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    Please use this identifier to cite or link to this item: https://ir.csmu.edu.tw:8080/ir/handle/310902500/3042


    Title: 基因氧化傷害之全面臨床分析檢驗方法開發及國際合作
    Clinical-Scale High-Throughput Analysis of 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine in Various Biological Samples by Isotope-Dilution LC-MS/MS with on-Line Solid-Phase Extraction & International Cooperation
    Authors: 胡瓊文;翁瑞宏
    Hu, Chiung-Wen;Wong, Ruey-Hong
    Contributors: 中山醫學院公共衛生系
    Keywords: 基因氧化傷害;液相層析串聯質譜儀;連線固相萃取;臨床級;DNA;體液
    Oxidatively damaged DNA;LC-MS/MS;On-line SPE;Clinical scale;DNA;Body fluid
    Date: 2008
    Issue Date: 2010-12-06T03:23:50Z (UTC)
    Abstract: 許多研究已證實生物體內反應性含氧物質(Reactive oxygen species, ROS)可能是造成老
    化、慢性疾病、癌症的原因之一。當生物體內無法適當移除ROS 則會造成氧化壓力進而攻
    擊生物體內的重要分子如核酸、蛋白質、脂肪，最後導致細胞或組織在結構及功能上產生
    改變。8-羥基去氧鳥糞嘌呤核 8-hydroxy-2’-deoxyguanosine (8-OHdG, so called 8-oxodGuo)，
    為主要的基因氧化性傷害產物。它是遭受最具反應性之氫氧自由基(OH．)攻擊guanine C-8
    位置時的產物。在基因複製時，此種基因氧化傷害會導致G → T 之反轉突變。過去十年，
    臨床研究已廣泛使用8-oxodGuo 的含量作為體內氧化壓力的生物指標。研究發現在許多疾
    病及癌症的病患上量測到體內極高的8-oxodGuo 含量，甚至某些疾病的嚴重程度與
    8-oxodGuo 含量呈正相關性。因此近年來，許多學者建議基因氧化傷害的量測未來可當做
    疾病發展的風險指標及評估治療的效標。然而在基因氧化傷害量測 (i.e. 8-oxodGuo)可完全
    作為臨床的指標之前，有些問題仍待釐清其中包括標準分析方法的建立及一般健康人的背
    景值範圍(reference range)確立。
    文獻中8-oxodGuo 的分析方法主要有包括高效液相層析儀配合電化學偵測
    (HPLC-ECD) ， 氣相層析質譜儀(GC-MS) 及酵素連結免疫吸附分析（ enzyme-link
    immunosorbent assay，簡稱ELISA）。然而這些分析法都有缺點且無法應用在大量樣本或每
    天例行的分析。最近由於質譜儀的快速發展學者開始運用LC-MS/MS 在8-oxodGuo 及其他
    基因損傷產物分析上。此分析方法其特異性及敏感性高可配合使用同位素稀釋法(isotope
    dilution)，所以可說是當前最精準的定量方法。同時可搭配線上固相萃取方法(on-line solid
    phase extraction, on-line SPE)。樣本注入後可自動萃取純化濃縮且連線直接進入LC-MS/MS
    進行分析，非常適用於臨床高通量的檢測。
    本研究計畫將建立一系列「連線固相萃取-液相層析串聯質譜儀配合同位素稀釋法」於
    各種生物檢體的8-oxodGuo(及8-oxoGua)定量以期未來能全面應用於臨床分析。同時並參
    與國際合作計畫藉由國際間不同分析方法的比較以確立基因氧化傷害之標準分析方法及一
    般人體內8-oxodGuo (及8-oxoGua)之基礎背景值範圍。
    Reactive oxygen species (ROS) in living cells have been suggested to be associated with the
    development of aging, cancer and some degenerative diseases since they cause oxidative damage
    to nucleic acids, proteins, and lipids. 8-hydroxy-2’-deoxyguanosine (8-OHdG or so called
    8-oxodGuo) is one of the most abundant DNA lesion formed by the addition of the hydroxyl
    radical to the C8 position of guanine in DNA. The presence of 8-oxodGuo residues in DNA can
    lead to GC to TA transversion unless repaired before DNA replication. In the past decade,
    8-oxodGuo measurement has been widely used to evaluate oxidative stress in patients with
    various diseases and cancers. It has been suggested that 8-oxodGuo measurement has a great
    potential in clinical use to serve as a biomarker of disease development risk and efficacy of
    therapy. However, there are some problems that need to be solved before markers could be used
    clinically. For example, what are the standard analytical methodology and reference ranges for
    8-oxodGuo ?
    In the past 20 years, several methods have been successfully applied for analyzing
    8-oxodGuo, such as high performance liquid chromatography with electrochemical detection
    (HPLC-ECD), gas chromatography with mass spectrometry (GC-MS) and enzyme-linked
    immunosorbent assay (ELISA). However, they have drawbacks and could be difficult to perform
    in the clinical laboratory for routine measurements, such as labor intensiveness, necessity for
    chemical derivatization, low sensitivity or a limited specificity due to possible interferences
    arising from the complex biological matrix (i.e. crude urine). Recently, liquid chromatography
    with tandem mass spectrometry (LC-MS/MS) has become a powerful technology to overcome
    the sensitivity and specificity issues in analysis of DNA lesions. With the use of isotope-dilution
    and on-line sample extraction (on-line SPE), LC-MS/MS has been proved to be the most accurate
    methodology and would allow for high-throughput analysis such as clinical trials. Therefore, the
    aims of this work are to develop a serial “on-line SPE LC-MS/MS” method for clinical scale
    high-throughput 8-oxodGuo analysis in various biological samples. In the meantime, we have
    been invited to participate「European Standards Committee for the Analysis of Urinary (DNA)
    Lesions (ESCULA)」. With our newly developed methods, we are going to compare multiple
    methods for analysis urinary 8-oxodGuo and 8-oxoGua and further establish reference ranges of
    8-oxodGuo/8-oxoGua.
    URI: https://ir.csmu.edu.tw:8080/handle/310902500/3042
    Appears in Collections:[公共衛生學系暨碩士班] 研究計劃

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