簡易檢索 / 詳目顯示

回結果列表
研究生： 陳景星
Chen, Ting-Hsing
論文名稱： 發展液相層析串聯式質譜儀的方法來定量分析雌素酮代謝物
Development of an LC/MS/MS method for the quantitative determination of estrone metabolites
指導教授： 廖寶琦
Liao, Pao-Chi
學位類別： 碩士
Master
系所名稱： 醫學院 - 環境醫學研究所
Department of Environmental and Occupational Health
論文出版年： 2003
畢業學年度： 91
語文別： 中文
論文頁數： 75
中文關鍵詞： 雌素酮 、大氣壓化學游離法離子源 、電灑游離法離子源 、液相層析串聯式質譜儀
外文關鍵詞： estrone metabolism, LC/MS/MS, Hep G2, ESI, APCI
相關次數： 點閱：122  下載：14 
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

    • 近年來，科學家發現環境中有些化學物質會干擾內分泌系統，美國環保署將這些化學物質稱為「環境內分泌干擾化合物（environmental endocrine disrupting chemicals）」，日本學術界則稱之為「環境荷爾蒙（environmental hormones）」。這些環境荷爾蒙如戴奧辛等會影響雌性素的代謝酵素CYP1A1，造成眾多雌性素之一的雌素酮（estrone, E1）代謝物量的改變，引起生物體內分泌失調，而可能產生疾病，因此對於E1及其代謝物的相關研究漸漸受到重視。在過去分析E1及其代謝物的工具主要以放射性免疫測定法（radioimmunoassay, RIA）和氣相層析-質譜儀（gas chromatography-mass spectrometry, GC-MS）居多，但是以上的方析工具在應用上分別有無法同時分析多個化合物，以及需要將分析物進一步衍生化、前處理時間冗長等缺點。因此，本研究以發展液相層析串聯式質譜儀（high performance liquid chromatography-tandem mass spectrometry, LC/MS/MS）的方法來同時定量分析E1及其代謝物E2、16α-OH E1、2-OH E1、4-OH E1、2-OMe E1、4-OMe E1、2-OH-3-OMe E1。由於各個分析物的物化性質不盡相同，在比較過電灑游離法離子源（electrospray ionization source, ESI）和大氣壓化學游離法離子源（atmospheric pressure chemical ionization source, APCI）後，決定採用APCI當作離子源，在層析系統參數方面，以40% acetonitrile/water且酸鹼值調為4的溶液為層析移動相。分析方法建立完成後，應用此方法偵測人類肝癌細胞（Hep G2）及人類血清中之E1及其代謝物，進行不同應用的結果顯示，此方析方法的確有可以應用於分析細胞代謝實驗，以及人類血液的分析上的潛力。

    Chemical substances in the enviroment that disturb hormonal homeostasis in biological systems are referred as environmental endocrine disrupting chemicals（EDCs）. For example, Dioxins are considered as environmental EDCs. They alter hepatic hydrozylase activity and result in disturbance of estrone metabolism profile. In order to assist clinical study to investigate the association between environmental chemical exposure and hormonal disorders, we have developed a quantitative analytical method using high performance liquid chromatography tandem mass spectrometer（LC/MS/MS）for the determination of estrone (E1) and its major metabolites, estradiol（E2）, 2-hydroxyestrone（2-OH E1）, 4-hydroxyestrone（4-OH E1）, 2-methoxyestrone（2-OMe E1）, 4-methoxyestrone（4-OMe E1）and 2-hydroxy -3-methoxyestrone （2-OH-3-OMe E1）. Comparison of signal responses obtained by electrospray ionization（ESI）and atmospheric pressure chemical ionization（APCI）, indicated that APCI gave most satifactory signal responses for the eight analytes. The calibration curve, precision, accuracy, recovery, and detection limit have been characterized. The analytical method was applied to analyze E1 and its major metabolites in cultured human hepatocarcinoma cell line (Hep G2) and human serum. The results showed that this quantitative analytical method can be used in cell culture and clinical study.

