The effect of human serum albumin (HSA), in its endogenous, free fatty acid free (FAF) and globulin free (GF) form, on the activity of CYP2C9 was studied in human liver microsomes using tolbutamide as the substrate. The widely used BSA was included to assess the differential effect of BSA and HSA. CYP2C9 activity was expressed as CLint (Vmax/Km). HSA(FAF) and BSA showed a concentration-dependent and biphasic (activation and inhibition) interaction with CYP2C9 activity. HSA(GF) and HSA exhibited an inhibitory effect, with an inhibition constant, Ki, of 19.9 microM (0.13% albumin) and 42.2 microM (0.35% albumin), respectively. Enzyme-kinetics revealed that the activation is accompanied by a decrease in Km values, while with inhibition Km values increased. A simplified method to calculate clearance, utilizing a single slope (V/S) determination based on V over the lowest linear range of [S] (designated as CLone) was assessed. Virtually identical values were obtained for CLint and CLone. The free-drug hypothesis was tested by comparing ratios of relative CLint/unbound fraction (FDH Test ratio). The FDH Test ratio for HSA was about 1, indicating that HSA binding of tolbutamide reduced the CYP2C9 activity in accord with the free-drug hypothesis. The FDH Test ratios for BSA and HSA(FAF) were 3.7 and 3.0, revealing a monophasic activation of CYP2C9. For 2%HSA(GF) the ratio of 0.3 confirmed inhibition. As revealed by their removal, free fatty acids and globulins, significantly alter the interaction of HSA with CYP2C9. In addition, HSA and BSA showed different effects on the oxidation of tolbutamide by CYP2C9.
Copyright 2004 Elsevier Inc.