Individual molecular species of different phospholipid classes. Part II. A method of analysis

Abstract

New analytical possibilities arise when glycerosphophatides are converted into diglyceride acetates or analogous compounds: In this less polar form the phospholipids can be subjected to the usual methods of triglyceride fractionation, including chromatography on silica gel mixed with silver nitrate. This opens a route to subfractionation of various glycerophosphatide classes, and makes analysis of the individual molecular species potentially possible in many cases. The same approach can also be applied to the analysis of sphingomyelins. Two methods are suitable for the conversion of glycerophosphatides into 舠diglyceride acetates舡: 1) Acetolysis in a mixture of acetic anhydride and acetic acid, and 2) treatment with phospholipase C (E.C. 3.1.4.3.) followed by acetylation. Acetolysis was used successfully with phosphatidyl choline, phosphatidyl ethanolamine and corresponding alkoxy phosphatide (native cephalin B), phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol, phosphatidic acid and cardiolipin. Phospholipase C from Clostridium welchii was used for native choline and ethanolamine plasmalogens as well as for sphingomyelins. Although this enzyme does not attack pure ethanolamine phosphatides it did so in the presence of added (dipalmitoyl) phosphatidyl choline or serum sphingomyelin. The mixed substrates containing sphingomyelin proved particularly valuable, since the plasmalogenic diglyceride acetates were easily separated from the usual diglyceride acetates and ceramide diacetates on silicic acid. The diglyceride acetates obtained from lecithins of hens舗 eggs, ox‐brain and human serum were subjected to preparative TLC on Kieselgel G mixed with silver nitrate. The resulting subfractions were characterized by GLC analysis of the fatty acid methyl esters. When these data were combined with enzymic analysis of the acids occupying the alpha and beta positions of the molecules a rather detailed description of the lecithins became possible. The use of diglyceride acetates provides a route to analysis of fatty acids in many phosphatidyl compounds which have been obtained previously only as mixtures with the alkoxy analogs.