Determination of stratum corneum lipid profile by tape stripping in combination with high-performance thin-layer chromatography

Intercellular lipids in the stratum corneum (SC) are responsible for the barrier function of mammalian skin. The main components of the SC lipids are ceramides, cholesterol, and free fatty acids, as established by thin-layer chromatographic analysis of lipids extracted from the human and mammalian SC. Up to now for lipid analysis the extracts of the entire SC has been used and information on whether the lipid composition changes with the depth in the SC is scarce. Tape stripping is a technique which removes corneocyte layers step by step with an adhesive film. The use of this technique for lipid analysis was hampered by the contamination of lipid extracts with compounds co-extracted from the tape with organic solvents used for the extraction of SC lipids. The aim of the present study was to establish a suitable analytical method for the determination of the local SC lipid composition. For this purpose, the SC samples were collected by sequential stripping with Leukoplex tape in five healthy volunteers. The lipids were extracted with ethyl acetate:methanol mixture (20:80) and separated by means of HPTLC. The results of this study revealed that the free fatty acid level is highest and the cholesterol and ceramide levels lowest in the uppermost SC layers (about 4 strippings). The levels remained unchanged in the underlying SC layers. In these layers, the ceramide level was about 60 wt% and the free fatty acid and cholesterol levels were about 20 wt% each. Ceramides could be separated into seven different fractions and the relative amounts of individual ceramide fractions did not significantly change with the SC depth. Cholesterol sulfate levels were about 5% of total cholesterol and did not change with the SC depth, except for the for the first strip where the level was about 1%. The method developed makes it possible to study the differences in the SC lipid profile in healthy and diseased human skin with relation to the SC lipid organization and to the skin barrier function in vivo.