DETERMINATION OF PHEOMELANIN BY MEASUREMENT OF AMINOHYDROXYPHENYLALANINE ISOMERS WITH HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

We describe an improved method for the analysis of pheomelanin in biol ogical samples. The method is based on a chemical degradation of the m elanin polymer and HPLC analysis of specific degradation products. Hyd riodic hydrolysis provides 4-amino-3-hydroxyphenylalanine (AHP) and 3- amino-L-tyrosine (AT) which are detected with an electrochemical detec tor. We have examined each step of the analysis and the results are pr esented in this paper. First the samples are hydrolyzed for 16 h. AT a nd AHP are then isolated from the hydrolysates by ion-exchange chromat ography and then separated and quantitated by HPLC and electrochemical detection. The method shows good reproducibility with a total impreci sion below 5.6%. The linearity of the method was shown from 0 to 490 n g AT and 0 to 850 ng AHP per sample, using a melanoma cell suspension (27 mg protein/ml) with up to 24-fold dilutions of the original sample . For cultured ''normal'' human melanocytes a minimal amount of 0.1 mg protein is sufficient for analysis of pheomelanin in the samples. Thi s method provides the opportunity to study the composition of the form ed melanin in cell lines, cultured in different growth media. (C) 1997 Academic Press.