Spectrophotometric Characterization of Eumelanin and Pheomelanin in Hair

Mammalian melanins exist in two chemically distinct forms: the brown to black eumelanins and the yellow to reddish-brown pheomelanins. They can be quantified by HPLC analysis of pyrrole-2,3,5-tricarboxylic acid (PTCA) and aminohydroxyphenylalanine (AHP). We recently developed a spectrophotometric method for assaying the total amount of eu- and pheomelanins by dissolving melanins in Soluene-350 plus water. In this study, we examined whether absorbance at 500 nm (A₅₀₀) of the Soluene-350 solution reflects the total amount of melanins obtained by the HPLC methods, and whether the ratio of absorbances between 650 and 500 nm reflects the eumelanin/total melanin ratio in mouse hair, sheep wool, and human hair. Our findings were as follows: (1) Total melanin levels calculated from A₅₀₀ values correlate well with those obtained from PTCA and AHP values by multiplying with the following factors: for mice, PTCA × 45 + AHP × 2.5; for sheep, PTCA × 40 + AHP × 15; and for humans, PTCA × 160 + AHP × 10. (2) The A₆₅₀/A₅₀₀ ratios were higher (0.25–0.33) in black to brown hair while they were significantly lower (0.10–0.14) in yellow to red hair. These results indicate that (1) the A₅₀₀ value can be used to quantify the total combined amount of eu- and pheomelanins, and (2) the A₆₅₀/A₅₀₀ ratio can serve as a parameter to estimate the eumelanin/total melanin ratio. The present method provides a convenient way to qualitatively characterize eu- and pheomelanins in melanins produced in follicular melanocytes.