The simultaneous analysis of 8-OHdG and 8-oxoG using HPLC with EC and MS detection
Principal Researcher: Michele Kelly
Oxidative DNA damage has been associated with Chronic Inflammation, Cancer, Neurodegeneration, and Heart Disease, as well as playing a central role in the Free Radical Theory of Aging. Diet, lifestyle and environmental conditions can alter the amount of oxidative stress that a body undergoes. The main causes of oxidative DNA damage are irradiation, chemical reactions, and oxidation by reactive oxygen species (ROS) including hydroxyl radicals. ROS are readily available in the body from respiratory and metabolic reactions occurring constantly. DNA is subject to thousands of oxidative attacks per day. The body has a series of repair and preventative mechanisms to defend against oxidative DNA damage, in the form of enzymes, radical scavengers and chain breaking chemicals. The effectiveness of these mechanisms is, however, limited. 8-oxoguanine and 8-hydroxy-2’-deoxyguanosine are the two most investigated DNA oxidation products. Guanine and 2-‘deoxyguanosine have the lowest oxidation potentials of the DNA bases and nucleosides respectively, making them easiest to oxidize. By identifying the nature and quantity of these oxidized DNA base adducts, the underlying mechanisms that generate oxidative DNA damage can be elucidated, the first step towards combating such damage.
This project investigates and monitors the formation of guanine and 2-‘deoxyguanosine oxidation products, 8-oxoguanine and 8-hydroxy-2’-deoxyguanosine by oxidation via the Fenton reaction over specific incubation periods. The Fenton reaction is the transition metal catalysed degradation of hydrogen peroxide, resulting in the formation of the hydroxyl radical, a ROS. The formation of the products over time is monitored by HPLC-EC and HPLC-MS/MS. HPLC-MS/MS also allows for the detection of products of further oxidation of 8-oxoguanine and 8-hydroxy-2’-deoxyguanosine The primary oxidation products also have low oxidation potentials and are therefore themselves easy to oxidise.
Currently, most research is based on either 8-oxoguanine or 8-hydroxy-2’deoxyguanosine, and not of both products simultaneously. The current research in this project involves the development of a HPLC-EC method incorporating monolithic columns to detect the formation of both simultaneously in samples. The research will allow for the elucidation of the mechanisms of formation of oxidative DNA damage lesions and the part that they play in diseases such as Chronic Inflammation, Cancer, Neurodegeneration, and Heart Disease.
Posters:
Electrochemical detection of 8-oxoG and 8OHdG in micodialysis samples.pdf
Electrochemical detection of copper induced oxidative DNA damage.pdf
Generation of 8-oxoguanine by carcinogenic metal nickel.pdf
Investigation of the nature and extentof oxidative DNA damage.pdf
