Investigations of the effects of ethanol on warfarin binding to human serum albumin

Abstract

Ethanol effects on warfarin binding to human serum albumin (HSA) have been studied by equilibrium dialysis and fluorescence methods at pH 7.4 in phosphate-buffered saline at 37°C. In the presence of various amounts of ethanol fluorescence intensity of bound warfarin decreased significantly but this intensity reduction was not solely from displacement of bound warfarin from HSA. By comparing fluorescence and equilibrium dialysis data we concluded that fluorescence intensity reduction of warfarin was mainly the result of changes in the surrounding environment of the warfarin binding site by ethanol interaction with HSA and that displacement of bound warfarin was not significant compared to the fluorescence intensity changes. The dissociation constant of warfarin binding to HSA decreased with an increasing amount of ethanol. From the changes in fluorescence intensity upon warfarin binding to HSA with the presence of ethanol ranging from 0 to 5.0% the following dissociation constants (Kd) were determined: 0% ethanol 5.39 ± 0.2 µM, 0.1% ethanol 5.86 ± 0.1 µM, 0.3% ethanol 5.83 ± 0.2 µM, 0.5% ethanol 6.76 ± 0.1 µM, 1% ethanol 7.01 ± 0.1 µM, 3% ethanol 9.9 ± 0.7 µM, 5% ethanol 13.01 ± 0.1 µM. From the equilibrium dialysis with the same ranges of ethanol presence the following Kd values were obtained: 0% ethanol 6.62 ± 1.6 µM, 0.1% ethanol 6.81 ± 1.1 µM, 0.3% ethanol 8.26 ± 2.5 µM, 0.5% ethanol 8.86 ± 1.9 µM, 1% ethanol 11.01 ± 4.2 µM, 3% ethanol 20.75 ± 2.4 µM, 5% ethanol 21.67 ± 2.2 µM. The results suggest that warfarin bound to HSA was displaced by ethanol. These data indicate that ethanol influence on warfarin binding to HSA may alter the pharmacokinetics of warfarin.