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Sandra Del Bino (1) • Shosuke Ito (2) • Kazumasa Wakamatsu (2) • Françoise Bernerd (1) (1) L'Oreal Research & Innovation, Aulnay-sous-Bois, France. (2) Fujita Health University School of Health Sciences, Toyoake, Japan CONSTITUTIVE PIGMENTATION AND INDIVIDUAL TYPOLOGY ANGLE MEASUREMENT: TOWARDS A BETTER CHARACTERIZATION OF MELANINS Skin constitutive pigmentation is given by the amount and distribution of melanins within the epidermis and in particular the photoprotective eumelanin and the phototoxic pheomelanin content. Constitutive pigmentation can be objectively classified through the Individual Typology Angle (ITA) based on colorimetric parameters of the L*a*b* system 1,2 . Experimentally, we showed a correlation between ITA values and biological markers of UV-induced erythema 3,4 reflecting the sensitivity to UV exposure. Nevertheless, a precise knowledge of melanins in human skin is lacking. The present work aimed at better analyzing melanin content in skin samples of variable constitutive pigmentations classified according to their ITA value using: i) image analysis of Fontana Masson stained sections ii) spectrophotometry following solubilization of samples with Soluene-350, and iii) high-performance liquid chromatography (HPLC) after chemical degradation of melanins. Results showed significant correlations between ITA and total melanin content assessed either by image analysis, spectrophotometry or HPLC. Furthermore a good correlation between ITA and PTCA or between ITA and TTCA, specific products of eumelanin and pheomelanin degradation was found. On the contrary, no correlation between ITA and 4-AHP another product of pheomelanin degradation was found. Altogether, our results contribute to a better charac- terization of the “chemical” melanin phenotype in skin with variable constitutive pigmentation. They also confirm the poor total melanin and photoprotective eumelanin content in Very light to Tan skin types, thereby explaining their higher sensitivity towards UV rays. INTRODUCTION AND OBJECTIVE MATERIAL AND METHODS CONCLUSIONS 1 Chardon et al. (1991). Int J Cosm Sc 13, 191-208 2 Del Bino and Bernerd (2013). Br. J. Dermatol. 169, S3, 33-40 3 Del Bino et al. (2006). Pigment Cell Res 19, 606-614 4 Del Bino et al. (2013). Br. J. Dermatol. 168, 1120-1123. 5 Ito et al. (2011). Pigment Cell Melanoma Res. 24, 605-613 6 Wakamatsu et al. (2002). Pigment Cell Res. 15, 225-232. 7 Ozeki et al. (1996). Pigment Cell Res. 9, 265-270 Conflict of interest: authors have no conflict of interest. ITA-based skin color classification of samples: ITA values were calculated from spectrocolorimeter (Datacolor) mea- surements of the L* (Luminance) and b* (yellow/blue component) parameters on 35 skin samples. The samples were classified into six color groups: Very light, Light, Intermediate, Tan, Brown and Dark 1,3 . Image analysis: The melanin index (MI) in the whole epidermis or in the basal layer was defined by image analysis on Fontana Masson stained sections as the surface covered by the melanin staining over the whole epidermis including the stratum corneum or the basal layer respectively. Chemical melanin analysis: Epidermal samples were homogenized and subjected to H 2 O 2 oxidation 5 and HI hydrolysis 6 . Specific degradation products of melanins were analyzed using HPLC. These included pyrrole-2,3,5- tricarboxylic acid (PTCA) and thiazole-2,4,5-tricarboxylic acid (TTCA) obtained after alkaline hydrogen peroxide oxidation of DHICA moiety of eumelanin and benzothiazole-type pheomelanin, respectively or 4-amino-3-hydroxyphenylalanine (4-AHP) obtained after reductive hydrolysis of benzothiazine-type pheomelanin with hydroiodic acid. Aliquots were additionally subjected to Soluene-350 solubilization. Total melanin content was estimated spectrophotometrically by analyzing absorbance at 500 nm 7 . 1 2 3 SIGNIFICANT CORRELATION BETWEEN ITA AND MELANIN INDEX (MI) IN THE WHOLE EPIDERMIS OR IN THE BASAL LAYER BY IMAGE ANALYSIS: 1 SIGNIFICANT CORRELATION BETWEEN ITA AND TOTAL MELANIN (TM) BY SPECTROPHOTOMETRY OR HPLC : 2 SIGNIFICANT CORRELATION BETWEEN ITA AND PTCA (EUMELANIN), TTCA (BENZOTHIAZOLE-TYPE PHEOMELANIN) BUT NOT 4-AHP (BENZOTHIAZINE-TYPE) BY HPLC: 3 EUMELANIN AND PHEOMELANIN CONTENT IN SKIN WITH DIFFERENT CONSTITUTIVE PIGMENTATION: 4 RESULTS REFERENCES
