Development of high Sensitive Methods for Analysis of Lipid Molecular Species

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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