Haque K, Cooper D P, Povey A C
Cancer Research Campaign Department of Carcinogenesis, Paterson Institute for Cancer Research, Manchester, UK.
Carcinogenesis. 1994 Nov;15(11):2485-90. doi: 10.1093/carcin/15.11.2485.
The 3' and 5'-monophosphates of O6-methyldeoxyguanosine and N7-methyldeoxyguanosine were chemically synthesized. Using these standards with deoxyguanosine-3'-monophosphate (dGp) as an internal standard, conditions were optimized to quantify O6-methyldeoxyguanosine-3'-monophosphate (O6-MedGp) and N7-methyldeoxyguanosine-3'-monophosphate (N7-MedGp) by 32P-postlabelling. Under optimal conditions, the labelling efficiencies of O6-MedGp and N7-MedGp were respectively approximately 100 and approximately 15%, with detection limits of approximately 1.1 and approximately 6.0 fmol respectively using 10 pmol dGp or 0.8 fmol of O6-MedGp if 2 pmol of dGp was used. The assay developed for O6-MedGp was then applied to the quantitation of [3H]-O6-MedGp and O6-MedGp isolated from DNA digests by immunoaffinity separation. The standard curve generated from the use of [3H]-O6-MedGp, thus isolated, was identical to that generated previously using the chemically synthesized O6-MedGp, indicating that no inhibitory factors co-eluted with the O6-MedGp. After passage through two immunocolumns, recovery of 4 and 40 fmol of O6-MedGp was approximately 30%. Four human stomach samples were analysed by combining this immunoaffinity purification with 32P-post-labelling: levels ranged from 0.21 to 0.86 mumol O6-MedGp/mol dG. Further DNA samples, isolated from the human colon, were fractionated by anion-exchange HPLC and the N7-MedGp and O6-MedGp containing fractions were purified by reverse-phase HPLC and immunoaffinity chromatography respectively. Adduct-containing fractions were dried and 32P-postlabelled. Whereas O6-MedGp was detected at levels between 0.3 and 3.4 mumol O6-MedGp/mol dG, no N7-MedGp was detected in these samples, probably due to depurination of N7-MedGp to N7-methylguanine or reduced assay sensitivity resulting from contaminating nucleotides and/or unidentified radioactivity eluting close to the N7-methyldeoxyguanosine-5'-monophosphate.
O6-甲基脱氧鸟苷和N7-甲基脱氧鸟苷的3'和5'-单磷酸酯已通过化学方法合成。以脱氧鸟苷-3'-单磷酸（dGp）作为内标，使用这些标准品对32P后标记法定量O6-甲基脱氧鸟苷-3'-单磷酸（O6-MedGp）和N7-甲基脱氧鸟苷-3'-单磷酸（N7-MedGp）的条件进行了优化。在最佳条件下，O6-MedGp和N7-MedGp的标记效率分别约为100%和约15%，若使用10 pmol dGp，检测限分别约为1.1 fmol和约6.0 fmol；若使用2 pmol dGp，则O6-MedGp的检测限为0.8 fmol。随后，将开发的用于检测O6-MedGp的方法应用于定量通过免疫亲和分离从DNA消化物中分离得到的[3H]-O6-MedGp和O6-MedGp。使用如此分离得到的[3H]-O6-MedGp生成的标准曲线与先前使用化学合成的O6-MedGp生成的标准曲线相同，这表明没有抑制因子与O6-MedGp共洗脱。通过两个免疫柱后，4 fmol和40 fmol的O6-MedGp的回收率约为30%。通过将这种免疫亲和纯化与32P后标记相结合，对四个人类胃样本进行了分析：O6-MedGp的水平范围为0.21至0.86 μmol O6-MedGp/mol dG。从人类结肠分离得到的进一步的DNA样本通过阴离子交换高效液相色谱进行分离，含有N7-MedGp和O6-MedGp的馏分分别通过反相高效液相色谱和免疫亲和色谱进行纯化。含有加合物的馏分干燥后进行32P后标记。虽然检测到O6-MedGp的水平在0.3至3.4 μmol O6-MedGp/mol dG之间，但在这些样本中未检测到N7-MedGp，这可能是由于N7-MedGp脱嘌呤生成N7-甲基鸟嘌呤，或者是由于污染的核苷酸和/或在接近N7-甲基脱氧鸟苷-5'-单磷酸处洗脱的未鉴定放射性导致检测灵敏度降低。
