Positioning the Cu/Zn Ratio within the Diagnostic Framework of Wilson Disease: Methodological and Conceptual Considerations

To the Editor:

We read with great interest the recent article by Begum et al. titled “Urinary Copper/Zinc Ratio for Diagnosing Wilson Disease,” published in the Pediatric Gastroenterology, Hepatology & Nutrition journal [1]. The authors’ efforts to propose a spot urinary copper/zinc (Cu/Zn) ratio as a surrogate for 24-hours urinary copper excretion in the diagnosis of pediatric Wilson disease (WD) are both timely and of potential clinical relevance. This simplified approach may indeed offer practical advantages, particularly in resource-limited settings. Nevertheless, we wish to highlight several methodological and interpretive considerations, particularly when evaluated against the backdrop of the current European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) recommendations [2] and recent advances in copper metabolism biomarkers [3].

One of the key methodological limitations is the lack of urinary creatinine normalization when calculating the Cu/Zn ratio. It is well established that spot urine-based measurements, particularly for trace elements, are significantly influenced by the patient’s hydration status and intra-individual variability in urine concentration. This issue is especially pronounced in pediatric patients because of the greater variation in body mass and diurnal fluctuations in urine output. Although the authors briefly acknowledge this issue, the absence of creatinine-adjusted ratios limits the reliability and reproducibility of the Cu/Zn metric as a diagnostic indicator. Current laboratory practice supports the use of copper-to-creatinine ratios in random urine samples to mitigate dilutional variability, especially in pediatric populations [3,4]. Without appropriate creatinine normalization, the Cu/Zn ratio may reflect random variability in urine concentrations rather than a disease-specific metabolic signal.

Another relevant concern pertains to the proposed positioning of the Cu/Zn ratio as a potential surrogate for 24-hours urinary copper excretion in the diagnostic workup of WD. Although the authors acknowledge that the current guidelines emphasize established markers, their proposal for the Cu/Zn ratio lacks contextual placement within a validated diagnostic framework. For instance, the Leipzig scoring system, endorsed by the ESPGHAN, does not incorporate this parameter [2]. Importantly, neither the 2018 ESPGHAN position paper [2] nor the 2022 biochemical review [3] currently recommend the Cu/Zn ratio as part of the diagnostic algorithm for WD. In contrast, recent literature underscores the limitations of non-validated surrogate markers, particularly when interpreted in isolation and without sufficient clinical and biochemical correlations [3,4]. Although exploratory studies on novel diagnostic markers are undoubtedly valuable, their interpretation should be carefully framed within existing diagnostic paradigms and validated against clinically relevant populations.

Another notable methodological concern relates to the 24-hours urine collections used as a reference standard in this study. The authors reported that these samples were collected without acidification, which differs from the well-established protocols for trace element measurement, particularly for copper. Acidification with nitric or hydrochloric acid is widely recommended to prevent copper loss through precipitation or adsorption to container walls, both of which can result in underestimated urinary copper concentrations [3,4]. Consequently, the use of non-acidified 24-hours samples may have introduced a systematic underestimation of copper excretion, potentially biasing the comparative assessment with the spot Cu/Zn ratio. While the Cu/Zn ratio was calculated from a separate morning spot urine sample, any inaccuracy in the reference standard inevitably influences the perceived diagnostic performance of surrogate markers. In this context, the lack of acidification calls into question the internal validity of the reported diagnostic metrics of the Cu/Zn ratio.

Finally, we believe that the diagnostic performance of the Cu/Zn ratio reported in this study may have been overestimated owing to the composition of the study population. While the comparison group included children with various hepatic disorders, no carriers of heterozygous ATP7B mutation or patients with borderline WD (for example, those with equivocal ceruloplasmin or urinary copper levels) were enrolled. These are precisely the patient categories in which diagnostic discrimination is most difficult and where emerging biomarkers must demonstrate their true utility [2,3]. The exclusion of such diagnostically challenging subgroups may have led to an overestimation of specificity and sensitivity values. A more representative cohort, including heterozygous carriers and early or mild phenotypes, would be essential towards evaluating the true clinical applicability of the Cu/Zn ratio, especially considering the phenotypic variability of pediatric WD.

In conclusion, we commend the authors for exploring an alternative and simplified approach for WD diagnosis through the use of the urinary Cu/Zn ratio. However, methodological limitations, including lack of creatinine normalization, non-acidified urine collection, and limited cohort diversity, raise concerns regarding the robustness and generalizability of the findings. We believe that these issues warrant further investigation and support cautious interpretation of the results before clinical application. Including such novel surrogate markers in future studies that employ more rigorous protocols and diagnostically complex patient groups will be essential to determine their true relevance within the diagnostic framework. We hope that this correspondence will contribute to the ongoing discussion and refinement of reliable and accessible biomarkers for WD in children.