MS/MS fragmentation pattern analysis confirms the production of the new esterified bile acids by the human gut microbiota
The pathophysiology of bile acids (BAs) has been widely studied. The BAs are signaling molecules that affect lipids and glucose homeostasis via activation of BAs FRX and TGR5 receptors in the liver and peripheral tissues. The ratio of conjugated/unconjugated BAs seems relevant to quantify these interactions and, therefore, the impact on the metabolism. The gut microbiota plays a key role because they convert the primary BAs into the secondary BAs, hydrolyzes the hepatically conjugated BAs and re-conjugates BAs with amino acids. New re-conjugated BAs with amino acids (MCBAs) in the form of amides or esters have been recently suggested, but it was not possible to confirm them. This study evaluates the production of MCBAs by human gut microbiota in in vitro colonic fermentations and designs a bioanalytical method to discriminate between amides and esters. Amides and seven new esters of re-conjugated BAs composed of lithocholic acid conjugated with leucine, valine and aminobutyric acid were identified and confirmed by MS/MS after incubation with chenodeoxycholic acid and lithocholic acid. There were no specific fragments in negative polarity to discriminate between amides and esters. However, in positive polarity the amides showed a characteristic MS/MS fragment consisting of the loss of water from the released amino acid, while the esters showed the loss of water plus carbon monoxide. This study confirmed for the first time the presence of esterified MCBAs, in addition to amides, and characterized the specific MS/MS fragmentation patterns to identify and discriminate them. These results show for the first time the existence of re-conjugated BAs by ester bond and the capability to produce them by the gut microbes. This bioanalytical method will allow including these new MCBAs in the BAs analysis.