Molecular species of glycerophospholipids and sphingomyelins of human erythrocytes: improved method of analysis

This study reports the application of modern methods of molecular species analysis in determination of the structure of both major and minor glycerophospholipids and sphingomyelins of human erythrocytes. Individual phospholipid classes were resolved from total lipid extracts by thin-layer chromatography. Diradylglycerols were released by Phospholipase C and converted into trimethylsilyl ethers, which were resolved into the alkenylacyl, alkylacyl and diacylglycerol subclasses by normal phase high performance liquid chromatography. Molecular species of diradylglycerols and ceramides were quantitated according to carbon and double bond number by gas liquid chromatography using a fused silica capillary column wall-coated with bonded RTx-2330. The molecular species of ceramides were determined by GC/MS. The diradyl glycerophosphocholines contained 93.0% diacyl, 4.6% alkylacyl and 2.5% alkenylacyl, while the diradyl glycerophosphoethanolamines were made up of 48.8% diacyl, 47.8% alkenylacyl and 3.4% alkylacyl subclasses. Analysis of the molecular species showed that the long chain polyunsaturated acids were mainly combined with C16 in all diradyl GPC subclasses and in diacyl GPE, while in the alkylacyl and alkenylacyl GPE and in diacyl glycerophosphoinositol and diacyl glycerophosphoserine they were combined mainly with C18 saturated fatty chains. In addition to the C16 and C18 alkyl and alkenyl, the ether fractions also contained significant proportions of C20, C22 and C24 chains. The molecular species of the ceramide moieties of the SPH were made up largely of mono- and diunsaturated species. Over 200 molecular species were identified and quantitated in a representative sample of human red blood cells.