Abstract
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.
Highlights
Free ceramide was characterized in human erythrocytes and ghosts
Free ceramide was demonstrated to be a component of human erythrocyte membrane
The concentration of free ceramide contributed by erythrocytes was much higher than that occurring in plasma (0.5 pmol) [7] or in platelets (0.08 pmol) [8]
Summary
Whole blood was drawn from normal humans on acidcitrate-dextrose anticoagulant (5 to 20 ml per subject, samples 1, 2,and 3). Erythrocytes and ghosts were prepared from outdated blood (1 month old) obtained at the local blood bank (samples to 7). Acetylated glycosphingolipids and free ceramide were recovered in the dichloroethane-acetone 1:1 (v/v) fractions They were analyzed by thin-layer chromatography (TLC)as their acetylated forms or after deacetylation. Native free ceramides were analyzed in solvent B: chloroform-methanol 90:10 (v/v). Free ceramide was hydrolyzed in methanol-concentrated hydrochloric acid-water 83:8.6:9.4 (v/v) at 80°C for 18 hr [11]. Fatty acid methyl esters were separated into hydroxy and nonhydroxy methyl esters by chromatography on a Florisil column [12]. They were analyzed by gas-liquid chromatography (GLC) on a SE-30wall-coatedcapillary column, 12 m long. Proteins were determined by the method of Lowry et al [18]
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