Abstract
Oxidative stress can disrupt the integrity of genetic material. Due to its importance in the pathogenesis of different kinds of disease, including neurodegenerative disease, cardiovascular disease and cancer, major efforts are put into the elucidation of mechanisms involved. Herein, the combination of electrochemistry/liquid chromatography/mass spectrometry (EC/LC/MS) is presented as convenient, fast and simple method to study nucleic acids oxidation. Guanosine was selected as test compound. 8-Hydroxyguanosine and (guanosine-H)2 were identified as primary oxidation products. Oxidation was accomplished in an electrochemical thin-layer cell integrated in the flow path of the autosampler of the chromatographic system. The reaction mixture was separated and mass analyzed by LC/MS. The use of LC was found to be particularly beneficial to resolve isobaric oxidation products. Another advantage of the setup used was the ability to decouple the electrochemical cell and the electrospray ionization source from each other eliminating any kind of cell potential interaction. Separation of EC from LC/MS, furthermore, facilitates method optimization. Experimental parameters were optimized for both techniques independently. Highest yields and best detectability of oxidation products were obtained with 10 mM ammonium formate at physiological pH delivered at a flow rate of 2.5-5 μL/min through the electrochemical cell.
Highlights
DNA damage has emerged as a major culprit in cancer and many diseases related to aging [1]
Guanosine was selected as test compound. 8-Hydroxyguanosine and2 were identified as primary oxidation products
EC/liquid chromatography (LC)/mass spectrometry (MS) was used for studying guanosine oxidation. 8hydroxyguanosine and2 were identified as primary oxidation products
Summary
DNA damage has emerged as a major culprit in cancer and many diseases related to aging [1]. DNA can be damaged by (i) spontaneous reactions, mostly hydrolysis; (ii) products of metabolism, such as reactive oxygen species (ROS) or reactive nitrogen species; and (iii) exogenous physical and chemical agents, such as ultraviolet light, ionizing radiation, toxins, pharmaceuticals, or pollutants. Produced lesions include base and sugar damages, strand brakes, crosslinks with proteins as well as the formation of bulky adducts. Endogenous and exogenous sources give rise to the formation of ROS (id .O2−, H2O2, .OH). ROS are constantly generated during oxidative respiration in mitochondria as a Abbreviations: EC, electrochemistry; ROS, reactive oxygen species consequence of ionizing radiation as well as exposure to transition metals, chemicals and pharmaceutical compounds. ROS are toxic giving rise to DNA damage. More than 100 different nucleosides resulting from oxidative stress have been isolated and characterized, which clearly indicates the complexity and diversity of nucleic acid oxidation reactions [9,10,11]
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