Murase Takayo, Oka Mitsuru, Nampei Mai, Miyachi Atsushi, Nakamura Takashi
Radioisotope and Chemical Analysis Center, Laboratory Management Department, Sanwa Kagaku Kenkyusho Co., Ltd., 363 Shiosaki, Hokusei-cho, Inabe-shi, Inabe, Mie, 511-0406, Japan.
API Development Group, Pharmaceutical Technology Laboratories, Sanwa Kagaku Kenkyusho Co., Ltd., 363 Shiosaki, Hokusei-cho, Inabe-shi, Inabe, Mie, 511-0406, Japan.
J Labelled Comp Radiopharm. 2016 May 15;59(5):214-20. doi: 10.1002/jlcr.3390. Epub 2016 Mar 22.
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.
在本研究中，我们开发了一种高灵敏度的黄嘌呤氧化还原酶（XOR）活性检测方法，该方法结合了[(13)C2,(15)N2]黄嘌呤与液相色谱（LC）/三重四极杆质谱（TQMS）。在该检测方法中，通过LC/TQMS测定XOR产生的[(13)C2,(15)N2]尿酸（UA）的量。为此检测方法，我们合成了[(13)C2,(15)N2]黄嘌呤作为底物、[(13)C2,(15)N2]UA作为分析标准品以及[(13)C3,(15)N3]UA作为内标。对在选定反应监测模式下使用LC/TQMS获得的[(13)C2,(15)N2]UA校准曲线进行了评估，结果表明在20至4000 nM范围内具有良好的线性（R(2) = 0.998，加权为1/x(2)）。作为活性较低样品的模型反应，测量了系列稀释小鼠血浆的XOR活性。由此，1024倍稀释小鼠血浆的XOR活性为4.49±0.44 pmol/100 μL/h（平均值±标准差，n = 3）。该值与健康人血浆的预测XOR活性值相当。因此，这种组合方法可用于获得对各种器官或人血浆进行XOR活性分析所需的高灵敏度测量结果。
