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UVA exposure elicits immediate and persistent pigment darkening of the skin, which is thought to result from the oxidation and polymerization of existing melanin and/or precursors. Melanocytes produce eumelanin and pheomelanin. Eumelanin consists of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), while pheomelanin consists of benzothiazine and benzothiazole units. Melanins can be analyzed by quantifying specific degradation products using HPLC. Specifically, eumelanin can be analyzed as pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA), specific degradation products of DHICA and DHI, respectively. Benzothiazole pheomelanin can be analyzed as thiazole-2,4,5-tricarboxylic acid (TTCA), whereas benzothiazine pheomelanin is analyzed as 4-amino-3-hydroxyphenylalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine (3-AHP). Upon UVA exposure, melanins undergo structural modifications. Eumelanin undergoes oxidative cleavage to free pyrrole-2,3,5-tricarboxylic acid (Free PTCA) and undergoes cross-linking to form pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA). UVA exposure of pheomelanin induces oxidative conversion from the benzothiazine to the benzothiazole. Nevertheless, these structural modifications have never been previously characterized in human skin. In this study, we exposed ex vivo skin to increasing UVA1 doses (60, 90 and 120 J/cm²; n = 6 in triplicate) and characterized the induced pigment darkening before, immediately, and 2 h after exposure through colorimetry, HPLC and spectrophotometry. The results showed changes in the CIELAB colorimetric parameters, namely a decrease in Luminance L*, the yellow-blue component b* and the Individual Typology Angle (ITA) in UVA1-exposed samples, indicative of skin darkening. In parallel, UVA1 exposure induced significant modifications of the levels of absorbance at 500 nm (A500) and melanin markers PTCA, PTeCA, PDCA, TTCA, and 4-AHP, as well as in the ratios of various markers, such as PTeCA/PTCA, Free/Total PTCA, and TTCA/4-AHP, indicative of photooxidation/degradation of melanins. Our study provides the first evidence of UVA1-induced modifications of melanins associated with pigment darkening occurring in human skin. Full article

Alkaline hydrogen peroxide oxidation (AHPO) of eumelanin and pheomelanin, two major classes of melanin pigments, affords pyrrole-2,3,5-tricarboxylic acid (PTCA), pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) from eumelanin and thiazole-2,4,5-tricarboxylic acid (TTCA) and thiazole-4,5-dicarboxylic acid (TDCA) from pheomelanin. Quantification of these five markers by HPLC provides useful information on the quantity and structural diversity of melanins in various biological samples. HPLC analysis of these markers using the original method of 0.1 M potassium phosphate buffer (pH 2.1):methanol = 99:1 (85:15 for PTeCA) on a reversed-phase column had some problems, including the short lifetime of the column and, except for the major eumelanin marker PTCA, other markers were occasionally overlapped by interfering peaks in samples containing only trace levels of these markers. These problems can be overcome by the addition of an ion pair reagent for anions, such as tetra-n-butylammonium bromide (1 mM), to retard the elution of di-, tri- and tetra-carboxylic acids. The methanol concentration was increased to 17% (30% for PTeCA) and the linearity, reproducibility, and recovery of the markers with this improved method is good to excellent. This improved HPLC method was compared to the original method using synthetic melanins, mouse hair, human hair, and human epidermal samples. In addition to PTCA, TTCA, a major marker for pheomelanin, showed excellent correlations between both HPLC methods. The other markers showed an attenuation of the interfering peaks with the improved method. We recommend this improved HPLC method for the quantitative analysis of melanin markers following AHPO because of its simplicity, accuracy, and reproducibility. Full article