Melanin standard method: titrimetric analysis.

Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin, and a standard mild isolation and purification protocol for Sepia melanin has been developed (Zeise, doctoral dissertation, Johns Hopkins University, 1991). The goal of the present work, developed using Sepia melanin, was to quantify the bioavailable carboxylic acid groups present in melanin particles. Bioavailability is governed by the accessibility of carboxy groups to the surrounding biological milieu, and is expressed as microequivalents of carboxy group per gram of melanin. The present work was carried out using an heterogeneous slurry of melanin in a nonaqueous system. A standard acidic titrant, and an automatic titrator operating in an equilibrium titration mode were used to characterize and quantify the carboxy group content of Sepia melanins and several other commonly used melanins purified by a standard method (Zeise et al., Pigment Cell Res. [Suppl] 2:48-53, 1992).

Melanin standard method: empirical formula 2.

Natural melanins are composed of two distinct portions; a protein fraction and a chromophoric backbone. There is no unequivocal evidence for covalent bonding between these two fractions, and standard protocols used in protein purification have failed to separate the protein fraction from the chromophoric fraction. In order to study the chromophoric backbone, many workers have resorted to harsh isolation and purification protocols that are now known to degrade and damage the chromophoric portion. These artifactual melanin preparations are poor models for valid chemical, physical, and biological studies. We have developed a mild isolation and purification protocol for melanins that takes into consideration both the particulate nature of natural melanins and the stability characteristics of the chromophoric fraction. Mathematical factoring of the quantitative amino acid data into the elemental analysis was used to obtain the empirical formula of the chromophoric backbone of melanins. The analyses have shown that melanins from various sources have significantly different amino acid compositions and contents, molar C/N ratios, and empirical formulae. This method successfully differentiates melanins from a variety of sources, namely, human hair, Sepia officinalis, Sigma Chemical Company (cat. no. M8631), autoxidation of dopa, and from the feathers of Rhode Island Red chickens. Analytical results from these studies are presented and discussed.

Melanin standard method: particle description.

Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin. There are no standard methods for the isolation, purification, and storage of melanins. Mild methods designed to preserve the native composition and structure of melanin are needed. The specific aim of the present work, using Sepia melanin, was to develop a mild and generally applicable protocol for the isolation and purification of melanins. It is well established that melanin polymers contain a large number of free carboxylic acid residues. These anionic residues are responsible for the cation exchange properties observed for melanins. Heating melanins with hydrochloric acid at reflux has been demonstrated to lead to extensive decarboxylation. Indeed, heat alone has been shown to cause decarboxylation, and care must be exercised to avoid such conditions. By analogy with cation exchange resins, melanins should be isolated and named according to the associated counterion (e.g., Sepia melanin--K+ form). The method reported here avoided extremes in pH and temperature, and was designed to yield melanin in the K+ form. Physical disaggregation of particulate melanin using a wet milling step was also found to facilitate removal of significant quantities of adsorbed protein. The following physical parameters were used to monitor the purification and to characterize the resultant melanin: pH, conductance, particle size, and diffuse reflectance spectroscopy.

Melanin standard method: empirical formula.

Melanin isolated from the ink sac of cuttle fish (Sepia melanin) is a proposed standard for natural eumelanin. Sepia melanin isolated by a standard protocol was submitted for both elemental analysis and quantitative amino acid analysis. The contribution of the detected amino acids to the elemental composition is subtracted from the total elemental analysis, and the resultant elemental composition reflects the composition of the Sepia melanin backbone chromophore. The assumption is made that, for eumelanins, there is only one nitrogen atom per monomeric unit, and thus, the empirical formula for the average monomeric Sepia melanin backbone chromophore was determined. Three key parameters can be determined for any melanin sample; namely, the molar C/N for the average monomeric unit, the formula weight of the average monomeric unit, and the total percent composition of amino acid residues. Three commonly used melanin preparations, namely, natural Sepia melanin, melanin prepared by the in vitro tyrosinase catalyzed polymerization of tyrosine (tyrosine-enzymatic melanin), and a polymer synthesized by the peroxide oxidative polymerization of tyrosine (tyrosine-chemical melanin), have been subjected to this standard method of characterization. Tyrosine-enzymatic and Sepia melanin are quite similar and tyrosine-chemical melanin is fundamentally different from the other two melanins.

