An easy-to-run method for routine analysis of eumelanin and pheomelanin in pigmented tissues

A procedure for analysis of melanin-pigmented tissues based on alkaline hydrogen peroxide degradation coupled with high-performance liquid chromatography (HPLC) ultraviolet determination of pyrrole-2,3,5-tricarboxylic acid (PTCA) for eumelanin and 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole (BTCA) and 1,3-thiazole-2,4,5-tricarboxylic acid for pheomelanin was recently developed. Despite advantages related to the degradation conditions and sample handling, a decrease of the reproducibility and resolution was observed after several chromatographic runs. We report herein an improved chromatographic methodology for simultaneous determination of PTCA and BTCA as representative markers of eumelanin and pheomelanin, respectively, based on the use of an octadecylsilane column with polar end-capping with 1% formic acid (pH 2.8)/methanol as the eluant. The method requires conventional HPLC equipments and gives very good peak shapes and resolution, without need of ion pair reagents or high salt concentrations in the mobile phase. The intra-assay precision of the analytical runs was satisfactory with CV values < or = 4.0% (n = 5) for the two markers which did not exceed 8% after 50 consecutive injections on the column over 1 week. The peak area ratios at 254 and 280 nm (A(280)/A(254): PTCA = 1.1, BTCA = 0.6) proved a valuable parameter for reliable identification of the structural markers even in the most complex degradation mixtures. The method can be applied to various eumelanin and pheomelanin pigmented tissues, including mammalian hair, skin and irides, and is amenable to be employed in population screening studies.     

Isomeric cysteinyldopas provide a (photo)degradable bulk component and a robust structural element in red human hair pheomelanin

Alkaline H₂O₂ degradation of red hair pheomelanin gave, besides 6‐(2‐amino‐2‐carboxyethyl)‐2‐carboxy‐4‐hydroxybenzothiazole (BTCA), a new product which was identified as 7‐(2‐amino‐2‐carboxyethyl)‐2‐carboxy‐4‐hydroxybenzothiazole (BTCA‐2) originating from 2‐S‐cysteinyldopa (2SCD) derived units. BTCA‐2 was also obtained from a variety of pheomelanic tissues and synthetic pigments. Simultaneous determination of BTCA and BTCA‐2 in segments of red hair locks taken at variable distances from the scalp in a group of 19 individuals indicated an abrupt drop of BTCA yields on passing from root to tip, whereas BTCA‐2 values remained virtually constant throughout hair length. Analysis of 4‐amino‐3‐hydroxyphenylalanine (AHP) and 3‐aminotyrosine (AT) in the same lock segments showed a closely similar trend, whereas yields of thiazole‐2,4,5‐tricarboxylic acid (TTCA) increased with increasing the distance from the scalp. Prolonged exposure of hair locks to sunlight caused a significant decrease in BTCA‐, but not BTCA‐2‐yielding elements. Finally, model studies showed a substantial degradation of 5SCD‐, but not 2SCD‐derived units, during pheomelanin synthesis in vitro. It is concluded that red hair pheomelanin consists of a degradable 5SCD‐derived bulk component associated with stable 2SCD‐derived units. Structural degradation occurs during hair growth probably as a result of oxidative processes related in part to sun exposure.     

A novel LC‐MS/MS method to quantify eumelanin and pheomelanin and their relation to UVR sensitivity – A study on human skin biopsies

Melanin in the skin can be divided into eumelanin and pheomelanin subtypes. Simultaneous quantification of these subtypes could clarify their relation to skin type and skin cancer development. We describe a novel, sensitive liquid chromatography–tandem mass spectrometry method to quantify two eumelanin markers, pyrrole‐2,3,5‐tricarboxylic acid (PTCA) and pyrrole‐2,3‐dicarboxylic acid (PDCA), and two pheomelanin markers, thiazole‐4,5‐dicarboxylic acid (TDCA) and thiazole‐2,4,5 tricarboxylic acid (TTCA), performed in a single run using the same biopsy. Volunteers with either Fitzpatrick skin type (FST) I/II or III/IV (n = 30) each provided a 4‐mm punch biopsy from the buttock. Upon analysis, the FST I + II group had significantly less of all four melanin biomarkers (PTCA, 0.75 ng/mm²; PDCA, 0.08 ng/mm²; TTCA, 0.24 ng/mm²; and TDCA, 0.10 ng/mm²) versus the FST III + IV group (PTCA, 4.89 ng/mm²; PDCA, 0.22 ng/mm²; TTCA, 2.61 ng/mm²; and TDCA, 0.72 ng/mm²), p ≤ 0.003. We find that this new LC‐MS/MS method is sensitive enough to quantify eumelanin and pheomelanin markers even in the lightest skin types.     

