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

doi:10.1016/j.freeradbiomed.2016.10.505

中山醫學大學機構典藏 CSMUIR > 醫學科技學院 > 生物醫學科學學系暨碩士班 > 期刊論文 > Item 310902500/18043

Please use this identifier to cite or link to this item: https://ir.csmu.edu.tw:8080/ir/handle/310902500/18043

Title:	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
Authors:	Hu, C.-W.;Chang, Y.-J.;Hsu, Y.-W.;Chen, J.-L.;Wang, T.-S.;Chao, M.-R.
Date:	2016
Issue Date:	2017-08-01T08:17:22Z (UTC)
ISSN:	08915849
Abstract:	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 of <10% 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 monodeoxynucleoside>single-stranded DNA>double-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 ~6 min at 25 °C and 2.3 h at ~0 °C. In single-stranded DNA, the half-life varied from 1.6 h at 37 °C to 533 h at −20 °C, whereas the half-life increased from 2.4 h at 37 °C to 1115 h 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. © 2016 Elsevier Inc.
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/18043 http://dx.doi.org/10.1016/j.freeradbiomed.2016.10.505
Relation:	Free Radical Biology and Medicine 101, 348-355
Appears in Collections:	[生物醫學科學學系暨碩士班] 期刊論文

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