Quantification of Metallothionein by Differential Pulse Polarography Overestimates Concentrations in Crustaceans

Abstract

If metallothionein concentrations in invertebrates are to be used as biomarkers for metal contamination in the aquatic environment, it is imperative thatthe methods used for quantitative analysis are reliable. A review of the literature concerned with quantification of crustacean metallothionein shows that utilization of differential pulse polarography generally results in higher concentrations than any other method. The obvious discrepancies were investigated by experimental comparison of three different methods (enzyme linked immuno sorbent assay (ELISA), a spectrophotometric assay, differential pulse polarography) for determination of metallothionein concentrations in the shore crab Carcinus maenas. Application of an ELISA to cytosolic tissue extracts of unexposed crabs gave basal metallothionein levels of approximately 180 and 80 microg g(-1) dw in midgut gland and gill, respectively; the levels increased 14-fold and 11-fold after exposure to 2 mg l(-1) Cd for 3 weeks. The spectrophotometric assay generally gave 2-fold higher results than the ELISA in unexposed crabs and similar results in Cd-exposed crabs. The determination of metallothionein by differential pulse polarography (successfully applied in vertebrate tissue) was found to be unsuitable for crustacean tissues due to unidentified interfering compounds which led to 5- to 20-fold overestimation of metallothionein levels. The method should not be used unless thoroughly validated in the group of organisms in question.