The histological analysis,colorimetric evaluation,and chemical quantification of melanin content in ‘suntanned’ fish

Human melanocytes respond to UV irradiation by increasing the synthesis of melanin. While much is now understood of the pathways governing this process and the nature of the melanin synthesized, little is known of melanins produced by lower vertebrates and their capacity to respond to UV. Here we report that a fish, red seabream, can undergo ‘suntanning’. Histological, colorimetric and chemical assays were performed for suntanned red seabream fish bred in net cages to analyse the melanins and compared with shaded or wild red seabream fish. For color evaluation, the L* values of suntanned fish were dramatically lower than those in the other two groups. Pyrrole‐2,3,5‐tricarboxylic acid (PTCA), an indicator of eumelanin, was detected in suntanned fish at five times higher levels than in shaded or wild fish while 4‐amino‐3‐hydroxyphenyl‐alanine (4‐AHP), a marker for pheomelanin, could not be detected in any of the samples. Histological analysis showed that melanocytes in the suntanned skin enlarged and increased in number to form a monolayer at the surface of the skin. Analysis of L* values and PTCA levels showed quite a high correlation coefficient (r = ?0.843). When comparing shaded and wild red seabream fish, the scores were closer but some significant differences were still found in some body areas. These results indicate that eumelanin accumulates in suntanned fish during the increase in skin color, which is induced by sunlight, presumably by ultraviolet radiation.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a G_s-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection ¹. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.     

The Interaction of Agouti Signal Protein and Melanocyte Stimulating Hormone to Regulate Melanin Formation in Mammals

Important regulatory controls of melanogenesis that operate at the subcellular level to modulate the structural and/or the functional nature of the melanins and melanin granules produced in melanocytes are reviewed. Melanocyte stimulating hormone and agouti signal protein have antagonistic roles and possibly opposing mechanisms of action in the melanocyte. In the mouse, melanocyte stimulating hormone promotes melanogenic enzyme function and elicits increases in the amount of eumelanins produced, while agouti signal protein reduces total melanin production and elicits the synthesis of pheomelanin rather than eumelanin. We are now beginning to understand the complex controls involved in regulating this switch at the molecular and biochemical levels. The quality and quantity of melanins produced by melanocytes have important physiological consequences for melanocyte function and undoubtedly play important roles in the various functions of the melanins per se, including hair and skin coloration and photoprotection.     

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.     

Mechanism of skin pigmentation

Melanin is a pigment that plays an important role in providing coloration and protecting human skin from the harmful effects of UV light radiation. Human skin color is determined by the type and amount of melanins that are synthesized and deposited within the melanosomes. In addition, the transfer of these specialized membrane-bound organelles from melanocytes to surrounding keratinocytes also plays a role in dictating human skin color. In order to investigate the principle features of skin pigmentation, the origin, function, and production ability of melanin should be highly understood in terms of biological and pathophysiological aspects. Furthermore, a deep understanding of melanin synthesis will also contribute to cosmetics and drugs development. In this review, the processes of melanin biosynthesis, such as survival, proliferation, and differentiation of melanin cells, as well as the biological regulation of human pigmentation were described.     

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.     

MC1R and the response of melanocytes to ultraviolet radiation

The constitutive color of our skin plays a dramatic role in our photoprotection from solar ultraviolet radiation (UVR) that reaches the Earth and in minimizing DNA damage that gives rise to skin cancer. More than 120 genes have been identified and shown to regulate pigmentation, one of the key genes being melanocortin 1 receptor (MC1R) that encodes the melanocortin 1 receptor (MC1R), a seven-transmembrane G protein-coupled receptor expressed on the surface of melanocytes. Modulation of MC1R function regulates melanin synthesis by melanocytes qualitatively and quantitatively. The MC1R is regulated by the physiological agonists alpha-melanocyte-stimulating hormone (alphaMSH) and adrenocorticotropic hormone (ACTH), and antagonist agouti signaling protein (ASP). Activation of the MC1R by binding of an agonist stimulates the synthesis of eumelanin primarily via activation of adenylate cyclase. The significance of cutaneous pigmentation lies in the photoprotective effect of melanin, particularly eumelanin, against sun-induced carcinogenesis. Epidermal melanocytes and keratinocytes respond to UVR by increasing their expression of alphaMSH and ACTH, which up-regulate the expression of MC1R, and consequently enhance the response of melanocytes to melanocortins. Constitutive skin pigmentation dramatically affects the incidence of skin cancer. The pigmentary phenotype characterized by red hair, fair complexion, inability to tan and tendency to freckle is an independent risk factor for all skin cancers, including melanoma. The MC1R gene is highly polymorphic in human populations, and allelic variation at this locus accounts, to a large extent, for the variation in pigmentary phenotypes and skin phototypes (SPT) in humans. Several allelic variants of the MC1R gene are associated with the red hair and fair skin (RHC) phenotype, and carrying one of these variants is thought to diminish the ability of the epidermis to respond to DNA damage elicited by UVR. The MC1R gene is considered a melanoma susceptibility gene, and its significance in determining the risk for skin cancer is of tremendous interest.     

