Platelet-activating factor acetylhydrolase activity in human tissues and blood cells

Human tissues, blood cells, and plasma have enzymes that catalyze the hydrolysis of PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). The activities are not due to phospholipases A₂ that hydrolyze long chain acyl groups at thesn-2 position of glycerophospholipids, since they are calcium-independent and are specific for hydrolysis of short chain acyl groups. We examined the biochemical properties of these PAF acetylhydrolase activities (EC 3.1.1.47) in homogenates of human liver and spleen, in white blood cells (neutrophils and monocytes), and in erythrocytes. The data suggest that the plasma and intracellular PAF acetylhydrolase activities are likely due to different proteins. Second, the intracellular PAF acetylhydrolase activities in liver and spleen share several biochemical features that differentiate them from the activities in blood cells. Third, the activities in monocytes and neutrophils have properties that differentiate them from the activity present in human erythrocytes. Finally, the erythrocyte activity has unique properties that place it in a separate category of short chain acylhydrolases. In conclusion, there is a family of distinct enzymes that can be identified as PAF acetylhydrolases based on their calcium-independence and specificity for a short residue at thesn02 position of phospholipids. 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.     

Stereospecific synthesis of antitumor active thioether PAF analogs

A novel stereospecific synthesis of antitumor active thioether analogs of platelet-activating factor (PAF) is reported. The synthesis is based upon: i) the use ofD-serine to provide the chiral center for the construction of the optically active phospholipid molecule; ii) development of thesn-1-thioalkyl function via thioacetate displacement of methanesulfonate-activated primary hydroxyl group followed by alkylation of thesn-1-thiolate function; and iii) introduction of the phosphocholine moiety through the 2-chloro-2-oxo-1,3,2-dioxaphospholane/trimethylamine sequence. The entire scheme relies on the use of a single protecting group. The synthetic thioether phospholipid 1-S-hexadecyl-2-N-acetamidodeoxy-sn-glycero-3-phosphocholine has been shown to be a potent antitumor active phospholipid, exhibiting tumor cytotoxicity against a lymphoblastoid lymphoma (Li-A) cell line and a malignant histiocytic (DHL-4) cell line of human origin at the same level of potency as ET-18-OMe and 1-O-octadecyl-2-N-acetamidodeoxy-sn-glycero-3-phosphocholine. The synthetic method described has a great deal of flexibility, providing a convenient general route to a wide range of thioether PAF analogs. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

A sensitive and specific radioimmunoassay for platelet-activating factor

A platelet-activating factor (PAF) analog with a reactive ω-aldehyde group at thesn-1 position was synthesized. The hapten-thyroglobulin conjugate was used to immunize rabbits to produce specific antibodies to PAF. The purified immunoglobulin G (IgG) fraction was found to bind stereo-specifically to tritiated PAF and to crossreact minimally with lysoPAF, plasmalogens, and other phospholipids. The radioimmunoassay detected as little as 20 pg of PAF per assay tube and was used to explore agonist-induced synthesis of PAF in rabbit neutrophils. Calcium ionophore A23187 at 1 μM induced PAF synthesis peaking at 2 min and reaching basal levels after 5 min.N-Formyl-Met-Leu-Phe (FMLP) at 0.1 μM also stimulated rapid synthesis and degradation of PAF with a peak at 5 min. Both A23187 and FMLP stimulated PAF synthesis in a dose-dependent manner. The radioimmunoassay should be applicable to the quantitation of PAF in biological samples. Based on a paper originally 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.     

Varietal differences in peanut triacylglycerol structure

Stereospecific analysis of triacylglycerols from six peanut varieties showed diversity in percent fatty acid placement. Distribution of the fatty acids among thesn-1,-2 and-3 positions was clearly nonrandom. The percentages of palmitic and stearic acids, generally very low at thesn-2 position, were more predominant at thesn-1 than thesn-3 position. Long chain fatty acids were located almost exclusively at thesn-3 position. Thesn-2 position of all varieties was high in unsaturated fatty acids. Triacyglycerols were sufficiently different to suggest that concentrations of specific triacylglycerol species may vary with variety. Mention of firm names or trade products does not imply that they are endorsed or recommended by the Department of Agriculture over other firms or similar products not mentioned.     

Separation of isomeric lysophospholipids by reverse phase HPLC

A reverse-phase high performance liquid chromatography (HPLC) method was developed which resolved isomers of lysophosphatidylcholine (LPC) differing in the location of the aliphatic chain (sn-1 orsn-2 position) and the position (Δ⁶ or Δ⁹) or geometric configuration (cis ortrans) of the olefin group in monounsaturated species. LPC isomers containing an acyl substituent at thesn-2 position eluted before their 1-acyl-sn-glycero-3-phosphocholine (1-acyl LPC) counterparts. The retention times of both thesn-1 andsn-2 isomers of monounsaturated species increased in the order Δ⁹-cis < Δ⁹-trans < Δ⁶-cis. The integrated ultraviolet absorbance (203 nm) in binary mixtures of the Δ⁹-cis and Δ⁶-cis 2-acyl lysophospholipid isomers correlated with the lipid phosphorus content of corresponding column eluates (r-0.994). Thus, the present method will facilitate synthesis of isomerically pure diradylphospholipids by providing homogeneous lysophospholipid precursors and help simplify the quantitative analysis of unsaturated lysophospholipid species.     

