Covalently bound lipids in reconstructed human epithelia

Abstract

The composition of free and covalently bound lipids in reconstructed epithelia generated with normal human keratinocytes, HaCaT cells and squamous carcinoma cells was investigated and compared with native skin. Stratum corneum isolated from native human and reconstructed epidermis was subjected to extensive extraction with chloroform-methanol mixtures followed by alkaline hydrolysis to release covalently bound lipids. High-performance thin layer chromatography was used for analysis of solvent-extractable and non-extractable lipids and gas liquid chromatography was performed to assess the fatty acid profile in extractable lipids. In both native and reconstructed tissue covalently bound lipids consisted of omega-hydroxyceramides, omega-hydroxyacids and free fatty acids. Small amounts of omega-hydroxyacids could already be detected in solvent-extractable fractions. omega-Hydroxyceramides consisted of Ceramide A, Ceramide B and a small fraction of unknown ceramides with intermediate polarity. The relative proportions of individual omega-hydroxyceramides were similar in both native and reconstructed stratum corneum. In contrast, differences were found in profiles of both solvent-extractable and non-extractable lipids isolated from epithelia reconstructed with transformed cell lines (HaCaT, SCC-12F2 and SCC-13 cells). Compared with native or reconstructed epidermis, in epithelia reconstructed with transformed cell lines the ceramide content was low, the most polar ceramides were missing and the content of free fatty acids was low. The same holds true for covalently bound lipids that were virtually absent in these epithelia. Marked similarities were demonstrated in the overall lipid composition of free and bound stratum corneum lipids in native epidermis and in epidermis reconstructed with normal human keratinocytes. The observed imbalance in fatty acid profile may account for differences in phase behaviour of stratum corneum lipids.