Bound Hydroxyceramide Hydrolysis in Response to Decreased Stratum Corneum Function

Objectives: Our previous studies demonstrated w-hydroxyceramides and w-hydroxyacids ester-linked to the outer surface of cornified cells in stratum corneum (SC). A recent study has demonstrated increased permeability of SC associated with lowered concentrations of covalently bound w-hydroxyceramide (S Meguro et al., Arch Derm Res 191:463, 2000). w-Hydroxyacid was not examined in this study. The present hypothesis is that the reduction of w-hydroxyceramide with decreasing barrier function results from hydrolysis of the amide linkage in this lipid by ceramidase. This should result in an increase in covalently bound w-hydroxyacid proportionate to the decrease in w-hydroxyceramide as barrier funcition decreases. Methods: Young pigs were placed on an essential fatty acid deficient diet. Transepidermal water loss (TEWL) was monitored, and SC samples were collected at intervals as TEWL increased. Free lipids were removed by extraction with chloroform-methanol mixtures. Residual tissue was hydrolysed, and bound lipids were recovered by extraction into chloroform. Lipid compositions were determined by thin-layer chromatography in conjunction with photodensitometry. Results: It was confirmed that w-hydroxyceramide decreased as TEWL increased. However, the decrease in hydroxyceramide was accompanied by a proportionate increase in w -hydroxyacid. Both the decrease in w-hydroxyceramide content with increasing TEWL (r² = 0.9952, p = 0.4) and the accompanying increase in w-hydroxyacid (r² = 0.9995, p = 0.01) were linear as judged by linear regression. Conclusion: It is concluded that hydrolysis of the amide linkage in covalently bound w-hydroxyceramide molecules to produce bound w-hydroxyacids correlates with increasing TEWL. This hydrolysis would be mediated by ceramidase, and would liberate free sphingosine, a broad acting antimicrobial. Together, the covalenly bound hydroxyceramide and ceramidase may constitute part of an innate immune system. Supported in part by NIH grant DEO0175 (JRH) and a Dows Student Research Award (JS).