Determination of pheomelanin by measurement of aminohydroxyphenylalanine isomers with high-performance liquid chromatography.
We describe an improved method for the analysis of pheomelanin in biological samples. The method is based on a chemical degradation of the melanin polymer and HPLC analysis of specific degradation products. Hydriodic hydrolysis provides 4-amino-3-hydroxyphenylalanine (AHP) and 3-amino-l-tyrosine (AT) which are detected with an electrochemical detector. We have examined each step of the analysis and the results are presented in this paper. First the samples are hydrolyzed for 16 h. AT and AHP are then isolated from the hydrolysates by ion-exchange chromatography and then separated and quantitated by HPLC and electrochemical detection. The method shows good reproducibility with a total imprecision below 5.6%. The linearity of the method was shown from 0 to 490 ng 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. This method provides the opportunity to study the composition of the formed melanin in cell lines, cultured in different growth media.
Kolb
A M
AM
Department of Clinical Chemistry, University Hospital Leiden, RC Leiden, 2300, The Netherlands.
Lentjes
E G
EG
Smit
N P
NP
Schothorst
A
A
Vermeer
B J
BJ
Pavel
S
S
D016475:3T3 Cells; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007536:Isomerism; D008543:Melanins / Q000032:analysis*; D051379:Mice; D008956:Models, Chemical; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis; D014466:Ultraviolet Rays
D008543:Melanins; C018362:pheomelanin; C019253:3-aminotyrosine; D014443:Tyrosine; C071227:4-amino-3-hydroxyphenylalanine