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

Abstract

Objectives:

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

Methods:

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

Results:

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

Conclusions:

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

Introduction

Wilson disease (WD), first described by Kinnear Wilson in 1912 [1] is an autosomal recessive genetic disorder which affects copper metabolism and has an estimated prevalence of between 1:30,000 and 1:50,000 [2]. Intracellular copper is normally incorporated into ceruloplasmin (Cp) during its biosynthesis in hepatocytes by a process that is enhanced by the copper transporter protein ATP7B [3]. In WD, ATP7B mutations cause the dysfunction of ATP7B leading to ineffective uptake of copper into the trans-Golgi-network [4] and a lack of incorporation of Cu into ceruloplasmin [5]. As Cp synthesis is unaffected in WD patients but less Cu is present in the organelle for incorporation, there is a higher proportion of the less stable apo-Cp form which leads to the lower steady state concentration of Cp in the circulation of most patients with WD. The ATP7B mutations also reduce the transfer of hepatocellular copper into bile, resulting in increased hepatic copper accumulation and a commensurate increase in non-ceruloplasmin copper (NCC) and increased urinary copper excretion. In WD patients hepatic copper accumulation occurs first, then increased copper accumulation may occur throughout the body. This results in copper-induced liver injury and eventual cirrhosis, and later neurological or psychiatric complications [6].

Ceruloplasmin was one of the first proteins identified as present at a lower concentration in patients with WD, and indeed the measurement of this protein in serum samples was useful as a diagnostic screen for patients with WD [7]. When trying to determine if there was excess copper in the circulation of patients with WD, the concept of NCC became relevant, as Cp does not exchange its copper under physiologic conditions. Thus the calculated NCC was determined by measuring the Cp concentration and estimating the Cp bound copper (Cp-Cu) by assuming the presence of six copper atoms per Cp molecule. This was then subtracted from the total copper to give the calculated NCC. However, with respect to the measurement of Cp, the routinely used immunochemical methods are not specific for the copper-containing holoprotein [8], and so the Cp concentration used in the calculation of NCC includes a contribution from the apo form. As a result, the calculated NCC is often misleading [8], particularly in WD patients. The ineffective incorporation of copper into Cp in and of itself does not lead to an elevated proportion of NCC, nor copper accumulation in the liver as evidenced by studies on patients with the rare disorder aceruloplasminemia where Cp synthesis is absent.

The current biochemical assays used for the investigation of WD either: lack clinical and diagnostic specificity, e.g. serum copper and Cp, or 24 h urinary copper excretion; or are invasive e.g. liver copper obtained by biopsy. Laboratory tests that do provide specificity for the disease e.g. Cp enzymatic activity [9], are not widely available and lack analytical traceability. Newer approaches to determining NCC can be classified into indirect methods, where the entities measured are defined by the reagents used to isolate them [10] and direct methods, where the copper containing compounds are identified and quantified individually by using a chromatographic separation prior to mass spectrometry [11, 12].

A major advantage of direct methods includes the effective analytical traceability they afford [13] compared to indirect methods, which involve chelating reagents, anti-bodies or tagging agents to work. Disadvantages of direct methods include: their cost and availability, which limit their use to specialist laboratories. Size exclusion chromatography (SEC) to separate serum proteins followed by elemental analysis (SEC-ICP-MS) has been investigated with variable success [14, 15] often limited by chromatographic resolution. The use of strong anion exchange (SAX) coupled to ICP-MS has also been described for the determination of copper speciation in a WD clinical trial [12]. Importantly, the work provided data for the concentration of Cu-Alb and Cu-Cp present in two commercially available human serum certified reference materials (CRM) materials (LGC8211 and ERM Da250a) developed and characterised by two European National Metrology Institutes.

We have previously reported an investigation into ethylenediaminetetraacetic acid (EDTA)-exchangeable copper and NCC in WD patients using SAX-ICP-MS/MS [11]. In the current work, we describe two new parameters, accurate non-ceruloplasmin copper (ANCC) and relative ANCC (RelANCC), for the assessment and monitoring of a cohort of 71 WD patients currently undergoing treatment.

