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Relative exchangeable copper: A new highly sensitive and highly specific biomarker for Wilson's disease diagnosis
Clinica Chimica Acta 412 (2011) 2254–2260
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Relative exchangeable copper: A new highly sensitive and highly specific biomarker for Wilson's disease diagnosis Souleiman El Balkhi a, d, Jean-Marc Trocello b, d, Joël Poupon a,⁎, Philippe Chappuis c, France Massicot e, Nadège Girardot-Tinant d, France Woimant b, d a
Laboratoire de toxicologie biologique, AP-HP, Hopital Lariboisière, 2, rue Ambroise Paré, 75475 Paris cedex 10, France Service de neurologie, AP-HP, Hopital Lariboisière, 2, rue Ambroise Paré, 75475 Paris cedex 10, France Service de biochimie et de biologie moléculaire, AP-HP, Hopital Lariboisière, 2, rue Ambroise Paré, 75475 Paris cedex 10, France d Centre de référence pour la maladie de Wilson, AP-HP, Hopital Lariboisière, 2, rue Ambroise Paré, 75475 Paris cedex 10, France e Laboratoire de Chimie-Toxicologie Analytique et celullaire, EA 4463, Université Paris Descartes, Faculté de Pharmacie, 4, avenue de l'Observatoire, 75006 Paris, France b c
a r t i c l e
i n f o
Article history: Received 30 June 2011 Received in revised form 5 August 2011 Accepted 11 August 2011 Available online 22 August 2011 Keywords: Exchangeable copper Relative exchangeable copper (REC) Labile copper ROC analysis Wilson disease Diagnosis
a b s t r a c t 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. © 2011 Elsevier B.V. All rights reserved.
1. 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 [3–5] 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 [7–9]. Mutations of ATP7B gene that completely prevent its function tend to produce more severe phenotypes [10–12]. 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
⁎ Corresponding author. Tel.: + 33 1 49 95 66 00; fax: + 33 1 49 95 65 71. E-mail address: [email protected] (J. Poupon). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.08.019
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
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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 [22–24]. 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-bindingproteins 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 [26–30]. Some authors used heavy extraction procedures and 65Cu stable isotope for the determination of this fraction [28–30]. 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.
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precisely one hour before ultrafiltration. The measurements of copper in ultrafiltrates were performed by Zeeman electrothermal atomic absorption spectrometry (ETAAS); 40 μL of sample was injected into the furnace. External calibration was conducted using NaCl 0.9% solution between 50 and 500 nM. All samples were determined in duplicates in different cups. Total serum and urine copper were measured by inductively coupled plasma optical emission spectrometry (ICP-OES) on a JY24 spectrometer (Jobin Yvon, Longjumeau, France). For better precision, serum copper concentrations under 5 μM were reanalyzed by ETAAS on a SIMAA 6100 spectrometer (Perkin Elmer, Courtaboeuf, France) using a 1:8 dilution of serum. Serum ceruloplasmin was determined using a rate immunonephelometric method (IMMAGE®, Beckman Coulter, Villepinte, France). The serum non-ceruloplasmin bound copper concentration (NCC) was calculated following the formula [NCC= total serum copper (μmol.L–1) − 0.049× ceruloplasmin (mg.L–1)]. Molecular genetic testing was performed on the whole coding and intron-exons boundary regions of ATP7B gene. Briefly, genomic DNA was extracted from peripheral blood leukocytes obtained from EDTA anticoagulated blood. dHPLC was used as screening tool for mutation detection followed by sequencing on both DNA strands on an automated sequencer. Most of mutations found (not indicated in the paper) are known as disease-causing mutations as reported in Wilson disease database [31]. 2.2. Repeatability and stability of CuEXC 2.2.1. Repeatability To evaluate the repeatability of the whole process of CuEXC determination, 30 patients were sampled twice and blood samples were separately treated as mentioned before (including EDTA dilution and incubation, ultrafiltration and measurement). The two values were compared as paired samples. In addition, an untreated patient and four healthy volunteers were sampled at 5 different times within 24 h to assess the circadian variation of CuEXC. 2.2.2. Short term stability To prove short term CuEXC stability (24 h), 25 blood samples were collected from 25 patients, centrifuged within 15 min and treated for CuEXC determination at T0. These samples were kept at room temperature and treated for CuEXC determination after 24 h. Values at T0 and T24 were compared as paired samples.
2. Materials and methods
2.2.3. Stability of CuEXC in frozen serum To test the stability of CuEXC in frozen serum, venous blood samples were taken from 80 patients. The serum was separated from cells by centrifugation, treated immediately and aliquots of the used serum were stored at − 40 °C. Serums were then thawed after several days (7–77 days). The frozen serums were thawed at room temperature (during 20–30 min) and treated for CuEXC determination. Values at T0 and Td were compared as paired samples (d is the number of freezing days).
2.1. Determination of biological parameters
2.3. Populations
For CuUF and CuEXC determination, blood samples were collected in Vacutainer®, trace elements dedicated tubes (Ref. 368380, BectonDickinson, 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 with EDTA 3 g.L –1 (1:1) and incubated for
Data were collected for subjects who were referred to the French National Wilson's disease Center in the Neurology Department at Lariboisière Hospital between January 2009 and May 2011 for diagnosis, investigation of features evoking WD, or for a familial screening for WD. We recruited 86 subjects during the study. Among them, 14 subjects (mean age 28.9 years, 5 females and 9 males) presented phenotypic and biological disturbances evoking WD and 72 subjects (mean age 34.4 years, 37 females and 35 males) were issued from general familial screening.
