Improvements in the analytical method for 8-hydroxydeoxyguanosine in nuclear DNA.
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/10(5) dG can be detected from 50 micrograms 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 pronase/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 degrees 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.
Karolinska Institute, Center for Nutrition and Toxicology, NOVUM, Huddinge, Sweden.
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Adachi
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D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D002138:Calibration; D002417:Cattle; D002467:Cell Nucleus / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D004355:Drug Stability; D004563:Electrochemistry; D005260:Female; D008297:Male; D010084:Oxidation-Reduction; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity
D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D003849:Deoxyguanosine