Usefulness of alkaline hydrogen peroxide oxidation to analyze eumelanin and pheomelanin in various tissue samples: application to chemical analysis of human hair melanins

Abstract

Eumelanin and pheomelanin in tissue samples can be specifically measured as the markers pyrrole-2,3,5-tricarboxylic acid (PTCA) and 4-amino-3-hydroxyphenylalanine after acidic permanganate oxidation and hydroiodic acid hydrolysis, respectively. Those degradation methods, although widely applied, are not easily performed in most laboratories. To overcome this difficulty, we developed alkaline H(2)O(2) oxidation in 1 M K(2)CO(3) that produces, in addition to the eumelanin marker PTCA, thiazole-2,4,5-tricarboxylic acid (TTCA) and thiazole-4,5-dicarboxylic acid (TDCA) as markers for pheomelanin and pyrrole-2,3-dicarboxylic acid (PDCA) as a marker for 5,6-dihydroxyindole-derived eumelanin. Those four degradation products can be easily separated by HPLC and analyzed with ultraviolet detection. The alkaline H(2)O(2) oxidation method is simple, reproducible and applicable to all pigmented tissues. Its application to characterize eumelanin and pheomelanin in human hair shows that PTCA and TTCA serve as specific markers for eumelanin and pheomelanin, respectively, although some caution is needed regarding the artificial production of TTCA from eumelanic tissue proteins.

Direct chemical evidence for eumelanin pigment from the Jurassic period

“…Under the oxidation conditions described by Ito and collaborators, melanin breaks down into distinct chemical markers associated with its monomeric precursors (16,17). A second fundamental method and the most used signature for melanin in physical chemistry, electron paramagnetic resonance spectroscopy (EPR), can verify the results of alkaline hydrogen peroxide oxidation.…”

“…Alkaline hydrogen peroxide degradation breaks this copolymer into unique markers ( Fig. 2A), pyrrole-2,3,5-tricarboxylic acid (PTCA), pyrrole-2,3-dicarboxylic acid (PDCA), pyrrole-2,3,4-tricarboxylic acid (iso-PTCA), and pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) (17,20,21,24). These products are common only to eumelanin and have never been produced by the alkaline hydrogen peroxide oxidation of any other biological material (17,21).…”

Mineralized rods and cones suggest colour vision in a 300 Myr-old fossil fish

“…To identify the presence of melanin in the fossil material, the alkaline hydrogen peroxide oxidation method [24][25][26][27] was used in the black to dark brown-coloured fossil eye fragments and the light brown-coloured rock matrix. Samples (8-17 mg) were ground to a fine powder using a mortar and pestle and added to water at a concentration of 10 mg per ml water and 100 ml aliquots were subjected to alkaline hydrogen peroxide oxidation 25 . PTCA and PDCA were analysed by the high-performance liquid chromatography (HPLC) conditions described previously 25 Morphological analysis of rods and cones.…”

“…Samples (8-17 mg) were ground to a fine powder using a mortar and pestle and added to water at a concentration of 10 mg per ml water and 100 ml aliquots were subjected to alkaline hydrogen peroxide oxidation 25 . PTCA and PDCA were analysed by the high-performance liquid chromatography (HPLC) conditions described previously 25 Morphological analysis of rods and cones. Generally speaking, the external contours of the rods and cones have been qualitatively described as rectangular and triangular, respectively.…”

“…These molecules are common only to eumelanin and have never been produced by the alkaline hydrogen peroxide oxidation of any other biological material [24][25][26] . The PTeCA/PTCA ratio of 2.7 is extremely high as compared with those of extant fish retina (0.10 in Table 1) and bovine retinal pigment epithelium melanosomes (0.13), indicating extensive thermal modification of eumelanine 26,27 .…”