Identification of human UDP-glucuronosyltransferases catalyzing hepatic 1α,25-dihydroxyvitamin D3 conjugation

Abstract

The biological effects of 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) are terminated primarily by P450-dependent hydroxylation reactions. However, the hormone is also conjugated in the liver and a metabolite, presumably a glucuronide, undergoes enterohepatic cycling. In this study, the identity of human enzymes capable of catalyzing the 1,25(OH) 2 D 3 glucuronidation reaction was investigated in order to better understand environmental and endogenous factors affecting the disposition and biological effects of vitamin D 3 . Among twelve different UGT isozymes tested, only UGT1A4 ≫ 2B4 and 2B7 supported the reaction. Two different 1,25(OH) 2 D 3 monoglucuronide metabolites were generated by recombinant UGT1A4 and human liver microsomes. The most abundant product was identified by mass spectral and NMR analyses as the 25-O-glucuronide isomer. The formation of 25-O-glucuronide by UGT1A4 Supersomes and human liver microsomes followed simple hyperbolic kinetics, yielding respective K m and V max values of 7.3 and 11.2 μM, and 33.7 ± 1.4 and 32.9 ± 1.9 pmol/min/mg protein. The calculated intrinsic 25-O-glucuronide M1 formation clearance for UGT1A4 was 14-fold higher than the next best isozyme, UGT2B7. There was only limited (4-fold) inter-liver variability in the 25-O-glucuronidation rate, but it was highly correlated with the relative rate of formation of the second, minor metabolite. In addition, formation of both metabolites was inhibited > 80% by the selective UGT1A4 inhibitor, hecogenin. If enterohepatic recycling of 1,25(OH) 2 D 3 represents a significant component of intestinal and systemic 1,25(OH) 2 D 3 disposition, formation of monoglucuronides by hepatic UGT1A4 constitutes an important initial step.

Glucuronidation of Dihydrotestosterone and trans-Androsterone by Recombinant UDP-Glucuronosyltransferase (UGT) 1A4: Evidence for Multiple UGT1A4 Aglycone Binding Sites

“…Atypical kinetics of UGT1A4-catalyzed glucuronidation have also been reported (Chouinard et al, 2006;Hashizume et al, 2008;Hyland et al, 2009). However, systematic kinetic studies to explore the existence of multiple aglycone binding sites in UGT1A4 have never been conducted.…”

Cytochrome P450 3A-Mediated Microsomal Biotransformation of 1α,25-Dihydroxyvitamin D3 in Mouse and Human Liver: Drug-Related Induction and Inhibition of Catabolism

“…This could explain the differential effects of the two glucocorticoids. Moreover, Hashizume et al (2008) homeostasis. Thus, it is critical to recognize that numerous commonly used drugs and herbal supplements are modulators of human and rodent PXR.…”

From Known Knowns to Known Unknowns: Predicting in Vivo Drug Metabolites

“…A large number of studies for identification of the CYP enzyme responsible for metabolism of some particular compound are reported and some of the most recent ones are cited here [93][94][95][96][97]. Similarly, articles describing the role of analytical techniques in the identification of UDP-glucuronosyltransferase enzyme isoforms responsible for glucuronide conjugation have been published [98][99][100].…”