Asano Tomomi, Taki Kentaro, Kitamori Kazuya, Naito Hisao, Nakajima Tamie, Tsuchihashi Hitoshi, Ishii Akira, Zaitsu Kei
Department of Human Life and Environment, Kinjo Gakuin University, 2-1723 Omori, Moriyama-ku, Nagoya 463-8521, Japan.
Department of Legal Medicine & Bioethics, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
ACS Omega. 2021 Mar 16;6(12):8588-8597. doi: 10.1021/acsomega.1c00403. eCollection 2021 Mar 30.
We developed a highly sensitive method for quantifying 21 bile acids (BAs) in the rat liver by capillary liquid chromatography tandem mass spectrometry (cLC/MS/MS) with one-pot extraction. High recovery rates were obtained for the one-pot methods with either methanol (MeOH) extraction or MeOH/acetonitrile (ACN) (1:1, v/v) mixture extraction; the results obtained for the MeOH/ACN mixture solution were better than the results obtained for MeOH. Thus, we determined that the one-pot method with MeOH/ACN was the most suitable method for the efficient extraction of BAs in the liver. Targeted BAs were well separated by cLC with gradient elution using ammonium acetate (NHOAc)-MeOH mobile phases. Method validation proved that the intra-day and inter-day accuracies and precisions were primarily less than ±20 and 20% relative standard deviation, respectively. Also, the limit of detection (LOD) and the limit of quantitation (LOQ) were 0.9-10 and 2.3-27 ng/g liver, which proves the high sensitivity of the method. Finally, we quantitated 21 BA concentrations in the liver samples of normal and nonalcoholic steatohepatitis (NASH) rats, both of which were derived from stroke-prone spontaneously hypertensive five (SHRSP5) /Dmcr rat. The hepatic BA profiles were found to be substantially different between the normal and NASH groups; the two groups were clearly separated along the first component axis in the score plots of the principal component analysis. In particular, 10 BAs (β-muricholic acid (MCA), glyco (G-) cholic acid (CA), G-chenodeoxycholic acid (CDCA), tauro (T-) CA, T-CDCA, T-ursodeoxycholic acid (UDCA), T-lithocholic acid (LCA), T-hiodeoxycholic acid (HDCA), T-α-MCA, and T-β-MCA) were significantly different between the two groups using Welch's -test with the false discovery rate correction method, demonstrating BA disruption in the NASH model rat. In conclusion, this method was able to quantify 21 BAs in the rat liver and will evaluate the hepatic BA pathophysiology of rat disease models.
我们开发了一种高灵敏度的方法，通过一锅法萃取的毛细管液相色谱串联质谱法（cLC/MS/MS）对大鼠肝脏中的21种胆汁酸（BAs）进行定量分析。甲醇（MeOH）萃取或MeOH/乙腈（ACN）（1:1，v/v）混合萃取的一锅法均获得了较高的回收率；MeOH/ACN混合溶液的结果优于MeOH的结果。因此，我们确定MeOH/ACN一锅法是肝脏中胆汁酸高效萃取的最合适方法。使用醋酸铵（NHOAc）-MeOH流动相进行梯度洗脱，通过cLC可将目标胆汁酸很好地分离。方法验证表明，日内和日间准确度和精密度的相对标准偏差分别主要小于±20%和20%。此外，检测限（LOD）和定量限（LOQ）分别为0.9 - 10和2.3 - 27 ng/g肝脏，证明了该方法的高灵敏度。最后，我们对正常和非酒精性脂肪性肝炎（NASH）大鼠肝脏样本中的21种胆汁酸浓度进行了定量分析，这两种大鼠均来源于易中风自发性高血压5型（SHRSP5）/Dmcr大鼠。发现正常组和NASH组之间的肝脏胆汁酸谱存在显著差异；在主成分分析的得分图中，两组沿第一成分轴明显分开。特别是，使用经错误发现率校正方法的Welch检验，发现两组之间有10种胆汁酸（β-鼠胆酸（MCA）、甘氨（G-）胆酸（CA）、G-鹅去氧胆酸（CDCA）、牛磺（T-）CA、T-CDCA、T-熊去氧胆酸（UDCA）、T-石胆酸（LCA）、T-猪去氧胆酸（HDCA）、T-α-MCA和T-β-MCA）有显著差异，表明NASH模型大鼠存在胆汁酸紊乱。总之，该方法能够对大鼠肝脏中的21种胆汁酸进行定量分析，并将评估大鼠疾病模型的肝脏胆汁酸病理生理学。
