Quantification of Plasma Bile Acids

Fasting blood samples were collected from consenting participants at recruitment, separated by components, and stored at -80 °C until measurement. After solvent extraction, plasma samples were detected for BAs in the multiple reaction monitoring mode on a ultraperformance liquid chromatography, coupled to tandem mass spectrometry (UPLC-MS/MS, Agilent Technologies, USA), according to the previously optimized method with minor modifications [26 (link)]. The target BAs included unconjugated primary BAs (cholate [CA] and chenodeoxycholate [CDCA]) and their amino acid conjugates (glycocholate [GCA], taurocholate [TCA], glycochenodeoxycholate [GCDCA], taurochenodeoxycholate [TCDCA]), as well as unconjugated secondary BAs (deoxycholate [DCA], lithocholate [LCA], hyocholic acid [HCA], and ursodeoxycholate [UDCA]) and conjugated secondary BAs (glycodeoxycholate [GDCA], taurodeoxycholate [TDCA], glycolithocholate [GLCA], taurolithocholic acid [TLCA], glycohyocholic acid [GHCA], taurohyocholic acid [THCA], glycoursodeoxycholate [GUDCA] and tauroursodeoxycholic acid [TUDCA]). BAs were quantified by calibration curves constructed from standards and corresponding isotopically labelled internal standards using MassHunter Workstation software (Agilent, Version B.08.00). BAs with detection rates below 80% (except for LCA) were omitted from subsequent analyses. The values below the limits of detection were imputed with the half minimum across all subjects. The intra-assay CVs ranged from 3.6 to 18.6% for all included BAs.