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In inner organs, glutamine contributes to proliferation, detoxification and establishment of a mechanical barrier, i.e., functions essential for skin, as well. However, the age-dependent and regional peculiarities of distribution of glutamine synthetase (GS), an enzyme responsible for generation of glutamine, and factors regulating its enzymatic activity in mammalian skin remain undisclosed. To explore this, GS localization was investigated using immunohistochemistry and double-labeling of young and adult human and rat skin sections as well as skin cells in culture. In human and rat skin GS was almost completely co-localized with astrocyte-specific proteins (e.g. GFAP). While GS staining was pronounced in all layers of the epidermis of young human skin, staining was reduced and more differentiated among different layers with age. In stratum basale and in stratum spinosum GS was co-localized with the adherens junction component beta-catenin. Inhibition of, glycogen synthase kinase 3beta in cultured keratinocytes and HaCaT cells, however, did not support a direct role of beta-catenin in regulation of GS. Enzymatic and reverse transcriptase polymerase chain reaction studies revealed an unusual mode of regulation of this enzyme in keratinocytes, i.e., GS activity, but not expression, was enhanced about 8-10 fold when the cells were exposed to ammonium ions. Prominent posttranscriptional up-regulation of GS activity in keratinocytes by ammonium ions in conjunction with widespread distribution of GS immunoreactivity throughout the epidermis allows considering the skin as a large reservoir of latent GS. Such a depository of glutamine-generating enzyme seems essential for continuous renewal of epidermal permeability barrier and during pathological processes accompanied by hyperammonemia.

The separation of the major stratum corneum lipids, i.e., ceramides, fatty acids, cholesterol and its esters by means of high-performance thin-layer chromatography is hereby presented. The used automated multiple development technique allows the reproducible development of a 17-step solvent gradient also capable of separating seven ceramide classes in the same run. Reliable quantification has been performed after visualisation and densitometric scanning. The present approach is less time and solvent-consuming than previously described procedures. The application to samples obtained by in vivo skin surface extraction with hexane-ethanol (2:1) demonstrates that the method can be routinely used for diagnostic purposes., (Copyright 2002 Elsevier Science B.V.)

Liposomes were incubated with an extract of human plantar stratum corneum. The liposomal lipids were hydrolysed, if composed of soy-bean phosphatidylcholine or phosphatidylglycerol. Rigid lipids were not degraded. The temperature optimum of the hydrolysis was between 30-35degC. CaCl enhanced, while EDTA reduced the rate of hydrolysis, indicating that the hydrolysis is due to a phospholipase A(2).

Reconstructed human skin was prepared from human keratinoblasts. After 1 week of cultivation at the air-liquid interface a stratified layer developed, similar to native human epidermis. Liposomes with an average diameter of 50 nm, made of phosphatidylcholine (PC), phosphatidylserine (PS) and human stratum corneum lipids (hSCL) were applied on top of this culture system. The rate of penetration through the reconstructed human epidermis was 1.38, 0.55 and 0.013 ng lipidh-1cm-2 for PC, hSCL and PS liposomes, respectively. Electron microscopy and confocal laser scanning microscopy showed that PS and hSCL liposomes aggregated at the skin surface, while PC liposomes remained homogeneously dispersed. Fluorescence measurements demonstrated that vesicles, made of native human stratum corneum lipids rapidly mixed with PS liposomes, weakly with hSCL liposomes and did not mix with PC liposomes.

Photoprotection against sunburn and associated irradiation-induced damages of the human skin is mainly attributed to the darkening of the biochrome melanin by its oxidation. Human skin lipids were examined for an additional protection by sterols. Lipid vesicles prepared from extracted human skin lipids as well as from mixtures of typical lipids of the stratum corneum were irradiated by UV light in the presence and absence of oxygen. The oxidative degradation of various lipids was measured by quantitative HPTLC, by the dichlorofluorescein fluorescent assay, by the thiobarbituric acid assay and a novel luminol-based chemiluminescence technique. Electron spin resonance was used to look for certain radical intermediates. The results indicate, that sterols, mainly free cholesterol, with their high concentration in the lipid barrier of the stratum corneum (up to 50 mol%) effectively compete with the peroxidation of other human skin lipids (ceramides and free fatty acids).

The stratum corneum lipids are unique in composition and have been used frequently as a model system of the skin's lipid barrier. Automated multiple development (AMD) of high-performance thin-layer chromatography plates in combination with a 25-step gradient, based on methanol, diethyl ether and n-hexane separated the six major human plantar stratum corneum lipids. Post-chromatographic staining of these lipids with a solution of MnCl2-H2SO4 at 130 degrees C or a solution of CuSO4-H3PO4 at 140 degrees C allowed visualization of the lipids and quantification. The MnCl2-H2SO4 solution stained saturated fatty acids less intensely. Therefore, the CuSO4-H3PO4 solution was used for quantification and we found, on average, 2.06% (w/w) cholesterol 3-sulphate, 20.16% (w/w) free fatty acids, 20.25% (w/w) ceramides, 43.53% (w/w) non-esterified sterols, 4.56% (w/w) triacylglycerols and 9.4% (w/w) sterolesters in the human plantar stratum corneum extracts. The concentration of phospholipids was less than 1% (w/w). In addition, the lipid composition of twenty different human plantar stratum corneum extracts was determined. Statistics revealed a correlation between the ratio of free fatty acids and non-esterified sterols (r=0.832, p<0.01, n=20). Several control experiments proved that this correlation is not due to the extraction method, the post-chromatographic staining procedure or bacterial contamination of the stratum corneum.

The interaction of dimyristoylphosphatidylcholine liposomes with the human stratum corneum was investigated by confocal laser scanning microscopy and differential scanning calorimetry. Human skin is characterized by a high autofluorescence. By introducing appropriate optical filters the autofluorescence of the skin was depressed and the penetration profile of fluorescence labelled vesicles was investigated. From optical sectioning it was obvious that neither the vesicles nor the fluorophore N-(lissamine rhodamine B sulfonyl)diacylphophatidylethanolamine (Rho-PE) penetrates in detectable amounts into the human skin. Differential scanning calorimetry of human stratum corneum revealed, that the peak positions of the human stratum corneum specific endothermic transitions at 10 degrees C, 35 degrees C, 50 degrees C, 62 degrees C, 73 degrees C and 81 degrees C did not change significantly after 18 h of non-occlusive vesicle application. However, the enthalpy of the transitions at 35 degrees C, 50 degrees C, 62 degrees C and 73 degrees C, estimated through peak heights increased, relative to the protein related peak at 81 degrees C. A novel transition at 10 degrees C was observed. From these data we conclude that DMPC liposomes do not penetrate intact into the human skin. We deduce, however, that the vesicles disintegrate at the surface of stratum corneum after non-occlusive application. The individual lipid molecules then interact with the lipid barrier of the stratum corneum and penetrate into the latter, which results in an increase of the enthalpy, related to the lipid components of the SC.