Liquid chromatograph mass spectrometer

For hepatic iron content, approximately 30 mg of liver tissue was homogenized by sonication (Fisher Scientific Sonic Dismembrator F60, Fair Lawn, NJ, USA) in 300 µL of RIPA buffer (Roche Complete Mini #11836153001, Roche, UK). After increasing the volume to 600 µL with distilled water, 150 µL of 40% nitric acid (Fisher Scientific metal free grade, CAS #7697-37-2) was added. Samples were digested for 90 min at 95 °C and then centrifuged at 12,000 rpm for 20 min. Supernatants were diluted to a volume of 4 mL using 1% nitric acid and filtered through 0.45 µm nylon syringe filters (Fisher Scientific #09-719-008), prior to loading for run in the MIP OES (Microwave-Induced Plasma Optical Emission Spectrometer, Agilent 4200 MP-AES, Santa Clara, CA, USA). To measure redox state in the livers, reduced glutathione (GSH) and glutathione disulfide (GSSG) were quantified using LC-MS/MS (Liquid Chromatograph Mass Spectrometer, Shimadzu 8050, Japan).

Serum and fecal BAs and serum 7α-hydroxy-4-cholesten-3-one (C4) concentrations were measured as previously described (22 (link), 23 (link)). Briefly, a deuterium-labeled internal standard was added to 20 μL of serum or 0.2–0.4 mg of the fecal sample that was solubilized in 5% potassium hydroxide in water at 80 °C for 20 minutes. After adding 2 mL of 0.5 M potassium phosphate buffer (pH 7.4), BAs were extracted with the Bond Elut C18 cartridges (200 mg; Agilent Technologies, Santa Clara, CA) and quantified using a liquid chromatograph mass spectrometer (Shimadzu, Kyoto, Japan).