Development and application of new enzymatic method for quantification of platelet activation factor (PAF).

点击翻译按钮获取中文摘要

Platelet activating factor (PAF), whose chemical structure is 1-alkyl-2- acetyl-sn-glycero-3-phosphocholine, shows a variety of physiological activities in a very small quantity. The possibility has been suggested that PAF is working in homeostasis of blod pressure, anaphylaxis or inflammation and so on in vivo. It has not, however, yet clarified the real significance in vivo, since no simple and reproducible methods for quantification of PAF is available. PAF has been quantified mainly by biological assay or by mass-spectrometry. The biological assay, which estimates the quantity of PAF by the activity to induce platelet aggregation (the limit of detection;10^<-13>mol), has a problem in reproducibility, since it is difficult to prepare platelets without any stimulation. The quantification by mass-spectrometry requires a mass-spectrometer (the limit of detection;2x10^<-11>mol). We developed a new quantitative system which are able to detect 10^<-11>mol of PAF easily and reproductively. … More In this system we applied a method for quantification of activity of cholineacetyltransferase. Namely, after separation of PAF fraction from samples by HPLC, choline obtained by treatment of the PAF fraction with phospholipase D is converted into [^3H]-acetylcholine by incubation of choline with cholineacetyltransferase in the presence of [^3H]-acetylCoA, radioactivity of extracted[^3D]-acetylcholine is measured. A variety of examination revealed that the good sensitivity was achieved by use of partially purified phospholipase D obtained from Streptomyces chromofuscus and cholineacetyltransferase from human pracenta. Phospholipase D, cholineacetyltransferase and [^3H]-acetylCoA are added to the reaction mixture simultaneausly, 6 hours of incubation finished the determination. We tested the ratio of the extraction cocktail and the best condition was set up. Applying this new enzymatic method for quantification of PAF, we showed the production and the release of PAF when J774.1 cells, a macrophage-like cell line, was stimulated by A23187. And we detected a lot of PAF in the peritoneal exudated fluid of rats injected intraperitoneally with casein. Less