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 o n the high performance liquid chromatography (HPLC) quantitation of sp ecific degradation products of each melanin type. The other requires i mage analysis, transmission electron microscopy (TEM), and stereology. This study was carried out in cultured human melanoma cells and for e ach line, melanins were measured by HPLC and cells were fixed and embe dded as pellets for TEM. Ultrathin sections were treated or not by the alkali elution method allowing the elimination of pheomelanins. The o btained micrographs were analyzed with our image analysis program perm itting the estimation of several primary parameters. Stereology was us ed for estimating melanosomal maturation, intracellular melanins conte nt, and number of melanized melanosomes per cell, for total melanin, e umelanins, or pheomelanins. Our results show a good correlation betwee n both methods for total melanin, particularly when using the cytoplas mic volume density of melanin (r=0.93). Moreover, we report that the n umber of melanized melanosomes per cell and not the melanosomal matura tion is responsible for the differences in total melanin content obser ved between the different cell lines. However, none of the stereologic al melanization parameters was correlated in the case of eumelanins or pheomelanins. In order to demonstrate the utter relevancy of this ste reological approach, utilization of more pigmented melanoma cells, com parative study of HPLC and stereology, in normal epidermal melanocytes and a new evaluation of the alkali elution method in appropriate anim al models would help us to explain the present results.