    第一章 序論 1 1-1 研究背景 1 1-2 研究目的 3 第二章 文獻回顧 4 2-1 環境荷爾蒙簡介 4 2-1-1 何謂環境荷爾蒙 4 2-1-2 環境荷爾蒙之毒性作用 6 2-2 環境荷爾蒙對E1代謝之影響 10 2-2-1 雌性素在生物體內之生成代謝途徑 10 2-2-2 E1及其代謝物與疾病之關係 13 2-3 過去分析E1及其代謝物之方法比較 15 第三章 研究方法與材料 18 3-1 研究架構 18 3-2 研究工具與材料 20 3-2-1 分析儀器與設備 20 3-2-2 藥品及試劑 21 3-3 分析參數之尋找方式 22 3-3-1 E1及其代謝物與內標準品前驅離子(precursor ion)與產物離子(production ion) 之尋找 22 3-3-2 電壓參數 25 3-3-3 離子源選擇及參數調整 25 3-3-4 層析系統參數 30 3-3-5 樣本前處理 30 3-4 驗證程序 33 3-4-1 檢量線之製備 33 3-4-2 方法偵測極限 33 3-4-3 前處理回收率 34 3-4-4 精密度 34 3-4-5 準確度 34 3-5 Hep G2細胞代謝E1之實驗 35 3-5-1 細胞培養 35 3-5-2 Ethoxyresorifin O-deethylase (EROD)測定分析 35 3-5-3 E1代謝實驗 38 3-6 分析人類血清E1及其代謝物之實驗 38 第四章 結果與討論 40 4-1 選定E1及其代謝物和內標準品之前驅離子與產物離子 40 4-2 離子源選擇 40 4-3 層析系統參數最佳化 43 4-4 分析程序參數之選擇 50 4-5 分析驗證之程序 50 4-5-1 檢量線 50 4-5-2 方法偵測極限 54 4-5-3 前處理回收率 54 4-5-4 精密度 54 4-5-5 準確度 57 4-6 Hep G2細胞代謝E1實驗之結果 57 4-6-1 EROD assay 57 4-6-2 E1代謝實驗 60 4-7 分析人類血清E1及其代謝物之實驗結果 66 第五章 結論 69 參考文獻 71 表目錄 表2-1 人類主要荷爾蒙分泌過多或不足時可能導致的疾病 5 表2-2 日本環境廳公佈之疑似環境荷爾蒙物質一覽表 7 表2-3 文獻中分析E1及其代謝物之方法比較 17 表4-1 E1及其代謝物和內標準品之前驅離子與產物離子 41 表4-2 方法偵測極限之結果 55 表4-3 回收率之結果 55 表4-4 精密度之結果 56 表4-5 準確度之結果 56 表4-6 女性血清中E1代謝物濃度 68 圖目錄 圖2-1雌性素在生物體內之生成代謝圖 11 圖2-2 E1在生物體內之代謝簡圖 12 圖2-3 戴奧辛毒性機制簡圖 14 圖3-1 研究架構 19 圖3-2 LC/MS/MS結構簡圖 23 圖3-3 MRM原理圖 24 圖3-4 電壓參數 26 圖3-5 電灑游離法原理 27 圖3-6 大氣壓化學游離法原理 28 圖3-7 前處理流程 32 圖3-8 EROD assay 原理 37 圖3-9 細胞代謝處理流程圖 39 圖4-1 ESI和APCI正、負離子掃描偵測對各個標準品產生之訊號強度比較 42 圖4-2 不同移動相對2-OH E1和4-OH E1解析度之影響 44 (a) MeOH/H2O (b) ACN/H2O 44 圖4-3 40 % ACN/H2O不同的pH值對2-OH E1和4-OH E1之解析度之影響圖 45 圖4-4 梯度流析動向示意圖 46 圖4-5 不同40 % ACN/H2O流速對訊號之影響 48 圖4-6 E1及其代謝物之層析位置 49 圖4-7 利用本分析所建立之檢量線(a) E1 (b) E2 (c) 2-OH E1 (d) 16α-OH E1 51 圖4-8 利用本分析所建立之檢量線(a) 4-OH E1 (b) 2-OMe E1 (c) 4-OMe E1 (d) 2-OH-3-OMe E1 52 圖4-9 (a) 空白實驗 (b) 含標準品2-OH E1、4-OHE1 0.9 ppb 53 圖4-10 暴露TCDD時間和CYP1A1活性的關係 58 圖4-11 暴露TCDD濃度和CYP1A1活性之關係 59 圖4-12 Hep G2細胞代謝E1實驗結果(a) 沒有添加E1 (b) 有添加E1 62 圖4-13 Hep G2細胞代謝E1實驗結果(a) E1 (b) 2-OH E1(c) 2-OMe E1(d) 4-OMe E163 圖4-14 Hep G2細胞代謝E1實驗之結果(a) E2 (b) 16a-OH E1 (c) 4-OH E1 64 圖4-15 Hep G2細胞E1代謝物間之比值分布(a) 2-OH E1/E2 (b) 2-OH E1/16a-OH E1 (c) 4-OMe E1/16a-OH E1(d) 4-OMe E1/4-OH E1 65 圖4-16 分析人類血清E1及其代謝物實驗之結果 67

