Substrate-dependent inhibition of human cytochrome P450 3A catalytic activity by specific isomers of vitamin E

摘要

OBJECTIVE Lithocholic acid,which is a secondary bile acid,has been reported to be hepatotoxic and carcinogenic.It is metabolized by human cytochrome P450 3A(CYP3A)to form 3-ketocholanoic acid.A previous study suggests that vitamin E isomers(tocotrienols and tocopherols)are metabolized by CYP3 A.Given that substrates of an enzyme may competitively inhibit the enzyme,we determined whether alpha-tocotrienol,gamma-tocotrienol,delta-tocotrienol,tocotrienol-rich mixture(a mixture consisting of 25.7% d-α-tocotrienol,2.6% d-β-tocotrienol,28.6% d-γ-tocotrienol,8.4% d-δ-tocotrienol,25.6% d-α-tocopherol,and 4.3% d-α-tocomonoenol),and alpha-tocopherol inhibit human liver microsomal CYP3Aactivity,as assessed by the enzymatic conversion of lithocholic acid to 3-ketocholanoic acid and of testosterone to6β-hydroxytestosterone.METHODS Enzymatic formation of 3-ketocholanoic acid via lithocholic acid 3-oxidation was determined in pooled human liver microsomes and recombinant CYP3A4 and CYP3A5.Enzyme inhibition assay was conducted in a mixture containing potassium phosphate buffer(pH 7.4),human liver microsomes,NADPH,lithocholic acid,and various concentrations of a test chemical.The amount of 3-ketocholanoic acid formed was quantified by a novel,validated ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS-MS)method.RESULTS Lithocholic acid was metabolized to 3-ketocholanoic acid by human recombinant CYP3A4 and CYP3A5enzymes and human liver microsomes.Alpha-tocotrienol,gamma-tocotrienol,delta-tocotrienol,and tocotriernol-rich mixture,but not alpha-tocopherol,inhibited 3-ketocholanoic acid formation in human liver microsomes.Concentration-response experiments indicated that tocotrienol-rich mixture and delta-tocotrienol inhibited 3-ketocholanoic acid formation with IC50 values of 6.6±2.1μg·mL-1 and 19.0±1.0μmol·L-1,respectively.CONCLUSION Tocotrienols inhibited CYP3A-catalyzed lithocholic acid 3-oxidation but not CYP3A-catalyzed testosterone 6-beta-hydroxylation.This suggests that lithocholic acid and testosterone bind to different CYP3 Abinding sites and that tocotrienols preferentially inhibit the lithocholic acid binding site on CYP3 Aenzymes.