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'.     

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.     

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.     

The neuromelanin of the human substantia nigra

The pigment of the human substantia nigra was isolated after extraction of lipids and proteins with 2% sodium cholate in 30% ethanol followed by 2% sodium dodecyl sulfate in 10% glycerol. The pigment was hydrolysed with HI or degraded by treatment with KMNO4 and the samples were examined for compounds known to derive from pheomelanin (4-amino-3-hydroxyphenylalanine, AHP and 4-amino-3-hydroxyphenylethylamine, AHPEA), or from eumelanin (pyrrole-2,3,5-tricarboxylic acid, PTCA). The HI hydrolysis yielded AHPEA in large quantities, indicating cysteinyldopamine as the main source of the pheomelanin moiety of the neuromelanin, but also trace amounts of AHP, derived from cysteinyldopa oxidation products. Dopamine and small quantities of dopa were also obtained by HI hydrolysis of the neuromelanin. The yield of PTCA was low, but the amounts observed show that part of the neuromelanin is of the eumelanin type, a fact compatible with an occasional exhaustion of the glutathione-cysteine reduction system at the site of neuromelanin formation.     

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.     

Relationship of melanin degradation products to actual melanin content: application to human hair

Methods not only for characterizing but also for quantitating melanin subtypes from the two types of melanin found in hair--eumelanin and pheomelanin--have been established. In relation to testing for drugs of abuse in hair, these methods will allow for correction of drug binding to specific melanin subtypes and will serve to improve drug measurement in hair. 5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) make up the majority of the eumelanin polymer while benzothiazene units derived from 2-cysteinyl-S-Dopa (2-CysDopa) and 5-cysteinyl-S-Dopa (5-CysDopa) compose the majority of the pheomelanin polymer. Our results show that: (1) pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA), markers for DHI and DHICA units, respectively, are produced in 0.37 and 4.8% yields, respectively, when melanins are subjected to alkaline hydrogen peroxide degradation, (2) 3-aminotyrosine (3AT) and 4-amino-3-hydroxyphenylalanine (AHP), markers for 2-CysDopa and 5-CysDopa, respectively, are produced in 16 and 23% yield, respectively, when subjected to hydriodic acid hydrolysis, and (3) that black human hair contains approximately 99% eumelanin and 1% pheomelanin, brown and blond hair contain 95% eumelanin and 5% pheomelanin; and red hair contains 67% eumelanin and 33% pheomelanin. These data will allow deeper investigation into the relationship between melanin composition and drug incorporation into hair.     

UVA-induced oxidative degradation of melanins: fission of indole moiety in eumelanin and conversion to benzothiazole moiety in pheomelanin

Eumelanin is photoprotective while pheomelanin is phototoxic to pigmented tissues. Ultraviolet A (UVA)-induced tanning seems to result from the photooxidation of pre-existing melanin and contributes no photoprotection. However, data available for melanin biodegradation remain limited. In this study, we first examined photodegradation of eumelanin and pheomelanin in human black hairs and found that the ratio of Free (formed by peroxidation in situ) to Total (after hydrogen peroxide oxidation) pyrrole-2,3,5-tricarboxylic acid (PTCA) increases with hair aging, indicating fission of the dihydroxyindole moiety. In red hair, the ratio of thiazole-2,4,5-tricarboxylic acid (TTCA) to 4-amino-3-hydroxyphenylalanine (4-AHP) increases with aging, indicating the conversion from benzothiazine to benzothiazole moiety. These photodegradation of melanins were confirmed by UVA (not UVB) irradiation of melanins from mice and human hairs and synthetic eumelanin and pheomelanin. These results show that both eumelanin and pheomelanin degrade by UVA and that Free/Total PTCA and TTCA/4-AHP ratios serve as sensitive indicators of photodegradation.     

HPLC analysis of pheomelanin degradation products in human urine

A sensitive and specific high performance liquid chromatography (HPLC) method was developed to quantify 4-amino-3-hydroxyphenylalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine (3-AHP) in urine. In degradation studies of melanin pigment, 4-AHP and 3-AHP are derived from benzothiazine units of pheomelanin and pheomelanin-related metabolites such as trichochromes. 5-S-Cysteinyldopa-derived benzothiazine products give 4-AHP while 2-S-cysteinyldopa-derived benzothiazine products give 3-AHP. 3-AHP is also derived from nitrotyrosine formed by nitration of tyrosine with reactive nitrogen species. For this reason, the influence of this biological process on the amount of 3-AHP found in biological material have been investigated. The method is based on hydriodic acid hydrolysis of the melanin polymer and reversed-phase HPLC with electrochemical detection of the degradation products 4-AHP and 3-AHP. The mobile phase consists of 25 mM ammonium acetate and sodium octanesulfonate as an ion-pairing reagent. The 4-AHP and 3-AHP peaks were well separated and the detector response was linear within the range 0-2 ng injected for both compounds. With the developed chromatographic system, 4-AHP and 3-AHP showed good separation in the biological samples. There was a strong correlation between 4-AHP and 3-AHP in the urine of 50 malignant melanoma patients and two healthy subjects (R0.977). The two compounds were also strongly correlated with 5-S-cysteinyldopa in urine, the correlation coefficients being 0.862 and 0.907, respectively. The method described is sensitive enough for analysis of pheomelanin in urine and in several other biological samples. The results indicate that 3-AHP in urine is not influenced by excreted 3-nitrotyrosine and the data indicate that pheomelanins are excreted in the urine of melanoma patients.     

