ABSTRACT
Purpose
Glucuronidation is a major barrier to flavonoid bioavailability; understanding its regiospecificity and reaction kinetics would greatly enhance our ability to model and predict flavonoid disposition. We aimed to determine the regioselective glucuronidation of four model flavonols using six expressed human UGT1A isoforms (UGT1A1, 1A3, 1A7, 1A8, 1A9, 1A10).
Methods
In vitro reaction kinetic profiles of six UGT1A-mediated metabolism of four flavonols (all with 7-OH group) were characterized; kinetic parameters (Km, Vmax and CLint = Vmax/Km) were determined.
Results
UGT1A1 and 1A3 regioselectively metabolized the 7-OH group, whereas UGT1A7, 1A8, 1A9 and 1A10 preferred to glucuronidate the 3-OH group. UGT1A1 and 1A9 were the most efficient conjugating enzymes with Km values of ≤1 μM and relative catalytic efficiency ratios of ≥5.5. Glucuronidation by UGT1As displayed surprisingly strong substrate inhibition. In particular, Ksi values (substrate inhibition constant) were less than 5.4 μM for UGT1A1-mediated metabolism.
Conclusion
UGT1A isoforms displayed distinct positional preferences between 3-OH and 7-OH of flavonols. Differentiated kinetic properties between 3-O- and 7-O- glucuronidation suggested that (at least) two distinct binding modes within the catalytic domain were possible. The existence of multiple binding modes should provide better “expert” knowledge to model and predict UGT1A-mediated glucuronidation.
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Abbreviations
- DHF:
-
dihydroxyflavone
- MS:
-
mass spectroscopy
- NMR:
-
nuclear magnetic resonance
- QHF:
-
tetrahydroxyflavone
- QSAR:
-
quantitative structure activity relationship
- SN2:
-
bimolecular nucleophilic substitution
- THF:
-
trihydroxyflavone
- UDPGA:
-
uridine diphosphoglucuronic acid
- UGTs:
-
UDP-glucuronosyltransferases
- UPLC:
-
ultra performance liquid chromatography
REFERENCES
Birt DF, Hendrich S, Wang W. Dietary agents in cancer prevention: flavonoids and isoflavonoids. Pharmacol Ther. 2001;90(2–3):157–77.
Ross JA, Kasum CM. Dietary flavonoids: bioavailability, metabolic effects, and safety. Annu Rev Nutr. 2002;22:19–34.
Chen J, Lin H, Hu M. Metabolism of flavonoids via enteric recycling: role of intestinal disposition. J Pharmacol Exp Ther. 2003;304(3):1228–35.
Setchell KD, Brown NM, Desai P, Zimmer-Nechemias L, Wolfe BE, Brashear WT, et al. Bioavailability of pure isoflavones in healthy humans and analysis of commercial soy isoflavone supplements. J Nutr. 2001;131(4 Suppl):1362S–75S.
Barve A, Chen C, Hebbar V, Desiderio J, Saw CL, Kong AN. Metabolism, oral bioavailability and pharmacokinetics of chemopreventive kaempferol in rats. Biopharm Drug Dispos. 2009;30(7):356–65.
Tukey RH, Strassburg CP. Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Annu Rev Pharmacol Toxicol. 2000;40:581–616.
Radominska-Pandya A, Ouzzine M, Fournel-Gigleux S, Magdalou J. Structure of UDP-glucuronosyltransferases in membranes. Methods Enzymol. 2005;400:116–47.
Mackenzie PI, Bock KW, Burchell B, Guillemette C, Ikushiro S, Iyanagi T, et al. Nomenclature update for the mammalian UDP glycosyltransferase (UGT) gene superfamily. Pharmacogenetics Genomics. 2005;15:677–85.
Fisher MB, Paine MF, Strelevitz TJ, Wrighton SA. The role of hepatic and extrahepatic UDP-glucuronosyltransferases in human drug metabolism. Drug Metab Rev. 2001;33(3–4):273–97.
Ohno S, Nakajin S. Determination of mRNA expression of human UDP-glucuronosyltransferases and application for localization in various human tissues by real-time reverse transcriptase-polymerase chain reaction. Drug Metab Dispos. 2009;37(1):32–40.
Miners JO, Knights KM, Houston JB, Mackenzie PI. In vitro-in vivo correlation for drugs and other compounds eliminated by glucuronidation in humans: pitfalls and promises. Biochem Pharmacol. 2006;71(11):1531–9.
Miners JO, Mackenzie PI, Knights KM. The prediction of drug-glucuronidation parameters in humans: UDP-glucuronosyltransferase enzyme-selective substrate and inhibitor probes for reaction phenotyping and in vitro-in vivo extrapolation of drug clearance and drug-drug interaction potential. Drug Metab Rev. 2010;42(1):189–201.
Aprile S, Del Grosso E, Grosa G. Identification of the human UDP-glucuronosyltransferases involved in the glucuronidation of combretastatin A-4. Drug Metab Dispos. 2010;38(7):1141–6.
