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

OBJECTIVE: To adapt and partially validate the accuracy and precision of serum exchangeable copper and 3-kDa ultrafiltered urinary copper concentrations as an analytical prerequisite to investigate the clinical utility of relative exchangeable copper and urinary small copper carrier-to-creatinine ratio as noninvasive biomarkers in dogs with copper-associated hepatitis. METHODS: Serum exchangeable copper and 3-kDa ultrafiltered urinary copper concentrations of surplus samples of dogs submitted for diagnostic purposes from October 2023 through March 2024 were measured by adapting a previously described ultracentrifugation method. Copper concentrations were quantified using inductively coupled plasma mass spectrometry. The optimal relative centrifugal force was selected based on minimizing the variability among replicates. Analytical performance was evaluated through spike recovery and intra- and interassay repeatability. RESULTS: Optimal parameters were identified as 5 g/L EDTA and 5,000 X g for serum exchangeable copper and 4,000 X g for 3-kDa ultrafiltered urinary copper. Spike recovery ranged from 97.9% to 108.5% for serum exchangeable copper and from 96.7% to 120% for ultrafiltered urinary copper. The median intra- and interassay coefficients of variation were 8.5% and 10.8% for exchangeable copper and 2.6% and 5.6% for ultrafiltered urinary copper, respectively. CONCLUSIONS: Ultrafiltration-based methods for measuring serum exchangeable copper and 3-kDa ultrafiltered urinary copper concentrations were successfully adapted for dogs, with spike recovery and intra- and interassay coefficients of variation within predefined acceptance criteria. CLINICAL RELEVANCE: The results provide the analytical foundation for future investigation of the diagnostic utility of relative exchangeable copper and urinary small copper carrier protein as noninvasive biomarkers for copper-associated hepatitis in dogs.