Abstract: AimTo identify the cytochrome P450 (CYP) isoform (s) involved in daidzein mono-hydroxylated metabolites using human liver microsomes. MethodsKinetic analysis of the rates of formation of mono-hydroxylated metabolites of daidzein, including 7,8,4′-trihydroxyisoflavone (7,8,4′-THI), 7,3′,4′-trihydroxyisoflavone (7,3′,4′-THI) and 6,7,4′-trihydroxyisoflavone (6,7,4′-THI), was performed using human liver microsomes (HLM) and recombinant enzymes at substrate concentrations ranging from 0.5 to 400 μmol·L^-1. Nine selective inhibitors or substrate probes specific for different CYP isoforms were applied for screening the isoform (s) responsible for mono-hydroxylated metabolism of daidzein. ResultsMichaelis-Menten kinetic parameters were best fitted to one-component enzyme kinetic model. The mean K_m(μmol·L^-1) and Vmax (μmol·g^-1·min^-1) values were 27±10 and 4.8±2.1, 54±22 and 2.3±1.0, 51±29 and 2.2±0.8, for the formation rates of 7,8,4′-THI, 7,3′,4′-THI, and 6,7,4′-THI, respectively. Furafylline, the CYP1A2 specific inhibitor, estrogen, and monoclonal antibody raised against human CYP1A2 (MAB-1A2) apparently inhibited the formation of mono-hydroxylated metabolites, The IC₅₀ of Fur for the formation of 7,3′,4′-THI, 6,7,4′-THI and 7,8,4′-THI was 1.0, 0.9 and 0.8 mol·L^-1, respectively. The IC₅₀ of estrogen for the formation of 7,3′,4′-THI, 6,7,4′-THI and 7,8,4′-THI were 51, 60 and 64 mol·L^-1, respectively. The IC₅₀ of MAB-1A2 for the formation of the mono-hydroxylated products was 1 mol·L^-1, but neither other selective inhibitor nor substrate probes, including coumarin (CYP2D6), sulphaphenzole (CYP2C9/10), omeprazole (CYP2C19), quinidine (CYP2D6), diethyldithiocarbamate (CYP2E1), troleandomycin (CYP3A4) and keteconazole (CYP3A4), did so with human liver microsomes. ConclusionThe in vitro studies indicated that daidzein mono-hydroxylated products were principally metabolized by CYP1A2 in human.