Correlations between Skin Condition Parameters and Ceramide Profiles in the Stratum Corneum of Healthy Individuals.

Ceramides are essential lipids for skin barrier function, and various classes and species exist in the human stratum corneum (SC). To date, the relationship between skin conditions and ceramide composition in healthy individuals has remained largely unclear. In the present study, we measured six skin condition parameters (capacitance, transepidermal water loss, scaliness, roughness, multilayer exfoliation, and corneocyte cell size) for the SC of the cheeks and upper arms of 26 healthy individuals and performed correlation analyses with their SC ceramide profiles, which we measured via liquid chromatography-tandem mass spectrometry. In the cheeks, high levels and/or ratios of two free ceramide classes containing an extra hydroxyl group in the long-chain moiety and a protein-bound ceramide class containing 6-hydroxysphingosine correlated with healthy skin conditions. In contrast, the ratios of two other free ceramide classes, both containing sphingosine, and a protein-bound ceramide class containing 4,14-sphingadiene correlated with unhealthy skin conditions, as did shortening of the carbon chain of the fatty acid portion of two ceramide classes containing non-hydroxy fatty acids. Thus, our findings help to elucidate the relationship between skin conditions and ceramide composition.

Alteration of epidermal lipid composition as a result of deficiency in the magnesium transporter Nipal4.

Lipids in the stratum corneum play an important role in the formation of the skin permeability barrier. The causative gene for congenital ichthyosis, NIPAL4, encodes a Mg2+ transporter and is involved in increases in intracellular Mg2+ concentrations that depend on keratinocyte differentiation. However, the role of this increased Mg2+ concentration in skin barrier formation and its effect on the lipid composition of the stratum corneum has remained largely unknown. Therefore, in the present study, we performed a detailed analysis of epidermal lipids in Nipal4 KO mice via TLC and MS. Compared with WT mice, the Nipal4 KO mice showed compositional changes in many ceramide classes (including decreases in ω-O-acylceramides and increases in ω-hydroxy ceramides), together with increases in ω-hydroxy glucosylceramides, triglycerides, and free fatty acids and decreases in ω-O-acyl hydroxy fatty acids containing a linoleic acid. We also found increases in unusual ω-O-acylceramides containing oleic acid or palmitic acid in the KO mice. However, there was little change in levels of cholesterol or protein-bound ceramides. The TLC analysis showed that some unidentified lipids were increased, and the MS analysis showed that these were special ceramides called 1-O-acylceramides. These results suggest that elevated Mg2+ concentrations in differentiated keratinocytes affect the production of various lipids, resulting in the lipid composition necessary for skin barrier formation.

Catalytic mechanism of trans-2-enoyl-CoA reductases in the fatty acid elongation cycle and its cooperative action with fatty acid elongases.

The fatty acid (FA) elongation cycle produces very-long-chain FAs with ≥C21, which have unique physiological functions. Trans-2-enoyl-CoA reductases (yeast, Tsc13; mammals, TECR) catalyze the reduction reactions in the fourth step of the FA elongation cycle and in the sphingosine degradation pathway. However, their catalytic residues and coordinated action in the FA elongation cycle complex are unknown. To reveal these, we generated and analyzed Ala-substituted mutants of 15 residues of Tsc13. An in vitro FA elongation assay showed that nine of these mutants were less active than WT protein, with E91A and Y256A being the least active. Growth complementation analysis, measurement of ceramide levels, and deuterium-sphingosine labeling revealed that the function of the E91A mutant was substantially impaired in vivo. In addition, we found that the activity of FA elongases, which catalyze the first step of the FA elongation cycle, were reduced in the absence of Tsc13. Similar results were observed in Tsc13 E91A-expressing cells, which is attributable to reduced interaction between the Tsc13 E91A mutant and the FA elongases Elo2/Elo3. Finally, we found that E94A and Y248A mutants of human TECR, which correspond to E91A and Y256A mutants of Tsc13, showed reduced and almost no activity, respectively. Based on these results and the predicted three-dimensional structure of Tsc13, we speculate that Tyr256/Tyr248 of Tsc13/TECR is the catalytic residue that supplies a proton to trans-2-enoyl-CoAs. Our findings provide a clue concerning the catalytic mechanism of Tsc13/TECR and the coordinated action in the FA elongation cycle complex.

Comparison of skin barrier abnormalities and epidermal ceramide profiles among three ω-O-acylceramide synthesis-deficient mouse strains.

BACKGROUND: The epidermis contains many structurally diverse ceramides, which form the skin permeability barrier (skin barrier). Mutations in genes involved in the synthesis of ω-O-acylceramides (acylceramides) and protein-bound ceramides cause ichthyosis. OBJECTIVE: We aimed to elucidate the relationship between the degree of skin barrier impairment and changes in epidermal ceramide profiles caused by mutations in acylceramide synthesis genes. METHODS: Knockout (KO) mice of three genes-fatty acid (FA) ω-hydroxylase Cyp4f39 (human CYP4F22 ortholog), FA elongase Elovl1, and acyl-CoA synthetase Fatp4-were subjected to transepidermal water loss measurement, toluidine blue staining, and epidermal ceramide profiling via liquid chromatography coupled with tandem mass spectrometry. RESULTS: Transepidermal water loss was highest in Cyp4f39 KO mice, followed by Elovl1 KO and Fatp4 KO mice, and Cyp4f39 KO mice also showed the strongest degree of toluidine blue staining. In Cyp4f39 KO, Elovl1 KO, and Fatp4 KO mice, acylceramide levels were 0.6%, 1.6%, and 12%, respectively, of those in wild-type mice. Protein-bound ceramide levels were 0.2%, 30%, and 33%, respectively, of those in wild-type mice. We also observed a near-complete absence of ω-hydroxy ceramides in Cyp4f39 KO mice, reduced total ceramide levels and shortened FA moieties in Elovl1 KO mice, and increased hydroxylated ceramide levels and slightly shortened FA moieties in Fatp4 KO mice. CONCLUSIONS: The degree of reduction in protein-bound ceramide levels is probably related to the severity of skin barrier defects in these three strains. However, reduced acylceramide levels and other changes in ceramide composition unique to each KO strain are also involved.