Establishing the background level of base oxidation in human lymphocyte DNA: results of an interlaboratory validation study
Establishing the background level of base oxidation in human lymphocyte DNA: results of an interlaboratory validation study
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DOI：
10.1096/fj.04-1767fje
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发表时间：
2005-01-01
期刊：
影响因子：
4.8
通讯作者：
Vaughan, N
中科院分区：
文献类型：
--
作者：
Gedik, CM;Collins, A;Vaughan, N
DNA中低水平8-氧代-7，8-二氢-2 '-脱氧鸟苷（8-oxodGuo）的精确测量受到鸟嘌呤在制备用于分析的DNA期间容易被氧化的阻碍。ESCODD是一个主要由欧洲实验室组成的联盟，它试图将这种人为因素减少到最低限度，并为样品制备和分析提供标准、可靠的协议。ESCODD现在已经分析了从几个欧洲国家健康年轻男性志愿者静脉血中分离的淋巴细胞DNA中的8-oxodGuo。采用了两种办法。在8个国家的10组志愿者的淋巴细胞中，通过HPLC电化学检测对8-oxodGuo进行分析。另一种酶法是基于用甲酰胺嘧啶DNA糖基化酶（FPG）消化DNA，将8-氧代-7，8-二氢鸟嘌呤（8-oxoGua）转化为脱嘌呤位点，随后使用彗星试验测量DNA断裂（6个国家的7组志愿者）。淋巴细胞DNA中8-oxodGuo的中位浓度（根据HPLC测定的各组受试者的平均值计算）为4.24/10（6）鸟嘌呤。用彗星试验测量的FPG敏感位点的中位浓度为0.34/10（6）鸟嘌呤。向所有参与者提供相同的HeLa细胞样品作为参考标准。HeLa细胞中8-oxodGuo的中值为2.78/10（6）鸟嘌呤（通过HPLC）和0.50/10（6）鸟嘌呤（通过酶法）。色谱法和基于FPG的方法之间的差异可能反映了HPLC法的高估（如果假氧化仍未完全控制）或酶法的低估。同时，很明显，DNA中碱基氧化的真实背景水平比过去经常声称的要低几个数量级。
Accurate measurement of low levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in DNA is hampered by the ease with which guanine is oxidized during preparation of DNA for analysis. ESCODD, a consortium of mainly European laboratories, has attempted to minimize this artifact and to provide standard, reliable protocols for sample preparation and analysis. ESCODD has now analyzed 8-oxodGuo in the DNA of lymphocytes isolated from venous blood from healthy young male volunteers in several European countries. Two approaches were used. Analysis of 8-oxodGuo by HPLC with electrochemical detection was performed on lymphocytes from 10 groups of volunteers, in eight countries. The alternative enzymic approach was based on digestion of DNA with formamidopyrimidine DNA glycosylase (FPG) to convert 8-oxo-7,8-dihydroguanine (8-oxoGua) to apurinic sites, subsequently measured as DNA breaks using the comet assay (7 groups of volunteers, in six countries). The median concentration of 8-oxodGuo in lymphocyte DNA, calculated from the mean values of each group of subjects as determined by HPLC, was 4.24 per 10(6) guanines. The median concentration of FPG-sensitive sites, measured with the comet assay, was 0.34 per 10(6) guanines. Identical samples of HeLa cells were supplied to all participants as a reference standard. The median values for 8-oxodGuo in HeLa cells were 2.78 per 10(6) guanines (by HPLC) and 0.50 per 10(6) guanines (by enzymic methods). The discrepancy between chromatographic and FPG-based approaches may reflect overestimation by HPLC (if spurious oxidation is still not completely controlled) or underestimation by the enzymic method. Meanwhile, it is clear that the true background level of base oxidation in DNA is orders of magnitude lower than has often been claimed in the past.