DETECTION OF 8-OXOGUANINE IN CELLULAR DNA USING 2,6-DIAMINO-8-OXOPURINE AS AN INTERNAL STANDARD FOR HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION

The quantitative aspect of the electrochemical detection method to det ect 8-oxo-7,8-dihydroguanine (8-oxoGua) has been improved by using an internal standard. In addition, emphasis was placed on the reduction o f artifactual oxidation of DNA during isolation and hydrolysis. Nuclea r DNA was isolated from Pat organs and purified on an anion-exchange c olumn following treatment with proteinase K and RNase. DNA hydrolysis to nucleobases or nucleosides was performed using either formic acid t reatment or enzymatic digestion, respectively. The levels of either 8- oxoGua or 8-hydroxy-7,8-dihydro-2'-deoxyguanosine were comparable. For accurate quantification, 2,6-diamino-8-oxopurine [(NH2)(2)-OH-Pur], a dded prior to hydrolysis, was used as an internal standard for the hig h-performance liquid chromatography with electrochemical detection ass ay. The baseline level of 8-oxoGua in DNA of Sprague-Dawley rats was e stimated to be 2 to 5 8-oxoGua residues per 10(6) DNA bases, with slig ht differences depending on the tissue origin. In agreement with the r esults of previous observations, the level of the oxidized base in the kidney of animal treated with iron complexed to nitrilotriacetic acid (Fe-NTA) (15 mg/kg) was three- to fourfold higher than that of untrea ted rats or animals treated with a saline solution, while there was no change in 8-oxoGua levels in the liver and colon of these treated ani mals. (C) 1998 Academic Press.