Selection of monoclonal antibodies against 6-oxo-M(1)dG and their use in an LC-MS/MS assay for the presence of 6-oxo-M(1)dG in vivo.
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(1)dG, is mutagenic in bacterial and mammalian cells and is repaired by the nucleotide excision repair pathway. In vivo, M(1)dG 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(1)dG, which is excreted in urine, bile, and feces. We have developed a specific monoclonal antibody against 6-oxo-M(1)dG and have incorporated this antibody into a procedure for the immunoaffinity isolation of 6-oxo-M(1)dG from biological matrices. The purified analyte is quantified by LC-MS/MS using a stable isotope-labeled analogue ([(15)N(5)]-6-oxo-M(1)dG) as an internal standard. Healthy male Sprague-Dawley rats excreted 6-oxo-M(1)dG 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(1)dG in rodents without exogenous introduction of M(1)dG.
Department of Biochemistry, Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine , Nashville, Tennessee 37232-0146, United States.
Dapo
D
Akingbade
Philip J
PJ
Kingsley
Sarah C
SC
Shuck
Tracy
T
Cooper
Robert
R
Carnahan
Jozef
J
Szekely
Lawrence J
LJ
Marnett
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D018736:DNA Adducts / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005243:Feces / Q000737:chemistry; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry
D000911:Antibodies, Monoclonal; D018736:DNA Adducts; D003849:Deoxyguanosine