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Selection of Monoclonal Antibodies Against 6-oxo-M1dG and Their Use in an LC-MS/MS Assay for the Presence of 6-oxo-M1dG in Vivo

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journal contribution
posted on 16.12.2015, 20:59 by Dapo Akingbade, Philip J. Kingsley, Sarah C. Shuck, Tracy Cooper, Robert Carnahan, Jozef Szekely, Lawrence J. Marnett
Oxidative stress triggers DNA and lipid peroxidation, leading to the formation of electrophiles that react with DNA to form adducts. A product of this pathway, (3-(2′-deoxy-β-d-erythro-pentofuranosyl)-pyrimido­[1,2-α]­purine-10­(3H)-one), or M1dG, is mutagenic in bacterial and mammalian cells and is repaired by the nucleotide excision repair pathway. In vivo, M1dG is oxidized to a primary metabolite, (3-(2-deoxy-β-d-erythro-pentofuranosyl)-pyrimido­[1,2-α]­purine-6,10­(3H,5H)-dione, or 6-oxo-M1dG, which is excreted in urine, bile, and feces. We have developed a specific monoclonal antibody against 6-oxo-M1dG and have incorporated this antibody into a procedure for the immunoaffinity isolation of 6-oxo-M1dG from biological matrices. The purified analyte is quantified by LC-MS/MS using a stable isotope-labeled analogue ([15N5]-6-oxo-M1dG) as an internal standard. Healthy male Sprague–Dawley rats excreted 6-oxo-M1dG at a rate of 350–1893 fmol/kg·d in feces. This is the first report of the presence of the major metabolite of M1dG in rodents without exogenous introduction of M1dG.