Quantification of metallothionein isoforms in fish liver and its implications for biomonitoring

A rapid, reproducible, and sensitive ion-exchange chromatography (IEC) meth od combined with graphite-furnace atomic absorption spectrometry (GF-AAS) w as developed to separate and quantify basal amounts of both metallothionein (MT) isoforms in dab (Limanda limanda) liver samples. Dab liver homogenate s were saturated with Cd, and obtained cytosols were purified by a:two-step acetone precipitation prior to chromatographic analysis. Metallothionein i soforms were separated by IEC and were subsequently quantified indirectly b y GF-AAS via their Cd contents. The amount of Cd needed for saturation was optimized. The efficiency of Cd saturation and acetone precipitation was pr oven by gel permeation chromatography (GPC) and metal distribution analysis . Based on the method of standard addition, a recovery fdr MT was 98% after acetone precipitation and 68% after IEC. The repeated determination of MT isoforms in a dab liver homogenate resulted in coefficients of variation of approximately 12% for both isoforms. Based on the detection limit for GF-A AS, the calculated detection limit for MT isoforms is 2 ng/mg protein. Ther efore, this method is suitable for monitoring purposes. The implications of isoform-specific measurement for biomarker monitoring are discussed.