Ma Bin, Jing Meng, Villalta Peter W, Kapphahn Rebecca J, Montezuma Sandra R, Ferrington Deborah A, Stepanov Irina
Masonic Cancer Center, University of Minnesota, Mayo Mail Code 806, 420 Delaware Street SE, Minneapolis, Minnesota 55455, United States.
Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Mayo Mail Code 493, 420 Delaware Street SE, Minneapolis, Minnesota 55455, United States.
Sci Rep. 2016 Mar 16;6:22375. doi: 10.1038/srep22375.
Age-related macular degeneration (AMD) is the leading cause of blindness among older adults in the developed world. Oxidative damage to mitochondrial DNA (mtDNA) in the retinal pigment epithelium (RPE) may play a key role in AMD. Measurement of oxidative DNA lesions such as 8-oxo-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-2'-deoxyadenosine (8-oxo-dA) in diseased RPE could provide important insights into the mechanism of AMD development. We have developed a liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry method for simultaneous analysis of 8-oxo-dG and 8-oxo-dA in human retinal DNA. The developed method was applied to the analysis of retinal DNA from 5 donors with AMD and 5 control donors without AMD. In mtDNA, the levels of 8-oxo-dG in controls and AMD donors averaged 170 and 188, and 8-oxo-dA averaged 11 and 17 adducts per 10(6) bases, respectively. In nuclear DNA, the levels of 8-oxo-dG in controls and AMD donors averaged 0.54 and 0.96, and 8-oxo-dA averaged 0.04 and 0.05 adducts per 10(6) bases, respectively. This highly sensitive method allows for the measurement of both adducts in very small amounts of DNA and can be used in future studies investigating the pathophysiological role of 8-oxo-dG and 8-oxo-dA in AMD and other oxidative damage-related diseases in humans.
年龄相关性黄斑变性（AMD）是发达国家老年人失明的主要原因。视网膜色素上皮（RPE）中线粒体DNA（mtDNA）的氧化损伤可能在AMD中起关键作用。测量病变RPE中氧化DNA损伤，如8-氧代-2'-脱氧鸟苷（8-氧代-dG）和8-氧代-2'-脱氧腺苷（8-氧代-dA），可为AMD发展机制提供重要见解。我们开发了一种液相色谱-纳升电喷雾电离-串联质谱法，用于同时分析人视网膜DNA中的8-氧代-dG和8-氧代-dA。所开发的方法应用于分析5名AMD供体和5名无AMD对照供体的视网膜DNA。在mtDNA中，对照和AMD供体中8-氧代-dG的水平平均分别为每10⁶个碱基170和188个加合物，8-氧代-dA平均分别为11和17个加合物。在核DNA中，对照和AMD供体中8-氧代-dG的水平平均分别为每10⁶个碱基0.54和0.96个加合物，8-氧代-dA平均分别为0.04和0.05个加合物。这种高灵敏度方法能够测量极少量DNA中的两种加合物，可用于未来研究8-氧代-dG和8-氧代-dA在人类AMD及其他氧化损伤相关疾病中的病理生理作用。