Degree of polymerization of 5,6‐dihydroxyindole‐derived eumelanin from chemical degradation study

Eumelanin is a brown‐black pigment comprising 5,6‐dihydroxyindole (DHI) and its 2‐carboxy derivative (DHICA), but the detailed structure of eumelanin is unclear. Chemical degradation is a powerful tool for analyzing melanin. H₂O₂ oxidation degradation of eumelanin affords pyrrole‐2,3,5‐tricarboxylic acid (PTCA) and pyrrole‐2,3‐dicarboxylic acid (PDCA). The ratio of PDCA to PTCA provides information about the eumelanin structure. In this article, we propose simple equations on the basis of previous experimental results on dimer yields for evaluating the yields of PTCA and PDCA from any DHI oligomers. Assuming the chemical disorder model of DHI‐melanin, we solve an equation where a theoretical expression for the ratio of PDCA to PTCA is set to the corresponding experimental value to obtain a plausible Poisson distribution of DHI oligomers. The results demonstrate that the main contributors to DHI‐melanin are tetramers and pentamers as shown by the mass spectrometry.     

Acid hydrolysis reveals a low but constant level of pheomelanin in human black to brown hair

We previously reported a constant ratio of the benzothiazole pheomelanin marker thiazole‐2,4,5‐tricarboxylic acid (TTCA) to the eumelanin marker pyrrole‐2,3,5‐tricarboxylic acid (PTCA) in eumelanic, black human hair. A constant level (20%–25%) of benzothiazole‐type pheomelanin was recently demonstrated in human skin with varying concentrations of melanin. Therefore, in this study, we aimed to investigate the origin of pheomelanin markers in black to brown human hair by developing a method to remove protein components from hair by heating with 6 M HCl at 110°C for 16 hr. For comparison, synthetic melanins were prepared by oxidizing mixtures of varying ratios of dopa and cysteine with tyrosinase. Hair melanins and synthetic melanins were subjected to acid hydrolysis followed by alkaline H₂O₂ oxidation. The results show that the hydrolysis leads to decarboxylation of the 5,6‐di‐hydroxyindole‐2‐carboxylic acid moiety in eumelanin and the benzothiazole moiety in pheomelanin and that eumelanic human hair contains 11%–17% benzothiazole‐type pheomelanin.     

Raman spectroscopy as a non‐invasive technique for the quantification of melanins in feathers and hairs

The quantification of melanins is a complex task due to the chemical heterogeneity of the pigments and the difficulty of their isolation. The best accepted procedure currently consists in the chemical cleavage of melanins and the subsequent detection of degradation products by HPLC, which implies the destruction of samples. Here, we show that Raman spectroscopy is a non‐invasive technique that can be used to quantify melanins. We made parallel analyses of the characteristics of pheomelanin and eumelanin Raman spectra as measured by confocal Raman microscopy and of degradation products of pheomelanin (4‐amino‐3‐hydroxyphenylalanine, 4‐AHP) and eumelanin (pyrrole‐2,3,5‐tricarboxylic acid, PTCA) as measured by HPLC in feathers of red‐legged partridges and hairs of wild boars and humans. We found strong correlations between the spectral Raman characteristics and 4‐AHP and PTCA levels, which indicates that the Raman spectra of melanins can be used to determine their content.     

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

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.     

