Wang Yuan, Wang Tao, Shi Xianzhe, Wan Dafang, Zhang Pingping, He Xianghuo, Gao Peng, Yang Shengli, Gu Jianren, Xu Guowang
National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China.
J Pharm Biomed Anal. 2008 Aug 5;47(4-5):870-5. doi: 10.1016/j.jpba.2008.02.022. Epub 2008 Feb 29.
The strong polar quaternary ammoniums, acetylcholine (ACh), choline (Ch) and butyrobetaine (BB, (3-carboxypropyl)trimethylammonium), are believed playing important roles in liver metabolism. These metabolites are at low levels and are weakly retained on reversed-phase liquid chromatographic (RP-LC) columns. Several hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) methods have been reported to analyze these compounds from different samples. However, no application to human liver tissues has been published. In this study, HILIC-MS/MS method was developed to simultaneously determine these three metabolites in human liver tissues. They were simply extracted from tissue, separated on a HILIC column, and detected by tandem MS in the mode of multiple reaction monitoring (MRM). Further studies on the recovery and repeatability based on real samples indicated the method was accurate and reliable. This method was successfully applied to measure the levels of ACh, Ch and BB in 61 human liver tissue samples including normal, hepatocellular carcinoma (HCC) and matched non-cancerous liver tissues. By comparison of Ch and ACh contents in 29 HCC with their matched non-cancerous liver tissues, it was found that ACh content increased in 11/29 HCC cases and decreased in 13/29 cases. Furthermore, the ACh/Ch ratio increased in 16/29 HCC cases, while it decreased in 8/29 cases. These results strongly indicated that there exist different patterns of ACh content in cancer tissues among HCC patients, thus highlighting the understanding of ACh and its relevant signal pathways in hepatic carcinogenesis and HCC progression.
强极性季铵盐类物质，如乙酰胆碱（ACh）、胆碱（Ch）和丁酸甜菜碱（BB，(3-羧丙基)三甲基铵），被认为在肝脏代谢中发挥着重要作用。这些代谢物含量较低，在反相液相色谱（RP-LC）柱上保留较弱。已有几种亲水作用液相色谱-串联质谱（HILIC-MS/MS）方法用于分析不同样品中的这些化合物。然而，尚未见有应用于人体肝脏组织的报道。在本研究中，开发了HILIC-MS/MS方法以同时测定人体肝脏组织中的这三种代谢物。它们只需从组织中简单提取，在HILIC柱上分离，然后通过多反应监测（MRM）模式的串联质谱进行检测。基于实际样品对回收率和重复性的进一步研究表明该方法准确可靠。该方法成功应用于测量61份人体肝脏组织样品（包括正常、肝细胞癌（HCC）及配对的癌旁肝组织）中ACh、Ch和BB的水平。通过比较29例HCC及其配对癌旁肝组织中Ch和ACh的含量，发现11/29例HCC病例中ACh含量升高，13/29例中降低。此外，16/29例HCC病例中ACh/Ch比值升高，而8/29例中降低。这些结果有力地表明，HCC患者癌组织中ACh含量存在不同模式，从而凸显了对ACh及其相关信号通路在肝癌发生和HCC进展中的理解。
