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Stereoselective Oxidation Kinetics of Deoxycholate in Recombinant and Microsomal CYP3A Enzymes: Deoxycholate
Yu-Jie Chen1, Jian Zhang1, Ping-Ping Zhu1
1Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry, West China School of Pharmacy, Sichuan University, Chengdu, People's Republic of China (Y.-J.C., J.Z., P.-P.Z., X.-W.T., Q.-H.L., W.W., S.-S.Y., L.-Z.G., L.X., K.L.); Metabolomics Shared Resource, University of Hawaii Cancer Center, Honolulu, Hawaii (M.-M.S., W.J.); Department of Molecular Biology and Biochemistry, University of California, Irvine, California (I.F.S.); State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, People's Republic of China (C.-X.L.); and Chengdu Health-Balance Medical Technology Co., Ltd., Chengdu, People's Republic of China (S.-S.Y., K.L.).
Abstract:
The primary bile acids (BAs) synthesized from cholesterol in the liver are converted to secondary BAs by gut microbiota. It was recently disclosed that the major secondary BA, deoxycholate (DCA) species, is stereoselectively oxidized to tertiary BAs exclusively by CYP3A enzymes. This work subsequently investigated the in vitro oxidation kinetics of DCA at C-1β, C-3β, C-4β, C-5β, C-6α, C-6β, and C-19 in recombinant CYP3A enzymes and naive enzymes in human liver microsomes (HLMs). The stereoselective oxidation of DCA fit well with Hill kinetics at 1-300 μM in both recombinant CYP3A enzymes and pooled HLMs. With no contributions or trace contributions from CYP3A5, CYP3A7 favors oxidation at C-19, C-4β, C-6α, C-3β, and C-1β, whereas CYP3A4 favors the oxidation at C-5β and C-6β compared with each other. Correlation between DCA oxidation and testosterone 6β-hydroxylation in 14 adult single-donor HLMs provided proof-of-concept evidence that DCA 19-hydroxylation is an in vitro marker reaction for CYP3A7 activity, whereas oxidation at other sites represents mixed indicators for CYP3A4 and CYP3A7 activities. Deactivation caused by DCA-induced cytochrome P450-cytochrome P420 conversion, as shown by the spectral titrations of isolated CYP3A proteins, was observed when DCA levels were near or higher than the critical micelle concentration (about 1500 μM). Unlike CYP3A4, CYP3A7 showed abnormally elevated activities at 500 and 750 μM, which might be associated with an altered affinity for DCA multimers. The disclosed kinetic and functional roles of CYP3A isoforms in disposing of the gut bacteria-derived DCA may help in understanding the structural and functional mechanisms of CYP3A.
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