Comparison of Different Methods 8-oxoguanine as a Marker of Oxidative DNA Damage of Measuring

ESCODD (European Standards Committee on Oxidative DNA Damage)* Accepted by Prof. B. Halliwell (Received 10 August 1999; In revised form 4 September 1999) We are attempting to resolve some of the problems encountered in measuring 8-hydroxy-2'-deoxyguano- sine (8-oxodG) in human cellular DNA as a marker of oxidative stress. Samples of authentic 8-oxodG were distributed, and participating laboratories undertook to analyse this material within a specified period. Most HPLC procedures gave values for 8-oxodG within :t:40% of the target, as did two of four GC-MS pro- cedures, and both LC-MS-MS methods. Calf thymus DNA samples containing increasing amounts of 8- oxodG were also distributed for analysis. Fewer than half the procedures tested were able to detect the dose response; those that were successful tended to be pro- cedures with low coefficients of variation. For the analysis of 8-oxodG in human cells, where it is likely to be present at much lower concentrations than in the calf thymus DNA, it is crucial to reduce analytical vari- ation to a minimum; a coefficient of variation of less than 10% should be the aim, to give reasonable pre- cision. HPLC with amperometric electrochemical detection is not recommended, as it is less sensitive than coulometric detection. Immunological detection, 32p-postlabelling and LC-MS-MS are alternative approaches to measurement of 8-oxodG in DNA that, on the grounds of precision ~ and detection of dose response, cannot at present be recommended. Keywords: Oxidative DNA damage, 8-hydroxy-2'- deoxyguanosine, HPLC, GC-MS, methods validation INTRODUCTION Measurement of oxidative DNA damage in human cells is crucial to an understanding of the consequences of oxidative stress in health and disease, and of the influence of dietary antioxi- dants. 8-Hydroxy-2'-deoxyguanosine (8-oxodG), or the corresponding base 8-oxoguanine (8-oxo- gua), are most commonly measured as a marker of oxidative damage, but estimates of the levels of damage in normal human cells by GC-MS and HPLC range over several orders of magnitude. [11 The European Standards Committee for Oxida- tive DNA Damage (ESCODD) was set up in 1997 to resolve methodological problems and to reach agreement on the basal level of oxidative damage in human cells. In phase 1, laboratories participat- ing in ESCODD received samples of calf thymus DNA and liver tissue, as well as standard 8-oxodG and 8-oxodG-containing oligonucleotides for analysis. Results confirmed the existence of wide variations between methods and indicated that even the determination of standard 8-oxodG * Address for Correspondence: Dr. Andrew Collins, DNA Instability Group, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Scotland, UK. Tel.: +44(0)1224 716634. Fax: +44(0)1224 716629. E-mail: a.collins@rri.sari.ac.uk. 333