Metal speciation analysis in eel (Anguilla anguilla) metallothioneins by anionic exchange-FPLC-isotope dilution-ICP-MS

Abstract

The role that different eel liver MT isoforms could play in metal sequestration was investigated here by “post-column” isotope dilution analysis (IDA) coupled to ICP-MS. Specimens of European eel (Anguilla anguilla) were exposed in aquarium tanks to 100 µg L⁻¹ of Cd for three weeks, while a control group was kept away from metal exposure. The separation of possible MT isoforms was carried out by initial size exclusion (SEC) fractionation of eel liver cytosol, followed by anionic exchange fast protein liquid chromatography (AE-FPLC). Once final separation is accomplished, a mixed solution containing the enriched isotopes ¹¹¹Cd, ⁶⁵Cu, and ⁶⁷Zn, was pumped at the exit of the AE column and the ratios ¹¹⁴Cd/¹¹¹Cd, ⁶³Cu/⁶⁵Cu, and ⁶⁴Zn/⁶⁷Zn were monitored “on line” using an ICP-(Q)MS (in order to carry out quantitative studies). Results demonstrated that those metals were preferably bound to one major MT isoform, which would accumulate the excess of Cd in the eels exposed to this toxic metal. Moreover, sulfur analysis by ICP-(DF)MS demonstrated a clear increase of apothionein in that major hepatic MT isoform, pointing to its induction in eels under cadmium exposure. As an example of application of the described metal speciation methodology to wild eels, a field sampling study was carried out in a river. Five eel liver MT isoforms proved to contain Cd, Cu and Zn, while an additional MT form (or a complex with an isoform having different metal stoichiometry), absent in the aquarium samples, did not contain Cu. The quantitative results obtained indicated, both in aquarium and wild conditions, the different preferences for metal sequestration of each eel liver MT. This information could be most valuable to assess the potential of eel liver MTs as biomarkers in environmental studies.