Measurement of 8-Oxo-deoxyguanosine in Lymphocytes, Cultured Cells, and Tissue Samples by HPLC with Electrochemical Detection
8-Oxoguanine is one of the most studied base oxidation products found in DNA. It has potential biological significance, because if present in DNA that is replicating, it can lead to incorporation of adenine rather than cytosine in the daughter strand. Thus it is considered as a premutagenic lesion. It occurs as a result of attack by reactive oxygen species released during the inflammatory response, and in small but significant amounts during normal respiration. The hydroxyl (OH) radical (arising from H2O2 by the transition metal ion-catalyzed Fenton reaction within the nucleus) is most likely responsible for the formation of 8-oxoguanine. Analytical methods—gas chromatography with mass spectrometric detection (GC-MS) and high-performance liquid chromatography (HPLC) —were developed for quantitation of oxidized bases produced in experimental studies of radiation and chemical damage to DNA, and these methods were naturally also applied to the measurement of background levels of oxidized bases in cellular DNA (1). With GC-MS, very high levels of 8-oxoguanine have been reported, typically between 10 and 100 for every 105 normal guanines. It has recently been recognized that spurious oxidation of DNA readily occurs during isolation and hydrolysis of DNA, and derivatization of the bases for analysis. HPLC, normally applied to measurement of the nucleoside, 8-oxo-deoxyguanosine (8-oxo-dG), has generally given values below those obtained with GC-MS; but with HPLC, too, oxidation artefacts have been identified.
KeywordsHomogenization Buffer Ammonium Sulfate Solution RNase IIIA Daughter Strand Isocratic System