Synthesis of platelet-activating factor by polymorphonuclear neutrophils stimulated with interleukin-8.

Human interleukin-8 (IL-8) was evaluated for its capability to induce the synthesis and release of platelet-activating factor (PAF) from human polymorphonuclear neutrophils (PMN). IL-8 promotes in a dose-dependent fashion (1-100 ng/ml) a rapid synthesis of PAF, which is only partially released. The synthesis of PAF is preceded by the activation of acetyl-CoA: 1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyl-transferase, suggesting that IL-8 activates the "remodeling pathway" of PAF synthesis. By thin layer chromatography and reverse-phase high pressure liquid chromatography, we demonstrated that PAF synthesized by human PMN stimulated with IL-8 is heterogeneous: the 2-acetylated phospholipids having the biological and physicochemical characteristics of PAF include the 1-O-alkyl form, which is produced in large extent (51%), and the 1-acyl form (20%). The analysis of the individual molecular species of radyl chain indicated nine peaks, 16:0 and 18:0 being the predominant forms. These results identify PAF as a direct product of IL-8 stimulation in PMN.     

Application of selected ion monitoring to determination of platelet-activating factor

The selected ion monitoring (SIM) technique was applied to determination of platelet-activating factor (PAF) or acetyl glyceryl ether phosphorylcholine (AGEPC). Two types of PAF, 1-hexadecyl- and 1-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (C16 = 0 AGEPC and C18 = 0 AGEPC), were found in human neutrophils on the challenge with ionophore A23187. The contents of C16 = 0 AGEPC in 1 X 10(7) neutrophil cells of four volunteers, respectively, were 47, 18, 59, and 73 ng and those of C18 = 0 AGEPC were 22, 4, 19, and 31 ng.     

Analysis of platelet-activating factor by GC-MS after direct derivatization with pentafluorobenzoyl chloride and heptafluorobutyric anhydride

Parallel analysis of platelet-activating factor (PAF) using chemical ionization gas chromatography-mass spectrometry after direct derivatization with pentafluorobenzoyl chloride (PFB) and heptafluorobutyric anhydride (HFB) provides a facile and highly sensitive means for detecting and elucidating the structure of the numerous alkyl-chain homologs of this acetylated phospholipid autacoid. In the present study, the PFB derivative was used for initial electron capture negative ion chemical ionization analysis of PAF candidate molecules in human PMN extracts of unknown composition. Subsequent pulsed positive ion/electron capture negative ion chemical ionization evaluation of the HFB derivative furnished a measure of the molecular weight from [MH]+ and yielded the required structural information from characteristic negative ions, in particular [M-(2HF + ketene)]- and [M-(HF + acetic acid)]-. These procedures easily permitted confirmation of the presence of C16:0-, C17:0-, C18:0-, and C18:1-AGEPC (acetyl glyceryl ether phosphocholine) in extracts of stimulated human PMN and also demonstrated that the C17:0- homolog was comprised of both straight-chain and branch-chain varieties.     

Biosynthesis of paf-acether. XVI. Acetyltransferase specificity determines composition of paf-acether molecular species in human polymorphonuclear neutrophils.

In human neutrophils, the velocity of the lyso paf-acether:acetyl-CoA acetyltransferase reaction was almost 2-fold higher in the presence of lyso paf-acether bearing a 16:0 alkyl chain at the sn-1 position of glycerol than in that of its 18:0 analog. The paf-acether produced from an equimolar mixture of the two substrates was a 5:1 mixture, respectively, of the 16:0 and 18:0 species. The ratio of 16:0/18:0 lyso paf-acether in microsomal fractions, as analyzed by gas chromatography, was close to 1, whereas the paf-acether formed in these fractions from endogenous phospholipids was nearly exclusively of the 16:0 form. We conclude that acetyltransferase possesses a higher affinity for 16:0 than for 18:0 lyso-PAF and thus might control the molecular composition of paf-acether synthesized by stimulated human polymorphonuclear neutrophils.     

