Free sphingosines of human skin include 6-hydroxysphingosine and unusually long-chain dihydrosphingosines

Ceramides containing 6-hydroxysphingosine, a previously unknown long-chain base, have recently been found in human skin. The present study investigated whether human skin also contains 6-hydroxysphingosine as the free base. Human skin surface lipids were obtained by washing with ethanol. A fraction enriched in sphingoid bases was isolated by preparative thin-layer chromatography and reacted with 2,4-dinitrofluorobenzene. The resulting N-dinitrophenyl derivatives were separated by thin-layer chromatography into three components, the most polar of which accounted for 15% of the total. After acetylation of the hydroxyl groups and repurification, each component was examined by nuclear magnetic resonance spectroscopy. The spectrum of the most polar of the derivatives indicated that it was 6-hydroxysphingosine or homologues of that substance. The spectra of the other two derivatives were virtually identical to those of derivatives prepared from authentic sphingosine and dihydrosphingosine. The chain-length distributions of the skin sphingoid bases were examined by gas chromatography after conversion of the dinitrophenyl acetates to dinitrophenyl trimethylsilyl derivatives. The analysis showed that the sphingosines and 6-hydroxysphingosines ranged from 17 to 22 carbons in length, with the 18- and 20-carbon species predominating. Surprisingly, the dihydrosphingosines included species with up to 26 carbons, with the 24-, 25-, and 26-carbon species accounting for about half of the total. Examination of the sphingoid bases of pig epidermis indicated that 6-hydroxysphingosine was not present and that the major chain length in the dihydrosphingosines was the 22-carbon species.