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

血小板活化因子（PAF）的化学结构为1-烷基-2-乙酰基-sn-甘油-3-磷酸胆碱，在极少量的情况下就显示出多种生理活性。提示PAF可能在体内起着调节血压、过敏反应或炎症等的作用。然而，它还没有阐明体内的真实的意义，因为没有简单的和可重复的方法来定量PAF。PAF主要通过生物测定法或质谱法进行定量。通过诱导血小板聚集的活性（检测极限;10 μ mol）估计PAF量的生物测定法<-13>在再现性方面存在问题，因为难以在没有任何刺激的情况下制备血小板。质谱法定量需要质谱仪（检测限：2x 10 μ <-11>mol）。我们建立了一个新的定量系统，它能够检测10 μ <-11>mol的PAF容易和重现性。 ...更多信息 在这个系统中，我们应用了一种方法来定量的胆碱乙酰转移酶的活性。即，在通过HPLC从样品中分离PAF组分后，通过在[^3H]-乙酰辅酶A存在下与胆碱乙酰转移酶孵育胆碱，将用磷脂酶D处理PAF组分获得的胆碱转化为[^3H]-乙酰胆碱，测量提取的[^3D]-乙酰胆碱的放射性。各种试验表明，用部分纯化的产色褐链霉菌的磷脂酶D和人血浆的胆碱乙酰转移酶可获得良好的灵敏度。将磷脂酶D、胆碱乙酰转移酶和[^3H]-乙酰辅酶A依次加入反应液中，孵育6小时，测定结束。对提取液的配比进行了试验，确定了最佳提取条件。应用这种新的定量PAF的酶法，我们显示了产生和释放PAF时，J774.1细胞，巨噬细胞样细胞系，A23187刺激。腹腔注射酪蛋白的大鼠腹腔渗出液中检测到大量PAF。少

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