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標題: 蛋白質胼合物作為生物指標評估雌性激素類化物組織劑量之研究
Applications of Protein Adducts as Biomarkers to Investigate the Dosimetry of Estrogen Quinonoids
作者: 吳勝雄
Wu, Sheng-hsiung
關鍵字: 蛋白質胼合物;alkaline permethylation;雌性激素;雌性激素類化物;氣相層析質譜儀;甲基碘;protein adducts;鹼性烷化;estrogen;estrogen quinone;GC/EIMS;CH3I
出版社: 環境工程學系
本研究係以發展一生物指標分析法,針對雌性激素類化物與蛋白質形成之共價鍵結產物-胼合物(protein adduct),作為推估雌性激素類化物於標的器官之組織劑量,進行一系列之探討。運用鹼性烷化分析法(alkaline permethylation),以甲基碘為衍生劑,並以氫氧化鈉為催化劑,將雌性激素之類化物與蛋白質中氨基酸-cysteine硫原子所生成之併合物自蛋白質結構移除,經由有機溶劑萃取與濃縮,進行氣相層析電子撞擊式質譜儀(GC/EIMS)分析。本研究已成功地建立此一分析法,並完成對雌性激素類化物包括estrogen-2,3-quinone及estrogen-3,4-quinone與cysteine、glutathione、及蛋白質形成之胼合物之定性分析。同時針此一分析方法進行反應條件之最佳化測試,並推估鹼性烷化之偵測極限在250 pmole至1 nmole之間。此外,運用高效能液相層析儀純化雌性激素之類化物之cysteine胼合物,作為定量所需檢量線之標準品。並針對bovine serum al-bumin於生理條件下與estrone-3,4-quinone反應後,經運用鹼性烷化分析法推估其estrone-3,4-quinone-BSA adducts生成量與添加之estrone-3,4-quinone濃度相關性約為2.92 nmole/g protein/nM,而鹼性烷化分析法可偵測至最低曝露濃度為10 nM。針對以estrone-3,4-quinone於相同曝露濃度(10 mM)與時間下於人類乳癌細胞T-47D反應,鹼性烷化分析法未偵測到E1-3,4-Quinone所生成protein adduct。

The objective of this research is to develop a biochemical assay using protein adducts as biomarker of exposure to assess the cumulative body burden of estrogen quinones in target organs. The alkaline permethyla-tion procedure which used methyl iodine and sodium hydroxide as cata-lysts to cleave cysteinyl adducts of estrogen quinones. The cleaved ad-ducts are recovered by organic solvent extractions and analyzed by gas chromatography/electron-impacted mass spectrometer (GC-EIMS). Cysteinyl adducts of estrogen-2,3- quinone and estrogen-3,4-quinone on cyteines (Cys), glutathiones (GSH) and bovine serum albumins (BSA) are characterized by the alkaline permethylation procedure. Results from the optimization of the alkaline permethylation procedure reveal that the optimal condition for adduct cleavage is when modified cysteines react with 6N NaOH and 1 mL of CH3I at 100℃ for 6 hours. The limit of detection is estimated to be between 250 pmole and 1 nmole on column. Additionally, the synthetic cysteinyl adduct of estrogen-3,4-quinone was further purified by HPLC-UV and was used to quantify modified proteins. Regression analysis of the concentration-adduct levels of indicates that the production of E1-3,4-quinone-modified BSA adducts was estimated to be 2.92 nmole/g protein/nM.
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