UBC Theses and Dissertations
Hepatic biotransformation of lithocholic acid : role of cytochrome P450 enzymes Deo, Anand K.
The purpose of the present study was to investigate the in vitro biotransformation of lithocholic acid (3α-hydroxy-5β-cholanoic acid, LCA). A validated and optimized method to analyze metabolite formation was developed using liquid chromatography-electrospray mass spectrometry (LC/MS). LCA (0.5-300 μM) was incubated with 0.5 mg/ml of hepatic microsomal protein (untreated male Wistar rats) for 30 min in a reaction mixture consisting of 1 mM NADPH and 46.5 mM potassium phosphate buffer at pH 7.4. LCA metabolites were resolved using a LC/MS and XTerra™ MS C₁₈ (2.1 mm ∗ 150 mm, 3.5 mm) column. The major metabolites were murideoxycholic acid (MDCA), isolithocholic acid (ILCA), and 3-ketocholanic acid (3KCA). Minor metabolites were β-muricholic acid (β-MCA), 6-ketolithocholic acid (6KLCA) and ursodeoxycholic acid (UDCA) and also included, M-1 to M-5, which were not identified. Recovery of all metabolite standards, except for 3KCA (60%), was between 80-100%. Metabolite formation was not observed in the absence of NADPH, with boiled microsomes, or following carbon monoxide treatment. Formation of MDCA, ILCA, UDCA and 6KLCA followed typical Michaelis-Menten kinetics. To identify the cytochrome P450 (CYP) enzymes involved in the formation of LCA metabolites, hepatic microsomes prepared from rats (Long Evans) treated with inducers including 3-methylcholanthrene (MC, 25 mg/kg bw/day ∗ 4 days), phenobarbital (PB, 75 mg/kg bw/day ∗ 4 days) and dexamethasone (DEX, 100 mg/kg bw/day x 4 days) were used. Formation of MDCA and ILCA was decreased with all three inducer treatments and formation of 3KCA was increased with DEX treatment suggesting that CYP3A is involved in formation of 3KCA, and non-inducible CYP enzymes catalyze formation of MDCA and ILCA. Metabolite formation was greater with male than female rat liver microsomes. Rabbit polyspecific anti-rat CYP2C IgG inhibited MDCA formation by 50% and polyspecific anti-rat CYP3A IgG inhibited 3KCA formation by 50%. Anti-rat CYP3A IgG inhibited MDCA, ILCA and 6KLCA formation partially, but inhibited M-1 and M-3 formation completely. Monospecific anti-rat CYP2C11 IgG was not effective in inhibiting the formation of any metabolites. In summary, the results demonstrate the involvement of CYP2C and CYP3A enzymes in rat hepatic LCA biotransformation.
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