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.

Fig. 1

Chemical structures of (A) 1,25(OH)₂D₃ and (B) 1α-hydroxy,25-glucuronide-D₃.

Fig. 2

Detection of monoglucuronides generated following incubation of 1,25(OH)₂D₃ with HLM. The reaction was performed for 30 min at 37°C in a 200-μL mixture containing 40 μM 1,25(OH)₂D₃, 1 mg/mL HLM, 50 μg/mL alamethicin, 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 5 mM saccharic acid 1,4-lactone, and 5 mM UDPGA. 1,25(OH)₂D₃ glucuronides (M1 and M2, panel A) and estradiol-17-glucuronide (internal standard (IS), panel B) were detected by selective ion monitoring at m/z 591 and m/z 447, respectively, as described under Materials and Methods.

Fig. 3

Mass spectrum of PTAD adduct of purified M1. Characterization of the chemical structure of M1 was performed using derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) as described previously [23]. The PTAD adduct of M1 gave a characteristic fragment ion of at m/z 314 in the API–positive ion mode.

Fig. 4

Identification of M1 by NMR as the 25-glucuronide metabolite of 1,25(OH)₂D₃. GHMBCAD spectrum with the proton spectrum shown as the trace across the top of the spectrum. The peak indicated by an arrow affords the major evidence of M1 being a 25-glucuronide (see results).

Fig. 5

Substrate concentration-rate profiles of 25-O-glucuronide (M1) and M2 formation by UGT1A4, UGT2B4 and UGT2B7. Each incubation contained 0.04 mg microsomal protein of Supersomes (UGT1A4, UGT2B4 or UGT2B7), 2 μg of alamethicin, 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 5 mM saccharic acid 1,4-lactone, 5 mM UDPGA, and 0.05, 1, 2, 5,10, 20, 30, or 40 μM 1,25(OH)₂D₃ in a total volume of 200 μL. After preincubation for 5 min at 37°C, the reaction was initiated by the addition of UDPGA. After 30-min incubation, the reaction was terminated by the addition of 125 μL of ice-cold acetonitrile containing 6% (v/v) acetic acid and 12.5 nmol β-estradiol-17-glucuronide (IS). Closed and open circles indicate the catalytic activities of 25-O-glucuronide (M1) formation and M2 formation, respectively.

Fig. 6

Inhibition of 25-O-glucuronide (M1) and M2 formation in human liver microsomes by hecogenin. Pooled HLM (0.5 mg/mL) were preincubated with alamethicin at 50 μg of alamethicin/mg of microsomal protein, and different concentrations (0, 5, 20, 50, 100, and 200 μM) of hecogenin for 10 min at room temperature, followed by the addition of 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 5 mM saccharic acid 1,4-lactone, and 1 μM 1,25(OH)₂D₃. The reaction was initiated by the addition of 5 mM UDPGA (a final volume of a mixture, 200 μL). After incubation at 37°C for 30 min, the reaction was terminated by the addition of 125 μL of ice-cold acetonitrile containing 6% (v/v) acetic acid and 12.5 nmol β-estradiol-17-glucuronide (IS). Closed and open circles indicate the catalytic activities of 25-O-glucuronide (M1) formation and M2 formation, respectively.

Fig. 7

Comparison of mRNA expression levels of UGT isozymes in paired (same donor) human liver and intestine. Paired (same donor) human liver and intestinal (jejunal) samples from nine Caucasian donors were used. Real-time quantitative PCR was performed as described in text. GAPDH was chosen as the internal control gene.