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Using ATP7B mutational analysis, the diagnosis of WD was definitely established only when two disease-causing mutations were found. A subject was considered as “wild-type homozygous” if no ATP7B mutation was found in the concerned exon of the family and as heterozygous if only one ATP7B mutation was found. A control group issue from general population included 62 presumably healthy subjects was used to set reference values (RV) of total Cu, CuUF, CuEXC and 40 of them for Cp and NCC (indicated in Table 1) [25]. The usual clinical features (hepatic, neurological) were investigated and total serum copper, ceruloplasmin concentration, and miction urinary copper were measured at the time of diagnosis, before the initiation of any therapy [32]. 2.4. Statistical analysis For all match paired samples, matched-pairs t-test was used to evaluate CuEXC repeatability and short term stability. Continuous variables (CuEXC, urinary Cu, Cp and NCC) were presented as numbers of patients, means, medians and standard deviation. All groups were compared to each other. Continuous variables which were normally distributed were compared between groups using student t-test. For continuous variables that were not normally distributed in the analyzed groups, comparison between groups was carried out by Kruskal–Wallis analysis of variance (Mann Whitney U test). Receiver operating curves (ROC) analysis was performed to determine sensitivity (Se), specificity (Sp) and predictive values of CuEXC, relative CuEXC (REC), serum Cp and miction urinary copper concentrations. Confidence intervals (CI) were fixed at 95%. Data from the control groups and the three groups of patients were used to perform the ROC analysis. All p values were based on two tailed comparisons and those less than 0.05 were considered to indicate statistical significance. All statistical analysis was performed with GraphPad Prism 5.04 for Windows (GraphPad Softwar, San Diego, CA). Analyse-it was used to statistically compare ROC curves (Analyse-it for Microsoft Excel (version 2.20) Analyse-it Software 2009, Ltd. http://www. analyse-it.com/). 3. Results 3.1. 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 paired CuEXC values was not different from 0, t = 0.991, p = 0.331). Finally, variation of CuEXC when sampled 5 times within 24 h from the same subject did not exceed 6% for the untreated patient and did not exceed 13% for control subjects as shown in Fig. 1. 3.2. Stability of CuEXC in frozen serum As shown in Fig. 2, CuEXC variation does not exceed 10% of the initial values when serum was frozen at −40 °C for 7 days (n = 16) and variations after 14 days (n = 15) were found to be slightly higher than 10% (13% maximum) for few samples. When storage duration was more than 30 days, variations of CuEXC were sporadic and largely exceeded 10% of the initial values. 3.3. Exchangeable copper in diagnosis Diagnosis for the 14 patients was confirmed by genetic WD mutation detection. In addition, 2 WD patients were discovered among the familial screening group yielding to 16 newly WD diagnosed patients in our study. On the other hand, among the 70 subjects of the familial screening: 25 subjects (mean age 37.6 years, 15 females) were identified as wild type homozygous, presenting no phenotypic nor biological disturbance regarding Wilson disease; and 45 subjects (mean age 32.6 years, 22 females) presented only one mutation (heterozygous). The CuEXC concentrations of the 16 newly diagnosed WD patients, before the initiation of any treatment, were at least 2 fold higher than RV obtained from the control group. Table 1 shows their presentation symptom profiles and their CuEXC, CuUF, total Cu, urine Cu, Cp, NCC concentrations and REC values. Only one patient had a CuEXC within the RV. One patient had a miction urinary Cu within RV and 4 of them had Cp concentrations above 0.14 g.L−1 which is the threshold found by Mak et al. (100% specificity and 93% sensitivity in diagnosing WD) [33]. CuUF values were above RV for 9 patients. In Fig. 3, WD patients, wild-type homozygous, heterozygous subjects and control subjects are plot-scattered with respect for each diagnostic test for WD. In Fig. 4, ROC curves for total Cu, Cp, NCC, miction urinary copper, CuEXC and the REC are shown. No difference was noticed between control subjects and wild-typehomozygous for total copper, Cp, CuEXC and REC. Urinary Cu for the control group was not determined but no significant difference between wild-type-homozygous and heterozygous groups was found. However,
Table 1 Presentation symptoms profiles for the 16 WD patients diagnosed by molecular genetic testing and values of CuEXC, CuUF, total Cu, urine Cu, Cp, NCC and REC concentrations.
Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Patient 11 Patient 12 Patient 13 Patient 14 Patient 15 Patient 16
Age
Presentation
52 17 14 39 20 14 51 20 19 34 36 19 54 32 29 13
Neurologic Familial screening Hepatic Neurologic Neurologic Neurologic Hepatic Neurologic Neurologic Neurologic Familial screening Neurologic Hepatic Hepatic Neurologic Hepatic
Total Cu μmol.L−1
Cp mg.L−1
Cu UF μmol.L−1
Cu Exc μmol.L−1
REC%
Urinary miction Cu μmol.L−1
NCC μmol.L−1
(12.5–22.5)
(0.2–0.5)
(0.06–0.16)
(0.62–1.15)
(3.0–8.1)
(0.07–0.80)
b1.6
8.5 7.2 8.4 2.5 13.8 4.6 3.9 3.9 3.4 6.4 7.4 6.9 3.2 9.0 10.7 3.3
0.15 0.12 0.13 0.02 0.20 0.03 0.06 0.05 0.03 0.07 0.14 0.21 0.07 0.15 0.21 0.05
0.22 0.15 0.26 0.16 0.17 0.14 0.24 0.19 0.18 0.12 0.15 0.15 0.18 0.15 0.22 0.13
3.52 2.02 2.04 2.08 3.58 3.34 1.16 3.32 1.94 3.70 1.71 5.06 2.11 2.12 2.54 1.12
41% 28% 24% 82% 26% 73% 30% 86% 57% 58% 23% 73% 65% 24% 24% 34%
3.8 7.4 8.9 2.7 10.5 11.0 0.4 3.4 2.5 1.8 2.9 9.4 2.1 1.5 3.7 1.7
1.15 1.075 1.883 1.55 4 3.08 0.97 2.9 1.94 3.46 0.54 − 3.39 − 0.21 1.65 0.41 0.8
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investigated subjects). CuUF ROC analysis is not shown but reveals to be irrelevant in diagnosis of WD and had poor Se and poor Sp. Except for NCC, specificity of these parameters using the cited cutoffs was higher than 98%. Sensitivities for total Cu, urinary Cu, Cp, NCC, CuEXC and REC were: 69%, 94%, 81%, 75%, 88% and 100%, respectively; which could be satisfying for diagnostic tests. However, in WD, false negatives and false positives could be problematic. Miction urinary Cu yielded to only one false negative and, with a cutoff 1.53 μmol.L−1, CuEXC gives one false positive and 2 false negatives. When taking into account the total Cu by calculating the ratio CuEXC/total Cu, the REC showed no false positive or false negative using 18.5% as cutoff. 4. Discussion
Fig. 1. Exchangeable copper variations within 24 h in four healthy subjects (continuous lines) and an untreated WD patient (broken line). A. CuEXC concentration variations within 24 h, B. Relative Exchangeable Copper (REC) variations within 24 h.
