• Eumelanin;
  • Pheomelanin;
  • PTCA;
  • PDCA;
  • TTCA;
  • TDCA;
  • AHP;
  • AT;
  • AHPEA;
  • HPLC


  1. Top of page
  2. Abstract
  9. References

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.
















5,6-dihydroxyindole-2-carboxylic acid


pyrrole-2,3,5-tricarboxylic acid


1,3-thiazole-4,5-dicarboxylic acid


1,3-thiazole-2,4,5-tricarboxylic acid


  1. Top of page
  2. Abstract
  9. References

It is now well recognized that animal melanins can be classified into two major groups: the brown-to-black insoluble eumelanin and the yellow-to-reddish-brown pheomelanin that is soluble in alkali. The other essential biopolymers – proteins, nucleic acids, and carbohydrates – are chemically well characterized. They are composed of distinct monomer units and these units are connected through covalent bonds that can be easily split either by chemical methods or by the action of enzymes. In contrast, melanin pigments are composed of many different types of monomer units that are connected through strong carbon–carbon bonds (1, 2), which makes their systemic characterization very difficult.

Both eumelanin and pheomelanin are derived from the common precursor dopaquinone, which is formed following the oxidation of tyrosine by tyrosinase (Fig. 1). Dopaquinone is a highly reactive intermediate and in the absence of thiol compounds it undergoes intramolecular cyclization, leading eventually to the formation of eumelanin. However, the intervention of thiols, such as glutathione and cysteine, with this process gives rise exclusively to thiol adducts of dopa, cysteinyldopas, among which 5-S-cysteinyldopa (5-S-CD) is the major isomer. Further oxidation of the thiol adducts leads to pheomelanin production via benzothiazine intermediates. Most melanin pigments present in pigmented tissues appear as mixtures or copolymers of eumelanin and pheomelanin.


Figure 1. . The biosynthetic pathways to eumelanin and pheomelanin. Note that activities of tyrosinase, Tyrp2, and Tyrp1 are involved in the production of eumelanin, while only tyrosinase activity (and the presence of cysteine) is necessary for the production of pheomelanin.

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Extensive studies carried out by Nicolaus's group at Naples (3) and Swan's group at Newcastle (4) led to the conclusion that eumelanin is a highly heterogeneous polymer consisting of different oxidative states of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) units, and the pyrrole units derived from their peroxidative cleavage. Two proteins related to tyrosinase, termed tyrosinase-related proteins (Tyrps), have been shown to further regulate eumelanin formation (5). Dopachrome tautomerase, or Tyrp2, catalyses the tautomerization of dopachrome to DHICA, and oxidative polymerization of DHICA is catalysed by Tyrp1, the brown locus protein. It is now clear that the activities of these Tyrps greatly affect the quantity and quality of the eumelanin produced. In contrast to eumelanin production, no enzymes (other than tyrosinase) appear to be directly involved in the course of pheomelanin production (6).


  1. Top of page
  2. Abstract
  9. References

Chemical Methods for Characterizing Eumelanin and Pheomelanin

Previous methods used for the quantification of melanins in pigmented tissues required the isolation of melanins. Moreover, none of those methods were suitable for distinguishing between eumelanin and pheomelanin. Extensive degradative studies provided a number of chemical degradative methods (1, 7). Among them, it should be noted that pyrrole-2,3,5-tricarboxylic acid (PTCA) was obtained from sepiomelanin in 6.5% yield using hydrogen peroxide at pH 7 followed by alkaline hydrolysis (8). Fattorusso et al. (8) reported that permanganate oxidation of pheomelanin yielded 1,3-thiazole-4,5-dicarboxylic acid (TDCA) and 1,3-thiazole-2,4,5-tricarboxylic acid (TTCA) derived from the benzothiazole units (Fig. 2).


Figure 2. . Structures of eumelanin, pheomelanin, products obtained by oxidation of eumelanin and pheomelanin with permanganate or hydrogen peroxide, and products obtained by reductive hydrolysis of pheomelanin with hydriodic acid.

