Pyrrole-2,3,5-tricarboxylic Acid Research Articles

Structural Investigation of DHICA Eumelanin Using Density Functional Theory and Classical Molecular Dynamics Simulations

Eumelanin is an important pigment, for example, in skin, hair, eyes, and the inner ear. It is a highly heterogeneous polymer with 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI) building blocks, of which DHICA is reported as the more abundant in natural eumelanin. The DHICA-eumelanin protomolecule consists of three building blocks, indole-2-carboxylic acid-5,6-quinone (ICAQ), DHICA and pyrrole-2,3,5-tricarboxylic acid (PTCA). Here, we focus on the self-assembly of DHICA-eumelanin using multi-microsecond molecular dynamics (MD) simulations at various concentrations in aqueous solutions. The molecule was first parameterized using density functional theory (DFT) calculations. Three types of systems were studied: (1) uncharged DHICA-eumelanin, (2) charged DHICA-eumelanin corresponding to physiological pH, and (3) a binary mixture of both of the above protomolecules. In the case of uncharged DHICA-eumelanin, spontaneous aggregation occurred and water molecules were present inside the aggregates. In the systems corresponding to physiological pH, all the carboxyl groups are negatively charged and the DHICA-eumelanin model has a net charge of -4. The effect of K+ ions as counterions was investigated. The results show high probability of binding to the deprotonated oxygens of the carboxylate anions in the PTCA moiety. Furthermore, the K+ counterions increased the solubility of DHICA-eumelanin in its charged form. A possible explanation is that the charged protomolecules favor binding to the K+ ions rather than aggregating and binding to other protomolecules. The binary mixtures show aggregation of uncharged DHICA-eumelanins; unlike the charged systems with no aggregation, a few charged DHICA-eumelanins are present on the surface of the uncharged aggregation, binding to the K+ ions.

5,6-Dihydroxyindole eumelanin content in human skin with varying degrees of constitutive pigmentation.

Human skin contains two distinct components: brown to black, insoluble eumelanin and light colored, alkaline-soluble pheomelanin. Eumelanin consists of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) moieties, while pheomelanin consists of benzothiazine (BT) and benzothiazole (BZ) moieties. These melanin monomer units can be quantitatively analyzed through specific degradation products by high-performance liquid chromatography (HPLC). Alkaline hydrogen peroxide oxidation (AHPO) of eumelanin gives rise to pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA) as specific degradation products of the DHICA and DHI moieties, respectively. BZ moiety in pheomelanin can be analyzed as thiazole-2,4,5-tricarboxylic acid (TTCA). By reductive hydrolysis with hydroiodic acid, BT moieties in pheomelanin can be analyzed as 4-amino-3-hydroxyphenylalanine (4-AHP). As a recently improved AHPO-HPLC method enabled a better characterization of PDCA, this prompted us to address the question of DHI to DHICA ratio in human skin samples with varying degrees of constitutive pigmentation ranging from very light to dark. Results showed for the first time the ratio of 4 moieties: DHI 35%, DHICA 41%, BZ 20%, and BT 4%. The ratio is constant regardless of the degree of pigmentation. The high content of DHICA moiety may impart an antioxidant property to the epidermis melanin.

Laccase Mediator Cocktail System as a Sustainable Skin Whitening Agent for Deep Eumelanin Decolorization.

The overproduction of eumelanin leads to a panel of unaesthetic hyper-pigmented skin diseases, including melasma and age spots. The treatment of these diseases often requires the use of tyrosinase inhibitors, which act as skin whitening agents by inhibiting the synthesis of eumelanin, with harmful side effects. We report here that laccase from Trametes versicolor in association with a cocktail of natural phenol redox mediators efficiently degraded eumelanin from Sepia officinalis, offering an alternative procedure to traditional whitening agents. Redox mediators showed a synergistic effect with respect to their single-mediator counterpart, highlighting the beneficial role of the cocktail system. The pro-oxidant DHICA sub-units of eumelanin were degraded better than the DHI counterpart, as monitored by the formation of pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA) degradation products. The most effective laccase-mediated cocktail system was successively applied in a two-component prototype of a topical whitening cream, showing high degradative efficacy against eumelanin.

