7,8-hydroxy-2'-deoxyguanosine/2'-deoxiguanosine ratio determined in hydrolysates of brain DNA by ultrachromatrography coupled to tandem mass spectrometry.

7,8-hydroxy-2'-deoxyguanosine (8-OHdG) is an abundant DNA lesion formed by oxidation of the nucleoside 2'-deoxyguanosine (2-dG) and one of the most studied and accepted oxidative stress biomarkers. 8-OHdG has a strong carcinogenic potential, and prolonged oxidative stress heightens pathological conditions and especially cancer risk. Our aim was to develop, validate and apply a reliable method to assess DNA oxidation in genomic cellular DNA of sensible target organs such as brain. A procedure to isolate and digest the DNA of brain tissue properly for further detection of 8-OHdG and 2-dG by Ultra Performance Liquid Chromatography-tandem Mass Spectrometry (UPLC-MS/MS) was optimized. The UPLC-MS/MS was validated following the American Food and Drug Administration (FDA) Guidelines using mice pups' brain samples. To demonstrate the applicability of the UPLC-MS/MS method, 8-OHdG/2-dG ratio was determined in brain tissue of 1day old newborn mice pups (P1) in a model of hypoxia pre-conditioning during fetal-to-neonatal transition. We found that hypoxia at birth (FiO 0.14) and for 8h thereafter induced lower levels of DNA oxidation in mice pups and rendered even protective against a postnatal asphyxia/reoxygenation insult compared with fetal to neonatal transition in room air. We conclude that the UPLC-MS/MS method developed has proven suitable for the analysis of DNA oxidation biomarker 8-OHdG/2-dG ratio in tissue samples from newborn mice pups. We aim to apply this method in future studies aiming to provide a deeper insight into the mechanisms of oxidation DNA caused during neonatal asphyxia and resuscitation.