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In 1983, we introduced a rapid method for quantitatively analysing eumelanin and pheomelanin in tissue samples, which made the isolation of melanin pigments unnecessary (9). This method is based on the formation of PTCA by permanganate oxidation of eumelanin and of aminohydroxyphenylalanine (AHP) isomers by hydriodic acid (HI) reductive hydrolysis of pheomelanin, respectively. These specific degradation products are determined by HPLC: PTCA is quantified with UV detection while AHP is determined with electrochemical detection. The original method was later improved to increase the sensitivity and to reduce the time for pre-HPLC work-up (10, 11). The improved method requires only 1–5 mg of tissue samples or 106 cultured cells for each PTCA and AHP measurement. Amounts of eumelanin and pheomelanin can be calculated by multiplying the amounts of PTCA and AHP by factors of 50 and 5, respectively. These conversion factors are based on the 2% yield of PTCA from sepiomelanin and the 20% yield of AHP from synthetic 5-S-CD-melanin, respectively (9, 10, 12).

Our HPLC method is relatively simple, fairly rapid and highly sensitive. It has been applied to quantitatively analyse eumelanin and pheomelanin, not only in synthetic melanins, isolated melanosomes, hair, feathers, skin, nevi and melanomas, but also in human epidermis and cultured melanocytes (2, 13, 14). Moreover, the same degradative methodology has been applied to characterize neuromelanin (15[16]–17; described later).

Formation of PTCA was interpreted in terms of the oxidative breakdown of indole units, either linked through the 2-position or bearing a carboxyl group at the same position (18). In our hands, permanganate oxidation of DHI-melanin and DHICA-melanin in acidic medium produced 0.03% and 2.8% PTCA, respectively (2). Similar results were also reported by Nicolaus's group (18). These results indicate that PTCA is a specific product arising from DHICA-derived structures. Amount of DHICA-melanin can thus be calculated by multiplying the amount of PTCA by a factor of 35 on the basis of the 2.8% yield of PTCA from DHICA-melanin.

A drawback in the acidic permanganate oxidation is that it does not produce appreciable amounts of pyrrole-2,3-dicarboxylic acid (PDCA) either from natural eumelanin or from DHI-melanin (4, 10). Napolitano et al. (19) have re-examined conditions for the oxidative degradation of eumelanin: Using alkaline hydrogen peroxide as the oxidizing agent, they improved the yield of PTCA to 6.1% from DHICA-melanin and obtained PDCA in a significant (0.46%) amount from DHI-melanin. It should be noted, however, that a comparable (0.39%) amount of PTCA was also obtained from DHI-melanin.

Spectrophotometric Methods for Analysing Eumelanin and Pheomelanin

Although our melanin assay based on chemical degradation and HPLC determination is relatively simple, it still requires an HPLC system equipped with UV and electrochemical detectors. In addition, PTCA arises from the DHICA-derived units but not from the DHI-derived units present in the eumelanin polymer. Therefore, we have developed a spectrophotometric method that is specific to eumelanin but does not discriminate between the DHI- and DHICA-derived units (20). In this method, hair and melanoma samples were hydrolysed in hot HI to remove pheomelanic components, and the insoluble eumelanic pigments were solubilized in hot NaOH in the presence of H2O2 and were analysed for absorbance at 350 nm. Although much less sensitive, this spectrophotometric method can substitute for the PTCA method for eumelanin assay when substantial amounts of samples are available.

Spectrophotometric measurement of melanin pigments usually employs hot NaOH solution to solubilize melanin from tissue samples. However, the method is not suitable for hair samples because of the insolubility of eumelanin. We found that hot Soluene-350 in the presence of 10% water is able to completely dissolve mouse hair, sheep wool, and human hair samples (21[22]–23). The resulting brown solutions were analysed for absorbances between 400 and 800 nm. It was found that soluene-350 solutions of eumelanin and pheomelanin differ considerably in visible absorption spectra, with eumelanin giving flatter spectra (24). The absorbance at 500 nm (A500) value may serve as an indicator of the total combined amount of eumelanin and pheomelanin. We have also found that when solubilized in Soluene-350, eumelanin (ratio 0.25–0.33) and pheomelanin (ratio 0.10–0.14) in hair show significantly different ratios of absorbances at 650–500 nm (A650/A500). The absorbance ratio can be used as a parameter to estimate the relative ratio of eumelanin to pheomelanin (24).

