SIMPLE AND RAPID QUANTITATION OF 21 BILE ACIDS IN RAT SERUM AND LIVER BY UPLC-MS-MS: EFFECT OF HIGH FAT DIET ON GLYCINE CONJUGATES OF RAT BILE ACIDS
SIMPLE AND RAPID QUANTITATION OF 21 BILE ACIDS IN RAT SERUM AND LIVER BY UPLC-MS-MS: EFFECT OF HIGH FAT DIET ON GLYCINE CONJUGATES OF RAT BILE ACIDS
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DOI：
10.18999/nagjms.75.1-2.57
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发表时间：
2013-02
影响因子：
0.9
通讯作者：
Yudai Suzuki;R. Kaneko;Mina Nomura;Hisao Naito;K. Kitamori;T. Nakajima;Tadashi Ogawa;H. Hattori;H. Seno;A. Ishii
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文献类型：
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作者：
Yudai Suzuki;R. Kaneko;Mina Nomura;Hisao Naito;K. Kitamori;T. Nakajima;Tadashi Ogawa;H. Hattori;H. Seno;A. Ishii
本文建立了一种以胆酸为内标物，采用超高效液相色谱-串联质谱（UPLC-MS-MS）负离子模式，快速测定大鼠血清和肝脏中21种胆汁酸及其结合物的方法。从血清和肝脏样品中液-液提取后，通过配备Acquity TQD串联四极杆质谱仪的UPLC分析标本。21种胆汁酸在5 min内完全分离，血清样品在0.25 ~ 5000 ng/mL范围内线性良好，肝脏样品在2.5 ~ 50 μg/g范围内线性良好。估计血清中的检测限（LOD）小于0.25至7.5 ng/mL，肝脏样品中的检测限小于2.5至10 ng/g。对本方法的重复性进行了验证;血清中的变异系数（CV）值小于26.7%，肝脏中的CV值小于25.9%。在动物实验中，我们比较了21种胆汁酸在易卒中的自发性高血压（SHRSP）大鼠的血清和肝脏样品中的含量，这些大鼠分别喂食对照（SP）饮食和高脂高胆固醇（HFC）饮食。饲喂HFC饲料后，血清和肝脏中某些胆汁酸的甘氨酸结合物显著增加，而牛磺酸结合物（TUDC）显著减少。我们的研究结果表明，胆汁酸谱的变化可用于非酒精性脂肪性肝病（NAFLD）的诊断。
ABSTRACT In this report, we present a simple and rapid method for analysis of 21 kinds of bile acids and the conjugates in rat serum and liver samples by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) in the negative ionization mode, using cholic-2, 2, 4, 4-d4 acid as internal standard. After liquid-liguid extraction from serum and liver samples, specimens were analyzed by UPLC equipped with an Acquity TQD tandem quadrupole mass spectrometer. All of the 21 bile acids were sufficiently separated within 5 min. For most bile acids, calibration curves showed good linearities in the range of 0.25 to 5000 ng/mL for serum samples, 2.5 ng/g to 50 μg/g for liver samples. The limits of detection (LOD) were estimated to be less than 0.25 to 7.5 ng/mL in serum, less than 2.5 to 10 ng/g in liver samples. The present method was validated with respect to repeatability; the coefficient of variation (CV) values were less than 26.7% in the serum and 25.9% in the liver. In the animal study, we compared 21 bile acids in the serum and liver samples of the stroke-prone spontaneously hypertensive (SHRSP) rats fed with control (SP) diet or high-fat and high-cholesterol-containing (HFC) diet. By feeding with HFC diet, the glycine conjugates of some bile acids significantly increased and the taurine conjugate of ulsodeoxicolate (TUDC) decreased in serum and liver samples. Our results suggest that the change of bile acid profiles could be applied for the diagnosis of non-alcoholic fatty liver disease (NAFLD).