Free sphingosine in human epidermis

Abstract

The aim of the present study is to determine the concentration and composition of free long-chain bases in human stratum corneum. Samples of foot callus obtained from a local chiropodist were extracted at room temperature with chloroform: methanol mixtures (2:1, 1:1, and 1:2). The extracts were concentrated, filtered to remove nonlipid debris, and dissolved in chloroform: methanol, 2:1, at a concentration of 25 mg/ml. Small portions of extract were treated with 2,4-dinitrofluorobenzene to convert free amino groups to N-dinitrophenyl derivatives, which were subsequently separated from other components by thin-layer chromatography (TLC) and quantitated by photodensitometry at a wave-length of 355 nm. The N-dinitrophenylsphingosines were then isolated by preparative TLC and oxidized with lead tetraacetate in benzene to produce aldehydes, which were analyzed by gas-liquid chromatography. It was found that callus lipid contained 3.39 +/- 0.57 mg free sphingosine, 1.60 +/- 0.72 mg dihydrosphingosine and traces of phytosphingosine per g lipid. The sphingosines and dihydrosphingosines ranged from 16 through 20 carbons in length, with 18:1 as the predominant constituent. These findings are similar to those recently reported for pig epidermis and support the possible role of free sphingosine as a regulator of epidermal differentiation.