Optimization of the workup procedure for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine with electrochemical detection.

Abstract

The artifactual generation of the biomarker for oxidative stress, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), during the workup procedure for its analysis is a difficult problem to solve, and the responsible factors are unclear. Here, peroxide removal and other antioxidant procedures during workup were compared using a limited amount of rat liver (50 mg) as starting material, with subsequent hydrolysis of 50 microg of DNA. A cold (0 degrees C) high salt GTC (4 M guanidine thiocyanate) nonphenol DNA extraction method was developed where DNA is quickly isolated. GSH (reduced glutathione) generated artifactual formation of 8-oxodG during the workup procedure, whereas H(2)O(2) removal using catalase, Fe(3+) removal and passivation using desferal, peroxide removal using glutathione peroxidase, ebselen and a peroxidase mimic lowered the 8-oxodG levels, all identifying peroxides as the responsible oxidants. Desferal was more protective when excluding Mg(2+) and Ca(2+) from buffers but was found to disturb the electrochemical detector when repeatedly injected five to six times, even at 100 microM. Addition of the OH(*) scavenger ethanol in all steps at 2% v/v had no protective effect. Zn(2+) was found necessary for efficient DNA hydrolysis using nuclease P(1), which was poor below 37 degrees C. Use of water substitutes was tested but inhibited DNA hydrolysis completely. H(2)(18)O could, however, work for mass spectrometry methods. Long-term (38 days) storage of 0.5% v/v Triton X-100 generated more 8-oxodG than Tween 20 when incubated with free dG. The cold GTC DNA extraction method was used for analysis of freshly isolated human lymphocytes/monocytes from 60 healthy men using catalase and TEMPO as antioxidants, giving a background level of 0.074 +/- 0.027 8-oxodG/10(5) dG (or 16 8-oxodG/10(8) nucleotides or 1943 8-oxodG/nuclei) which is probably the lowest value obtained yet. No increase with age was seen. Oxidation of dG to 8-oxodG during workup was found to fit a mathematically defined curve, and a calculated background level of 0.047 8-oxodG/10(5) dG was obtained. To obtain more reliable results it is recommended that control samples are included during the workup procedure, having an equal amount of cells (or DNA) as the exposed samples.