An improved assay for platelet-activating factor using HPLC-tandem mass spectrometry

Abstract

We describe an improved assay for platelet-activating factor (PAF; 1-O -alkyl-2-acetyl-sn -glycero-3-phosphocholine) using HPLC-tandem mass spectrometry (LC-MS/MS). The present method can readily detect as little as 1 pg (1.9 fmol) of PAF, a significant improvement over previously described LC-MS/MS methods, and gives a linear response up to 1,000 pg of PAF. Our method also overcomes the artifacts from isobaric lipids that have limited the usefulness of certain existing LC-MS/MS assays for PAF. In the course of these studies, we detected three novel lipid species in human neutrophils. One of the novel lipids appears to be a new molecular species of PAF, and the other two have chromatographic and mass spectrometric properties consistent with stearoylformyl-glycerophosphocholine and oleoyl-formyl-glycerophosphocholine.These observations identify previously unknown potential interferences in the measurement of PAF by LC-MS/MS. Moreover, our data suggest that the previously described palmitoyl-formyl-glycerophosphocholine is not unique but rather is a member of a new and poorly understood family of formylated lipids. Platelet-activating factor (PAF) is a phospholipid autacoid with the chemical structure 1-O -alkyl-2-acetyl-sn -glycero-3-phosphocholine (1-3). The alkyl chain at sn -1 is most commonly hexadecyl, octadecyl, or octadec-cis -9-enyl, designated 16:0, 18:0, or 18:1, respectively. Other molecular species with different alkyl moieties occur in minor amounts (4-7). PAF has been implicated in the inflammatory response that follows injury (8-11) as well as in inflammatory conditions, including asthma and anaphylaxis (12, 13). More recently, PAF has been implicated in the growth and metastasis of epithelial cancers (14-17). Physiological roles have been proposed for PAF in fertility and embryonic development as well as in learning and memory (18).Like most lipid mediators, PAF is not stored but is rapidly synthesized on demand in appropriately stimulated cells through the remodeling of membrane lipids (19,20) and is rapidly broken down by acetylhydrolases in cells and in blood plasma (21). In consequence, PAF exists at very low concentrations in biological specimens, and studies on the biology of PAF have been limited by investigators' ability to quantify PAF with accuracy and precision. Early PAF measurements relied on bioassay. Nearly a decade passed between the discovery of PAF and the elucidation of its chemical structure, making physicochemical assay possible.Currently, MS-based methods represent the state of the art in PAF assays in terms of sensitivity, precision, specificity, dynamic range, and versatility. The most sensitive approach described to date involves removal of the phosphocholine head group and its replacement with a nonpolar group. The resulting volatile derivative is then analyzed by GC-MS (22-28). As little as 50 fg of PAF can be detected using this approach (25). However, the requirement for derivatization makes these assays time-consuming, expensive, and susceptible to losses and...

Role of platelet-activating factor in pneumolysin-induced acute lung injury

“…Delipidated solids were removed by centrifugation and reserved for total protein assay. After separation (16), the chloroform phase of the solvent was transferred to a fresh tube, and the methanolic phase was washed with 4 mL of chloroform. Both chloroform extracts were pooled, evaporated under a stream of argon, and reconstituted in 1 mL of chloroform/methanol (1:1 vol/vol).…”

“…Lungs were snap frozen 5 mins after PLY infusion, pulverized, weighed, and stored at Ϫ80°C. Homogenates were incubated essentially as described (16). Delipidated solids were removed by centrifugation and reserved for total protein assay.…”

Ion-trap tandem mass spectrometric analysis of Amadori-glycated phosphatidylethanolamine in human plasma with or without diabetes

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Chronic Alcohol Exposure Increases Circulating Bioactive Oxidized Phospholipids

“…PAF was purified by silica normal phase HPLC before reversed phase LC/MS/MS (18). Each sample in 85% methanol was injected onto a reverse phase C18 HPLC column (2 ϫ 150 mm, 5-m ODS(2) Phenomenex) equilibrated with 85% methanol containing 0.2% formic acid at a flow rate of 0.2 ml/min.…”