Identification of human UDP-glucuronosyltransferases catalyzing hepatic 1α,25-dihydroxyvitamin D3 conjugation
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.
References (41)
- et al.
Liganded VDR induces CYP3A4 in small intestinal and colon cancer cells via DR3 and ER6 vitamin D responsive elements
Biochem Biophys Res Commun
(2002) - et al.
In vivo metabolism of 24R,25-dihydroxyvitamin D3: structure of its major bile metabolite
Biochim Biophys Acta
(1997) - et al.
In vitro and in vivo glucuronidation of 24,25-dihydroxyvitamin D3
Steroids
(1999) - et al.
Characterization of new conjugated metabolites in bile of rats administered 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3
Steroids
(2000) - et al.
Characterization of urinary metabolites of vitamin D3 in man under physiological conditions using liquid chromatography–tandem mass spectrometry
J Pharm Biomed Anal
(2002) - et al.
Protein measurement with the Folin phenol reagent
J Biol Chem
(1951) - et al.
Characterization of two UDP glucuronosyltransferases that are predominantly expressed in human colon
Biochem Biophys Res Commun
(1998) - et al.
Evidence for a monoglucuronide of 1,25-dihydroxyvitamin D3 in rat bile
J Biol Chem
(1982) - et al.
UDP-glucuronosyltransferases and clinical drug-drug interactions
Pharmacol Ther
(2005) - et al.
The tissue-specific distribution of 3H-seocalcitol (EB 1089) and 3H-calcitriol in rats
J Steroid Biochem Mol Biol
(2004)
Tissue-specific, inducible, and hormonal control of the human UDP-glucuronosyltransferase-1 (UGT1) locus
J Biol Chem
Interpatient heterogeneity in expression of CYP3A4 and CYP3A5 in small bowel: lack of prediction by the erythromycin breath test
Drug Metab Dispos
Cytochrome P450 3A4 and P-glycoprotein expression in human small intestinal enterocytes and hepatocytes: a comparative analysis in paired tissue specimens
Clin Pharmacol Ther
Expression of enzymatically active CYP3A4 by Caco-2 cells grown on extracellular matrix-coated permeable supports in the presence of 1α,25-dihydroxyvitamin D3
Mol Pharmacol
Transcriptional control of intestinal cytochrome P-4503A by 1α,25-dihydroxy vitamin D3
Mol Pharmacol
Vitamin D receptor as an intestinal bile acid sensor
Science
Vitamin D metabolism and mechanisms of calcium transport
J Am Soc Nephrol
Expression of genes involved in calcium absorption in human duodenum
Eur J Clin Invest
Vitamin D-inducible calcium transport and gene expression in three Caco-2 cell lines
Am J Physiol Gastrointest Liver Physiol
Intestinal and hepatic CYP3A4 catalyze hydroxylation of 1α,25-dihydroxyvitamin D3: implications for drug-induced osteomalacia
Mol Pharmacol
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