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.     

Cutaneous Photobiology. The Melanocyte vs. the Sun: Who Will Win the Final Round?

Solar ultraviolet radiation (UV) is a major environmental factor that dramatically alters the homeostasis of the skin as an organ by affecting the survival, proliferation and differentiation of various cutaneous cell types. The effects of UV on the skin include direct damage to DNA, apoptosis, growth arrest, and stimulation of melanogenesis. Long‐term effects of UV include photoaging and photocarcinogenesis. Epidermal melanocytes synthesize two main types of melanin: eumelanin and pheomelanin. Melanin, particularly eumelanin, represents the major photoprotective mechanism in the skin. Melanin limits the extent of UV penetration through the epidermal layers, and scavenges reactive oxygen radicals that may lead to oxidative DNA damage. The extent of UV‐induced DNA damage and the incidence of skin cancer are inversely correlated with total melanin content of the skin. Given the importance of the melanocyte in guarding against the adverse effects of UV and the fact that the melanocyte has a low self‐renewal capacity, it is critical to maintain its survival and genomic integrity in order to prevent malignant transformation to melanoma, the most fatal form of skin cancer. Melanocyte transformation to melanoma involves the activation of certain oncogenes and the inactivation of specific tumor suppressor genes. This review summarizes the current state of knowledge about the role of melanin and the melanocyte in photoprotection, the responses of melanocytes to UV, the signaling pathways that mediate the biological effects of UV on melanocytes, and the most common genetic alterations that lead to melanoma.     

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.     

Cutaneous photobiology. The melanocyte vs. the sun: who will win the final round?

Solar ultraviolet radiation (UV) is a major environmental factor that dramatically alters the homeostasis of the skin as an organ by affecting the survival, proliferation and differentiation of various cutaneous cell types. The effects of UV on the skin include direct damage to DNA, apoptosis, growth arrest, and stimulation of melanogenesis. Long-term effects of UV include photoaging and photocarcinogenesis. Epidermal melanocytes synthesize two main types of melanin: eumelanin and pheomelanin. Melanin, particularly eumelanin, represents the major photoprotective mechanism in the skin. Melanin limits the extent of UV penetration through the epidermal layers, and scavenges reactive oxygen radicals that may lead to oxidative DNA damage. The extent of UV-induced DNA damage and the incidence of skin cancer are inversely correlated with total melanin content of the skin. Given the importance of the melanocyte in guarding against the adverse effects of UV and the fact that the melanocyte has a low self-renewal capacity, it is critical to maintain its survival and genomic integrity in order to prevent malignant transformation to melanoma, the most fatal form of skin cancer. Melanocyte transformation to melanoma involves the activation of certain oncogenes and the inactivation of specific tumor suppressor genes. This review summarizes the current state of knowledge about the role of melanin and the melanocyte in photoprotection, the responses of melanocytes to UV, the signaling pathways that mediate the biological effects of UV on melanocytes, and the most common genetic alterations that lead to melanoma.     

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.     

Current challenges in understanding melanogenesis: bridging chemistry,biological control,morphology, and function

Melanin is a natural pigment produced within organelles, melanosomes, located in melanocytes. Biological functions of melanosomes are often attributed to the unique chemical properties of the melanins they contain; however, the molecular structure of melanins, the mechanism by which the pigment is produced, and how the pigment is organized within the melanosome remains to be fully understood. In this review, we examine the current understanding of the initial chemical steps in the melanogenesis. Most natural melanins are mixtures of eumelanin and pheomelanin, and so after presenting the current understanding of the individual pigments, we focus on the mixed melanin systems, with a critical eye towards understanding how studies on individual melanin do and do not provide insight in the molecular aspects of their structures. We conclude the review with a discussion of important issues that must be addressed in future research efforts to more fully understand the relationship between molecular and functional properties of this important class of natural pigments.