Jenner A, England T G, Aruoma O I, Halliwell B
International Antioxidant and Neurodegenerative Disease Research Centres, Pharmacology Group, University of London, King's College, Manresa Road, London. SW3 6LX, U.K.
Biochem J. 1998 Apr 15;331 ( Pt 2)(Pt 2):365-9. doi: 10.1042/bj3310365.
Analysis of oxidative damage to DNA bases by GC-MS enables identification of a range of base oxidation products, but requires a derivatization procedure. However, derivatization at high temperature in the presence of air can cause 'artifactual' oxidation of some undamaged bases, leading to an overestimation of their oxidation products, including 8-hydroxyguanine. Therefore derivatization conditions that could minimize this problem were investigated. Decreasing derivatization temperature to 23 degrees C lowered levels of 8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-(hydroxymethyl)uracil measured by GC-MS in hydrolysed calf thymus DNA. Addition of the reducing agent ethanethiol (5%, v/v) to DNA samples during trimethylsilylation at 90 degrees C also decreased levels of these four oxidized DNA bases as well as 5-hydroxyuracil. Removal of guanine from hydrolysed DNA samples by treatment with guanase, prior to derivatization, resulted in 8-hydroxyguanine levels (54-59 pmol/mg of DNA) that were significantly lower than samples not pretreated with guanase, independent of the derivatization conditions used. Only hydrolysed DNA samples that were derivatized at 23 degrees C in the presence of ethanethiol produced 8-hydroxyguanine levels (56+/-8 pmol/mg of DNA) that were as low as those of guanase-pretreated samples. Levels of other oxidized bases were similar to samples derivatized at 23 degrees C without ethanethiol, except for 5-hydroxycytosine and 5-hydroxyuracil, which were further decreased by ethanethiol. Levels of 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxycytosine measured in hydrolysed calf thymus DNA by the improved procedures described here were comparable with those reported previously by HPLC with electrochemical detection and by GC-MS with prepurification to remove undamaged base. We conclude that artifactual oxidation of DNA bases during derivatization can be prevented by decreasing the temperature to 23 degrees C, removing air from the derivatization reaction and adding ethanethiol.
通过气相色谱 - 质谱联用（GC - MS）分析DNA碱基的氧化损伤能够鉴定一系列碱基氧化产物，但需要进行衍生化处理。然而，在有空气存在的情况下高温衍生化会导致一些未受损碱基发生“人为”氧化，从而导致其氧化产物（包括8 - 羟基鸟嘌呤）被高估。因此，研究了能够将此问题最小化的衍生化条件。将衍生化温度降至23摄氏度可降低通过GC - MS在水解小牛胸腺DNA中测得的8 - 羟基鸟嘌呤、8 - 羟基腺嘌呤、5 - 羟基胞嘧啶和5 - （羟甲基）尿嘧啶的水平。在90摄氏度进行三甲基硅烷化期间向DNA样品中添加还原剂乙硫醇（5%，v/v）也降低了这四种氧化DNA碱基以及5 - 羟基尿嘧啶的水平。在衍生化之前，通过用鸟嘌呤酶处理水解的DNA样品来去除鸟嘌呤，导致8 - 羟基鸟嘌呤水平（54 - 59 pmol/mg DNA）显著低于未用鸟嘌呤酶预处理的样品，这与所用的衍生化条件无关。只有在乙硫醇存在下于23摄氏度衍生化的水解DNA样品产生的8 - 羟基鸟嘌呤水平（56±8 pmol/mg DNA）与用鸟嘌呤酶预处理的样品一样低。除了5 - 羟基胞嘧啶和5 - 羟基尿嘧啶被乙硫醇进一步降低外，其他氧化碱基的水平与在没有乙硫醇的情况下于23摄氏度衍生化的样品相似。通过本文所述的改进方法在水解小牛胸腺DNA中测得的8 - 羟基鸟嘌呤、8 - 羟基腺嘌呤和5 - 羟基胞嘧啶水平与先前通过高效液相色谱 - 电化学检测以及通过带有预纯化以去除未受损碱基的GC - MS报道的水平相当。我们得出结论，通过将温度降至23摄氏度、从衍生化反应中去除空气以及添加乙硫醇，可以防止衍生化过程中DNA碱基的人为氧化。
