Propofol hydroxylation by dog liver microsomes: Assay development and dog breed differences

Pharmacokinetic studies indicate that clearance of propofol, an anesthetic agent, is slower in greyhounds compared with other dog breeds. Biotransform ation of propofol to 2,6-diisopropyl-1,4-quinol (4-hydroxypropofol) by cyto chrome P-450 in the liver is proposed as a critical initial step in the eli mination of this drug in dogs. Breed differences in the activity of this en zyme could therefore explain pharmacokinetic differences. An in vitro propo fol hydroxylase assay was developed and then used to compare enzyme activit ies in liver microsomes from male greyhound, beagle, and mixed-breed dogs ( five each). HPLC of incubate identified only one NADPH-dependent metabolite , which had a chromatographic retention time and UV absorbance, fluorescenc e, and mass spectra that were identical with authentic 4-hydroxypropofol st andard. HPLC with fluorescence detection provided a highly sensitive quanti tation method for 4-hydroxypropofol with a quantitation limit of 8 ng/ml us ing optimized excitation/emission wavelengths (288 nm/330 nm, respectively) . Estimates of apparent K-m and V-max for propofol hydroxylation by microso mes from a male beagle dog were 7.3 mu M and 3.8 nmol/mg/min, respectively. At a substrate concentration of 20 mu M, propofol hydroxylase activity was significantly lower (p = .032) in greyhound microsomes (1.7 +/- 0.4 nmol/m g/min) compared with beagle microsomes (5.1 +/- 1.3 nmol/mg/min) but was no t statistically different (p = .42) compared with mixed-breed microsomes (3 .1 +/- 1.2 nmol/mg/min). These results indicate that there are breed differ ences in propofol hydroxylase activity and that deficient hydroxylation of propofol by one or more hepatic cytochrome P-450 isoforms may contribute to slow pharmacokinetic clearance of propofol by greyhounds.