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.     

Formation of trierucoylglycerol (trierucin) from 1,2-Dierucoylglycerol by a homogenate of microspore-derived embryos ofBrassica napus L

Homogenates of microspore-derived embryos of rape (Brassica napus L.) incubated with [1-¹⁴C]erucoyl-CoA and 1,2-dierucoylglycerol are able to assemble trierucoyl-glycerol (trierucin). In addition, radioactive triacylglycerols are formed by transferring [1-¹⁴C]-erucoyl moieties to endogenous lipid precursors. Stereospecific analysis of radioactive triacylglycerols revealed that labeled erucoyl moieties had been incorporated exclusively into thesn-1,3 positions with more than 95% of radioactivity in thesn-3 position. No incorporation of labeled erucic acid into thesn-2 position has been observed. All data agree with the involvement of 1,2-diacylglycerol acyltransferase (E.C. 2.3.1.20), which utilized 1,2-dierucoylglycerol as well as endogenous 1,2-diacylglycerols as acceptors of erucoyl moieties. This result is of particular interest for the genetic modification of rape and other Cruciferae for producing trierucin in their seed oils. NRCC No. 33513.     

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.     

Stereospecific analysis of triacyl-sn-glycerols in Docosahexaenoic acid-rich fish oils

This paper presents the positional distribution of fatty acids in docosahexaenoic acid (22∶6n-3)-rich fish oil triacyl-sn-glycerols (TG). Stereospecific analysis of TG was carried out by a nonenzymatic method. The TG of bonito head oil, obtained after a winterization process, contained 22∶6n-3 at concentrations of 28,7, and 49 mole % in thesn-1,sn-2, andsn-3 positions, respectively. In the TG of oil before the winterization process, 22∶6n-3 was concentrated in thesn-3 position, followed evenly by thesn-1 andsn-2 positions. Tuna orbital oil, obtained after winterization, showed the preferential association of 22∶6n-3 to thesn-3 position, followed by thesn-1 position. This distribution pattern was similar to that observed for seal oil TG rather than sardine oil TG. The bonito head and tuna orbital oils are useful as fish oils with characteristics different from those of common fish oils, such as menhaden, sardine, and herring oils.     

Platelet-activating factor (PAF) stimulates the lysoPAF acetyltransferase in leukocyte-rich plasma: Use in PAF antagonist studies

Addition of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to leukocyte-rich plasma from several species resulted in the rapid and pronounced activation of the PAF biosynthetic enzyme acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (EC 2.3.1.67). Activation of acetyltransferase by PAF occurred in leukocyte-rich plasma from human, chimpanzee, rhesus monkey, and dog. The neutrophil was indicated to be the major cellular source of the activabable acetyltransferase in leukocyte-rich plasma. The induction of acetyltransferase was substantial with 10 nM PAF, and maximal at 10–30 seconds. Measurable acetyltransferase activation was significantly greater when the PAF-activated cells were separated from the plasma by centrifugation before the acetyltransferase assay. This may be due in part to the removal of the PAF-specific acetylhydrolase present in plasma which can cleave the acetyl group from PAF. Measuring PAF activation of acetyltransferase in leukocyte-rich plasma can be useful to determine the potency of PAF antagonists with neutrophils in plasma compared to isolated neutrophils in aqueous buffer, and as anex vivo assay to determine the efficacy and plasma concentration equivalents of antagonists administered to whole animals. The PAF antagonist L-659,989 was shown to be 3–5 times more potent in inhibiting PAF induction of acetyltransferase in isolated human neutrophils than in human leukocyte-rich plasma, with IC₅₀ values of 10 nM and 40 nM, respectively. In theex vivo assay, oral administration of the PAF antagonist L-667, 131 to dogs resulted in very substantial inhibition of PAF induction of acetyltransferase in the leukocyte-rich plasma. Utilizing theex vivo assay, oral administration of 1 mg/kg L-659,989 to rats was found to result in plasma concentration equivalents of approximately 200–300 nM L-659,989. Our findings offer a new approach for charagerizing thein vitro andin vivo efficacy of PAF receptor antagonists and demonstrate that PAF may be able to activate neutrophils in the bloodin vivo, further enhancing PAF synthesis. 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 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.     

