The ω-hydroxyceramides of pig epidermis are attached to corneocytes solely through ω-hydroxyl groups

The cornified outer cells of mammalian epider- mis possess a monolayer of � -hydroxyceramides that are ester-linked to the exterior of a cross-linked protein envelope. In the present study, conclusive evidence was sought on which of the ceramide hydroxyl groups are involved in the linkage to protein. This was obtained by derivatizing all free hydroxyl groups in isolated solvent-extracted porcine stratum corneum using triisopropylsilyl (TIPS) chloride in pyridine in the presence of silver nitrate. After an 18-h reaction, the tis- sue was recovered, rinsed, and the derivatized ceramides were then released from protein linkage by hydrolysis with 1M KOH in 95% methanol. This gave a single ceramide prod- uct that was shown by nuclear magnetic resonance to contain two triisopropyl groups. Acetylation of the product using ace- tic anhydride in pyridine resulted in a downfield shift of the NMR signal for the � -methylene protons, showing that it was the � -hydroxyl that was free in the initial reaction product, and subsequently was acetylated. These results show that all of the � -hydroxyceramides of corneocyte lipid envelopes are attached to protein through their � -hydroxyl groups. — Stewart, M. E., and D. T. Downing. The � -hydroxyceramides of pig epidermis are attached to corneocytes solely through � -hydroxyl groups. J. Lipid Res. 2001. 42: 1105-1110.