The Usefulness of 4‐Amino‐3‐hydroxyphenylalanine as a Specific Marker of Pheomelanin

Reductive hydrolysis of pheomelanin with hydriodic acid (HI) gives two aminohydroxyphenylalanine isomers, 4‐amino‐3‐hydroxyphenylalanine (`specific AHP') and 3‐amino‐4‐hydroxyphenylalanine (3‐aminotyrosine, AT), which derive from the oxidative polymerization of 5‐S‐cysteinyldopa, and 2‐S‐cysteinyldopa, respectively. Since we first introduced this analytical method, the combined amount of AHP and AT (`total AHP') has been extensively used as a marker of pheomelanin. However, one problem with using total AHP as a marker is that background levels originate from precursors other than pheomelanin. Considerable and variable amounts of background AT are produced from other sources, most likely nitrotyrosine residues in proteins. In order to overcome this problem, we developed HPLC conditions which enable the direct injection of the HI reduction products into the HPLC system allowing good separation of AHP and AT. In this way we could study the importance of both degradation products separately and their specificity as markers for pheomelanin. The usefulness of the present method is validated using human hair samples of various colours which were divided into dark, fair or red colours. The combined amount of specific AHP and AT shows an excellent correlation with total AHP, and the amount of specific AHP also correlates with the amount of total AHP. We also examined total AHP and specific AHP values against pyrrole‐2,3,5‐tricarboxylic acid (PTCA) values in the human hair samples. These results show that specific AHP measurement gives a more prominent segregation for the ratio of specific AHP to PTCA among hairs of various colours than the ratio of total AHP to PTCA. Thus, we conclude that `specific AHP' is a more specific marker of pheomelanin than is `total AHP'.     

Changes in the Proliferation and Differentiation of Neonatal Mouse Pink‐Eyed Dilution Melanocytes in the Presence of Excess Tyrosine

Changes in the proliferation and differentiation of epidermal melanocytes derived from newborn mice wild‐type at the pink‐eyed dilution (p) locus (P/P) and from congenic mice mutant at that locus (p/p) were investigated in serum‐free primary culture, with or without the addition of L‐Tyr. Incubation with added L‐Tyr inhibited the proliferation of P/P melanocytes in a concentration‐dependent manner and inhibition was gradually augmented as the donor mice aged. In contrast, L‐Tyr stimulated the proliferation of p/p melanoblasts–melanocytes derived from 0.5‐day‐old mice, but inhibited their proliferation when derived from 3.5‐ or 7.5‐day‐old mice. L‐Tyr stimulated the differentiation of P/P melanocytes. However, almost all cells were undifferentiated melanoblasts in control cultures derived from 0.5‐, 3.5‐ and 7.5‐day‐old p/p mice, but L‐Tyr induced their differentiation as the age of the donor mice advanced. The content of the eumelanin marker, pyrrole‐2,3,5‐tricarboxylic acid as well as the pheomelanin marker, 4‐amino‐3‐hydroxyphenylalanine in p/p melanocytes was greatly reduced compared with P/P melanocytes. However, the contents of eumelanin and its precursor, 5,6‐dihydroxyindole‐2‐carboxylic acid, as well as the contents of pheomelanin and its precursor, 5‐S‐cysteinyldopa in culture media from p/p melanocytes were similar to those of P/P melanocytes at all ages tested. L‐Tyr increased the content of eumelanin and pheomelanin two‐ to threefold in cultured cells and media derived from 0.5‐, 3.5‐ and 7.5‐day‐old mice. These results suggest that the proliferation of p/p melanoblasts–melanocytes is stimulated by L‐Tyr, and that the differentiation of melanocytes is induced by L‐Tyr as the age of the donor mice advanced, although eumelanin and pheomelanin fail to accumulate in p/p melanocytes and are released from them at all ages of skin development.     

Quantitative Analysis of Eumelanin and Pheomelanin in Humans,Mice, and Other Animals: a Comparative Review

The color of hair, skin, and eyes in animals mainly depends on the quantity, quality, and distribution of the pigment melanin, which occurs in two types: black to brown eumelanin and yellow to reddish pheomelanin. Microanalytical methods to quantify the amounts of eumelanin and pheomelanin in biological materials were developed in 1985. The methods are based on the chemical degradation of eumelanin to pyrrole‐2,3,5‐tricarboxylic acid and of pheomelanin to aminohydroxyphenylalanine isomers, which can be analyzed and quantitated by high performance liquid chromatography. This review summarizes and compares eumelanin and pheomelanin contents in various pigmented tissues obtained from humans, mice, and other animals. These methods have become valuable tools to study the functions of melanin, the control of melanogenesis, and the actions and interactions of pigmentation genes. The methods have also found applications in many clinical studies. High levels of pheomelanin are found only in yellow to red hairs of mammals and in red feathers of birds. It remains an intriguing question why lower vertebrates such as fishes do not synthesize pheomelanin. Detectable levels of pheomelanin are detected in human skin regardless of race, color, and skin type. However, eumelanin is always the major constituent of epidermal melanin, and the skin color appears to be determined by the quantity of melanin produced but not by the quality.     

