Limits...
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.

Akingbade D, Kingsley PJ, Shuck SC, Cooper T, Carnahan R, Szekely J, Marnett LJ - Chem. Res. Toxicol. (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine , Nashville, Tennessee 37232-0146, United States.

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(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.

Show MeSH
6-oxo-M1dG is present in rat feces. A representativeLC-MS/MS chromatogram of 6-oxo-M1dG and the internal standard[15N5]-6-oxo-M1dG isolated from ratfeces is displayed. The inset represents a chromatogram of internalstandard alone, purified from PBS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3285145&req=5

fig6: 6-oxo-M1dG is present in rat feces. A representativeLC-MS/MS chromatogram of 6-oxo-M1dG and the internal standard[15N5]-6-oxo-M1dG isolated from ratfeces is displayed. The inset represents a chromatogram of internalstandard alone, purified from PBS.

Mentions: While the amounts of 6-oxo-M1dG are low,the assay provideda sufficient signal-to-noise ratio to permit accurate quantification.Figure 6 displays a sample chromatogram froma processed fecal sample. The upper trace (m/z 320 → 204) shows the analyte, while the bottomtrace (m/z 325 → 209) representsthe [15N5]-6-oxo-M1dG internal standard.Both compounds gave chromatographic peaks that were well above thebackground noise. The inset of Figure 6 isthe internal standard alone after recovery from PBS. There is no peakin the 6-oxo-M1dG trace of the inset, which is illustrativeof the fact that there was no isotopic impurity in the internal standardthat could contribute to the 6-oxo-M1dG signal.


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.

Akingbade D, Kingsley PJ, Shuck SC, Cooper T, Carnahan R, Szekely J, Marnett LJ - Chem. Res. Toxicol. (2012)

6-oxo-M1dG is present in rat feces. A representativeLC-MS/MS chromatogram of 6-oxo-M1dG and the internal standard[15N5]-6-oxo-M1dG isolated from ratfeces is displayed. The inset represents a chromatogram of internalstandard alone, purified from PBS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285145&req=5

fig6: 6-oxo-M1dG is present in rat feces. A representativeLC-MS/MS chromatogram of 6-oxo-M1dG and the internal standard[15N5]-6-oxo-M1dG isolated from ratfeces is displayed. The inset represents a chromatogram of internalstandard alone, purified from PBS.
Mentions: While the amounts of 6-oxo-M1dG are low,the assay provideda sufficient signal-to-noise ratio to permit accurate quantification.Figure 6 displays a sample chromatogram froma processed fecal sample. The upper trace (m/z 320 → 204) shows the analyte, while the bottomtrace (m/z 325 → 209) representsthe [15N5]-6-oxo-M1dG internal standard.Both compounds gave chromatographic peaks that were well above thebackground noise. The inset of Figure 6 isthe internal standard alone after recovery from PBS. There is no peakin the 6-oxo-M1dG trace of the inset, which is illustrativeof the fact that there was no isotopic impurity in the internal standardthat could contribute to the 6-oxo-M1dG signal.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine , Nashville, Tennessee 37232-0146, United States.

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(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.

Show MeSH