臺灣博碩士論文加值系統

Dehydroepiandrosterone (3β-hydroxyandrost-5-ene-17-one; DHEA)是內生性類固醇亦是合成之同化性類固醇之一在運動競賽上被禁止使用。文獻指出在血中或是尿液中都証實了DHEA會進一步轉化代謝形成epitestosterone (E)、testosterone (雄性激素；T) 、androsterone (A)以及etiocholanolone (ETIO)，並且會影響Testosterone/ Epitestosterone (T/ E) ratio。本研究將26位健康男性受試者分成四組，包括：placebo組(6人)、50 mg組(8人)、100 mg組(7人)及200 mg組(5人)。每一受試者各服用同劑量之DHEA二次，每一次間隔三天，受試者之尿液收集在第一次服用前24小時、第一服用後及第二次服用藥物隨即參與長距離賽跑後之72小時，收集時間為4, 8, 12, 24, 48, 72小時。對於尿液樣品在GC-MS分析之前是應用酵素（從E. coli.�n而得之β-glucuronidase）和化學水解 (trimethylchlorosilane；TMCS)二種方法來進行水解，目的為去除葡萄糖醛酸與硫酸共軛代謝產物。本研究結果顯示：(1) 經兩種水解方法(β-glucuronidase與TMCS)處理後，得知口服DHEA後受試者之尿液DHEA、A以及ETIO之濃度在4至24小時呈現增加趨勢，其後逐漸回復至原來之值；(2)使用高劑量DHEA後DHEA、E、T、A以及ETIO之濃度及T/E ratio，在兩種水解方法中皆有呈現增加的現象；(3) 使用高劑量DHEA後硫酸型代謝物(DHEA、E、T、A及ETIO)比葡萄糖醛酸型代謝物之濃度高；(4)使用DHEA後隨即長距離賽跑結果顯示跑步對於尿液之DHEA、E、T、A、ETIO之濃度及T/E ratio皆無影響。

Dehydroepiandrosterone (3β-hydroxyandrost-5-ene-17-one; DHEA) is one of the synthetic anabolic steroids banned in sports. Studies done in serum or urine have shown that DHEA can convert to epitestosterone (E), testosterone (T), androsterone (A), etiocholanolone (ETIO) and will influence Testosterone/ Epitestosterone (T/ E ) ratio in the metabolic pathway. In this study, twenty-six healthy male volunteers were randomly divided into four groups (i.e., placebo, 50, 100 and 200 mg). Each subject orally ingested DHEA twice with 3 days interval between each dosing. Immediately following the second dosing, all the subjects participated a 10-kilometer run and their urine samples were collected at 4, 8, 12, 24, 48 and 72 hrs. Enzymatic (β-glucuronidase from E. coli) hydrolysis was used to deconjugate glucuronide-conjugated metabolites; chemical (trimethyliodosilane; TMCS) hydrolysis was applied to simultaneously deconjugate both glucuronides and sulfates of the metabolites. All the samples were subjected to gas chromatography-mass spectrometry (GC-MS) analysis. The results showed: (1) The concentrations of DHEA, A, ETIO in urine reached peak between 4 to 24 hours after DHEA administration and declined thereafter until approximately 48 to 72 hrs when the profiles were analyzed by either β-glucuronidase or TMCS; (2) The concentrations of DHEA, A, ETIO, T, E and T/E ratio were increased when higher dose of DHEA was ingested and the urine samples were hydrolyzed by either β-glucuronidase or TMCS; (3) Sulfate-conjugated DHEA, A, ETIO, T, E showed higher concentration than glucuronide-conjugated metabolites, particularly when higher doses DHEA were taken; (4) Long-distance race did not show any effects on DHEA, A, ETIO, T, E concentrations and T/E ratio when it was performed immediately after DHEA administration.

1. 背景介紹 (Background and significance) 頁次
1.1 Dehydroepiandrosterone之發現 (The discovery of dehydroepiandrosterone)---1
1.2 DHEA之藥理與細胞生理學 (The pharmacology and cell physiology of DHEA)----2
1.3 DHEA之生物合成與代謝 (The biosynthesis and metabolism of DHEA)-----------3
1.4 DHEA之功用與效用 (The functions and effects of DHEA)---------------------5
1.5 DHEA在運動員中的使用 (The use of DHEA in athletes)-----------------------6
1.6 運動藥檢在的研究 (The studies of DHEA in sports drug testing)------------6
1.7 研究目的 (The purposes of the study)-------------------------------------9
2. 實驗 (Experimental)
2.1 實驗材料 (Materials)-----------------------------------------------------10
2.1.1 標準品 (Reference standards)-------------------------------------------10
2.1.2 試劑與溶劑 (Reagents and solvents)-------------------------------------10
2.1.3 溶液製備 (Preparation of solutions)------------------------------------11
2.1.4 儲備溶液 (Stock solutions)---------------------------------------------12
2.2 實驗方法 (Methods)-------------------------------------------------------13
2.2.1 尿液檢體及取樣時間 (Urine samples and urine collecting time)-----------13
2.2.2 樣品前處理 (Urine sample preparation)----------------------------------14
2.3 實驗儀器 (Instrumentation)-----------------------------------------------17
2.4 定量離子之評估 (Evaluation of quantitation ions)-------------------------19
2.5 方法確效 (Method validation)---------------------------------------------19
2.5.1 偵測極限及定量極限 (Limit of detection and limit of quantitation)------19
2.5.2 回收率 (Recovery)------------------------------------------------------20
2.5.3 準確性與精確性 (Accuracy and precision)--------------------------------21
2.6 檢量線 (Calibration curve)-----------------------------------------------22
3. 結果 (Results)
3.1 特徵離子和強度 (Characteristic ions and their abundances)----------------23
3.2 代謝產物在尿液中之鑑定 (Identification of the DHEA metabolites in urine)-23
3.3 偵測極限與定量極限 (Limit of detection and limit of quantitation)--------24
3.4 檢量線 (Calibration Curve)-----------------------------------------------24
3.5 回收率之評估 (Evaluation of recovery)------------------------------------26
3.6 準確性與精確性之評估 (Evaluation of accuracy and precision)--------------27
3.7 DHEA、T、E、A以及ETIO之尿液中濃度變化及代謝情形 (Metabolic profiles and concentrations of DHEA, T, E, A and ETIO in TMCS-and β-glucuronidase-hydrolyzed urine samples)----------------------------------------------------28
3.8 DHEA、T、E、A以及ETIO之共軛代謝物之評估 (Evaluation of Sulfate conjugates in DHEA, T, E, A and ETIO metabolites)---------------------------------------30
3.9 T與E代謝物濃度比率之變化 (Profiles of T/E ratio in TMCS-and β-glucuronidase-hydrolyzed urine samples)--------------------------------------32
3.10 DHEA、T、E、A以及ETIO之尿液中代謝累積量 (Cumulative amounts of DHEA, T, E, A and ETIO in TMCS-and β-glucuronidase-hydrolyzed urine samples)----------32
3.11 尿液中DHEA、T、E、A以及ETIO之代謝濃度之統計分析 (Statistical analyses of DHEA, T, E, A and ETIO in TMCS-and β-glucuronidase-hydrolyzed urine samples)-33
4. 討論 (Discussion)---------------------------------------------------------35
5. 結論 (Conclusions)--------------------------------------------------------37
6. 參考文獻 (References)-----------------------------------------------------59
7. 附件 (Appendix)-----------------------------------------------------------62

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