An Improved 32P-Postlabeling/High-Performance Liquid Chromatography Method for the Analysis of the Malondialdehye-Derived 1,N2-Propanodeoxyguanosine DNA Adduct in Animal and Human Tissues

Abstract

Malondialdehyde (MDA) is a major lipid peroxidation product that is mutagenic and tumorigenic. The MDA-modified DNA adduct, 3-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1, 2-alpha]purin-10(3H)-one (M1G), has been detected in human tissues and may be a marker of human cancer risk. In this paper, we describe an improved 32P-postlabeling/HPLC method for sensitive detection and quantitation of this MDA-modified 2'-deoxyribonucleotide adduct. Specific improvements include (i) unequivocal structural identification of the postlabeling products, both the 3', 5'-bisphosphate of M1G (MDA-3',5'-dGDP) and the 5'-monophosphate of M1G (MDA-5'-dGMP); (ii) efficient separation of the 32P-postlabeling products by HPLC; and (iii) the incorporation of a synthetically prepared MDA-modified DNA (or the 3'-monophosphate of M1G) with a known modification level as an internal standard. This improved quantitative methodology provides high intra- and inter-assay reproducibility and has been applied to the analysis of this adduct in rodent and human samples.