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.

Neuromelanin of the human substantia nigra: a mixed-type melanin.

Model melanins, synthesized with different cysteinyldopamine/dopamine ratios in the incubates, were oxidized with KMnO4 and the resulting compounds were analyzed by HPLC. The ratios between a phaeomelanin-derived compound, thiazole-4,5-dicarboxylic acid (TDCA), and a compound derived from eumelanin, pyrrole-2,3,5-tricarboxylic acid (PTCA), reflected the composition of the model melanins. The neuromelanin of the human substantia nigra was isolated, and the pigment, as well as intact brain tissue from human substantia nigra was oxidized with KMnO4 and the TDCA/PTCA ratios were determined. Analysis of the isolated neuromelanin showed it to contain 2.3% sulfur and 8.1% nitrogen. The sulfur content indicates the pigment is a mixed-type melanin, and the TDCA/PTCA ratio indicates that it consists of units derived from benzothiazines and from indoles in about equal amounts.

The neuromelanin of the human substantia nigra.

The pigment of the human substantia nigra was isolated after extraction of lipids and proteins with 2% sodium cholate in 30% ethanol followed by 2% sodium dodecyl sulfate in 10% glycerol. The pigment was hydrolysed with HI or degraded by treatment with KMNO4 and the samples were examined for compounds known to derive from pheomelanin (4-amino-3-hydroxyphenylalanine, AHP and 4-amino-3-hydroxyphenylethylamine, AHPEA), or from eumelanin (pyrrole-2,3,5-tricarboxylic acid, PTCA). The HI hydrolysis yielded AHPEA in large quantities, indicating cysteinyldopamine as the main source of the pheomelanin moiety of the neuromelanin, but also trace amounts of AHP, derived from cysteinyldopa oxidation products. Dopamine and small quantities of dopa were also obtained by HI hydrolysis of the neuromelanin. The yield of PTCA was low, but the amounts observed show that part of the neuromelanin is of the eumelanin type, a fact compatible with an occasional exhaustion of the glutathione-cysteine reduction system at the site of neuromelanin formation.

5-S-cysteinyldopac in human urine.

5-S-Cysteinyldopac, a compound hitherto demonstrated only in brain tissue, has been isolated and quantified in urine. The urines from 12 individuals were found to contain 20 +/- 9.1 micrograms 5-S-cysteinyldopac/24 hours. Incubation of 5-S-cysteinyldopamine with MAO-containing tissue did not give any formation of 5-S-cysteinyldopac, indicating that this compound is formed by nucleophilic addition of cysteine directly to dopac. The findings give further evidence for a small but significant non-specific oxidation of endogenous catechol derivatives in vivo, a fact to be considered when using 5-S-cysteinyldopa as a measure of pigment metabolism.

Tyrosinase activity in serum from patients with malignant melanoma.

A preparation procedure is presented for the determination of tyrosinase-catalysed stereo-specific dopa oxidase activity in serum. Purification is obtained by separation on a Phenyl-Sepharose hydrophobic interaction column, followed by Con-A-Sepharose chromatography. Five out of seven sera from patients with widespread melanoma metastases were found to contain detectable quantities of tyrosinase. There was no tyrosinase activity in seven sera from patients with other malignancies, nor in six other control sera from individuals without malignancies. One serum which showed high tyrosinase activity was processed as above and studied by SDS-PAGE. A dopa-reactive band with an apparent MW of 66 kD was present in the gel, i.e. at the same place as that of the soluble tyrosinase of cultured human malignant melanoma cells. The protein was found to have the same pI at isoelectric focusing, and eluted in the same way from the preparation columns used, as did soluble tyrosinase.

Dopaquinone addition products in cultured human melanoma cells.

