Chemical Characterization of Melanins in Sheep Wool and Human Hair

The color of hair and wool in mammals and feathers in birds is mostly determined by the quantity and quality of melanins that are synthesized in follicular melanocytes and transferred to keratinocytes. There are two chemically distinct types of melanin pigments: the black to brown eumelanins and the yellow to reddish pheomelanins. Melanins in sheep wool and human hair of various colors were characterized by HPLC methods to estimate 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-derived units in eumelanins and benzothiazine units in pheomelanins. Melanins were also characterized by spectrophotometric methods after differential solubilization in alkalies. It was demonstrated that 1) black wool in Asiatic sheep contains eumelanin with the DHICA content similar to black mouse melanin, while black to brown melanins from human hair contain much lower ratios of DHICA-derived units, comparable to the slaty mutation in mice, 2) dark brown to brown hair in human contains eumelanin whose chemical properties are indistinguishable from those of black hair, 3) dark red wool and red human hair contain pheomelanic pigments whose chemical properties are rather different from those of yellow pheomelanins in mice, and 4) light brown, blonde, and red hairs in human can be differentiated from each other with this methodology.     

Comparative Analysis of Hair Melanins by Chemical and Electron Spin Resonance Methods

Eighteen hair samples from Karakul newborn lambs with various colors were estimated for eumelanin and pheomelanin contents (C_e and C_p, respectively) by electron spin resonance (ESR) spectrometry and by high-performance liquid chromatography (HPLC). Correlation coefficients between the values estimated by the ESR and HPLC methods were 0.96, 0.93, and 0.99 for C_e, C_p, and C_e/C_p, respectively. The high correlation coefficients show that both methods fit well for estimation of relative values of these parameters. The absolute values of C_e and C_e/C_p coincide rather well when C_e is high, but considerable discrepancies appear when C_e is low. The reasons for these discrepancies are discussed. The HPLC method appears to be more sensitive for detection of low concentrations of pheomelanin, while the ESR method fits well for mass selection purposes.     

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.     

Effects of Melanogenesis‐Inducing Nitric Oxide and Histamine on the Production of Eumelanin and Pheomelanin in Cultured Human Melanocytes

Melanin pigments produced in human melanocytes are classified into two categories; black coloured eumelanin and reddish‐yellow pheomelanin. Stimulation of melanocytes with α‐melanocyte‐stimulating hormone (α‐MSH), one of several melanogenic factors, has been reported to enhance eumelanogenesis to a greater degree than pheomelanogenesis, which contributes to hyperpigmentation in skin. Nitric oxide (NO) and histamine are also melanogenesis‐stimulating factors that are released from cells surrounding melanocytes following ultraviolet (UV) irradiation. In this study, the effects of NO and histamine on the ratio of eumelanin and pheomelanin were examined in human melanocytes, and then compared with that of α‐MSH. The amounts of eumelanin and pheomelanin were quantified using high‐performance liquid chromatography analysis after oxidation and hydrolysis of melanin. Melanogenesis was induced by the addition of α‐MSH, NO, or histamine to melanocytes. The amount of eumelanin production significantly increased with independent stimulation by these melanogenic factors, especially histamine, while that of pheomelanin significantly increased with α‐MSH and NO, but only slightly with histamine. As a result, the ratio of eumelanin and pheomelanin increased significantly with the addition of NO or histamine. These results suggest that NO and histamine, as in the case of α‐MSH, may contribute to UV‐induced hyperpigmentation by enhancing eumelanogenesis.     

