Rapid analysis of bile acids in different biological matrices using LC-ESI-MS/MS for the investigation of bile acid transformation by mammalian gut bacteria

Abstract

Bile acids are important signaling molecules that regulate cholesterol, glucose, and energy homoeostasis and have thus been implicated in the development of metabolic disorders. Their bioavailability is strongly modulated by the gut microbiota, which contributes to generation of complex individual-specific bile acid profiles. Hence, it is important to have accurate methods at hand for precise measurement of these important metabolites. Here, a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous identification and quantitation of primary and secondary bile acids as well as their taurine and glycine conjugates was developed and validated. Applicability of the method was demonstrated for mammalian tissues, biofluids, and cell culture media. The analytical approach mainly consists of a simple and rapid liquid-liquid extraction procedure in presence of deuterium-labeled internal standards. Baseline separation of all isobaric bile acid species was achieved and a linear correlation over a broad concentration range was observed. The method showed acceptable accuracy and precision on intra-day (1.42-11.07 %) and inter-day (2.11-12.71 %) analyses and achieved good recovery rates for representative analytes (83.7-107.1 %). As a proof of concept, the analytical method was applied to mouse tissues and biofluids, but especially to samples from in vitro fermentations with gut bacteria of the family Coriobacteriaceae. The developed method revealed that the species Eggerthella lenta and Collinsella aerofaciens possess bile salt hydrolase activity, and for the first time that the species Enterorhabdus mucosicola is able to deconjugate and dehydrogenate primary bile acids in vitro.

Metabolism of Oxo-Bile Acids and Characterization of Recombinant 12α-Hydroxysteroid Dehydrogenases from Bile Acid 7α-Dehydroxylating Human Gut Bacteria

“…The Actinobacteria represented in the phylogeny, from the family Coriobacteriaceae, are of particular interest due to previous reports of 12␣-HSDH activity. Wegner et al demonstrated that mouse fecal isolate Enterorhabdus mucosicola DSM 19490 T expresses bile salt hydrolase, as well as 3␣-HSDH and 12␣-HSDH activities (36). Our phylogenetic analysis suggests that the NAD(P)-dependent oxidoreductase (accession no.…”

Analysis of human C24 bile acids metabolome in serum and urine based on enzyme digestion of conjugated bile acids and LC-MS determination of unconjugated bile acids

“…Some unusual BA species with hydroxylation sites on C-1, 2, 4, 6 and 19 have been detected in biological samples in early human life and in patients with liver diseases. However, these challenges have not yet been addressed by LC-MS techniques currently used in BA analysis [20–24]. Unusual species have seldom been detected using LC-MS, except for a recently-characterized CYP3A-mediated hydroxylated metabolite of DCA, 1β, 3α, 12α-trihydroxy-5β-cholan-24-oic acid (DCA-1β-ol) [25–27].…”

Targeted Synthesis and Characterization of a Gene Cluster Encoding NAD(P)H-Dependent 3α-, 3β-, and 12α-Hydroxysteroid Dehydrogenases from Eggerthella CAG:298, a Gut Metagenomic Sequence

“…Eggerthella lenta (formerly Eubacterium lentum) is a Gram-positive, non-sporeforming, nonmotile, obligate anaerobic bacterium whose growth is stimulated by arginine (17,18). E. lenta is a particularly important participant in the gut sterolbiome, various strains of which have the potential to encode enzymes that metabolize cardiac glycosides (19,20) and 21-dehydroxylate corticosteroids (21), hydrolyze conjugated bile acids (22), and oxidize bile acid hydroxyl groups (23,24). E. lenta strains have previously been grouped according to their pattern of hydroxysteroid dehydrogenase (HSDH) activities, with some strains having no HSDH activity (group V) and others expressing 3␣-HSDH, 3␤-HSDH, 7␣-HSDH, and 12␣-HSDH (group III) (24).…”