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 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 alpha 2-macroglobulin-zinc and ceruloplasmin-copper (firmly-bound species) remained in the serum. The zinc and copper bound with alpha 2-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 alpha 2-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 alpha 2-macroglobulin-zinc and 1% for ceruloplasmin-copper in intra-assay measurements, and 5% for alpha 2-macroglobulin-zinc and 4% for ceruloplasmin-copper in inter-assay measurements), and there is less risk of contamination during separation.
Inagaki
K
K
Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Japan.
Mikuriya
N
N
Morita
S
S
Haraguchi
H
H
Nakahara
Y
Y
Hattori
M
M
Kinosita
T
T
Saito
H
H
D001798:Blood Proteins / Q000378:metabolism*; D002614:Chelating Agents; D003300:Copper / Q000097:blood / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D011485:Protein Binding; D012116:Resins, Plant; D015032:Zinc / Q000097:blood / Q000378:metabolism*
D001798:Blood Proteins; D002614:Chelating Agents; D012116:Resins, Plant; D003300:Copper; D015032:Zinc