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

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

Species-specific variations in the molecular heterogeneity of the platelet-activating factor

The molecular heterogeneity of platelet-activating factor (PAF) synthesized by unstimulated and Ca2+ ionophore (A23187)-stimulated PMN from rat, mouse, and guinea pig and by rat basophilic leukemia (RBL) cells was investigated by gas chromatography-negative ion chemical ionization mass spectrometry. Several molecular species of PAF ranging from C14:0 to C19:0 were detected in all of the cells studied. PAF produced by each cell type exhibited a unique pattern of molecular species distribution. Although C16:0 was the major PAF molecular species of rat PMN and RBL cells representing 96% and 85% of the total PAF, respectively, PAF from mice PMN contained 81% of C16:0, 10% of C18:1, and 6% of C18:0. Alternatively, A23187-stimulated guinea pig PMN yielded PAF molecular species 35% in C16:0, 35% in C17:0, 8% in C18:1, and 3% in C18:0. Small but significant differences in the PAF molecular species distribution of resting and ionophore stimulated cells were also observed. In contrast to the PAF molecular species composition, the precursor 1-O-alkyl-2-acyl-glycero-3-phosphocholine of all the cell types was predominantly hexadecyl (C16:0) alkyl chain in the sn-1 position, representing 60 to 80% of the total 1-O-alkyl-2-acyl-glycero-3-phosphocholine. Thus, these results not only indicate a high degree of selectivity for utilization of precursor substrates for PAF biosynthesis, but also demonstrate that the selectivity is species specific.     

Identification of the major metabolite of 12-HETE produced by renal tubular epithelial cells

The identification and polarity of release of the major metabolite of 12-HETE produced by cultured canine renal tubular epithelial cells was determined. When incubated with 1.0 microM [3H]12-HETE for 1 h, cultured Madin Darby Canine Kidney (MDCK) cells converted 35% of the radiolabeled 12-HETE to a more polar metabolite. Following high performance liquid chromatography isolation and chemical derivatization, gas-liquid chromatography combined with mass spectrometry was used to identify the compound as 8-hydroxyhexadecatrienoic acid [16:3(8-OH)]. The electron impact mass spectrum of the hydrogenated derivative contained major ions at m/z = 215 and 245, corresponding to cleavage on either side of the trimethylsilyl group, and chemical ionization with NH3 yielded a major ion at m/z = 359, corresponding to the protonated molecular weight of the methyl ester. Incubation with 25 mM alpha-naphthoflavone, 20 microM nordihydroguaiaretic acid, and 0.1 mM 4-pentenoic acid failed to inhibit the formation 16:3 (8-OH), suggesting that the formation of 16:3 (8-OH) is not mediated by the cytochrome P-450, lipoxygenase, or mitochondrial beta-oxidation pathways. When grown on fibronectin-treated polycarbonate filters, MDCK cells released the 16:3 (8-OH) in both the apical and basolateral directions, irrespective of which side the 12-HETE was encountered. These results demonstrate the conversion of 12-HETE to a 16-carbon monohydroxy derivative by renal tubular epithelium and suggest that this product can be released to either the potential urinary space or the kidney parenchyma and renal microcirculation.     

Quantitation of lyso-platelet activating factor molecular species from human neutrophils by mass spectrometry

Two physicochemical methods have been developed for the quantitative analysis of lyso-platelet activating factor (lyso-PAF) based on gas-liquid chromatography-mass spectrometry (GLC/MS) and fast atom bombardment-mass spectrometry (FAB/MS) using stable isotope dilution. After addition of deuterated internal standards, lyso-PAF produced from neutrophils was purified by silicic acid chromatography and thin-layer chromatography (TLC). The GLC/MS assay employed phospholipase C or hydrofluoric acid for hydrolysis of the phosphocholine moiety to yield ether monoglycerides. Condensation of monoglycerides with acetone yielded the 1-O-alkyl-2,3-isopropylidene glycerol which could be analyzed by GLC/MS. The ions corresponding to M-15 fragments for both the labeled and unlabeled derivatives were monitored in a selected ion recording mode. Standard curves were found to be linear over the range tested (10-2000 ng) with a limit of detection found to be below 200 pg injected on column. For the FAB/MS assay, the unmodified lyso-PAF was well suited for direct analysis; however, the limit of detection (S/N greater than 3) using a glycerol matrix was found to be 5 ng placed on the probe tip. It was found that human neutrophils contain approximately 300 pg/10(6) cells which increased 2-3-fold during the 5-min period following challenge with 1.9 microM calcium ionophore, A23187. Two molecular species of lyso-PAF were identified as hexadecyl and octadecyl ethers at sn-1 with the octadecyl molecular species of lyso-PAF predominating in abundance after stimulation.     