The concentrations of dopa, cysteinyldopas, 5-S-glutathionyldopa, gamma-glutamyl-5-S-cysteinyldopa and 5-S-cysteinylglycinedopa, were analysed in homogenates of cultured human melanoma cells and in culture media. Cysteinyldopas were found to be the major catechol in the cells, with a molar concentration more than a hundred times that of dopa. 5-S-Glutathionyldopa was found in the same amount as dopa, while the quantity of 5-S-cysteinylglycinedopa was one order of magnitude less. gamma-Glutamyl-5-S-cysteinyldopa was not present in detectable amounts. In the medium the concentrations of dopa, 5-S-cysteinylglycinedopa and of 5-S-glutathionyldopa were about one half of those in the cells, while the concentration of cysteinyldopas was about 2%. The ratio between 2-S-cysteinyldopa and 5-S-cysteinyldopa when incubating dopa and cysteine with tyrosinase was identical with the ratio between the analogically synthetised isomers of glutathionyldopa. Consequently, from the calculation of these ratios in cells and media one cannot deduce whether cysteinyldopas arise from the direct addition of cysteine to dopaquinone, or from degradation of glutathionyldopa. Oxidation of 5-S-glutathionyldopa gives a red chromophore with maximum absorption at 480 nm which develops into a black pigment.

Metabolism of 5-S-glutathionyldopa.

5-S-Glutathionyldopa is oxidized at incubation with tyrosinase and dopa producing a black pigment. The reaction proceeds with the formation of two chromophores with absorption spectra similar to those of dopachrome and melanochrome, respectively. Zn2+ catalyses the formation of the melanochrome-like compound. The oxidation of 5-S-glutathionyldopa by dopaquinone, formed by the action of human tyrosinase and mushroom tyrosinase, is considerably slower than that of 5-S-cysteinyldopa. The higher oxidation potential of 5-S-glutathionyldopa, and/or the greater number of dopaquinone molecules necessary for pigment formation from 5-S-glutathionyldopa and/or the formation of tyrosinase-inhibiting products from 5-S-glutathionyldopa can explain the slower oxidation of this compound. The oxidative pathways suggested for 5-S-glutathionyldopa by the present findings may be relevant both in the melanocyte and in non-specific oxidation of cathechols occurring in other cells.

Oxidation of dopa in the skin of black and albino mice.

The dopa oxidase activity of tyrosinase in the skin from albino and black mice was assayed using a technique based on the formation of two diastereomers of 5-S-cysteinyldopa when incubating tissue extracts with both L-dopa and D-dopa as substrates in the presence of cysteine. The amounts of 5-S-L-cysteinyl-L-dopa and 5-S-L-cysteinyl-D-dopa formed were then determined. In the extract from black mouse skin the L-L-diastereomer was produced in more than ten times the amount of the L-D-diastereomer. This stereospecific dopa-oxidation is indicative of the presence of tyrosinase and corresponds well with earlier determinations of the rates of oxidation for human tyrosinase, using L-dopa and D-dopa as substrates. Stereospecific dopa oxidation was absent in albino skin, and the nonspecific dopa-oxidation was two orders of magnitude less than the dopa oxidation in black skin. The study demonstrates the lack of tyrosinase activity in albino skin, and quantifies the non-specific dopa oxidation. The lack of tyrosinase activity in the eluates from albinotic skin was found not to be due to the presence of a tyrosinase inhibitor.

Oxidation of dopa in human albinism.

The urine of an albino woman contained small quantities of 5-S-cysteinyldopa; 6-hydroxy-5-methoxyindole-2-carboxylic acid, a melanin precursor metabolite, was lacking. The 5-S-cysteinyldopa excretion observed may reflect non-specific oxidation of dopa. Two other albino patients showed normal values for the excretion of 5-S-cysteinyldopa and of 6-hydroxy-5-methoxyindole-2-carboxylic acid.

Urinary excretion of 5-S-cysteinyldopa and 6-hydroxy-5-methoxyindole-2-carboxylic acid: differences between pigmented and albino mice.

Urinary excretion of the phaeomelanin precursor 5-S-cysteinyldopa (5-S-CD) and the eumelanin metabolite 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI-2-C) was studied in black and albino mice. The urinary concentration of 5-S-CD was 31.7 ng/ml in black and 16.1 ng/ml in albino mice. The concentration of 6H5MI-2-C was 21.0 ng/ml in the urine of black mice. The compound could not be demonstrated in the urine of albino mice.

Urinary excretion of melanocytic metabolites in fertile women.

Pregnant women and women taking oral contraceptives show urinary excretion values of 5-S-cysteinyldopa and of 6-hydroxy-5-methoxyindole-2-carboxylic acid in the same range as nonpregnant women not taking oral contraceptives. The excretion of these melanoma markers can therefore be used in in the biochemical diagnosis of metastatic melanoma in pregnancy and in women taking oral contraceptives.