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A highly sensitive assay for xanthine oxidoreductase activity using a combination of [13C2,15N2]xanthine and liquid chromatography/triple quadrupole mass spectrometry

Murase, Takayo ; Oka, Mitsuru ; Nampei, Mai ; Miyachi, Atsushi ; Nakamura, Takashi

Journal of labelled compounds & radiopharmaceuticals, 2016-05, Vol.59 (5), p.214-220 [Periódico revisado por pares]

England: Blackwell Publishing Ltd

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  • Título:
    A highly sensitive assay for xanthine oxidoreductase activity using a combination of [13C2,15N2]xanthine and liquid chromatography/triple quadrupole mass spectrometry
  • Autor: Murase, Takayo ; Oka, Mitsuru ; Nampei, Mai ; Miyachi, Atsushi ; Nakamura, Takashi
  • Assuntos: Animals ; Carbon Radioisotopes - chemistry ; Chromatography, Liquid ; Enzyme Assays - methods ; Humans ; LC/TQMS ; Mass Spectrometry ; Mice ; Nitrogen Radioisotopes - chemistry ; stable isotope-labeled substrate ; uric acid ; Uric Acid - chemistry ; Uric Acid - metabolism ; Uric Acid - pharmacology ; xanthine ; Xanthine Dehydrogenase - blood ; Xanthine Dehydrogenase - metabolism ; xanthine oxidoreductase activity
  • É parte de: Journal of labelled compounds & radiopharmaceuticals, 2016-05, Vol.59 (5), p.214-220
  • Notas: ark:/67375/WNG-N5H20LSK-Z
    istex:8D61BBB4E7ED07B62A9CB1A69FBA1E3F306807B3
    ArticleID:JLCR3390
    ObjectType-Article-1
    SourceType-Scholarly Journals-1
    ObjectType-Feature-2
    content type line 23
  • Descrição: In this study, we developed a highly sensitive assay for xanthine oxidoreductase (XOR) activity utilizing a combination of [13C2,15N2]xanthine and liquid chromatography (LC)/triple quadrupole mass spectrometry (TQMS). In this assay, the amount of [13C2,15N2]uric acid (UA) produced by XOR was determined by using LC/TQMS. For this assay, we synthesized [13C2,15N2]xanthine as a substrate, [13C2,15N2]UA as an analytical standard, and [13C3,15N3]UA as an internal standard. The [13C2,15N2]UA calibration curve obtained using LC/TQMS under the selected reaction monitoring mode was evaluated, and the results indicated good linearity (R2 = 0.998, weighting of 1/x2) 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. We developed a highly sensitive assay of xanthine oxidoreductase (XOR) activity, using [13C2,15N2]xanthine as the substrate, and determined the amount of [13C2,15N2]uric acid (UA) produced by XOR through liquid chromatography(LC)/triple quadrupole mass spectrometry (TQMS). A [13C2,15N2]UA calibration curve was formed using LC/TQMS under the selected reaction monitoring mode, which showed good linearity (R2 = 0.998, weighting of 1/x2) in the range of 20 to 4000 nM and LLOQ 20 nM.
  • Editor: England: Blackwell Publishing Ltd
  • Idioma: Inglês;Francês;Alemão
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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