以穩定同位素稀釋液相層析奈電噴灑游離串聯質譜法分析(1)人類白血球DNA中乙二醛與DNA的交聯產物與 (2)人類尿液中乙烯基與8-羥基-2’-去氧鳥糞嘌呤核苷

Analysis of (1) Glyoxal-Induced DNA Cross-Links in Human White Blood Cell DNA and (2) Etheno Adducts and 8-Hydroxy-2’-deoxyguanosine in Human Urine by Stable Isotope Dilution Liquid Chromatography-Nanospray Ionization Tandem Mass Spectrometry

張雅嵐(Ya-lan chang)

指導教授 ： 陳皓君

國立中正大學/理學院/化學暨生物化學系/碩士(2012年)

摘要

人類生活環境中充滿了許多對人體具有危害性的反應性物質。而這些化學物質進入人體當中會與DNA反應而造成DNA的損害，進而產生DNA加成產物。在本論文所描述的研究中，主要分為兩部分。第一部分為發展出一套具有高靈敏度與高特異性的穩定同位素稀釋奈升流速液相層析奈電噴灑游離串聯質譜法(nanoLC-NSI/MS/MS)用來評估組織DNA受到乙二醛損害的程度。實驗結果顯示於市售的人類胎盤DNA所測得的乙二醛交聯產物含量為在108個正常鹼基中分別有5.92個dG-gx-dC和6.39個dG-gx-dA，而在人類白血球DNA中分析結果為在11個吸煙者之乙二醛交聯產物含量為在108個正常鹼基中有1.44 ± 1.17個dG-gx-dC和1.70 ± 1.08 dG-gx-dA；在9個非吸煙者則有0.81 ± 0.62個dG-gx-dC和0.79 ± 0.85 dG-gx-dA。第二部分則是利用穩定同位素稀釋毛細管液相層析奈電噴灑游離串聯質譜法(capillary LC-NSI/MS/MS)同時量測人類尿液中乙烯基腺嘌呤(1,N6-ethenoadenine, εAde)、乙烯基胞嘧啶(3,N4-ethenocytosine, εCyt)、乙烯基鳥糞嘌呤(1,N2-ethenoguanine, 1,N2-εGua)及8羥基鳥糞嘌呤去氧核苷(8-hydroxy-2’-deoxyguanosine, 8OHdG)的含量。實驗結果顯示，在4個吸菸者尿液樣品中，其所測得εAde，εCyt和1,N2-εGua的平均濃度分別為52.2 ± 82.4, 21.2 ± 24.7和76.6 ± 53.9 pg/mL，而8OHdG的平均濃度為2.82 ± 1.52 ng/mL；而在8個非吸菸者尿液樣品中，其所測得εAde，εCyt和1,N2-εGua的平均濃度分別為40.8 ± 44.8, 55.6 ± 89.5和69.6 ± 116.7 pg/mL，而8OHdG的平均濃度為2.64 ± 2.67 ng/mL。希望藉此分析方法可以比較出εAde、εCyt、1,N2-εGua及8OHdG與癌症的相關性，以此做為癌症風險評估的指標。

關鍵字

奈升流速液相層析奈電噴灑游離串聯質譜法

並列摘要

There are many reactive chemicals in our living environment which could reactive with DNA forming DNA adducts. There are two parts in my thesis. In the first part, we developed a highly sensitive and specific quantitative assay for simultaneous detection and quantification of dG-gx-dA and dG-gx-dC cross-links by stable isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) under the highly selective reaction monitoring (H-SRM) mode. Levels of dG-gx-dC and dG-gx-dA in human placenta DNA are 5.92 and 6.39 in 108 normal nucleotides, respectively. Levels of dG-gx-dC and dG-gx-dA are 1.44 ± 1.17 and 1.70 ± 1.08 in 108 normal nucleotides ,respectively, in 11 smokers’ white blood cell (WBC) DNA, and those in 9 nonsmokers’ WBC DNA are 0.81 ± 0.62 and 0.79 ± 0.85 in 108 normal nucleotides, respectively. Thus, it is clinically feasible using this highly sensitive assay to investigate the potential of these cross-links as low-invasive biomarkers for glyoxal-induced DNA damage and in disease development and prevention. In the second part, we developed a highly specific and sensitive method for simultaneous quantification of etheno DNA adducts: εAde, εCyt, 1,N2-εGua, and 8OHdG in human urine by stable isotope dilution capillary LC-NSI/MS/MS under the H-SRM mode. The urine concentration of εAde, εCyt, 1,N2-εGua, and 8OHdG are 52.2 ± 82.4, 21.2 ± 24.7, 76.6 ± 53.9 pg/mL and 2.82 ± 1.52 ng/mL in 4 smokers’ urine samples, respectively, and those in 8 nonsmokers’ urine samples are 40.8 ± 44.8, 55.6 ± 89.5, 69.6 ± 116.7 pg/mL and 2.64 ± 2.67 ng/mL, respectively. This capillary LC-NSI/MS/MS method provides a useful assay in measuring etheno and oxidative adducts as noninvasive biomarkers in cancer risk assessment.

並列關鍵字

nanoLC-NSI/MS/MS

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