Identification of free ceramide in human erythrocyte membrane

Free ceramide was characterized in human erythrocytes and ghosts. Its concentration was found to be 5.6 mumol/100 ml of packed cells. It was isolated by thin-layer chromatography of its acetylated form and purified by thin-layer chromatography after deacetylation. It was constituted mainly of C16, C22, C24:0, and C24:1 nonhydroxy fatty acids and of C18:1 sphingosine. A small amount of 2-hydroxy fatty acids was also detected, containing mainly C24:0 hydroxy fatty acid. The structures of the ceramides and identification of the minor bases were confirmed by electron-impact and chemical ionization mass spectra of the trimethylsilylated ceramides.