Comprehensive Bile Acid Profiling in Mice

Bile acids were extracted from liver, whole gallbladder bile, whole small intestine with content, and dried feces in 90% ethanol. Briefly, solid tissues and feces were homogenized in 90% ethanol and incubated at 50°C overnight. After centrifugation, the clear supernatant was used for total bile acid measurement with a bile acid assay kit. To collect fecal samples, an individual mouse was placed in a jar briefly and fresh feces were collected. Bile acid pool was calculated as the total bile acids in liver, gallbladder, and small intestine. For LC-MS analysis of bile acid composition, ethanol extracts were dried and then resuspended in injection buffer containing 0.1% formic acid in 1:1 water: mixture of acetonitrile and methanol (1:1) and subsequently analyzed on a Thermo Fisher Scientific UltiMate 3000 UHPLC with a Waters Cortecs C18 column (Waters Acquity UPLC HSS T3 1.8 um, 2.1x150 mm, part No. 186003540) and a TSQ Quantis triple quadrupole mass spectrometer. The running condition is as follow: Solvent A: 0.1% formic acid; Solvent B: 0.1% formic acid in 1:1 methanol: acetonitrile. Flow rate: 0.3 ml/min. Gradient: 52%–90% B in 18 min, 90% to 52%B in 0.1 min, hold for 4 min. Run time: 22 min. TSQ Quantis triple quadrupole mass spectrometer Ion Mode: Ion Source Type: H-ESI; Spray Voltage: Static; Negative Ion (V): 2500; Sheath Gas (Arb): 50; Aux Gas (Arb): 10; Sweep Gas (Arb): 1; Ion Transfer Tube Temp (°C): 325; Vaporizer Temp (°C): 350; Polarity: Negative; Cycle Time (sec): 0.8. Other LC-MS parameters (Retention time, ion monitoring) are listed in supplemental Table S1. Standard curves for bile acids and internal standard glyco-CDCA-d4 (G-CDCA-d4) were generated with purified compounds and relative area under the curve was calculated. To measure T-CDCA-d4 metabolism in fecal slurry mixtures, fresh fecal sample was resuspended in a reaction buffer consisting of 10% PBS (pH=7.4), and 90% 3 mM sodium acetate (pH = 5.2) to a final suspension of 4 mg fecal sample/ml. T-CDCA-d4 was added to a final concentration of 20 μg/ml and the mixture was incubated at 37°C for 6 h. An equal amount of methanol was added and the mixture was incubated on ice for 1 h to precipitate protein. After centrifugation, an aliquot of the supernatant was vacuum dried and resuspended in injection buffer. LC-MS measurement of bile acids was performed as described above.