Effect of ascorbic acid (vitamin C) on the ESR spectra of the red and black hair: pheomelanin free radicals are not always present in red hair

Increased incidence of melanoma in the population with red hair is conditioned by synthesis of pheomelanin pigments in the skin and their phototoxic properties. The recent research has shown that free radicals of pheomelanin are produced not only by the influence of UV irradiation, but also in UV‐independent pathways of oxidative stress. It has been ascertained, that the color of the hair is not always determinant of the amount of pheolemanin radicals in red hair. Therefore, in order to evaluate the risk of melanoma in different individuals, it is necessary to define the amount of free radicals of pheomelanin in red hair using ESR spectroscopy method. Besides, it is very important to find effective antioxidant, capable of neutralizing free radicals of pheomelanin. It was proved that ascorbic acid neutralizes free radicals of pheomelanin very effectively. The main goal of our research was to define the presumably optimal concentration of ascorbic acid as an antioxidant and study the kinetics of the influence of this concentration on red and black hair. It has been found out, that ascorbic acid influences the free radicals of red and black hair, and its appropriate optimal concentration is 10 mM. The obtained results can be considered in dermatology and cosmetology. Copyright © 2015 John Wiley & Sons, Ltd.     

The role of melanin as protector against free radicals in skin and its role as free radical indicator in hair

Throughout the body, melanin is a homogenous biological polymer containing a population of intrinsic, semiquinone-like radicals. Additional extrinsic free radicals are reversibly photo-generated by UV and visible light. Melanin photochemistry, particularly the formation and decay of extrinsic radicals, has been the subject of numerous electron spin resonance (ESR) spectroscopy studies. Several melanin monomers exist, and the predominant monomer in a melanin polymer depends on its location within an organism. In skin and hair, melanin differs in content of eumelanin or pheomelanin. Its bioradical character and its susceptibility to UV irradiation makes melanin an excellent indicator for UV-related processes in both skin and hair. The existence of melanin in skin is strongly correlated with the prevention against free radicals/ROS generated by UV radiation. Especially in the skin melanin (mainly eumelanin) ensures the only natural UV protection by eliminating the generated free radicals/ROS. Melanin in hair can be used as a free radical detector for evaluating the efficacy of hair care products. The aim of this study was to investigate the suitability of melanin as protector of skin against UV generated free radicals and as free radical indicator in hair.     

Spectrophotometric methods for quantifying pigmentation in human hair-influence of MC1R genotype and environment.

Eumelanin (brown/black melanin) and pheomelanin (red/yellow melanin) in human hair can be quantified using chemical methods or approximated using spectrophotometric methods. Chemical methods consume greater resources, making them less attractive for epidemiological studies. This investigation sought to identify the spectrophotometric measures that best explain the light-dark continuum of hair color and the measure that is best able to distinguish red hair from nonred hair. Genetic analysis was performed on these two measures to determine the proportion of genetic and environmental influences on variation in these traits. Reflectance curves along the visible spectrum and subjective ratings of hair color were collected from 1730 adolescent twin individuals. Discriminant class analyses were performed to determine the spectrophotometric measure that could best proxy for eumelanin and pheomelanin quantities. The ratio of light reflected in the green portion of the spectrum to that reflected in the red portion of the spectrum was best able to distinguish red hair from nonred hair. Melanocortin 1 receptor (MC1R) genotype explained some, but not all, variation in this measure. Light absorbed in the red portion of the spectrum was best able to explain the light-dark continuum of hair color. Variance components analysis showed that there were qualitatively different genetic influences between males and females for the light-dark continuum of hair. Our results show that spectrophotometric measures approximating variation in eumelanin and pheomelanin may be considered as an alternative to chemical methods in larger epidemiological studies.     

