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Measurements of serum non-ceruloplasmin copper by a direct fluorescent method specific to Cu(II)

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Published/Copyright: January 11, 2017
Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 55 Issue 9

Abstract

Background:

Meta-analyses indicated the breakdown of copper homeostasis in the sporadic form of Alzheimer’s disease (AD), comprising copper decreases within the brain and copper increases in the blood and the pool not bound to ceruloplasmin (non-Cp Cu, also known in the literature as “free” copper). The calculated non-Cp Cu (Walshe’s) index has many limitations.

Methods:

A direct fluorescent method for non-Cp Cu detection has been developed and data are presented herein. The study included samples from 147 healthy subjects, 36 stable mild cognitive impairment (MCI) and 89 AD patients, who were tested for non-Cp Cu through the direct method, total serum copper, ceruloplasmin concentration and o-dianisidine ceruloplasmin activity. The indirect non-Cp Cu Walshe’s index was also calculated.

Results:

The direct method was linear (0.9–5.9 μM), precise (within-laboratory coefficient variation of 9.7% for low and 7.1% for high measurements), and had a good recovery. A reference interval (0–1.9 μM) was determined parametrically in 147 healthy controls (27–84 years old). The variation of non-Cp Cu was evaluated according to age and sex. Non-Cp Cu was 1.5 times higher in AD patients (regarding the upper value of the reference interval) than in healthy controls. Healthy, MCI and AD subjects were differentiated through the direct non-Cp Cu method [areas under the curve (AUC)=0.755]. Considering a 95% specificity and a 1.91 μmol/L cut-off, the sensitivity was 48.3% (confidence interval 95%: 38%–58%). The likelihood ratio (LR) was 9.94 for positive test results (LR+) and 0.54 for negative test result (LR−).

Conclusions:

The direct fluorescent test reliably and accurately measures non-Cp Cu, thereby determining the probability of having AD.

Keywords: Alzheimer’s disease; ceruloplasmin; copper; metal; mild cognitive impairment; non-ceruloplasmin copper

Acknowledgments

The authors thank Patrizio Pasqualetti, PhD who wrote the statistical results of current investigation and Valentine Negrouk, PhD for precision test replication and measurements of non-Cp Cu in healthy US subjects at IGEA Research Corporation, Miami, FL, USA.

  1. Author contributions: R. Squitti, M. Siotto, E. Cassetta, I. Ghafir El Idrissi and N.A. Colabufo contributed to the experimental design and writing of the manuscript. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: National Research Council, Aging Program 2012–2014, ‘A low-copper diet as a preventive strategy for cognitive disability in Aging’; Italian Health Department 5XMille project ‘Un metodo sensibile, diretto e preciso per misurare il rame Non-legato alla Ceruloplasmina nel siero per applicazione in ambiente clinico’ 02/09/2013 al 31/08/2015; ‘Tolerability and efficacy of Zinc therapy in Mild Cognitive Impairment for treatment and prevention of Alzheimer’s disease: a prospective, randomized, double-blind, parallel, placebo controlled Phase II clinical trial’ (Project Code: CO-2013-02358488); Canox4drug SpA 2013–2016 ‘Non-Ceruloplasmin copper in Alzheimer’s disease’ (Prot. 30/2013). Italian Ministry of Health, Ricerca Corrente (RS).

  3. Employment or leadership: Rosanna Squitti is consultant of IGEA Research Corporation, Miami, FL, USA and owner of 3% stocks Canox4drug SPA; Emanuele Cassetta is consultant of Canox4Drug SPA; other author have no conflict of interests to declare.

  4. Honorarium: None declared.

  5. Competing interests: The funding organization(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.

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Received: 2016-9-19
Accepted: 2016-12-5
Published Online: 2017-1-11
Published in Print: 2017-8-28

©2017 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/cclm-2016-0843
Keywords for this article
Alzheimer’s disease; ceruloplasmin; copper; metal; mild cognitive impairment; non-ceruloplasmin copper

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Mass spectrometry or immunoassay: est modus in rebus
  4. Reviews
  5. The use of liquid chromatography-tandem mass spectrometry for therapeutic drug monitoring of antibiotics in cancer patients
  6. Tackling serum folate test in European countries within the health technology assessment paradigm: request appropriateness, assays and health outcomes
  7. Genetics and Molecular Diagnostics
  8. Genetic diagnosis of α1-antitrypsin deficiency using DNA from buccal swab and serum samples
  9. General Clinical Chemistry and Laboratory Medicine
  10. Serum triglyceride measurements: the commutability of reference materials and the accuracy of results
  11. Variant peptide detection utilizing mass spectrometry: laying the foundations for proteogenomic identification and validation
  12. Evaluation of two fully automated immunoassay based tests for the measurement of 1α,25-dihydroxyvitamin D in human serum and comparison with LC-MS/MS
  13. Parallel diurnal fluctuation of testosterone, androstenedione, dehydroepiandrosterone and 17OHprogesterone as assessed in serum and saliva: validation of a novel liquid chromatography-tandem mass spectrometry method for salivary steroid profiling
  14. Determination of oxycodone and its major metabolites noroxycodone and oxymorphone by ultra-high-performance liquid chromatography tandem mass spectrometry in plasma and urine: application to real cases
  15. Identification and quantitation of phosphatidylethanols in oral fluid by liquid chromatography-tandem mass spectrometry
  16. Relationship between plasma and salivary melatonin and cortisol investigated by LC-MS/MS
  17. Paramagnetic micro-particles as a tool for rapid quantification of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma by UHPLC-MS/MS
  18. Measurements of serum non-ceruloplasmin copper by a direct fluorescent method specific to Cu(II)
  19. The serum concentrations of leptin and MCP-1 independently predict low back pain duration
  20. Immunoassay screening in urine for synthetic cannabinoids – an evaluation of the diagnostic efficiency
  21. Cancer Diagnostics
  22. Study of kallikrein-related peptidase 6 (KLK6) and its complex with α1-antitrypsin in biological fluids
  23. Cardiovascular Diseases
  24. A candidate liquid chromatography mass spectrometry reference method for the quantification of the cardiac marker 1-32 B-type natriuretic peptide
  25. The natriuretic peptide MR-proANP predicts all-cause mortality and adverse outcome in community patients: a 10-year follow-up study
  26. CASZ1 loss-of-function mutation contributes to familial dilated cardiomyopathy
  27. Diabetes
  28. Evaluating new HbA1c methods for adoption by the IFCC and NGSP reference networks using international quality targets
  29. Infectious Diseases
  30. Analytical and diagnostic performance of two automated fecal calprotectin immunoassays for detection of inflammatory bowel disease
  31. Letters to the Editor
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  33. Precision of nonfasting lipid profiles should focus on clinical relevance rather than necessarily obtaining the least variation
  34. Triglyceride concentrations should be measured after elimination of free glycerol to exclude interindividual variations due to adiposity and fasting status
  35. Estimation of the reference interval for serum folate measured with assays traceable to the WHO International Standard
  36. Implausible elevation of peripheral thyroid hormones during therapy with a protein supplement
  37. Interference in Na+ measurements on the Siemens RAPIDPoint® 500 after nortriptyline intoxication: a case report
  38. Usefulness of maternal red cell antibodies to predict hemolytic disease of the fetus and newborn and significant neonatal hyperbilirubinemia: a retrospective study
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