An infographic is available for this article at:https://links.lww.com/MPG/C26.
What Is Known
- Twenty-four hours urinary copper excretion (UCE) is recommended to be used for diagnosis of Wilson disease.
- Twenty-four hours urinary copper excretion may be a useful tool to assess treatment efficacy during follow-up.
- In adults’ recommendations, during maintenance therapy, 24-hour urinary copper excretion should be between 3 and 8 μmol/day, and nonceruloplasmin-bound copper concentration (NCC) should be between 0.8 and 2.4 μmol/L.
What Is New
- The level of nonceruloplasmin-bound copper concentration drops to <0.8 μmol/L within 1 year of treatment.
- In the absence of progressive liver disease or signs of copper deficiency, 24-hour urinary copper excretion decreases to ≤8 μmol/day and <6 μmol/day after 1 and 5 years of treatment, respectively.
Wilson disease (WD) is a rare genetic disorder of copper metabolism with a reported prevalence of 1:30,000–50,000 (1–5). The disease is caused by mutations in the ATP7B gene encoding enzyme, which is critical for copper excretion into the bile, resulting in a progressive accumulation and deposition of copper in the liver and other organs, such as the nervous system, corneas, kidneys, and heart. Clinical presentations in childhood vary from asymptomatic liver disease to cirrhosis or acute liver failure, with or without neurological and psychiatric symptoms (1). Approximately 20% of WD patients present acutely with liver dysfunction, whereas most of the patients have presentation as chronic liver disease or detected on family screening (6).
Recently, it has been recommended that the diagnostic workup of children with liver disease, apart from liver function tests and coagulogram, should include serum ceruloplasmin, 24-hour urinary copper and liver copper content from hepatic tissue (if the diagnosis is inconclusive), and mutational analysis where available (1). These suggested investigations are based on copper metabolism, which involves renal and gastrointestinal (via hepatic circulation) excretion (7).
The 24-hour urinary copper excretion (UCE) is not only accepted as the most specific method in the diagnosis of WD (8,9), but can also be used to assess the efficacy of treatment during the follow-up period (1). In adults, it is suggested that during maintenance therapy, the value of 24-hour UCE should be 200–500 μg/day or 3–8 μmol/day (10–12). However, there are limited data regarding the cutoff value of 24-hour UCE and its clinical utility during follow-up in children with WD. Studies in WD adults (one included both children and adults) reported that the level of UCE was significantly reduced following 1–2 years of therapy, as compared to the level tested before treatment (13,14).
We retrospectively reviewed the urine copper values that were tested as part of clinical management of our patients on the follow-up.
PATIENTS AND METHODS
Medical records of children diagnosed with WD at Kings’ College Hospital (KCH) from 2005 to 2018 were retrospectively reviewed. The demographic data and laboratory investigations were collected. Both data from the initial presentation and during follow-up visits at KCH were included. Among 55 WD children, 51 patients (92.8%) had been confirmed by genetic testing. Only patients who had either 24-hour or spot urinary copper test during follow-up visits were included in the present study. Patients who switched between different therapies were not excluded; however, 2 patients treated with zinc monotherapy were excluded.
Treatment with penicillamine (D-pen) and trientine was started at 5 mg · kg−1 · day−1 for approximately 2–3 weeks. If patients could tolerate at this dose (without any sign and symptoms of adverse effects), the dosage was increased to 15–20 mg · kg−1 · day−1 (full dose). In this study, 24-hour UCE was collected in acid washed plastic containers provided by the hospital laboratory and measured by flame atomic absorption spectrophotometer. The amount of nonceruloplasmin-bound copper concentration (NCC) is calculated from total serum copper and ceruloplasmin concentrations using the method described previously (7).
Statistical Analysis
Data were analyzed by SPSS version 24.0. The numeric data were displayed as median and interquartile range (IQR). The quantitative data were displayed as proportion and percentage. Of note, the analyses were performed not only per patient but also per follow-up visits. The duration of follow-up was grouped by year. To compare the levels of 24-hour UCE among different durations of follow-up, Kruskal-Wallis with pairwise comparison was used. Significance values have been adjusted by the Bonferroni correction for multiple tests. The level of significance was defined as P < 0.05.
