Colonic Bile Acid Extraction and Quantification

Colonic bile acid extraction was performed as previously described with proper modification [20 (link)]. Briefly, colonic content (20 mg) was homogenized in 400 μL methanol and centrifuged at 12,000× g and 4 °C for 15 min. The supernatant was collected and evaporated in a centrifuge (Eppendorf, Hamburg, Germany) to remove the solvent. The obtained solute was resolved in 400 μL methanol and centrifuged under the same conditions before use. Bile acid standards (Sigma-Aldrich, St. Louis, MO, USA), including deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), tauroursodeoxycholic acid (TUDCA), hyodeoxycholic (HDCA), lithocholic acid (LCA), β-muricholic acid (β-MCA), taurocholic acid (TCA), and cholic acid (CA), were used as internal standards. A UPLC-MS system (Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze the prepared samples. The instrumental settings were set and data processing was performed as previously described [21 (link)].