High-throughput copper speciation with SEC-UV-ICPMS: Insights from Wilson's disease mouse models

Wilson's disease (WD) is a genetic disorder caused by ATP7B gene mutations, impairing copper excretion and leading to toxic copper accumulation in vital organs. Inductively coupled plasma mass spectrometry (ICPMS)-based serum copper analysis has shown reliability in WD studies, with relative exchangeable copper (REC, proportion of exchangeable copper in the blood relative to the total copper) and accurate non-ceruloplasmin-bound copper (ANCC) emerging as promising biomarkers. However, challenges persist in applying these methods in live animal studies because of limited sample volumes and impractical copper specifications, necessitating alternative analytical approaches.

This study comprehensively evaluated a size-exclusion chromatography coupled with ultraviolet (SEC-UV)-ICPMS method for precise copper speciation in mouse serum. This method effectively separated major copper-binding proteins while minimizing sample preparation and consumption. As a comparison, the direct injection-based ICPMS method was optimized to determine relative exchangeable copper (REC), and strong anion exchange (SAX)-ICPMS-based copper speciation approaches were investigated to validate the accuracy of the SEC-UV-ICPMS method, further confirming its robustness for live mouse studies. Further application to WD mouse models (ATP7B^tx-J) revealed distinct copper distribution differences between diseased and healthy states. Relative non-ceruloplasmin-bound copper (RNCC) was newly identified as a promising potential biomarker, defining a diagnostic threshold of 52-58 %. By accurately quantifying copper species in mouse serum, this study established a reliable analytical framework that greatly improved our understanding of copper distribution in Wilson's disease research. Our approach demonstrated high specificity, reproducibility, and throughput, suitable for live mouse studies.

The SEC-UV-ICPMS platform offers a robust and efficient approach for serum copper speciation and quantification that addresses current limitations in live animal studies. By establishing RNCC as a reliable potential biomarker and enabling detailed copper profiling, this method enhances the precision of copper measurement and supports therapeutic monitoring. The applicability to WD research and capability of absolute quantification of copper species underscore its potential as a critical tool in clinical diagnostics and mechanistic studies, advancing the understanding and treatment of copper metabolism disorders.