Combining ultrafiltration and diffusive gradients in thin films techniques for speciation/fractionation of Cu and Zn in cytosol of liver of Nile tilapia (Oreochromis niloticus)

Abstract

This work aims to evaluate the size and lability of Cu and Zn bound to proteins in the cytosol of fish liver of Oreochromis niloticus by employing solid phase extraction (SPE), Diffusive gradients in thin films (DGT) and ultrafiltration (UF). SPE was carried out using Chelex-100. DGT containing Chelex-100 as binding agent was employed. Analyte concentrations were determined by ICP-MS. Total Cu and Zn concentration in cytosol (1g of fish liver in 5mL of TRIS-HCl) ranged from 39.6 to 44.3 ng m L−1 and 1498 to 2106 ng mL−1, respectively. Data from UF (10-30 kDa) suggested that Cu and Zn in cytosol was associated to about 70% and 95%, respectively, with high-molecular weight proteins. Cu-metallothionein was not selectively detected (although 28% of Cu was associated with low-molecular weight proteins). However, information about the specific proteins in cytosol will require coupling UF with organic mass spectrometry. Data from SPE showed the presence of labile Cu species of about 17%, while the fraction of labile Zn species was higher than 55%. However, data from DGT suggested a fraction of labile Cu species only of 7% and a labile Zn fraction of 5%. This data, as compared with previous data from literature, suggests that the DGT technique gave a more plausible estimation of the labile pool of Zn and Cu in cytosol. The combination of results from UF and DGT is capable to contribute to the knowledge about the labile and low-molecular pool of Cu and Zn.