Targeted analysis of bile acids in ileal and cecal content, fecal pellets, serum, and bile were performed with an ACQUITY ultraperformance liquid-chromatography (UPLC) system using a C8 BEH column (2.1 × 100 mm, 1.7 μm) coupled with a Xevo TQ-S triplequadrupole mass spectrometer equipped with an electrospray ionization (ESI) source operating in negative ionization mode (All Waters, Milford, MA) as previously described [33]. The sample was thawed on ice and 25 mg was added to 1 mL of pre-cooled methanol containing 0.5 μM stable-isotope-labeled bile acids as internal standards (IS), followed by homogenization (1.0-mm-diameter zirconia/silica beads added) and centrifugation. Supernatant (200 μl) was transferred to an autosampler vial. 20 μL of serum was extracted by adding 200 μL pre-cooled methanol containing 0.5 μM IS. 5 μL of gall bladder bile was extracted with 500 μL pre-cooled methanol containing 0.5 μM IS. Following centrifugation, the supernatant of the extract was transferred to an autosampler vial for quantitation. Following centrifugation, the supernatant of the extract was transferred to an autosampler vial for quantitation. Bile acids were detected by either multiple reaction monitoring (MRM) (for conjugated bile acid) or selected ion monitoring (SIM) (for non-conjugated bile acid). MS methods were developed by infusing individual bile acid standards. Calibration curves were used to quantify the biological concentration of bile acids. Bile acid quantitation was performed in the laboratory of Dr. Andrew Patterson at Penn State University.

Random Forest analysis was performed in MetaboAnalyst 3.0 (http://www.metaboanalyst.ca/faces/ModuleView.xhtml) [34]. Briefly, the data were uploaded in the Statistical Analysis module with default settings and no further data filtering. Random Forest analysis Ward clustering algorithm and Euclidean distance were used to identify top bile acids within ursodiol treatment groups. Heatmaps and box and whisker plots of bile acid concentrations, and nonmetric multidimensional scaling (NMDS) depicting the dissimilarity indices via Horn distances between bile acid profiles were generated using R packages (http://www.R-project.org).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.