We measured the A500 values of the Soluene-350 solution from hairs and compared the amounts of eumelanin and pheomelanin calculated from values of PTCA and AHP using conversion factors that fit best (24). We obtained the following conversion factors: for PTCA, factors are 45, 40, and 160 in mice, sheep, and humans, respectively; for AHP, factors are 2.5, 15, and 10. These conversion factors indicate that the yields of PTCA from eumelanin are 2.2, 2.5, and 0.6% in mice, sheep, and humans, respectively. Natural eumelanin is produced by copolymerization of DHI and DHICA in various ratios which appear to be governed chiefly by the activity of Tyrp2 (25, 26). Thus, the low yield of PTCA in human hairs suggests a low activity of Tyrp2 in humans as compared with other species. Alaluf et al. (27) recently reported that the HPLC method underestimates the melanin content of human epidermis by a factor of 3 as compared with the spectrophotometric method (NaOH solubilization). This discrepancy can be solved using a conversion factor of 160 instead of 50. The conversion factors for AHP also suggest that the yields of AHP from pheomelanin are 40, 7, and 10% in mice, sheep, and humans. However, it should be cautioned that what we measure with the spectrophotometric methods (regardless of solubilizing agents used) is not the concentration of the pigment but the intensity of absorbance of the pigment (24). The large difference in the conversion factors for AHP might be ascribed to the difference in the colour intensity (22). In this regard, it is interesting to note that the colours of pheomelanic sheep wool and human hairs are dark red while those of lethal yellow and recessive yellow mouse hairs are yellowish.

The total amount of melanin, as measured by A500, is useful in characterizing synthetic and natural melanins in combination with PTCA and AHP measurements (21[22]–23, 28, 29). For example, the PTCA/total melanin ratio in slaty mouse hair was only one-fifth that of black hair, suggesting that slaty hair melanin contains DHICA-derived units at a level one-fifth that of black (21). This is caused by the decreased activity of Tyrp2 in slaty mice. The AHP/total melanin ratio is also useful in characterizing synthetic as well as natural pheomelanic pigments (21[22]–23, 29).

Although the spectrophotometric method is useful, it has some disadvantages: 1) Background absorbance arises from tissue components such as proteins, as indicated by the low, but significant absorbance from white or albino hairs (21[22]–23), and 2) the viscosity of Soluene-350 requires caution for reproducible measurements.


  1. Top of page
  2. Abstract
  9. References

Necessity to Separate AHP and 3-aminotyrosine

In previous reports from our laboratory, the HPLC conditions used were such that AHP and 3-aminotyrosine (AT) eluted in a single peak. This was based on the assumption that any natural pheomelanin pigment consists of a fixed ratio of 5-S-CD and 2-S-CD because the ratio of these CD isomers in biological materials is chemically controlled to be approximately 5:1 (30, 31). In fact, the estimation of pheomelanin as the combined amount of AHP and AT (in this report, we call this combined amount `total' AHP to discriminate from `specific' AHP) did not impose any serious problem in most cases. However, small amounts of background values of total AHP were found in hairs from tyrosinase-negative, albino mice (21, 23) and humans (unpublished results). In addition, red hair was found to be closely associated with loss-of-function mutations of the melanocortin 1 receptor (MC1R) (32). After this discovery, there has been a growing interest in red hair as a risk factor for melanoma and for non-melanoma skin cancer (33, 34).

Reductive hydrolysis of pheomelanin with HI gives two AHP isomers, 4-amino-3-hydroxyphenylalanine (specific AHP) and 3-amino-4-hydroxyphenylalanine (3-aminotyrosine, AT), which derive from the oxidative polymerization of 5-S-CD and 2-S-CD, 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 of AHP seem to originate from precursors other than pheomelanin. Considerable and variable amounts of background AT are produced from other sources, most likely 3-nitrotyrosine residues in proteins. The nitration of tyrosine appears to be a common biological phenomenon originating from nitric oxide (35, 36) and this process seems to occur also in the skin, especially after oxidative stress such as UV radiation (37).

