC was quite different from that of 1-alkyl-2-acyl-GPC in both the antrum and corpus, demonstrating a high degree of selectivity of alkyl chain utilization in PAF biosynthesis. The amount of 1-acyl-2-acetyl-GPC was much greater than that of 1-alkyl-2-acetyl-GPC. The molecular heterogeneity of 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC in the corpus was similar to that in the antrum. Water-immersion stress affected not only the amount of 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC, but also their molecular heterogeneity in the antrum and corpus. Whereas the amounts of 1-hexadecyl-2-acetyl-GPC and 1-acyl [16∶0, 18∶0 and 18∶1]-2-acetyl-GPC decreased markedly (to less than one-fifth) in the antrum after such stress for 1 hr, the amount of 1-octadecyl-2-acetyl-GPC increased markedly (up to 4-fold) in the corpus and severe lesions were observed after stress for 7 hr. The changes may be associated with the pathogenicity of gastric ulcers. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

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.     

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.     

Polymerizable glycerophosphocholines containing terminal 2,4-hexadienyloxy groups and their polymerized vesicles(1)

Summary Polymerizable glycerophosphocholines containing one or two 2,4-hexadienyloxy groups at the terminal of the acyl chains were prepared. Those were 1-[11-(2,4-hexadienyloxy)undecanoyl]-2-0-alkyl-rac-glycero-3-phosphocholines 1, 1-acyl-2-[11-(2,4-hexadienyloxy)undecanoyl]-sn-glycero-3-phosphocholines 2 and 1,2-bis[11-(2,4-hexadienyloxy)undecanoyl]-sn-glycero-3-phosphocholine 3. Those having one hexadienyloxy group formed small unilamellar vesicles. One having two groups formed lipid bilayers, but not unilamellar vesicles. 1 and 2 could form stable microcapsules (polymerized vesicles) with the diameters ranging from 20 to 40 nm.     

Effect of platelet-activating factor on cortisol and corticosterone secretion by perfused dog adrenal

Administration of platelet-activating factor (PAF) to perfused adrenal increased cortisol and corticosterone secretion. With hexadecyl PAF (C₁₆PAF; 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine), the increase was significant at 1 nM and maximal at 10 nM. The responses to 10 nM octadecyl PAF (C₁₈PAF; 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine) were one fourth of those to 10 nM C₁₆PAF. The addition of C₁₆PAF to dispersed adrenal cells significantly increased cortisol and corticosterone production at 0.1 nM and 10 nM, respectively. C₁₆PAF was about 1000 times more potent than histamine on a molar basis in respect to cortisol response in both perfused adrenal and dispersed adrenal cells. The results suggest that PAF induces cortisol release from dog adrenal. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989. The present data were also reported at the VIIth International Congress on Hormonal Steroids, Madrid, Spain, September, 1986 (J. Steroid Biochem. 25, 76S, 1986, Abstract).     

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.     

Biosynthesis of triacylglycerols containing ricinoleate in castor microsomes using 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine as the substrate of oleoyl-12-hydroxylase

We have examined the biosynthetic pathway of triacylglycerols containing ricinoleate to determine the steps in the pathway that lead to the high levels of ricinoleate incorporation in castor oil. The biosynthetic pathway was studied by analysis of products resulting from castor microsomal incubation of 1-palmitoyl-2-[¹⁴C]oleoyl-sn-glycero-3-phosphocholine, the substrate of oleoyl-12-hydroxylase, using high-performance liquid chromatography, gas chromatography, mass spectrometry, and/or thin-layer chromatography. In addition to formation of the immediate and major metabolite, 1-palmitoyl-2-[¹⁴C]rici-noleoyl-sn-glycero-3-phosphocholine, ¹⁴C-labeled 2-linoleoyl-phosphatidylcholine (PC), and ¹⁴C-labeled phosphatidylethanolamine were also identified as the metabolites. In addition, the four triacylglycerols that constitute castor oil, triricinolein, 1,2-diricinoleoyl-3-oleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linoleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linolenoyl-sn-glycerol, were also identified as labeled metabolites in the incubation along with labeled fatty acids: ricinoleate, oleate, and linoleate. The conversion of PC to free fatty acids by phospholipase A₂ strongly favored ricinoleate among the fatty acids on the sn-2 position of PC. A major metabolite, 1-palmitoyl-2-oleoyl-sn-glycerol, was identified as the phospholipase C hydrolyte of the substrate; however, its conversion to triacylglycerols was blocked. In the separate incubations of 2-[¹⁴C]ricinoleoyl-PC and [¹⁴C]ricinoleate plus CoA, the metabolites were free ricinoleate and the same triacylglycerols that result from incubation with 2-oleoyl-PC. Our results demonstrate the proposed pathway: 2-oleoyl-PC. Out results demonstrate the proposed pathway: 2-oleoyl-PC→2-ricinoleoyl-PC→ricinoleate →triacylglycerols. The first two steps as well as the step of diacylglycerol acyltransferase show preference for producing ricinoleate and incorporating it in triacylglycerols over oleate and linoleate. Thus, the productions of these triacylglycerols in this relatively short incubation (30 min), as well as the availability of 2-oleoyl-PC in vivo, reflect the in vivo drive to produce triricinolein in castor bean.     

