Comparison of high performance liquid chromatography and stereological image analysis for the quantitation of eumelanins and pheomelanins in melanoma cells.
The aim of the study was to compare two methods quantifying eumelanins and pheomelanins, pigments synthesized by melanocytes. One is based on the high performance liquid chromatography (HPLC) quantitation of specific degradation products of each melanin type. The other requires image analysis, transmission electron microscopy (TEM), and stereology. This study was carried out in cultured human melanoma cells and for each line, melanins were measured by HPLC and cells were fixed and embedded as pellets for TEM. Ultrathin sections were treated or not by the alkali elution method allowing the elimination of pheomelanins. The obtained micrographs were analyzed with our image analysis program permitting the estimation of several primary parameters. Stereology was used for estimating melanosomal maturation, intracellular melanins content, and number of melanized melanosomes per cell, for total melanin, eumelanins, or pheomelanins. Our results show a good correlation between both methods for total melanin, particularly when using the cytoplasmic volume density of melanin (r=0.93). Moreover, we report that the number of melanized melanosomes per cell and not the melanosomal maturation is responsible for the differences in total melanin content observed between the different cell lines. However, none of the stereological melanization parameters was correlated in the case of eumelanins or pheomelanins. In order to demonstrate the utter relevancy of this stereological approach, utilization of more pigmented melanoma cells, comparative study of HPLC and stereology, in normal epidermal melanocytes and a new evaluation of the alkali elution method in appropriate animal models would help us to explain the present results.
Donois
E
E
Unité Facteurs de Défense et de Régulation Cellulaire, UFR de Biologie, Talence, France.
Del Marmol
V
V
Wakamatsu
K
K
Ito
S
S
Ghanem
G
G
Surlève-Bazeille
J E
JE
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D016014:Linear Models; D008543:Melanins / Q000032:analysis*; D008544:Melanocytes / Q000737:chemistry*; D008545:Melanoma; D014407:Tumor Cells, Cultured
D008543:Melanins; C018362:pheomelanin; C041877:eumelanin