Identification of black sturgeon caviar pigment as eumelanin

Reported herein is the purification of the pigment of black sturgeon caviar and its unambiguous identification as a typical eumelanin by means of chemical degradation coupled with electron paramagnetic resonance (EPR) evidence. HPLC and LC-MS analysis of oxidative degradation mixtures revealed the formation of pyrrole-2,3,5-tricarboxylic acid (PTCA), a specific marker of eumelanin pigments, in yields compatible with a 6.5% w/w pigment content. EPR spectral features and parameters were in close agreement with those reported for a typical natural eumelanin such as Sepia melanin from squid ink. The identification for the first time of eumelanin in a fish roe is expected to provide a novel molecular basis for the valorization of black caviar and production wastes thereof in food chemistry and diet.

Photobleached Oxidative Degradation of Melanins: Chemical Characterization of Melanins Present in Alpaca Fiber.

In order to characterize the phenotype and to examine the effects of sun exposure on the color and structure of eumelanin (EM) and pheomelanin (PM) in alpaca fibers, we applied Soluene-350 solubilization, alkaline hydrogen peroxide oxidation (AHPO) and hydroiodic acid (HI) hydrolysis to the base and tip fibers of 20 true-black (TB) and 20 warm-black (WB) alpacas. We analyzed absorbances at 500 nm (A500) and 650 nm (A650), Free and Total pyrrole-2,3,5-tricarboxylic acid (PTCA), 2,3,4,5-tetracarboxylic acid (PTeCA) as degradative products from EM, and 4-amino-3-hydroxyphenylalanine (4-AHP), 3-amino-4-hydroxyphenylalanine (3-AHP) and thiazole-2,4,5-tricarboxylic acid (TTCA) as degradative products from PM. We found that the ratio of PTeCA/Total PTCA increased significantly from the base to the tip in both colors of alpaca fibers, while the ratios of A650/A500 and 4-AHP/3-AHP decreased significantly. These results show that structures made of both EM and PM in alpaca fibers are modified significantly by sun exposure inducing color change. This study indicates that the ratios of A650/A500, PTeCA/Total PTCA and 4-AHP/3-AHP are highly sensitive markers of color change and photodegradation of EM and PM, respectively.

Does Eumelanin Oxidation Play a Role on the Photostability of Ethyl Glucuronide in Hair Exposed to Simulated Solar Radiation?

The action of solar radiation on the concentration of the ethanol metabolite ethyl glucuronide (EtG) in 40 hair samples of nonabstinent subjects was investigated. Hair samples of different colors were analyzed before and after irradiation with artificial sunlight under a light dose corresponding to 3 months of sun exposure. After irradiation, an increase of EtG concentration was detected in 55% of the samples ranging from 5% to 141%. In 16 cases, a concentration reduction ranging from -2% to -74% was observed. The measure of the level of pyrrole-2,3,5-tricarboxylic acid (PTCA), a marker of eumelanin oxidation, demonstrated the largest increase in oxidation in light brown hair where the greatest degradation of EtG was observed after irradiation. However, the rise of PTCA in all hair tested was accompanied by increase in EtG concentration in 8/10 samples and by decrease in 2/10, suggesting no correlation between the two markers. To verify if hair structure was modified by light, scanning electron microscopy (SEM) analysis was performed on irradiated hair of different colors and compared with the dark samples. SEM revealed modification of hair structure in all samples showing partial shaft exfoliation and reduction of hair thickness under the treatment with solar radiation.

Improved HPLC Conditions to Determine Eumelanin and Pheomelanin Contents in Biological Samples Using an Ion Pair Reagent.

Alkaline hydrogen peroxide oxidation (AHPO) of eumelanin and pheomelanin, two major classes of melanin pigments, affords pyrrole-2,3,5-tricarboxylic acid (PTCA), pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) from eumelanin and thiazole-2,4,5-tricarboxylic acid (TTCA) and thiazole-4,5-dicarboxylic acid (TDCA) from pheomelanin. Quantification of these five markers by HPLC provides useful information on the quantity and structural diversity of melanins in various biological samples. HPLC analysis of these markers using the original method of 0.1 M potassium phosphate buffer (pH 2.1):methanol = 99:1 (85:15 for PTeCA) on a reversed-phase column had some problems, including the short lifetime of the column and, except for the major eumelanin marker PTCA, other markers were occasionally overlapped by interfering peaks in samples containing only trace levels of these markers. These problems can be overcome by the addition of an ion pair reagent for anions, such as tetra-n-butylammonium bromide (1 mM), to retard the elution of di-, tri- and tetra-carboxylic acids. The methanol concentration was increased to 17% (30% for PTeCA) and the linearity, reproducibility, and recovery of the markers with this improved method is good to excellent. This improved HPLC method was compared to the original method using synthetic melanins, mouse hair, human hair, and human epidermal samples. In addition to PTCA, TTCA, a major marker for pheomelanin, showed excellent correlations between both HPLC methods. The other markers showed an attenuation of the interfering peaks with the improved method. We recommend this improved HPLC method for the quantitative analysis of melanin markers following AHPO because of its simplicity, accuracy, and reproducibility.

