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研究生: 陳美娟
Mei-Chuan Chen
論文名稱: 線上掃集濃縮-毛細管微胞電動層析法對血清及尿液樣品中微量皮質固酮及氫基皮質酮之分析研究
Determination of corticosterone and 17-hydroxycorticosterone in plasma and urine samples by sweeping techniques using micellar electrokinetic chromatography
指導教授: 林震煌
Lin, Cheng-Huang
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2004
畢業學年度: 92
語文別: 中文
論文頁數: 109
中文關鍵詞: 掃集濃縮毛細管微胞電動層析法皮質固酮氫基皮質酮
論文種類: 學術論文
相關次數: 點閱:110下載:2
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  • 利用毛細管電泳/紫外光吸收偵測法(CE/UV),結合線上濃縮的技
    術,分析代謝性壓力實驗下,注射去氧葡萄糖(2-deoxy-D-glucose)試
    劑前後,老鼠血清中皮質固酮(corticosterone),以及運動前後人體尿
    液中氫基皮質酮(17-hydroxycorticosterone)的含量變化。本研究以一
    般微胞電動層析法(normal-MEKC)、毛細管電泳掃集法(sweeping-
    MEKC)以及陽離子選擇性完全注射掃集法(CSEI-sweep-MEKC),
    三種分析方法進行偵測極限的比較,發現normal-MEKC 只能偵測至7
    μg/mL(S/N=3),而sweeping-MEKC 和CSEI- sweep-MEKC 可分別達
    3 ng/mL 及5 ng/mL(S/N=3)的偵測極限。在真實樣品分析中,檢測
    代謝性壓力實驗下,老鼠血清中皮質固酮的平均含量,其中慢性受壓
    老鼠濃度為552 ± 152 ng/mL;急性受壓老鼠濃度為375 ± 105 ng/mL,
    皆較未受壓老鼠濃度153 ± 109 ng/mL,明顯增高;而運動性壓力實驗
    下,人體尿液的檢測,在5~10 分鐘內跑完800 公尺後,人體尿液中微
    量氫基皮質酮的平均濃度,男性為137 ± 55 ng/mL、女性為128 ± 25
    ng/mL,相較於未跑前所測量的平均濃度,男性63 ± 37 ng/mL,女性
    60 ± 20 ng/mL,亦有升高的趨勢。實驗中所研發之分析技術,只需要
    極少量的樣品,即可檢測壓力指標荷爾蒙-糖皮質類固醇的濃度,有
    助於醫學上用來了解身體對壓力因子的反應機制,做為個體健康狀態
    的參考指標,並可應用於醫藥學上,評估各種天然或合成藥物對於減
    緩壓力之療效。

    The analysis of corticosterone in mouse blood serum (metabolic-stress
    experiment) and 17-hydroxycorticosterone in human urine (exercise-stress
    experiment) samples by means of capillary electrophoresis/UV absorbance
    in conjunction with online sample concentration techniques is described.
    The use of normal MEKC had an analyte detection limit of 7 μg/mL (S/N
    =3); whereas when online sample concentration methods, including
    sweeping-micellar electrokinetic chromatography (sweeping-MEKC)
    and cation-selective exhaustive injection-sweep-micellar electrokinetic
    chromatography (CSEI-sweep-MEKC) were used, the detection limits
    could be improved to 3 and 5 ng/mL, respectively. In the analysis of actual
    samples from animal metabolic-stress experiments (39 mouse), chronically
    stressed animals showed a higher level (552 ± 152 ng/mL) and acute
    stressed animals showed an intermediate level (375 ± 105 ng/mL).
    In comparison, normal animals show a lower concentration level of
    corticosterone (153 ± 109 ng/mL). In addition, based on a human
    exercise-stress experiment (seven volunteers), the acute stressed humans
    (after exercise, 800m of running) show a higher concentration of
    17-hydroxycorticosterone (137 ± 55 ng/mL for males; 128 ± 25 ng/mL for
    females) and the non-stressed humans (before exercise) show a lower
    concentration (63 ± 37 ng/mL for male; 60 ± 20 ng/mL for female),
    respectively. Furthermore, this method requires only small amounts of
    plasma and could be used as a routine tool in pharmacological studies
    dealing with different types of drugs, whether derived from natural herbs or
    by synthesis.