Ethical Approval
This analysis was registered as an audit of clinical practice as per our hospital research governance framework and hence did not require ethical approval (audit reference number CH038).
RESULTS
Demographic Data
Among 55 children diagnosed with WD at KCH between January 2005 and December 2018, 28 patients with a median age of 11.4 years old (ranged 3.8–17.3) had urinary copper tests during follow-up visits. The median duration of follow-up was 6.8 years (range 1.4–14.4). Of these, 9 patients (32.1%) had acute liver failure like presentation, where 2 required liver transplantation. At the initial visit, all patients had been treated with penicillamine. However, 8 had been changed to trientine on experiencing adverse effects from penicillamine. Following 1 year of treatment, liver function tests (aspartate aminotransferase, alanine aminotransferase) remained abnormal in 7 patients, which gradually improved in the second year of follow-up. Demographic data and initial presentation together with laboratory results including Wilson prognostic index (15) of 28 studied patients are presented in Table 1 and Suppl Table 1, https://links.lww.com/MPG/C27. Additionally, the doses of chelating therapy are shown in Suppl Figure 1, https://links.lww.com/MPG/C29 and Suppl Figure 2, https://links.lww.com/MPG/C30.
TABLE 1 -
Demographic data of 28 studied patients
| Demographic data |
All (n = 28) |
| Male, n (%) |
17 (60.7) |
| Age, y |
11.4 (9.4–13.9) |
| Duration of follow-up, y |
6.9 (3.8–10.4) |
| Presentation, n (%) |
| Asymptomatic |
8 (28.6) |
| Chronic liver disease |
11 (39.1) |
| Acute liver failure |
9 (32.1) |
| Laboratory results at first visit |
| Total bilirubin, μmol/L |
17.5 (9.3–33.8) |
| AST, IU/L |
116.0 (71.8–198.3) |
| ALT∗, IU/L |
115.0 (42.3–436.0) |
| Albumin, g/L |
42.5 (26.5–45.0) |
| INR |
1.14 (1.03–1.99) |
| White cell count, 109/L |
6.2 (5.1–8.4) |
| Ceruloplasmin, g/L |
0.07 (0.05–0.17) |
| NCC†, μmol/L |
3.1 (0.6–5.6) |
| Liver copper content‡, μg/g dry weight liver |
445.0 (194.3–1028.5) |
| 24-h urinary copper excretion |
| Pre-penicillamine∗, μmol/day |
3.4 (2.3–8.8) |
| Post-penicillamine§, μmol/day |
26.2 (13.4–35.9) |
| Wilson prognostic index |
4.0 (1.0–7.0) |
| Penicillamine, n (%) |
18 (64.3) |
| Trientine, n (%) |
8 (28.6) |
| Liver transplantation, n (%) |
2 (7.1) |
ALT = alanine aminotransferase; AST = aspartate aminotransferase; INR = international normalized ratio; IU = international units; NCC = nonceruloplasmin-bound copper concentration.
∗n = 22.
†n = 26.
‡n = 20.
§n = 24.
Urinary Copper Excretion
Over the median follow-up duration of 7 years, 21 patients had at least one 24-hour urinary copper test. Of those, 4 had been tested for both 24-hour and spot UCE during follow-up. The remained 7 children had only spot UCE tests.
At the first visit, the median of 24-hour urinary copper collected before penicillamine challenge test (PCT) was 3.7 μmol/day (IQR: 2.3–8.7), and this value increased to 26.2 μmol/day (IQR: 13.4–35.9) after 1000 mg penicillamine administration. There is a significant decrease in the concentration of 24-hour UCE overtime when analyzed with Kruskal-Wallis (P < 0.001) (Fig. 1, Suppl Table 2, https://links.lww.com/MPG/C28). As compared to the post-PCT level, the excretion rate was significantly reduced to 2.2 μmol/day at 3–4 years (P = 0.009). Comparisons have been made between patients with and without acute liver failure as well as between patients treated with penicillamine and trientine, and only 24-hour UCE at the first visit (post-PCT) were significantly higher in the acute liver failure and penicillamine groups (Suppl Figures 3, https://links.lww.com/MPG/C31 and 4, https://links.lww.com/MPG/C32).
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FIGURE 1: Twenty-four hours urinary copper excretion in 24 WD patients. WD = Wilson disease.
