Quantitation of Lipid Peroxidation Product DNA Adducts in Human Prostate by Tandem Mass Spectrometry: A Method That Mitigates Artifacts

Reactive oxygen species (ROS) and chronic inflammation contribute to DNA damage of many organs, including the prostate. ROS cause oxidative damage to biomolecules, such as lipids, proteins, and nucleic acids, resulting in the formation of toxic and mutagenic intermediates. Lipid peroxidation (LPO) products covalently adduct to DNA and can lead to mutations. The levels of LPO DNA adducts reported in humans range widely. However, a large proportion of the DNA adducts may be attributed to artifact formation during the steps of isolation and nuclease digestion of DNA. We established a method that mitigates artifacts for most LPO adducts during the processing of DNA. We have applied this methodology to measure LPO DNA adducts in the genome of prostate cancer patients, employing ultrahigh-performance liquid chromatography electrospray ionization ion trap multistage mass spectrometry. Our preliminary data show that DNA adducts of acrolein, 6-hydroxy-1,N²-propano-2′-deoxyguanosine (6-OH-PdG) and 8-hydroxy-1,N²-propano-2′-deoxyguanosine (8-OH-PdG) (4–20 adducts per 10⁷ nucleotides) are more prominent than etheno (ε) adducts (<0.5 adducts per 10⁸ nucleotides). This analytical methodology will be used to examine the correlation between oxidative stress, inflammation, and LPO adduct levels in patients with benign prostatic hyperplasia and prostate cancer.