title: Metabolism of isoniazid by neutrophil myeloperoxidase leads to isoniazid-NAD(+) adduct formation: A comparison of the reactivity of isoniazid with its known human metabolites.
abstract: The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD(+)) via the mycobacterial catalase-peroxidase enzyme, KatG, has been described as the major component of the mode of action of isoniazid (INH). However, there are numerous human peroxidases that may catalyze this reaction. The role of neutrophil myeloperoxidase (MPO) in INH-NAD(+) adduct formation has never been explored; this is important, as neutrophils are recruited at the site of tuberculosis infection (granuloma) through infected macrophages' cell death signals. In our studies, we showed that neutrophil MPO is capable of INH metabolism using electron paramagnetic resonance (EPR) spin-trapping and UV-Vis spectroscopy. MPO or activated human neutrophils (by phorbol myristate acetate) catalyzed the oxidation of INH and formed several free radical intermediates; the inclusion of superoxide dismutase revealed a carbon-centered radical which is considered to be the reactive metabolite that binds with NAD(+). Other human metabolites, including N-acetyl-INH, N-acetylhydrazine, and hydrazine did not show formation of carbon-centered radicals, and either produced no detectable free radicals, N-centered free radicals, or superoxide, respectively. A comparison of these free radical products indicated that only the carbon-centered radical from INH is reducing in nature, based on UV-Vis measurement of nitroblue tetrazolium reduction. Furthermore, only INH oxidation by MPO led to a new product (λmax=326nm) in the presence of NAD(+). This adduct was confirmed to be isonicotinyl-NAD(+) using LC-MS analysis where the intact adduct was detected (m/z=769). The findings of this study suggest that neutrophil MPO may also play a role in INH pharmacological activity.
authors: Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada.
authors: Saifur R
authors: SR
authors: Khan
authors: Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada.
authors: Andrew G M
authors: AG
authors: Morgan
authors: Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada.
authors: Karim
authors: K
authors: Michail
authors: Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada.
authors: Nutan
authors: N
authors: Srivastava
authors: Department of Chemistry, Faculty of Sciences, University of Alberta, Edmonton, Canada.
authors: Randy M
authors: RM
authors: Whittal
authors: Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada; Pharmacology and Toxicology Department, Faculty of Pharmacy, Taibah University, Madinah, Saudi Arabia.
authors: Naif
authors: N
authors: Aljuhani
authors: Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada. Electronic address: siraki@ualberta.ca.
authors: Arno G
authors: AG
authors: Siraki
mesh_terms: D000995:Antitubercular Agents / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D006834:Hydrazines / Q000737:chemistry; D007538:Isoniazid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D009243:NAD / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D018375:Neutrophil Activation / Q000187:drug effects; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D009195:Peroxidase / Q000737:chemistry / Q000378:metabolism*; D061251:Primary Cell Culture; D013482:Superoxide Dismutase / Q000737:chemistry / Q000378:metabolism; D013481:Superoxides / Q000737:chemistry; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D000995:Antitubercular Agents; D006834:Hydrazines; C525017:isonicotinyl-NAD; D009243:NAD; D013481:Superoxides; C029424:hydrazine; D009195:Peroxidase; D013482:Superoxide Dismutase; D013755:Tetradecanoylphorbol Acetate; C014949:acetylhydrazine; D007538:Isoniazid
keywords: Free radicals; INH-NAD(+); Isoniazid; Myeloperoxidase; Neutrophil