    Adlercreutz H, Gorbach SL, Goldin BR, Woods MN, Dwyer JT. Estrogen metabolism and excretion in oriential and Caucasian women. J Natl Cancer. 86: 1076-1082 (1994).

    Berg D, Thaler, Kuss E. Concentrations of 2-hydroxyoestrogens in human serum measures by a heterologous immunoassay with an 125I-labelled ligand. Acta Endocrinologica. 100:154-160 (1982).

    Berg D, Sonsalla R, Kuss E. Concentrations of 2-methoxyoestrogens in human serum measures by a heterologous immunoassay with an 125I-labelled ligand. Acta Endocrinologica. 103:282-288 (1983).

    Boffetta P, Jourenkova N, Gustavsson P. Cancer risk from occupational and environmental exposure to polycyclic aromatic hydrocarbons. Cancer Causes Control. 8:444-472 (1997).

    Brown NM, Lamartiniere CA. Xenoestrogens alter mammary gland differentiation and cell proliferation in the rat. Environ Health Perspect. 103: 708-713 (1995).

    Bucala R, Fishman J, Cerami A. The reaction of 16α-hydroxyestrone with erythrocytes in vitro and in vivo. Eur J Biochemistry. 140: 593-598 (1984).

    Christian J, Gruber, Walter T, Christian S, Johannes C, Huber. Production and actions of estrogens. The New England J Med. 346:340-352 (2002).

    Coumoul X, Diry M, Robillot C, Barouki R. Differential regulation of cytochrome P450 1A1 and 1B1 by a combination of dioxin and pesticides in the breast tumor cell line MCF-7. Cancer Res. 61:3942-3948 (2001).

    Clark E J, Norris D O., Jones R E. Interactions of gonadal steroids and pesticides (DDT, DDE) on gonaduct growth of larval tiger salamanders, Ambystoma tigrinum. General and Comparative Endocrinology. 109: 94-105 (1998).

    Fishman J, Osborne MP, Telang NT. The role of estrogen in mammary
    carcinogenesis. Ann NY Acad Sci. 768:91-100 (1995).

    Fishman J, Schneider J, Hershcope RJ, Bradlow HL. Increased estrogen-16-alpha-hydroxylase activity in women with breast and endometrial cancer. J Steroid Biochem. 20:1077-1081 (1984).

    Floyd RA. The role of 8-Hydroxyguanine in carcinogenesis. Carcinogenesis. 11:1447-1450 (1990).

    Franklin H, Epstein MD. Estrogen and the risk of breast. The New England J Med. 334:276-285 (2001).

    George RM, David DB. Rapid metabolic clearance of the catechol estrogen 2-hydroxyestrone. J Clini Endocrinol Metab. 51:1211-1213 (1980).

    Hiroshi N, Richard J, Hershcopf J. Radioimmunassay of 16α-hydroxyestrone in human urine. Steroids. 53:37-48 (1989).

    Kazutake S, Kuniko M, Miho S, Kenichiro Y. Studies on neurosteroids characterization of estrogens in rat brains using gas chromatography
    -tandem mass spectrometry. J chromatogr A. 847:171-178 (1999).

    Kime DE. The effects of pollution on reproduction in fish. Rev Fish Biol Fisheries. 5:52-96 (1995).

    Kuniko M, Misako Y, Kazutake S. Studies on neurosteroids characterization of catechol estrogens in rat brains using liquid chromatography-mass spectrometry-mass spectrometry. Analyst. 125:811-814 (2000).

    Kuniko M, Misako Y, Kazutake S. Studies on neurosteroids determination of enzymatically formed catechol estrogens and guaiacol estrogens by rat brains using liquid chromatography-mass spectrometry-mass spectrometry. J chromatogr B. 748:89-96 (2000).

