Phase II metabolism of warfarin in primary culture of adult rat hepatocytes.
We used adult rat hepatocytes in primary culture (HPC) as a model system to study the hepatic phase II metabolism of the anticoagulant warfarin. Hepatocytes were isolated by a collagenase perfusion technique and maintained for 24 hr in Waymouth's medium containing 0.1 mM (R)-warfarin. When HPC medium was analyzed by reverse phase high performance liquid chromatography with diode-array detection, 4'-, 6-, and 7-hydroxywarfarin were identified. Several putative conjugates were observed eluting between 13 and 18 min. Treatment of hepatocyte medium with beta-glucuronidase and sulfatase resulted in the loss of five putative conjugates and concomitant increases in 4'-, 6-, and 7-hydroxywarfarin and warfarin, suggesting that these metabolites and warfarin were conjugated. Use of the beta-glucuronidase inhibitor saccharic acid 1,4-lactone enabled the determination of the relative extents of conjugation of each metabolite by glucuronic acid and sulfate. Glucuronidation was the predominant pathway for 4'-hydroxywarfarin, whereas 6-hydroxywarfarin and warfarin occurred mainly as sulfate conjugates. In contrast, 7-hydroxywarfarin was converted to both glucuronide and sulfate conjugates. Exposure of HPC to phenobarbital resulted in a decrease in cytochrome P-450-mediated production of hydroxylated warfarin metabolites; however, an increase in the production of 8-hydroxywarfarin was observed when HPC were exposed to beta-naphthoflavone. Unique conjugation patterns were found when hydroxylated warfarins were substituted for warfarin in HPC medium. Both 7- and 8-hydroxywarfarin were converted to one sulfate and two glucuronide conjugates, whereas 4'-hydroxywarfarin was converted to a single glucuronide conjugate. A spectral library of these conjugates was used to identify the major conjugates of warfarin formed by rat HPC.
Jansing
R L
RL
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
Chao
E S
ES
Kaminsky
L S
LS
D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005965:Glucuronates / Q000378:metabolism; D005966:Glucuronidase / Q000378:metabolism; D006900:Hydroxylation; D008099:Liver / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D051381:Rats; D013429:Sulfatases / Q000378:metabolism; D013440:Sulfides / Q000378:metabolism; D014859:Warfarin / Q000378:metabolism*
D005965:Glucuronates; D013440:Sulfides; D014859:Warfarin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013429:Sulfatases; D005966:Glucuronidase