total Cu and Cp were significantly lower in heterozygous (14.3± 3.7 μmol.L−1 and 0.33 ±0.07 mg.L–1 respectively) and mostly in WD patients (6.4 ±3.2 μmol.L−1 and 0.104 ±0.07 mg.L–1 respectively) versus wild-type-homozygous (16.4 ±3.6 μmol.L−1 and 0.37 ± 0.12 mg.L respectively) and control group subjects (16.8 ±4.9 μmol.L−1 and 0.33 ±0.07 mg.L–1 respectively). Urinary Cu in WD patients was significantly higher compared to other studied groups. The ROC curves analysis performed for WD patients and all other groups (including control subjects) suggested that the most useful cutoffs for total Cu, urinary Cu, Cp, NCC, CuEXC and REC are respectively: N7.8 μmol.L −1, N0.84 μmol.L −1, b0.157 g.L −1, N0.8 μmol.L −1, N1.53 μmol.L −1 and b18.5%. No significant differences were found between areas (AUC) of total Cu (0.969), CuEXC (0.999), Cp (0.981), urinary Cu (0.992) and REC (1.00) (p N 0.09 at least). This demonstrates that CuEXC and REC are at least as discriminatory for WD diagnosis as Cp or miction urinary Cu in a population of patients presenting features evoking WD. NCC could be a statistically discriminating marker with NCC cutoff value N 0.8 μmol.L −1 but yielded 2 false negatives and a very large number of false positives (n = 25 namely 27% of
Fig. 2. CuEXC stability in frozen serum. Circles indicate mean values of variation after different days of freezing. Bars indicate SD. Results are expressed as percentages of zero time values.
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 REC has an ideal prognostic accuracy in identifying WD and yielded 100% Se and 100% Sp. REC was the only biomarker without false negatives or positives. Moreover, ROC curve analysis of CuEXC demonstrates that the sensitivity and the specificity of this diagnostic tool are better than those of Cp and comparable to those of urinary Cu. The determination method was successfully applied in routine framework to a large number of subjects. Estimating the “free” copper has been a subject of interest since the incrimination of copper in WD. At present, simple methods of determination of the total body load of copper are not widely available. The NCC was suggested to estimate the Cu load in WD but it revealed to be unreliable as a diagnostic test and had many draw backs as a monitoring test [21]. Therefore, we carried out and validated a method for the determination of both CuUF and CuEXC that allowed us to establish preliminary RV in healthy subjects [25]. This method was then applied in routine framework and CuEXC has been evaluated as a diagnostic tool in WD. Since the toxic copper was thought to be “free”, attention was first directed to CuUF because this fraction represents protein unbound copper. In our study, CuUF values were rather close to RV for all WD patients in the absence of chelator intake and the test was not discriminatory in diagnosing WD. However, we observed that CuUF values vary upon chelator intakes but remain within RV under zinc treatment (not shown). Recently, ultrafiltrable copper has been determined in healthy subjects and WD patients by McMillin et al. [34]. RV in healthy subjects obtained by authors (0–1.6 μmol.L −1) were comparable to our CuEXC values. This is certainly due to the use of EDTA containing tubes for blood sampling. However, results reported in WD patients were intriguingly very elevated, ranging between 11.0 and 45.5 μmol. L −1. The instability of CuUF required the treatment of blood within 15 min after sampling, which hampered our ability to track patients from other institutions. Therefore, CuEXC short term stability and CuEXC stability in frozen serum had to be tested as well as the repeatability of the test when blood is sampled at different times of the day. The presented results show the analytical reliability of CuEXC determination. Indeed, CuEXC determination revealed to be repeatable and the CuEXC in serum was stable at room temperature for 24 h (only stability for delays N48 h was studied in our previous work) and in frozen serum for at least 7 days. Thus, CuEXC could be measurable whenever serum is either immediately frozen after sampling-centrifugation or has less that 24 h. It should be noted that method-dependent variation of ceruloplasmin measurement is still significant and that cutoffs can vary upon laboratory, standardization and the age of the reference subjects [23]. In our study,
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Fig. 3. Diagnostic tests for control subjects (n = 62), wild-type-homozygous (n = 25), heterozygous (n = 45) and for patients with WD (n = 16) depicting: total copper, miction urinary copper, ceruloplasmin, non ceruloplasmin copper, exchangeable copper, Relative CuEXC/total copper. *, $ and § indicate statistical significant difference (p b 0.05) vs controls, homozygous and heterozygous, respectively. Broken lines represent the cutoffs obtained from ROC analysis.
the Cp cutoff offering the best Se and Sp was found to be 0.16 g.L−1 and is in agreement with manufacturer recommendation. In contrast, the Cp cutoff (0.14 g.L−1) suggested by Mak et al. [33] yielded poor sensitivity (62%). Miction urinary copper cutoff determined in our study at 0.84 μmol.L −1 offered the best sensitivity and specificity and is in agreement with 24 h-urinary copper cutoff reported in literature [35]. This suggests that miction urine copper could be comparable to 24-h urine copper as a diagnosis tool. However, Roberts et al. have recently suggested lower cutoff to better diagnose WD due to some atypical presentations in ≈20% of patients but admitted that
the low cutoff led to a reduced specificity [16]. Moreover, contamination of the urinary collection apparatus can readily occur and urine collection is often uncompleted which complicate the interpretation of urinary results. In addition, urinary Cu was reported to increase in some liver diseases involving copper metabolism disturbances [36,37]. Further studies should focus on the use of this new biomarker in a cohort of patients with severe hepatic disturbances background and reference values in children and teenagers have to be established. The contribution of REC and CuEXC in improving the monitoring of WD treated patients is another subject to be studied.
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Fig. 4. ROC curves for different diagnostic tests for WD: total copper (AUC = 0.9699, 95%-CI 0.929–1.01), miction urinary copper (AUC = 0.9937, 95%-CI 0.98–1.007), ceruloplasmin AUC = 0.9808, 95%-CI 0.958–1.0003), non ceruloplasmin copper (AUC = 0.7827, 95%-CI 0.66–0.91), CuEXC (AUC = 0.9841, 95%-CI 0.96–1.00, p b 0.0001) and relative exchangeable copper (AUC = 1.00). Broken lines indicate nonsignificance.
