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Accurate non-ceruloplasmin bound copper: a new biomarker for the assessment and monitoring of Wilson disease patients using HPLC coupled to ICP-MS/MS

  • Chris F. Harrington EMAIL logo , Geoff Carpenter , James P.C. Coverdale , Leisa Douglas , Craig Mills , Karl Willis and Michael L. Schilsky
Published/Copyright: July 30, 2024
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 63 Issue 2

Abstract

Objectives

Assessment of Wilson disease is complicated, with neither ceruloplasmin, nor serum or urine copper, being reliable. Two new indices, accurate non-ceruloplasmin copper (ANCC) and relative ANCC were developed and applied to a cohort of 71 patients, as part of a Wilson Disease Registry Study.

Methods

Elemental copper-protein speciation was developed for holo-ceruloplasmin quantitation using strong anion exchange chromatography coupled to triple quadrupole inductively coupled plasma mass spectrometry. The serum proteins were separated using gradient elution and measured at m/z 63 (63Cu+) and 48 (32S16O+) using oxygen reaction mode and Cu-EDTA as calibration standard. The ANCC was calculated by subtraction of the ceruloplasmin bound copper from the total serum copper and the RelANCC was the percentage of total copper present as the ANCC.

Results

The accuracy of the holo-ceruloplasmin measurement was established using two certified reference materials, giving a mean recovery of 94.2 %. Regression analysis between the sum of the copper containing species and total copper concentration in the patient samples was acceptable (slope=0.964, intercept=0, r=0.987) and a difference plot, gave a mean difference for copper of 0.38 μmol/L. Intra-day precision for holo-ceruloplasmin at serum copper concentrations of 0.48 and 3.20 μmol/L were 5.2 and 5.6 % CV and the intermediate precision at concentrations of 0.80 and 5.99 μmol/L were 6.4 and 6.4 % CV, respectively. The limit of detection (LOD) and lower limit of quantification (LLOQ) for holo-ceruloplasmin were 0.08 and 0.27 μmol/L as copper, respectively.

Conclusions

ANCC and Relative ANCC are important new diagnostic and monitoring biomarker indices for Wilson disease (WD).

Keywords: ceruloplasmin; copper; holo-ceruloplasmin; exchangeable copper: inductively coupled plasma mass spectrometry; strong anion exchange chromatography; Wilson disease
Corresponding author: Chris F. Harrington, Supra-Regional Assay Service (SAS), Trace Element Laboratory, 15-16 Frederick Sanger Road, Surrey Research Park, Guildford, Surrey, GU2 7YD, UK; and Department of Clinical Biochemistry, Royal Surrey NHS Foundation Trust, Guildford, Surrey, UK, E-mail:

Funding source: Association for Laboratory Medicine

Funding source: Wilson Disease Association (USA)

Acknowledgments

The Wilson Disease Association (USA) for funding to support the Yale Registry Study and analytical development work. The Association for Laboratory Medicine for provision of a research and development award (CH). Dr T. Maheswarans, Countess of Chester Hospital (UK), for samples from a treatment naive WD patient. Dr Ayse Coskun for help with collation of patient samples and details.

  1. Research ethics: The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

  2. Informed consent: Informed consent was obtained from all individuals included in this study, or their legal guardians or wards.

  3. Author contributions: The authors have accepted responsibility for the entire content of this manuscript and approved its submission. The study was conceived by C.F.H. and M.S.; work with the Wilson patients was undertaken by M.S. and other clinicians involved in the Yale Registry Study; experimental data was obtained by G.C., L.D., C.M. and K.W.; data processing was performed by J.P.C.C., G.C., L.D., C.M. and C.F.H.; C.F.H., J.P.C.C. and M.S. wrote the manuscript; all authors contributed to the discussion of the data and revision of the manuscript.

  4. Competing interests: The authors state no conflict of interest.

  5. Research funding: The Wilson Disease Association (USA) for funding to support the Yale Registry Study and analytical development work. The Association for Laboratory Medicine for provision of a research and development award.

  6. Data availability: Not applicable.

  7. Consultant or advisory role: C.H. has membership of the consulting and advisory board of Arbormed Ltd and has received payment for this work. M.S. has received support from Wilson Disease Association, USA, Alexion Ltd, Vivet Therapeutics and Orphalan; he has membership of the consulting and advisory board of Arbormed Ltd; he is Chair (unpaid) of the Medical Advisory Committee of the Wilson Disease Association, USA.

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/cclm-2024-0213).

Received: 2024-02-13
Accepted: 2024-07-12
Published Online: 2024-07-30
Published in Print: 2025-01-29

© 2024 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/cclm-2024-0213
Keywords for this article
ceruloplasmin; copper; holo-ceruloplasmin; exchangeable copper: inductively coupled plasma mass spectrometry; strong anion exchange chromatography; Wilson disease

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  2. Editorial
  3. CD34+ progenitor cells meet metrology
  4. Reviews
  5. Venous blood collection systems using evacuated tubes: a systematic review focusing on safety, efficacy and economic implications of integrated vs. combined systems
  6. The correlation between serum angiopoietin-2 levels and acute kidney injury (AKI): a meta-analysis
  7. Opinion Papers
  8. Advancing value-based laboratory medicine
  9. Clostebol and sport: about controversies involving contamination vs. doping offence
  10. Direct-to-consumer testing as consumer initiated testing: compromises to the testing process and opportunities for quality improvement
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  13. Genetics and Molecular Diagnostics
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