Relative exchangeable copper: A new highly sensitive and highly specific biomarker for Wilson's disease diagnosis
Highlights
► A rapid and reliable biological test for Wilson disease (WD) diagnosis is still needed. ► Excheangeable Copper (CuEXC) determination revealed to be stable and analytically reliable. ► Relative exchangeable copper (REC=CuEXC/total copper %) is a new biomarker for diagnois of WD. ► REC has 100% specificity and 100% sensitivity in diagnosis of WD.
Introduction
Wilson disease (WD) is an autosomal recessive inherited disorder of copper metabolism resulting in a pathological accumulation of this metal in various organs, mostly liver and brain [1], [2]. The culprit gene, identified as ATP7B in 1993 [3], [4], [5] encodes an ATP-driven copper transporter of the same name. It transports copper at the trans-Golgi network for incorporation into apo-ceruloplasmin (apo-Cp) to form ceruloplasmin (Cp) and regulates copper exocytosis into the bile duct [6]. In WD patients, apo-Cp is still produced by hepatocytes but failure to incorporate Cu during its biosynthesis results in the secretion of an unstable polypeptide with a half-life of 3–5 h instead of 3–5 days for holo-Cp [7], [8], [9]. Mutations of ATP7B gene that completely prevent its function tend to produce more severe phenotypes [10], [11], [12]. In absence of controlled regulation of Cu exocytosis, Cu accumulates in cells and induces widespread free radical-mediated damages [1]. This is followed by hepatocyte apoptosis and the liberation of non-ceruloplasmin-bound copper in the blood. Percentage of copper bound to ceruloplasmin in plasma is debated. It is usually reported to fluctuate between 70 and 95% [13]. We have recently reported that this percentage turned out to be 87% in healthy subjects [14].
Failure to diagnose a WD patient can result in lost opportunities for instauration of treatment leading to irreversible clinical damages and death [15]. Clinical diagnosis (hepatic and neurological signs, detectable Kayser–Fleischer rings), MRI imaging and biological tests such as low serum ceruloplasmin concentration, increased urinary copper excretion, calculated non-ceruloplasmin-bound copper (NCC) and ATP7B mutation testing are common steps used for a diagnosis of WD [16]. However, it is difficult to interpret some patient's results owing to various clinical and biochemical phenotypes. Normal serum ceruloplasmin concentrations may occur in some hepatic or neurologic presentations of WD patients. Kayser–Fleischer (KF) ring is undetectable in many of them [17]. Although hepatic copper measurement is quite discriminatory for the diagnosis, sampling is invasive and the distribution of copper within the liver is often inhomogeneous [16], [18]. Interpreting 24-hour urinary copper excretion can be difficult due to overlap with findings in other types of liver disease. Indeed, patients with certain chronic liver diseases, including autoimmune hepatitis, may have increased basal 24-hour copper excretion [16]. Heterozygous may also have intermediate levels.
In support of the diagnosis of Wilson disease, compared to biochemical testing, molecular genetic analysis gives the definite proof of the disease by identifying the underlying genetic defect but is cumbersome, time-consuming and requires tedious work. Number of reported unknown ATP7B gene variants whose significance is highly questioned is continuously increasing. To date, over 600 mutations have been described with 508 suspected to be disease-causing [19], which makes testing more challenging.
The serum non-ceruloplasmin bound copper concentration (NCC) [NCC = total serum copper (μmol.L–1) − 0.049 × ceruloplasmin (mg.L–1)] has been proposed as a diagnostic test for WD [16], [20]. This fraction, improperly called “free copper”, is supposed to estimate the toxic copper concentration in the blood [21]. But large variations and even negatives values are often encountered due to methods imprecision for low concentrations and to the multitude of factors influencing Cp concentrations in serum [22], [23], [24]. Therefore, a rapid and reliable biological test for the diagnosis of WD is still needed.
In a previous work [25], we proposed a method for analytical determination of two loosely bound copper fractions in plasma/serum:
- –
ultrafiltrable Cu (CuUF) representing Cu bound to low molar mass molecules, such as amino acids, is determined by ultrafiltration of plasma through a membrane able to retain copper-binding-proteins such as albumin (67 kDa), Cp (132 kDa), and transcuprein (270 kDa);
- –
exchangeable Cu (CuEXC) is thought to correspond to the labile fraction of copper complexed to albumin [26], [27], [28], [29], [30]. Some authors used heavy extraction procedures and 65Cu stable isotope for the determination of this fraction [28], [29], [30]. However, CuEXC is easily exchangeable in the presence of high-copper-affinity chelators such as EDTA and it could be determined after the incubation of serum with EDTA during 1 h followed by ultrafiltration of the diluted serum.
CuUF dramatically decreased within few hours after blood sampling and the CuEXC slightly increased within few days [25]. Thus, blood samples had to be treated within less than 15 min after sampling to prevent the reported CuUF decrease. As we noticed during this study, CuUF was not relevant for diagnosis and focus was put on CuEXC.
Here, CuEXC repeatability as well as short term stability (at room temperature) and long term stability (after serum freezing) is examined.
