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
journal contributionposted on 16.12.2015, 20:59 by Dapo Akingbade, Philip J. Kingsley, Sarah C. Shuck, Tracy Cooper, Robert Carnahan, Jozef Szekely, Lawrence J. Marnett
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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.