High-performance liquid chromatography (HPLC) analysis of eu- and pheomelanin in melanogenesis control

Two types of melanogenesis, eumelanogenesis and pheomelanogenesis, can be switched from one type to another under certain physiologic or pathologic conditions. To study the regulation of melanogenesis, we developed a high-performance liquid chromatography method to analyze quantitatively the contents of eu- and pheomelanin in tissue samples without any isolation procedures. The rationale is that permanganate oxidation of eumelanin yields pyrrole-2,3,5-tricarboxylic acid, which may serve as a quantitatively significant indicator of eumelanin, whereas hydriodic acid hydrolysis of pheomelanin yields aminohydroxyphenylalanine as a specific indicator of pheomelanin. The method has been successfully applied to the analysis of eu- and pheomelanin not only in synthetic melanins, melanosomes, hair, feathers, and melanomas, but also in human epidermis and cultured melanocytes. These studies indicate that there exists an inverse relationship between the contents of eu- and pheomelanin. We propose that the switching between the two types of melanogenesis is mainly controlled by the level of tyrosinase activity: higher activity leads to eumelanogenesis and lower activity leads to pheomelanogenesis. When tyrosinase activity is low, dopaquinone, a reactive intermediate in melanogenesis, is quantitatively converted to glutathionyldopa, which gives rise exclusively to pheomelanin. When tyrosinase activity is high, an excess of dopaquinone is produced, which results in the inactivation of glutathione reductase and gamma-glutamyl transpeptidase, enzymes essential for pheomelanogenesis. These biochemical events eventually leads to eumelanogenesis.