HPLC Analysis of Pheomelanin Degradation Products in Human Urine

Abstract

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.

Quantitative fluorescence spectra and quantum yield map of synthetic pheomelanin

“…spectral features are simply superpositions of narrower peaks, each corresponding to a particular species within the eumelanin compound. Because the synthesis pathway for pheomelanin is similar to that of eumelanin (the major difference being the presence of cysteine), 29 it is not unreasonable to suspect that a similar chemical disorder model, involving benzothiazine variants rather than 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) units, may also apply to pheomelanin.…”

Selective and Sensitive Determination of Pheomelanin in Biological Samples Using MEKC with Laser-Induced Fluorescence Detection Based on Intramolecular Excimer-Forming Fluorescence Derivatization

“…The pretreatment of the cell cultures, hair samples and melanoma tissue and the determination of the concentration of the protein in the cell suspensions were measured as described previously [8,14]. For the analysis of 4-AHP and 3-AHP, chemical degradation of the samples was performed by hydriodic acid (HI) reduction according to the method of S. Ito et al [11,13].…”

“…gene key for melanin biosynthesis and the subsequent gene manipulation were performed as described previously [18]. Synthetic pheomelanin was prepared by oxidation of L-dopa with cysteine, following the standard procedure with tyrosinase [7,8], and the resulting pheomelanin was centrifuged, dried and stored in a desiccator at room temperature.…”

“…These LC methods, however, usually require complicated mobile phases to completely separate the two marker aminohydroxyphenylalanine (AHP) isomers for the determination of pheomelanin [6][7][8][9][10][11][12][13]. Furthermore, these methods do not provide sufficient sensitivity and selectivity required for the trace analysis of pheomelanin in biological samples.…”

“…Currently, several liquid chromatography (LC) methods based on electrochemical detection (ECD) [6][7][8][9][10][11], UV detection [12,13] for the quantitative analysis of pheomelanin have been reported. These LC methods, however, usually require complicated mobile phases to completely separate the two marker aminohydroxyphenylalanine (AHP) isomers for the determination of pheomelanin [6][7][8][9][10][11][12][13].…”