Chemical characterization and quantitative analysis of oxidative DNA damage, induced by free hydroxyl radicals, in tumour tissue by chromatographic and mass spectrometric techniques.

摘要

Throughout the last two-tree decades, many analytical methods for the measurement of oxidative DNA base damage have been developed. Besides the GC-MS technique, which is reported in a large number of articles, a view other possible techniques were explored for the monitoring of oxidative DNA damage, i.e. capillary electrophoresis with UV- or MS-detection and HPLC-FLUO.;The SIM-GC-MS technique provides not only low LOD, with the LLOQ approximately 1 ng/ml for each of the four modified bases, but also high selectivity by virtue of monitoring a few selected ions that are characteristic of only one analyte in a complex mixture. This unique characteristic of mass spectrometry (SIM, qualifier/quantifier ion monitoring), combined with the precise measurement of retention times by capillary gas chromatography makes unequivocal identification of these compounds possible. Furthermore, the assay exhibits excellent accuracy and reproducibility. Currently, the SIM-GC-MS technique remains the option of choice for the measurement of oxidized DNA bases in tumour DNA, with or without labeled internal standards, but more recently the LC-MS/(MS) technique is becoming increasingly important.;The possibility of capillary electrophoresis for the analysis of 8-OH-A and 8-OH-G in urine and DNA is investigated. Separation of 8-OH-A and 8-OH-G from the unmodified DNA bases is possible by both CZE and MEKC. However, the analysis of 8-OH-A and 8-OH-G in a DNA hydrolysis mixture, which is described with GC-MS, is not feasible because the sample is not "clean" enough. 8-OH-A and 8-OH-G are not resolved from the sample matrix. The same problem is encountered in the analysis of 8-OH-A and 8-OH-G in urine. Isolation of 8-OH-A and 8-OH-G from the sample matrix (DNA hydrolysate and urine) by RP-HPLC is the solution to this problem.;A few applications are reported in which the monitoring of oxidative DNA damage plays an useful tool in fundamental cancer research.;Further optimization and validation of the various chromatographic and enzymatic assays will require extensive and specific inter-laboratory trials (ring tests). This could be achieved through the ESCODD group, or direct cooperation between two or several groups.