Kolb et al. (38) and Borges et al. (39) described good separation of AHP and AT in HI hydrolysates of various tissue samples. However, their reported methods require ion-exchange chromatography prior to the HPLC separation, which is not only time-consuming but is also very costly (because of using commercial, disposable ion-exchange columns). In order to overcome this problem, we developed HPLC conditions that enable the direct injection of the HI reduction products into the HPLC system allowing good separation of AHP and AT (40). In this way, we can study the importance of both degradation products separately and their specificity as markers for pheomelanin.

We also modified the procedure for evaporating HI, which makes it unnecessary to use a special evaporation apparatus (10). Thus, a 100 μl portion of the hydrolysate is transferred to a test tube and evaporated to dryness using a vacuum pump connected to an ice-cooled vacuum trap and two filter flasks containing NaOH pellets. The residue is then dissolved in 200 μl 0.1 M HCl. Usually, 10–20 μl of the solution was analysed on the HPLC system: injecting 50 μl caused broadening of peaks of AHP and AT.

HPLC Separation of AHP and AT

Figure 3 shows typical chromatograms of AHP and AT standards, HI reduction products of synthetic pheomelanin and typical examples of human red, black, and albino hair. After removal of HI, the samples were injected directly into the HPLC system without any pre-treatment. The AHP and AT peaks were well separated with retention times around 20 min. AHP was a major peak in pheomelanic samples. Before achieving this optimal resolution, we examined various pHs of 0.1 M sodium citrate buffers containing 1 mM sodium octanesulfonate. The best results were obtained with the pH 3.0 buffer, whereas the pH of 5.7 described by Kolb et al. (38) was not applicable to our column because a sharp negative peak appeared between the peaks of AHP and AT. This negative peak seems to be derived from a trace amount of acid remaining in the HI hydrolysate. We found that the pH 3.0 buffer is the most suitable for the separation of AHP and AT, because the negative peak appeared prior to the peaks of AHP and AT with this buffer. The same HPLC conditions are now being used to determine the levels of pheomelanin in the sera of melanoma patients (unpublished results). Takasaki et al. (41) was also able to separate AHP and AT under different HPLC conditions and applied that method to the determination of pheomelanin in the urine of melanoma patients.


Figure 3. . HPLC chromatograms of standard and HI reduction products. (A) Specific AHP (peak 1) and AT (peak 2) standards (1 ng each). (B) HI hydrolysate of synthetic pheomelanin. (C) HI hydrolysate of human red hair. Taken from (40).

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Comparison of Specific AHP and Total AHP as a Marker of Pheomelanin

The usefulness of specific AHP was validated on 77 human hairs of various colours (40). The combined amount of specific AHP and AT showed an excellent correlation to the amount of total AHP (r=0.991), and the amount of specific AHP also correlated well with the amount of total AHP (r=0.963). The amount of specific AHP showed only a modest correlation to the amount of AT (r=0.782) because of the background values in AT. From these results, we conclude that 1) specific AHP is a more specific marker of pheomelanin than is total AHP, and 2) total AHP values that we have previously used reflect well the production of pheomelanin in most cases. The AT to specific AHP ratio is highly variable, because the background AT value varies from one sample to another. Kolb et al. (38) also observed a wide variation in the AT to AHP ratios among different types of hair and types of skin. They also proposed that the AHP isomer is a specific marker for the presence of pheomelanin. Wenczl et al. (42) described a large difference in the AT to AHP ratio in cultured human melanocytes of extreme skin types.

