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Improvements in the analytical method for 8-hydroxydeoxyguanosine in nuclear DNA
Karolinska Institute, Center for Nutrition and Toxicology NOVUM, S-141 57 Huddinge, Sweden
2To whom correspondence should be addressed
Modifications at two points in the sequence of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) analysis have contributed to a more accurate and simplified determination of 8-OH-dG in DNA. The first was an improvement in the detection limit for 8-OH-dG in high-performance liquid chromatography analysis and the second was a pronase digestion and ethanol precipitation method (pronase/ethanol method) for DNA isolation which could minimize artificial formation of 8-OH-dG. Since the changes in background current from electrochemical detection are regularly periodical, it was possible to reduce this background change by connecting a pressure damper, degassing the eluent before use and finally subtracting its theoretical function. After this background correction, the detection limit for 8-OH-dG was improved one order of magnitude, from 20 fmol (5.68 pg) to 1.76 fmol (0.5 pg). Therefore, 0.005 8-OH-dG/105 dG can be detected from 50 µg DNA. This improvement will allow the analysis of small samples, tissues from needle biopsies, <5 ml whole blood, etc., and will contribute to the accuracy of 8-OH-dG measurements. The pronasd ethanol method resulted in lower levels of 8-OH-dG than the phenol method in analyses of both rat liver and calf thymus DNA, even after 6 h incubation at 45°C. The level obtained by the pronase/ethanol method with butylated hydroxytoluene was approximately equal to or lower than the 8-OH-dG levels reported in normal rat liver. The pronase/ethanol method for DNA isolation can replace the phenol or other methods in 8-OH-dG analysis. This method also omits the use of highly toxic organic solvents.
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