Materials and methods

Patient samples

Human serum samples were collected from 71 individuals being treated for WD over a four year period, who consented to be part of a Wilson Disease Registry Study (Yale Medical School, USA). A serum sample from a newly diagnosed WD patient, prior to treatment, was obtained with patient consent. Serum samples from 31 non-WD individuals, which were submitted for trace element assessment as part of a health and wellbeing screen were used as control samples. The trace element results for these individuals were all within the normal reference ranges, which would indicate a normal C-reactive protein, a normal total protein level, a normal albumin, and the chromatograms showed a normal protein profile. Samples were taken into blue-topped BD Vacutainer trace element tubes (Fisher Scientific, Loughborough, UK). After 45–60 min at room temperature, samples were centrifuged at 1,500 rpm for 10 min, and serum was removed and stored at −80 °C. When required, samples were thawed at room temperature (+25 °C) for 30 min prior to analysis.

Speciation standards, chemicals, and reagents

Ammonium acetate (Fisher Scientific, Loughborough, UK), EDTA, concentrated ultrapure nitric acid, Tris base and Tris-HCl (Merck, Gillingham, UK). Mobile phase buffers were prepared using ultrapure water (18.2 MΩ cm) obtained using a MilliQ system (Millipore, Gillingham, UK) and pH measured using a 3,200 series pH electrode (Agilent Technologies, Didcot, UK). Human metalloprotein standards for ceruloplasmin (Cp), albumin (Alb), alpha-2-macroglobulin (Mcg) and transferrin (Tf) (Merck, UK) were used without further purification. Inorganic copper and germanium standards (1,000 mg/L) (Inorganic Ventures, Southend, UK) were used as calibration and internal standard respectively.

Two human serum CRMs, LGC8211 and ERM DA250a (LGC Reference Materials, Teddington, UK) with certified values for total copper and published values [12] for copper bound to Cp were used for validation of method accuracy.

Determination of total elemental concentration

Total serum copper concentrations were determined by ICP-MS using an in-house developed and validated clinical method. Samples were diluted 1 in 50 with nitric acid (1 % v/v) diluent containing germanium (Ge) internal standard added to a final concentration of 15.0 μg/L. Analysis was carried out using an aqueous calibration curve and the ICP-MS instrument (iCapQ, ThermoFisher Scientific, Hemel Hempstead, UK) was operated in collision cell mode (He gas) with kinetic energy discrimination. Internal quality control materials were evaluated at three concentrations μmol/L: low 5.0–6.1; medium 10.3–12.6; and high 20.3–24.7 (these ranges were determined using n>20 measurements in each case). The laboratory demonstrates acceptable performance in an external quality assessment (EQA) scheme for copper in serum (UK NEQAS for Trace Elements, Guildford, UK).

Speciation instrumentation

Liquid chromatographic separation of serum proteins was achieved using a Dionex ICS-5000 HPLC system (Thermo-Fisher Scientific, Hemel Hempstead, UK) equipped with polyetheretherketone (PEEK) tubing. Metalloproteins were detected using an ICP-MS/MS instrument (iCapTQ, Thermo-Fisher Scientific, Hemel Hempstead, UK) fitted with nickel cones, a Peltier-cooled (+2.6 °C) cyclone spray chamber and a glass concentric nebulizer (1 mL/min).

Speciation of copper-containing proteins

The protein separation was achieved by gradient elution using a SAX column (Q-STAT column, 4.6 mm I.D. × 10 cm, 7 μm particle size, TSKgel, Tosoh, VWR, Lutterworth, UK), with a 25 μL injection volume at a flow rate of 0.7 mL min⁻¹. Buffer A: 50 mM Tris, pH 7.4. Buffer B: 50 mM Tris, 1 M NH₄OAc, pH 7.4. Gradient (linear): 0.0–1.0 min 0% B, 1.0–3.0 min 20 % B, 3.0–4.5 min 50 % B, 4.5–5.5 min 100 % B, 5.5–7.5 min 100 % B (acquisition end), 7.5–9.0 min 0% B, 9.0–10.0 min 0 % B (re-equilibration). The HPLC system was hyphenated to ICP-MS/MS using a short length of PEEK tubing (50 × 0.25 mm ID). Data for m/z=63 (⁶³Cu⁺) and 48 (³²S¹⁶O⁺) were obtained in reaction cell mode using an oxygen flow rate of 0.3 mL /min. The ⁶³Cu isotope was used to identify and quantitate the Cu-containing proteins and the ³²S¹⁶O polyatomic was generated in the collision cell to overcome the highly abundant ¹⁶O¹⁶O interference on the ³²S isotope. This proxy for the ³²S isotope was used to indicate the presence of proteins containing cysteine and methionine amino acids.

