Elsevier

Clinica Chimica Acta

Volume 412, Issues 23–24, 20 November 2011, Pages 2254-2260
Clinica Chimica Acta

Relative exchangeable copper: A new highly sensitive and highly specific biomarker for Wilson's disease diagnosis

https://doi.org/10.1016/j.cca.2011.08.019Get rights and content

Abstract

Wilson disease (WD) is an autosomal recessive inherited disorder of copper metabolism. Failure to diagnose WD can be dramatic leading to irreversible damages. The molecular genetic analysis of ATP7B gene is the reference test for diagnosis but the number of reported mutations of the ATP7B gene is on the rise. The analysis is cumbersome and requires tedious work. Other clinical and biological tests are proposed but it is often difficult to interpret some patients' results. A rapid and reliable biological test for WD diagnosis is still needed.

Analytical reliability of Exchangeable copper (CuEXC) determination procedure is examined by studying the repeatability, the short term stability and stability in frozen serum. Relative exchangeable copper (REC = CuEXC/total copper%) is proposed and evaluated as a new diagnostic test and compared to classic tests used for WD diagnosis.

Sixteen new Wilson disease patients were diagnosed in our institution between January 2009 and May 2011. The different biological tests used for WD diagnosis yielded lower sensitivity and specificity compared to our new biomarker, the REC.

We show that REC is an excellent discriminatory tool for the diagnosis of WD offering 100% sensitivity and 100% specificity.

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.

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