Mono- and dimethyl phosphatidates from different subtypes of choline and ethanolamine glycerophosphatides

Abstract

Preparation and some properties of phosphatidic acids and their mono- and dimethyl esters from natural phosphatides were studied. 1. 1. Hydrolysis with cabbage phospholipase D (EC 3.1.4.4) converted all three subclasses of ox heart lecithin, viz. phosphatidyl choline, choline plasmalogen and alkyl acyl lecithin, into phosphatidic acids. Phospholipase D catalysed methanolysis converted phosphatidyl choline to phosphatidyl methanol. The same reaction appeared to take place also with choline plasmalogens and alkyl acyl lecithins. The hydrolysis, and quite likely also the methanolysis, was a fast reaction with phosphatidyl cholines and alkyl acyl lecithins but very slow with the plasmalogens. 2. 2. The three subclasses of phosphatidic acid, the diacyl, the plasmalogenic, and the alkyl acyl forms, were completely separated from each other by thin-layer chromatography of the dimethyl esters. 3. 3. The Chromatographie properties of phosphatidic acid were shown to depend on the cations present ; the Ca 2+ salt was not retained by DEAE-cellulose or alumina from neutral solvents, but the Na + salt was retained appreciably. Also on silicic acid columns these two salts gave remarkably different elution patterns. The behaviour of phosphatidyl methanol on DEAE-cellulose was affected much less than that of phosphatidic acid by the presence of Ca 2+. 4. 4. Diazomethanolysis converted all three subclasses of ox heart lecithin into the corresponding dimethyl phosphatidates. Phosphatidyl cholines and alkyl acyl lecithins seemed to react without complication, but choline plasmalogens gave an unidentified product in addition to the dimethyl phosphatidate. Diazomethanolysis of the same three subtypes present in ox brain ethanolamine phosphatides also gave the different dimethyl phosphatidates. 5. 5. The more polar products formed in the diazomethanolysis of the lecithins and ethanolamine lipids appeared to contain phosphatidyl methanol and O-methylated lecithins and chephalins, which are probably intermediate products of the reaction. 6. 6. The dimethyl phosphatidates could be effectively separated into different molecular species by argentation chromatography. The positional analysis of fatty acids in these lipids could be carried out with pancreatic lipase which attacks preferentially the ester groups on C-1 of glycerol. The plasmalogenic dimethyl phosphatidates and their alkyl analogs were virtually stable towards this enzyme. 7. 7. NMR spectra of phosphatidic acid and its mono- and dimethyl esters were recorded.