Advanced chemical methods in melanin determination

Among the biopolymers, melanins are unique in many respects. The other essential biopolymers - proteins, nucleic acids, and carbohydrates - are chemically well characterized; their precursors (monomer units) and modes of connection between the monomer units are known, and sequences of their connection can be determined with well-established methodologies. In contrast, we still do not have a method to determine accurately the ratio of various units present in melanins. This is largely because of the chemical properties of melanins, such as their insolubility over a broad range of pH, the heterogeneity in their structural features, and also because of the lack of methods that can split melanin polymers into their monomer units (all other biopolymers can be hydrolysed to the corresponding monomer units). To overcome this difficulty, we developed a rapid and sensitive method for quantitatively analysing eumelanin and pheomelanin in biological samples by chemical degradation methods followed by HPLC determination. This HPLC microanalytical method for characterizing eumelanin and pheomelanin has become a useful tool for the study of melanogenesis. This review will summarize the usefulness and limitations of the various chemical and spectrophotometric methods used to analyse melanins at the biochemical, cellular, and tissue levels. Emphasis is given on the usefulness of 4-amino-3-hydroxyphenylalanine as a specific marker of pheomelanin.     

Spectrophotometric Characterization of Eumelanin and Pheomelanin in Hair

Mammalian melanins exist in two chemically distinct forms: the brown to black eumelanins and the yellow to reddish-brown pheomelanins. They can be quantified by HPLC analysis of pyrrole-2,3,5-tricarboxylic acid (PTCA) and aminohydroxyphenylalanine (AHP). We recently developed a spectrophotometric method for assaying the total amount of eu- and pheomelanins by dissolving melanins in Soluene-350 plus water. In this study, we examined whether absorbance at 500 nm (A₅₀₀) of the Soluene-350 solution reflects the total amount of melanins obtained by the HPLC methods, and whether the ratio of absorbances between 650 and 500 nm reflects the eumelanin/total melanin ratio in mouse hair, sheep wool, and human hair. Our findings were as follows: (1) Total melanin levels calculated from A₅₀₀ values correlate well with those obtained from PTCA and AHP values by multiplying with the following factors: for mice, PTCA × 45 + AHP × 2.5; for sheep, PTCA × 40 + AHP × 15; and for humans, PTCA × 160 + AHP × 10. (2) The A₆₅₀/A₅₀₀ ratios were higher (0.25–0.33) in black to brown hair while they were significantly lower (0.10–0.14) in yellow to red hair. These results indicate that (1) the A₅₀₀ value can be used to quantify the total combined amount of eu- and pheomelanins, and (2) the A₆₅₀/A₅₀₀ ratio can serve as a parameter to estimate the eumelanin/total melanin ratio. The present method provides a convenient way to qualitatively characterize eu- and pheomelanins in melanins produced in follicular melanocytes.     

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.     

Sensitive determination of pheomelanin after 5-carboxyfluorescein succinimidyl ester precapillary derivatization and micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

The suitability of micellar electrokinetic capillary chromatography (MEKC) with laser-induced fluorescence (LIF) detection for the determination of pheomelanin in biological materials has been investigated. 5-Carboxyfluorescein succinimidyl ester was chosen as the labeling reagent to precapillary derivatize the two marker aminohydroxyphenylalanine (AHP) isomers produced after reductive hydrolysis of pheomelanin with hydriodic acid (HI). Various parameters affecting derivatization and separation were systematically studied. Under optimal conditions, the analytes could be separated within 18 min, and the relative standard deviations (R.S.D.) of migration time and corrected peak areas were less than 5.5%. Compared with the conventional high-performance liquid chromatography (HPLC) method with electrochemical detection, the 100-fold improvements in sensitivity were achieved by applying LIF detection. As a preliminary application, this method has been successfully applied to the determination of pheomelanin in two human melanoma cell cultures, black hair, melanoma tissue and urine samples of human melanoma patients with the spiked recoveries in the range of 88-96%.     

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.     

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.     