Triacylglycerol structure of human colostrum and mature milk

Because triacylglycerol (TAG) structure influences the metabolic fate of its component fatty acids, we have examined human colostrum and mature milk TAG with particular attention to the location of the very long chain polyunsaturated fatty acid on the glycerol backbone. The analysis was based on the formation of various diacylglycerol species from human milk TAG upon chemical (Grignard degradation) or enzymatic degradation. The structure of the TAG was subsequently deduced from data obtained by gas chromatographic analysis of the fatty acid methyl esters in the diacylglycerol subfractions. The highly specific TAG structure observed was identical in mature milk and colostrum. The three major fatty acids (oleic, palmitic and linoleic acids) each showed a specific preference for a particular position within milk TAG: oleic acid for thesn-1 position, palmitic acid for thesn-2 position and linoleic acid for thesn-3 position. Linoleic and α-linolenic acids exhibited the same pattern of distribution and they were both found primarily in thesn-3 (50%) andsn-1 (30%) positions. Their longer chain analogs, arachidonic and docosahexaenoic acids, were located in thesn-2 andsn-3 positions. These results show that polyunsaturated fatty acids are distributed within the TAG molecule of human milk in a highly specific fashion, and that in the first month of lactation the maturation of the mammary gland does not affect the milk TAG structure.     

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.     

Lipase-catalyzed transesterification of phosphatidylcholine at controlled water activity

The incorporation of a free fatty acid into thesn-1 position of phosphatidylcholine by lipase-catalyzed transesterification was investigated. The thermodynamic water activity of both the enzyme preparation and the substrate solution was adjusted to the same value prior to the reaction. The reaction rate increased with increasing water activity but the yield of modified phosphatidylcholine decreased due to hydrolysis. By using a large excess of the free fatty acid (heptadecanoic acid), the hydrolysis reaction was slowed down, so a higher yield was obtained at a given degree of incorporation. The best results were obtained withRhizopus arrhizus lipase immobilized by adsorption on a polypropylene support. With this preparation, a yield of 60% and nearly 50% incorporation of heptadecanoic acid (100% incorporation in thesn-1 position) was obtained at a water activity of 0.064. The enzyme preparation had good operational stability and position specificity. Little incorporation (<1%) was observed in thesn-2 position, when almost all the fatty acid in thesn-1 position was exchanged.     

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%).     

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).     

Antitumor activity of synthetic alkylphospholipids with or without PAF activity

1-O-Octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OMe) has been reported to possess definite antitumor activity in vivo. Twenty-two alkyl lysophospholipid analogs were chemically synthesized, and their antitumor activity against mouse experimental tumors (Sarcoma 180, MM46, P388) was examined. Among them, 1-O-octadecyl-2-O-acetoacetyl-rac-glycerol-3-phosphocholine was found to show antitumor activity similar to ET-18-OMe with less acute toxicity. Intravenous injection of the ET-18-OMe withsn-3 configuration retarded the subcutaneous growth of Sarcoma 180 cells effectively, while the growth inhibition by thesn-1 isomer was much less effective. This stereospecificity was similar to that observed in their activities as platelet-activating factor (PAF) agonists. The acetoacetyl compound, another PAF agonist, showed similar stereospecific antitumor action in vivo. These findings suggest that some alkyl lysophospholipids may activate host cells to a cytostatic stage against tumor cells in vivo through binding to a PAF receptor. Our preliminary results indicated that the responsible cells under these conditions might be primarily immature macrophages present in the bone marrow. No appreciable or even adverse stereospecificity was observed in the different sets of experiments where the activity of ET-18-OMe against MM46 tumor cells in vivo or the direct cytotoxicity against human promyelocytic leukemia HL-60 cells in vitro was examined. Under, some conditions, the antitumor activity of ET-18-OMe in vivo may be revealed through direct cytotoxicity and/or modulation of the host defense system by “nonspecific” mechanisms. Some alkylphospholipids without PAF activity may also show antitumor activity through similar, “nonspecific” mechanisms.     

Distribution of the major fatty acids of human milk betweensn-2 andsn-1,3 positions of triacylglycerols

Human milk triacylgycerols (TAG) were analyzed by tandem mass spectrometry. The SIMPLEX method and a simple linear model were used to interpret the distribution of fatty acids between thesn-2 andsn-1,3 positions in 24 major molecular weight groups of TAG. The number of regio-isomeric pairs of TAG varied between 3 and 18 in each of these groups. Hexadecanoic (16∶0), tetradecanoic (14∶0) and dodecanoic acids (12∶0) typically occupied thesn-2 position in TAG containing less than 54 acyl carbons, whereas long-chain C18 and C20 acids were predominantly located at the primary positions. The positions of the three fatty acids within a TAG molecule were shown to depend on the fatty acid combination. The maximum of 12∶0 in thesn-2 position appeared at acyl carbon number (ACN) 48, the maxima of 14∶0 were at ACN 44 and ACN 50, and for 16∶0 at ACN 46 and 52.