Novel Insights into the Human Gut Microbially Conjugated Bile Acids: The New Diversity of the Amino Acid-Conjugated Derivatives.

Bile acids (BAs) are biomolecules involved in lipids and glucose metabolism, and recently discovered microbially conjugated BAs (MCBAs) play an important role. Although the production of MCBAs amidated with amino acids (AA), similarly to those hepatically produced, has been confirmed, new structural isomers, conjugated with non-proteinogenic AA, remain unidentified. This study evaluates the production of MCBAs by human gut microbiota and discriminates, for the first time, between structural isomers. Thirteen MCBAs composed of lithocholic acid conjugated either with the AA valine and leucine or the non-proteinogenic AA, 5-amino valeric acid, and different aminobutyric acid derivatives (GABA and 2-amino-butyric) were confirmed by MS/MS fragmentation patterns and authentic standards. Only the fragmentation patterns in positive polarity confirmed the occurrence of different amino acid-conjugated derivatives. This study showed the microbiota's ability to produce MCBAs from both proteinogenic and non-proteinogenic AAs and disclosed, for the first time, the MS fragmentation rules to differentiate the structural isomers of MCBAs with amino groups at different positions. The MCBA content in fresh stool samples was 3-fold that of hepatically conjugated BAs, confirming their relevance. These findings will introduce the methodology for the analysis of this new MCBA family in BA analysis.