Figure 4 illustrates the usefulness of specific AHP in analysing pheomelanin in tissue samples. The ratios of total AHP to PTCA and specific AHP to PTCA were compared in the 77 human hair samples (40). The hair samples were divided into dark, fair or red colours. The average ratios of total AHP to PTCA of red, fair, and dark hairs were 15.5 (median, 8.87), 1.61 (median, 1.09), and 0.39 (median, 0.27), respectively, while those of specific AHP to PTCA of red, fair, and dark hairs were 8.55 (median, 2.81), 0.45 (median, 0.25), and 0.048 (median, 0.044), respectively. In both ratios there were significant differences among these three colors. The background of values found in total AHP of dark hairs became negligible in specific AHP values. Thus, the ratio of specific AHP to PTCA is more prominently segregated among red, fair, and dark colors than is the ratio of total AHP to PTCA as shown in mean and median values. Eumelanin is more specifically produced in melanocytes of dark hairs than we originally thought (14). The switch between the production of eumelanin and pheomelanin in follicular melanocytes of humans now appears to be controlled as precisely as in the mouse (21, 23).


Figure 4. . (A) Relationship between the ratio of total AHP to PTCA in 77 human hair samples. (B) Relationship between the ratio of specific AHP to PTCA in human hair. Taken from (40) with modification. Differences were analysed for statistical significance using the Mann–Whitney U-test.

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Source of Background AT

It was expected that 3-nitrotyrosine could be a source resulting in the background value of AT. We therefore subjected 3-nitrotyrosine to HI reduction. 3-Nitrotyrosine was reduced to give AT in 83% yield within 1 h of the hydrolysis conditions, with yields remaining constant up to 20 h (80–85%). Thus, the source of background AT is most likely the 3-nitrotyrosine residues in proteins. The simplicity and sensitivity of the present HPLC method also makes it useful to analyse contents of 3-nitrotyrosine-containing proteins in the form of AT in non-pigmented tissues. The formation of 3-nitrotyrosine-containing proteins has been observed in many pathological conditions such as neurodegenerative disease, acute lung injury, atherosclerosis, bacterial and viral infections and chronic inflammation (43). In addition, it was recently reported that expression of 3-nitrotyrosine by melanoma cells strongly correlated with poor survival in patients with stage 3 disease (44). One has to caution, however, that AT in melanoma cells as measured by our method also comes from pheomelanin of 2-S-CD-derived units.


Our HPLC methods to analyse specific degradation products of eumelanin and pheomelanin have found many applications (2, 13, 14). The contents of pheomelanin have been analysed in human red hair (22, 40, 45) and in mouse yellow hair (21, 23). In an earlier report (45), we cautioned that phenotypically red hair was not necessarily pheomelanic according to our HPLC method of total AHP determination. By using the more specific marker (the AHP isomer), a more reliable chemical classification of red hair can now be expected.

The amount of pheomelanin can be obtained by multiplying the amount of specific AHP by a conversion factor of 9, based on the 11% yield from synthetic pheomelanin (40). The 13% yield of total AHP in the latest study (40) is a little lower than previously obtained [18% in (10), 16–20% in (12)], and the difference might be because of the preparation of synthetic pheomelanin.

Napolitano et al. (46) recently described other markers of pheomelanin, i.e. 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole (BTCA) and TTCA, which are produced by alkaline hydrogen peroxide treatment of various hair samples. They suggested that BTCA represents a new biogenetic marker for predicting individuals at high risk for skin cancer and melanoma.


  1. Top of page
  2. Abstract
  9. References

Alkaline Hydrogen Peroxide Oxidation (and HI Reduction)

Dark brown pigments, similar to eumelanin and pheomelanin, are also produced in cells other than melanocytes. For example, humans and primates produce neuromelanin in dopaminergic nigrostriatal neurones (47). Recently, Napolitano et al. (48) have shown that H2O2 oxidation of DHI-melanin in 1 M K2CO3 produces PDCA in a yield (∼0.5%) which is much higher than that produced by acidic KMnO4 oxidation. To characterize the diverse types of melanins, especially to identify dopamine (DA)-derived melanin, we have improved the alkaline H2O2 oxidation method of Napolitano et al. in terms of speed and sample size required and we have re-examined the HI hydrolysis method of Wakamatsu et al. (15). Figure 5 summarizes the results with alkaline H2O2 oxidation in comparison with those with acidic KMnO4 oxidation (49). The results with H2O2 oxidation show that 1) PDCA, a specific marker of DHI units in eumelanin, is produced in yields 10 times higher than by acidic KMnO4 oxidation, 2) PTCA is produced in higher yields as well, but it is also artificially produced from pheomelanin (see data for 5-S-CD-melanin and lethal yellow hair), and 3) the PDCA/PTCA ratio may be useful in characterizing eumelanin with various ratios of DHI/DHICA. The results with HI reductive hydrolysis show that DA-melanin produces a low (0.3%) yield of 1:1 mixture of 4-amino and 3-amino isomers of AHPEA while cys-DA-melanin produces a high (12%) yield of 5:1 mixture of AHPEA isomers. 4-AHPEA may thus serve as a specific indicator of cys-DA-derived melanin.