Cyclic Oscillations in Melanin Composition within Hairs of Baboons

The wild‐type agouti‐banding pattern for hair is well characterized in lower mammals such as mice. The switch between eumelanin and pheomelanin in bands in the hair results from the interaction of α‐melanocyte stimulating hormone and agouti signal protein through the melanocortin 1 receptor on melanocytes. However, such banding patterns have not been described to date in higher mammals. We now report such ‘agouti’‐banding patterns that occur in several subspecies of baboons, and characterize those hairs using chemical and immunohistochemical methods. Hair and skin samples were obtained from the dorsa of adult male baboons of different subspecies (Papio cynocephalus hamadryas (PCH) and Papio cynocephalus anubis (PCA)). The hairs were excised with scissors into the gray and the white bands of the PCH subspecies and into the black and the yellow bands of the PCA subspecies, and were analyzed for total melanin, eumelanin, and pheomelanin by spectrophotometric and chemical methods. Hairs in the PCA subspecies oscillate between a eumelanic band (with high melanin content) and a pheomelanic band, while hairs in the PCH subspecies oscillate between a eumelanic band (with low melanin content) and a non‐pigmented band. Those chemical data are consistent with the histological appearance of the hair bulbs stained by the Fontana‐Masson technique. The difference in the melanin content between PCH and PCA subspecies is most likely related to tyrosinase levels, as suggested by the presence of unpigmented muzzle in the PCH subspecies compared with the black muzzle in the PCA subspecies.     

Characterization of Melanogenesis in Normal Human Epidermal Melanocytes by Chemical and Ultrastructural Analysis

Chemical and ultrastructural studies were conducted to define the relationship between type of melanogenesis and fine structures of melanosomes in normal human epidermal melanocytes. Chemical analysis of epidermal melanin demonstrated that the ratio of eumelanin/pheomelanin varied individually, ranging from 1.31 to exclusively eumelanic. Ultrastructural analysis of fine structures of melanosomes revealed that spheroid melanosomes were frequently observed in melanocytes of the epidermis whose eumelanin/pheomelanin ratio was less than 5. Conversely, ellipsoid melanosomes predominated in melanocytes of the epidermis whose ratio was more than 10. On the basis of these findings, it seems reasonable to conclude that 1) normal human epidermal melanocytes synthesize both eumelanin and pheomelanin and 2) pheomelanin synthesis may be characterized by the presence of spheroid melanosomes whereas eumelanin synthesis is ascribed to ellipsoid melanosomes.     

Tyrosinase-related proteins suppress tyrosinase-mediated cell death of melanocytes and melanoma cells

The synthesis of melanin intermediates through tyrosinase (TYR) involves the production of cytotoxic free radicals. By using recombinant adenoviruses that express TYR, tyrosinase-related protein 1 (TYRP1) or DOPAchrome tautomerase (DCT), we analyzed the biological function of these proteins with regard to melanin production and the growth of melanocytes, fibroblasts, melanoma cells and nonmelanoma cancer cells. High-level expression of TYR produced newly synthesized melanin and induced cell death in all of these cells. However, when TYRP1 or DCT was coexpressed with TYR in melanocytes and melanoma cells, TYR-mediated cell death was clearly decreased. This decrease was not observed in nonmelanocytic cells. Western blot analysis and measurement of enzyme activity revealed that the expression of TYRP1 or DCT had little effect on the amount or activity of cointroduced TYR in either the melanocytic or nonmelanocytic cells. In cells expressing both TYR and TYRP1 or TYR and DCT, the total amount of melanin and/or eumelanin increased substantially more than that in cells expressing TYR alone. On the other hand, the level of pheomelanin was similar in these three cell types. These findings suggest that TYRP1 and DCT play an important role in suppressing TYR-mediated cytotoxicity in melanocytic cells without decreasing TYR expression and/or activity. These biological activities of TYRP1 and DCT may work through the interaction with TYR in melanosomal compartment.     

