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 muM) 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 μM) 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 ∼3.2 μM. Similarly, the metabolism of TLB (50 μM) was enhanced (∼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 μM) 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.