Over the follow-up period, 11 children with WD experienced a decreasing level of median spot urine copper to creatinine ratio (UCCR) from 6.3 μmol/mmol creatinine (Cr) at the time of diagnosis to 1.7 μmol/mmol Cr within 1 year of treatment, and then decrease to 1.1 and 0.4 μmol/mmol Cr at 1–5 years and 5–10 years, respectively (Fig. 2, Suppl Table 2, https://links.lww.com/MPG/C28). Of note, within a small group of patients, we divided the duration of follow-up to before and after 5 years of therapy.
FIGURE 2: Spot urinary copper to creatinine ratio in 11 followed-up patients.
Nonceruloplasmin-Bound Copper Concentration
Among 28 pediatric WD patients, 26 had the followed-up values of serum copper available to calculate for NCC. Figure 3 demonstrates the median values of serum free copper at the first visit and during the follow-up period.
FIGURE 3: Nonceruloplasmin-bound copper concentration in 26 patients.
When taking the level of NCC into account, 24 patients had both 24-hour UCE and NCC during a total number of 48 follow-up visits. We found that before receiving any treatments, a majority of patients (69.6%) had high levels of both NCC (>2.4 μmol/L) and 24-hour UCE post PCT (median 28.8 μmol/day). Both levels had been decreasing over time with the treatment of copper chelating agents. This can be seen as a majority of followed-up patients were in the group of NCC <0.8 μmol/L (accounted for 71.4%, 77.8%, and 66.7% within 1 year and after 1 and 5 years of follow-up, respectively). The median levels of 24-hour UCE in this group (NCC <0.8 μmol/L) were 13.3, 8.3, and 5.9 μmol/day within 1 year of treatment and after 1 and 5 years of follow-up, respectively.
DISCUSSION
Urinary copper measurement for 24 hours is a useful parameter for the diagnosis and monitoring treatment of WD. In untreated patients, 24-hour UCE can reflect the concentration of nonceruloplasmin-bound copper in the circulation (10). It is reported that the cutoff value of baseline 24-hour UCE is 0.65 μmol/day with a sensitivity of 78.9% and specificity of 87.9% (16); predominantly, this value is normal in asymptomatic or mild liver disease patients (10). Moreover, 86% of WD patients had basal UCE >1.6 μmol/day (9). Urinary copper data in our study showed a median baseline UCE of 3.4 μmol/day (range 0.4–128.7) with only 2 of 22 patients having the value between 0.65 and 1.6 μmol/day, where approximately 50% of patients were asymptomatic and only 7% required liver transplantation (Table 1). Our finding is comparable with the study of Walshe (13); he reported a median basal 24-hour UCE of 3.3 μmol/day in presymptomatic patients, suggesting the high utility of baseline urine copper measurement with a caveat that normal value does not exclude WD (1).
Wang et al (17) studied in 31 children with WD and reported a significant correlation between 24-hour UCE and UCCR; the mean ratio was 0.408 ± 0.401 and 0.135 ± 0.060 μg/mmol Cr in children with WD and other liver diseases, respectively (17). Another study investigated on the screening performance of spot urinary copper in 153 healthy preschool children (18). With a comparison between spot urine samples and 24-hour copper excretion in 6 WD patients, they proposed that the cutoff values for the differentiation of WD were 0.5 μmol/L for spot urine copper concentration and 0.1 μmol/mmol Cr for the ratio with creatinine. They also found that among 35 spot urine samples from 6 WD patients, the median UCCR was reduced over time from 0.23 (within 1 year of treatment) to 0.17 μmol/mmol Cr (after 1 year of treatment). Their findings are consistent with ours; among 12 followed-up patients with 17 spot urine samples, the median UCCR was 1.7 (IQR 0.8–2.2) and 0.9 (IQR 0.3–2.5) μmol/mmol Cr within and after 1 year of treatment, respectively. Two WD patients had UCCR measured after penicillamine administration (without a comparative value with 24-hour urinary copper concentration); this revealed a median of 6.3 μmol/mmol Cr (Suppl Table 2, https://links.lww.com/MPG/C28). Although our data showed a decreasing trend of spot UCCR after treatment, our WD patients had an extremely higher level of UCCR during follow-up, as compared to those in Tang's study (18) (Fig. 2). Therefore, the use of UCCR may require more critical evaluation and interpretation, as this method has not been widely accepted.
