Comprehensive analysis of the formation and stability of peroxynitrite-derived 8-nitroguanine by LC-MS/MS: Strategy for the quantitative analysis of cellular 8-nitroguanine
| Autor: | Jian-Lian Chen, Chiung-Wen Hu, Yu-Wen Hsu, Yuan-Jhe Chang, Mu-Rong Chao, Tsu-Shing Wang |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Guanine Lysis DNA damage CHO Cells Biochemistry Cell Line 03 medical and health sciences chemistry.chemical_compound Cricetulus 0302 clinical medicine Limit of Detection Tandem Mass Spectrometry Peroxynitrous Acid Physiology (medical) Animals Humans Chromatography High Pressure Liquid Observer Variation Endothelial Cells Reproducibility of Results RNA DNA Hydrogen-Ion Concentration DNA extraction Kinetics 030104 developmental biology chemistry 030220 oncology & carcinogenesis Nucleic acid Depurination Cattle Peroxynitrite Half-Life |
| Zdroj: | Free Radical Biology and Medicine. 101:348-355 |
| ISSN: | 0891-5849 |
| Popis: | Peroxynitrite is a major oxidizing and nitrating biological agent formed at sites of inflammation. Peroxynitrite can cause DNA damage and is thought to contribute to inflammation-related carcinogenesis. This study describes a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the direct determination of peroxynitrite-derived 8-nitroguanine (8-nitroGua) in DNA hydrolysates. This method exhibited a sensitive detection limit of 3 fmol and inter- and intraday imprecision of10% and was applied to systemically examine the formation and stability of peroxynitrite-derived 8-nitroGua in different DNA substrates under various conditions. The 8-nitroGua formation was maximal at pH 8. The formation rate of 8-nitroGua in different DNA substrates decreased in the order of monodeoxynucleosidesingle-stranded DNAdouble-stranded DNA. A stability test revealed that the half-life for the depurination of 8-nitroGua from DNA was short and affected by both the temperature and DNA structure. When present in monodeoxynucleoside, the half-life of 8-nitroGua was estimated to be ~6min at 25°C and 2.3h at ~0°C. In single-stranded DNA, the half-life varied from 1.6h at 37°C to 533h at -20°C, whereas the half-life increased from 2.4h at 37°C to 1115h at -20°C in double-stranded DNA. We demonstrated that the measurement of 8-nitroGua in isolated DNA is not practicable because 8-nitroGua is unstable and lost during DNA extraction from cell. Therefore, we suggest that directly detecting cellular 8-nitroGua following nuclear membrane lysis is an alternative measure of the nitrative damage of nucleic acids, accounting for both DNA and RNA lesions within cells. |
| Databáze: | OpenAIRE |
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