Microdetermination of the 6,15-diketo-13,14-dihydroprostaglandin F1alpha in human plasma using gas chromatography/selected ion monitoring with [18O]6,15-diketo-13,14-dihydro-prostaglandin F1alpha as an internal standard

We devised a simple and effective purification method for the microdetermination of 6,15-diketo-13,14-dihydro-prostaglandin F1alpha (DK), a metabolite of prostacyclin (PGI2). [18O]DK was synthesized from the repeated base-catalyzed hydrolysis of methyl ester derivatives in [18O] water to obtain an internal standard for the gas chromatography/selected ion monitoring (GC/SIM) of DK. The methyl ester-methoxime-tert-butyldimethylsilyl ether derivative was prepared, then gas chromatography/selected ion monitoring was carried out by monitoring the ion at m/z 613.4 for DK and at m/z 617.4 for an internal standard. A good linear response over the range of 10 pg to 10 ng was demonstrated. We detected DK to a level of about 297.8 pg/ml in human plasma. This method can be used to determine DK in biological samples.     

Formation and metabolism of leukotriene C4 in macrophages exposed to bacterial lipopolysaccharide

Lipopolysaccharide (10 micrograms/ml) was found to stimulate resident mouse peritoneal macrophages to produce leukotriene C4 (36 +/- 1.3 ng/10(6) cells, SEM, n = 20) within 16 h. Spontaneous synthesis in control cultures without lipopolysaccharide was less than 1.6 ng/10(6) cells. Leukotriene C4 was characterized by reversed-phase high-performance liquid chromatography, ultraviolet spectrometry and radioimmunoassay. When the macrophages, prelabeled with [3H]arachidonic acid, were treated with lipopolysaccharide radioactivity was incorporated into leukotriene C4. The amount produced varied with the method of macrophage preparation and incubation conditions and was dependent on the amount of lipopolysaccharide added (0.5-60 micrograms/ml), on cell counts and on the incubation time (4-16 h). The released leukotriene C4 was converted to a compound identified as a C6-cysteinylleukotriene, indicating metabolism of the leukotriene by the macrophages. Parallel determinations of prostaglandins E2 and F2 alpha by radioimmunoassay demonstrated that leukotriene C4 and prostaglandin E2 are formed by mouse peritoneal macrophages to a similar degree.     

Synthesis of lipoxins and other lipoxygenase products by macrophages from the rainbow trout, Oncorhynchus mykiss

Rainbow trout macrophages maintained in short term culture when incubated with either calcium ionophore, A23187, or opsonized zymosan synthesize a range of lipoxygenase products including lipoxins and leukotrienes. These cells are unusual in that they generate more lipoxin than leukotriene following such challenge. The main lipoxin synthesized was lipoxin (LX) A4. This compound was identified by cochromatography with authentic standard during reversephase high performance liquid chromatography, by ultra violet spectral analysis, radiolabeling following incorporation of [14C]arachidonic acid substrate into macrophage phospholipids, and gas chromatography electron impact mass spectrometry of the methyl ester, trimethylsilyl ether derivative. Other 4-series lipoxins synthesized by trout macrophages were identified as 11-trans-LXA4, 7-cis-11-trans-LXA4, and 6(S)-LXA4. These cells also produced 5-series lipoxins tentatively identified as LXA5, 11-trans-LXA5 and possibly 6(S)-LXA5. No LXB4 or LXB5 was, however, detected. The dynamics of leukotriene and lipoxin release were also determined. Lipoxin generation was slower than leukotriene generation the latter reaching a maximum after 30 min of exposure to ionophore (5 microM, 18 degrees C) compared with 45 min for the former.     

Electrophoretic analysis of the cytoplasmic and nuclear protein changes after induction of differentiation in WEHI-3B myelomonocytic leukemia cells

