Xanthine oxidoreductase activity assay in tissues using stable isotope-labeled substrate and liquid chromatography high-resolution mass spectrometry

• New XOR activity assay was developed using [15N2]-xanthine as substrate.
• [15N2]-UA production was measured by LC/HRMS.
• [15N2]-UA determination was validated with good linearity, accuracy, and precision.
• HRMS full-scan mode was used to examine endogenous inhibitor effects.
• XOR assay results showed good correlation with conventional LC/FL assay results.

Studies of pathological mechanisms and XOR inhibitor characterization, such as allopurinol, febuxostat, and topiroxostat, require accurate and sensitive measurements of XOR activity. However, the established assays have some disadvantages such as susceptibility to endogenous substances such as uric acid (UA), xanthine, or hypoxanthine. Here, we aimed to develop a novel XOR activity assay utilizing a combination of high-performance liquid chromatography (LC) and high-resolution mass spectrometry (HRMS) for tissues such as the liver, kidney, and plasma. Stable isotope-labeled [15N2]-xanthine was utilized as substrate and the production of [15N2]-uric acid was determined. [15N2]-UA production by XOR was dependent on the amounts of [15N2]-xanthine and enzyme and the time of reaction. Because high concentrations of endogenous xanthine and hypoxanthine affect XOR activities, we employed a multi-component analysis using LC/HRMS to improve the accuracy of XOR activity assay. Quantification of [15N2]-UA was validated and showed good linearity, accuracy, and precision. We measured the XOR activities of retired ICR mice using [15N2]-xanthine and LC/MS. The XOR activities in plasma, kidney, and liver samples were 38.1 ± 0.7, 158 ± 5, 928 ± 25 pmol/min/mg of protein, respectively (mean ± SD, n = 5). Furthermore, we measured the XOR activities in the same samples using the LC/ultraviolet and LC/fluorescence (FL) methods. The level of [15N2]-xanthine oxidation by XOR was equal to that of xanthine oxidation and approximately 7.9–8.9 times higher than that of pterin oxidation. We found a good correlation between XOR activities examined using LC/MS assay with [15N2]-xanthine and those examined using LC/FL assay with pterin. This result suggested that although both the LC/MS assay with [15N2]-xanthine and the LC/FL assay with pterin were useful, the former provided information regarding XOR activities that more directly reflected the physiological condition than the latter.