    第一章緒論 1-1 內分泌(endocrine)簡介…………………………………………………………..01 1-1.1 激素(hormone)的分泌……………………………………………….01 1-1.2 激素的種類……………………………………………………………...01 1-1.3 激素的作用機制………………………………………………………...02 1-1.4 激素分泌的調節與負回饋控制………………………………………...05 1-2 分析物簡介……………………………………………………………………..06 1-2.1 腎上腺皮質類固醇的分類……………………………………………...06 1-2.2 皮質類固醇的結構和生物合成途徑…………………………………...07 1-2.3 皮質類固醇的分泌和調節……………………………………………...08 1-2.4 皮質固酮和氫基皮質酮的生理作用…………………………………...11 1-3 研究目的………………………………………………………………………..12 1-4 類固醇激素的分析法…………………………………………………………..13 第二章研究原理和方法 2-1 毛細管電泳層析法的發展………………………………………………………..16 2-2 毛細管電泳層析的分離原理……………………………………………………..20 2-2.1 電泳遷移率………………………………………………………………..20 2-2.2 電滲流……………………………………………………………………..21 2-2.3 分離效率與解析度………………………………………………………..26 2-2.4 電分散作用………………………………………………………………..27 2-3 毛細管電泳層析的分離模式……………………………………………………..29 2-3.1 毛細管區帶電泳法………………………………………………………29 2-3.2 微胞電動層析法…………………………………………………………32 2-4 毛細管電泳線上濃縮技術………………………………………………………..34 2-4.1 毛細管電泳掃集法………………………………………………………..36 2-4.2 陽離子選擇性完全注射掃集法…………………………………………39 第三章研究儀器和藥品 3-1 實驗儀器…………………………………………………………………………..41 3-1.1 毛細管電泳儀器…………………………………………………………41 3-1.2 訊號放大器……………………………………………………………..…43 3-1.3 儀器及週邊設備列表……………………………………………………45 3-2 實驗藥品…………………………………………………………………………..47 3-2.1 藥品列表…………………………………………………………………..47 3-2.2 藥品性質介紹……………………………………………………………48 3-3 分析物的取得與處理……………………………………………………………..49 3-3.1 老鼠血清樣品來源………………………………………………………..49 3-3.2 人體尿液樣品來源………………………………………………………..50 3-3.3 血清及尿液樣品的前處理………………………………………………..50 第四章研究過程和結果討論 4-1 標準品最佳分離條件的確立……………………………………………………52 4-1.1 分析物的吸收光譜………………………………………………………52 4-1.2 一般微胞電動層析法及訊號放大器……………………………………52 4-1.3 毛細管電泳掃集法的最佳條件…………………………………………56 4-1.4 掃集法對濃度的線性關係及再現性探討………………………………..65 4-1.5 陽離子選擇性完全注射掃集法…………………………………………69 4-1.6 比較不同電泳模式的堆積效率和偵測極限……………………………73 4-2 真實樣品的定量分析……………………………………………………………..75 4-2.1 老鼠血清中皮質固酮的定量分析………………………………………75 4-2.2 人體尿液中氫基皮質酮的定量分析……………………………………..80 4-2.3 血清及尿液樣品萃取回收率的探討……………………………………...86 第五章結論和展望 5-1 最佳電泳層析條件方面…………………………………………………………..87 5-2 真實樣品定量分析方面…………………………………………………………..87 5-3 應用與展望………………………………………………………………………..88 論文發表……………………………………………………………………………….89 參考資料………………………………………………………………………………90 附件︰期刊論文

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