Agouti Protein Inhibits the Production of Eumelanin and Phaeomelanin in the Presence and Absence of α-Melanocyte Stimulating Hormone

Melanocytes synthesise two types of melanin: the brown-black eumelanin and the red-yellow phaeomelanin. In mice, the relative proportions of these two melanins are regulated by α-MSH, which preferentially increases the synthesis of eumelanin and by the Agouti protein (AP), the expression of which correlates with the growth of yellow phaeomelanin-containing hair. It has been proposed that AP acts by antagonizing the action of α-MSH at the MCI receptor, although it has been suggested that it may also act independently of α-MSH. In the present study we show that AP inhibits melanogenesis in B16F1 melanoma cells in the presence and absence of α-MSH and also causes dose-related decreases in the synthesis of both eumelanin and phaeomelanin. In the presence of α-MSH AP had a greater effect on eumelanin production and this is consistent with an antagonistic action at the MCI receptor. In the absence of α-MSH however, AP produced similar reductions in the synthesis of both melanins. These changes were not seen in B16G4F cells which lack the MCI receptor, suggesting that even in the absence of α-MSH AP acts at the MCI receptor. How this action is mediated at the intracellular level is not yet clear, although it appears to be associated with a decrease in tyrosinase activity.     

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'.     

Chemical Degradation of Melanins: Application to Identification of Dopamine-melanin

Melanocytes produce two chemically distinct types of melanin pigments, eumelanins and pheomelanins. These pigments can be quantitatively analyzed by acidic KMnO₄ oxidation or reductive hydrolysis with hydriodic acid (HI) to form pyrrole-2,3,5-tricarboxylic acid (PTCA) or aminohydroxyphenylalanine (AHP), respectively. Dark brown melanin-like pigments are also widespread in nature, for example, in the substantia nigra of humans and primates (neuromelanin), in butterfly wings and in the fungus Cryptococcus neoformans. To characterize such diverse types of melanins, we have improved the alkaline H₂O₂ oxidation method of Napolitano et al. (Tetrahedron, 51: 5913–5920, 1995) and re-examined the HI hydrolysis method of Wakamatsu et al. (Neurosci. Lett., 131: 57–60, 1991). The results obtained with H₂O₂ oxidation show that 1) pyrrole-2,3-dicarboxylic acid (PDCA), a specific marker of 5,6-dihydroxyindole units in melanins, is produced in yields ten times higher than by acidic KMnO₄ oxidation, and 2) PTCA is artificially produced from pheomelanins. The results with HI hydrolysis show that dopamine-melanin produces a 1:1 mixture of 3-amino and 4-amino isomers of aminohydroxyphenylethylamine, while the isomer ratio is about 0.2 in melanins prepared from dopamine and cysteine. These results indicate that alkaline H₂O₂ oxidation is useful in characterizing synthetic and natural eumelanins and that reductive hydrolysis with HI can be applied to analyzing oxidation products of dopamine such as neuromelanin.     

Eumelanin and Phaeomelanin Contents of Human Epidermis and Cultured Melanocytes

There are two chemically distinct types of melanin: the red-yellow phaeomelanins and the brown-black eumelanins. While both melanins have been detected in human epidermis and cultured melanocytes, it is unknown how the phaeomelanin/eumelanin ratio in human melanocytes maintained in vitro relates to that in the epidermis from which they were isolated. This study uses high-performance liquid chromatography to quantify the eumelanin and phaeomelanin contents of epidermis and/or cultured melanocytes from 12 Europeans with lightly pigmented skin and 9 non-Europeans with more deeply pigmented skin. Epidermis from non-Europeans contained the highest levels of both eumelanin and phaeomelanin and had the lowest phaeomelanin/eumelanin ratios. In contrast, while cultured melanocytes from non-Europeans also had higher levels of eumelanin and phaeomelanin than melanocytes from Europeans, there was no difference in the phaeomelanin/eumelanin ratios in the two groups. However, the phaeomelanin/eumelanin ratios were higher in the cultured melanocytes than in the corresponding epidermis so that while eumelanin was the predominant melanin in the epidermis, phaeomelanin was the major melanin in the cultured melanocytes. These observations may have important implications for the use of cultured human melanocytes in the study of melanogenesis in man.     

