A novel HPLC procedure for the analysis of 8-oxoguanine in DNA.
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.
Herbert
K E
KE
Division of Chemical Pathology, University of Leicester, UK.
Evans
M D
MD
Finnegan
M T
MT
Farooq
S
S
Mistry
N
N
Podmore
I D
ID
Farmer
P
P
Lunec
J
J
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
D018736:DNA Adducts; D007202:Indicators and Reagents; D014970:Xanthines; D019820:Xanthine; D014527:Uric Acid; C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; D006148:Guanine Deaminase