Pheomelanin-related benzothiazole isomers in the urine of patients with diffuse melanosis of melanoma.

BACKGROUND: Currently used as structural markers for pheomelanin identification and quantitation, benzothiazole compounds derived from isomers of cysteinyldopa have been indicated by recent in vitro studies as new potential pheomelanogenesis intermediates. The presence of benzothiazole compounds in the urine of patients with melanoma with or without diffuse melanosis was investigated. METHODS: Hydrophilic interaction liquid chromatography with zwitterionic stationary phase (ZIC-HILIC) and photo-diode array (PDA) detection was used for analysis of 6-(2-amino-2-carboxyethyl)-4-hydroxybenzothiazole-2-carboxylic acid (BTCA-5), and 7-(2-amino-2-carboxyethyl)-4-hydroxybenzothiazole-2-carboxylic acid (BTCA-2), derived from 5-S-cysteinyldopa (5-S-CD) and 2-S-cysteinyldopa (2-S-CD) isomers, respectively. After minimal sample preparation, isocratic chromatography allowed efficient separation of the compounds, which were safely identified by their typical absorption features. RESULTS: Three patients with diffuse melanosis, 16 patients with melanoma (stages III and IV) and three healthy subjects were investigated. The urinary BTCAs were found to be highly associated with melanosis but more loosely to excreted 5-S-CD. Analysis of the pigmented fraction of urine following alkaline hydrogen peroxide degradation and quantitation of BTCAs provided evidence for the presence of pheomelanins at high levels in patients with melanosis. CONCLUSION: Identification of free BTCA isomers in urine provides a significant contribution in the field of urinary melanogens, and has important implications for biosynthetic activity of normal and pathologic melanocytes.

The effect of peripheral enzyme inhibitors on levodopa concentrations in blood and CSF.

Levodopa combined with a dopa-decarboxylase inhibitor, such as carbidopa, shifts the metabolism to the COMT pathway. Adding the peripheral acting COMT inhibitor entacapone provides improvement for patients with PD suffering from motor fluctuations. We studied the effects of the enzyme inhibitors entacapone and carbidopa on the levodopa concentrations in CSF and in blood. Five PD patients with wearing-off underwent lumbar drainage and intravenous microdialysis. Samples were taken 12 h daily for 3 days. Day 1; intravenous levodopa was given, day 2; additional oral entacapone 200 mg tid, day 3; additional oral entacapone 200 mg tid and carbidopa 25 mg bid. Levodopa in CSF and in dialysates was analysed. The AUC for levodopa increased both in blood and CSF when additional entacapone was given alone and in combination with carbidopa. The C(max) of levodopa in both CSF and blood increased significantly. Additional entacapone to levodopa therapy gives an increase of C(max) in CSF and in blood. The increase is more evident when entacapone is combined with carbidopa.

Hydrophilic interaction liquid chromatographic analysis of aminohydroxyphenylalanines from melanin pigments.

Malignant melanomas are more often seen in subjects with light colored skin who tan poorly than in persons who tan more rapidly. This has been attributed to the structure of their pigment, pheomelanin, which differs markedly from the eumelanin of persons with darker skin. To study the hydrolysis products of pheomelanin pigments a new method was developed for analysis of 4-amino-3-hydroxyphenylalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine (3-AHP). Pheomelanin samples were hydrolyzed and extracted with solid-phase extraction columns using strong cation-exchange (SCX) cartridges. Separation of 4-AHP and 3-AHP was achieved on a ZIC-HILIC column (150 mm x 2.1mm I.D.) with a mobile phase consisting of acetonitrile: 0.1 M ammonium acetate buffer, pH 4.5 (82:18, v/v). Detection was performed with an electrochemical detector at +400 mV. Run time was 30 min. The limits of detection were 73 pg and 51 pg for 4-AHP and 3-AHP respectively, using 2 microl injections. Good linearity was found within the range 0.05-5.0 microg/ml. Absolute recovery was 70% and relative recovery was 100%. The AHPs were stable for 1 year in the hydrolyzed samples, for 4 days in the eluates from solid-phase sorbents stored in the refrigerator, and for 2 days diluted with mobile phase and stored in the autosampler at 10 degrees C. The within-day imprecision was <5% and the between-day imprecision was <7% for the two analytes. The method, applied to the analysis of pheomelanin in urine from human melanoma patients, allows the analysis of 30 samples in one set and is suitable for routine work with human hair and melanoma cells. By using the ZIC-HILIC stationary phase, ion-pairing reagents could be avoided, which makes the method suitable to further analysis of degradation products from pheomelanins using mass spectrometric detection.

HPLC analysis of pheomelanin degradation products in human urine.

A sensitive and specific high performance liquid chromatography (HPLC) method was developed to quantify 4-amino-3-hydroxyphenylalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine (3-AHP) in urine. In degradation studies of melanin pigment, 4-AHP and 3-AHP are derived from benzothiazine units of pheomelanin and pheomelanin-related metabolites such as trichochromes. 5-S-Cysteinyldopa-derived benzothiazine products give 4-AHP while 2-S-cysteinyldopa-derived benzothiazine products give 3-AHP. 3-AHP is also derived from nitrotyrosine formed by nitration of tyrosine with reactive nitrogen species. For this reason, the influence of this biological process on the amount of 3-AHP found in biological material have been investigated. The method is based on hydriodic acid hydrolysis of the melanin polymer and reversed-phase HPLC with electrochemical detection of the degradation products 4-AHP and 3-AHP. The mobile phase consists of 25 mM ammonium acetate and sodium octanesulfonate as an ion-pairing reagent. The 4-AHP and 3-AHP peaks were well separated and the detector response was linear within the range 0-2 ng injected for both compounds. With the developed chromatographic system, 4-AHP and 3-AHP showed good separation in the biological samples. There was a strong correlation between 4-AHP and 3-AHP in the urine of 50 malignant melanoma patients and two healthy subjects (R0.977). The two compounds were also strongly correlated with 5-S-cysteinyldopa in urine, the correlation coefficients being 0.862 and 0.907, respectively. The method described is sensitive enough for analysis of pheomelanin in urine and in several other biological samples. The results indicate that 3-AHP in urine is not influenced by excreted 3-nitrotyrosine and the data indicate that pheomelanins are excreted in the urine of melanoma patients.