engleski

HPLC method for the determination of NMFs and applications

Filaggrin (filament-aggregating protein), a structural protein responsible for the mechanical strength and integrity of the stratum corneum, is proteolytically degraded into a pool of free amino acids called natural moisturizing factors. Natural moisturizing factors, including histidine and glutamine which are further degraded into 2-pyrrolidone-5-carboxylic acid and urocanic acid have substantial part in regulating hydration of the stratum corneum, pH of the skin, lipid synthesis etc. We have developed a simple reversed phase high performance liquid chromatography method for determination of free amino acids and their metabolites in the stratum corneum including histidine, 2-pyrrolidone-5-carboxylic acid, tyrosine, trans- and cis- urocanic acid. The method is based on non-invasive stratum corneum detachment by tape stripping and subsequent extraction of tape strips in concentrated ammonia. Advantage of the method is the use of volatile extraction solvent, ammonia, and therefore neutralization step prior to chromatographic performance is omitted. Method was easily applied on normal and damaged (e. g. atopic dermatitis) human stratum corneum, mice skin and reconstructed human epidermis samples. It was shown to be sensitive enough to quantify the analytes in homozygous filaggrin gene carriers and reduced natural moisturizing factors levels were found in the individuals after exposure to skin irritants as sodium lauryl sulfate and sodium hydroxide. Moreover, method has successfully followed conversion of trans- to cis- urocanic acid in both, stratum corneum and interstitial fluid, after exposing subjects to UVB irradiation.

NMF; stratum corneum; atopic dermatitis

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