Detection and Quantification of Endogenous Cyclic DNA Adducts Derived from trans-4-Hydroxy-2-nonenal in Human Brain Tissue by Isotope Dilution Capillary Liquid Chromatography Nanoelectrospray Tandem Mass Spectrometry

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Abstract
A sensitive and selective capillary liquid chromatography nanoelectrospray isotope dilution mass spectrometric method was developed to identify and quantify the endogenous cyclic DNA adducts derived from trans-4-hydroxy-2-nonenal with 2‘-deoxyguanosine (HNE-dG) in human brain tissues. Authentic and 13C and 15N stable isotope-labeled HNE-dG were synthesized to serve as standards. The in vitro HNE-modified calf-thymus DNA as well as the DNA samples isolated from human brain tissues of normal and Alzheimer's disease subjects were enzymatically digested to nucleosides in vitro with the presence of internal standard (HNE-dG-13C10, 15N5). The enzymatic digests were cleaned up by solid phase extraction. Only 1−2 μg of DNA digests was loaded on a laboratory-constructed reversed phase capillary chromatography column, and the HNE-dG adducts were separated from intact nucleosides and quantified by a high capacity ion trap mass spectrometer in the MS/MS mode. This method was able to quantify an adduct level of approximately 40 lesions/109 normal DNA nucleosides. The detected level of HNE-dG adducts in hippocampus/parahippocampal gyrus and inferior parietal regions of postmortem brains from AD subjects were 556 ± 379 and 238 ± 72 adducts per 109 normal nucleosides, respectively. These results were consistent with the 32P postlabeling results, which detected 400−600 adducts per 109 normal nucleotides in the hippocampus.

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