以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在人類白血球DNA和尿液中定量乙基嘌呤加成產物

Quantitative Assay for Ethylpurine Adducts in Human White Blood Cells DNA and Human Urine by Stable Isotope Dilution Capillary LC-Nanospray Ionization Tandem Mass Spectrometry

劉彥甫(Liu, Yen Fu)

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

香煙中含有許多烷基化試劑，研究顯示抽菸會導致DNA受到乙基化試劑的傷害而產生3-乙基腺嘌呤(3-ethyladenine)及7-乙基鳥糞嘌(7-ethylguanine) DNA加成產物。為了準確定量在DNA上的乙基加成產物，我利用中性熱水解方法使3-乙基腺嘌呤和7-乙基鳥糞嘌呤從白血球DNA上被釋出，透過固相萃取管柱的樣品前處理步驟，再以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，在高選擇反應追蹤模式(H-SRM)下去偵測這些被釋出的3-EtA和7-EtG。3-乙基腺嘌呤及7-乙基鳥糞嘌呤的偵測極限分別為30.7 和55.9 amol。在20位吸菸者的白血球DNA中，108個正常核苷酸中有16.0 ± 7.8個3-乙基腺嘌呤與9.7 ± 8.3個7-乙基鳥糞嘌呤；在20位非吸菸者中，108個正常核苷酸中有5.4 ± 2.6個3-乙基腺嘌呤與0.3 ± 0.8個7-乙基鳥糞嘌呤，比較此二加成產物在吸菸者與非吸菸者之含量，其p值皆小於0.0001。經由體內修復系統修復的加成產物則被排放到尿液當中，因此我透過固相萃取管柱的樣品前處理步驟，再以毛細管液相層析奈電噴灑游離串聯質譜法，在H-SRM下同時分析尿液中3-乙基腺嘌呤及7-乙基鳥糞嘌呤的含量。在5位吸菸者的尿液中，3-乙基腺嘌呤濃度為120.9 ± 33.5 pg/mL，7-乙基鳥糞嘌呤濃度為61.0 ± 8.5 pg/mL。我希望藉由此分析方法來評估DNA受到乙基化損害的程度，比較出3-乙基腺嘌呤和7-乙基鳥糞嘌呤與疾病的相關性，做為癌症風險評估的生化指標。

關鍵字

7-乙基鳥糞嘌呤 ； 3-乙基腺嘌呤

並列摘要

Cigarette smoke contains many alkylating agents which damage DNA producing DNA adducts, such as 3-ethyladenine (3-EtA) and 7-ethylguanine (7-EtG). To quantify ethylated adducts on DNA, I used neutral thermal hydrolysis to release 3-EtA and 7-EtG from DNA. The adducts were purified by a reversed-phase solid-phase extraction column and analyzed by stable isotope dilution capillary liquid chromatography nanospray ionization tandem mass spectrometry (capLC-NSI/MS/MS) under the highly selected reaction monitoring (H-SRM) mode. The amount of ethylated DNA adducts represents the steady-state levels of DNA adducts resulting from the ethylating agent after repair in vivo. The detection limit of 3-EtA and 7-EtG was 30.7 and 55.9 amol, respectively. Levels of 3-EtA and 7-EtG are 16.0 ± 7.8 and 9.7 ± 8.3 in 108 normal nucleotides, respectively, in white blood cells (WBC) DNA of 20 smokers, which are statistically significantly higher than those in 20 nonsmokers with 5.4 ± 2.6 3-EtA and 0.3 ± 0.8 7-EtG in 108 normal nucleotides (p < 0.0001). These DNA adducts are repaired in vivo and then released in human urine. The urine samples were purified by a mixed-mode cation exchange followed by a reversed-phase solid-phase extraction column and analyzed by capLC-NSI/MS/MS under the H-SRM. The urinary concentration of 3-EtA and 7-EtG are 120.9 ± 33.5 pg/mL and 61.0 ± 8.5 pg/mL in 5 smokers. The highly sensitive and specific stable isotope dilution nanoLC-NSI/MS/MS assay should be clinically useful in assessing the possibility of measuring ethylpurines in human WBC DNA and in urine as risk biomarkers for smoking-related cancers.

並列關鍵字

7-ethylguanine ； 3-ethyladenine

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