COMPARISON OF HPLC AND STEREOLOGIC IMAGE-ANALYSIS FOR THE QUANTITATION OF EUMELANINS AND PHEOMELANINS IN NEVUS CELLS AND STIMULATED MELANOMA-CELLS

The aim of the study was to compare two methods of quantitating eumela nins and pheomelanins, pigments synthesized by melanocytes, One is bas ed on the high performance liquid chromatography quantitation of speci fic degradation products of each melanin type. The other requires imag e analysis, transmission electron microscopy, and stereology. In a pre vious study, we showed good correlations between both methods for tota l melanin but not for eumelanins or pheomelanins. We describe here the same comparison in more pigmented cells (nevus cells and stimulated H BL melanoma cells). Transmission electron microscopy micrographs were image analyzed to generate several primary parameters. Stereology was used for estimating melanosomal maturation, intracellular melanin cont ent, and the number of melanized melanosomes per cell, for total melan in, eumelanins, or pheomelanins. Our results showed a good correlation between both methods for total melanin, eumelanins, and pheomelanins with an r equal to 0.99, 0.91, and 0.93, respectively, when all the po ints were used in the linear regression analyses. In the melanoma cell group (HBL cells cultured in media of different compositions), the ch emical and morphometric estimations were not parallel in the case of e umelanins and pheomelanins. In addition, the stereologic and high perf ormance liquid chromatography pheomelanins to eumelanins ratios were s till not correlated. These results demonstrate the relevancy of the st ereologic method, but the low level of melanization, the possible lack of specificity of melanogenesis in melanoma cells, and a problem of s ensitivity of the stereologic method in this context seem to be obstac les in obtaining better results, The utilization of normal human melan ocytes could give some answers to our hypotheses.