Abstract
Flavonoids are inactivated by phase II metabolism and occur in the body as glucuronides. Mammalian β-glucuronidase released from neutrophils at inflammatory sites may be able to deconjugate and thus activate flavonoid glucuronides. We have studied deconjugation kinetics and pH optimum for four sources of β-glucuronidase (human neutrophil, human recombinant, myeloid PLB-985 cells, Helix pomatia) with five flavonoid glucuronides (quercetin-3-glucuronide, quercetin-3′-glucuronide, quercetin-4′-glucuronide, quercetin-7-glucuronide, 3′-methylquercetin-3-glucuronide), 4-methylumbelliferyl-β-D-glucuronide, and para-nitrophenol-glucuronide. All substrate-enzyme combinations tested exhibited first order kinetics. The optimum pH for hydrolysis was between 3.5-5, with appreciable hydrolysis activities up to pH 5.5. At pH 4, the Km ranged 44-fold from 22 μM for quercetin-4′-glucuronide with Helix pomatia β-glucuronidase, to 981 μM for para-nitrophenol-glucuronide with recombinant β-glucuronidase. Vmax (range: 0.735-24.012 μmol·min−1·unit−1 [1 unit is defined as the release of 1 μM 4-methylumbelliferyl-β-D-glucuronide per min]) and the reaction rate constants at low substrate concentrations (k) (range: 0.002-0.062 min−1·(unit/L)−1 were similar for all substrates-enzyme combinations tested. In conclusion, we show that β-glucuronidase from four different sources, including human neutrophils, is able to deconjugate flavonoid glucuronides and non-flavonoid substrates at fairly similar kinetic rates. At inflammatory sites in vivo the pH, neutrophil and flavonoid glucuronide concentrations seem favorable for deconjugation. However, it remains to be confirmed whether this is actually the case.
