Abstract

We have investigated the effects of methanol, acetonitrile, dimethyl sulphoxide or dioxane on enzyme kinetic parameters (K_m and V_max) of p-nitrophenol hydroxylation, phenacetin deethylation and metoprolol metabolism in rat liver microsomes, at different solvent concentrations. In the case of p-nitrophenol hydroxylation, methanol (0.1 %-0.75 % v/v) and dimethyl sulphoxide (0.01 %-0.075 % v/v) showed 1.3 and 2.5-fold increase respectively, in the K_m with a less marked effect on the V_max. In contrast, dimethyl sulphoxide (0.1 %-0.75% v/v) showed a 1.96-fold decrease in the V_max. Unlike, methanol and dimethyl sulphoxide, acetonitrile showed activation of the p-nitrophenol hydroxylation activity as indicated by a 0.7-fold decrease in the K_m. In the case of metoprolol metabolism, dioxane resulted in a 1.5 to 3.1-fold increase in the K_m of all the three metabolite formation pathways in concentration dependent manner, with less marked effect on V_max of the reaction. Interestingly, while acetonitrile did not affect the K_m for metabolite 1 and 2 formation, the K_m of metabolite 3 formation was decreased in a concentration dependent manner again with little effect on all the three V_max values. Further, methanol had little to no effect on the V_max of metabolite 1 and 2 formation while metabolite 3 formation was increased by 2-fold. The K_m of all three metabolite formations was found to be decreased in presence of methanol. The phenacetin deethylation activity in rat liver microsomes followed atypical kinetics. Methanol and dimethyl sulphoxide did not affect the auto activation kinetics at concentration range studied. Dimethyl sulphoxide and dioxane appeared to be unsuitable for characterizing the Cytochrome450 mediated reactions because they showed a very significant effect on the V_max/K_m ratio, starting at concentrations of 0.025 % v/v and 0.25 % v/v, respectively. Methanol and acetonitrile at concentration <0.5 % v/v appeared to be acceptable solvents for solubilization of substrates metabolized by Cytochrome450s.