In response to a differentiation factor (G-CSF) the myelomonocytic leukemia cell line (WEHI-3B(D+) differentiates to form mature macrophages and neutrophils. The effect of G-CSF on WEHI-3B(D+) differentiation was augmented by low concentrations (5 ng/ml) of actinomycin D. Quantitative binding of an antineutrophil serum was used to segregate the differentiated cells from the leukemic blast cells. Molecular markers of later myeloid differentiation were detected in myelocytes and macrophages purified from differentiating WEHI-3B(D+) cells. To study the initial molecular processes associated with the initiation of WEHI-3B(D+) cells to differentiation, the protein changes were analyzed using gel electrophoresis. Quantitative analysis of the fluorographs from the two-dimensional (2D) electrophorograms of the 35S-labeled proteins revealed major changes in the biosynthetic rates for 16 proteins within 5 hr: The biosynthesis of six proteins was increased and another ten proteins were synthesized at a reduced rate. Two of the proteins (17K and 36K daltons) were located in the nucleus. Pulse-chase experiments indicated that protein turnover for these proteins was rapid but the degradation of four proteins was suppressed. At least six of the proteins (16K to 120K daltons) were acidic and were associated with the cytoplasm. Electrophoretic analysis of the 35S-labeled proteins indicated that a 35K protein induced by G-CSF was found in high abundance only in purified cells of intermediate differentiation (eg, myelocytes). Other proteins (eg, a very high molecular weight protein, and a 16K dalton protein) were obviously late markers of differentiated neutrophils or macrophages.     

m-Synephrine: Normal Occurrence in Adrenal Gland

Abstract: Gas chromatography-mass spectrometry-selected ion monitoring has been used to identify m-synephrine (phenylephrine) in bovine adrenal gland. After ion exchange chromatography, m-synephrine was converted to its N-trifluoroacetyl-O-trifluoroacetoxy derivative and identified by retention time and relative intensities of the two characteristic ions at m/e 140 and m/e 455. Deuterated m-synephrine was synthesized and used as an internal standard for quantitative analysis. Bovine adrenal gland contains 83 ng g-¹ (range, 33-131) of endogenous m-synephrine.     

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

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

Quantitation of fatty acyl-coenzyme As in mammalian cells by liquid chromatography-electrospray ionization tandem mass spectrometry

Fatty acyl-CoAs participate in numerous cellular processes. This article describes a method for the quantitation of subpicomole amounts of long-chain and very-long-chain fatty acyl-CoAs by reverse-phase LC combined with electrospray ionization tandem mass spectrometry in positive ion mode with odd-chain-length fatty acyl-CoAs as internal standards. This method is applicable to a wide range of species [at least myristoyl- (C14:0-) to cerotoyl- (C26:0-) CoA] in modest numbers of cells in culture ( approximately 10(6)-10(7)), with analyses of RAW264.7 cells and MCF7 cells given as examples. Analysis of these cells revealed large differences in fatty acyl-CoA amounts (12 +/- 1.0 pmol/10(6) RAW264.7 cells vs. 80.4 +/- 6.1 pmol/10(6) MCF7 cells) and subspecies distribution. Very-long-chain fatty acyl-CoAs with alkyl chain lengths > C20 constitute <10% of the total fatty acyl-CoAs of RAW264.7 cells versus >50% for MCF7 cells, which somewhat astonishingly contain approximately as much C24:0- and C26:0-CoAs as C16:0- and C18:0-CoAs and essentially equal amounts of C26:1- and C18:1-CoAs. This simple and robust method should facilitate the inclusion of this family of compounds in "lipidomics" and "metabolomics" studies.     

Determination of phenelzine in human plasma with gas chromatography—mass spectrometry using an isotope labeled internal standard

A quantitative gas chromatographic—mass spectrometric assay was developed for the determination of phenelzine in human plasma. Phenelzine, in aqueous solution or in plasma reacts at room temperature with pentafluorobenzaldehyde to form quantitatively a hydrazone derivative. The derivative has good gas chromatographic characteristics. The assay utilizes selected ion monitoring in a gas chromatographic effluent, the molecular ion generated by electron impact ionization of phenelzine derivative. Phenelzine-d₇ was synthesized and used as an internal standard. The assay can measure 2 ng/ml of the drug with about 10% precision.The method was used for the determination of steady state levels of phenelzine in the plasma of patients taking a therapeutic dose of the drug.     