The potent pro‐oxidant activity of rhododendrol–eumelanin induces cysteine depletion in B16 melanoma cells

RS‐4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD), a skin‐whitening agent, is known to induce leukoderma in some people. To explore the mechanism underlying this effect, we previously showed that the oxidation of RD with mushroom or human tyrosinase produces cytotoxic quinone oxidation products. We then examined the metabolism of RD in B16F1 melanoma cells in vitro and detected RD‐pheomelanin and RD‐quinone bound to non‐protein and protein thiols. In this study, we examined the changes in glutathione (GSH) and cysteine in B16 cells exposed to RD for up to 24 h. We find that the levels of cysteine, but not those of GSH, decrease during 0.5‐ to 3‐h exposure, due to oxidation to cystine. This pro‐oxidant activity was then examined using synthetic melanins. Indeed, we find that RD‐eumelanin exerts a pro‐oxidant activity as potent as Dopa‐pheomelanin. GSH, cysteine, ascorbic acid, and NADH were oxidized by RD‐eumelanin with a concomitant production of H₂O₂. We propose that RD‐eumelanin induces cytotoxicity through its potent pro‐oxidant activity.     

Photoaging of human retinal pigment epithelium is accompanied by oxidative modifications of its eumelanin

Although photodegradation of the retinal pigment epithelium (RPE) melanin may contribute to the etiology of age‐related macular degeneration, the molecular mechanisms of this phenomenon and the structural changes of the modified melanin remain unknown. Recently, we found that the ratio of pyrrole‐2,3,4,5‐tetracarboxylic acid (PTeCA) to pyrrole‐2,3,5‐tricarboxylic acid (PTCA) is a marker for the heat‐induced cross‐linking of eumelanin. In this study, we examined UVA‐induced changes in synthetic eumelanins to confirm the usefulness of the PTeCA/PTCA ratio as an indicator of photo‐oxidation and compared changes in various melanin markers and their ratios in human melanocytes exposed to UVA, in isolated bovine RPE melanosomes exposed to strong blue light and in human RPE cells from donors of various ages. The results indicate that the PTeCA/PTCA ratio is a sensitive marker for the oxidation of eumelanin exposed to UVA or blue light and that eumelanin and pheomelanin in human RPE cells undergo extensive structural modifications due to the life‐long exposure to blue light.     

Visible light accelerates the ultraviolet A‐induced degradation of eumelanin and pheomelanin

Exposure to excess ultraviolet (UV) A radiation induces the degradation/modification of both eumelanin and pheomelanin that may be deleterious to pigmented tissues. Although the spectral distribution of solar energy comprises nearly half of visible light (VL), few studies have been conducted to examine the role of VL in the photodegradation of both types of melanin, either VL alone or in combination with UVA. In this study, we examined the effects of physiological doses of VL (150 to 300 J cm^?2) alone or in combination with a physiological dose of UVA (20 J cm^?2) in normal human epidermal melanocytes. The degradation/modification of melanin structures was evaluated by our chemical degradation—high performance liquid chromatography methods. The results show that VL accelerates UVA‐induced changes in the structural features of both eumelanin and pheomelanin, although VL or UVA alone induced only minor changes in melanin structure. The differential spectral method provides support for the additive effects of VL.     

