2.6. Bile Acid (BA) Analysis

The serum and liver tissues were processed for bile acid analysis, as described previously [19]. The processed samples were filtered using Whatman^TM Mini-UniPrepTM syringeless filters (0.2 µm PTFE, Cat. #09-923-102). High-throughput profiling of BAs was performed using a Shimadzu liquid chromatography/mass spectrometry (LC–MS) 8600 system. BA metabolite data in the original scale in terms of raw area counts were normalized by median centering. Missing values were imputed with the lower limit of detection for a given metabolite. When ≥30% of the values were missing, data were excluded from analyses. The peak and area under the curve for individual analytes were related to validated library standards to compute BA levels, which are read out as related to these library standards.

The bile acids, including deuterium (d4)-labeled internal standards (IS), used in this study, are listed in Table S7. GβMCA, TβMCA, ωMCA, TωMCA, GHCA, THCA, GHDCA, d₄-GCA, d₄-TCA, d₄-CDCA, d₄-GDCA, d₄-DCA, d₄-LCA were purchased from Cayman Chemical Co. (Ann Arbor, MI). αMCA, TαMCA, βMCA, HCA, THDCA, MDCA, GLCA, TLCA, isoDCA, isoLCA, 7-KetoDCA, 7-KetoLCA, 12-KetoLCA, Allo-isoLCA, and DhLCA were available from Steraloids Inc (Newport, RI). All other bile acid standards were obtained from Sigma-Aldrich (St Louis, MO). LC–MS-grade solvents and chemicals were purchased from Fisher Scientific (New Lawn, NJ), unless indicated otherwise. The stock solutions of the bile acid standards at a concentration of 100 μM (each bile acid) were prepared in 70% ethanol. Standard bile acid mixtures for calibration curves were prepared at the concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, 10, and 30 µM in acetonitrile/methanol/water (25:25:50, v/v). IS solution, which was a mixture of 10 stable isotope-labeled bile acids (d₄-CA, d₄-GCA, d₄-TCA, d₄-CDCA, d₄-DCA, d₄-GCDCA, d₄-TCDCA, d₄-LCA, d₄-GLCA, d₄-TLCA), was also prepared at 0.5 µM (each bile acid) in acetonitrile/methanol/water (25/25/50, v/v/v). To construct the calibration curves, 10 μL of each standard solution was mixed with 20 μL of IS solution and diluted to 400 µL with acetonitrile/methanol/water (25/25/50, v/v/v). A 2 μL aliquot was injected into the LC/MS/MS system.

For quantification of bile acids in the liver, 20 mg of liver tissue was homogenized with 500 µL of acetonitrile/methanol/water (25/25/50, v/v/v) using the Precellys Evolution Tissue Homogenizer with Cryolys (Bertin Corp, Rockville, MD). After centrifugation at 12,000× g for 2 min at room temperature, the supernatant (10 µL) was spiked with IS solution (20 µL, 10 pmol). The mixture was diluted to 400 µL with methanol/acetonitrile/water (25/25/50, v/v/v). For the serum specimen, 10 µL was spiked with IS solution (20 µL, 10 pmol), and the mixture was diluted with 250 µl of acetonitrile/methanol (1:1, v/v). After centrifugation at 12,000× g for 2 min at room temperature, the supernatant (200 µL) was diluted with water (200 µL). All samples were filtered through 0.2 µm PTFE membrane, and 2 μL aliquots were injected into the LC/MS/MS system. The Shimadzu LCMS-8600 CL liquid chromatography triple-quadrupole tandem mass spectrometer equipped with a dual ion source (DUIS) interface was used. Data were collected and processed using Lab Solutions software. Table S8 lists the multiple reaction monitoring (MRM) transitions, collision energy (CE), and retention time (RT) data for the measured bile acids.