Investigation of Hg species binding biomolecules in dolphin liver combining GC and LC-ICP-MS with isotopic tracers

Hg stable isotopically labeled species were used to assist the investigation of Hg species (i.e. IHg and MeHg) binding biomolecules in the aqueous soluble fraction of white-sided dolphin (Lagenorhynchus acutus) liver homogenate (QC04LH4). By using stable enriched isotopic tracers (199IHg and 201MeHg), the transformation yields of both mercury species (i.e. methylation and demethylation) were evaluated during the different sample preparation steps, such as the tissue lyophilization and the extraction by ultrasonication. Hg species partitioning after fractionation of the sample (cytosolic fraction and residual fraction) was also studied and showed that MeHg is mainly present in the cytosol (60%), while IHg is preferentially associated to the solid residue (88%). The analysis of the water-soluble fraction by size exclusion chromatography-ICP MS revealed the association of Hg species with biomolecules in a wide molecular weight range. The ratio of IHg and MeHg was successively determined in each SEC fraction by GC-ICP-MS. Furthermore the use of isotopic tracers allowed demonstration of specific Hg species affinity with different biomolecule molecular weight fractions. The results indicate a specific affinity of the IHg with a biomolecule at an elution time corresponding to a metallothionein whereas MeHg is associated to a biomolecule of larger molecular weight.