There is also a discrepancy among the experts in terms of their views on how to measure the amount of copper excreted in urine during follow-up, whether it should be done during on or off medication. Although it is recommended that 24-hour urinary copper should be monitored on therapy with the acceptable range of 3–8 μmol/day (10,12), some suggested to measure this during off-treatment and proposed a value of <0.5 μmol/day as a “normal” value (19). Another recommended that the concentration of 24-hour urinary copper measured after 48-hour off-treatment should be ≤1.6 μmol/day; noncompliance should be suspected if the patients’ urinary copper concentration is >1.6 μmol/day coupled with the level of NCC of >2.4 μmol/L (10).
A recent study in adults reported the levels of 24-hour UCE during follow-up period after “48-hour off-treatment” and the levels during “on treatment”; they found 24-hour UCE during on therapy can be approximately 10 times higher than those obtained after a 48-hour dose interruption (14).
In our study, we aimed to evaluate the clinical use of UCE in WD patients during the follow-up period. Unlike adults where chelator dose is usually reduced over time, in children their body growth indirectly contributes to weaning as the chelator dose is not increased with increasing body weight. A sequential urine copper measurement may be of value to assess urine copper losses and to estimate body copper load and rarely iatrogenic copper deficiency.
We found that within 1 year of treatment, there is a huge variation of the urinary copper concentration. In 1 patient, the excretion rate can be up to more than 200 μmol/day, measured at 6 months after treatment. This result is compatible with that of the study of Pfeiffenberger (14) and may be explained by a high copper load in the body before therapy. We noted that chelating agents achieve their best effectiveness after 3 to 4 years of treatment, as the median level of 24-hour UCE significantly reduced from 26.2 μmol/day before treatment (but post PCT) to 2.2 μmol/day (P = 0.009) (Fig. 1, Suppl Table 2, https://links.lww.com/MPG/C28). The reduction of 24-hour UCE during long-term WD therapy reflects the depletion of the copper load in the body over years (14). Although the value of UCE was rising again after 48 months, one of the reasons could have been due to nonadherent patients taking medication just before the tests (Suppl Figures 1, https://links.lww.com/MPG/C29 and 2, https://links.lww.com/MPG/C30).
Similarly, the amount of NCC dramatically decreased after the administration of treatment (Fig. 3, Suppl Table 2, https://links.lww.com/MPG/C28). Interestingly, although NCC level remained significantly low over time, the median copper concentration measured from 24-hour urine was conversely higher than the recommended range (3–8 μmol/day) (10,12) after 4 years of treatment in 2 patients (Fig. 1). This can be explained by patients’ nonadherence, which needed a frequent monitoring so that the median concentration of 24-hour UCE was reduced to 5.6 μmol/day at >5–10 years (Fig. 1). Likewise, the variation of the copper excretion rate was also detected in the studies of both Pfeiffenberger and Walshe; they explained this by a small sample size at some time points (14). In the present study, however, the long-term variation is less than those observed in the other two studies (13,14). This probably means that a majority of our patients were less likely to be nonadherent (meaning taking medication before clinic appointment if they were not adherent before).
Our study suggests that within 1 year of treatment, the level of NCC drops to <0.8 μmol/L and the 24-hour UCE decreases to ≤8 μmol/day and <6 μmol/day after 1 and 5 years of treatment, respectively, without any progression of liver disease or signs of copper deficiency (eg, anemia, neutropenia). However, there is a limitation in the use of NCC level. Although it is an easy method that may represent an estimate value for unbound copper, NCC is calculated from the levels of ceruloplasmin and serum copper. Therefore, its reliability solely depends on the other 2 tests; the validity of this method needs to be established. Moreover, recent studies (20–22) proposed a new promising tool, namely, exchangeable copper, which can provide an exact estimation of copper overload and importantly this method is not dependent on the level of ceruloplasmin. This novel method was not available in our center.
To our knowledge, we report the first experience in pediatric WD addressing the clinical utility of urinary copper concentration measured on therapy during follow-up visits. A prospective evaluation of these parameters in multicenter setting is suggested.
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