The action spectrum for generation of the primary intermediate revealed by ultrafast absorption spectroscopy studies of pheomelanin

The observation that fair-skinned individuals are more susceptible to skin cancers is commonly explained by invoking an enhanced photoreactivity of the red melanin, pheomelanin compared with the black melanin, eumelanin. For the wavelength range from 500 to 1000 nm, pump-probe spectroscopic measurements reveal the photoexcitation of pheomelanin by UVA light that generates an immediate (< 100 fs) transient absorption centered at 780 nm. Using a tunable femtosecond excitation source, the action spectrum between 300 and 390 nm for generation of the primary intermediate was measured. Similar action spectra are found for the sample with molecular weight (MW) between 1000 and 10 000 and the one with MW > 10 000 fractions of pheomelanin, indicating that the reactive chromophore has a low MW but is present and its photophysics is similar in the aggregated pigment. The shape of the action spectrum differs from the absorption spectrum of bulk melanin and mass-selected fractions but resembles reported absorption spectrum of benzothiazines, oxidation products of 5-S-cysteinyl-dopa, which are formed along the biosynthetic pathway of pheomelanin.     

MC1R,Eumelanin and Pheomelanin: Their Role in Determining the Susceptibility to Skin Cancer

Skin pigmentation is due to the accumulation of two types of melanin granules in the keratinocytes. Besides being the most potent blocker of ultraviolet radiation, the role of melanin in photoprotection is complex. This is because one type of melanin called eumelanin is UV absorbent, whereas the other, pheomelanin, is photounstable and may even promote carcinogenesis. Skin hyperpigmentation may be caused by stress or exposure to sunlight, which stimulates the release of α‐melanocyte stimulating hormone (α‐MSH) from damaged keratinocytes. Melanocortin 1 receptor (MC1R) is a key signaling molecule on melanocytes that responds to α‐MSH by inducing expression of enzymes responsible for eumelanin synthesis. Persons with red hair have mutations in the MC1R causing its inactivation; this leads to a paucity of eumelanin production and makes red‐heads more susceptible to skin cancer. Apart from its effects on melanin production, the α‐MSH/MC1R signaling is also a potent anti‐inflammatory pathway and has been shown to promote antimelanoma immunity. This review will focus on the role of MC1R in terms of its regulation of melanogenesis and influence on the immune system with respect to skin cancer susceptibility.     

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 (PT eCA , 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.     

An electron spin resonance study of gamma-ray irradiated ginseng

Using electron spin resonance (ESR) spectroscopy, we revealed the presence of four radical species in gamma-ray irradiated ginseng (Agaliaceae). Before irradiation, the representative ESR spectrum of ginseng is composed of a sextet centered at g = 2.0, a sharp singlet at the same g-value, and a singlet at about g = 4.0. The first one is attributable to a hyperfine (hf) signal of Mn2+ ion (hf constant: 7.4 mT). The second one is due to an organic free radical. The third one is originated from Fe3+. Upon gamma-ray irradiation, a new ESR (the fourth) signal was detectable in the vicinity of g = 2.0 region. The progressive saturation behaviors of the ESR signals at various microwave power levels were indicative of different relaxation time for those radicals. The anisotropic ESR spectra were detected by the angular rotation of the sample tube. This is due to the existence of anisotropic microcrystalline in the ginseng powder sample.     

PHOTOINDUCED OXYGEN CONSUMPTION IN MELANIN SYSTEMS–II. ACTION SPECTRA and QUANTUM YIELDS FOR PHEOMELANINS

Abstract— Photoinduced oxygen consumption in systems containing synthetic and natural pheomelanins has been studied by ESR spectroscopy using a nitroxide spin probe to monitor oxygen concentration. Action spectra and quantum yields have been determined for melanin from 5-S-cysteinyldopa and for pheomelanins extracted from red human hair and red chicken feathers. For comparison, data also were obtained for eumelanins from black hair and black feathers. The action spectrum for oxygen consumption by cysteinyldopa melanin is closely related to that previously obtained for eumelanins except that it shows slightly less efficiency at intermediate wavelengths (ca. 300–400 nm). Action spectra for the natural pheomelanins resemble either that for cysteinyldopa melanin or that for eumelanin. In general pheomelanins are no more effective than eumelanins in promoting oxygen consumption, i.e. they are no more susceptible to net photooxidation.     

