title: A highly sensitive assay for xanthine oxidoreductase activity using a combination of [(13) C2 ,(15) N2 ]xanthine and liquid chromatography/triple quadrupole mass spectrometry.
abstract: In this study, we developed a highly sensitive assay for xanthine oxidoreductase (XOR) activity utilizing a combination of [(13) C2 ,(15) N2 ]xanthine and liquid chromatography (LC)/triple quadrupole mass spectrometry (TQMS). In this assay, the amount of [(13) C2 ,(15) N2 ]uric acid (UA) produced by XOR was determined by using LC/TQMS. For this assay, we synthesized [(13) C2 ,(15) N2 ]xanthine as a substrate, [(13) C2 ,(15) N2 ]UA as an analytical standard, and [(13) C3 ,(15) N3 ]UA as an internal standard. The [(13) C2 ,(15) N2 ]UA calibration curve obtained using LC/TQMS under the selected reaction monitoring mode was evaluated, and the results indicated good linearity (R(2)  = 0.998, weighting of 1/x(2) ) in the range of 20 to 4000 nM. As a model reaction of less active samples, the XOR activity of serial-diluted mouse plasma was measured. Thereby, the XOR activity of the 1024-fold-diluted mouse plasma was 4.49 ± 0.44 pmol/100 μL/h (mean ± standard deviation, n = 3). This value is comparable to the predicted XOR activity value of healthy human plasma. Hence, this combination method may be used to obtain high-sensitivity measurements required for XOR activity analysis on various organs or human plasma.
authors.affiliation: Radioisotope and Chemical Analysis Center, Laboratory Management Department, Sanwa Kagaku Kenkyusho Co., Ltd., 363 Shiosaki, Hokusei-cho, Inabe-shi, Inabe, Mie, 511-0406, Japan.
authors.forename: Takayo
authors.initials: T
authors.lastname: Murase
authors.affiliation: API Development Group, Pharmaceutical Technology Laboratories, Sanwa Kagaku Kenkyusho Co., Ltd., 363 Shiosaki, Hokusei-cho, Inabe-shi, Inabe, Mie, 511-0406, Japan.
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Oka
authors.affiliation: Pharmacological Study Group, Pharmaceutical Research Laboratories, Sanwa Kagaku Kenkyusho Co., Ltd., 363 Shiosaki, Hokusei-cho, Inabe-shi, Inabe, Mie, 511-0406, Japan.
authors.forename: Mai
authors.initials: M
authors.lastname: Nampei
authors.affiliation: Radioisotope and Chemical Analysis Center, Laboratory Management Department, Sanwa Kagaku Kenkyusho Co., Ltd., 363 Shiosaki, Hokusei-cho, Inabe-shi, Inabe, 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-shi, Inabe, Mie, 511-0406, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakamura
mesh_terms: D000818:Animals; D002250:Carbon Radioisotopes / Q000737:chemistry*; D002853:Chromatography, Liquid; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D009590:Nitrogen Radioisotopes / Q000737:chemistry*; D014527:Uric Acid / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D014968:Xanthine Dehydrogenase / Q000097:blood / Q000378:metabolism*
chemical_list: D002250:Carbon Radioisotopes; D009590:Nitrogen Radioisotopes; D014527:Uric Acid; D014968:Xanthine Dehydrogenase
keywords: LC/TQMS; stable isotope-labeled substrate; uric acid; xanthine; xanthine oxidoreductase activity