title: A novel HPLC procedure for the analysis of 8-oxoguanine in DNA.
abstract: The chromatographic quantitation of 8-oxoguanine adducts in DNA is widespread in the literature, although results obtained by HPLC of 8-oxodeoxyguanosine do not always agree with levels determined by GC-MS. To help explain this discrepancy, here we describe a novel procedure for the analysis of 8-oxoguanine adducts in DNA. Although it proved difficult to directly quantitate 8-oxoguanine in the presence of high levels of endogenous guanine using conventional reversed-phase HPLC, a simple preincubation of DNA acid hydrolysates with guanase allowed such analyses. The assay relied on our observation that 8-oxoguanine was not a substrate for guanase, and on sensitive electrochemical detection. The limit of detection for 8-oxoguanine was 5 nM or 250 fmol on column. Using this procedure, the background level of 8-oxoguanine in commercially available calf thymus DNA was 0.4 nmol/mg DNA or 3.2 mol/10(5) mol guanine.
authors.affiliation: Division of Chemical Pathology, University of Leicester, UK.
authors.forename: K E
authors.initials: KE
authors.lastname: Herbert
authors.forename: M D
authors.initials: MD
authors.lastname: Evans
authors.forename: M T
authors.initials: MT
authors.lastname: Finnegan
authors.forename: S
authors.initials: S
authors.lastname: Farooq
authors.forename: N
authors.initials: N
authors.lastname: Mistry
authors.forename: I D
authors.initials: ID
authors.lastname: Podmore
authors.forename: P
authors.initials: P
authors.lastname: Farmer
authors.forename: J
authors.initials: J
authors.lastname: Lunec
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004247:DNA / Q000737:chemistry* / Q000528:radiation effects; D018736:DNA Adducts; D004563:Electrochemistry / Q000379:methods; D005720:Gamma Rays; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis; D006148:Guanine Deaminase; D007202:Indicators and Reagents; D008099:Liver / Q000201:enzymology; D011817:Rabbits; D012680:Sensitivity and Specificity; D014527:Uric Acid / Q000032:analysis; D019820:Xanthine; D014970:Xanthines / Q000032:analysis
chemical_list: D018736:DNA Adducts; D007202:Indicators and Reagents; D014970:Xanthines; D019820:Xanthine; D014527:Uric Acid; C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; D006148:Guanine Deaminase