Continuum of Host-Gut Microbial Co-metabolism: Host CYP3A4/3A7 are Responsible for Tertiary Oxidations of Deoxycholate Species

Abstract

The gut microbiota modifies endogenous primary bile acids (BAs) to produce exogenous secondary BAs, which may be further metabolized by cytochrome P450 enzymes (P450s). Our primary aim was to examine how the host adapts to the stress of microbe-derived secondary BAs by P450-mediated oxidative modifications on the steroid nucleus. Five unconjugated tri-hydroxyl BAs that were structurally and/or biologically associated with deoxycholate (DCA) were determined in human biologic samples by liquid chromatographytandem mass spectrometry in combination with enzyme-digestion techniques. They were identified as DCA-19-ol, DCA-6b-ol, DCA-5bol, DCA-6a-ol, DCA-1b-ol, and DCA-4b-ol based on matching in-laboratory synthesized standards. Metabolic inhibition assays in human liver microsomes and recombinant P450 assays revealed that CYP3A4 and CYP3A7 were responsible for the regioselective oxidations of both DCA and its conjugated forms, glycodeoxycholate (GDCA) and taurodeoxycholate (TDCA). The modification of secondary BAs to tertiary BAs defines a host liver (primary BAs)-gut microbiota (secondary BAs)-host liver (tertiary BAs) axis. The regioselective oxidations of DCA, GDCA, and TDCA by CYP3A4 and CYP3A7 may help eliminate host-toxic DCA species. The 19-and 4b-hydroxylation of DCA species demonstrated outstanding CYP3A7 selectivity and may be useful as indicators of CYP3A7 activity. 283

Species Differences of Bile Acid Redox Metabolism: Tertiary Oxidation of Deoxycholate is Conserved in Preclinical Animals

“…Authentic standards of 25 unconjugated BAs and 4 stable isotope-labeled internal standards were obtained from Steraloids (Newport, RI), TRC (Toronto, Canada), Santa Cruz (Dallas, TX, USA) or Sigma-Aldrich (St. Louis, MO) as previously described (Zhu et al, 2018). Six DCA metabolites, DCA-1β-ol, DCA-4β-ol, DCA-5β-ol, DCA-6β-ol, DCA-6α-ol and DCA-19-ol, were synthesized as described in our recent report (Zhang et al, 2019).…”

“…In vitro metabolism was performed using the protocol described in our recent report (Chen et al, 2019;Zhang et al, 2019). In brief, stock solutions of unconjugated BAs were prepared in DMSO.…”

“…The mass spectrometer was operated in the negative mode with a 3.0 kV capillary voltage. Selected ion recording for quantification and identification of unconjugated BAs was described in our previous reports (Yin et al, 2017;Zhu et al, 2018;Zhang et al, 2019). The source and desolvation temperatures were set at 150 and 550 °C, respectively.…”

“…We have recently characterized both in vitro and in vivo the tertiary oxidations of DCA, GDCA and TDCA regioselectively at C-1β, C-3β, C-4β, C-5β, C-6α, C-6β and C-19, in which the 3β-hydroxylated metabolite rapidly dehydrates into 3-dehydroDCA (Zhang et al, 2019). This finding was also confirmed in this study that 3-dehydroDCA, DCA-5β-ol, DCA-6α-ol, DCA-4β-ol and DCA-6β-ol were also detected in the feces of dogs, rats and mice (Figure 2).…”

“…We have recently extended the scope of tertiary BA metabolism in human based on BA metabolome analysis in combination with in vitro metabolism assays. As shown in Figure 1, a series of tertiary oxidation reactions of DCA, glycodeoxycholic acid (GDCA) and taurodeoxycholic acid (TDCA) regioselectively at C-3β, C-1β, C-5β, C-6α, C19, C-4β and C-6β were characterized to be exclusively catalyzed by CYP3A4 and CYP3A7 (Zhang et al, 2019). The 19-hydroxylation of DCA, GDCA and TDCA was identified as a novel in vitro marker of CYP3A7 activity (Chen et al, 2019).…”