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FUNCTIONAL ACTIVITY IN HUMAN HEART EXPRESSING CYP1A1
Author(s) -
Huguet Jade,
Gaudette Fleur,
Michaud Véronique,
Turgeon Jacques
Publication year - 2013
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.27.1_supplement.672.2
Subject(s) - metabolite , western blot , microsome , cytosol , chemistry , metabolism , monoclonal antibody , human heart , biochemistry , active metabolite , microbiology and biotechnology , pharmacology , biology , antibody , enzyme , medicine , gene , immunology
Background Drug metabolism by CYP450s expressed in cardiac myocytes can modulate cardiac drug levels and actions. CYP1A1 and 2C8 are importantly expressed in the human heart. A cocktail approach with 7‐Ethoxyresorufin (7‐ET) and repaglinide (REP) were chosen to study selective metabolism of CYP1A1 and 2C8, respectively. Methods Incubations were first performed in Supersome™ of each CYP450s founded in the heart. Then, cytosolic (S1) and human heart microsomes (HHM) were isolated by differential centrifugations from one human heart. Resorufin and REP metabolite were quantified by LC‐MS/MS. Western blot analysis was performed using a monoclonal antibody raised against CYP1A1. Results REP metabolism was selective towards 2C8 and 7‐ET for 1A1 in Supersome™. For 7‐ET, Km was 0.36 vs. 0.4μM and Vmax was 1640 vs. 1944 pmoles of resorufin/mg of protein/min when incubated alone vs. with REP, respectively. For REP, Km was 3.7 vs. 3.35μM and Vmax was 1283 vs. 1120 pmoles REP metabolite/mg of protein/min when incubated alone vs. with 7‐ET, respectively. No activity was observed when 7‐ET and REP were incubated in HHM but there was a low activity in the S1 fraction (0.3 pmoles of resorufin/mg prot/min). This was confirmed by Western blot analysis where CYP1A1 protein was found in S1 but not in HHM. Conclusion This cocktail approach allows us to study the CYP450 activities in the heart. Research funded by the CIHR, HSF and FRSQ.