Yukiko Nakanishi

Usefulness of alkaline hydrogen peroxide oxidation to analyze eumelanin and pheomelanin in various tissue samples: application to chemical analysis of human hair melanins

Eumelanin and pheomelanin in tissue samples can be specifically measured as the markers pyrrole‐2,3,5‐tricarboxylic acid (PTCA) and 4‐amino‐3‐hydroxyphenylalanine after acidic permanganate oxidation and hydroiodic acid hydrolysis, respectively. Those degradation methods, although widely applied, are not easily performed in most laboratories. To overcome this difficulty, we developed alkaline H2O2 oxidation in 1 M K2CO3 that produces, in addition to the eumelanin marker PTCA, thiazole‐2,4,5‐tricarboxylic acid (TTCA) and thiazole‐4,5‐dicarboxylic acid (TDCA) as markers for pheomelanin and pyrrole‐2,3‐dicarboxylic acid (PDCA) as a marker for 5,6‐dihydroxyindole‐derived eumelanin. Those four degradation products can be easily separated by HPLC and analyzed with ultraviolet detection. The alkaline H2O2 oxidation method is simple, reproducible and applicable to all pigmented tissues. Its application to characterize eumelanin and pheomelanin in human hair shows that PTCA and TTCA serve as specific markers for eumelanin and pheomelanin, respectively, although some caution is needed regarding the artificial production of TTCA from eumelanic tissue proteins.

Pigmentation effects of solar simulated radiation as compared with UVA and UVB radiation

Different wavelengths of ultraviolet (UV) radiation elicit different responses in the skin. UVA induces immediate tanning and persistent pigment darkening through oxidation of pre‐existing melanin or melanogenic precursors, while UVB induces delayed tanning which takes several days or longer to develop and requires activation of melanocytes. We compared the effects of a 2‐week repetitive exposure of human skin to solar‐simulated radiation (SSR), UVA or UVB at doses eliciting comparable levels of visible tanning and measured levels of melanins and melanin‐related metabolites. Levels of eumelanin and pheomelanin were significantly higher in the order of SSR, UVB, UVA or unexposed control skin. Levels of free 5‐S‐cysteinyldopa (5SCD) were elevated about 4‐fold in SSR‐ or UVB‐exposed skin compared with UVA‐exposed or control skin. Levels of protein‐bound form of 5SCD tended to be higher in SSR‐ or UVB‐exposed skin than in UVA‐exposed or control skin. Total levels of 5‐hydroxy‐6‐methoxyindole‐2‐carboxylic acid (5H6MI2C) and 6H5MI2C were higher in SSR‐ than in UVB‐exposed or control skin. These results show that SSR is more effective in promoting delayed tanning than UVB radiation alone, suggesting a synergistic effect of UVA radiation. Furthermore, free 5SCD may serve as a good marker of the effect of SSR and UVB.

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.

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.

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.

Tyrosinase-catalyzed metabolism of rhododendrol (RD) in B16 melanoma cells: production of RD-pheomelanin and covalent binding with thiol proteins

RS‐4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD) was reported to induce leukoderma of the skin. To explore the mechanism underlying that effect, we previously showed that oxidation of RD with mushroom tyrosinase produces RD‐quinone, which is converted to secondary quinone products, and we suggested that those quinones are cytotoxic because they bind to cellular proteins and produce reactive oxygen species. We then confirmed that human tyrosinase can oxidize both enantiomers of RD. In this study, we examined the metabolism of RD in B16F1 melanoma cells in vitro. Using 4‐amino‐3‐hydroxy‐n‐butylbenzene as a specific indicator, we detected moderate levels of RD‐pheomelanin in B16F1 cells exposed to 0.3 to 0.5 mM RD for 72 h. We also confirmed the covalent binding of RD‐quinone to non‐protein thiols and proteins through cysteinyl residues. The covalent binding of RD‐quinone to proteins was 20‐ to 30‐fold greater than dopaquinone. These results suggest that the tyrosinase‐induced metabolism of RD causes melanocyte toxicity.

Serum Levels of Pigmentation Markers Are Elevated in Patients Undergoing Hemodialysis

Background: Diffuse hyperpigmentation is common among patients with chronic renal failure undergoing hemodialysis (HD). We have examined serum levels of 5-S-cysteinyldopa (5SCD, a pheomelanin precursor), pheomelanin, eumelanin, and protein-bound (PB-) 3,4-dihydroxyphenylalanine (DOPA) and PB-5SCD in HD patients. Methods: Pheomelanin and eumelanin were assayed by chemical degradation methods. Results: Serum levels of free 5SCD in HD patients (n = 16) were 9-fold higher than in healthy controls (n = 16). Levels of pheomelanin in HD patients were 2.6-fold higher than in controls, while levels of eumelanin did not differ between HD patients and controls. Levels of PB-DOPA and PB-5SCD in HD patients were approximately 1.5-fold higher than in controls. Serum levels of free 5SCD were positively correlated to the duration of HD therapy. Conclusions: The high constitutive levels of free 5SCD, pheomelanin, and PB-DOPA in the blood may be deteriorating in HD patients through the production of reactive oxygen species.

Acid hydrolysis reveals a low but constant level of pheomelanin in human black to brown hair

We previously reported a constant ratio of the benzothiazole pheomelanin marker thiazole‐2,4,5‐tricarboxylic acid (TTCA) to the eumelanin marker pyrrole‐2,3,5‐tricarboxylic acid (PTCA) in eumelanic, black human hair. A constant level (20%–25%) of benzothiazole‐type pheomelanin was recently demonstrated in human skin with varying concentrations of melanin. Therefore, in this study, we aimed to investigate the origin of pheomelanin markers in black to brown human hair by developing a method to remove protein components from hair by heating with 6 M HCl at 110°C for 16 hr. For comparison, synthetic melanins were prepared by oxidizing mixtures of varying ratios of dopa and cysteine with tyrosinase. Hair melanins and synthetic melanins were subjected to acid hydrolysis followed by alkaline H2O2 oxidation. The results show that the hydrolysis leads to decarboxylation of the 5,6‐di‐hydroxyindole‐2‐carboxylic acid moiety in eumelanin and the benzothiazole moiety in pheomelanin and that eumelanic human hair contains 11%–17% benzothiazole‐type pheomelanin.

Serum levels of 5-s-cysteinyldopa are correlated with skin colors in hemodialysis patients but not in peritoneal dialysis patients.

Background: Diffuse hyperpigmentation is common in patients with chronic renal failure undergoing hemodialysis (HD) or peritoneal dialysis (PD). We previously reported that serum levels of 5-S-cysteinyldopa (5SCD, a pheomelanin precursor) and pheomelanin were significantly elevated in HD patients. Methods: Skin color was assessed using a Mexameter that measures the melanin index (MI) and the erythema index (EI). The upper inner arms (non-sun-exposed site) and the foreheads (sun-exposed site) of HD and PD patients and control subjects were analyzed. Results: MI values on the upper inner arms and on the foreheads of HD and PD patients were significantly higher than in controls. In HD patients, significant correlations were found for serum 5SCD levels with MI and EI on the upper inner arm, and for EI on the forehead. In PD patients, no such correlations were found. Conclusions: Hyperpigmentation in HD patients results partly from accumulation of pheomelanin in the skin.