Bile acid profiling in BALF
acid-3α,12α-diol-2,2,4,4-d4
Bile acids were profiled from the supernatants obtained after pelleting microbial cells for DNA extraction using high-speed centrifugation and blinded to the associated clinical data. Samples were spiked with 500 pmol of each internal standard and alkalinized with 0.1 volumes of 1 M NaOH solution. Extraction of bile acid analytes was achieved through solid-phase extraction using OASIS HLB® reversed-phase sorbent (Waters, batch 163A). Cartridges containing 60 mg of OASIS® HLB sorbent were conditioned with 1 mL of methanol and then with 3 mL of water. Samples were passed twice through the column, after which two washes with 1 mL of water were performed. Retained analytes were then eluted with 1 mL of 90% acetonitrile in water, dried in a stream of nitrogen and reconstituted with 50% methanol in water. Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry and bile acid identification were performed as we previously described [For quantifying bile acid load in BALF we spiked the samples with a mix of deuterized internal standards 5β-cholanic acid-3α,12α-diol-2,2,4,4-d4 (CA-D4, C1070-015 Steraloids), and 5β-cholanic acid-3α,7α-diol-2,2,4,4-d4 (CDCA-D4, C0940-015 Steraloids) and one analogous BA 23-Nor-5β-cholanic acid-3α, 7α, 12α triol (23-NCA, N2450-000 Steraloids). To quantitate those bile acid species for which no deuterium-labelled internal standard was available we used 23-NCA. Quantification represents the ratio between the peak area of the analyte and the peak area of the internal standard.The odds of detecting BAs did not change across batches, except for batch 4 in which we observed a significantly reduced probability of detecting BAs with respect to the reference batch (batch 1) (OR (95% confidence interval), 0.18(0.04–0.76), 