Spectrophotometric Characterization of Eumelanin and Pheomelanin in Hair

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Abstract
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.