Lovell M A, Gabbita S P, Markesbery W R
Sanders-Brown Center on Aging, and Department of Chemistry, University of Kentucky, Lexington 40536-0230, USA.
J Neurochem. 1999 Feb;72(2):771-6. doi: 10.1046/j.1471-4159.1999.0720771.x.
One of the leading etiologic hypotheses regarding Alzheimer's disease (AD) is the involvement of free radical-mediated oxidative stress in neuronal degeneration. Although several recent studies show an increase in levels of brain DNA oxidation in both aging and AD, there have been no studies of levels of markers of DNA oxidation in ventricular CSF. This is a study of levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), the predominant marker of oxidative DNA damage, in intact DNA and as the "free" repair product that results from repair mechanisms. Free 8-OHdG was isolated from CSF from nine AD and five age-matched control subjects using solid-phase extraction columns and measured using gas chromatography/mass spectrometry with selective ion monitoring. Intact DNA was isolated from the same samples and the levels of 8-OHdG determined in the intact structures. Quantification of results was carried out using stable isotope-labeled 8-OHdG. By using this sensitive methodology, statistically significant elevations (p < 0.05) of 8-OHdG were observed in intact DNA in AD subjects compared with age-matched control subjects. In contrast, levels of free 8-OHdG, removed via repair mechanisms, were depleted significantly in AD samples (p < 0.05). Our results demonstrate an increase in unrepaired oxygen radical-mediated damage in AD DNA as evidenced by the increased presence of 8-OHdG in intact DNA and decreased concentrations of the free repair product. These data suggest that the brain in AD may be subject to the double insult of increased oxidative stress, as well as deficiencies in repair mechanisms responsible for removal of oxidized bases.
关于阿尔茨海默病（AD）的主要病因假说之一是自由基介导的氧化应激参与神经元变性。尽管最近的几项研究表明，衰老和AD患者脑内DNA氧化水平均有所升高，但尚未有关于脑室脑脊液中DNA氧化标志物水平的研究。本研究检测了完整DNA中8-羟基-2'-脱氧鸟苷（8-OHdG）的水平，8-OHdG是氧化性DNA损伤的主要标志物，同时也检测了作为修复机制产生的“游离”修复产物的8-OHdG水平。使用固相萃取柱从9例AD患者和5例年龄匹配的对照者的脑脊液中分离出游离8-OHdG，并采用气相色谱/质谱联用选择性离子监测法进行测定。从相同样本中分离出完整DNA，测定其中8-OHdG的水平。结果定量采用稳定同位素标记的8-OHdG。通过这种灵敏的方法，与年龄匹配的对照者相比，AD患者完整DNA中的8-OHdG水平有统计学意义的显著升高（p<0.05）。相反，通过修复机制去除的游离8-OHdG水平在AD样本中显著降低（p<0.05）。我们的结果表明，AD患者DNA中未修复的氧自由基介导的损伤增加，完整DNA中8-OHdG的存在增加以及游离修复产物浓度降低证明了这一点。这些数据表明，AD患者的大脑可能受到氧化应激增加以及负责去除氧化碱基的修复机制缺陷的双重损害。
