Production of New Microbially Conjugated Bile Acids by Human Gut Microbiota

Abstract

Gut microbes have been recognized to convert human bile acids by deconjugation, dehydroxylation, dehydrogenation, and epimerization of the cholesterol core, but the ability to re-conjugate them with amino acids as an additional conversion has been recently described. These new bile acids are known as microbially conjugated bile acids (MCBAs). The aim of this study was to evaluate the MCBAs diversity produced by the gut microbiota through a metabolomics approach. In this study, fresh fecal samples from healthy donors were evaluated to explore the re-conjugation of chenodeoxycholic and 3-oxo-chenodeoxycholic acids by the human gut microbiota. No significant differences were found between the conversion trend of both BAs incubations. The in vitro results showed a clear trend to first accumulate the epimer isoursochenodeoxycholic acid and the dehydroxylated lithocholic acid derivatives in samples incubated with chenodeoxycholic and 3-oxo-chenodeoxycholic acid. They also showed a strong trend for the production of microbially conjugated dehydroxylated bile acids instead of chenodeoxycholic backbone conjugates. Different molecules and isomers of MCBAs were identified, and the new ones, valolithocholate ester and leucolithocholate ester, were identified and confirmed by MS/MS. These results document the gut microbiota's capability to produce esters of MCBAs on hydroxyls of the sterol backbone in addition to amides at the C24 acyl site. This study opens a new perspective to study the BAs diversity produced by the human gut microbiota.

Keywords: MCBAs; bile acids; gut microbiota; metabolomics.

Figure 1

Summary of the expected conversion of secondary BAs by human gut microbiota.

Figure 2

BAs trend identified after samples incubation. Black color: non-detected; Red color: detected after incubation; Blue color: incubated and detected. CDCA: Chenodeoxycholic acid; LCA: Lithocholic acid; UDCA: Ursodeoxycholic acid; iDCA Isodeoxycholic acid. 1: Identified with authentic standard; 2: Identified according retention time based on Reiter et al., 2021; 3: Identified by MS/MS fragments.

Figure 3

Conversion possibilities of microbially conjugated BAs by the human gut microbiota. (a) Position possibilities for the amino acid along the cholic acid backbone; (b) Position possibilities for the amino acid along the cholic acid backbone in case of esterification reaction; (c) Position possibilities for the amino acid in case of hydroxylated, dehydroxylated and oxidized BAs; (d) Amino acid used to cover the conjugation possibilities.

Figure 4

MCBAs of valine identified. (a) valoisolithocholic acid; (b) valoiso7β-Hydroxy-5β-cholan-24-oic acid; (c) valolithocholic acid; (d) valoisolithocholate ester; (e) valoiso7β-Hydroxy-5β-cholate ester.

Figure 5

MS/MS spectra of valine conjugates. (a) microbially conjugated BA in the 24 acyl site; (b) microbially conjugated BA by esterification of C3; (c) MS/MS fragments of valine conjugate confirmed at 17.15 min; (d) MS/MS fragments of valine conjugate confirmed at 16.80 min; (e) MS/MS fragments of valine conjugate confirmed at 16.16 min; (f) MS/MS fragments of valine conjugate confirmed at 15.48 min; (g) MS/MS fragments of valine conjugate confirmed at 14.83 min.