We present the distribution of CuEXC in three different groups of subjects according to their ATP7B mutation status to be compared to a control group (issue from general population). These subjects were referred to our institution (the French National Wilson's disease Center) for a diagnostic investigation of WD and for a familial screening. Finally, CuEXC and the relative exchangeable copper (REC) (namely the CuEXC/total copper ratio) are evaluated as diagnosis biomarkers for WD in this cohort of subjects and are then compared to the usual biomarkers: total Cu, Cp, urinary Cu and NCC.
Section snippets
Determination of biological parameters
For CuUF and CuEXC determination, blood samples were collected in Vacutainer®, trace elements dedicated tubes (Ref. 368380, Becton-Dickinson, Le Pont de Claix, France) and instantaneously transferred to our laboratory to be treated within 15 min [25]. Blood was centrifuged at 3 000 rpm for 10 min and serum was prepared immediately for ultrafiltration.
Serum was immediately ultrafiltered on Amicon® Ultra-4® (Millipore, Molsheim, France) to determine CuUF. For CuEXC determination, serum was diluted
Repeatability and short term stability
CuEXC determination (values ranging between 0.18 and 2.56 μmol.L−1) revealed to be repeatable when it was carried out on 30 paired blood samples separately issued from the same patient (difference between all paired CuEXC values was not different from 0, t = 0.798, p = 0.431). In addition, CuEXC (ranging between 0.49 and 2.58 μmol.L−1) was found to be stable when blood sample was kept at room temperature for 24 h and no difference was noticed between CuEXC values at T0 and T24 (difference between all
Discussion
Clinical and biological manifestations of WD may show considerable variations. Genetic analysis of ATP7B gene remains the most decisive tool but it is greatly hampered by its process length and an increasing number of reported gene mutations. The relative exchangeable copper (REC), proposed and studied in this article, offers an excellent alternative to confirm the diagnosis of WD and to appreciate the toxic fraction of copper in the blood of WD patients.
The results presented here indicate that
Conclusion
The determination of CuEXC, which represents the labile copper, offers great contributions in diagnosing WD. Exchangeable copper offers an accurate view of the copper overload. It was found to be analytically reliable and have good sensitivity and specificity in diagnosing WD. We propose the use of the REC (ratio CuEXC/total copper) as a new biomarker since it revealed to be highly specific and highly sensitive in diagnosing Wilson disease.
References (37)
Wilson's disease
Clin Gastroenterol Hepatol
(2005)- et al.
Isolation and characterization of a human liver cDNA as a candidate gene for Wilson disease
Biochem Biophys Res Commun
(1993) - et al.
Mechanisms of copper incorporation into human ceruloplasmin
J Biol Chem
(2002) - et al.
Kayser–Fleischer ring as the presenting sign of Wilson disease
Am J Ophthalmol
(2002) - et al.
Wilson's disease in patients presenting with liver disease: a diagnostic challenge
Gastroenterology
(1997) - et al.
Diagnostic value of quantitative hepatic copper determination in patients with Wilson's Disease
Clin Gastroenterol Hepatol
(2005) - et al.
Non-ceruloplasmin-bound copper in routine clinical practice in different laboratories
J Trace Elem Med Biol
(2008) Monitoring copper in Wilson's disease
Adv Clin Chem
(2010)- et al.
Value of an enzymatic assay for the determination of serum ceruloplasmin
J Lab Clin Med
(2004) - et al.
Role of copper in thermal stability of human ceruloplasmin
Biophys J
(2008)
Stable isotope pilot study of exchangeable copper kinetics in human blood plasma
J Trace Elem Med Biol
Wilson disease
Presse Med
Abnormal copper metabolism in adult
Rev Med Interne
Systems biology approach to Wilson's disease
Biometals
The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene
Nat Genet
The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene
Nat Genet
Cellular copper distribution: a mechanistic systems biology approach
Cell Mol Life Sci
Turnover of the copper and protein moieties of ceruloplasmin
Nature
Cited by (99)
Semi-synthetic human albumin isoforms: Production, structure, binding capacities and influence on a routine laboratory test
2023, International Journal of Biological MacromoleculesWilson's disease: overview
2023, Medicina ClinicaTrientine tetrahydrochloride versus penicillamine for maintenance therapy in Wilson disease (CHELATE): a randomised, open-label, non-inferiority, phase 3 trial
2022, The Lancet Gastroenterology and HepatologyMaintenance therapy simplification using a single daily dose: A preliminary real-life feasibility study in patients with Wilson disease
2022, Clinics and Research in Hepatology and GastroenterologyCitation Excerpt :Due to the immunological determination of serum ceruloplasmin used herein, non ceruloplasmin-bound copper concentration could not be calculated reliably and was thus not used to assess the effectiveness of SDD. Herein, CuEXC, a biomarker usually used for the diagnosis of WD along with the relative exchangeable copper (REC), was directly measured as it has recently been suggested to be a useful tool for the follow-up of chronic patients [24, 39–43]. Interestingly, in the present study, CuEXC remained stable after years of SDD, which is in favor of the effectiveness of this treatment regimen.
Considerations for optimizing Wilson's disease patients’ long-term follow-up
2022, Gastroenterologia y HepatologiaNon-invasive diagnosis and follow-up of rare genetic liver diseases
2022, Clinics and Research in Hepatology and Gastroenterology