Identification and quantitation of free ceramides in human platelets

Abstract

Free ceramides were isolated from human platelets. Their structures were unequivocally determined by gas–liquid chromatography–mass spectrometry of the trimethylsilyl ether derivatives. The major components were N-(palmitoyl) sphingosine, N-(stearoyl) sphingosine, N-(eicosanoyl) sphingosine, N-(docosanoyl) sphingosine, N-(tetracosanoyl) sphingosine, and N-(tetracosenoyl) sphingosine. Sphinganine- and sphingadienine-containing ceramides as well as ceramides containing other unsaturated acids were also present. The amount of ceramides was determined by quantitative gas–liquid chromatography, using radioactive ceramide as internal standard and synthetic crystalline ceramides for comparison of peak areas. The concentration of ceramides was found to be 1.31 μg/lO9 platelets or 0.47 μg/mg of platelet protein.

Keywords

• gas–liquid chromatography–mass spectrometry
• trimethylsilyl ethers