Elsevier

Biochemical Pharmacology

Volume 75, Issue 5, 1 March 2008, Pages 1240-1250
Biochemical Pharmacology

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

https://doi.org/10.1016/j.bcp.2007.11.008Get rights and content

Abstract

The biological effects of 1α,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 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 (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.

Introduction

Variable CYP3A4 catalytic activity in the small intestine contributes to inter-individual differences in the oral bioavailability and efficacy/toxicity of many drugs. Much of this variability results from differences in CYP3A4 protein content, which can vary more than eight-fold even in mucosal epithelium obtained from healthy volunteers [1], [2]. Although the source of that variability is probably multi-factorial, some studies have documented dynamic changes in CYP3A4 transcription that can occur following activation of the vitamin D receptor (VDR) by its natural ligand, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) [3], [4], [5], [6]. The intestinal mucosa is an important target tissue for 1,25(OH)2D3, where it regulates the expression of calcium binding and transport proteins and helps maintain calcium homeostasis throughout the body [7], [8], [9]. In addition, we have recently shown that up-regulation of CYP3A4 gene expression by 1,25(OH)2D3 may provide negative feedback control of the hormone's biological effects in the intestine through CYP3A4-catalyzed metabolism of 1,25(OH)2D3 to inactive oxidation products [10].

Delivery of 1,25(OH)2D3 to the intestinal mucosa can occur by either vascular or luminal routes. Although 1,25(OH)2D3 is highly bound to plasma proteins, diffusion of free 1,25(OH)2D3 and/or its uptake across the basolateral enterocyte cell surface are thought to occur [11]. Alternatively, studies utilizing radiolabeled 1,25(OH)2D3 have shown that a putative glucuronide conjugate of the hormone is excreted into bile and, in some chemical form, gets reabsorbed through intestinal enterocytes into portal blood [12], [13]. Glucuronidation of different vitamin D species has been well documented [14], [15], [16], [17], [18], and it is reasonable to hypothesize that a glucuronide metabolite of 1,25(OH)2D3 is formed in human liver, exported into the bile, and delivered to the duodenum. Once there, it could be hydrolyzed by β-glucuronidases, permitting absorption of the active hormone across the apical membrane of enterocytes and transcriptional activation of VDR gene targets such as CYP3A4. This mechanism has significant appeal, because it might explain the preferential expression of CYP3A4 in proximal sections of the small intestine, compared to more distal regions [19].

A necessary first step in the enterohepatic recycling of 1,25(OH)2D3 and regulation of intestinal CYP3A4 by the mechanism we propose would likely involve a conjugation reaction catalyzed by hepatic UDP-glucuronosyltransferase(s). In this study, experiments were conducted to characterize the glucuronidation of 1,25(OH)2D3 and to identify which of the human liver UGT isozymes is responsible for catalyzing the reaction(s).

Section snippets

Chemicals

Uridine-5′-diphosphoglucuronic acid (UDPGA), saccharic acid 1,4-lactone, alamethicin, trifluoperazine, β-estradiol-17-glucuronide, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) and β-glucuronidase (Helix pomatia) were purchased from Sigma–Aldrich (St. Louis, MO, USA). 1,25(OH)2D3 and hecogenin were purchased from Tetrionics (Madison, WI, USA) and Science Lab. Com. (Kingwood, TX, USA), respectively. 1,25(OH)2D3 used for Semi-preparative biosynthesis of the 25-O-glucuronide metabolite was obtained

Identification of 1,25(OH)2D3 monoglucuronide metabolites formed by HLM

After incubation of 1,25(OH)2D3 (chemical structure shown in Fig. 1) with pooled HLM and UDPGA, two metabolites (M1 and M2) were identified by UV absorbance (not shown) and selective mass detection at m/z 591, which corresponds to [M–H] under the API-negative ion mode (Fig. 2). Both metabolites were generated in a UDPGA-, microsomal protein- and time-dependent manner at substrate concentrations between 0.005 and 40 μM (not shown). After treatment with β-glucuronidase, LC-UV analysis (265 nm)

Discussion

Previous investigators have shown that, in humans, the most active form of vitamin D3, 1,25(OH)2D3, can be exported into bile as a polar conjugate, including glucuronide(s), and reabsorbed in the proximal small intestine [12], [13]. Based on the extensive enterohepatic recycling of glucuronides of bile acids and related steroids, it is likely that 1,25(OH)2D3 undergoes similar metabolism, biliary transport and intestinal reabsorption processes. In this study, we demonstrated that two different

Acknowledgements

This work was supported in part by the National Institutes of Health (GM63666, GM32165 and ES07033), Eli Lilly & Co., and Dainippon Sumitomo Pharma Co., Ltd.

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