The use of enriched 111Cd as tracer to study de novo cadmium accumulation and quantitative speciation in Anguilla anguilla tissues

In order to determine de novo incorporation of cadmium into fish liver and kidney, the stable isotope Cd-111 was used as tracer and the European eel ( Anguilla anguilla) as model organism. The exposure of animals to Cd-111 ( 100 ng L-1) gave rise to the in vivo dilution of the natural previously existing Cd in the selected tissues and therefore a change of Cd isotope ratios can be measured. The measurement of this new Cd-114/Cd-111 ratio by ICP-MS, and the subsequent application of mathematical calculations based on the isotope dilution methodology, allowed us to quantitatively discriminate between the previously accumulated natural Cd and the isotopically enriched Cd ( de novo incorporated). This was observed for total cadmium in liver and kidney and also for speciated Cd (e.g., that bound to different cytosolic fractions, such as the Metallothionein fraction (MT fraction). In addition, the quantitative results observed pointed out that in liver natural Cd decreased, while kidney received an appreciable amount of endogenous cadmium, supporting the idea that a redistribution of Cd between liver and kidney in fish occurs, as has previously been described in mammals. Moreover, Cd speciation analysis provided the real cadmium balance ( natural Cd versus enriched Cd) in the MT fraction, demonstrating quantitatively the Cd mobilization in hepatic and renal MT fractions. On the other hand, since MTs bind Cd, they were capable of in vivo incorporation of the enriched and stable isotope Cd-111 during the period of exposure. The separation of the Cd-111 labelled MTs was accomplished by anion-exchange (AE) chromatography, which was coupled on-line with ICP-MS detection to investigate the in vivo ability of individual liver MT isoforms for binding cadmium. The Cd-114/Cd-111 ratio, measured in three individual MT isoforms, was comparatively lower in the predominant MT- 2a isoform, which seems to act in the preferential incorporation of the new incoming Cd. On the other hand, the determination of Cd in separated MT isoforms ( by an on-line post-column isotope dilution technique) in conjunction with the calculated Cd isotope ratios has allowed for the first time the determination of both recently ( exogenous) and previously ( endogenous) Cd bound to individual MT isoforms. The results shown here could open the way to the study of other metalloproteins metals preferences, where radioactive techniques are inadequate for metal speciation analysis.