Part of the Methods in Pharmacology and Toxicology book series (MIPT)
Bioassay of 2′-Deoxyguanosine/8-Hydroxy-2′-Deoxyguanosine by HPLC With Electrochemical/Photodiode Array Detection
Living organisms exposed to reactive oxygen species (ROS) on a continual basis will promote oxidative stress, thereby forming mutations in DNA and damage to cells. As an end result, it has been shown that modified forms of damaged DNA can lead to mutagenesis, carcinogenesis, degenerative diseases, cancer, diabetes, and aging (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16). With this in mind, the DNA nucleoside 2′-deoxyguanosine (dG) undergoes a hydroxylation reaction at the C-8 position to yield a useful biomarker, 8-hydroxy-2′-deoxyguanosine (8-OH-dG) that has been employed to measure oxidative damage as illustrated in Fig. 1. Other disease/nondisease related events resulting in the formation of this oxidation product, 8-OH-dG, are reported by Möller et al. (5). There have been a wide variety of assays used in the detection of free radical-mediated DNA oxidation products such as: high performance liquid chromatography coupled with electrochemical and ultraviolet detection (HPLC-ECD and HPLC-UV) (2, 3, 4, 5, 6, 7, 8, 9,12,13,16, 17, 18, 19, 20, 21,24), liquid and gas chromatography-mass spectrometry (LC-MS and GC-MS) (4,10,12,22, 23, 24, 25, 26, 27), postlabeling techniques (32P-HPLC , 32P-TLC [thin-layer chromatography] [30, 31, 32, 33], or fluorescent probe-HPLC ), antibody assays (35, 36, 37), and lastly, HPLC using tandem mass spectrometry (HPLC-MS-MS) (1,23,25,26,28,29). R. A. Floyd, H. Kasai, and others (9,17,18,38,39) were some of the early pioneers for accurately measuring DNA oxidation adducts using HPLC in series with electrochemical, spectrophotometric, or fluoro-metric detection in order to determine the level of oxidative DNA damage in cells.
KeywordsMethylene Blue Continual Basis Early Pioneer Picomole Level Respective Retention Time
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