High-Performance Liquid Chromatography (HPLC) Analysis of Eu- and Pheomelanin in Melanogenesis Control.

Abstract

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.

A validated LC/MS/MS method for the quantification of pyrrole-2,3,5-tricarboxylic acid (PTCA), a eumelanin specific biomarker, in human skin punch biopsies

“…In human skin, eumelanin and pheomelanin are produced in the basal layer of the epidermis by neural crest derived melanocytes, packed in cytoplasmic melanosomes, and transferred to neighboring epithelial keratinocytes [2,3,[6][7][8][9]. Melanogenesis of these two pigments is initiated with the common rate limiting step of l-tyrosine hydroxylation to dopaquinone catalyzed by the copper containing enzyme tyrosinase [2,4,[9][10][11][12]. Following this step, however, their synthetic pathways diverge (Fig.…”

“…These dihydroxyindoles are then converted to their respective quinone derivatives and undergo oxidative polymerization to eumelanin via tyrosinase related protein 1 (Tyrp1). However, with the intervention of thiols such as glutathione and cysteine, and the activation of glutathione reductase and gamma-glutamyl transpeptidase, further oxidation leads to pheomelanin production via cysteinyldopa and benzothiazine intermediates [4][5][6]12].…”

“…Techniques to quantify the degradation products of melanin in biological samples after chemical oxidation, like those developed by Ito et al, have become popular with researchers because they can be used as a measurement of melanin content while making its isolation unnecessary [2][3][4]12]. These methods are based on the formation of monomers such as pyrrole-2,3,5-tricarboxylic acid (PTCA), a quantitatively significant indicator of eumelanin, followed by HPLC or spectrophotometric analysis.…”

Increased dietary intake of tyrosine upregulates melanin deposition in the hair of adult black-coated dogs

“…Cysteine is another amino acid which plays an important role in melanin biosynthesis, specifically by facilitating the generation of cysteinyl-DOPA in the pathway to pheomelanin production. There is evidence that greater availability of Cys can favour red/yellow pigmentation over black ( Ito, 1993 ). Despite the moderately increased Cys found in diet A, this did not appear to translate to plasma concentration and so is unlikely to have had a bearing here.…”

“…Follicular melanocytes produce black eumelanin and brown pheomelanin at differential concentrations, a process principally controlled by genetic factors, resulting in the myriad of colours and patterns seen in the natural world ( Ozeki et al., 1995 ). Ito (1993) showed that the relative proportion of eumelanin and pheomelanin was controlled by activity of the enzyme tyrosinase. Lower levels of activity favour production of pheomelanin, while higher levels favour eumelanin.…”