Identification of metabolites in human hepatic bile using 800 MHz 1H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS
Identification of metabolites in human hepatic bile using 800 MHz 1H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS
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DOI：
10.1039/b814426e
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发表时间：
2009-01-01
影响因子：
--
通讯作者：
Lindon, John C.
中科院分区：
文献类型：
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作者：
Duarte, Iola F.;Legido-Quigley, Cristina;Lindon, John C.
首次应用高场核磁共振波谱（800 MHz的1 H观察）的人肝胆汁（而不是胆囊胆汁）的报告。用于详细研究的胆汁样本来自移植前器官取出期间收集的具有轻度脂肪浸润的供体肝脏。此外，为了特别关注胆汁酸的检测，通过800 MHz 1 H NMR光谱、HPLC-NMR/MS和UPLC-MS分析胆汁提取物。在整个胆汁样品中，借助于二维H-1-H-1 TOCSY和H-1-C-13 HSQC光谱，已经指定了40个化合物。这些包括磷脂酰胆碱、14种氨基酸、10种有机酸、4种碳水化合物和多元醇（葡萄糖、葡萄糖醛酸、甘油和肌醇）、胆碱、磷酸胆碱、甜菜碱、三甲胺-N-氧化物和其他小分子。对一些关键代谢物的浓度范围进行了基于NMR的初步评估。一些观察到的化学位移与预期的数据库值不同，可能是由于体抗磁化率的差异。整个提取物的NMR光谱鉴定了主要胆汁酸（胆酸、脱氧胆酸和鹅脱氧胆酸），但无法区分给定胆汁酸的甘氨酸和牛磺酸结合物。然而，这是通过HPLC-NMR/MS实现的，其能够分离和鉴定相对丰度从约0.1%变化的10种结合胆汁酸。（牛磺石胆酸）至34.0%（甘氨胆酸），其中只有五种最丰富的酸可以通过核磁共振检测到，包括异构体甘氨脱氧胆酸和甘氨鹅去氧胆酸，其难以通过常规LC-MS分析区分。在一项单独的实验中，使用UPLC-MS可以检测和鉴定13种胆汁酸。这项工作显示了互补的潜力，NMR光谱，MS和联用NMR/MS阐明复杂的代谢概况的人肝胆汁。这将是正在进行的肝脏排泄功能和器官移植研究的有用基线信息。
The first application of high field NMR spectroscopy (800 MHz for 1 H observation) to human hepatic bile (as opposed to gall bladder bile) is reported. The bile sample used for detailed investigation was from a donor liver with mild fat infiltration, collected during organ retrieval prior to transplantation. In addition, to focus on the detection of bile acids in particular, a bile extract was analysed by 800 MHz 1 H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS. In the whole bile sample, 40 compounds have been assigned with the aid of two-dimensional H-1-H-1 TOCSY and H-1-C-13 HSQC spectra. These include phosphatidylcholine, 14 amino acids, 10 organic acids, 4 carbohydrates and polyols (glucose, glucuronate, glycerol and myo-inositol), choline, phosphocholine, betaine, trimethylamine-N-oxide and other small molecules. An initial NMR-based assessment of the concentration range of some key metabolites has been made. Some observed chemical shifts differ from expected database values, probably due to a difference in bulk diamagnetic susceptibility. The NMR spectra of the whole extract gave identification of the major bile acids ( cholic, deoxycholic and chenodeoxycholic), but the glycine and taurine conjugates of a given bile acid could not be distinguished. However, this was achieved by HPLC-NMR/MS, which enabled the separation and identification of ten conjugated bile acids with relative abundances varying from approximately 0.1% (taurolithocholic acid) to 34.0% ( glycocholic acid), of which, only the five most abundant acids could be detected by NMR, including the isomers glycodeoxycholic acid and glycochenodeoxycholic acid, which are difficult to distinguish by conventional LC-MS analysis. In a separate experiment, the use of UPLC-MS allowed the detection and identification of 13 bile acids. This work has shown the complementary potential of NMR spectroscopy, MS and hyphenated NMR/MS for elucidating the complex metabolic profile of human hepatic bile. This will be useful baseline information in ongoing studies of liver excretory function and organ transplantation.