Calibration standards for ⁶³Cu were prepared from a 1,000 mg/L Cu standard using 50 mM Tris solution (pH 7.4) containing 0.1 mM EDTA. Patient serum samples were typically prepared with 5-fold dilution in 50 mM Tris solution (pH 7.4). Method performance was monitored using human serum CRM LGC8211 and ERM DA250a.

Data processing

Speciation data were acquired and processed using Chromeleon ver. 7 software and the ChromControl plug-in for speciation experiments. All chromatograms were blank subtracted. Additional data processing was carried out using Microsoft Excel 2011 (Microsoft, Seattle, USA).

Calculation of ANCC and RelANCC

The ANCC was calculated by subtraction of the Cp bound copper from the total serum copper (Cu_ANCC=Cu_total − Cu_Cp). and the RelANCC was the percentage of the total copper present as ANCC (RelANCC=ANCC/Total Cu × 100 %).

Results and discussion

Biomedical copper speciation

Protein standards were used to establish retention times via specific isotopes (in brackets): Tf (⁵⁷Fe⁺)=216 s; Cu-EDTA (also known as exchangeable Cu) (⁶³Cu⁺)=240 s; Mcg (³¹P¹⁶O⁺)=228 s; Alb (⁶³Cu⁺)=300 s; and Cp (⁶³Cu⁺)=320 s, see Supplementary Figure S1. All proteins were chromatographically resolved and most importantly, Cp-Cu was well separated from both EDTA-Cu and albumin bound Cu (Alb-Cu). The chromatograms for ⁶³Cu⁺ (m/z=63) and ³²S¹⁶O⁺ (m/z=48) in Figure 1, show the elution of the copper-containing proteins in a typical WD patient compared to non-WD.

Figure 1:

Example serum copper speciation achieved using SAX-ICP-MS/MS with continuous monitoring of ⁶³Cu⁺ and ³²S¹⁶O⁺. (A) and (B) show non-WD, (C) and (D) show WD patient. Protein assignment was achieved using comparison to single protein retention time standards.

The accuracy of the method was established by determining the recovery for total copper and Cp-Cu for two human serum CRMs, LGC8211 and ERM DA250a. The total copper is certified in both materials, but because the Cp-Cu is not certified, comparison was made to recently published data for a similar method using HPLC-ICP-ID-MS [12]. The determined total copper concentration (mean ± SD) for LGC8211 was 16.2 ± 0.9 μmol/L recovery 89.0 ± 5 % and for ERM DA250a was 19.5 ± 0.1 μmol/L recovery 97.7 ± 1 %. In comparison to values determined for LGC8211 when distributed as part of the UK NEQAS for Trace Element EQA scheme the total copper recovery was 91.8 ± 5 % (value shown on the certificate was a mean of 47 participant results). The determined Cp-Cu concentration for LGC8211 was 15.3 ± 0.5 μmol/L recovery 93.6 ± 3 % and for ERM DA250a was 14.7 ± 0.5 μmol/L recovery 94.8 % ± 3 %. These values are shown in Table 1 and indicate an excellent level of accuracy and precision for the method.

Table 1:

Comparison of Cu-Cp determined by HPLC-ICP-MS/MS in two CRMs to the indicative published values [12].

