Effect of albumin on phenytoin and tolbutamide metabolism in human liver microsomes: an impact more than protein binding.

The cytochrome P450 (P450)-dependent conversion of phenytoin (PHT) to p-hydroxy phenytoin (pHPPH), and tolbutamide (TLB) to 4-hydroxy tolbutamide (hydroxy-TLB), in human liver microsomes was studied in the presence of increasing concentrations (0-4%) of bovine serum albumin (BSA). Therefore, the free fraction (f(u)) of PHT and TLB varied. Whereas the f(u) of PHT (5 microM) decreased, an increase (3-fold), rather than a decrease in the pHPPH formation rate was observed when BSA (<1%) was present. The stimulation was attributed to a significant decrease in apparent K(m). The change, however, was diminished as the BSA concentration reached 4% (PHT f(u) = 0.2), in which the reaction velocity remained the same as that measured in the absence of BSA. Therefore, unchanged K(m) (16.2 +/- 0.7 microM) and V(max) (9.4 +/- 0.2 pmol/min/mg of protein) values were determined based on total PHT concentrations, whereas correction for f(u) led to an unbound K(m) (K(mu)) of approximately 3.2 microM. Similarly, the metabolism of TLB (50 microM) was enhanced (approximately 2-fold) in the presence of 0.25% BSA but remained only 35% of the control activity (no BSA) at 1% BSA. However, the remaining activity was higher (3-fold) than that determined with an equivalent free concentration of TLB (4 microM) calculated according to its f(u) (0.08). The difference became less significant when BSA concentration was 4% (f(u) < 0.02). Collectively, the results suggest a 2-fold effect of BSA on PHT and TLB hydroxylation: first, facilitation of the reactions via a decrease in K(m); second, a decrease in f(u) leading to a drop in reaction rate. For a given P450 reaction, therefore, the effect of BSA may depend upon enzyme affinity, catalytic capacity, and the extent of protein binding.