Individual molecular species of phospholipids: III. Molecular species of ox-brain lecithins

Abstract

1. 1. A mixture of native choline plasmalogens and alkoxy lecithins was isolated from ox brain by partial alkaline methanolysis of the total lecithin; the preparation appeared to be free from phosphatidyl cholines. 2. 2. Hydrolysis of total ox-brain lecithins with phospholipase C (EC 3.1.4.3) from Clostridium welchii converted all three subclasses of the glycerylphosphorylcholine lipids into diglycerides; these were subsequently acetylated. The resulting mixture of diglyceride acetates was separated by chromatography on kieselgel G into ordinary diglyceride acetates (95.2%), plasmalogenic diglyceride acetates (1.9%) and corresponding alkoxy compounds (2.2%). Chromatographic evidence and observations on partial hydrolysis with pancreatic lipase (EC 3.1.1.3) proved that the fraction of the ordinary diglyceride acetates contained about 99% 1,2-diglyceride acetates and about 1% 1,3-diglyceride acetates. Hydrolysis of the plasmalogenic diglyceride acetates and the alkoxy analogs with pancreatic lipase suggested that their long-chain acyl ester groups were located on C-2, and the acetyl group on C-3, of glycerol. 3. 3. All three subclasses of diglyceride acetates were fractionated on silver nitratecontaining kieselgel G into four subfractions which contained 0, 1, 2 and “many” double bonds, respectively, in their long fatty acid chains proper. Furthermore, the dienoic subfractions of the type 1-oleoyl-2-oleoyl-3-acetate and 1-stearoyl-2-linoleyl-3-acetate could be separated from each other. The plasmalogen diglyceride acetates appeared to form silver complexes even though they had vinyl ether double bonds. 4. 4. The fatty acid chains at the C-1 and C-2 positions in the twelve main subfractions of diglyceride acetates were analyzed by gas-liquid chromatography. The composition of the major molecular species could be predicted from the results by assuming that the number of double bonds is constant within each subfraction. 5. 5. The ordinary 1,2-diglyceride acetates were also subjected in intact form to gasliquid chromatography whereby 3–4 fractions of different molecular sizes were revealed in each of the main subfractions. The results confirmed the predictions made by argentation, fractionation and chain analysis. 6. 6. All three subclasses of ox-brain lecithins, the phosphatidyl cholines, the plasmalogens and the alkoxy lecithins, appeared to consist of remarkably similar populations of molecular species. 7. 7. The partial hydrolysis of five different types of diglyceride acetates with pancreatic lipase is described. The primary ester groups appeared to be cleaved faster than the secondary ones in 1,2-diglyceride acetates as well as 1,3-diglyceride acetates, and also in the corresponding two types of plasmalogen compounds having vinyl ether chains on C-1; even the alkoxy analogs of 1,2-diglyceride acetates were deacetylated.