Material	Indicative value, μmol/L	Found value, μmol/L	n	SD, μmol/L	RSD, %	Recovery, %
LGC8211	16.3	15.3	19	0.5	3.0	93.6
ERMDA250a	15.5	14.7	8	0.5	3.3	94.8
					Mean	94.2

To establish that the method was not affected by loss of copper from Cp-Cu during the chromatographic separation, the sum of copper in all the copper-containing peaks in the chromatogram, was compared to the total serum copper concentration determined by ICP-MS, across the range for WD and non-WD patients, which was 0.50–24.4 μmol/L. The sum of the copper peaks by speciation analysis was plotted against the total serum copper (Figure 2A) and a Bland-Altman difference plot was also used to determine the agreement (Figure 2B). Regression analysis showed excellent agreement between these parameters (slope 0.964, intercept 0, r 0.987), as did the difference plot (mean difference 0.38 μmol/L). The results confirm that all the copper-containing species are quantitatively eluted from the column, that no copper was irreversibly retained on the column and that no unbound copper was sequestered by the eluting proteins.

Figure 2:

Comparison of total copper to summation of copper containing species. (A) Linear regression for total copper by summation of copper-containing peaks by HPLC-ICP-MS/MS and total copper measured by ICP-MS in WD and non-WD patient samples (dotted line shows y=x). (B) Bland-Altman difference plot for the total Cu concentration determined directly by ICP-MS and sum of the copper-containing species (dotted lines show ± 1.96 × SD).

This data, together with the excellent recovery values for Cp-Cu and total copper in the two CRMs, indicates that the method provides accurate results and is suitably robust across the range of serum copper encountered in WD and non-WD patient samples.

Non-ceruloplasmin copper (NCC)

The measurement of NCC levels in WD patients can provide clinicians with a greater understanding of treatment effectiveness, drug safety and adherence. It can also provide more reliable monitoring information than urinary copper excretion, highlighting possible under or over treatment. Several different techniques have been used to determine NCC, including calculation based on total serum copper levels and immunochemically determined Cp concentrations and directly using molecular weight cut-off spin filters in conjunction with measurement of copper by ICP-MS. However, the use of calculated NCC is known to be inaccurate. This is because the immunochemical method overestimates the copper-containing holo-form of Cp because it does not discriminate it from apo-Cp [8], which is present in a greater proportion in WD patients.

In the absence of a well-validated method for the measurement of NCC, a reference range for a non-WD population is not well defined. However, because it is directly related to the proportion of circulating Cp-Cu, a theoretical range can be calculated based on published serum copper reference ranges. Even so, commonly reported values for the proportion of Cp-Cu in serum are inconsistent [12, 16–18]. For example, an early report using Sephadex G-150 gel filtration found 71 % of total serum copper to be Cp-bound [19], while more recent work reported Cp-bound copper to make up 95 % of total serum copper [20]. It remains unclear as to whether these reported differences are a consequence of biological variation, experimental approach, or a combination of both. In the current study, the proportion of copper in the serum present as Cp-Cu was found to have a mean and median value respectively of 90.1 and 90.3 % (absolute range 78.2–98.5 %) in the non-WD samples and 64.1 and 69.4 % (absolute range 22.0–93.1 %) in the treated WD patients, which illustrates the much greater variation of Cp-Cu in the treated WD patients.

Based on the assumption of the proportion of Cp-bound copper being 90 % of total serum copper and given a total serum copper reference range of 7.9–25.1 μmol/L [21], a predicted NCC reference range of 0.79–2.51 μmol/L can be calculated. Some experimentally determined NCC ranges are shown in comparison to the predicted range in Table 2. Reference ranges were determined using 30 kDa molecular weight cut-off spin filters 0.6–1.1 μmol/L [14, 22], while methodological improvements using 10 kDa spin filters and EDTA treatment yielded similar results 0.48–1.63 μmol/L [23]. Meta-analysis of a cohort of 338 patients who had historic testing data for both total serum copper and Cp levels calculated retrospective NCC concentrations, assuming 0.30 μg/L (0.047 μmol/L) Cu per 1 mg Cp; though the wide range of calculated results −7.8–12 μmol/L highlights once again the inaccuracy of measuring Cp immunochemically [24].

