Human UGT1A4 and UGT1A3 conjugate 25-hydroxyvitamin D3: metabolite structure, kinetics, inducibility, and interindividual variability

Abstract

25-Hydroxyvitamin D3 (25OHD3) is used as a clinical biomarker for assessment of vitamin D status. Blood levels of 25OHD3 represent a balance between its formation rate and clearance by several oxidative and conjugative processes. In the present study, the identity of human uridine 5'-diphosphoglucuronyltransferases (UGTs) capable of catalyzing the 25OHD3 glucuronidation reaction was investigated. Two isozymes, UGT1A4 and UGT1A3, were identified as the principal catalysts of 25OHD3 glucuronidation in human liver. Three 25OHD3 monoglucuronides (25OHD3-25-glucuronide, 25OHD3-3-glucuronide, and 5,6-trans-25OHD3-25-glucuronide) were generated by recombinant UGT1A4/UGT1A3, human liver microsomes, and human hepatocytes. The kinetics of 25OHD3 glucuronide formation in all systems tested conformed to the Michaelis-Menten model. An association between the UGT1A4*3 (Leu48Val) gene polymorphism with the rates of glucuronide formation was also investigated using human liver microsomes isolated from 80 genotyped livers. A variant allele dose effect was observed: the homozygous UGT1A4*3 livers (GG) had the highest glucuronidation activity, whereas the wild type (TT) had the lowest activity. Induction of UGT1A4 and UGT1A3 gene expression was also determined in human hepatocytes treated with pregnane X receptor/constitutive androstane receptor agonists, such as rifampin, carbamazepine, and phenobarbital. Although UGT mRNA levels were increased significantly by all of the known pregnane X receptor/constitutive androstane receptor agonists tested, rifampin, the most potent of the inducers, significantly induced total 25OHD3 glucuronide formation activity in human hepatocytes measured after 2, but not 4 and 24 hours, of incubation. Finally, the presence of 25OHD3-3-glucuronide in both human plasma and bile was confirmed, suggesting that the glucuronidation pathway might be physiologically relevant and contribute to vitamin D homeostasis in humans.

Figures

Figure 1.

Formation of 25OHD3 monoglucuronides with HLMs. The proposed chemical structure of 25OHD3 monoglucuronide, conjugated at either C-25 (A) or C-3 (B) position of 25OHD3 (M1 and M2) is shown. C, Representative chromatogram of 3 glucuronide isomers. Glucuronides were analyzed by LC-MS at m/z 575 under the negative mode. D, Chromatogram of the isolated d6–25OHD3-25-glucuronide as an internal standard. The corresponding peak was isolated by LC-UV and then analyzed by LC-MS at m/z 581 under the negative mode.

Figure 2.

Mass spectra of the PTAD derivatives of 25OHD3 glucuronides. A, Mass spectrum of the PTAD-25OHD3-3-glucuronide standard. B, Mass spectrum of the PTAD-M2. C, Proposed fragmentation of M2 to generate ion m/z 474. D, Mass spectrum of the PTAD-M1. The PTAD-derivatized 25OHD3 glucuronides were ionized and scanned from m/z 250 to 800 in the positive ion mode. Experimental details have been described in Materials and Methods.

Figure 3.

Substrate concentration-rate profiles of 25OHD3 monoglucuronides by pooled HLMs (A), UGT1A3 (B), and UGT1A4 (C). Each set of data was fit to a simple hyperbolic model using nonlinear regression data analysis (Prism v.5). Experimental details have been described in Materials and Methods. Closed circle, open circle, and closed square indicate the catalytic activities of 25OHD3-25-glucuronide (M1), 25OHD3-3-glucuronide (M2), and 5,6-trans-25OHD3-glucuronide (M3) formation, respectively.

Figure 4.