Determination of eumelanin in human urine

Normal and malignant melanocytes produce melanins and melanin-related metabolites, most of which are retained in the cells but some are secreted into the blood and then excreted in the urine. In this study, we developed a method to measure levels of eumelanin in urine samples and evaluated its clinical significance in comparison with the melanin-related metabolites 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C) and 5-S-cysteinyldopa (5-S-CD), and with pheomelanin, measured after degradation as 4-amino-3-hydroxyphenylalanine (4-AHP). The method is based on the production of pyrrole-2,3,5-tricarboxylic acid (PTCA) on permanganate oxidation of eumelanin, followed by quantification by liquid chromatography. For 118 urine samples from 10 control subjects, mean urinary excretions of PTCA, 6H5MI2C, 5-S-CD and 4-AHP were 19, 67, 37 and 59 micromol/mol creatinine respectively. In melanoma patients (n = 45), the mean urinary excretions of PTCA, 6H5MI2C, 5-S-CD, and 4-AHP were 91, 926, 4070 and 3530 micromol/mol creatinine respectively. Median level of PTCA in melanoma patients was elevated 2.1-fold compared with control subjects. The degrees of elevation for 6H5MI2C, 5-S-CD, and 4-AHP were 1.8-, 22- and 6.2-fold respectively. Thus, although urinary PTCA is of little clinical value in following the progression of melanoma, urinary 4-AHP appears to be of considerable value in this respect.     

Stimulation of the proliferation and differentiation of mouse pink-eyed dilution epidermal melanocytes by excess tyrosine in serum-free primary culture

The epidermal cell suspensions of the neonatal dorsal skin derived from wild type mouse at the pink-eyed dilution (p) locus (black, C57BL/10JHir-P/P) and their congenic mutant mouse (pink-eyed dilution, C57BL/10JHir-p/p) were cultured with a serum-free melanocyte growth medium supplemented with additional L-tyrosine (Tyr) from initiation of the primary culture. L-Tyr inhibited the proliferation of P/Pmelanocytes in a dose-dependent manner, whereas L-Tyr stimulated the proliferation of p/p melanoblasts and melanocytes regardless of dose. On the other hand, L-Tyr stimulated (P/P) or induced (p/p) the differentiation of epidermal melanocytes in a dose-dependent manner. In both P/P and p/p melanoblasts and melanocytes cultured with 2.0 mM L-Tyr for 14 days, slight increases in contents of eumelanin marker, pyrrole-2,3,5-tricarboxylic acid (PTCA) and pheomelanin marker, aminohydroxyphenylalanine (AHP) were observed. The average number of total melanosomes (stages I, II, III, and IV) per P/P melanocyte was not changed by L-Tyr treatment, but the proportion of stage IV melanosomes in the total melanosomes was increased. On the contrary, in p/p melanoblasts and melanocytes L-Tyr increased dramatically the number of stage II, III, and IV melanosomes as well as the proportion of stage III melanosomes. Contents of PTCA and eumelanin precursor, 5,6-dihydroxyindole-2-carboxylic acid (DHICA) of cultured media in p/p melanocytes were much more greatly increased than in P/P melanocytes. However, contents of AHP and pheomelanin precursor, 5-S-cysteinyldopa (5-S-CD) of cultured media in p/p melanocytes were increased in a similar tendency to P/Pmelanocytes. These results suggest that p/p melanocytes in the primary culture are induced to synthesize eumelanin by excess L-Tyr, but difficult to accumulate them in melanosomes.     

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.     

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.     

Optimization of conditions for preparing synthetic pheomelanin

Synthetic melanins are often used as the standards for natural melanins. Conditions for preparing synthetic pheomelanin from L-dopa and L-cysteine were studied under different temperatures (25 and 37 degrees C), different amounts of cysteine (1.5 and 2.0 eq), and different amounts of tyrosinase (10 and 20 mg). Effects of addition of catalase and of replacement of oxygen with air were also examined. The best preparation of pheomelanin was obtained in a good yield, when 1 mmol of L-dopa was oxidized at 25 degrees C under oxygen current in 100 ml of a pH 6.8 buffer by 20 mg of mushroom tyrosinase inn the presence of 1.5 mmol of L-cysteine. The pheomelanin thus obtained had an elemental composition and an absorption spectra that were very similar to those from a pheomelanin prepared by tyrosinase oxidation of 5-S-cysteinyldopa. Hydrolysis of synthetic pheomelanins and pheomelanic tissues with hydriodic acid gave aminohydroxyphenylalanines (AHP) as a major product and hydroxybenzothiazolylalanines as a minor product. It is proposed that AHP can be used as a specific indicator of purity of pheomelanins.     

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 ≤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 (A₂₈₀/A₂₅₄: 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.