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 pigmente d tissues based on alkaline hydrogen peroxide degradation has been develope d and accomplishes simultaneous determination of eumelanins and pheomelanin s. Pyrrole-2,3,5- tricarboxylic acid, the typical eumelanin marker, was obt ained in higher yields than in previous procedures. A benzothiazole acid, 6 -(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole, characterized i n 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 reac tion time. Pyrrole-2,3,5-tricarboxylic acid, 6-(2-amino-2-carboxyethyl)-2-c arboxy-4-hydroxybenzothiazole, and 1,3-thiazole-2,4,5- tricarboxylic acid w ere quantified in a single chromatographic analysis without fractionation o r work up of the degradation mixture. The linearity (linearity coefficient from 0.997 to 0.999) was excellent and the inter-assay (percentage coeffici ent of variation values in the range 0.2-2, n = 6) and intra-assay (percent age coefficient of variation values less than or equal to 0.4) reproducibil ity of the method was very satisfactory. A variety of mammalian hairs inclu ding coat color mutant mice and human hairs were used to evaluate the metho d. All the phenotypically pheomelanic hairs gave 1,3-thiazole-2,4,5-tricarb oxylic acid in variable yields, whereas 6-(2-amino-2-carboxyethyl)-2-carbox y-4-hydroxybenzothiazole was obtained only from some of the red hair sample s examined. Notably, a marked tendency to actinic damage, as evidenced by d etermination of the minimal erythema dose, was exhibited by those subjects whose hair afforded the highest 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydr oxybenzothiazole yields. This suggests that 6-(2-amino-2-carboxyethyl)-2-ca rboxy-4-hydroxybenzothiazole represents a new biogenetic marker for predict ing individuals at high risk for skin cancer and melanoma.