Quantification of the metal distribution in metallothioneins of the human liver by HPLC coupled with ICP-AES

Fractions containing metallothioneins (MT's), extracted from the liver cyto sol of humans, were analysed to determine the complete distribution pattern of the metals copper, cadmium and zinc. Samples of cirrhotic livers which had come from organs removed during transplantation were examined for diffe rences in the trace-element binding pattern. After the extraction of supern atants from the tissue samples, membrane ultrafiltration of the cytosolic s olution was carried out to separate all high-molecular proteins with molecu lar weights >100 kDa. This procedure retains the metal content of the MT's in its initial form, in contrast to the often-used heat treatment of sample s, which changes the copper distribution significantly. The MT's themself w ere isolated using size exclusion and anion exchange chromatography. Their metal content was determined simultaneously on-line by combination with an ICP-AES as element detector. Calibration of the procedure was performed by means of a column by-pass-injection of elemental standards into the separat ion system. The MT content in the samples was calculated using the determin ed metal concentrations and the generally accepted metal/protein ratios for Cu (12:1), Cd (7:1) and Zn (7:1). These values were compared with values r esulting from a Cd-109-saturation-assay. When various liver samples of diff erent pathogenesis were compared, the highest level of Cu-MT was found in p rimary biliary cirrhosis.