title: Identification of human UDP-glucuronosyltransferases catalyzing hepatic 1alpha,25-dihydroxyvitamin D3 conjugation.
abstract: The biological effects of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) 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)2D3 glucuronidation reaction was investigated in order to better understand environmental and endogenous factors affecting the disposition and biological effects of vitamin D3. Among 12 different UGT isozymes tested, only UGT1A4 >> 2B4 and 2B7 supported the reaction. Two different 1,25(OH)2D3 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 Km and Vmax values of 7.3 and 11.2 microM 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 (four-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)2D3 represents a significant component of intestinal and systemic 1,25(OH)2D3 disposition, formation of monoglucuronides by hepatic UGT1A4 constitutes an important initial step.
authors.affiliation: Department of Pharmaceutics, University of Washington, Seattle, WA, United States.
authors.forename: Takanori
authors.initials: T
authors.lastname: Hashizume
authors.forename: Yang
authors.initials: Y
authors.lastname: Xu
authors.forename: Michael A
authors.initials: MA
authors.lastname: Mohutsky
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Alberts
authors.forename: Chad
authors.initials: C
authors.lastname: Hadden
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Kalhorn
authors.forename: Nina
authors.initials: N
authors.lastname: Isoherranen
authors.forename: Margaret C
authors.initials: MC
authors.lastname: Shuhart
authors.forename: Kenneth E
authors.initials: KE
authors.lastname: Thummel
mesh_terms: D015870:Gene Expression; D020719:Glucuronides / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007583:Jejunum / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D020719:Glucuronides; D007527:Isoenzymes; D012333:RNA, Messenger; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; D014453:Glucuronosyltransferase