@article {Tang648, author = {Tang, Cuyue and Lin, Yuh and Rodrigues, A. David and Lin, Jiunn H.}, title = {Effect of Albumin on Phenytoin and Tolbutamide Metabolism in Human Liver Microsomes: An Impact More Than Protein Binding}, volume = {30}, number = {6}, pages = {648--654}, year = {2002}, doi = {10.1124/dmd.30.6.648}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {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{\textendash}4\%) of bovine serum albumin (BSA). Therefore, the free fraction (fu) of PHT and TLB varied. Whereas the fu of PHT (5 μM) 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 apparentKm. The change, however, was diminished as the BSA concentration reached 4\% (PHTfu = 0.2), in which the reaction velocity remained the same as that measured in the absence of BSA. Therefore, unchanged Km (16.2 {\textpm} 0.7 μM) and Vmax (9.4 {\textpm} 0.2 pmol/min/mg of protein) values were determined based on total PHT concentrations, whereas correction for fu led to an unboundKm (Kmu) 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 itsfu (0.08). The difference became less significant when BSA concentration was 4\% (fu \< 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 inKm; second, a decrease infu 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. The American Society for Pharmacology and Experimental Therapeutics}, issn = {0090-9556}, URL = {http://dmd.aspetjournals.org/content/30/6/648}, eprint = {http://dmd.aspetjournals.org/content/30/6/648.full.pdf}, journal = {Drug Metabolism and Disposition} }