Irradiated (15N)DNA as an internal standard for analysis of base-oxidized DNA constituents by isotope dilution mass spectrometry.
A simple and convenient procedure for the preparation of isotopically labeled DNA enriched in oxidized deoxynucleosides is described. 15N-Labeled DNA was isolated from Escherichia coli cells grown in an isotopically enriched medium, and the level of oxidative damage was increased by in vitro irradiation under oxygen. The resulting DNA was hydrolyzed and subsequently analyzed by GC/MS. Results indicated that the DNA was 99% 15N-enriched and that 1% of the total 2'-deoxyguanosine was converted into 8-hydroxy-2'-deoxyguanosine (8-OHdG). When applied to the analysis of 8-OHdG, [15N]DNA as internal standard gave a better reproducibility (CV, 7.9%; n = 5) as compared to the monomeric 8-[18O]hydroxy-2'-deoxyguanosine (CV, 16%; n = 4). Background levels of 8-OHdG in rat colon DNA determined with [15N]DNA and 8-18OHdG as internal standard were 26 +/- 11 and 15 +/- 7 8-OHdG per 10(6) deoxynucleosides, respectively.
Laboratory of Organic-Analytical Chemistry, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands.
A J
AJ
Teixeira
J C
JC
Klein
H A
HA
Heus
A P
AP
de Jong
D000818:Animals; D001483:Base Sequence; D003106:Colon / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D007554:Isotopes; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010084:Oxidation-Reduction; D051381:Rats; D012015:Reference Standards
D007554:Isotopes; D009587:Nitrogen Isotopes; D004247:DNA