Pheomelanin Production by the Lipoxygenase-Catalyzed Oxidation of 5-S-Cysteinyldopa and 5-S-Cysteinyldopamine

5-S-cysteinyl-dopa (cysdopa) and 5-S-cysteinyl-dopamine (cysdopamine) are oxidized in vitro by soybean lipoxygenase (LOX) in the presence of hydrogen peroxide giving rise to the corresponding pheomelanins. The reaction is activated by caffeic acid and other catechols, suggesting a cofactor role for these compounds. The activating effect is proportional to the concentration of the cofactor, with a saturation profile. The activation extent of the various cofactors is directly related to LOX affinity for the same compounds. The possible implications of the peroxidative action of LOX in Parkinson's disease and in aging are discussed.     

An Improved Modification of Permanganate Oxidation of Eumelanin That Gives a Constant Yield of Pyrrole-2,3,5-Tricarboxylic Acid

Microanalysis of eumelanin is based on the formation of pyrrole-2,3,5-tricarboxylic acid (PTCA) on permanganate oxidation followed by its HPLC determination. A problem in this method was that the oxidation gave concave, exponential curves when the amounts of PTCA formed were plotted against the amounts of sample oxidized. The problem has been mostly overcome by adding a homogenate of 5 mg of a mouse liver to the oxidation medium. Sepia melanin, C57BL black mouse hair, B16 mouse melanoma, and MM418 human melanoma cells were oxidized in the absence or presence of the liver homogenate. The yields of PTCA increased about 1.5-fold by adding the liver homogenate and the calibration curves became linear or almost linear. With the improved method the PTCA values from various types of samples can be reliably compared.     

Dysplastic Melanocytic Nevi Contain High Levels of Pheomelanin: Quantitative Comparison of Pheomelanin/Eumelanin Levels Between Normal Skin,Common Nevi,and Dysplastic Nevi

The degree and type of melanogenesis, i.e., either eumelanin of pheomelanin, has been shown to be a reliable marker for the differentiation of the melanocyte. If exposed to UV light, these two melanins were reported to behave differently; eumelanin was photoprotective whereas pheomelanin was phototoxic to cultured tumor cells. Our previous study indicated that dysplastic melanocytic nevus (DMN) undergoes altered melanogenesis, forming pheomelanosome-like granules. The present study examined chemically the type and degree of melanin synthesized in 31 melanocytic nevi excised from 27 patients as compared with that occurring in the surrounding normal skin. The tissue content of eumelanin and pheomelanin was expressed by the amounts of pyrrole-2,3,5-tricarboxylic acid (PTCA) and aminohydroxyphenylalanine (AHP), respectively. We found that DMN lesions contain significantly higher amounts of pheomelanin than either common melanocytic nevus (CMN) or normal skin. Differences in pheomelanin content between DMN and CMN could not be accounted for by inherently higher levels of pheomelanin within the skin in general from DMN patients. Our present finding substantiates our previous claim that epidermal melanocytes in DMN undergo deranged melanogenesis.     

High SLC7A11 expression in normal skin of melanoma patients

BackgroundMelanoma is one of the highest metastatic cancers and its incidence is rapidly increasing. A great effort has been devoted to determine gene mutations and expression profiles in melanoma cells, but less attention has been given to the possible influence of melanin synthesis in melanocytes on melanomagenesis. SLC7A11 encodes the cystine/glutamate antiporter xCT and its expression increases the antioxidant capacity of cells by providing cysteine that may be used for glutathione (GSH) synthesis. Melanocytes, however, can also use cysteine for pheomelanin synthesis and pigmentation. Therefore, pheomelanin synthesis may lead to chronic oxidative stress. Possible consequences of this for melanomagenesis have never been explored.MethodsWe quantified the expression of SLC7A11 and other genes that are involved in the synthesis of pheomelanin but do not regulate the transport of cysteine from the extracellular medium to the cytosol (CTNS, MC1R, ASIP and SLC45A2) in non-tumorous skin of 45 patients of cutaneous melanoma and 50 healthy individuals. We controlled for the effects of Fitzpatrick skin type, age, gender, body mass, frequency of sun exposure and sunburns and number of melanocytic nevi, as well as for the intrinsic antioxidant capacity as given by the expression of the gene NFE2L2.ResultsThe expression of SLC7A11, but not of the other genes, was significantly higher in melanoma patients than in healthy individuals. This was independent of phenotypic factors and antioxidant capacity, thus supporting an effect of pheomelanin-induced oxidative stress on melanomagenesis.ConclusionOur findings indicate that SLC7A11 downregulation in normal epidermal melanocytes may represent a preventive treatment against melanoma.     

