P-32-postlabeling high-performance liquid chromatography (P-32-HPLC) adapted for analysis of 8-hydroxy-2 '-deoxyguanosine

8-Hydroxy-2' -deosyguanosine (8-OH-dG) is a promutagenic lesion in DNA caus ed by reactive oxygen species. It normally exists at a level of 0.1-1 per 1 0(5) 2'-deoxyguanosines (dG), To analyze the lesion in easily obtainable bi ological samples, a very sensitive analytical method is required. The metho d should also handle the problem with potential oxidation of dG to 8-OH-dG during workup and analysis, P-32-postlabeling high-performance liquid chrom atography (P-32-HPLC) is an analytical method previously used to analyze li pophilic DNA adducts at levels as low as 1 per 10(9) normal nucleotides whe n 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 hig h sensitivity and good reproducibility, prevented oxidation of dG present i n 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 ca lf thymus DNA, the method had a detection limit of 0.1 8-OH-dG per 105 dG w hen 1 mu g of DNA was used. The standard deviation of repeated analyses of the same sample was +/-10% and the result corresponded well with the establ ished analytical method using HPLC with electrochemical detection. P-32-HPL C is sensitive enough to enable analysis of low levels of 8-OH-dG in biolog ical samples such as small volumes of blood, needle biopsies and tissue swa bs. It also substantially reduces oxidation of dG to 8-OH-dG during sample workup and analysis.