Red clover (Trifolium pratense L.) is used as a source for isoflavone (IF) dietary supplements. In this study, we focused on the red clover IF irilone (IRI), because of its reported comparatively high bioavailability. Because the conjugative metabolism plays a key role in the elimination of IF, we investigated the species-specific differences and glucuronidation kinetics of IRI using different liver microsomes as well as the recombinant UDP-glucuronosyltransferases (UGTs) 1A1, 1A7, 1A8, 1A9, 1A10, and 2B15. Both possible monoglucuronides, the IRI-O-4′-monoglucuronide (IRI-G4′) and the IRI-O-5-monoglucuronide (IRI-G5), were detected. Human liver microsomes (HLM) as well as rat liver microsomes predominantly formed IRI-G5, whereas for porcine liver microsomes, IRI-G4′ prevailed. HLM showed an apparent Vmax value of 0.43 nmol/min · mg and an apparent Km value of 9.8 μM for the formation of IRI-G5 and a Vmax of 0.35 nmol/min · mg and a Km of 64.7 μM in the case of IRI-G4′. Formation of both glucuronides was best fit using the substrate inhibition equation. The glucuronidation of IRI by UGTs led to values for the intrinsic clearance varying between 4 and 100 ml/min · mg, with UGT1A7 showing the lowest and UGT1A10 the highest IRI conversion rate. The results indicate that IRI undergoes an efficient glucuronidation, presumably in the intestine and liver, following atypical kinetic profiles.
Footnotes
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.033076.
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The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- IF
- isoflavone
- DAD
- diode array detector
- HLM
- human liver microsomes
- HPLC
- high-performance liquid chromatography
- IRI
- 5,4′-dihydroxy-6,7-methylenedioxy-IF
- IRI-G4′
- IRI-O-4′-monoglucuronide
- IRI-G5
- IRI-O-5-monoglucuronide
- HPLC-DAD
- high-performance liquid chromatography-diode array detector
- MUG
- 4-(trifluoromethyl) umbelliferone-glucuronide
- PLM
- porcine liver microsomes
- RLM
- rat liver microsomes
- UGT
- UDP glucuronosyltransferase
- UDPGA
- uridine-5′-diphosphate-β,d-glucuronic acid.
- Received March 2, 2010.
- Accepted December 21, 2010.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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