Vol. 27, Issue 11, 1293-1299, November 1999

Propofol Hydroxylation by Dog Liver Microsomes: Assay Development and Dog Breed Differences

Michael H. Court, Bonnie L. Hay-Kraus, Dennis W. Hill, Albert J. Kind, and David J. Greenblatt

Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, Massachusetts (M.H.C., D.J.G.); Department of Clinical Sciences, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts (M.H.C., B.L.H-K.); and Microchemistry Laboratory, Pathobiology Department, University of Connecticut, Storrs, Connecticut (D.W.H., A.J.K.)

Pharmacokinetic studies indicate that clearance of propofol, an anesthetic agent, is slower in greyhounds compared with other dog breeds. Biotransformation of propofol to 2,6-diisopropyl-1,4-quinol (4-hydroxypropofol) by cytochrome P-450 in the liver is proposed as a critical initial step in the elimination of this drug in dogs. Breed differences in the activity of this enzyme could therefore explain pharmacokinetic differences. An in vitro propofol hydroxylase assay was developed and then used to compare enzyme activities 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, fluorescence, and mass spectra that were identical with authentic 4-hydroxypropofol standard. HPLC with fluorescence detection provided a highly sensitive quantitation method for 4-hydroxypropofol with a quantitation limit of 8 ng/ml using optimized excitation/emission wavelengths (288 nm/330 nm, respectively). Estimates of apparent K_m and V_max for propofol hydroxylation by microsomes from a male beagle dog were 7.3 µM and 3.8 nmol/mg/min, respectively. At a substrate concentration of 20 µM, propofol hydroxylase activity was significantly lower (p = .032) in greyhound microsomes (1.7 ± 0.4 nmol/mg/min) compared with beagle microsomes (5.1 ± 1.3 nmol/mg/min) but was not statistically different (p = .42) compared with mixed-breed microsomes (3.1 ± 1.2 nmol/mg/min). These results indicate that there are breed differences 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.