Influences of Sex,Castration, and Androgens on the Eumelanin and Pheomelanin Contents of Different Feathers in Wild Mallards

In mallards the bright nuptial plumage of the drake represents the neutral, sex hormone-independent coloration of the species that both sexes eventually exhibit after castration. We compared the pheo- and eumelanin contents of feathers from the head, breast, flank, and under-tail coverts in five groups of mallards after the post-nuptial molt in summer: intact hens, intact drakes, castrated drakes, castrated drakes injected with testosterone during the spring, and castrated drakes injected with 5α-dihydrotestosterone during the spring. In the head feathers and under-tail coverts, the gonadal hormones of the intact birds and the testosterone injections into castrates significantly reduced the eumelanin content, tended to increase the pheomelanin content, and, thereby, changed the melanin type from eumelanic in the untreated castrates to mixed melanic in the other three groups. The eumelanin contents of the flank feathers did not differ among the groups, but the pheomelanin contents at this site was significantly elevated in the two intact groups and the testosterone-treated compared to the uninjected castrates. Again, the melanin type changed from eumelanic in the castrates to mixed melanic in the other three groups. The high pheomelanin content of the breast feathers in the castrated birds was significantly reduced in the hens, intact drakes, and testosterone-injected castrates with a concomitant tendency for elevated eumelanin contents. At this site, a change occurred from pheomelanic to mixed melanic. 5α-dihydrotestosterone was clearly less effective than testosterone in affecting the melanin contents in castrates and resulted in an intermediate coloration. The differing effects of the two androgens might be a result of differences in their conversion to estrogens.     

毛干DNA提取方法概述

毛干作为最容易取得的一种无创、运输和储存方便的生物样本,对从核酸分子水平上进行各方面研究有着十分重要的意义。但毛干中DNA含量低,不易提取,而且存在大量角蛋白和色素,纯化不净会对下游PCR扩增等反应产生抑制作用。基于毛干DNA提取现状,综述并比较了近三十年动物及人类毛发的毛干DNA提取、纯化等相关方法,拟为毛干DNA提取在分子生物学各领域的推广应用提供充分的文献支持和参考。     

Composition of Mammalian Eumelanins: Analyses of DHICA-Derived Units in Pigments From Hair and Melanoma Cells

The proportions in which two eumelanin monomers, namely 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI), compose the eumelanin polymer are believed to determine properties of the pigment including its color. These proportions are, however, not well elucidated for naturally occurring eumelanins, largely because of methodological difficulties. In this study we estimate the content of DHICA-derived units in mammalian eumelanins using a combination of two analytical techniques: 1) quantitation of DHICA-derived eumelanin by measuring the yield of pyrrole-2,3,5-tricarboxylic acid (PTCA index) and 2) spectrophotometrical quantitation of total (DHI + DHICA) eumelanin at 350 nm (A₃₅₀ index). The ratio of PTCA/A₃₅₀ measured for melanins synthesized from DHI and DHICA mixed in various molar proportions correlates well with the content of DHICA in synthetic polymers. Using this relationship as a standard curve we estimated the proportion of DHICA-derived units in mammalian eumelanins from hair and melanoma cells and found it to be much higher in rodent pigments (58.8%-98.3%; two species, mouse and hamster were examined) as compared to human eumelanins (19.2%-41.8%; one Caucasian and one Oriental individual were examined). No relationship between proportion of DHICA-derived units in eumelanin and hair color is found. The latter seems to be determined predominantly by the ratio of pheo- to eumelanin synthesis.