Photoprotection by melanin.

This paper is an attempt to summarize the current state of information on melanin and epidermal melanin pigmentation (EMP) as photoprotective agents. The chemistry and biochemistry of melanin (the particle) and its interaction, in its various forms, with UV radiation are considered. Methods of attenuation of UV radiation are discussed in terms of structure and chemical constituents. Photoprotection by constitutive and facultative pigmentation is reviewed with minimum erythema dose (MED) as the end point. The issue of acclimatization to UV radiation is discussed in terms of UVB phototherapy for psoriasis. Finally, skin cancer is considered as an end point and the reduction of its incidence with pigment level is discussed. It is concluded that whilst EMP provides protection, its extent depends on the end point chosen for evaluation. MED is a convenient photobiological end point but is rather insensitive, whereas skin cancer is sensitive but impractical for laboratory studies. Our current state of knowledge of melanin lacks information on its absorption and scattering coefficients and its refractive index. Methods for the quantitative measurement of EMP are also urgently required.

A pulse radiolysis investigation of the oxidation of indolic melanin precursors: evidence for indolequinones and subsequent intermediates.

The rate constants associated with the series of successive transient absorptions initiated by one-electron oxidation of 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA), precursors of melanin, and N-methyl-5,6-dihydroxyindole (NMDHI), a model compound, have been studied by pulse radiolysis. The initial transient species resulting from N3. oxidation reaction at pH 7.3-7.4 are assigned as the corresponding semiquinones. In each case, these radicals decayed, probably by disproportionation, into products most readily monitored in the 400-430 nm region. For DHI, the decay in this region could be fitted by two parent concentration independent first-order processes. These may correspond to transformations between 5,6-indolequinone, and its quinone-imine and quinone-methide tautomers. With NMDHI, on the other hand, a single longer-lived product with a peak around 430 nm predominated after decay of the corresponding radical, due almost certainly to N-methyl-5,6-indolequinone. The data appear to exclude significant melanin polymerisation by condensation of semiquinones, reaction of semiquinones with dihydroxyindoles, self-addition of indolequinones or tautomers, or reaction of indolequinones or tautomers with the parent dihydroxyindoles. It is suggested that polymerisation of melanin may rather occur by stepwise addition of indolequinone methide/imine to reduced oligomeric species.

Chemically induced Parkinson's disease. II: Intermediates in the oxidation and reduction reactions of the 1-methyl-4-phenyl-2,3-dihydropyridinium ion and its deprotonated form.

The one-electron reduction product of 1-methyl-4-phenyl-2,3-dihydropyridinium ion has been generated by pulse radiolysis and its absorption spectrum recorded. This radical was found to decay by second-order kinetics (2k = 9.5 x 10(8) M-1 s-1) to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenyl-2,3-dihydropyridinium ion. Reactions of the above radical species and that formed by one-electron reduction of 1-methyl-4-phenylpyridinium ion, which can also be generated by one-electron oxidation of 1-methyl-4-phenyl-1,2-dihydropyridine, with a number of molecules of biochemical interest have been studied. The one-electron reduction product of oxidised nicotinamide adenine dinucleotide efficiently reduced 1-methyl-4-phenyl-2,3-dihydropyridinium ion (k = 2.2 x 10(9) M-1 s-1). The relevance of these results in relation to redox cycling, a possible mechanism for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity, is discussed.

Sunlight, melanogenesis and radicals in the skin.

Melanocytes are cells of neural crest origin residing at the dermal-epidermal juncture. They produce specialized organelles called melanosomes within which the biochemical processes of melanization occurs. UV radiation is capable of inducing melanogenesis and, during the biosynthesis of melanins, several of the putative precursors "leak out" of the melanosome and can be detected in the skin, serum and urine of individuals undergoing active melanogenesis. Most notable are the cysteinyldopas (formed by nucleophilic addition of cysteine to dopaquinone) and several dihydroxyindoles (formed by intramolecular cyclization of dopaquinone). These catechols often are methylated in the melanocyte to afford a mixture of the monomethoxy derivatives and, in some cases, the dimethoxy species. Recent investigations in our laboratories have demonstrated that the cysteinyldopas, dihydroxyindoles, and their various methylated derivatives are photochemically unstable. Irradiation with biologically relevant ultraviolet radiation (i.e. wavelengths greater than 300 nm) results in the rapid destruction of the precursors/metabolites and the production of a variety of free radical species. The photochemistry and potential photobiological significance of melanogenic intermediates is discussed.

