Most of the natural
melanin pigments consist of not only indolic
eumelanin but also
sulfur-containing
pheomelanin. Previous methods for spectrophotometric assay of
melanins use solubilization in alkaline media; a major disadvantage of these procedures is that they do not distinguish between
eumelanin and
pheomelanin. A spectrophotometric method for assaying
eumelanin in tissue samples is described. Sepia
melanin serves as a standard. Hair and
melanoma samples were hydrolyzed in hot
hydriodic acid, and insoluble eumelanic pigments were solubilized in hot
sodium hydroxide in the presence of
hydrogen peroxide and analyzed for absorbance at 350 nm (A350). The detection limit of
eumelanin was ca. 2 micrograms.
Eumelanins prepared from
dopa,
5,6-dihydroxyindole and its carboxy derivative gave similar A350 values. Mixed-type
melanins prepared from
dopa and various ratios of
cysteine gave A350 values inversely proportional to their
sulfur contents. Excellent correlations were observed between A350 values and contents of
pyrrole-2,3,5-tricarboxylic acid, an oxidation product specific for
eumelanin, in hair samples from sheep and humans of various colors and in
melanomas and eyes from mice. The present method provides a specific and direct measurement of
eumelanin contents in tissue samples.