閱譜之視知覺處理歷程---事件誘發電位研究

中山醫學大學機構典藏 CSMUIR > 健康管理學院 > 公共衛生學系暨碩士班 > 研究計劃 > Item 310902500/3915

Please use this identifier to cite or link to this item: https://ir.csmu.edu.tw:8080/ir/handle/310902500/3915

Title:	閱譜之視知覺處理歷程---事件誘發電位研究 Development of Multiple Mass Spectrometric Biomarkers for Assessing Rns/Ros and Its Application in Human Lung Inflammation
Authors:	胡瓊文;施穎銘
Contributors:	中山醫學大學：公共衛生學系
Keywords:	連線固相萃取;液相層析串聯質譜儀;氧化壓力;硝化壓力;發炎 on-line SPE;LC-MS/MS;oxidative stress;nitrative stress;inflammation
Date:	2010
Issue Date:	2011-06-24T07:20:39Z (UTC)
Abstract:	體內的自由基主要分成?大?：活性含氧物質(Reactive oxygen species, ROS)及活性含氮物質 (reactive nitrogen species, RNS)，如超氧陰?子(O2 -)、氫氧自由基(OH)、過氧亞硝酸根(ONOO-)及硝?氯(NO2Cl)等；ROS 及RNS 間可交互生成。當體內ROS/RNS 生成?超過抗氧化機制時，則會造成體內的氧化壓?(oxidative stress)或硝化壓?(nitrative stress)。由於ROS/RNS 本身活性高，很容?攻擊生物體內的重要分子如核酸、蛋白質、脂肪等，這些損傷是造成?化、慢性病及多種癌症的重要原因。ROS/RNS 極?穩定(半衰期極短)，因此?測ROS/RNS 所形成的「基因、蛋白及脂質修飾產物」?相形重要，同時也能幫助探究ROS/RNS 引起疾病的相關機制。ROS 可造成DNA 氧化傷害，8-oxo-7,8-dihydro-2’-deoxyguanosin (8-oxodGuo)是主要的產物之ㄧ，其經鹼基移除修?的產物為8-oxo-7,8-dihydroguanine (8-oxoGua)，而經核酸?除修?則產物為8-oxodGuo。體內多元?飽和脂肪酸也?被ROS 氧化，造成脂質過氧化傷害，丙二醛(malondialdehyde, MDA)是穩定且具代表性的產物。RNS 能直接造成DNA 硝化傷害，8-nitroguanine (8-NO2-G)及8-nitroxanthine (8-NO2-X) 是重要產物。RNS ?先與體內的二級胺反應可生成亞硝胺(N-nitrosamines)，亞硝胺再經代謝將造成DNA 烷基化修飾；?如N7-alkylguanine 及N3-alkyladenine。RNS 也會造成蛋白質硝基化修飾，其中以硝基化?胺酸(3-nitrotyrosine, 3-NTYR)是重要的穩定產物，其可被進一步代謝成主要代謝物 3-nitro-4-hydroxyphenylacetic acid (NHPA)。目前除?DNA 氧化傷害產物外，其餘指標仍然少有研究運用質譜技術(mass spectrometry)?建?高特?性及高敏感?的快速分析方法。本研究的目的在於運用液相層析??質譜儀(LC-MS/MS)搭配?線固相萃取(on-line solid phase extraction, on-line SPE)及同位素稀釋法(isotope-dilution)建?各式『硝化及氧化損傷綜合指標』的?床分析方法，並運用於探討肺部發炎反應，期以分子的角?探究ROS/RNS 在肺部發炎所造成的危害。本研究所開發的方法以非侵入性且?收集的?液樣本為主，並以開發?膜積液樣本分析法為輔。本計畫執?時間共需三?：第一?：運用on-line SPE LC-MS/MS 搭配isotope-dilution 開發體內脂質過氧化傷害(MDA)及蛋白質硝化傷害(3-NTYR 及NHPA)之?床分析方法。第二?：運用 on-line SPE LC-MS/MS 搭配isotope-dilution 開發體內基因硝化傷害(8-NO2-G 及8-NO2-X)、體內亞硝胺(9 種常?N-nitrosamines)及基因烷基化(N3-methyladenine)之?床分析方法，並展開肺部發炎者?液及?膜積液(pleural effusion)樣本之先期運用測試。第三?：將擴大?液收樣並將各式ROS/RNS 生物偵測指標(8-oxodGuo、8-oxoGua、MDA、3-NTYR、NHPA、8-NO2-G、8-NO2-X、N-nitrosamines、 N7-alkylguanine 及N3-alkyladenine)廣泛運用於樣本分析，以期探討發炎病患是否遭受比較高的 ROS/RNS 損傷或體內亞硝胺生成；同時比較各種ROS/RNS 生物偵測指標間的相關性及適用性，並跟常?的?床發炎指標比對。此外，本研究也將收集?膜積液，以探討我們所建?的各式 ROS/RNS 生物偵測指標能否幫助區分發炎引起的滲出液(exudate)及非發炎引起的?出液 (transudate)。 responsible for a variety of the degenerative processes of human diseases. Since ROS/RNS themselves are very reactive with an extremely short half-life, the measurement of oxidatively/nitratively modified DNA, protein and lipids in biological samples has been suggested to be more appropriate biomakers for diseases in which ROS/RNS are involved. 8-Hydroxy-2’-deoxyguanosine (8-OHdG or so called 8-oxodGuo) is the most used biomarker for oxidative DNA lesions. Malondialdehyde (MDA) is one of the end products of lipid peroxidation and has also been used to assess the oxidative stress in human. As for nitrative stress, 8-nitroguanine (8-NO2-G) and 8-nitroxanthine (8-NO2-X) are representative DNA nucleobase products of nitrative lesion by RNS, while 3-nitrotyrosine (3-NTYR) and its metabolite 3-nitro-4-hydroxyphenylacetic acid (NHPA) are recognized to be useful biomarkers for assessing nitration of protein. Meanwhile, RNS (i.e. N2O3) can also react with secondary amines to form N-nitrosamines, that are capable of further alkylating nucleobase to form mutagenic lesions (i.e. N7-alkylguanine and N3-alkylguanine). Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is a powerful analytical technique. It is noted that except for oxidaitvely modified DNA lesions, there was little mass spectrometric-based methods available for quantitaion of other RNS/ROS stress biomarkers. Therefore, the aims of this study are to develop serial “on-line SPE LC-MS/MS” methods to quantitate various nitrative/oxidative stress biomarkers in biological samples. These methods will be further applied in healthy subjects and patients with lung inflammation to investigate the possible roles of RNS and ROS in lung inflammation. This project will last three years with several objects as follows: 1st year: To develop isotope dilution LC-MS/MS with on-line SPE methods for determining MDA and 3-NTYR/NHPA; 2nd year: To develop isotope dilution LC-MS/MS with on-line SPE methods for determining 8-NO2-G/8-NO2-X, N-nitrosamines and N3-methyladenine. Meanwhile, these methods will be firstly applied in the urine and pleural effusion samples of patients with lung inflammation; 3rd year: To comprehensively measure various nitrative/oxidative stress biomarkers in urine samples of healthy subjects and patients with lung inflammation to assess whether theses RNS/ROS stress biomarkers were significantly increased in patients. The methods will also be applied in the patients who had pleural effusion to investigate whether these RNS/ROS stress biomarkers could help to distinguish between transudate and inflammatory exudate.
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/3915
Appears in Collections:	[公共衛生學系暨碩士班] 研究計劃

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