Tang L, Singh R, Liu Z, Hu M. Structure and concentration changes affect characterization of UGT isoform-specific metabolism of isoflavones. Mol Pharm. 2009;6(5):1466–82.
Zhou Q, Zheng Z, Xia B, Tang L, Lv C, Liu W, et al. Use of Isoform-Specific UGT Metabolism to Determine and Describe Rates and Profiles of Glucuronidation of Wogonin and Oroxylin A by Human Liver and Intestinal Microsomes. Pharm Res. 2010;27(8):1568–83.
Sorich MJ, Smith PA, Miners JO, Mackenzie PI, McKinnon R. Recent advances in the in silico modelling of UDP glucuronosyltransferase substrates. Curr Drug Metab. 2008;9(1):60–9.
Sorich MJ, Smith PA, McKinnon RA, Miners JO. Pharmacophore and quantitative structure activity relationship modelling of UDP-glucuronosyltransferase 1A1 (UGT1A1) substrates. Pharmacogenetics. 2002;12(8):635–45.
Smith PA, Sorich MJ, McKinnon RA, Miners JO. In silico insights: chemical and structural characteristics associated with uridine diphosphate glucuronosyltransferase substrate selectivity. Clin Exp Pharmacol Physiol. 2003;30(11):836–40.
Tang L, Ye L, Singh R, Wu B, Zhao J, Lv C, et al. Use of Glucuronidation Fingerprinting to Describe and Predict mono- and di- Hydroxyflavone Metabolism by Recombinant UGT Isoforms and Human Intestinal and Liver Microsomes. Mol Pharm. 2010;7(3):664–79.
Chohan KK, Paine SW, Waters NJ. Quantitative structure activity relationships in drug metabolism. Curr Top Med Chem. 2006;6(15):1569–78.
Wong YC, Zhang L, Lin G, Zuo Z. Structure-activity relationships of the glucuronidation of flavonoids by human glucuronosyltransferases. Expert Opin Drug Metab Toxicol. 2009;5(11):1399–419.
Boersma MG, van der Woude H, Bogaards J, Boeren S, Vervoort J, Cnubben NH, et al. Regioselectivity of phase II metabolism of luteolin and quercetin by UDP-glucuronosyl transferases. Chem Res Toxicol. 2002;15(5):662–70.
Davis BD, Brodbelt JS. Regioselectivity of human UDP-glucuronosyl-transferase 1A1 in the synthesis of flavonoid glucuronides determined by metal complexation and tandem mass spectrometry. J Am Soc Mass Spectrom. 2008;19(2):246–56.
Miners JO, Smith PA, Sorich MJ, McKinnon RA, Mackenzie PI. Predicting human drug glucuronidation parameters: application of in vitro and in silico modeling approaches. Annu Rev Pharmacol Toxicol. 2004;44:1–25.
Wu B, Morrow J, Singh R, Zhang S, Hu M. Three-Dimensional Quantitative Structure-Activity Relationship Studies on UGT1A9-Mediated 3-O-Glucuronidation of Natural Flavonols Using a Pharmacophore-Based Comparative Molecular Field Analysis Model. J Pharmacol Exp Ther. 2011;336(2):403–13.
Singh R, Wu BJ, Tang L, Liu ZQ, Hu M. Identification of the Position of Mono-O-Glucuronide of Flavones and Flavonols by Analyzing Shift in Online UV Spectrum (λmax) Generated from an Online Diode-arrayed Detector. J Agric Food Chem. 2010;58(17):9384–95.
Christopoulos A, Lew MJ. Beyond eyeballing: fitting models to experimental data. Crit Rev Biochem Mol Biol. 2000;35(5):359–91.
Luukkanen L, Taskinen J, Kurkela M, Kostiainen R, Hirvonen J, Finel M. Kinetic characterization of the 1A subfamily of recombinant human UDP-glucuronosyltransferases. Drug Metab Dispos. 2005;33(7):1017–26.
Williamson G, Barron D, Shimoi K, Terao J. In vitro biological properties of flavonoid conjugates found in vivo. Free Radic Res. 2005;39(5):457–69.
Hutzler JM, Tracy TS. Atypical kinetic profiles in drug metabolism reactions. Drug Metab Dispos. 2002;30(4):355–62.
Segel IH. Enzyme kinetics: behavior and analysis of rapid equilibrium and steady state enzyme systems, New edn. New York: Wiley; 1993.
Li C, Wu Q. Adaptive evolution of multiple-variable exons and structural diversity of drugmetabolizing enzymes. BMC Evol Biol. 2007;7:69.
Laakkonen L, Finel M. A molecular model of the human UGT1A1, its membrane orientation and the interactions between different parts of the enzyme. Mol Pharmacol. 2010;77(6):931–9.
ACKNOWLEDGMENTS
This work was supported by grants from the National Institutes of Health (GM070737) to MH.
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Wu, B., Xu, B. & Hu, M. Regioselective Glucuronidation of Flavonols by Six Human UGT1A Isoforms. Pharm Res 28, 1905–1918 (2011). https://doi.org/10.1007/s11095-011-0418-5
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DOI: https://doi.org/10.1007/s11095-011-0418-5