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-SN-甘油-3-磷胆碱，具有多种生理活性，含量极少。有研究表明，PAF在体内可能处于血压、过敏反应或炎症等的动态平衡状态。然而，它还没有阐明在活体内的真正意义，因为还没有简单和可重复的方法来定量PAF。PAF的定量方法主要有生物测定法和质谱法。通过诱导血小板聚集的活性(检测下限；10^~(-13)~(-13)~(gt；mol))来估计PAF的量的生物测定法存在重复性问题，因为在没有任何刺激的情况下很难制备血小板。用质谱仪进行定量需要质谱仪(检出限：2x10^&lt；-11&gt；摩尔)。我们开发了一种新的定量体系，能够简便、重复性地检测PAF的10^~(-11)~(-11)~(-1)摩尔。…此外，在该系统中，我们还应用了一种胆碱乙酰转移酶活性的定量方法。也就是说，用高效液相色谱法从样品中分离PAF组分后，用磷脂酶D处理PAF组分得到的胆碱在[~3H]-乙酰辅酶A存在下与胆碱乙酰转移酶孵育转化为[~3H]-乙酰胆碱，测定提取的[~3d]-乙酰胆碱的放射性。多种检测结果表明，利用部分纯化的发色链霉菌磷脂酶D和人胆碱乙酰转移酶可获得良好的敏感性。同时向反应混合物中加入磷脂酶D、胆碱乙酰转移酶和[~3H]-乙酰辅酶A，孵育6h完成测定。对提取鸡尾酒的配比进行了试验，确定了最佳提取条件。应用这种新的定量PAF的方法，我们显示了A23187刺激巨噬细胞系J774.1细胞时PAF的产生和释放。大鼠腹腔注射酪蛋白后，在腹腔液中检测到大量PAF。较少

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