Photo-induced radical formation in cellulose

The characteristics of cellulose radicals produced by photo-irradiation were studied from their ESR spectra. All spectra of the irradiated samples contained a singlet spectrum with a line width of 28 gauss. Spectra consisting mainly of a single absorption line were observed in the initial stage of irradiation. With the elapse of irradiation time, two absorption lines with a line width of 8 gauss were added to the original singlet spectrum, leading to the three absorption lines widths of 8, 28, and 8 gauss, respectively, of which the middle line with a line width of 28 gauss showed the maximum intensity. This three-line spectrum was easily produced when hydrogen peroxide or metallic ions such as Fe³⁺, Ag⁺, Fe²⁺, and Ce⁴⁺ were used as sensitizers. The observed ESR spectra consisted of a singlet with a line width of 28 gauss and a triplet with a line width of 8 gauss and a splitting factor of 28 gauss. The triplet component was more unstable toward warming, as compared with the singlet component. Based on the experimental results, it was suggested that cellulose radicals due to the scissions of cellulose chains corresponding to the ESR spectra with a single line could be the most important factor for the initiation of graft copolymerization.     

Comparison of structural and chemical properties of black and red human hair melanosomes

Melanosomes in black and red human hair are isolated and characterized by various chemical and physical techniques. Different yields of 4-amino-hydroxyphenolanaline by HI hydrolysis (a marker for pheomelanin) and pyrrole-2,3,5-tricarboxylic acid by KMnO(4)/H(+) oxidation (a marker for eumelanin) indicate that the melanosomes in black hair are eumelanosomes, whereas those in red hair are mainly pheomelanosomes. Atomic force microscopy reveals that eumelanosomes and pheomelanosomes have ellipsoidal and spherical shapes, respectively. Eumelanosomes maintain structural integrity upon extraction from the keratin matrix, whereas pheomelanosomes tend to fall apart. The black-hair eumelanosomes have an average of 14.6 +/- 0.5% amino acids content, which is attributed to the internal proteins entrapped in the melanosomes granules. The red-hair melanosomes contain more than 44% of amino acid content even after extensive proteolytic digestion. This high content of amino acids and the poorly reserved integrity of red-hair melanosomes suggest that some proteins are possibly covalently bonded with the melanin constituents in addition to those that are entrapped inside the melanin species. Soluene solubilization assay indicates the absorbance of melanin per gram of sample, adjusted for the amino acid content, is a factor of 2.9 greater for the black-hair melanosomes than the red-hair melanosomes. Metal analysis reveals significant amounts of diverse heavy metal ions bound to the two types of melanosomes. The amount of Cu(II) and Zn(II) are similar but Fe(III) content is four times higher in the red-hair melanosomes. (13)C solid-state nuclear magnetic resonance spectra and infrared spectra are presented and are shown to be powerful techniques for discerning differences in the amino acid contents, the 5,6-dihydroxyindole-2-carboxylic acid:5,6-dihydroxyindole ratio, and the degree of cross-linking in the pigment. Excellent agreement is observed between these spectral results and the chemical degradation data.     

Probing the Motional Behavior of Eumelanin and Pheomelanin with Solid‐State NMR Spectroscopy: New Insights into the Pigment Properties

