Investigation of metal complexes with metallothionein in rat tissues by hyphenated techniques

The potential of hyphenated techniques based on a combination of microbore reversed-phase (RP) HPLC or capillary zone electrophoresis (CZE) with inductively coupled plasma (ICP) or electrospray (ES) mass spectrometry (MS) was demonstrated for the characterization of metal complexes with metallothionein in rat liver and kidney. The mixture of MT complexes was isolated from the tissues by size-exclusion LC and further characterized in neutral pH conditions (pH 6.8-7.2) by RP-HPLC or CZE. The metal stoichiometry and the molar mass of the eluted complexes was measured by ICP-MS and ES-MS, respectively. An additional dimension to the analysis was achieved by post-column acidification of the chromatographic eluent that allowed the determination of the molecular weight of the demetallated complexes with 10-fold higher sensitivity. The approach allowed the detection of two major metallothionein (MT) isoforms (MT-1 and MT-2) in liver and one MT isoform in kidney. The actual number of peaks in chromatograms and electropherograms was bigger because of the formation of mixed Cd-Cu complexes of the same MT isoform that showed different hydrophobicities.