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.
D000001:Calcimycin / Q000494:pharmacology; D002855:Chromatography, Thin Layer / Q000379:methods; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D008244:Lysophosphatidylcholines / Q000097:blood / Q000302:isolation & purification; D013058:Mass Spectrometry / Q000379:methods; D009504:Neutrophils / Q000032:analysis* / Q000187:drug effects; D010972:Platelet Activating Factor / Q000031:analogs & derivatives / Q000032:analysis / Q000096:biosynthesis
D008244:Lysophosphatidylcholines; C029271:O-deacetyl platelet activating factor; D010972:Platelet Activating Factor; D000001:Calcimycin