5. 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 [1] Burkhead JL, Gray LW, Lutsenko S. Systems biology approach to Wilson's disease. Biometals 2011;24:455–66. [2] Ferenci P. Wilson's disease. Clin Gastroenterol Hepatol 2005;3:726–33. [3] Bull PC, Thomas GR, Rommens JM, Forbes JR, Cox DW. The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nat Genet 1993;5:327–37. [4] Tanzi RE, Petrukhin K, Chernov I, et al. The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nat Genet 1993;5:344–50.
[5] Yamaguchi Y, Heiny ME, Gitlin JD. Isolation and characterization of a human liver cDNA as a candidate gene for Wilson disease. Biochem Biophys Res Commun 1993;197:271–7. [6] Banci L, Bertini I, Cantini F, Ciofi-Baffoni S. Cellular copper distribution: a mechanistic systems biology approach. Cell Mol Life Sci 2010;67:2563–89. [7] Gitlin D, Janeway CA. Turnover of the copper and protein moieties of ceruloplasmin. Nature 1960;185:693. [8] Hellman NE, Gitlin JD. Ceruloplasmin metabolism and function. Annu Rev Nutr 2002;22:439–58. [9] Hellman NE, Kono S, Mancini GM, Hoogeboom AJ, De Jong GJ, Gitlin JD. Mechanisms of copper incorporation into human ceruloplasmin. J Biol Chem 2002;277:46632–8. [10] Cox DW. Molecular advances in Wilson disease. Prog Liver Dis 1996;14:245–64. [11] Liu M, Cohen EJ, Brewer GJ, Laibson PR. Kayser–Fleischer ring as the presenting sign of Wilson disease. Am J Ophthalmol 2002;133:832–4. [12] Deguti MM, Genschel J, Cancado EL, et al. Wilson disease: novel mutations in the ATP7B gene and clinical correlation in Brazilian patients. Hum Mutat 2004;23: 398. [13] Sedlak E, Wittung-Stafshede P. Discrete roles of copper ions in chemical unfolding of human ceruloplasmin. Biochemistry 2007;46:9638–44. [14] El Balkhi S, Poupon J, Trocello JM, Massicot F, Woimant F, Laprevote O. Human plasma copper proteins speciation by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry. Solutions for columns calibration by sulfur detection. Anal Chem 2010;82:6904–10. [15] Walshe JM. Cause of death in Wilson disease. Mov Disord 2007;22:2216–20.
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[16] Roberts EA, Schilsky ML. Diagnosis and treatment of Wilson disease: an update. Hepatology 2008;47:2089–111. [17] Steindl P, Ferenci P, Dienes HP, et al. Wilson's disease in patients presenting with liver disease: a diagnostic challenge. Gastroenterology 1997;113:212–8. [18] Ferenci P, Steindl-Munda P, Vogel W, et al. Diagnostic value of quantitative hepatic copper determination in patients with Wilson's Disease. Clin Gastroenterol Hepatol 2005;3:811–8. [19] Luoma LM, Deeb TM, Macintyre G, Cox DW. Functional analysis of mutations in the ATP loop of the Wilson disease copper transporter, ATP7B. Hum Mutat 2010;31:569–77. [20] Twomey PJ, Viljoen A, Reynolds TM, Wierzbicki AS. Non-ceruloplasmin-bound copper in routine clinical practice in different laboratories. J Trace Elem Med Biol 2008;22:50–3. [21] Walshe JM. Monitoring copper in Wilson's disease. Adv Clin Chem 2010;50: 151–63. [22] Twomey PJ, Viljoen A, House IM, Reynolds TM, Wierzbicki AS. Relationship between serum copper, ceruloplasmin, and non-ceruloplasmin-bound copper in routine clinical practice. Clin Chem 2005;51:1558–9. [23] Mak CM, Lam CW. Diagnosis of Wilson's disease: a comprehensive review. Crit Rev Clin Lab Sci 2008;45:263–90. [24] Macintyre G, Gutfreund KS, Martin WR, Camicioli R, Cox DW. Value of an enzymatic assay for the determination of serum ceruloplasmin. J Lab Clin Med 2004;144: 294–301. [25] El Balkhi S, Poupon J, Trocello JM, et al. Determination of ultrafiltrable and exchangeable copper in plasma: stability and reference values in healthy subjects. Anal Bioanal Chem 2009;394:1477–84.
[26] Musci G, Fraterrigo TZ, Calabrese L, McMillin DR. On the lability and functional significance of the type 1 copper pool in ceruloplasmin. J Biol Inorg Chem 1999;4:441–6. [27] Sedlak E, Zoldak G, Wittung-Stafshede P. Role of copper in thermal stability of human ceruloplasmin. Biophys J 2008;94:1384–91. [28] Buckley WT, Vanderpool RA. Analytical variables affecting exchangeable copper determination in blood plasma. Biometals 2008;21:601–12. [29] Venelinov TI, Beattie JH, Dainty JR, Hollands WJ, Fairweather-Tait SJ, Harvey LJ. Stable isotope pilot study of exchangeable copper kinetics in human blood plasma. J Trace Elem Med Biol 2007;21:138–40. [30] Venelinov TI, Davies IM, Beattie JH. Dialysis-Chelex method for determination of exchangeable copper in human plasma. Anal Bioanal Chem 2004;379:777–80. [31] Cox DW, Bugbee D, Davies L, Kenney S. WD mutation database; 2011. [32] Trocello JM, Chappuis P, Chaine P, et al. Wilson disease. Presse Med 2009;38:1089–98. [33] Mak CM, Lam CW, Tam S. Diagnostic accuracy of serum ceruloplasmin in Wilson disease: determination of sensitivity and specificity by ROC curve analysis among ATP7B-genotyped subjects. Clin Chem 2008;54:1356–62. [34] McMillin GA, Travis JJ, Hunt JW. Direct measurement of free copper in serum or plasma ultrafiltrate. Am J Clin Pathol 2009;131:160–5. [35] Dhawan A, Taylor RM, Cheeseman P, De Silva P, Katsiyiannakis L, Mieli-Vergani G. Wilson's disease in children: 37-year experience and revised King's score for liver transplantation. Liver Transpl 2005;11:441–8. [36] Trocello JM, Chappuis P, El Balkhi S, et al. Abnormal copper metabolism in adult. Rev Med Interne 2010;31:750–6. [37] Nicastro E, Ranucci G, Vajro P, Vegnente A, Iorio R. Re-evaluation of the diagnostic criteria for Wilson disease in children with mild liver disease. Hepatology 2010;52:1948–56.
