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Inhibition of Human CYP3A‐Catalyzed Lithocholic Acid 3‐Oxidation by Specific Vitamin E Isomers
Author(s) -
Lau Aik Jiang,
Chai Swee Fen,
Teo Josephine Si Min
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.934.5
Subject(s) - lithocholic acid , chemistry , tocotrienol , biochemistry , bile acid , microsome , cyp3a4 , enzyme , cytochrome p450 , vitamin e , antioxidant , tocopherol
Lithocholic acid is a hepatotoxic and carcinogenic bile acid. Previous studies suggested that CYP3A4 but not CYP3A5 catalyzed the oxidation of lithocholic acid to 3‐ketocholanoic acid and that CYP3A metabolized vitamin E isomers (tocotrienols and tocopherols). Given that a substrate of an enzyme may also be an inhibitor of that enzyme, we determined whether α‐tocotrienol, γ‐tocotrienol, δ‐tocotrienol, a tocotrienol‐rich mixture (a mixture consisting of 25.7% α‐tocotrienol, 2.6% β‐tocotrienol, 28.6% γ‐tocotrienol, 8.4% δ‐tocotrienol, 25.6% α‐tocopherol, and 4.3% α‐tocomonoenol), and α‐tocopherol inhibit the oxidation of lithocholic acid to 3‐ketocholanoic acid. Enzymatic formation of 3‐ketocholanoic acid via lithocholic acid 3‐oxidation was determined in pooled human liver microsomes and recombinant CYP3A4 and CYP3A5 enzymes. Enzyme inhibition assay was conducted in an incubation mixture containing potassium phosphate buffer, lithocholic acid, NADPH, enzyme (human liver microsomes, recombinant CYP3A4, or recombinant CYP3A5), and a putative inhibitor (a tocotrienol, a tocotrienol‐rich mixture, or α‐tocopherol). The amount of 3‐ketocholanoic acid was quantified by ultra‐high performance liquid chromatography‐tandem mass spectrometry. Lithocholic acid was metabolized to 3‐ketocholanoic acid by human recombinant CYP3A4 and CYP3A5 enzymes and human liver microsomes. Human liver microsomal‐catalyzed lithocholic acid 3‐oxidation exhibited Michaelis‐Menten kinetics at substrate concentrations less than 100 μM. Substrate inhibition kinetics was evident at lithocholic acid concentrations greater than 100 μM. The K m , V max , and V max /K m values for human liver microsomal LCA 3‐oxidation were 26 μM, 304 pmol/min/mg, and 12 μl/min/mg, respectively. Enzyme kinetics experiments indicated that human recombinant CYP3A4 and CYP3A5 catalyzed lithocholic acid 3‐oxidation to a similar extent. α‐Tocotrienol, γ‐tocotrienol, δ‐tocotrienol, and a tocotrienol‐rich mixture, but not α‐tocopherol, inhibited lithocholic acid 3‐oxidation by human liver microsomes. Concentration‐response experiments indicated that a tocotrienol‐rich mixture and δ‐tocotrienol inhibited human liver microsomal lithocholic acid 3‐oxidation with IC 50 of 6.6 ± 2.1 μg/ml and 19.0 ± 1.0 μM, respectively. Overall, lithocholic acid 3‐oxidation is catalyzed by both CYP3A4 and CYP3A5 enzymes. It is differentially inhibited by tocotrienols, but not α‐tocopherol, suggesting vitamin E isomer‐specific inhibition of human liver microsomal lithocholic acid 3‐oxidation. Support or Funding Information Supported by National University of Singapore Research Fund.