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.
Acrolein is a highly reactive alpha,beta-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 mug of DNA hydrolysates was 1.5 fmol, which corresponded to 50 adducts/10(9) normal nucleosides. Application of this method to the analysis of Acro-dG adducts in acrolein (10-fold)-treated calf thymus DNA showed approximately 830 lesion/10(6) DNA nucleosides using as low as 50 ng of DNA. Application of this method to DNA samples (1-2 mug) isolated from brain tissues from Alzheimer's disease subjects and age-matched controls demonstrated 2800-5100 Acro-dG adducts/10(9) 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.
Liu
Xinli
X
Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055, USA.
Lovell
Mark A
MA
Lynn
Bert C
BC
D000171:Acrolein / Q000737:chemistry*; D001921:Brain; D002196:Capillaries; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods*; D018736:DNA Adducts / Q000032:analysis* / Q000737:chemistry*; D003849:Deoxyguanosine / Q000737:chemistry*; D006801:Humans; D007201:Indicator Dilution Techniques; D007477:Ions / Q000737:chemistry; D007554:Isotopes / Q000737:chemistry; D015394:Molecular Structure; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
D018736:DNA Adducts; D007477:Ions; D007554:Isotopes; D000171:Acrolein; D003849:Deoxyguanosine