    Luigi AC, Orazia MG, Felice PA. Gas chromatography/mass spectrometry of catechol estrogens. Steroids. 57:437-443 (1992).

    Longcope GW, Edman CD, Porter JC, Mavdonald PL. Metabolism of catecholestrogen by human erythrocytes. J Clin Endocrinol Metab. 57:277-282 (1983).

    McLachlan J, Newbold R, Korach K, Lamb J, Suzuki Y. Transplacental toxicology: prenatal factors influencing postnatal fertility. In: Developmental Toxicology (Kimmel C, Buelke-Sam J, eds). New York:Raven Press. 213-232 (1981).

    Miksicek RJ. Estrogenic flavonoids：structural requirements for biological activity, Pro Soc Exptl Biol Med. 208:44-50 (1995).

    Muti P, Bradlow HL, Micheli A. Estrogen metabolism and risk of
    breast cancer：a prospective study of the 2:16 alpha-hydroxyestrone
    ratio in premenopausal and postmenopausal women. Epidemiol.
    11:635-640 (2000).

    Nagel SC, Thayer KA. Relative binding affinity-serum modified
    access(RBA-SMA) assay predicts the relative in vivo bioactivity of the
    xenoestreogens bisphenol A and octylphenol. Environ Health Perspect.
    105:70-76 (1997).

    Poland A, Knutson JC. 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons：examination of the mechanism of toxicity. Annu Rev Pharmacol. 22:517-54 (1982).

    Reyes H, Resiz-Porszasz S, Hankinson O. Identification of Ah receptor nuclear translocator protein（Arnt）as a component of the DNA binding from of the Ah receptor Sci. 256:1193-1195 (1992).

    Safe S, Astroff B, Harris M, Zacharewski T, Dickerson R. 2,3,7,8-tetra-
    chlorodibenzo-p-dioxin(TCDD) and related compounds as antiestrogens
    ：Characterization and mechanism of action. Pharmacol Toxicol.
    69:400-409 (1991).

    Santodonato J. Review of the estrogenic and antiestrogenic activity
    of polycyclic aromatic hydrocarbins: relationship to carcinogenicity.
    Chemosphere. 34:835-848 (1997).

    Schneider SL., Alks V, Morreal CE. Estrogen properties of 3,9-dihy- droxybenz[a]anthracene, a potential metabolite of benz[a]anthracene. J Natl Cancer Inst. 57:1351-1354 (1976).

    Schneider J, Kinne D, Fracchia A. Abnormal oxidative metabolism of estradiol in women with breast cancer：a case control study. Proc Natl Acad Sci USA. 79:3047-3051 (1982).

    Shigeo I, Robert L, Jack F. Concentration of 16α-hydroxyestrone in human plasma as measured by a specific RIA. J Steroid Biochem. 18:329-332 (1983).

    Sumpter JP. Feminized responses in fish to environmental estrogens. Toxicol Lett. 82-83: 737-742 (1995).

    Telang NT, Suto A, Wong GY. Introduction by estrogen metabolite
    16-alpha-hydroxyestrone of genotoxic damage and aberrant proliferation
    in mouse mammary epithelial cells.J Natl Cancer Inst. 84:634-638 (1992).

    Tiwari RK, Guo L, Bradlow HL. Selective responsiveness of human
    breast cancer cells to indole-3-carbinol, a chemopreventive agent. J Natl
    Cancer Inst. 86:126-131 (1994).
    Toppari J, Larsenc JC, Christiansen P, Giwercman A, Grandjean P. Male reproductive health and environmental xenoestrogens. Environ Health Perspect. 104:741-803 (1996)

    Uma R, Daniel WS. Estrone and estradiol metabolism in vivo in human breast cysts. Steroids. 65:883-888 (2000).

    Wolff MS, Toniolo PG, Leel EW, Rivera M, Dubin N. Blood levels of organochlorine residues and risk of breast cancer. J Natl Cancer Inst. 85: 648-652 (1993).

    Xiao-yao X, David M. Quantitative analysis of estrogens in human urine using gas chromatography/negative chemical ionization mass spectrometry. Rapid Commun Mass Spectrom. 14:1991-2001 (2000).

    下載圖示 校內：立即公開
    校外：2003-07-04公開
    QR CODE