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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• 作者：Dapo Akingbade ; Philip J. Kingsley ; Sarah C. Shuck ; Tracy Cooper ; Robert Carnahan ; Jozef Szekely ; Lawrence J. Marnett
• 刊名：Chemical Research in Toxicology
• 出版年：2012
• 出版时间：February 20, 2012
• 年：2012
• 卷：25
• 期：2
• 页码：454-461
• 全文大小：381K
• 年卷期：v.25,no.2(February 20, 2012)
• ISSN：1520-5010

文摘

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鈥?deoxy-尾-d-erythro-pentofuranosyl)-pyrimido[1,2-伪]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-尾-d-erythro-pentofuranosyl)-pyrimido[1,2-伪]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鈥揇awley rats excreted 6-oxo-M₁dG at a rate of 350鈥?893 fmol/kg路d in feces. This is the first report of the presence of the major metabolite of M₁dG in rodents without exogenous introduction of M₁dG.