Metabolism of platelet-activating factor by blood platelets and plasma

High performance liquid chromatography in combination with a radioactivity detector was used to study the metabolism of platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by washed platelets, platelet-free plasma and platelet-rich plasma obtained from rabbits and humans. Degradation of platelet-activating factor in plasma was completely inhibited by diisopropylfluorophosphate and was partially inhibited by ethylenediamine tetraacetic acid. Washed platelets metabolized platelet-activating factor not only to the 2-lyso compound but also, by reacylation of this lyso intermediate, to an analogue of platelet-activating factor probably containing a long-chain acyl group at thesn-2 position. These transformations occurred, but to a lesser extent, in platelet-rich plasma.     

Specific binding of antibodies to platelet-activating factor (PAF) as demonstrated by thin-layer chromatography/Immunostaining

The specificity of rabbit antibodies produced by injection of 1-O-(15'-carboxypentadecyl)-2-N,N-dimethylcar-bamoyl-sn-glycero-3-phosphocholine bovine serum albumin (BSA) conjugates was examined by a thin-layer chromatography (TLC)/immunostaining method. Phosphatidylcholine (PC), lysophosphatidylcholine (lysoPC), lyso platelet-activating factor (lysoPAF), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), sphingomyelin (SM), phosphatidylinositol (PI), phosphatidic acid (PA) and cardiolipin (CL) were not immunostained. Among several synthetic PAF-related compounds, the antibodies only bound to PAF agonists which have the activity to induce washed rabbit platelet aggregation. The results suggest that the binding sites of the antibodies on the PAF molecule are the acetyl group at thesn-2 position and the choline moiety at thesn-3 position of glycerol, both of which are essential for exerting the biological function of PAF and for binding to the PAF receptors located on cellular membranes. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

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.     

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.     

Platelet-activating factor detected in bronchoalveolar lavage fluids from an asthmatic patient

It recently has been recognized that platelet-activating factor (PAF) may be a mediator of asthma exacerbation. We had the opportunity to analyze bronchoalveolar lavage fluids from an asthmatic infant, which were characterized by neutrophil infiltration. The patient's lungs were washed on three occasions with saline during asthmatic attacks. PAF was found in each case on the basis of its ability to cause the immediate aggregation of washed rabbit platelets. The PAF detected was equivalent to 1–1.4 pmol of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, three quarters of which were recovered in cell-associated form. By contrast, we did not detect PAF in bronchoalveolar exudates from patients with larungeal stenosis or with respiratory distress syndrome. LysoPAF, the direct precursor as well as initial metabolite of PAF, was also analyzed after being converted to PAF by acetylation. There was a wide variation in the amount of lysoPAF present in individual patients, suggesting that lysoPAF levels cannot be taken as an indicator for the presence of PAF. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

The effect of inhibitors of platelet aggregation on the metabolism of platelet-activating factor (PAF) in washed rabbit platelets

The rabbit platelet metabolizes platelet-activating factor (PAF) intracellulary. PAF is deacetylated to produce lysoPAF which, in turn, can be acylated to produce 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl GPC). Some PAF receptor antagonists have been shown to inhibit this metabolic conversion. In the present study we examined whether the PAF receptor antagonists SRI 63-441 and WEB 2086 would inhibit the metabolism of PAF by intact rabbit platelets. In addition, we examined whether iloprost, a stable analogue of prostaglandin I₂ (PGI₂), and a potent inhibitor of platelet activation induced by a range of agonists, would also inhibit PAF metabolism. We found that SRI 63-441 and WEB 2086 caused an almost complete inhibition of the conversion of PAF to alkylacyl GPC. Iloprost caused up to a 50% inhibition of PAF metabolism compared to antagonist-free controls. Iloprost (and PGI₂) is thought to inhibit platelet response by elevation of cAMP, while receptor antagonists act by blocking PAF binding to its receptor. Since iloprost caused partial inhibition of PAF metabolism, the results of this study suggest that inhibition of PAF metabolism does not occur solely due to competitive inhibition of PAF binding to its receptor. Based on a paper presented at the Third International Conference on Platelet Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

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.