Kojic acid inhibited melanin synthesis by tyrosinase pathway in Pteria penguin

Kojic acid has significant anti-melanogenic activity in mammals. However, few reports showed its function and mechanism in bivalves. In this study, by survival rate investigation and colour identification, 0.2–5 g/L kojic acid treatment for 24–48 hr was verified to be safe and effective to lighten shell colour of Pteria penguin (Röding; 1798). The liquid chromatograph–tandem mass spectrometer (LC-MS/MS) analysis showed that the content of PDCA (pyrrole-2, 3-dicarboxylic acid) and PTCA (pyrrole-2,3,5-tricarboxylic acid), main alkaline oxidation products of eumelanin, were significantly decreased from 651.0 to 275.7 ng/mg (by 57.7%) after kojic acid treatment. Consistent with decrease in melanin content, the tyrosinase activity was also significantly inhibited by up to 54.7% in a dose-dependent and time-dependent manner. Different from the mammals, the qRT-PCR (quantitative real-time PCR) showed that kojic acid inhibited the expression of genes in tyrosinase pathway, including Tyr (Tyrosinase), Mitf (microphthalmia-associated transcription factor), Yel (Yellow) and Cdk2 (cyclin-dependent kinase 2), but had no effect on PpBcl2 (B-cell lymphoma 2). These data indicated that kojic acid changed shell colour by blocking tyrosinase activity and negatively regulating transcription of tyrosinase pathway genes in P. penguin. The effectiveness of kojic acid in modifying nacre colour suggested potential for its application in the aquaculture production of higher quality cultured pearls in P. penguin.

Quantitation of eumelanin and pheomelanin markers in diverse biological samples by HPLC-UV-MS following solid-phase extraction

Eumelanin and pheomelanin are well known and common pigments found in nature. However, their complex polymer structure and high thermostability complicate their direct chemical identification. A widely used analytical method is indirect determination using HPLC with UV detection of both types of melanin by their most abundant oxidation products: pyrrole-2,3-dicarboxylic acid (PDCA), pyrrole-2,3,5-tricarboxylic acid (PTCA), thiazole-4,5-dicarboxylic acid (TDCA), and thiazole-2,4,5-tricarboxylic acid (TTCA). An increasing interest in pigmentation in biological research led us to develop a highly sensitive and selective method to identify and quantify these melanin markers in diverse biological samples with complex matrices. By introducing solid-phase extraction (SPE, reversed-phase) following alkaline oxidation we could significantly decrease background signals while maintaining recoveries greater than 70%. Our HPLC-UV-MS method allows for confident peak identification via exact mass information in corresponding UV signals used for quantitation. In addition to synthetic melanin and Sepia officinalis ink as reference compounds eumelanin markers were detected in brown human hair and a brown bivalve shell (Mytilus edulis). Brown feathers from the common chicken (Gallus g. domesticus) yielded all four eumelanin and pheomelanin markers. The present method can be easily adapted for a wide range of future studies on biological samples with unknown melanin content.

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

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

Determination of eumelanin and pheomelanin in melanomas using solid-phase extraction and high performance liquid chromatography–diode array detection (HPLC-DAD) analysis

Determination of eumelanin and pheomelanin in melanomas that exhibit different pigmentation was carried using a solid-phase extraction (SPE) preparation method based on weak anion exchange chemistry. This extraction significantly enhanced the chromatographic profile obtained by reverse phase high performance liquid chromatography-diode array detection (RP-HPLC-DAD). The SPE method was developed using aqueous standards of melanin markers: thiazole-2,4,5-tricarboxylic acid (TTCA), thiazole-4,5-dicarboxylic acid (TDCA), pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA) and non-pigmented cell lines spiked with those markers. An excellent average recovery, above 90%, was obtained for the four markers with a relative standard deviation below 7%. We have also optimized the stationary phase and the mobile phase (phosphate concentration and pH) to improve sensitivity and to reduce the analysis time. Elution of the four markers is achieved in 5 min and total analysis of biological samples is completed in 15 min. The quantification limits for TDCA, TTCA, PDCA and PTCA are 60, 50, 47 and 48 ng/mL respectively. Furthermore, DAD detection improves the marker identification in complex matrices through the analysis of UV spectra. We have successfully applied this method to melanoma tumors and cells. Murine B16BL6 tumor are highly pigmented with mostly eumelanin (98.1% of eumelanin) while human SK-MEL-3 tumor contain about 30% pheomelanin. B16BL6 and B16F10 are eumelanic cells lines and NHEM melanocytes contain about 24% of pheomelanin.