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Relative exchangeable copper: A new highly sensitive and highly specific biomarker for Wilson's disease diagnosis Souleiman El Balkhi a, d, Jean-Marc Trocello b, d, Joël Poupon a,⁎, Philippe Chappuis c, France Massicot e, Nadège Girardot-Tinant d, France Woimant b, d a
Laboratoire de toxicologie biologique, AP-HP, Hopital Lariboisière, 2, rue Ambroise Paré, 75475 Paris cedex 10, France Service de neurologie, AP-HP, Hopital Lariboisière, 2, rue Ambroise Paré, 75475 Paris cedex 10, France Service de biochimie et de biologie moléculaire, AP-HP, Hopital Lariboisière, 2, rue Ambroise Paré, 75475 Paris cedex 10, France d Centre de référence pour la maladie de Wilson, AP-HP, Hopital Lariboisière, 2, rue Ambroise Paré, 75475 Paris cedex 10, France e Laboratoire de Chimie-Toxicologie Analytique et celullaire, EA 4463, Université Paris Descartes, Faculté de Pharmacie, 4, avenue de l'Observatoire, 75006 Paris, France b c
a r t i c l e
i n f o
Article history: Received 30 June 2011 Received in revised form 5 August 2011 Accepted 11 August 2011 Available online 22 August 2011 Keywords: Exchangeable copper Relative exchangeable copper (REC) Labile copper ROC analysis Wilson disease Diagnosis
a b s t r a c t 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. © 2011 Elsevier B.V. All rights reserved.
1. 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 [3–5] 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 [7–9]. Mutations of ATP7B gene that completely prevent its function tend to produce more severe phenotypes [10–12]. 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
⁎ Corresponding author. Tel.: + 33 1 49 95 66 00; fax: + 33 1 49 95 65 71. E-mail address: [email protected] (J. Poupon). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.08.019
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
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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 [22–24]. 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-bindingproteins 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 [26–30]. Some authors used heavy extraction procedures and 65Cu stable isotope for the determination of this fraction [28–30]. 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.
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precisely one hour before ultrafiltration. The measurements of copper in ultrafiltrates were performed by Zeeman electrothermal atomic absorption spectrometry (ETAAS); 40 μL of sample was injected into the furnace. External calibration was conducted using NaCl 0.9% solution between 50 and 500 nM. All samples were determined in duplicates in different cups. Total serum and urine copper were measured by inductively coupled plasma optical emission spectrometry (ICP-OES) on a JY24 spectrometer (Jobin Yvon, Longjumeau, France). For better precision, serum copper concentrations under 5 μM were reanalyzed by ETAAS on a SIMAA 6100 spectrometer (Perkin Elmer, Courtaboeuf, France) using a 1:8 dilution of serum. Serum ceruloplasmin was determined using a rate immunonephelometric method (IMMAGE®, Beckman Coulter, Villepinte, France). The serum non-ceruloplasmin bound copper concentration (NCC) was calculated following the formula [NCC= total serum copper (μmol.L–1) − 0.049× ceruloplasmin (mg.L–1)]. Molecular genetic testing was performed on the whole coding and intron-exons boundary regions of ATP7B gene. Briefly, genomic DNA was extracted from peripheral blood leukocytes obtained from EDTA anticoagulated blood. dHPLC was used as screening tool for mutation detection followed by sequencing on both DNA strands on an automated sequencer. Most of mutations found (not indicated in the paper) are known as disease-causing mutations as reported in Wilson disease database [31]. 2.2. Repeatability and stability of CuEXC 2.2.1. Repeatability To evaluate the repeatability of the whole process of CuEXC determination, 30 patients were sampled twice and blood samples were separately treated as mentioned before (including EDTA dilution and incubation, ultrafiltration and measurement). The two values were compared as paired samples. In addition, an untreated patient and four healthy volunteers were sampled at 5 different times within 24 h to assess the circadian variation of CuEXC. 2.2.2. Short term stability To prove short term CuEXC stability (24 h), 25 blood samples were collected from 25 patients, centrifuged within 15 min and treated for CuEXC determination at T0. These samples were kept at room temperature and treated for CuEXC determination after 24 h. Values at T0 and T24 were compared as paired samples.
2. Materials and methods
2.2.3. Stability of CuEXC in frozen serum To test the stability of CuEXC in frozen serum, venous blood samples were taken from 80 patients. The serum was separated from cells by centrifugation, treated immediately and aliquots of the used serum were stored at − 40 °C. Serums were then thawed after several days (7–77 days). The frozen serums were thawed at room temperature (during 20–30 min) and treated for CuEXC determination. Values at T0 and Td were compared as paired samples (d is the number of freezing days).
2.1. Determination of biological parameters
2.3. Populations
For CuUF and CuEXC determination, blood samples were collected in Vacutainer®, trace elements dedicated tubes (Ref. 368380, BectonDickinson, 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 with EDTA 3 g.L –1 (1:1) and incubated for
Data were collected for subjects who were referred to the French National Wilson's disease Center in the Neurology Department at Lariboisière Hospital between January 2009 and May 2011 for diagnosis, investigation of features evoking WD, or for a familial screening for WD. We recruited 86 subjects during the study. Among them, 14 subjects (mean age 28.9 years, 5 females and 9 males) presented phenotypic and biological disturbances evoking WD and 72 subjects (mean age 34.4 years, 37 females and 35 males) were issued from general familial screening.
