Dizdaroglu M
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Free Radic Res. 1998 Dec;29(6):551-63. doi: 10.1080/10715769800300591.
Recently, several papers reported an artifactual formation of a number of modified bases from intact DNA bases during derivatization of DNA hydrolysates to be analyzed by gas chromatography-mass spectrometry (GC/MS). These reports dealt with 8-hydroxyguanine (8-OH-Gua), 5-hydroxycytosine (5-OH-Cyt), 8-hydroxyadenine (8-OH-Ade), 5-hydroxymethyluracil (5-OHMeUra) and 5-formyluracil that represent only a small percentage of the 20 or so modified DNA bases that can be analyzed by GC/MS. Removal of intact DNA bases by prepurification of calf thymus DNA hydrolysates using HPLC was shown to prevent artifactual formation of these modified bases during derivatization. It needs to be emphasized that the procedures for hydrolysis of DNA and derivatization of DNA hydrolysates used in these papers substantially differed from the established procedures previously described. Furthermore, a large number of relevant papers reporting the levels of these modified bases in DNA of various sources have been ignored. Interestingly, the levels of modified bases reported in the literature were not as high as those reported prior to prepurification. Most values for the level of 5-OH-Cyt were even lower than the level measured after prepurification. Levels of 8-OH-Ade were quite close to, or even the same as, or smaller than the level reported after prepurification. The same holds true for 5-OHMeUra and 8-OH-Gua. All these facts raise the question of the validity of the claims about the measurement of these modified DNA bases by GC/MS. A recent paper reported a complete destruction of 2, 6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-Gua) and 4,6-diamino-5-formamidopyrimidine (FapyAde) by formic acid under the conditions of DNA hydrolysis prior to GC/MS. The complete destruction of FapyGua and FapyAde by formic acid is in disagreement with the data on these compounds in the literature. These two compounds were measured by GC/MS following formic acid hydrolysis for many years in our laboratory and by other researchers with no difficulties. These facts clearly raise the question of the validity of the claims made about the previous measurements of these compounds by GC/MS.
最近，几篇论文报道了在对用于气相色谱-质谱联用（GC/MS）分析的DNA水解产物进行衍生化过程中，从完整的DNA碱基人工形成了一些修饰碱基。这些报道涉及8-羟基鸟嘌呤（8-OH-Gua）、5-羟基胞嘧啶（5-OH-Cyt）、8-羟基腺嘌呤（8-OH-Ade）、5-羟甲基尿嘧啶（5-OHMeUra）和5-甲酰基尿嘧啶，而它们在可通过GC/MS分析的约20种修饰DNA碱基中仅占一小部分。使用高效液相色谱（HPLC）对小牛胸腺DNA水解产物进行预纯化以去除完整的DNA碱基，结果表明这可防止在衍生化过程中这些修饰碱基的人工形成。需要强调的是，这些论文中使用的DNA水解和DNA水解产物衍生化程序与先前描述的既定程序有很大不同。此外，大量报道各种来源DNA中这些修饰碱基水平的相关论文被忽视了。有趣的是，文献中报道的修饰碱基水平并不像预纯化之前报道的那么高。5-OH-Cyt的大多数值甚至低于预纯化后测得的水平。8-OH-Ade的水平与预纯化后报道的水平相当接近，甚至相同，或者更低。5-OHMeUra和8-OH-Gua也是如此。所有这些事实都引发了关于通过GC/MS测量这些修饰DNA碱基说法的有效性问题。最近一篇论文报道，在GC/MS分析之前的DNA水解条件下，甲酸会使2,6-二氨基-4-羟基-5-甲酰胺基嘧啶（Fapy-Gua）和4,6-二氨基-5-甲酰胺基嘧啶（FapyAde）完全分解。甲酸使FapyGua和FapyAde完全分解这一情况与文献中关于这些化合物的数据不一致。多年来，在我们实验室以及其他研究人员的实验室中，在甲酸水解后通过GC/MS对这两种化合物进行测量都没有困难。这些事实清楚地引发了关于先前通过GC/MS测量这些化合物说法的有效性问题。
