Submicellar aggregates of deoxycholate and glycodeoxycholate suppress in vitro CYP3A oxidation via membrane...

Cytochrome P450 3A4 (CYP3A4) and 3A7 (CYP3A7) specifically catalyze the 1β- and 5β-hydroxylation of deoxycholic acid (DCA) and glyco-DCA (GDCA). Their oxidation rates in microsomal and recombinant CYP3A4/3A7 conform to the Hill kinetic model at low substrate concentrations of up to 400 and 250 μM for DCA and GDCA, respectively, but decline progressively at submicellar concentrations, culminating in complete enzyme deactivation at or above the critical micelle concentration. We hypothesized that this atypical kinetic behavior originates from the disruption of microsomal vesicles by submicellar assemblies of DCA/GDCA. Transmission electron microscopy revealed that submicellar DCA did not compromise vesicle integrity, whereas membrane disruption became evident at or above 1000 μM. Spectroscopic analyses (UV, NMR, and bilirubin-probed fluorescence) determined the critical micelle concentration of both bile salts at approximately 1000 μM and informed the presence of oligomers below this threshold. Notably, at the concentrations of oligomer formation, DCA/GDCA exhibited no inhibitory effect on cytosolic sulfotransferase; however, they markedly suppressed the membrane-associated activities of CYP1A2 and CYP2D6, as probed by phenacetin and dextromethorphan oxidation, respectively. This differential susceptibility between cytosolic and vesicle-bound enzymes corroborates the proposed vesicle membrane-dependent mechanism. Collectively, our findings strongly suggest that submicellar DCA and GDCA autoinhibit their CYP3A-catalyzed oxidation through a mechanism that involves a vesicle membrane-dependent mechanism, with their self-assembly being intrinsically linked to the observed anomalous kinetics. SIGNIFICANCE STATEMENT: This work demonstrates that submicellar deoxycholate and glycodeoxycholate autoinhibit CYP3A oxidation via membrane perturbation, not substrate sequestration. Spectroscopic detection of bile salt oligomers below the critical micelle concentration correlates with suppression of multiple membrane-bound P450s (CYP1A2/2D6/3A) while sparing cytosolic sulfotransferase, indicating that submicellar bile salts impair electron transfer among P450, P450 reductase, and cytochrome b5. This membrane-dependent mechanism resolves the long-standing atypical kinetics of secondary bile acids and has implications for bile acid-drug interaction assessment, particularly in cholestatic liver disease.