Figure 5. . Chemical characterization of synthetic and natural melanins by (A) alkaline H2O2 oxidation and (B) acidic KMnO4 oxidation. Taken from Table 1 of (49) with modification.

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Applications of Hydrogen Peroxide Oxidation (and HI Reduction)

It is generally accepted that neuromelanin is produced from DA (47). Cysteine may be incorporated into neuromelanin in a mechanism similar to pheomelanin production. However, our group and Rorsman's group reached opposite conclusions as to whether cysteine (cysteinyl-DA) is actually incorporated (15[16]–17). To solve this discrepancy, a more accurate method needs to be developed to chemically characterize neuromelanin (our unpublished results). We prepared synthetic models of neuromelanin by tyrosinase oxidation of DA and cysteine in various ratios (15). Alkaline peroxide oxidation of these model neuromelanins produces thiazole carboxylic acids, TTCA and TDCA, in addition to PTCA and PDCA (Fig. 6). TTCA and TDCA are produced from benzothiazole intermediates formed by ring contraction of benzothiazine units of pheomelanin. In the course of this study, we noticed that TTCA and TDCA are extremely susceptible to acid. Therefore, we avoided the ether extraction of acidified reaction mixtures that we usually perform in the oxidation of melanins. We found that the yield of PDCA is fairly constant in synthetic melanins with various DA and cysteine ratios, while the yield of TTCA is proportional to the sulfur to nitrogen ratio. Therefore, the degree of incorporation of cysteine (the S/N ratio) in neuromelanin can be determined from its TTCA/PDCA ratio against a standard curve for synthetic models. These results also suggest that the same methodology should be useful for analysing eumelanin and pheomelanin in various tissues. Melanin pigments may be characterized by contents of PTCA, PDCA, TTCA, TDCA and the ratios among them. Especially, TTCA may serve as a specific marker of pheomelanin (46).


Figure 6. . HPLC chromatograms of standard and alkaline H2O2 oxidation products. (A) Standards of TDCA (peak 1), PDCA (peak 2), TTCA (peak 3), and PTCA (peak 4,50 ng each). (B) Oxidation products of synthetic melanin prepared from a 1:0.1 mixture of DA and cysteine. (C) Oxidation products of neuromelanin (a kind gift from Dr Luigi Zecca, Milan, Italy).

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The dark pigment in butterfly wings is another example of a natural melanin whose chemical nature was mostly unknown. The grey and black pigments have been assumed to be DA-melanin by virtue of their solubility properties and the incorporation of radiolabeled precursors into the melanin produced (50, 51). We applied H2O2 oxidation and HI reduction to follow the developmental increase of melanin in wings from Precis coenia (52). The results showed that the production of melanin was low at the first stage, followed by the production of cysteinyl-DA-melanin, and at the later stage most of the melanin produced was of the mixed type with a shift to a more eumelanic, DA-melanin.

Certain bacteria and fungi also produce insoluble, dark brown melanin-like pigments. We have applied alkaline H2O2 oxidation to the analysis of melanin pigments produced in Cryptococcus neoformans, an opportunistic fungal pathogen that causes life-threatening infections in brains of about 10% of AIDS patients (53). Cryptococcus neoformans produces dark pigments on its cell wall when grown in media containing a diphenolic substrate such as dopa or DA. A principal enzyme responsible for the pigment production in C. neoformans has been identified as a laccase (53). We performed H2O2 oxidation of C. neoformans cells grown on DA or dopa agar. Cells grown on DA agar gave a high ratio of PDCA/PTCA while cells grown on dopa agar gave a high ratio of PTCA/PDCA. The production of AHPEA and AHP by HI reduction was not high. These data provide direct chemical evidence for the formation of eumelanic pigments by oxidation of catecholic precursors by C. neoformans laccase (53).


