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

A method for the speciation of zinc and copper binding with proteins in hum an serum was explored by chelating resin (Chelex-100) pre-treatment and ind uctively 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 a dsorption on the Chelex-100 resin after the chelating resin pre-treatment, while alpha(2)-macroglobulin-zinc and ceruloplasmin-copper (firmly-bound sp ecies) remained in the serum. The zinc and copper bound with alpha(2)-macro globulin and ceruloplasmin, respectively, were then determined by ICP-MS af ter batch treatment of the serum samples with the Chelex-100 resin. In addi tion, the total concentrations of zinc and copper were also determined by I CP-MS after a 20-fold dilution with 0.1 M HNO3. The albumin-zinc and -coppe r were estimated as the differences between the concentrations of total and firmly-bound species. The present batch pre-treatment method was applied t o 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 m yelodysplastic syndrome patient. The observed concentrations of alpha(2)-ma croglobulin-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 c opper), respectively. The present method is simple (only addition of the ch elating resin and centrifugation is required) and reproducible (average RSD = 2% for alpha(2)-macroglobulin-zinc and 1% for ceruloplasmin-copper in in tra-assay measurements, and 5% for alpha(2)-macroglobulin-zinc and 4% for c eruloplasmin-copper in inter-assay measurements), and there is less risk of contamination during separation.