Selection of monoclonal antibodies against 6-oxo-M 1dG and their use in an LC-MS/MS assay for the presence of 6-oxo-M 1dG in vivo

Abstract

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 M 1dG, is mutagenic in bacterial and mammalian cells and is repaired by the nucleotide excision repair pathway. In vivo, M 1dG 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-M 1dG, which is excreted in urine, bile, and feces. We have developed a specific monoclonal antibody against 6-oxo-M 1dG and have incorporated this antibody into a procedure for the immunoaffinity isolation of 6-oxo-M 1dG from biological matrices. The purified analyte is quantified by LC-MS/MS using a stable isotope-labeled analogue ([ 15N 5]-6-oxo-M 1dG) as an internal standard. Healthy male Sprague-Dawley rats excreted 6-oxo-M 1dG at a rate of 350-1893 fmol/kg·d in feces. This is the first report of the presence of the major metabolite of M 1dG in rodents without exogenous introduction of M 1dG. © 2012 American Chemical Society.