Method for simultaneous isolation and quantitation of platelet activating factor and multiple arachidonate...

A novel method has been developed for the extraction and simultaneous separation and quantitation of key arachidonate metabolites and platelet activating factor (PAF) from plasma samples of limited size. Aqueous solutions of these metabolites were added onto a solid phase C-18 cartridge and arachidonate metabolites and PAF were eluted, successively, with acetonitrile-methanol (85:15, v/v), followed by 100% methanol. Arachidonate metabolites (first eluate) were fractionated by C-18 reverse-phase high-performance liquid chromatography using a program designed for the resolution of 31 arachidonate metabolites (3-min separation). The fractions were collected and assayed by radioimmunoassay or radiography, if radioactively labeled. Two internal standards were added to each sample, 15-hydroxyeicosadienoic acid (detected at 235 nm) to determine gradient shifts and [1-14C]eicosatrienoic acid to estimate the recoveries of arachidonate metabolites at any stage of the process. This method was developed to answer specific questions concerning the mode of action of Staphylococcus aureus enterotoxin B (SEB). Plasma samples from mice challenged with SEB were analyzed and major differences were seen in 5-lipoxygenase metabolites. Low doses of SEB vs saline stimulated 5-hydroxyeicosatetraenoic acid production, while high doses of SEB stimulated leukotriene D4 production.