Development of high Sensitive Methods for Analysis of Lipid Molecular Species

建立了一种简便、灵敏的磷脂分子种类定量分析方法。以磷脂为原料，经磷脂酶C处理制得芳基甘油，转化为相应的二硝基苯甲酰衍生物，可在254 nm波长处进行检测。用十八烷基硅烷反相色谱柱对36个分子物种的衍生物进行了拆分。所有的衍生物都有相同的峰面积，并且梨的面积与衍生物的量成正比。在两种溶剂体系的组合下，双酰基分子物种同时拆分了烷基和烷基分子物种的衍生物。定量是在皮摩尔水平上进行的。该方法用于测定凝血酶和胶原刺激后不同分子种类的人血小板的质量变化。在刺激下，磷脂酰肌醇和磷脂酰丝氨酸的每个分子物种都有…其中磷脂酰胆碱和含有花生四烯酸的二酰基和烯基磷脂酰乙醇胺的分子物种被选择性地水解。在低Ca~(2+)浓度下，磷脂酰肌醇、磷脂酰胆碱和二酰基磷脂酰乙醇胺在凝血酶抑制后被水解，而在胶原刺激下只有磷脂酰肌醇被水解并产生血栓素B_2。在高钙浓度下，S磷脂酰胆碱和二酰基磷脂酰乙醇胺在胶原刺激下被降解，而磷脂酰丝氨酸和烯基磷脂酰乙醇胺在凝血酶和胶原刺激下被降解。(1-^&lt；14&gt；C)花生四烯酸被异相结合到各种磷脂类的单个分子物种中，表明质量的测定对于准确描绘磷脂代谢是必不可少的。较少

A simple and sensitive method was developed for quantitative analysis of phospholipid molecular species. Firadylglycerols were prepared from phospholipids by phospholipase C treatment and converted to corresponding dinitrobenzoyl derivatives, which could be detected at 254 nm. The derivatives or 36 molecular species were resolved by high performance luquid chromatography with on octadecylsily) reversed-phase colum. All the derivatives had the same peak area per mo., and pear areas were proportional to the amounts to the derivatives. The derivatives of alkenylacyl- and alkylacylmolecular species were resolved concomitantly with diacyl molecular species using a combination of two solvent systems. Quantification was carried out at the picomole level.The method was used for determination of mass changes in the various molecular species of human platelets after stimulation with thrombin and collagen. Upon stimulation, every molecular species of phosphatidylionositol and phosphatidylserine w … More as equally hydrolyzed, where the molecular species of phosphatidylcholine and diacyl-and aleknylacylphosphatidylethanolamine containing areachidoni acid were selectivey hydrolyzed. At low Ca^<2+> concentrations, which results from the release from internal stores, phosphatidylinositol, phosphatidylcholine and diacylphosphatidylethanolamine were hydrolyzed after stinulation with thrombin, wheras only phosphatidylinositol was hyrolyzed with production of thromboxane B_2 after stimulation with collagen. At high Ca^<2+> concentration,s phosphatidylcholine and diacylphosphatidylethanolamine were hydrolyzed after stimulation with collagen, and phosphatidylserine and alkenylacylphosphatidylethanolamine were degraded after stimulation with both thrombin and collagen. (1-^<14> C) Arachidonic acid was heterogeneously incorporated into the individual molecular species of the vatious phospholipid classes, indicating that the determination of mass is essential for accurate picture of phospholipid metabolism. Less