  1. Top of page
  2. Abstract
  9. References

Our HPLC methods for assaying eumelanin and pheomelanin are highly sensitive and specific and possess many advantages but also have certain disadvantages. Table 1 briefly illustrates some advantages and disadvantages of our methods.

Table 1.  . Advantages and disadvantages of the two oxidation methods Thumbnail image of

The acidic KMnO4 oxidation method that we have been using for quantitative analysis of eumelanin has a number of advantages: 1) PTCA is formed primarily from DHICA-derived units in eumelanin, thus making PTCA a specific marker of DHICA content (21), and 2) PTCA is not artificially formed from pheomelanin. However, this method also has some disadvantages: 1) the yields of PDCA are too low to be used as a marker of DHI-derived units, and 2) the amount of PTCA formed gives a slightly concave calibration curve against the amount of melanin oxidized despite recent improvements (11).

The alkaline H2O2 oxidation has several advantages (49): 1) PDCA is an excellent indicator of DHI-derived units in eumelanin and the PDCA/PTCA ratio is useful in characterizing various types of eumelanin, a ratio >1 indicating DA-melanin. 2) The calibration curves for PDCA and PTCA are linear. 3) It is easier to perform than the KMnO4 oxidation. However, the alkaline H2O2 oxidation method also has a certain disadvantage: yields of PTCA and PDCA from 5-S-CD-melanin are abnormally high compared with those with KMnO4 oxidation. This indicates that indole units are artificially formed during the oxidation, because the postulated structure of 5-S-CD-melanin does not contain an indole unit (4). Prota et al. (54) and our group (49) also found abnormally high yields of PTCA and PDCA when lethal yellow and recessive yellow mouse hairs were subjected to alkaline H2O2 oxidation. We therefore recommend that special care be taken when alkaline H2O2 oxidation is used to analyse pigmented tissues containing pheomelanin. It should be added that DA-protein conjugate also gives rise to a high yield of PDCA upon alkaline H2O2 oxidation (55). Smit et al. (56) also reported that melanin degradation by H2O2 oxidation of epidermal skin and melanoma cells yielded considerable amounts of PDCA and PTCA. They also indicated the usefulness of fluorescent detection (280/335 nm) compared with UV, especially for the detection of PDCA.


  1. Top of page
  2. Abstract
  9. References

In this review, we presented the advances in chemical analysis of melanins, with special emphasis on the methodology used to determine the quality and quantity of melanins present in pigmented tissues. We also presented new methodology of separately analysing specific AHP and AT. Preliminary results from neuromelanin characterization are also included.

Our HPLC microanalytical methods coupled with acidic KMnO4, alkaline H2O2 oxidation and HI reductive hydrolysis to characterize eumelanin and pheomelanin have become a useful tool for the chemical study of various types of melanin pigments including those derived from DA. The Soluene-350 solubilization method is also useful in analysing the total amount of melanin pigments, especially in otherwise insoluble samples such as hair. However, we should keep in mind to select a proper method that suits a given sample, because each method had advantages and disadvantages.

Although we used the abbreviations AHP and AT in the main text of this review, we now propose to use 4-AHP and 3-AHP to abbreviate 4-amino-3-hydroxyphenylalanine and 3-amino-4-hydroxyphylalanine, respectively. This is to discriminate more clearly total AHP (a combined amount of 4-AHP and 3-AHP) and specific AHP (4-AHP).


  1. Top of page
  2. Abstract
  9. References
  • 1
    Prota G. Melanins and Melanogenesis. New York: Academic Press; 1992. pp. 1–290
  • 2
    Ito S. Advances in chemical analysis of melanins. In: Nordlund JJ, Boissy RE, Hearing VJ, King RA, Ortonne JP. The Pigmentary System: Physiology and Pathophysiology. New York: Oxford University Press, 1998. pp. 439–450
  • 3
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