Insect cuticular melanins are distinctly different from those of mammalian epidermal melanins

Melanin from several insect samples was isolated and subjected to chemical degradation and HPLC analysis for melanin markers. Quantification of different melanin markers reveals that insect melanins are significantly different from that of the mammalian epidermal melanins. The eumelanin produced in mammals is derived from the oxidative polymerization of both 5,6‐dihydroxyindole and 5,6‐dihydroxyindole‐2‐carboxylic acids. The pheomelanin is formed by the oxidative polymerization of cysteinyldopa. Thus, dopa is the major precursor for both eumelanin and pheomelanin in mammals. But insect eumelanin appears to be mostly made from 5,6‐dihydroxyindole and originates from dopamine. More importantly, our study points out the wide spread occurrence of pheomelanin in many insect species. In addition, cysteinyldopamine and not cysteinyldopa is the major precursor for insect pheomelanin. Thus, both eumelanin and pheomelanin in insects differ from higher animals using dopamine and not dopa as the major precursor.     

The mouse ruby‐eye 2d (ru2d/Hps5ru2‐d) allele inhibits eumelanin but not pheomelanin synthesis

The novel mutation named ru2^d/Hps5^ru2‐d, characterized by light‐colored coats and ruby‐eyes, prohibits differentiation of melanocytes by inhibiting tyrosinase (Tyr) activity, expression of Tyr, Tyr‐related protein 1 (Tyrp1), Tyrp2, and Kit. However, it is not known whether the ru2^d allele affects pheomelanin synthesis in recessive yellow (e/Mc1r^e) or in pheomelanic stage in agouti (A) mice. In this study, effects of the ru2^d allele on pheomelanin synthesis were investigated by chemical analysis of melanin present in dorsal hairs of 5‐week‐old mice from F₂ generation between C57BL/10JHir (B10)‐co‐isogenic ruby‐eye 2^d and B10‐congenic recessive yellow or agouti. Eumelanin content was decreased in ruby‐eye 2^d and ruby‐eye 2^d agouti mice, whereas pheomelanin content in ruby‐eye 2^d recessive yellow and ruby‐eye 2^d agouti mice did not differ from the corresponding Ru2^d/‐ mice, suggesting that the ru2^d allele inhibits eumelanin but not pheomelanin synthesis.     

A Chemist's View of Melanogenesis

The significance of our understanding of the chemistry of melanin and melanogenesis is reviewed. Melanogenesis begins with the production of dopaquinone, a highly reactive o‐quinone. Pulse radiolysis is a powerful tool to study the fates of such highly reactive melanin precursors. Based on pulse radiolysis data reported by Land et al. (J Photochem Photobiol B: Biol 2001;64:123) and our biochemical studies, a pathway for mixed melanogenesis is proposed. Melanogenesis proceeds in three distinctive steps. The initial step is the production of cysteinyldopas by the rapid addition of cysteine to dopaquinone, which continues as long as cysteine is present (1 μM). The second step is the oxidation of cysteinyldopas to give pheomelanin, which continues as long as cysteinyldopas are present (10 μM). The last step is the production of eumelanin, which begins only after most cysteinyldopas are depleted. It thus appears that eumelanin is deposited on the preformed pheomelanin and that the ratio of eu‐ to pheomelanin is determined by the tyrosinase activity and cysteine concentration. In eumelanogenesis, dopachrome is a rather stable molecule and spontaneously decomposes to give mostly 5,6‐dihydroxyindole. Dopachrome tautomerase (Dct) catalyses the tautomerization of dopachrome to give mostly 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA). Our study confirmed that the role of Dct is to increase the ratio of DHICA in eumelanin and to increase the production of eumelanin. In addition, the cytotoxicity of o‐quinone melanin precursors was found to correlate with binding to proteins through the cysteine residues. Finally, it is still unknown how the availability of cysteine is controlled within the melanosome.     

Melanins in IGR 1 Melanoma Cells

Information on the composition of melanins is obtained by analysis both of 4-amino-3-hydroxyphenylalanine (AHP) after hydriodic acid degradation and of pyrrole-2,3,5-tricarboxylic acid (PTCA) after potassium permanganate oxidation. Analysis of thiazole-4,5-dicarboxylic acid (TDCA) and pyrrole-2,3-dicarboxylic acid (PDCA) after permanganate oxidation, provides additional information on the composition, TDCA on pheomelanin residues, and PDCA on indolic residues without carboxy groups. Using model melanins formed from dopa and cysteinyldopa in different proportions, we found the TDCA/(PTCA+PDCA) ratio to yield a reliable estimate of the relative proportions of pheomelanin and eumelanin. The PDCA/PTCA ratio reflects the relationship between indole residues with and without carboxy groups. We have analyzed degradation products from cultures of IGR 1, an extensively studied melanoma cell line. Cell cultures were harvested after 2, 4, and 7 days. Culture media were changed after 2 days in all series, and also after 4 days in one series harvested at 7 days. Cells without medium change had seven times the amount of melanin found in cultures with medium change. The PDCA/PTCA ratio decreased with increasing amounts of melanin. With increased melanization, eumelanin is increased relatively more than pheomelanin. The cell content of 5-S-cysteinyldopa (5-S-CD) was similar in all cultures, while 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MICA), a eumelanin precursor metabolite, was found in increased amounts of media of heavily pigmented cultures.     

