Stable Isotope Labeling-mass Spectrometry For The Analysis Of Bile Acids

Bile acids（BAs）are a class of fatty acids,involved in many important pathophysiological processes,including maintaining lipid,energy,and carbohydrate homeostasis.However,due to differences in their chemical structures,low contents in biological samples and large differences in abundance,accurate quantitative analysis of BAs is still challenging.With the development of analytical instruments,liquid chromatography-mass spectrometry（LC-MS）has become an important technique for the analysis of BAs.However,the analysis of BAs still has some difficulties,such as the low ionization efficiency and complex matrix effects.Therefore,it is necessary to develop a highly sensitive and selective analytical method for the determination of BAs in biological samples.Obesity has developed worldwide and has been reported to associate with cardiovascular diseases,hypertension,hyperlipidemia,type 2 diabetes mellitus and fatty liver.Based on the role of BAs in maintaining lipid,carbohydrate and energy metabolism homeostasis,BAs is also closely related to obesity.There have been many reports on the effects of BAs on obesity and the changes of BAs in obesity.However,the relationship between enterohepatic circulation of BAs and obesity remains unclear.Therefore,it would be of importance to explore the metabolism of BAs related to the pathogenesis of obesity.In this study,we mainly focuses on the development of a LC-MS method for the analysis of BAs in biological samples and exploring the role of BAs metabolism associated with obesity by the developed LC-MS method.The contents in this study can be summarized as follows:1.Based on stable isotope labeling and ultra high performance liquid chromatography-mass spectrometry technique,a high sensitive method for the simultaneous determination of 20 BAs including unconjugated and conjugated bile acids in biological samples was established.In the developed method,two stable isotope labeling reagents,2-dimethylaminoethylamine（DMED）and d₄-DMED were used to label biological samples and BAs standards,respectively.The samples were mixed followed by UHPLC-MS/MS analysis.The results showed that the sensitivity of BAs increased by 1-1539 times after DMED labeling.The separation of bile acid isomers was achieved within 16 min on C18 column by UHPLC.The method has high sensitivity,specificity,wide linear dynamic range（three orders of magnitude）,and can be applied to a variety of complex biological samples,including serum,mice liver,feces and cells.The method was also applied to the determination of BAs in different kinds of cells.The quantitative analysis of BAs in 293T,Hela,HL-7702 and Huh7.5 cells was achieved for the first time.2.BAs metabolism in obese model mice was explored by our developed LC-MS method.BAs in livers,small intestines,colons and feces of mice were determined by the LC-MS method,and 19,19,19 and 17 BAs could be detected and quantified,respectively,in livers,small intestines,colons and feces of mice.Compared with the wild type（WT）mice,the total BAs contents in feces of obese mice increased;while the total BAs contents in small intestines decreased.DCA/CDCA in livers,small intestines,colons and feces of obese mice increased all.In feces,the contents of DCA,CA and HDCA increased significantly in obese mice;in colons,the contents of DCA,HDCA and ACA increased significantly in obese mice;in small intestines,the contents of CA and 7-KDCA decreased significantly in obese mice;in livers,the contents of CA,CDCA,ACA and 7-KDCA decreased significantly in obese mice,while the contents of HDCA increased significantly.These results make it possible for these BAs to be potential markers of obesity.In addition,we found significant differences in intestinal bacterial communities between obese and WT mice:in obese mice the abundance of Clostridia increased,but the abundance of Bacteroidia decreased,suggesting that the metabolism of BAs may be related to the pathogenesis of obesity and plays an important role in the occurrence of obesity.