Melanin is the most widespread pigment in the animal kingdom. Despite its importance, its detailed structure and overall molecular architecture remain elusive. Both eumelanin (black) and pheomelanin (red) occur in the human body. These two melanin compounds show very different responses to UV‐radiation exposure, which could relate to their microscopic features. Herein, the structural properties and motional behavior of natural eu‐ and pheomelanin extracted from black and red human hair are investigated by means of solid‐state NMR spectroscopy. Several 1D and 2D NMR spectroscopic techniques were combined to highlight the differences between the two forms of the pigment. The quantitative analysis of the ¹H NMR wide‐line spectra extracted from 2D ¹H–¹³C LG‐WISE experiments revealed the presence of two dynamically distinguishable components in both forms. Remarkably, the more mobile fraction of the pigment showed a higher mobility with respect to the proteinaceous components that coexist in the melanosome, which is particularly evident for the red pigment. An explanation of the observed effects takes into account the different architecture of the proteinaceous matrix that constitutes the physical substrate onto which melanin polymerizes within the eu‐ and pheomelanosomes. Further insight into the molecular structure of the more mobile fraction of pheomelanin was also obtained by means of the analysis of 2D ¹H–¹³C INEPT experiments. Our view is that not only structural features inherent in the pure pigment, but also the role of the matrix structure in defining the overall melanin supramolecular arrangement and the resulting dynamic behavior of the two melanin compounds should be taken into account to explain their functions. The reported results could pave a new way toward the explanation of the molecular origin of the differences in the photoprotection activity displayed by black and red melanin pigments.     

COMPARISON OF THE EFFECTS OF UV-VISIBLE IRRADIATION OF MELANINS and MELANIN-HEMATOPORPHYRIN COMPLEXES FROM HUMAN BLACK and RED HAIR

Clinical evidence indicates that people with light skin and red hair have a higher incidence of UV radiation-induced diseases including cancer. It is not known whether this is because of the lower protection due to the smaller amounts of eumelanin present in the skin of these people or whether the presence of pheomelanin in their skin is responsible for the higher susceptibility to carcinogenesis. Irradiation of melanoproteins from red hair with UV-visible light has been reported to produce superoxide. Comparative studies on the formation of superoxide during the irradiation of the melanins isolated from human black and red hair (BHM and RHM, respectively) are reported in this paper. These showed that no superoxide formation could be detected in the case of the BHM. whereas the formation of superoxide during the irradiation of RHM could be definitely demonstrated. Irradiation of the RHM with NADH resulted in the oxidation of more NADH than the irradiation of NADH with BHM. The observation that RHM is an active photosensitizer indicates that this property of pheomelanin present in light skin may at least partly be responsible for the harmful effect of radiation on people with light skin and/or red hair. Administration of hematoporphyrin followed by visible irradiation is a currently used mode of therapy for cancer. The present studies have shown that hematoporphyrin is bound to both BHM and RHM. The binding of hematoporphyrin to the melanins increased the formation of superoxide by RHM and the oxidation of NADH by both the melanins. The binding of porphyrins to melanins may have an influence upon the photosensitivity in diseases such as porphyrias and may also affect the therapeutic efficacy of hematoporphyrin.     

Formation of free radicals in photoirradiated cellulose. VII. Radical decay

The ESR spectra of untreated and photosensitized celluloses irradiated with three different ultraviolet light sources, i.e., λ > 2537 Å, λ > 2800 Å, λ > 3400 Å, at 77°K under vacuum were studied. Based on the warm-up process, that is, warming the sample from 77°K to 273°K for a certain time and recorded at 77°K, the decay behavior of free radicals of celluloses was examined for changes of the pattern and the intensities of ESR spectra. For the untreated samples irradiated with light of λ > 2537 Å and λ > 2800 Å, beside the two doublet spectra originating from hydrogen atoms (508 gauss splitting) and formyl radicals (129 gauss splitting), the observed sevenline spectrum was resolved to be a superposition of a singlet (ΔH_msl = 16 gauss), a doublet (24 gauss splitting), a triplet (34 gauss splitting), and a quartet (overall width, 88 gauss) spectrum. For the photosensitized samples irradiated with light of λ > 3400 Å, the 1:1:1 three-line spectrum was resolved to be a superposition of a singlet (ΔH_msl = 27 gauss), a doublet (43 gauss splitting), and a triplet (34 gauss splitting) spectrum. The five-line spectra of the photosensitized samples irradiated with light of λ > 2537 Å and λ > 2800 Å were resolved to be a superposition of a singlet (ΔH_msl = 27 gauss), a doublet (43 gauss splitting), and a triplet (34 gauss splitting) spectrum. Based on these findings, the conclusion was drawn that at least six kinds of spectra, generated from six kinds of radical species, were formed in cellulose irradiated with ultraviolet light under appropriate experimental conditions.     