Table 2:

Reference ranges for non-ceruloplasmin bound copper in non-WD patients (in some cases termed “exchangeable copper”, “reactive exchangeable copper”, “NCC” or “ANCC”).

Approach	Lower limit, μmol/L	Upper limit, μmol/L	Ref.
Calculated from reference values for total serum copper, assuming NCC=10 % total Cu	0.79	2.51	[21]a
ANCC: subtraction of Cp-Cu from total serum copper measurementsb	0.25	5.37	This work
Clinical trial: Ultrafiltration (MW cut-off spin filters) to determined NCC values.	0.8	2.3	[29]
Incubation with EDTA, separation using 30 kDa MW cut-off spin filters	0.62	1.15	[22]
Incubation with EDTA, separation using 30 kDa MW cut-off spin filters	0.6	1.1	[14]
Incubation with EDTA, separation using 10 kDa MW cut-off spin filters	0.48	1.63	[23]
Pooled plasma from adults with normal copper concentrations, analysed by ultrafiltration without EDTA	−0.39	6.14	[30]
Retrospective review of 338 patients. NCC calculated as 0.0472 μmol Cu/mg Cp	−7.8	12.0	[24]

^aCitation refers to total serum copper reference ranges.

^bAbsolute range shown.

Accurate non-ceruloplasmin copper (ANCC) and relative ANCC (RelANCC)

The current work used the direct quantification of Cp-Cu and total copper using elemental standards with metrological traceability and then calculation of a new biomarker index, the Accurate NCC (ANCC) value. This approach, facilitated by the excellent separation and resolution of Cp from other serum proteins, allowed for an analytically preferable subtraction of Cp-Cu from a robust measurement of total serum copper (Cu_ANCC=Cu_total − Cu_Cp). This was undertaken for 126 patient visits across a 4 year treatment period of 71 WD patients (Table 3). The experimentally determined ANCC values for the WD patients undergoing treatment was 0.25–5.37 μmol/L, with mean and median values of 1.46 and 1.25 μmol/L, respectively. The values determined for the Relative ANCC (RelANCC=ANCC/Total Cu × 100 %) were range 6.89–77.96 %, with mean and median values of 35.95 and 30.64 %, respectively.

Table 3:

Summary of results for the analysis of samples from a cohort of WD patients treated over a four year period (n=126), a newly diagnosed treatment naive WD patient (n=1) and a cohort of non-WD patients (n=31) being nutritionally assessed for trace elements.

Measurement parameter	WD untreateda (n=2)	WD treated (n=126)	Non-WD (n=31)
Total patient number	1	71	31
Mean total Cu, μmol/L	1.64	5.26	15.45
Median total Cu, μmol/L	–	4.96	14.40
Absolute range lower, μmol/L	–	0.50	9.60
Absolute range upper, μmol/L	–	15.28	24.40
Mean sum Cu species, μmol/L	1.64	4.88	15.37
Median sum Cu species, μmol/L	–	4.11	15.25
Absolute range lower, μmol/L	–	0.57	9.90
Absolute range upper, μmol/L	–	16.54	23.37
Mean Cu recovery, %	99.96	95.33	100.20
Mean Cp-Cu, μmol/L	1.03	3.81	13.86
Median Cp-Cu, μmol/L	–	3.05	13.86
Absolute range lower, μmol/L	–	0.22	8.85
Absolute range upper, μmol/L	–	12.77	21.92
Mean proportion of Cu as Cp-Cu, %	62.93	64.05	90.12
Median proportion of Cu as Cp-Cu, %	–	69.36	90.34
Absolute range lower %	–	22.04	78.2
Absolute range upper %	–	93.11	98.5
Mean ANCC, μmol/L	0.61	1.46	1.58
Median ANCC, μmol/L	–	1.25	1.41
Absolute rangelower, μmol/L	–	0.25	0.22
Absolute range upper, μmol/L	–	5.37	3.97
Mean RelANCC, %	37.07	35.95	9.88
Median RelANCC, %	–	30.64	9.66
Absolute range lower %	–	6.89	1.54
Absolute range upper %	–	77.96	21.83

^aMeasured in duplicate.

