Speciation of protein-binding zinc and copper in human blood serum by chelating resin pre-treatment and inductively coupled plasma mass spectrometry

Abstract

A method for the speciation of zinc and copper binding with proteins in human serum was explored by chelating resin (Chelex-100) pre-treatment and inductively coupled plasma mass spectrometry (ICP-MS). It was shown by a SEC (size-exclusion chromatography)-ICP-MS system that albumin-zinc and albumin-copper (loosely-bound species) could be selectively removed from serum by adsorption on the Chelex-100 resin after the chelating resin pre-treatment, while α₂-macroglobulin-zinc and ceruloplasmin-copper (firmly-bound species) remained in the serum. The zinc and copper bound with α₂-macroglobulin and ceruloplasmin, respectively, were then determined by ICP-MS after batch treatment of the serum samples with the Chelex-100 resin. In addition, the total concentrations of zinc and copper were also determined by ICP-MS after a 20-fold dilution with 0.1 M HNO₃. The albumin-zinc and -copper were estimated as the differences between the concentrations of total and firmly-bound species. The present batch pre-treatment method was applied to the speciation analysis of zinc and copper binding with proteins in sera donated from 25 healthy volunteers as well as from a pregnant woman and a myelodysplastic syndrome patient. The observed concentrations of α₂-macroglobulin-zinc and ceruloplasmin-copper were in the ranges 109–202 ng ml⁻¹ (12.4–31.3% of total zinc) and 513–880 ng ml⁻¹ (90.6–99.7% of total copper), respectively. The present method is simple (only addition of the chelating resin and centrifugation is required) and reproducible (average RSD = 2% for α₂-macroglobulin-zinc and 1% for ceruloplasmin-copper in intra-assay measurements, and 5% for α₂-macroglobulin-zinc and 4% for ceruloplasmin-copper in inter-assay measurements), and there is less risk of contamination during separation.