Eumelanin Precursor Research Articles

New pyrrole acids by oxidative degradation of eumelanins with hydrogen peroxide. Further hints to the mechanism of pigment breakdown

Oxidative degradation of natural and synthetic eumelanins with alkaline H 2O 2 afforded a complex mixture of low molecular weight products which comprised, besides 1 and 2, three novel pyrrole acids. These were isolated and identified as 2-carboxyhydroxymethylpyrrole-3,5-dicarboxylic acid ( 3), 2-carboxymethylpyrrole-3,5-dicarboxylic acid ( 4) and 2-hydroxymethylpyrrole-3,5-dicarboxylic acid ( 5). Investigation of the oxidative degradation of the eumelanin precursors 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid, as well as of the oligomers 6–10 showed that pyrrole acids 3–5 originate by peroxidative disruption of both carboxylated and non-carboxylated indole units, and that pyrrole 4 arises mainly from indole units not substituted at the 7-position. None of the new pyrroles was converted to 1 by treatment with alkaline H 2O 2, suggesting that they are formed by different degradation routes. These results can be accommodated into an improved mechanistic scheme for rationalising the origin of pyrrole acids by H 2O 2 degradation of eumelanins.

Enzyme-linked immunosorbent assay to measure eumelanin precursors in human serum

Lehé, C. L.1; Oomen, L.1; Kammeyer, A.1; Pavel, S.2; Taat, C.3; Westerhof, W.4; Das, P. K.1 Author Information

Melanins in IGR 1 Melanoma Cells

Information on the composition of melanins is obtained by analysis both of 4-amino-3-hydroxyphenylalanine (AHP) after hydriodic acid degradation and of pyrrole-2,3,5-tricarboxylic acid (PTCA) after potassium permanganate oxidation. Analysis of thiazole-4,5-dicarboxylic acid (TDCA) and pyrrole-2,3-dicarboxylic acid (PDCA) after permanganate oxidation, provides additional information on the composition, TDCA on pheomelanin residues, and PDCA on indolic residues without carboxy groups. Using model melanins formed from dopa and cysteinyldopa in different proportions, we found the TDCA/(PTCA+PDCA) ratio to yield a reliable estimate of the relative proportions of pheomelanin and eumelanin. The PDCA/PTCA ratio reflects the relationship between indole residues with and without carboxy groups. We have analyzed degradation products from cultures of IGR 1, an extensively studied melanoma cell line. Cell cultures were harvested after 2, 4, and 7 days. Culture media were changed after 2 days in all series, and also after 4 days in one series harvested at 7 days. Cells without medium change had seven times the amount of melanin found in cultures with medium change. The PDCA/PTCA ratio decreased with increasing amounts of melanin. With increased melanization, eumelanin is increased relatively more than pheomelanin. The cell content of 5-S-cysteinyldopa (5-S-CD) was similar in all cultures, while 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MICA), a eumelanin precursor metabolite, was found in increased amounts of media of heavily pigmented cultures.

The effects of glutathione and ascorbic acid on the oxidations of 6-hydroxydopa and 6-hydroxydopamine

The interactions of ascorbic acid (AA) and reduced glutathione (GSH) in the oxidations of the catecholaminergic neurotoxins 6-hydroxydopa (TOPA) and 6-hydroxydopamine (6-OHDA) were investigated by both high performance liquid chromatography with electrochemical detection (HPLC-ED) and spectrometric methods. These comparative studies showed TOPA and 6-OHDA to be extremely AA nor GSH was found capable of significantly impeding the oxidations of these trihydroxyphenyls, or of regenerating these substances by reducing back their oxidation products, even though such a redox exchange mechanism was demonstrated for AA and the dihydroxyphenyl dopamine. Although ineffective in keeping TOPA and 6-OHDA as reduced molecules, GSH may nevertheless influence the neurotoxicity of trihydroxyphenyls by interacting with their oxidation products forming glutathionyl conjugates, thereby switching the reaction pathway away from potentially toxic eumelanin precursors and toward the production of pheomelanin. Electrochemical analyses established the formation of two oxidation products derived from each trihydroxyphenyl, one detected at −100 mV and the other at + 700 mV. AA had no effect on either oxidation product, where GSH significantly decreased the levels of both oxidation products. The component detected at + 700 mV is the cyclized, reduced leukochrome. The identity of the component detected at −100 mV was not established, but it is considered to be either the p-quinone or the cyclized, oxidized aminochrome.