Analysis and quantification of pheomelanins by radioimmunoassay.

An antiserum against alpha-amino-(4-hydroxy-6-benzothiazolyl)propionic acid (AHBP), a major product obtained after hydriodic acid hydrolysis of pheomelanin, was raised in rabbits immunized with AHBP coupled to bovine serum albumin (BSA). The antiserum was used to develop a radioimmunoassay (RIA) to AHBP. The limit of detection of the RIA is 0.1 ng of AHBP. The antiserum does not crossreact with other major hydriodic acid degradation products, and the assay has been used to estimate the amounts of AHBP in synthetic and natural melanins.

Photogeneration of free radicals from eumelanogenic intermediates and metabolites.

We have recently demonstrated that cysteinyldopas, pheomelanogenic precursors and excreted eumelanogenic metabolites, are photolabile and initiate DNA damage in vitro. In this study we have extended our photochemical investigations to eumelanogenic indole intermediates and metabolites. Continuous-wave photolysis of 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA), or its 5-methoxylated metabolite (HMICA) with biologically relevant ultraviolet radiation (i.e., wavelengths greater than 300 nm) resulted in rapid destruction of starting material. Using ESR spin-trapping techniques we observed the initial production of free radical species; prolonged photolysis resulted in the formation of polymeric photoproducts. Radicals were trapped by the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and characterized by their ESR spectra as hydrated electrons and hydrogen atoms. Experiments further demonstrated that while DHI photoionizes, the two indole-2-carboxylic acid derivatives do not ionize appreciably upon irradiation, rather homolysis of X-H bonds appears to be a significant photochemical pathway. The potential photobiological significance of melanogenic indole intermediates is discussed.

Biosynthetic and structural studies on pheomelanin.

13C-NMR spectroscopy of pheomelanin biopolymers, prepared from isotopically enriched precursors, has been developed as a tool for structure elucidation of melanins. By employing large pulse-widths and short cycle time, only the signals originating from labeled carbons are observed in the high-resolution spectra of these polymers.

Chemically induced Parkinson's disease: intermediates in the oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to the 1-methyl-4-phenyl-pyridinium ion.

Various unstable intermediate oxidation states have been postulated in the metabolic activation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to the 1-methyl-4-phenyl pyridinium ion. We now report the first direct observation of these free radical intermediates by pulse radiolysis and flash photolysis. Studies are described of various reactions of such species, in particular with dopamine whose autoxidation to dopamine quinone is reported to be potentiated by 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine.

A pulse radiolysis investigation of the oxidation of the melanin precursors 3,4-dihydroxyphenylalanine (dopa) and the cysteinyldopas.

The unstable quinones of 3,4-dihydroxyphenylalanine (dopa) and the most abundant cysteinyldopa isomers (2S-, 5S- and 2,5S,S'-) have been generated rapidly via disproportionation of their respective semiquinones prepared pulse radiolytically by one-electron oxidation of the corresponding dopas with azide radicals. Dopaquinone decays via a base-catalysed unimolecular cyclisation yielding leucodopachrome which, under the present conditions, is immediately oxidised by remaining dopaquinone to form dopachrome and dopa back again. Addition of cysteine increased the rate of dopaquinone decay and precluded dopachrome formation. By contrast, the cysteinyldopa quinones decayed via an acid-catalysed unimolecular cyclisation involving the cysteine side chain to form a cyclic quinone-imine observed directly for the first time. These quinone-imine intermediates subsequently rearranged to more stable phenolic benzothiazine isomers. The addition of cysteine had little effect on cysteinyldopa quinone decay and did not prevent quinone-imine formation. The absorption spectra, extinction coefficients and rate constants for formation and decay of these various transient species involved in melanisation are reported.