Identification of a tyrosinase gene and its functional analysis in melanin synthesis of Pteria penguin

Tyrosinase is a key rate-limiting enzyme in melanin synthesis. In this study, a new tyrosinase gene (Tyr) was identified from Pteria penguin and its effect on melanin synthesis was deliberated by RNA interference (RNAi). The cDNA of PpTyr was 1728 bp long, containing a 5′untranslated region (UTR) of 11 bp, a 3′UTR of 295 bp, and an open reading fragment of 1422 bp encoding 473 amino acids. Amino acid alignment showed PpTyr had the highest (50%) identity to tyrosinase-like protein 1 from Pinctada fucata. Phylogenetic tree analysis classified PpTyr into α-subclass of type-3 copper protein. Tissue expression analysis indicated that PpTyr was highly expressed in mantle, a nacre formation related tissue. After PpTyr RNA interference, PpTyr mRNA was significantly inhibited by 71.0% (P < 0.05). For other melanin-related genes, PpCreb2 and PpPax3 expression showed no significant change, but PpBcl2 was obviously increased. By liquid chromatograph-tandem mass spectrometer (LC-MS/MS) analysis, the total content of PDCA (pyrrole-2, 3-dicarboxylic acid) and PTCA (pyrrole-2,3,5-tricarboxylic acid), as main markers for eumelanin, was sharply decreased by 66.6% after PpTyr RNAi (P < 0.05). The percentage of PDCA was also obviously decreased from 20.1% to 13.9%. This indicated that tyrosinase played a key role in melanin synthesis and color formation of P. penguin.

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 6M HCl at 110°C for 16hr. 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 H2 O2 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.

Photodegradation of Eumelanin and Pheomelanin and Its Pathophysiological Implications.

Eumelanin is photoprotective for pigmented tissues while pheomelanin is phototoxic. In this review, we summarize current understanding of how eumelanin and pheomelanin structures are modified by ultraviolet A (UVA) and also by visible light and how reactive oxygen species participate in those processes. Alkaline hydrogen peroxide oxidation was employed to characterize eumelanin and benzothiazole-type pheomelanin, giving pyrrole-2,3,5-tricarboxylic acid (PTCA) and thiazole-2,4,5-tricarboxylic acid (TTCA), respectively. Reductive hydrolysis with hydroiodic acid gives 4-amino-3-hydroxyphenylalanine (4-AHP) from the benzothiazine moiety of pheomelanin. The results show that the photoaging of eumelanin gives rise to free PTCA (produced by peroxidation in situ) and pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA, produced by cross-linking). The TTCA/4-AHP ratio increases with photoaging, indicating the conversion of benzothiazine to the benzothiazole moiety. Analysis of those markers and their ratios show that both eumelanin and pheomelanin in human retinal pigment epithelium melanosomes undergo extensive structural modifications due to their lifelong exposure to blue light. Using synthetic melanins, we also found that singlet oxygen, in addition to superoxide anions, is photogenerated and quenched upon UVA irradiation. The (patho)physiological significance of those findings is discussed in relation to the tanning process, to melanomagenesis in the skin and to age-related macular degeneration in the eyes.

Analysis of Antiretrovirals in Single Hair Strands for Evaluation of Drug Adherence with Infrared-Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry Imaging.