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Using ATP7B mutational analysis, the diagnosis of WD was definitely established only when two disease-causing mutations were found. A subject was considered as “wild-type homozygous” if no ATP7B mutation was found in the concerned exon of the family and as heterozygous if only one ATP7B mutation was found. A control group issue from general population included 62 presumably healthy subjects was used to set reference values (RV) of total Cu, CuUF, CuEXC and 40 of them for Cp and NCC (indicated in Table 1) [25]. The usual clinical features (hepatic, neurological) were investigated and total serum copper, ceruloplasmin concentration, and miction urinary copper were measured at the time of diagnosis, before the initiation of any therapy [32]. 2.4. Statistical analysis For all match paired samples, matched-pairs t-test was used to evaluate CuEXC repeatability and short term stability. Continuous variables (CuEXC, urinary Cu, Cp and NCC) were presented as numbers of patients, means, medians and standard deviation. All groups were compared to each other. Continuous variables which were normally distributed were compared between groups using student t-test. For continuous variables that were not normally distributed in the analyzed groups, comparison between groups was carried out by Kruskal–Wallis analysis of variance (Mann Whitney U test). Receiver operating curves (ROC) analysis was performed to determine sensitivity (Se), specificity (Sp) and predictive values of CuEXC, relative CuEXC (REC), serum Cp and miction urinary copper concentrations. Confidence intervals (CI) were fixed at 95%. Data from the control groups and the three groups of patients were used to perform the ROC analysis. All p values were based on two tailed comparisons and those less than 0.05 were considered to indicate statistical significance. All statistical analysis was performed with GraphPad Prism 5.04 for Windows (GraphPad Softwar, San Diego, CA). Analyse-it was used to statistically compare ROC curves (Analyse-it for Microsoft Excel (version 2.20) Analyse-it Software 2009, Ltd. http://www. analyse-it.com/). 3. Results 3.1. 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 paired CuEXC values was not different from 0, t = 0.991, p = 0.331). Finally, variation of CuEXC when sampled 5 times within 24 h from the same subject did not exceed 6% for the untreated patient and did not exceed 13% for control subjects as shown in Fig. 1. 3.2. Stability of CuEXC in frozen serum As shown in Fig. 2, CuEXC variation does not exceed 10% of the initial values when serum was frozen at −40 °C for 7 days (n = 16) and variations after 14 days (n = 15) were found to be slightly higher than 10% (13% maximum) for few samples. When storage duration was more than 30 days, variations of CuEXC were sporadic and largely exceeded 10% of the initial values. 3.3. Exchangeable copper in diagnosis Diagnosis for the 14 patients was confirmed by genetic WD mutation detection. In addition, 2 WD patients were discovered among the familial screening group yielding to 16 newly WD diagnosed patients in our study. On the other hand, among the 70 subjects of the familial screening: 25 subjects (mean age 37.6 years, 15 females) were identified as wild type homozygous, presenting no phenotypic nor biological disturbance regarding Wilson disease; and 45 subjects (mean age 32.6 years, 22 females) presented only one mutation (heterozygous). The CuEXC concentrations of the 16 newly diagnosed WD patients, before the initiation of any treatment, were at least 2 fold higher than RV obtained from the control group. Table 1 shows their presentation symptom profiles and their CuEXC, CuUF, total Cu, urine Cu, Cp, NCC concentrations and REC values. Only one patient had a CuEXC within the RV. One patient had a miction urinary Cu within RV and 4 of them had Cp concentrations above 0.14 g.L−1 which is the threshold found by Mak et al. (100% specificity and 93% sensitivity in diagnosing WD) [33]. CuUF values were above RV for 9 patients. In Fig. 3, WD patients, wild-type homozygous, heterozygous subjects and control subjects are plot-scattered with respect for each diagnostic test for WD. In Fig. 4, ROC curves for total Cu, Cp, NCC, miction urinary copper, CuEXC and the REC are shown. No difference was noticed between control subjects and wild-typehomozygous for total copper, Cp, CuEXC and REC. Urinary Cu for the control group was not determined but no significant difference between wild-type-homozygous and heterozygous groups was found. However,
Table 1 Presentation symptoms profiles for the 16 WD patients diagnosed by molecular genetic testing and values of CuEXC, CuUF, total Cu, urine Cu, Cp, NCC and REC concentrations.
Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Patient 11 Patient 12 Patient 13 Patient 14 Patient 15 Patient 16
Age
Presentation
52 17 14 39 20 14 51 20 19 34 36 19 54 32 29 13
Neurologic Familial screening Hepatic Neurologic Neurologic Neurologic Hepatic Neurologic Neurologic Neurologic Familial screening Neurologic Hepatic Hepatic Neurologic Hepatic
Total Cu μmol.L−1
Cp mg.L−1
Cu UF μmol.L−1
Cu Exc μmol.L−1
REC%
Urinary miction Cu μmol.L−1
NCC μmol.L−1
(12.5–22.5)
(0.2–0.5)
(0.06–0.16)
(0.62–1.15)
(3.0–8.1)
(0.07–0.80)
b1.6
8.5 7.2 8.4 2.5 13.8 4.6 3.9 3.9 3.4 6.4 7.4 6.9 3.2 9.0 10.7 3.3
0.15 0.12 0.13 0.02 0.20 0.03 0.06 0.05 0.03 0.07 0.14 0.21 0.07 0.15 0.21 0.05
0.22 0.15 0.26 0.16 0.17 0.14 0.24 0.19 0.18 0.12 0.15 0.15 0.18 0.15 0.22 0.13
3.52 2.02 2.04 2.08 3.58 3.34 1.16 3.32 1.94 3.70 1.71 5.06 2.11 2.12 2.54 1.12
41% 28% 24% 82% 26% 73% 30% 86% 57% 58% 23% 73% 65% 24% 24% 34%
3.8 7.4 8.9 2.7 10.5 11.0 0.4 3.4 2.5 1.8 2.9 9.4 2.1 1.5 3.7 1.7
1.15 1.075 1.883 1.55 4 3.08 0.97 2.9 1.94 3.46 0.54 − 3.39 − 0.21 1.65 0.41 0.8
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investigated subjects). CuUF ROC analysis is not shown but reveals to be irrelevant in diagnosis of WD and had poor Se and poor Sp. Except for NCC, specificity of these parameters using the cited cutoffs was higher than 98%. Sensitivities for total Cu, urinary Cu, Cp, NCC, CuEXC and REC were: 69%, 94%, 81%, 75%, 88% and 100%, respectively; which could be satisfying for diagnostic tests. However, in WD, false negatives and false positives could be problematic. Miction urinary Cu yielded to only one false negative and, with a cutoff 1.53 μmol.L−1, CuEXC gives one false positive and 2 false negatives. When taking into account the total Cu by calculating the ratio CuEXC/total Cu, the REC showed no false positive or false negative using 18.5% as cutoff. 4. Discussion
Fig. 1. Exchangeable copper variations within 24 h in four healthy subjects (continuous lines) and an untreated WD patient (broken line). A. CuEXC concentration variations within 24 h, B. Relative Exchangeable Copper (REC) variations within 24 h.
