Methodological considerations and factors affecting 8-hydroxy-2-deoxyguanosine analysis

Oxidative stress is related to a number of diseases due to the formation of reactive oxygen species (ROS). There are also several substances found in the occupational environment or as life style related situations that generates ROS. A stable biomarker for oxidative stress on DNA is 8-hydroxy-2'-deoxyguanosine (8-OH-dG). A potential problem in the work-up and analysis of 8-OH-dG is oxidation of dG with false high levels as a result of analysis. This paper summarizes and discusses some of the critical moments in terms of auto-oxidation. The removal of transition metals, low temperatures, absence of isotopes (or 2'-deoxyguanosine) and incubation times are all important factors. Removal of oxygen is complicated while the problem is reduced if a nitroxide (TEMPO) is added during work-up. Certain reducing agents and enzymes could be critical if added during work-up. The application of the 32p-HPLC method to analyze 8-OH-dG is discussed. The 32P-HPLC method is suitable for 8-OH-dG analysis and avoids several factors that oxidizes dG by removal of dG before addition of isotopes. Factors of crucial importance (columns, eluents, gradients and detection of 32p) for the analysis of 8-OH-dG are commented upon and certain recommendations are made to make it possible to apply the 32P-HPLC methodology for this type of analysis.