Development of a Method for Quantification of Acrolein−Deoxyguanosine Adducts in DNA Using Isotope Dilution-Capillary LC/MS/MS and Its Application to Human Brain Tissue
- 18 August 2005
- journal article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 77 (18), 5982-5989
- https://doi.org/10.1021/ac050624t
Abstract
Acrolein is a highly reactive α,β-unsaturated aldehyde and is known to react with DNA forming exocyclic acrolein−deoxyguanosine adducts (Acro−dG). These aldehyde−DNA lesions may play a role in mutagenesis, carcinogenesis, and neurodegenerative diseases. In the present work, we described the development and evaluation of a highly sensitive and selective capillary liquid chromatography nanoelectrospray isotope dilution tandem mass spectrometry method for quantitatively analyzing Acro−dG in DNA hydrolysates. This was achieved by applying a stable isotope-labeled analogue Acro−dG-13C10,15N5 as an internal standard to the DNA to be analyzed and then hydrolyzing the DNA enzymatically to nucleosides. The acrolein-modified nucleosides were separated from normal nucleosides by capillary liquid chromatography and quantified by a high-capacity ion trap mass spectrometer in the MS/MS mode. The developed method achieved attomole-level sensitivity (limit of detection was 10 fg, 31 amol on column) for detection of pure Acro−dG adduct standards. The limit of quantification of Acro−dG adducts obtained in 10 μg of DNA hydrolysates was 1.5 fmol, which corresponded to 50 adducts/109 normal nucleosides. Application of this method to the analysis of Acro−dG adducts in acrolein (10-fold)-treated calf thymus DNA showed ∼830 lesion/106 DNA nucleosides using as low as 50 ng of DNA. Application of this method to DNA samples (1−2 μg) isolated from brain tissues from Alzheimer's disease subjects and age-matched controls demonstrated 2800−5100 Acro−dG adducts/109 DNA nucleosides. Statistically significant differences (P < 0.05) in levels of Acro−dG between AD subjects and controls were observed in DNA isolated from the hippocampus/parahippocampal gyrus.Keywords
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