Journal of Lipid Research, Vol 28, 42-49, Copyright © 1987 by Lipid Research, Inc.
Quantitation of lyso-platelet activating factor molecular species from human neutrophils by mass spectrometry
PE Haroldsen, KL Clay and RC Murphy
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.
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