Melanin from epidermal human melanocytes: Study by pyrolytic GC/MS

Pigmentation of human skin is determined by the presence of melanin, the polymeric pigment that is produced in melanocytes and transferred to adjacent keratinocytes. Epidermal melanocytes produce two distinct types of melanin pigments: eumelanin, composed mainly of indole-type monomers, and pheomelanin that contains benzothiazine-type backbone. Eumelanin protects skin against UV-induced damages, whereas pheomelanin is believed to act as a potent UV photosensitizer and promote carcinogenesis. In this study, pyrolysis in combination with gas chromatography and mass spectrometry (Py-GC/MS) was applied for structural studies of the epidermal pigment isolated from the cultured human melanocytes. The analysis was preceded by investigations of DOPA-originated synthetic eumelanin and pheomelanin standards. This allowed determination of pyrolytic markers for both types of melanin pigments. To obtain additional information on the natural pigment structure, the samples were thermally degraded in the presence of tetramethylammonium hydroxide as the derivatizing agent. It was shown that the analyzed pigment from normal human epidermal melanocytes derived from moderately pigmented skin is of eumelanin type with little incorporation of a pheomelanin component. The results indicate that Py-GC/MS is a rapid and efficient technique for the differentiation of epidermal melanin types and may be an alternative to commonly used methods based on chemical degradation.     

Photoionization thresholds of melanins obtained from free electron laser-photoelectron emission microscopy, femtosecond transient absorption spectroscopy and electron paramagnetic resonance measurements of oxygen photoconsumption

Free electron laser-photoelectron emission microscopy (FEL-PEEM), femtosecond absorption spectroscopy and electron paramagnetic resonance (EPR) measurements of oxygen photoconsumption were used to probe the threshold potential for ionization of eumelanosomes and pheomelanosomes isolated from human hair. FEL-PEEM data show that both pigments are characterized by an ionization threshold at 282 nm. However, pheomelanosomes exhibit a second ionization threshold at 326 nm, which is interpreted to be reflective of the benzothiazine structural motif present in pheomelanin and absent in eumelanin. The lower ionization threshold for pheomelanin is supported by femtosecond transient absorption spectroscopy. Unlike photolysis at 350 nm, following excitation of solubalized synthetic pheomelanin at 303 nm, the transient spectrum observed between 500 and 700 nm matches that for the solvated electron, indicating the photoionization threshold for the solubalized pigment is between 350 and 303 nm. For the same synthetic pheomelanin, EPR oximetry experiments reveal an increased rate of oxygen uptake between 338 nm and 323 nm, narrowing the threshold for photoionization to sit between these two wavelengths. These results on the solubalized synthetic pigment are consistent with the FEL-PEEM results on the human melanosomes. The lower ionization potential observed for pheomelanin could be an important part of the explanation for the greater incidence rate of UV-induced skin cancers in red-haired individuals.     

Detection and quantitation of a pheomelanin component in melanin pigments using pyrolysis–gas chromatography/tandem mass spectrometry system with multiple reaction monitoring mode

Here, we describe the reliable method for the detection and quantitation of a pheomelanin component in melanin pigments. Synthetic melanins with various contents of pheomelanin‐type structural units were thermally degraded, and the multiple reaction monitoring mode was applied to detect the pheomelanin markers in the pyrolysates by GC/MS/MS. The method allowed the specific detection and quantitation of a pheomelanin component in melanin with the incorporation of pheomelanin‐type units as low as 0.05%. Considering highly universal character of the pheomelanin markers, the method could be applied for structural studies of natural melanin pigments being mixtures of eumelanin and pheomelanin. Copyright © 2012 John Wiley & Sons, Ltd.     

On the structure of human hair melanins from an infrared spectroscopy analysis of their interactions with Cu2+ ions.

