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Review
. 2023 May 5;24(9):8305.
doi: 10.3390/ijms24098305.

Recent Advances in Characterization of Melanin Pigments in Biological Samples

Kazumasa Wakamatsu  1 , Shosuke Ito  1
Affiliations
Review

Recent Advances in Characterization of Melanin Pigments in Biological Samples

Kazumasa Wakamatsu et al. Int J Mol Sci. .

Abstract

The melanin pigments eumelanin (EM) and pheomelanin (PM), which are dark brown to black and yellow to reddish-brown, respectively, are widely found among vertebrates. They are produced in melanocytes in the epidermis, hair follicles, the choroid, the iris, the inner ear, and other tissues. The diversity of colors in animals is mainly caused by the quantity and quality of their melanin, such as by the ratios of EM versus PM. We have developed micro-analytical methods to simultaneously measure EM and PM and used these to study the biochemical and genetic fundamentals of pigmentation. The photoreactivity of melanin has become a major focus of research because of the postulated relevance of EM and PM for the risk of UVA-induced melanoma. Our biochemical methods have found application in many clinical studies on genetic conditions associated with alterations in pigmentation. Recently, besides chemical degradative methods, other methods have been developed for the characterization of melanin, and these are also discussed here.

Keywords: LC–MS; Raman spectroscopy; alkaline hydrogen peroxide oxidation (AHPO); electron paramagnetic resonance spectroscopy; eumelanin; hydroiodic acid hydrolysis; pheomelanin; pump–probe microscopy; tape stripping.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 9
Figure 9
Melanin characterization using AHPO, HI hydrolysis, and soluene-350 solubilization for chemical analysis; TEM and SEM for surface morphology; MALDI-MS, HPLC, Pump–probe, Raman, EPR, NMR, FTIR, Elemental analysis, Pyrolysis, and UV/visible spectroscopy for structural elucidation. Modified with permission from ref. [230]. Copyright 2011, Elsevier.
Figure 1
Figure 1
Biosynthetic pathways leading to the production of EM and PM. Note that the activities of tyrosinase, Tyrp1 and Dct/Tyrp2, and the precursor tyrosine are involved in the production of EM, while only tyrosinase (and the precursors tyrosine and cysteine) is necessary for the production of PM. While mouse Tyrp1 acts as a DHICA oxidase as shown in the Figure, the function of human TYRP1 has not been established. In the reaction of DQ and cysteine, 5SCD and 2SCD are produced at a ratio of 5:1, respectively [33]. Benzothiazine and Benzothiazole intermediates are made from 5SCD–quinone and 2SCD–quinone, respectively. MFSD12 (major facilitator superfamily domain-containing protein 12; depicted by double lines) is a component of the melanosomal cysteine import system. Modified from ref. [4].
Figure 2
Figure 2
Alkaline hydrogen peroxide oxidation (AHPO) and hydroiodic acid (HI) hydrolysis of eumelanin (EM) and pheomelanin (PM) yielding various melanin markers. 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA), pyrrole-2,3-dicarboxylic acid (PDCA), pyrrole-2,3,5-tricarboxylic acid (PTCA), pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA), thizole-4,5-dicarboxylic acid (TDCA), thiazole-2,4,5-tricarboxylic acid (TTCA), 4-amino-3-hydroxyphenylalanine (4-AHP), 3-amino-4-hydroxyphenylalanine (3-AHP). Adapted from ref. [48].
Figure 3
Figure 3
Correlation of the PTCA amounts generated using AHPO and acidic KMnO4 oxidation from human hair samples. The correlation was significant (p < 0.0001). n = 38. Adapted with permission from ref. [43]. Copyright 2011, John Wiley and Sons.
Figure 4
Figure 4
HPLC chromatograms of standard melanin markers (A), and AHPO mixtures from human black hair (B), from human red hair (C), and from dark human epidermis (D). Adapted from ref. [48].
Figure 5
Figure 5
Correlation between TM using a spectrophotometric method and EM + PM using an HPLC method. (A) Six different colors of human hair samples (a total of 228 samples). TM was measured at 500 nm. Bars represent SEM. Adapted with permission from ref. [43]. Copyright 2011, John Wiley and Sons. (B) A total of 35 human epidermis samples with diverse pigmentation. Adapted with permission from ref. [52]. Copyright 2015, John Wiley and Sons.
Figure 6
Figure 6
Correlation between EPR and HPLC estimates of TM contents (EM + PM) in hair samples. n = 17. Adapted with permission from ref. [105]. Copyright 2006, John Wiley and Sons.
Figure 7
Figure 7
Intensity of the low-field component (P) of the EPR signal of 5SCD-M vs. content of 4-AHP in selected samples subjected to photolysis. Adapted with permission from ref. [94]. Copyright 2018, John Wiley and Sons.
Figure 8
Figure 8
Relationship between EM and PM levels measured using HPLC and the scores of PLSR factors collecting information on characteristics of the Raman spectra. (A,B) are the feathers of red-legged partridges. Adapted with permission from ref. [160]. Copyright 2013, John Wiley and Sons.
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