32P-postlabeling high-performance liquid chromatography (32P-HPLC) adapted for analysis of 8-hydroxy-2'-deoxyguanosine.
8-Hydroxy-2'-deoxyguanosine (8-OH-dG) is a promutagenic lesion in DNA caused by reactive oxygen species. It normally exists at a level of 0.1-1 per 10(5) 2'-deoxyguanosines (dG). To analyze the lesion in easily obtainable biological samples, a very sensitive analytical method is required. The method should also handle the problem with potential oxidation of dG to 8-OH-dG during workup and analysis. 32P-postlabeling high-performance liquid chromatography (32P-HPLC) is an analytical method previously used to analyze lipophilic DNA adducts at levels as low as 1 per 10(9) normal nucleotides when analyzing microgram amounts of DNA. This method was adapted for analysis of 8-OH-dG. The aim was to develop an analytical method that provided a high sensitivity and good reproducibility, prevented oxidation of dG present in samples to 8-OH-dG, was capable of analyzing DNA from very small samples and still offered high sample throughput and ease of use. In analysis of calf thymus DNA, the method had a detection limit of 0.1 8-OH-dG per 10(5) dG when 1 microgram of DNA was used. The standard deviation of repeated analyses of the same sample was +/-10% and the result corresponded well with the established analytical method using HPLC with electrochemical detection. 32P-HPLC is sensitive enough to enable analysis of low levels of 8-OH-dG in biological samples such as small volumes of blood, needle biopsies and tissue swabs. It also substantially reduces oxidation of dG to 8-OH-dG during sample workup and analysis.
Zeisig
M
M
Karolinska Institutet, Department of Biosciences at Novum, Unit for Analytical Toxicology, SE-141 57 Huddinge, Stockholm, Sweden.
Hofer
T
T
Cadet
J
J
Möller
L
L
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D002021:Buffers; D002417:Cattle; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000737:chemistry; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008214:Lymphocytes / Q000737:chemistry; D010084:Oxidation-Reduction; D010761:Phosphorus Radioisotopes; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D002021:Buffers; D010761:Phosphorus Radioisotopes; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D003849:Deoxyguanosine