total Cu and Cp were significantly lower in heterozygous (14.3± 3.7 μmol.L−1 and 0.33 ±0.07 mg.L–1 respectively) and mostly in WD patients (6.4 ±3.2 μmol.L−1 and 0.104 ±0.07 mg.L–1 respectively) versus wild-type-homozygous (16.4 ±3.6 μmol.L−1 and 0.37 ± 0.12 mg.L respectively) and control group subjects (16.8 ±4.9 μmol.L−1 and 0.33 ±0.07 mg.L–1 respectively). Urinary Cu in WD patients was significantly higher compared to other studied groups. The ROC curves analysis performed for WD patients and all other groups (including control subjects) suggested that the most useful cutoffs for total Cu, urinary Cu, Cp, NCC, CuEXC and REC are respectively: N7.8 μmol.L −1, N0.84 μmol.L −1, b0.157 g.L −1, N0.8 μmol.L −1, N1.53 μmol.L −1 and b18.5%. No significant differences were found between areas (AUC) of total Cu (0.969), CuEXC (0.999), Cp (0.981), urinary Cu (0.992) and REC (1.00) (p N 0.09 at least). This demonstrates that CuEXC and REC are at least as discriminatory for WD diagnosis as Cp or miction urinary Cu in a population of patients presenting features evoking WD. NCC could be a statistically discriminating marker with NCC cutoff value N 0.8 μmol.L −1 but yielded 2 false negatives and a very large number of false positives (n = 25 namely 27% of
Fig. 2. CuEXC stability in frozen serum. Circles indicate mean values of variation after different days of freezing. Bars indicate SD. Results are expressed as percentages of zero time values.
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 REC has an ideal prognostic accuracy in identifying WD and yielded 100% Se and 100% Sp. REC was the only biomarker without false negatives or positives. Moreover, ROC curve analysis of CuEXC demonstrates that the sensitivity and the specificity of this diagnostic tool are better than those of Cp and comparable to those of urinary Cu. The determination method was successfully applied in routine framework to a large number of subjects. Estimating the “free” copper has been a subject of interest since the incrimination of copper in WD. At present, simple methods of determination of the total body load of copper are not widely available. The NCC was suggested to estimate the Cu load in WD but it revealed to be unreliable as a diagnostic test and had many draw backs as a monitoring test [21]. Therefore, we carried out and validated a method for the determination of both CuUF and CuEXC that allowed us to establish preliminary RV in healthy subjects [25]. This method was then applied in routine framework and CuEXC has been evaluated as a diagnostic tool in WD. Since the toxic copper was thought to be “free”, attention was first directed to CuUF because this fraction represents protein unbound copper. In our study, CuUF values were rather close to RV for all WD patients in the absence of chelator intake and the test was not discriminatory in diagnosing WD. However, we observed that CuUF values vary upon chelator intakes but remain within RV under zinc treatment (not shown). Recently, ultrafiltrable copper has been determined in healthy subjects and WD patients by McMillin et al. [34]. RV in healthy subjects obtained by authors (0–1.6 μmol.L −1) were comparable to our CuEXC values. This is certainly due to the use of EDTA containing tubes for blood sampling. However, results reported in WD patients were intriguingly very elevated, ranging between 11.0 and 45.5 μmol. L −1. The instability of CuUF required the treatment of blood within 15 min after sampling, which hampered our ability to track patients from other institutions. Therefore, CuEXC short term stability and CuEXC stability in frozen serum had to be tested as well as the repeatability of the test when blood is sampled at different times of the day. The presented results show the analytical reliability of CuEXC determination. Indeed, CuEXC determination revealed to be repeatable and the CuEXC in serum was stable at room temperature for 24 h (only stability for delays N48 h was studied in our previous work) and in frozen serum for at least 7 days. Thus, CuEXC could be measurable whenever serum is either immediately frozen after sampling-centrifugation or has less that 24 h. It should be noted that method-dependent variation of ceruloplasmin measurement is still significant and that cutoffs can vary upon laboratory, standardization and the age of the reference subjects [23]. In our study,
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Fig. 3. Diagnostic tests for control subjects (n = 62), wild-type-homozygous (n = 25), heterozygous (n = 45) and for patients with WD (n = 16) depicting: total copper, miction urinary copper, ceruloplasmin, non ceruloplasmin copper, exchangeable copper, Relative CuEXC/total copper. *, $ and § indicate statistical significant difference (p b 0.05) vs controls, homozygous and heterozygous, respectively. Broken lines represent the cutoffs obtained from ROC analysis.
the Cp cutoff offering the best Se and Sp was found to be 0.16 g.L−1 and is in agreement with manufacturer recommendation. In contrast, the Cp cutoff (0.14 g.L−1) suggested by Mak et al. [33] yielded poor sensitivity (62%). Miction urinary copper cutoff determined in our study at 0.84 μmol.L −1 offered the best sensitivity and specificity and is in agreement with 24 h-urinary copper cutoff reported in literature [35]. This suggests that miction urine copper could be comparable to 24-h urine copper as a diagnosis tool. However, Roberts et al. have recently suggested lower cutoff to better diagnose WD due to some atypical presentations in ≈20% of patients but admitted that
the low cutoff led to a reduced specificity [16]. Moreover, contamination of the urinary collection apparatus can readily occur and urine collection is often uncompleted which complicate the interpretation of urinary results. In addition, urinary Cu was reported to increase in some liver diseases involving copper metabolism disturbances [36,37]. Further studies should focus on the use of this new biomarker in a cohort of patients with severe hepatic disturbances background and reference values in children and teenagers have to be established. The contribution of REC and CuEXC in improving the monitoring of WD treated patients is another subject to be studied.
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Fig. 4. ROC curves for different diagnostic tests for WD: total copper (AUC = 0.9699, 95%-CI 0.929–1.01), miction urinary copper (AUC = 0.9937, 95%-CI 0.98–1.007), ceruloplasmin AUC = 0.9808, 95%-CI 0.958–1.0003), non ceruloplasmin copper (AUC = 0.7827, 95%-CI 0.66–0.91), CuEXC (AUC = 0.9841, 95%-CI 0.96–1.00, p b 0.0001) and relative exchangeable copper (AUC = 1.00). Broken lines indicate nonsignificance.
5. 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 [1] Burkhead JL, Gray LW, Lutsenko S. Systems biology approach to Wilson's disease. Biometals 2011;24:455–66. [2] Ferenci P. Wilson's disease. Clin Gastroenterol Hepatol 2005;3:726–33. [3] Bull PC, Thomas GR, Rommens JM, Forbes JR, Cox DW. The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nat Genet 1993;5:327–37. [4] Tanzi RE, Petrukhin K, Chernov I, et al. The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nat Genet 1993;5:344–50.
