A highly sensitive assay of human plasma xanthine oxidoreductase activity using stable isotope-labeled xanthine and LC/TQMS
A highly sensitive assay of human plasma xanthine oxidoreductase activity using stable isotope-labeled xanthine and LC/TQMS
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DOI：
10.1016/j.jchromb.2016.10.033
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发表时间：
2016-12-15
影响因子：
3
通讯作者：
Nakamura, Takashi
中科院分区：
文献类型：
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作者：
Murase, Takayo;Nampei, Mai;Nakamura, Takashi
血浆黄嘌呤氧化还原酶（XOR）活性升高和各种病理之间的关联已被广泛报道。然而，由于人的XOR活性低于动物如小鼠的XOR活性，因此难以精确测量人血浆XOR活性。我们利用[C-13（2），N-15（2）]黄嘌呤和液相色谱/三重四极杆质谱的组合开发了一种高灵敏度的XOR活性测定法。在本研究中，我们建立并验证了一种新的人血浆XOR活性测定利用这种技术。[C-13（2），N-15（2）]尿酸的校准曲线在4-4000 nM范围内呈线性（r（2）> 0.995），定量下限为4 nM，对应于6.67 pmol/h/mL血浆的XOR活性。合并人血浆XOR活性的试验内和试验间变异系数分别为6.5%和9.1%。20名健康志愿者的血浆XOR活性范围为32.8至227 pmol/h/mL（平均值+/- SD = 89.1 +/- 55.1，n = 20），与丙氨酸转氨酶（r= 0.827）、天冬氨酸转氨酶（r = 0.487）和尿酸（r= 0.502）相关。该方法有望用于研究XOR的功能及其抑制剂在多种疾病中的作用。(C)2016爱思唯尔B. V.保留所有权利。
Associations between elevated plasma xanthine oxidoreductase (XOR) activity and various pathologies have been widely reported. However, it has been difficult to accurately measure human plasma XOR activity because the XOR activity of humans is lower than that of animals such as mouse. We developed a highly sensitive assay for XOR activity utilizing a combination of [C-13(2),N-15(2)] xanthine and liquid chromatography/triple quadrupole mass spectrometry. In the present study, we established and validated a novel human plasma XOR activity assay utilizing this technique. The calibration curve of [C-13(2),N-15(2)]uric acid showed linearity over the range of 4-4000 nM (r(2) > 0.995) with a lower limit of quantitation of 4 nM which corresponds to an XOR activity of 6.67 pmol/h/mL plasma. Intra- and inter-assay coefficients of variation of pooled human plasma XOR activity were 6.5% and 9.1%, respectively. Plasma XOR activities of 20 healthy volunteers ranged from 32.8 to 227 pmol/h/mL (mean +/- SD = 89.1 +/- 55.1, n = 20), which correlated with alanine transaminase (r= 0.827), aspartate transaminase (r = 0.487), and uric acid (r= 0.502). The established assay is expected to be useful for investigating the function of XOR and the effect of its inhibitors in various diseases. (C) 2016 Elsevier B.V. All rights reserved.