Melanins were isolated from dark and red human hair and complexed with copper ions at various pH values in a complexing medium. IR spectra of melanins and their Cu2+-complexes for pellets with KBr were obtained. The IR spectra indicate that Cu2+ ions bound to melanins are fixed by different carboxyl and hydroxyl (phenolic and/or alcoholic) groups in the macromolecule. From these results it is concluded that, generally, melanin carboxyl groups are responsible for interactions of metal ions with the melanin molecule. Complexes of melanins isolated from dark and red human hair show structural differences when analysed by IR spectroscopy. Conclusions from these investigations assist in the differentiation of structures of analysed hair melanins. IR spectral analysis of melanin samples and their complexes suggest that melanin samples obtained from red hair may contain eumelanin.     

PHOTOSENSITIZATION OF MELANINS: A COMPARATIVE STUDY

Abstract— Measurements of photosensitized free-radical production and oxygen consumption have been made in several melanin systems. The melanins studied were cysteinyldopa melanin (a model for pheomelanin), natural pheomelanins extracted from red hair and from red chicken feathers, and eumelanin extracted from bovine eyes. Rose Bengal was used as photosensitizer. A significant enhancement in the rates of free radical production and oxygen consumption was found in all systems when sensitizer was present. The largest effects were found with cysteinyldopa melanin and effects on pheomelanins were shown to be greater (by factors of4–6) than on eumelanins.     

Probing the motional behavior of eumelanin and pheomelanin with solid-state NMR spectroscopy: new insights into the pigment properties

Melanin is the most widespread pigment in the animal kingdom. Despite its importance, its detailed structure and overall molecular architecture remain elusive. Both eumelanin (black) and pheomelanin (red) occur in the human body. These two melanin compounds show very different responses to UV-radiation exposure, which could relate to their microscopic features. Herein, the structural properties and motional behavior of natural eu- and pheomelanin extracted from black and red human hair are investigated by means of solid-state NMR spectroscopy. Several 1D and 2D NMR spectroscopic techniques were combined to highlight the differences between the two forms of the pigment. The quantitative analysis of the (1) H?NMR wide-line spectra extracted from 2D (1) H-(13) C LG-WISE experiments revealed the presence of two dynamically distinguishable components in both forms. Remarkably, the more mobile fraction of the pigment showed a higher mobility with respect to the proteinaceous components that coexist in the melanosome, which is particularly evident for the red pigment. An explanation of the observed effects takes into account the different architecture of the proteinaceous matrix that constitutes the physical substrate onto which melanin polymerizes within the eu- and pheomelanosomes. Further insight into the molecular structure of the more mobile fraction of pheomelanin was also obtained by means of the analysis of 2D (1) H-(13) C INEPT experiments. Our view is that not only structural features inherent in the pure pigment, but also the role of the matrix structure in defining the overall melanin supramolecular arrangement and the resulting dynamic behavior of the two melanin compounds should be taken into account to explain their functions. The reported results could pave a new way toward the explanation of the molecular origin of the differences in the photoprotection activity displayed by black and red melanin pigments.     

Melanocortin 1 receptor variants: functional role and pigmentary associations

The significance of human cutaneous pigmentation lies in its protective role against sun-induced DNA damage and photocarcinogenesis. Fair skin and red hair are characterized by a low eumelanin to pheomelanin ratio, and have been associated with increased risk of skin cancer. Cutaneous pigmentation is a complex genetic trait, with more than 120 genes involved in its regulation, among which the melanocortin 1 receptor gene (MC1R) plays a key role. Although a large number of single nucleotide polymorphisms (SNPs) have been identified in pigmentation genes, very few SNPs have been examined in relation to human pigmentary phenotypes and skin cancer risk. Recent GWAS have identified new candidate determinants of pigmentation traits, but MC1R remains the best characterized genetic determinant of human skin and hair pigmentation as well as the more firmly validated low-penetrance skin cancer susceptibility gene. In this review, we will address how the melanocortin system regulates pigmentation, the effect of MC1R variants on the physiologic function of the MC1 receptor, and how specific MC1R variants are associated with distinct human pigmentation phenotypes.