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

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

Relative degradation of different arachidonoyl molecular species of choline glycerophospholipids in opsonized zymosan-stimulated rabbit alveolar macrophages

The relative degradation of arachidonoyl molecular species of glycerophospholipids prelabeled with [3H]20:4 caused by opsonized zymosan was studied in rabbit alveolar macrophages using a recently developed high-performance liquid chromatographic method. The opsonized zymosan caused the release of [3H]20:4 only from choline glycerophospholipids, no significant changes being observed in the radioactivities of other glycerophospholipids and triacylglycerol. Choline glycerophospholipids were resolved into seven arachidonoyl molecular species, which differed as to the alkyl ether or acyl residue bound at the 1-position, by high-performance liquid chromatography. Arachidonate was predominantly located in the alkyl type having 16:0 at the 1-position which comprised more than half of the total arachidonoyl molecular species of choline glycerophospholipids. The radioactivities of all arachidonoyl molecular species of choline glycerophospholipids, except for the 18:2-20:4 and 18:1-20:4 species of diacylglycerophosphocholine, decreased to 80-85% of the control values as a result of the challenge with opsonized zymosan for 1 h. However, 50% of the released 20:4 came from the 16:0-20:4 species of alkylacylglycerophospholipids, which were the most predominant species of choline glycerophospholipids. The present results indicate that the 16:0-20:4 species of alkylacylglycerophosphocholine is a significant source of arachidonate and 1-O-alkyl-2-lysoglycerophosphocholine, the precursor of the platelet-activating factor, relative to other arachidonoyl species in activated alveolar macrophages.     

Molecular species of platelet-activating factor generated by human neutrophils challenged with ionophore A23187

Two species of platelet-activating factor (PAF), 1-hexadecyl- and 1-octadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16 = 0 AGEPC and C18 = 0 AGEPC) were detected in ionophore A23187-stimulated human neutrophils. The amount of AGEPC in 1 x 10(7) neutrophil cells was 80 +/- 26 pmol (mean +/- standard error) with a range of 14 to 223 pmol (n = 8), and it consisted of 80% of the C16 = 0 species and 20% of the C18 = 0 species. Most of the AGEPC derived from ionophore-treated neutrophils remained cell associated rather than being secreted into the medium, even when the medium contained ample albumin protein, which can trap AGEPC. These results were obtained by a technique of gas chromatography-mass spectrometry coupled with selected ion monitoring.     

Influence of immunologic activation and cellular fatty acid levels on the catabolism of platelet-activating factor within the murine mast cell (PT-18)

The present study has examined the catabolism of 1-O-[3H]hexadecyl-2-acetyl-GPC (C16-PAF) and of 1-O-octadecyl-2-acetyl-GPC (C18-PAF) in spleen-derived PT-18 murine mast cells (mast cells). Mast cells catabolized exogenous PAF into two inactive metabolites, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lysoPAF) and 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acyl-GPC). The rate of conversion of C16-PAF to metabolites was more rapid than that of C18-PAF. Analysis of the acyl composition of 1-O-alkyl-2-acyl-GPC formed during the metabolism of PAF revealed that arachidonic acid (20:4) was the major fatty acyl chain incorporated at the sn-2 position. However, 25% of newly formed 1-O-alkyl-2-acyl-GPC was reacylated with docosahexaenoic acid (22:6). The influence of cellular fatty acid content on PAF catabolism was further explored in mast cells in which the ratio of fatty acids within cellular phosphoglycerides had been altered by supplementing the cells with various fatty acids in culture. Mast cells supplemented with 20:4 or 22:6 converted PAF to 1-O-alkyl-2-acyl-GPC at a significantly higher rate than non-supplemented cells. In contrast, cells supplemented with linoleic acid (18:2) metabolized PAF at rates similar to non-supplemented cells. Analysis of the acyl composition of 1-O-alkyl-2-acyl-GPC derived from the metabolism of PAF in 20:4-supplemented cells indicated that 20:4 was incorporated exclusively into the sn-2 position. Conversely, 22:6-supplemented cells incorporated predominantly 22:6 at the sn-2 position of 1-alkyl-2-lyso-GPC. Supplementation with 18:2 had no effect on the acylation pattern seen in newly formed 1-O-alkyl-2-acyl-GPC. Activation of passively sensitized mast cells with antigen or with ionophore A23187 significantly enhanced the rate of catabolism of exogenously-provided PAF but had no effect on the acylation pattern of 1-O-alkyl-2-acyl-GPC. Experiments performed with the soluble fraction of the cells showed that acetyl hydrolase activity was increased in mast cells stimulated with antigen. In addition, supernatant fluids from antigen or ionophore-treated mast cells converted PAF to lysoPAF, suggesting that acetyl hydrolase activity was released during cell activation. These data indicate that the ability of mast cells to catabolize PAF to inactive metabolites is influenced by cell activation and by the cellular levels of certain fatty acids.