For comparison to the WD patients (Figure 3), speciation analysis was carried out on 31 serum samples from non-WD individuals submitted for trace element assessment as part of a health and wellbeing screen, obtaining an ANCC mean and median of 1.58 and 1.41 μmol/L, respectively and a range of 0.22–3.97 μmol/L (Table 3). The RelANCC values were mean and median of 9.88 and 9.66 %, respectively and a range 1.54–21.83 %.

Figure 3:

Comparison of developed biomarkers between one untreated newly diagnosed WD patient shown as a single horizontal line (n=1), 71 WD patients on treatment (n=126) and 31 non-WD patients (n=31). Confidence intervals were calculated as 1.5 × interquartile range (IQR). (A) Comparison of ANCC concentrations (μmol/L) and (B) Rel ANCC (%).

The ANCC levels in WD patients undergoing treatment (Figure 3A) were significantly lower (p<0.05, two-tailed t-test assuming unequal sample variances) than non-WD patients. The RelANCC values (Figure 3B) for the WD patients undergoing treatment were significantly higher (p<0.05, two-tailed t-test assuming unequal sample variances) than non-WD patients.

Clinical interpretation of ANCC and Rel ANCC

The WD Registry samples were obtained over a four year period, from individuals currently undergoing treatment; typically either a chelating agent (Trientine or D-penicillamine) to facilitate urinary copper excretion [27, 28] or a zinc salt (zinc acetate, zinc gluconate or zinc picolinate) to induce the production of intestinal cell metallothionein, reducing copper absorption [29]. These treatment regimens (see Supplementary Table S3A–D) act to reduce the elevated proportion of NCC caused by WD.

Total serum copper alone is unsatisfactory for determining WD treatment status (Supplementary Table S1A–D), in fact, 22 of the 71 patients in the WD cohort were found to be within the non-WD reference range for total serum copper 7.9–25.1 μmol/L, for at least one of their treatment visits. Instead, copper speciation was used to specifically quantify Cp-Cu and thus calculate the ANCC and RelANCC. For example, the ANCC level determined for sample WDR01_002 (month 36) 1.25 μmol/L was within the theoretical non-WD NCC reference range, while total serum copper 5.98 μmol/L was low. The RelANCC value of 21.0 % was within the experimentally determined values for the non-WD population. This patient was administered 150 mg zinc acetate daily and so the ANCC and RelANCC would suggest that, although this individual has low serum copper, the current treatment was effective in normalising the NCC. In contrast, patient WDR01_060 (month 36) had a normal serum copper of 11.74 μmol/L but exhibited an elevated ANCC of 3.48 μmol/L and RelANCC of 29.7 %, higher than the values for the non-WD population. This patient was prescribed 1,000 mg Trientine daily and the ANCC and RelANCC indicate that this dose may have been insufficient, dietary copper intake may be high, or the patient was not taking the medication as frequently as prescribed or apart from food as recommended. Adherence to chelation therapy is recognised as poor in some patient groups [30].

As can be seen by the value for a treatment naive WD patient (Table 3), the RelANCC is a useful biomarker index for the diagnosis of WD. In this patient the RelANCC was 37.1 % compared to the maximum value of 24.4 % for the non-WD patient group. The RelANCC values for the WD registry samples (Figure 3B) indicate that the majority of treated patients (74 %) had values greater than the maximum for the non-WD patient group. This might indicate that these patients are either not adhering to their medication, are not receiving sufficient treatment, or that treatment has only recently been initiated.

Conclusions

The work presents a new and robust procedure for use in the clinical investigation of copper metabolism, in particular in patients with WD. ANCC may be an important indicator in the management of WD patients, providing clinicians with insight into potential under and overtreatment, or non-adherence. The approach requires validation in specimens from patients in all phases of their treatment and suitable reference ranges established in these populations. Importantly, we put forth RelANCC as a potential diagnostic tool for WD, which will need to be validated in other non-WD populations, in particular those with heterozygosity for WD where copper parameters may be mildly altered. Future studies will address the biochemical consequences of serum copper re-distribution in WD with acute liver failure and also the neurological presentation of WD.