Covalently bound lipids in reconstructed human epithelia

The composition of free and covalently bound lipids in reconstructed epithe lia generated with normal human keratinocytes, HaCaT cells and squamous car cinoma cells was investigated and compared with native skin. Stratum corneu m isolated from native human and reconstructed epidermis was subjected to e xtensive 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-extract able lipids and gas liquid chromatography was performed to assess the fatty acid profile in extractable lipids. In both native and reconstructed tissu e covalently bound lipids consisted of omega-hydroxyceramides, omega-hydrox yacids and free fatty acids. Small amounts of w-hydroxyacids could already be detected in solvent-extractable fractions, omega-Hydroxyceramides consis ted of Ceramide A, Ceramide B and a small fraction of unknown ceramides wit h intermediate polarity. The relative proportions of individual omega-hydro xyceramides 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 tran sformed cell lines (HaCaT, SCC-12F2 and SCC-13 cells). Compared with native or reconstructed epidermis, in epithelia reconstructed with transformed ce ll lines the ceramide content was low, the most polar ceramides were missin g and the content of free fatty acids was low. The same holds true for cova lently bound lipids that were virtually absent in these epithelia, Marked s imilarities were demonstrated in the overall lipid composition of free and bound stratum corneum lipids in native epidermis and in epidermis reconstru cted with normal human keratinocytes, The observed imbalance in fatty acid profile may account for differences in phase behaviour of stratum corneum l ipids.