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(22-34) Determination of metallothionein and related protein levels in fish tissues for monitoring purposes. Van Campenhout, Karen*,1, Goenaga Infante, Heidi2, Adams, Freddy2, Blust, Ronny1, 1 University of Antwerp (RUCA), Groenenborgerlaan 171, Antwerp, Belgium2 Micro and Trace Analysis Center, University of Antwerp (UIA), Universiteitsplein 1, Antwerp, Belgium ABSTRACT- Heavy metal exposure has been shown to interfere with the metabolism and the intracellular speciation of essential metals altering the cytosolic levels of the different metalloprotein fractions, including metallothioneins (MTs). Fish MT is known to occur in some species in multiple isoforms, while other species only have 1 MT isoform. For integrating MTs in a biomonitoring concept the present information is however incomplete. The biological significance of the different MT isoforms still remains unclear and isoform specific induction and metal binding capacities were reported. As a consequence, there is a growing awareness of the need of combining the popular techniques for determining total MT contents (such as saturation assays) with complementary hybrid techniques capable of separating and detecting the different metalloprotein fractions that are present. The aim of this work was to combine an existing method for determining total MT levels (thiomolybdate saturation method) with potent complementary high performance liquid chromatographic techniques such as Size Exclusion (SE) and Anion-Exchange (AE) coupled 'on-line' with ICP TOF MS (Inductively Coupled Time-of-flight Plasma Mass Spectrometry). Using SE HPLC ICP TOF MS the quasi-simultaneous speciation of 16 isotopes bound to metal-binding proteins of distinct molecular weight was demonstrated using minute volumes of samples. The DLs for Cd, Cu and Zn bound to MT showed that metal speciation at pg levels could be performed in samples from uncontaminated sampling sites. Further speciation studies of the MT isoforms were conducted using AE HPLC ICP TOF MS showing tissue specific induction of the different MT isoforms. Key words: metallothionein, fish, isoform, metal-binding protein |
