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.
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We thank Xun Ming for his help with the mass spectrometry analysis; Adam Zarth for useful discussions in this study; the Minnesota Lions Eye Bank for procuring donor eyes; and Kathy Goode and Sung Lee for photographing and processing eye tissue. We also thank Robert Carlson for editorial assistance. This study was supported by startup funds to IS from the Masonic Cancer Center, University of Minnesota via NCI grant P30 CA077598 and support from Minnesota Masonic Charities; the University of Minnesota Academic Health Center – Faculty Development Award; Arnold and Mabel Beckman Initiative for Macular Research; an anonymous benefactor for AMD Research; and an unrestricted grant to the Department of Ophthalmology and Visual Neurosciences from the Research to Prevent Blindness. Mass-spectrometry analyses were carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, supported in part by grant CA-77598 from the NCI.