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

The biological effects of 1 alpha,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 D3. Among 12 different UGT isozymes tested, only UGT1A4 >> 2B4 and 2137 supported the reaction. Two different 1,25(OH)(2)D-3 monoglucuronicle 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 mu M 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)(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. (c) 2007 Elsevier Inc. All rights reserved.