Roles of reactive oxygen species in UVA‐induced oxidation of 5,6‐dihydroxyindole‐2‐carboxylic acid‐melanin as studied by differential spectrophotometric method

Eumelanin photoprotects pigmented tissues from ultraviolet (UV) damage. However, UVA‐induced tanning seems to result from the photooxidation of preexisting melanin and does not contribute to photoprotection. We investigated the mechanism of UVA‐induced degradation of 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA)‐melanin taking advantage of its solubility in a neutral buffer and using a differential spectrophotometric method to detect subtle changes in its structure. Our methodology is suitable for examining the effects of various agents that interact with reactive oxygen species (ROS) to determine how ROS is involved in the UVA‐induced oxidative modifications. The results show that UVA radiation induces the oxidation of DHICA to indole‐5,6‐quinone‐2‐carboxylic acid in eumelanin, which is then cleaved to form a photodegraded, pyrrolic moiety and finally to form free pyrrole‐2,3,5‐tricarboxylic acid. The possible involvement of superoxide radical and singlet oxygen in the oxidation was suggested. The generation and quenching of singlet oxygen by DHICA‐melanin was confirmed by direct measurements of singlet oxygen phosphorescence.     

Chemical analysis of constitutive pigmentation of human epidermis reveals constant eumelanin to pheomelanin ratio

The skin constitutive pigmentation is given by the amount of melanin pigment, its relative composition (eu/pheomelanin) and distribution within the epidermis, and is largely responsible for the sensitivity to UV exposure. Nevertheless, a precise knowledge of melanins in human skin is lacking. We characterized the melanin content of human breast skin samples with variable pigmentations rigorously classified through the Individual Typology Angle (ITA) by image analysis, spectrophotometry after solubilization with Soluene‐350 and high‐performance liquid chromatography (HPLC) after chemical degradation. ITA and total melanin content were found correlated, ITA and PTCA (degradation product of DHICA melanin), and TTCA (degradation product of benzothiazole‐type pheomelanin) as well but not 4‐AHP (degradation product of benzothiazine‐type pheomelanin). Results revealed that human epidermis comprises approximately 74% of eumelanin and 26% pheomelanin, regardless of the degree of pigmentation. They also confirm the low content of photoprotective eumelanin among lighter skins thereby explaining the higher sensitivity toward UV exposure.     

Microanalysis of eumelanin and pheomelanin in hair and melanomas by chemical degradation and liquid chromatography

A method for the quantitative analysis of eumelanin and pheomelanin in tissues, e.g., hair and melanoma, is described. The method is simple and rapid because it does not require the isolation of melanins from the tissues. The rationale is that permanganate oxidation of eumelanin yields pyrrole-2,3,5-tricarboxylic acid (PTCA) which may serve as a quantitatively significant indicator of eumelanin, while hydriodic acid hydrolysis of pheomelanin yields aminohydroxyphenylalanine (AHP) as a specific indicator of pheomelanin. The degradation products, PTCA and AHP, can be readily analyzed by high-performance liquid chromatography. Chemical degradations of synthetic melanins, prepared from dopa, 5-S-cysteinyldopa, and their mixtures in various ratios, gave PTCA and AHP in yields that correlated with the dopa/5-S-cysteinyldopa ratio. The PTCA/AHP ratio as well as the contents of PTCA and AHP reflected well the type of melanogenesis in hair and melanomas. The amounts needed for each degradation were 0.5 mg of melanin, 2 mg of hair, and 5 mg of tissue samples. As many as 20 samples can be analyzed within 3 working days.