High Plasma Level of a Eumelanin Precursor, 6-Hydroxy-5-Methoxyindole-2-Carboxylic Acid as a Prognostic Marker for Malignant Melanoma

Melanin synthesis is a biologic property unique to the melanocyte. It is highly elevated in malignant melanoma with the production of both eumelanin (brown/black pigment) and pheomelanin (yellow/red pigment), dihydroxyindole (DHI) and cysteinyldopa (CD), respectively, being major precursors. Melanin metabolites are often released in the urine of patients with disseminated melanoma metastasis (melanuria). To establish a better method for the detection of occult melanoma this study compares the plasma levels of a pheomelanin metabolite, 5-S-CD, and a eumelanin metabolite, 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C), in melanoma and non-melanoma patients and correlates them with tumor thickness and melanoma metastasis. We found a) that the normal plasma levels of 5-S-CD and 6H5M12C are less than 2.22 ng/ml and 1.04 ng/ml, respectively; b) that the group with the normal 6H5MI2C plasma level does not have any metastasis, whereas a normal 5-S-CD level is seen in both non-melanoma and melanoma patients with and without metastasis; c) that a high plasma 6H5MI2C level is seen in all melanoma patients with tumor thickness more than 3.0 mm regardless of the presence or absence of metastasis, whereas in thinner melanoma patients this is seen only in positive metastasis group; and d) that all melanoma patients with positive metastases showed a high plasma 6H5MI2C level (more than 1.75 ng/ml). We conclude that the measurement of plasma levels of melanin metabolites provides a method for detecting occult melanoma metastasis and estimating the prognosis of melanoma patients, plasma 6H5MI2C level being more sensitive and reliable than that of 5-S-CD, and its increased level being a high risk factor.

Preparation of monoclonal mouse antibodies against two specific eu-melanin related compounds

Two eu-melanin precursors, 6-hydroxy-5-methoxyindole-2-carboxylic acid (HM12C) and 5,6-dihydroxyindole-2-carboxylic acid (DHI2C) were synthesized and coupled to bovine serum albumin, hemocyanin and polylysine by the combined action of carbodiimide and succinimide. These indole-carrier conjugates served as antigens for the production of specific antibodies against DHI2C and HMI2C in BALB/c mice. The specificity of these antibodies was tested using a combination of affinity chromatography and ELISA procedures. Polyclonal mouse antibodies reacted with the indole-carrier conjugates, but not with the unbound indole compounds. Monoclonal antibodies from two hybridoma cell lines were obtained from a HMI2C-immunized mouse after a fusion with four subclonings. They reacted with free HMI2C and to a lesser extent with unbound DHI2C. One monoclonal showed 50% inhibition in the ELISA test at concentrations of 0.6 μmol·l −1 and 5 μmol·l −1 for HMI2C and DHI2C, respectively. These antibodies did not show any cross-reactivity with nine structurally related compounds and should be valuable reagents for the detection and quantification of HMI2C and other eu-melanin related compounds.

Measurement of eumelanin precursor metabolites in the urine as a new marker for melanoma metastases

Measurement of eumelanin precursor metabolites in the urine as a new marker for melanoma metastases

Measurement of Eumelanin Precursor Metabolites in the Urine as a New Marker for Melanoma Metastases

This article introduces a rapid high-performance liquid chromatographic assay to measure urinary pheomelanin and eumelanin metabolites, 5-S-cysteinyldopa and indoles, 5(6)-hydroxy-6(5)-methoxyindole-2-carboxylic acid. Our high-performance liquid chromatographic study clearly showed (1) urine of melanoma patients with positive metastasis revealed significant amounts of 5-S-cysteinyldopa and indoles (5,6-dihydroxyindole-2-carboxylic acid plus 6-hydroxy-5-methoxyindole-2-carboxylic acid) above 1 mumol/d and 2 mumol/d, respectively; and (2) in patients with metastasis-free melanoma these melanin metabolites might be excreted into the urine but always less than the two values cited above. As there is a discrepancy regarding the specificity of 5-S-cysteinyldopa as a marker for estimation of melanoma metastasis, high-performance liquid chromatographic measurement of urinary indoles will provide an additional assay in the detection of melanoma metastasis from an early stage. Both melanoma markers were increased in the urine of patients with metastatic melanoma.

Solid-state analysis of eumelanin biopolymers by electron spectroscopy for chemical analysis

The single most misleading aspect of structure elucidation of naturally occurring biopolymers lies in the removal and preparation of the sample for chemical and physical composition analysis. We present a method for the application of electron spectroscopy for chemical analysis (ESCA or XPS (X-ray photoelectron spectroscopy)) to elucidate the chemical composition and organic functional group distribution within one type of biopolymer, eumelanin. In addition, the relative proportions of the different carbon, oxygen, and nitrogen functionalities within these eumelanin precursor model compounds and the polymers generated from them are presented

ChemInform Abstract: Improved Syntheses of 6‐Hydroxy‐5‐methoxy‐ and 5‐Hydroxy‐6‐methoxyindoles and Their O‐Acetates, Analogs of Natural Eumelanin Precursors.

Abstract4‐Benzyloxy‐3‐methoxybenzaldehyde (I) is nitrated and debenzylated to form the derivative (IIa).

Improved syntheses of 6‐hydroxy‐5‐methoxy‐ and 5‐hydroxy‐6‐methoxyindoles and their O‐acetates, analogs of natural eumelanin precursors

AbstractImproved routes for syntheses of gram quantities of the isomeric 5,6‐acetoxymethoxyindoles, and milligram amounts of the 5,6‐hydroxymethoxyindoles, have been developed. They depend on regiospecific nitration of the benzyl ethers of vanillin and isovanillin as the first steps. Improved condensations of 4,5‐acetoxymethoxy‐2‐nitrobenzaldehydes with nitromethane gave 4,5‐acetoxymethoxy‐2,β‐dinitrostyrenes as the key intermediates to all four indoles.