[5] Yamaguchi Y, Heiny ME, Gitlin JD. Isolation and characterization of a human liver cDNA as a candidate gene for Wilson disease. Biochem Biophys Res Commun 1993;197:271–7. [6] Banci L, Bertini I, Cantini F, Ciofi-Baffoni S. Cellular copper distribution: a mechanistic systems biology approach. Cell Mol Life Sci 2010;67:2563–89. [7] Gitlin D, Janeway CA. Turnover of the copper and protein moieties of ceruloplasmin. Nature 1960;185:693. [8] Hellman NE, Gitlin JD. Ceruloplasmin metabolism and function. Annu Rev Nutr 2002;22:439–58. [9] Hellman NE, Kono S, Mancini GM, Hoogeboom AJ, De Jong GJ, Gitlin JD. Mechanisms of copper incorporation into human ceruloplasmin. J Biol Chem 2002;277:46632–8. [10] Cox DW. Molecular advances in Wilson disease. Prog Liver Dis 1996;14:245–64. [11] Liu M, Cohen EJ, Brewer GJ, Laibson PR. Kayser–Fleischer ring as the presenting sign of Wilson disease. Am J Ophthalmol 2002;133:832–4. [12] Deguti MM, Genschel J, Cancado EL, et al. Wilson disease: novel mutations in the ATP7B gene and clinical correlation in Brazilian patients. Hum Mutat 2004;23: 398. [13] Sedlak E, Wittung-Stafshede P. Discrete roles of copper ions in chemical unfolding of human ceruloplasmin. Biochemistry 2007;46:9638–44. [14] El Balkhi S, Poupon J, Trocello JM, Massicot F, Woimant F, Laprevote O. Human plasma copper proteins speciation by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry. Solutions for columns calibration by sulfur detection. Anal Chem 2010;82:6904–10. [15] Walshe JM. Cause of death in Wilson disease. Mov Disord 2007;22:2216–20.
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S. El Balkhi et al. / Clinica Chimica Acta 412 (2011) 2254–2260
[16] Roberts EA, Schilsky ML. Diagnosis and treatment of Wilson disease: an update. Hepatology 2008;47:2089–111. [17] Steindl P, Ferenci P, Dienes HP, et al. Wilson's disease in patients presenting with liver disease: a diagnostic challenge. Gastroenterology 1997;113:212–8. [18] Ferenci P, Steindl-Munda P, Vogel W, et al. Diagnostic value of quantitative hepatic copper determination in patients with Wilson's Disease. Clin Gastroenterol Hepatol 2005;3:811–8. [19] Luoma LM, Deeb TM, Macintyre G, Cox DW. Functional analysis of mutations in the ATP loop of the Wilson disease copper transporter, ATP7B. Hum Mutat 2010;31:569–77. [20] Twomey PJ, Viljoen A, Reynolds TM, Wierzbicki AS. Non-ceruloplasmin-bound copper in routine clinical practice in different laboratories. J Trace Elem Med Biol 2008;22:50–3. [21] Walshe JM. Monitoring copper in Wilson's disease. Adv Clin Chem 2010;50: 151–63. [22] Twomey PJ, Viljoen A, House IM, Reynolds TM, Wierzbicki AS. Relationship between serum copper, ceruloplasmin, and non-ceruloplasmin-bound copper in routine clinical practice. Clin Chem 2005;51:1558–9. [23] Mak CM, Lam CW. Diagnosis of Wilson's disease: a comprehensive review. Crit Rev Clin Lab Sci 2008;45:263–90. [24] Macintyre G, Gutfreund KS, Martin WR, Camicioli R, Cox DW. Value of an enzymatic assay for the determination of serum ceruloplasmin. J Lab Clin Med 2004;144: 294–301. [25] El Balkhi S, Poupon J, Trocello JM, et al. Determination of ultrafiltrable and exchangeable copper in plasma: stability and reference values in healthy subjects. Anal Bioanal Chem 2009;394:1477–84.
[26] Musci G, Fraterrigo TZ, Calabrese L, McMillin DR. On the lability and functional significance of the type 1 copper pool in ceruloplasmin. J Biol Inorg Chem 1999;4:441–6. [27] Sedlak E, Zoldak G, Wittung-Stafshede P. Role of copper in thermal stability of human ceruloplasmin. Biophys J 2008;94:1384–91. [28] Buckley WT, Vanderpool RA. Analytical variables affecting exchangeable copper determination in blood plasma. Biometals 2008;21:601–12. [29] Venelinov TI, Beattie JH, Dainty JR, Hollands WJ, Fairweather-Tait SJ, Harvey LJ. Stable isotope pilot study of exchangeable copper kinetics in human blood plasma. J Trace Elem Med Biol 2007;21:138–40. [30] Venelinov TI, Davies IM, Beattie JH. Dialysis-Chelex method for determination of exchangeable copper in human plasma. Anal Bioanal Chem 2004;379:777–80. [31] Cox DW, Bugbee D, Davies L, Kenney S. WD mutation database; 2011. [32] Trocello JM, Chappuis P, Chaine P, et al. Wilson disease. Presse Med 2009;38:1089–98. [33] Mak CM, Lam CW, Tam S. Diagnostic accuracy of serum ceruloplasmin in Wilson disease: determination of sensitivity and specificity by ROC curve analysis among ATP7B-genotyped subjects. Clin Chem 2008;54:1356–62. [34] McMillin GA, Travis JJ, Hunt JW. Direct measurement of free copper in serum or plasma ultrafiltrate. Am J Clin Pathol 2009;131:160–5. [35] Dhawan A, Taylor RM, Cheeseman P, De Silva P, Katsiyiannakis L, Mieli-Vergani G. Wilson's disease in children: 37-year experience and revised King's score for liver transplantation. Liver Transpl 2005;11:441–8. [36] Trocello JM, Chappuis P, El Balkhi S, et al. Abnormal copper metabolism in adult. Rev Med Interne 2010;31:750–6. [37] Nicastro E, Ranucci G, Vajro P, Vegnente A, Iorio R. Re-evaluation of the diagnostic criteria for Wilson disease in children with mild liver disease. Hepatology 2010;52:1948–56.