Adherence to a drug regimen can be a strong predictor of health outcomes, and validated measures of adherence are necessary at all stages of therapy from drug development to prescription. Many of the existing metrics of drug adherence (e.g., self-report, pill counts, blood monitoring) have limitations, and analysis of hair strands has recently emerged as an objective alternative. Traditional methods of hair analysis based on LC-MS/MS (segmenting strands at ≥1 cm length) are not capable of preserving a temporal record of drug intake at higher resolution than approximately 1 month. Here, we evaluated the detectability of HIV antiretrovirals (ARVs) in hair from a range of drug classes using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI) with 100 μm resolution. Infrared laser desorption of hair strands was shown to penetrate into the strand cortex, allowing direct measurement by MSI without analyte extraction. Using optimized desorption conditions, a linear correlation between IR-MALDESI ion abundance and LC-MS/MS response was observed for six common ARVs with estimated limits of detection less than or equal to 1.6 ng/mg hair. The distribution of efavirenz (EFV) was then monitored in a series of hair strands collected from HIV infected, virologically suppressed patients. Because of the role hair melanin plays in accumulation of basic drugs (like most ARVs), an MSI method to quantify the melanin biomarker pyrrole-2,3,5-tricarboxylic acid (PTCA) was evaluated as a means of normalizing drug response between patients to develop broadly applicable adherence criteria.

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

Eumelanin is a brown-black pigment comprising 5,6-dihydroxyindole (DHI) and its 2-carboxy derivative (DHICA), but the detailed structure of eumelanin is unclear. Chemical degradation is a powerful tool for analyzing melanin. H2 O2 oxidation degradation of eumelanin affords pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA). The ratio of PDCA to PTCA provides information about the eumelanin structure. In this article, we propose simple equations on the basis of previous experimental results on dimer yields for evaluating the yields of PTCA and PDCA from any DHI oligomers. Assuming the chemical disorder model of DHI-melanin, we solve an equation where a theoretical expression for the ratio of PDCA to PTCA is set to the corresponding experimental value to obtain a plausible Poisson distribution of DHI oligomers. The results demonstrate that the main contributors to DHI-melanin are tetramers and pentamers as shown by the mass spectrometry.

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

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

High-performance liquid chromatography estimation of cross-linking of dihydroxyindole moiety in eumelanin

Eumelanin pigments consist of various ratios of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI). On alkaline hydrogen peroxide oxidation, these indole moieties give rise to pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA), respectively. In a recent study, we detected considerable amounts of other pyrrole acids, pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) and pyrrole-2,3,4-tricarboxylic acid (isoPTCA), among the oxidation products of fossil ink sacs more than 160million years old. PTeCA and isoPTCA arise from the cross-linking of the DHI moiety of eumelanin at the C2 and/or C3 positions. We mimicked the process of cross-linking by heating synthetic eumelanins prepared from various ratios of DHICA and DHI at 100°C for 18days (or at 40°C for 180days). The heated eumelanins were analyzed after alkaline peroxide oxidation as PTCA, PDCA, PTeCA, and isoPTCA by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. On heating, PTCA decreased rapidly due to decarboxylation, whereas PDCA decreased gradually. Concurrently, PTeCA increased gradually to levels close to PTCA. IsoPTCA also increased gradually at lower levels. Similar changes were observed at 40°C at a much slower rate. These findings suggest that the PTeCA/PTCA ratio may serve as a good indicator of aging (cross-linking) of eumelanin.

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.

Age‐dependent changes in eumelanin composition in hairs of various ethnic origins

Hair pigmentation is one of the most conspicuous phenotypes of humans. From a chemical point of view, however, data remain scarce regarding human hair pigmentation characteristics. To determine melanin content and composition in human eumelanic hair from individuals of different ethnic origins and at different ages, we collected hair from 56 subjects with eumelanic hair from each group of African-American, East Asian, and Caucasian origin. The 56 subjects consist of 14, seven each of males and females, each from four age classes of younger than 11, between 12 and 19, between 20 and 45, and older than 46. We analysed hair colour scale, total melanin value, and contents of pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA). We measured age-dependent increases in the relative quantity of eumelanin in pigmented human hairs in the three ethnic groups. Regarding melanin composition, we observed an increase in the PDCA/PTCA ratio with age in African-American and Caucasian hairs until approaching the quite constant level of the ratio in East Asian hairs in the elderly individuals. Our results evidence differences in the content and composition of eumelanin in human hair among African-American, Caucasian and East Asian individuals. Furthermore, we show evidence of age-dependent changes in the quantity and quality of eumelanin in pigmented human hairs. In particular, the age-dependent modification of the PDCA/PTCA ratio, a marker for 5,6-dihydroxyindole units in eumelanin, suggests a chronological evolution of hair follicle melanocyte phenotype (e.g. decrease in dopachrome tautomerase expression).