Exchangeable copper concentration and 3-kilodalton ultrafiltered urinary copper measurements are successfully adapted and partially validated in dogs.

Ultrafiltration methods for serum and urinary copper measurement are analytically validated for future CAH biomarker studies in dogs.

This study adapted and partially validated two ultrafiltration-based analytical methods for measuring serum exchangeable copper (ExCu) and 3-kDa ultrafiltered urinary copper concentrations in dogs, as a prerequisite for evaluating their clinical utility as noninvasive biomarkers for copper-associated hepatitis (CAH). Surplus diagnostic serum and urine samples from dogs were collected between October 2023 and March 2024. Copper concentrations were quantified using inductively coupled plasma mass spectrometry (ICP-MS), adapting a previously described ultracentrifugation protocol. Optimal parameters were determined to be 5 g/L EDTA with centrifugation at 5,000 × g for serum ExCu and 4,000 × g for ultrafiltered urinary copper. Spike recovery was 97.9–108.5% for serum ExCu and 96.7–120% for urinary copper, both within predefined acceptance criteria. Intra- and interassay coefficients of variation (CV) were 8.5% and 10.8% for serum ExCu, and 2.6% and 5.6% for urinary copper, respectively, indicating acceptable precision. These findings establish the analytical foundation needed to investigate the diagnostic performance of relative exchangeable copper (RExCu) and the urinary small copper carrier-to-creatinine ratio as noninvasive alternatives to liver biopsy for diagnosing CAH in dogs. Future clinical studies are warranted to evaluate the sensitivity, specificity, and reference intervals of these biomarkers in affected versus healthy canine populations.

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