Microanalysis of Melanins in Mammalian Hair by Alkaline Hydrogen Peroxide Degradation: Identification of a New Structural Marker of Pheomelanins

A highly sensitive, easy-to-perform method for melanin analysis in pigmented tissues based on alkaline hydrogen peroxide degradation has been developed and accomplishes simultaneous determination of eumelanins and pheomelanins. Pyrrole-2,3,5- tricarboxylic acid, the typical eumelanin marker, was obtained in higher yields than in previous procedures. A benzothiazole acid, 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole, characterized in our previous studies as a specific marker of pheomelanins, and the newly identified 1,3-thiazole-2,4,5-tricarboxylic acid were also used for pigment analysis. Optimal yields of the pigment markers were obtained at 24 h reaction time. Pyrrole-2,3,5-tricarboxylic acid, 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole, and 1,3-thiazole-2,4,5- tricarboxylic acid were quantified in a single chromatographic analysis without fractionation or work up of the degradation mixture. The linearity (linearity coefficient from 0.997 to 0.999) was excellent and the inter-assay (percentage coefficient of variation values in the range 0.2–2, n = 6) and intra-assay (percentage coefficient of variation values ≤ 0.4) reproducibility of the method was very satisfactory. A variety of mammalian hairs including coat color mutant mice and human hairs were used to evaluate the method. All the phenotypically pheomelanic hairs gave 1,3-thiazole-2,4,5-tricarboxylic acid in variable yields, whereas 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole was obtained only from some of the red hair samples examined. Notably, a marked tendency to actinic damage, as evidenced by determination of the minimal erythema dose, was exhibited by those subjects whose hair afforded the highest 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole yields. This suggests that 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole represents a new biogenetic marker for predicting individuals at high risk for skin cancer and melanoma