Interindividual difference in 25OHD3 monoglucuronide formation and inductive expression of UGT1A3 and UGT1A4 genes in human hepatocytes. A, Twenty HLMs isolated from different donors were incubated with 25OHD3 (10μM) as described above. Three monoglucuronides 25OHD3-25-glucuronide (25OHD3-25-G), 25OHD3-3-glucuornide (25OHD3-3-G), and 5,6-trans-25OHD3-25-glucuronide (t-25OHD3-25-G) were detected, and the formation rates represent the total amounts of product formed per minute per milligram of protein. B, Effects of the UGT1A4*3 genotype on the rates of 25OHD3 glucuronidation. UGT1A4*3 (Leu48Val) variant gene product exhibits an increased glucuronidation activity. Subject (n = 80) characteristics and UGT1A4*3 genotype are summarized in Table 2, with 43 wild type (TT), 32 heterozygous (TG), and 5 homozygous (GG). The major product, 25OHD3-25-glucruonide, was quantified using LC-MS, and the formation rate represents the total amounts of product generated per minute per milligram of protein. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparison test; *, P < .05 was considered statistically significant. C, Inductive expression of UGT1A3 and UGT1A4 genes in human hepatocytes. Human hepatocytes from 3 different donors were treated with 10μM rifampin (RIF), 400μM phenobarbital (PB), 0.5μM hyperforin (HF), 50μM carbamazepine (CBZ), 200μM levitiracetam (LEV), or vehicle control (CTR, 0.1% vol/vol) for 48 hours, as indicated. Total RNA from each sample was isolated, and the expression of UGT1A3 and UGT1A4 was determined by quantitative RT-PCR assay. Data represent mean ± SE (from 4 replicate determinations of 3 different liver donors) of the fold induction in treated cells, compared with those of vehicle treated cells, after normalization to the 18s ribosomal RNA level. Statistical analysis was performed using an unpaired t test. *, P < .05 for the inductive effect of drugs, compared with corresponding control group.

Figure 5.

Measurement of 25OHD3 monoglucuronides in human hepatocytes and plasma. A, Representative chromatograms of 3 monoglucuronides in human hepatocytes treated with 25OHD3. Solid line, cell culture medium extracts after a 24-hour incubation; dash line, cell culture medium extracts after a 2-hour incubation. Arrows indicate the presence of 3 25OHD3 monoglucuronides. B, Time-dependent formation of 25OHD3 glucuronides in human hepatocytes with or without rifampin (10μM) pretreatment. Statistical analysis was performed using an unpaired t test (rifampin vs vehicle treated at different time points). C, Representative chromatogram of 25OHD3-3-glucuronide (m/z 575) in human plasma. Human plasma (0.5 mL) was subjected to protein precipitation, solid-phase extraction using Oasis WAX anion exchange columns, and then the eluates were dried and reconstituted for LC-MS analysis. Solid line, plasma extracts indicating the presence of 25OHD3-3-glucuronide; dash line, reaction products from HLM incubations with 25OHD3 indicating 3 major monoglucuronides.

Figure 6.

The presence of 25OHD3-3-glucuronide in human bile and its binding to DBP. Representative chromatograms from human bile (A), 25OHD3-3-glucuronide (B), and 25OHD3-25-glucuronide (C) after PTAD derivatization. Human bile (5 mL) was subjected to liquid-liquid extraction and solid-phase extraction using Waters Oasis WAX anion exchange cartridges as described in Materials and Methods. The bile extracts were derivatized with PTAD and analyzed by LC-MS/MS. Putative 25OHD3 monoglucuronides were detected by multiple reaction monitoring (MRM) channels of m/z 734 → 298 and 734 → 474. Specifically, m/z 734 → 474 is a characteristic fragment from 25OHD3-3-glucuronide. The standards 25OHD3-3-glucuronide and 25OHD3-25-glucuronide were also analyzed under the same conditions. D, Binding of 25OHD3-3-glucuronide and 25OHD3 to rat plasma DBP. The competitive binding assay was performed as described in Materials and Methods. Various concentrations of standards 25OHD3 and 25OHD3-3-glucuronide were used in the assay, and the percentage of bound 3H-25OHD3 was calculated after competitive displacement of 3H-25OHD3 by addition of the tested compounds.