以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同時定量人類尿液中五種DNA加成產物與古丁尼

Simultaneous Quantification of Five DNA Adducts and Cotinine in Human Urine by Stable Isotope Dilution Nanoflow Liquid Chromatography Nanospray Ionization Tandem Mass Spectrometry

陳勝宏(Sheng-Hong Chen)

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(2014年)

摘要

人類暴露於環境中外來的化學物質以及體內生成之具反應性物質的破壞會導致體內DNA鹼基產生共價性的修飾，稱為DNA加成產物(DNA adducts)，它在多階段性的致癌過程中扮演一個關鍵性的角色，是導致細胞癌化的起始步驟。假如DNA加成產物沒有被體内自身修復機制所修復，這些修飾過的DNA鹼基則會在DNA複製過程中產生鹼基配對錯誤，導致基因突變，最終發展成癌症。由於生物體內存在對受損DNA的切除修復酵素，而被修復後的DNA加成產物會流至體液(biological fluids)，如尿液或血液。在此研究中，我們發展出一套具有高靈敏度及高特異性的奈升流速液相層析奈電噴灑游離串聯質譜法(nanoLC-NSI-MS/MS)，在高選擇反應追蹤(H-SRM)模式下搭配穩定同位素稀釋法同時量測人類尿液中1,N6-乙烯基腺嘌呤(1,N6-ethenoadenine；εAde)、3,N4-乙烯基胞嘧啶(3,N4-ethenocytosine；εCyt)、1,N2-乙烯基鳥糞嘌呤(1,N2-ethenoguanine；1,N2-εGua)、7-乙基鳥糞嘌呤(7-ethylguanine；7-EtG)、8-羥基-2’-去氧鳥糞嘌呤核苷(8-hydroxy-2’-deoxyguanosine；8-OH-dG)與古丁尼(cotinine；COT)之含量，尿液樣品前處理只需要經過Strata-X逆相固相萃取管柱純化，且不需要經過衍生化的步驟。目前已量測了13個人類尿液樣品，在5個吸菸者尿液樣品中所偵測到的εAde、εCyt、1,N2-εGua、7-EtG、8-OH-dG、COT平均濃度分別為171.2 ± 77.1 pg/mL、285.8 ± 193.5 pg/mL、104.0 ± 61.2 pg/mL、110.1 ± 64.6 pg/mL、3.38 ± 1.14 ng/mL、784.8 ± 536.3 ng/mL；而在8位非吸菸者尿液中所偵測到的εAde、εCyt、1,N2-εGua、7-EtG、8-OH-dG、COT平均濃度分別為85.2 ± 144.1 pg/mL、202.3 ± 247.3 pg/mL、31.5 ± 31.6 pg/mL、56.6 ± 68.5 pg/mL、2.31 ± 2.35 ng/mL、0.47 ± 0.43 ng/mL。希望藉此分析方法可以比較出εAde、εCyt、1,N2-εGua、7-EtG、8-OH-dG及COT與癌症的相關性，以此做為癌症風險評估的指標。

關鍵字

固相萃取管柱 ； 穩定同位素稀釋 ； DNA加成產物 ； 串聯質譜儀

並列摘要

Exposure to environmental exogenous chemicals and endogenous reactive species in human can lead to the formation of structurally modified DNA bases (DNA adducts), which play a key role on multi-stage carcinogenesis process, and are the initial step of carcinogenesis. If DNA adducts have not been repaired by the body, these modified DNA bases could mispair by base to base, and lead to mutation during DNA replication, and develop into cancer eventually. These promutagenic DNA adducts are excised by base excision repair and nucleotide excision repair mechanisms and they are excreted into urine or blood as adducted bases and the nucleosides. In this report, we have developed an assay of high sensitivity and high specificity for simultaneous quantification of the five DNA adducts and cotinine by isotope dilution nanoflow liquid chromatography-nanospray ionization-tandem mass spectrometry (nanoLC-NSI/MS/MS)in human urine. Sample purification in urine before analysis by MS only requires a Strata-X reversed phase solid phase extraction column. The urine concentration of εAde、εCyt、1,N2-εGua、7-EtG、8-OH-dG and COT in 5 smokers are 171.2 ± 77.1 pg/mL、285.8 ± 193.5 pg/mL、104.0 ± 61.2 pg/mL、110.1 ± 64.6 pg/mL、3.38 ± 1.14 ng/mL and 784.8 ± 536.3 ng/mL, respectively；those in 8 nonsmokers are 85.2 ± 144.1 pg/mL、202.3 ± 247.3 pg/mL、31.5 ± 31.6 pg/mL、56.6 ± 68.5 pg/mL、2.31 ± 2.35 ng/mL and 0.47 ± 0.43 ng/mL, respectively. This highly sensitive and specific assay based on stable isotope dilution nanoLC-NSI/MS/MS assay provides a useful assay in measuring etheno、ethylpurine and oxidative adducts as noninvasive biomarkers in cancer risk ssessment.

並列關鍵字

SPE ； stable isotope dilution ； DNA adduct ； nanoLC-NSI-MS/MS

參考文獻

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