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(22-42) Quantitation by Capillary Electrophoresis of Metallothionein in Liver of Aquatic Animal Species. Eberini, Ivano1, Gianazza, Elisabetta1, Regondi, Simona1, McKenzie, David2, Agradi, Elisabetta*,1, 1 Universita' degli Studi di Milano, Milano, Italia2 University of Birmingham, Birmingham, U.K. ABSTRACT- Metallothionein is a stress protein whose synthesis is upregulated by the exposure to heavy metals. It can thus be regarded as a biomarker and its quantitation in organisms from a specific area in the wild used to assess the level of inorganic pollution. Analytical procedures whose performance is marginally affected by changes in protein sequence along the evolutionary scale are suitable for the wide scope investigations required by environmental toxicology. Its peculiar solubility properties allow metallothionein to be specifically enriched in tissue extracts and easily detected by capillary electrophoresis irrespective of the species samples derive from. When analyzed by micellar electrokinetic capillary chromatography (MECC), metallothionein migration lasts approx. 15 min. Several narrow peaks are detected, that elute over a time span of approx. 2 min. To validate the analytical procedure based on MECC, identification of metallothionein in fish liver extracts was by comparison with the migration of a purified rabbit standard. All peaks with migration time around 15 min disappeared when the run was monitored at 280 nm, since metallothionein contain no aromatic aminoacid. Quantitation of metallothionein by MECC closely correlates with results with other analytical procedures. Metallothionein was thus quantitated in liver extracts from fish either living in the wild or raised in aquaculture and exposed to environmental pollution for a short period of time while restrained/confined in net-lined cages. Methallothionein levels were found to correlate with inorganic pollutant concentration in the test area as evaluated by chemical procedures. Correlation was higher for experimentally exposed animals than for natural samples. Indeed free living fish swim relatively long distances and is thus exposed to a varying concentration of different chemicals for different length of time. Research sponsored by EC (CITYFISH) Key words: metallothionein, biomarkers, heavy metals, capillary electrophoresis |
