Effect of albumin on the estimation, in vitro, of phenytoin Vmax and Km values: implications for clinical correlation.
The effect of bovine serum albumin (BSA) on human liver metabolism, in vitro, of 14C-phenytoin (PHT) was studied. Michaelis Menten parameters were determined for the conversion of PHT to p-hydroxy phenytoin in seven different microsomal preparations with the addition of 0, 2, and 4% BSA. The unbound Km (Kmu) values were 30.8 +/- 18.6, 1.57 +/- 0.21 and 1.50 +/- 0.17 microM (mean +/- S.D.), respectively; however, there was excellent agreement among the Vmax values (29.1, 31.8 and 31.5 pmol/min/mg). With intact tissue slices, BSA (4%) added to incubations of PHT had a minimal effect on the Vmax values in two of the four livers studied and resulted in a mean Kmu value of 2.20 +/- 0.59 microM, although the Kmu in the absence of BSA was 6.64 +/- 3.17. In scaling-up to the whole body, Vmax values were 3.9 and 1.0 mg/kg/day for microsomes and slices, respectively, compared to 5.9 mg/kg/day, in vivo. The Kmu values determined in the presence of albumin in both microsomes and slices were similar to those based on in vivo human steady state data (Kmu = 2-3 microM), and the intersubject variation, in vitro, was decreased in the presence of BSA. These findings for phenytoin metabolism suggest that the addition of albumin to incubation media for slices or microsome experiments may yield Km estimates that are more representative of in vivo values.
Ludden
L K
LK
Division of Clinical Pharmacology, Center for Drug Evaluation and Research, Food and Drug Administration, Rockville, Maryland, USA.
Ludden
T M
TM
Collins
J M
JM
Pentikis
H S
HS
Strong
J M
JM
D000927:Anticonvulsants / Q000493:pharmacokinetics*; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism; D010672:Phenytoin / Q000493:pharmacokinetics*; D012710:Serum Albumin, Bovine / Q000494:pharmacology*
D000927:Anticonvulsants; D012710:Serum Albumin, Bovine; D010672:Phenytoin