Non-invasive Assessment of Oxidatively Damaged DNA: Liquid Chromatography-Tandem Mass Spectrometry Analysis of Urinary 8-Oxo-7,8-Dihydro-2′-Deoxyguanosine
The ability to non-invasively assess DNA oxidation and its repair, has significant utility in large-scale, population-based studies. Such studies could include the assessments of: the efficacy of antioxidant intervention strategies, pathological roles of DNA oxidation in various disease states and population or interindividual differences in antioxidant defence and DNA repair. The most popular method, to non-invasively assess oxidative insult to the genome is by the analysis of urine for 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), using chromatographic techniques or immunoassay procedures. The provenance of extracellular 8-oxodG remains a subject for debate. However, previous studies have shown that factors, such as diet and cell death, do not appear to contribute to extracellular 8-oxodG, leaving processes, such as the repair of DNA and/or the 2′-deoxyribonucleotide pool, as the sole source of endogenous 8-oxodG. The method in this chapter describes a non-invasive approach for assessing oxidative stress, via the efficient extraction of urinary 8-oxodG using a validated solid-phase extraction procedure. Subsequent analysis by liquid chromatography-tandem mass spectrometry provides the advantages of sensitivity, internal standardisation, and robust peak identification, and is widely considered to be the “gold standard”.
Key wordsOxidative stress DNA damage DNA repair Chromatography Solid phase extraction Urine Liquid chromatography Mass spectrometry
RS and PBF are supported by the U.K. Medical Research Council. Some development of this methodology in the laboratory of MDE and MSC was supported by using a University of Leicester miscellaneous income fund held by MSC. RS, MDE, PBF, and MSC are partners of ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a network of excellence operating within the European Union 6th Framework Program, Priority 5: “Food Quality and Safety” (Contract No. 513943). JKS is supported by (ECNIS, Contract No. FOOD-CT-2005-513943); FT is supported by Cancer Research UK (CRUK Programme Grant C325/A6691) and Experimental Cancer Medicine Centre Network (ECMC Grant C325/A7241).
The formation of a European-centred laboratory consortium, the European Standards Committee on Urinary (DNA) Lesion Analysis (ESCULA) is enabling an examination of methodology and issues surrounding the analysis of urinary 8-oxodG in the first instance. Further information about this consortium, including activities and membership can be obtained by contacting Dr. M.S. Cooke (firstname.lastname@example.org; http://escula.org).
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