Abstract
The assay in serum of non-caeruloplasmin copper, as exchangeable copper after complexation with EDTA (ExCu) and total copper has been evaluated and compared in patients with varying c-reactive protein(CRP).
Measurement of ExCu and total copper, range 0.2–47.2 µmol/L, was developed using ICP-MS. The chelating agents EDTA and TEPA were compared over 0.0–10 g/L after incubation with serum for 60 mins followed by ultrafiltration with Amicon 10 kDa filter. The assay for ExCu was optimised with EDTA 3 g/L (8.1 mmol/L) maintained at pH 7.0–8.0 before ultrafiltration. TEPA was not as selective in chelation of copper. Patients n = 82 were studied in relation to changes in inflammatory marker CRP and a group of patients n = 37 with normal CRP.
The ExCu assay gave excellent recoveries (94–102 % but poor recovery for free uncomplexed copper), good repeatability, limit of quantitation 0.19 µmol/l with a provisional reference range 0.48 to 1.63 µmol/L (n = 37 patients). The range for relative exchangeable copper (exchangeable copper divided by total serum copper) was 2.49 to 9.96 %. ExCu was elevated in conditions with increased CRP greater than 100 mg/L suggesting an effect of inflammation on the free copper fraction.
A reliable and reproducible assay for ExCu and total copper has been developed. The upregulated inflammatory state increases the ExCu suggesting excess free copper.
Acknowledgments
We would like to thank colleagues in the department of Clinical Biochemistry of The Royal Liverpool and Broadgreen Hospitals Trust for encouragement during this study. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Research funding: None declared. Employment or leadership: None declared.Honorarium: None declared.
Disclosure statement
The funding organisation(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.