Regioselective glucuronidation of flavonols by six human UGT1A isoforms.

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 (K(m), V(max) and CL(int) = V(max)/K(m)) 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 K(m) values of ≤1 μM and relative catalytic efficiency ratios of ≥5.5. Glucuronidation by UGT1As displayed surprisingly strong substrate inhibition. In particular, K(si) 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.