Abstract
To confirm that platelet activating factor (PAF) plays an important role as a mediator in acute inflammation and allergic reacton, it is necessary to develop a more sensitive and stable method, capable of measuring trace amounts of PAF in various biological samples.For that reason, we have improved gas chromatography/negative ion chemical ionization mass spectrometry (GC/NICI-MS), (established by Ramesha and Pickett in 1985) by employing SPB-1 column and isobutane as the reagent gas. Furthermore, by using this method, we investigated the time course of hexadecyl-PAF production and release from human polymorphonuclear leukocytes (PMNs) stimulated by Ca-ionophore A23187 in this experiment. PMNs were obtained from venous blood from a human donor by centrifugation and were stimulated by 5 μM of Ca-ionophore A23187. PAF was purified by SEP-PAK silica column and thin layer chromatography, and then it was hydrolyzed by phospholipase C. The extract was treatd with pentafluorobenzoyl chloride. We obtained the following results: the production of hexadecyl-PAF in cell pellet reached peak (8.1 ng/107 cells) at 2 minutes after stimulation and that in the supernatant reached peak (6.3 ng/107 cells) at about 7.5 minutes after stimulation.We conclude that the procedure of GC/NICI-MS improved by us will be useful for the structural identification of trace amounts of unknown PAF synthesized by cells.
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