title: Xanthine oxidoreductase activity assay in tissues using stable isotope-labeled substrate and liquid chromatography high-resolution mass spectrometry.
abstract: 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 [(15)N2]-xanthine was utilized as substrate and the production of [(15)N2]-uric acid was determined. [(15)N2]-UA production by XOR was dependent on the amounts of [(15)N2]-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 [(15)N2]-UA was validated and showed good linearity, accuracy, and precision. We measured the XOR activities of retired ICR mice using [(15)N2]-xanthine and LC/MS. The XOR activities in plasma, kidney, and liver samples were 38.1±0.7, 158±5, 928±25pmol/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 [(15)N2]-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 [(15)N2]-xanthine and those examined using LC/FL assay with pterin. This result suggested that although both the LC/MS assay with [(15)N2]-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.
authors.affiliation: Radioisotope and Chemical Analysis Center, Laboratory Management Department, Sanwa Kagaku Kenkyusho Co., Ltd., 363 Shiosaki, Hokusei-cho, Inabe-city, Mie 511-0406, Japan. Electronic address: ta_murase@skk-net.com.
authors.forename: Takayo
authors.initials: T
authors.lastname: Murase
authors.affiliation: Pharmacological Study Group, Pharmaceutical Research Laboratories, Sanwa Kagaku Kenkyusho Co., Ltd., 363 Shiosaki, Hokusei-cho, Inabe-city, Mie 511-0406, Japan.
authors.forename: Mai
authors.initials: M
authors.lastname: Nampei
authors.affiliation: API Development Group, Pharmaceutical Technology Laboratories, Sanwa Kagaku Kenkyusho Co., Ltd., 363 Shiosaki, Hokusei-cho, Inabe-city, Mie 511-0406, Japan.
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Oka
authors.affiliation: Pharmacology Research Group1, Research Department, Medical R&D Division, Fuji Yakuhin Co., Ltd., 636-1, Iidasinden, Nishi-ku, Saitama-city, Saitama 331-0068 Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Ashizawa
authors.affiliation: Pharmacology Research Group1, Research Department, Medical R&D Division, Fuji Yakuhin Co., Ltd., 636-1, Iidasinden, Nishi-ku, Saitama-city, Saitama 331-0068 Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Matsumoto
authors.affiliation: Radioisotope and Chemical Analysis Center, Laboratory Management Department, Sanwa Kagaku Kenkyusho Co., Ltd., 363 Shiosaki, Hokusei-cho, Inabe-city, Mie 511-0406, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyachi
authors.affiliation: Pharmacological Study Group, Pharmaceutical Research Laboratories, Sanwa Kagaku Kenkyusho Co., Ltd., 363 Shiosaki, Hokusei-cho, Inabe-city, Mie 511-0406, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakamura
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D008813:Mice, Inbred ICR; D014968:Xanthine Dehydrogenase / Q000378:metabolism*
chemical_list: D014968:Xanthine Dehydrogenase
keywords: High-resolution mass spectrometry; Stable isotope-labeled substrate; Xanthine oxidoreductase activity; Xanthine oxidoreductase inhibitor