Laboratory analysis

Serum samples were analyzed for the following 15 of the most abundant BAs at Metabolon Inc.: chenodeoxycholic acid (CDCA), cholic acid (CA), GCDCA, taurochenodeoxycholic acid (TCDCA), GCA, taurocholic acid (TCA), DCA, LCA, glycodeoxycholic acid (GDCA), taurodeoxycholic acid (TDCA), glycolithocholic acid (GLCA), taurolithocholic acid (TLCA), ursodeoxycholic acid (UDCA), glycoursodeoxycholic acid (GUDCA), and tauroursodeoxycholic acid (TUDCA).^{[ 33 ]} Case and matched‐control samples were placed next to each other in the same batch but in random order. Blinded quality control (QC) samples (n = 40 study duplicates) were regularly spaced throughout each batch. Intraclass correlation coefficients for the measured BAs all exceeded 0.99, indicating excellent technical reproducibility. Samples were spiked with a solution of corresponding labeled internal standards for each BA and were subjected to protein precipitation with acidified methanol. Samples were centrifuged, and a portion of the clear supernatant was evaporated to dryness in a stream of nitrogen at 40°C. The dried extract was reconstituted, and an aliquot was injected onto an Agilent Infinity II/Sciex QTrap 6500 liquid chromatography–tandem mass spectrometry system equipped with a C18 reverse‐phase high‐performance liquid chromatography column with acquisition in negative ion mode using electrospray ionization. The peak area of each parent (pseudo‐multiple reaction monitoring mode) or product ion was measured against the peak area of the respective internal standard. Quantitation was performed using a weighted linear least squares regression analysis generated from fortified calibration standards prepared immediately before each run.