以GC-MS及GC-IRMS檢測DHEA、雞睪丸的攝取與素食者之T/E比值與δ13C‰值_

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題名：	以GC-MS及GC-IRMS檢測DHEA、雞睪丸的攝取與素食者之T/E比值與δ13C‰值
作者：	徐谷甫
作者(外文)：	Ku-Fu Hsu
校院名稱：	國立體育大學
系所名稱：	體育研究所
指導教授：	許美智張簡國平
學位類別：	博士
出版日期：	2013
主題關鍵詞：	脫氫表雄甾酮；雞睪丸；素食者；氣相層析同位素比值質譜儀；睪固酮/表睪固酮；DHEA；Chicken testis；Vegetarians；GC-IRMS；T/E
原始連結：	連回原系統網址
相關次數：	被引用次數:期刊(0) 博士論文(0) 專書(0) 專書論文(0) 排除自我引用:0 共同引用:0 點閱:42

脫氫表雄甾酮(Dehydroepiandrosterone，DHEA)是世界運動禁藥管制機構(World Anti-Doping Agency，WADA)所禁用的物質。然而DHEA在美國是合法的藥物，可經由超市或網路購買，容易造成運動員長期的使用或濫用。WADA規定的陽性閾值如下，睪固酮(testosterone)及表睪固酮(epitestosterone)的濃度為200 ng/mL；睪固酮/表睪固酮比值(T/E值)為4；本膽烷醇酮(etiocholanolone)及雄甾酮(androsterone)的濃度為10,000 ng/mL；DHEA的濃度為100 ng/mL，任何一個項目超過上述陽性閾值時，需進一步利用氣相層析同位素比值質譜儀(Gas Chromatography Isotope Ratio Mass Spectrometry，GC-IRMS)檢測睪固酮前驅物及代謝物之δ13C‰數值，δ13C‰數值小於-28‰時判定為陽性。
長期服用DHEA是否造成尿液中testosterone、epitestosterone、etiocholanolone、androsterone、DHEAtestosterone代謝物之δ13C‰數值的改變，目前的研究仍相當缺乏。因此，本實驗用氣相層析質譜儀(Gas Chromatography Mass Spectrometry，GC-MS)及GC-IRMS檢測多次服用DHEA後，尿液中之DHEA及其代謝物的濃度與δ13C‰數值。9名受試者分別參與服用單次50 mg的DHEA(DHEA組)及每日服用50 mg的DHEA持續服用7天(週DHEA組)。GC-MS的實驗結果發現，DHEA的服用在DHEA組及週DHEA組中並不會增加testosterone及epitestosterone的濃度，不過服用DHEA後會在體內轉變成etiocholanolone及androsterone，所以可從etiocholanolone、androsterone及DHEA的濃度發現陽性的結果。另外，本研究建議訂定陽性閾值Etiocholanolone/ Testosterone為550及Androsterone/ Testosterone為650，能協助陽性率的結果。GC-IRMS的實驗結果發現，在DHEA組及週DHEA組從第2小時到第72小時，其前驅物及代謝物的δ13C‰數值小於-28‰，可判定受試者違規服用DHEA。
睪丸是製造雄性賀爾蒙的主要組織，而雞睪丸是東方民族常用於進補的食材。然而運動員食用雞睪丸是否會提升體內的雄性激素而造成運動禁藥陽性結果，目前未有實驗證實。因此，本實驗受試者食用500克雞睪丸，並利用GC-MS檢測testosterone、epitestosterone、etiocholanolone、androsterone及DHEA，GC-IRMS檢測testosterone的前驅物及代謝物之δ13C‰數值，以瞭解食用雞睪丸是否超過陽性閾值。從GC-MS及GC-IRMS的結果發現，食用雞睪丸後僅會提升尿液中etiocholanolone及androsterone的排出量，其餘的數值皆未明顯改變，此外Etiocholanolone/Testosterone及Androsterone/ Testosterone也並未超過建議之陽性閾值，所以食用雞睪丸並不會造成陽性結果的判定。
GC-IRMS是檢測同化性物質的重要工具，但飲食型態會影響δ13C‰數值，例如： C3型的植物其δ13C‰數值比人體數值低，素食者會食用大量的蔬菜，而可能降低體內δ13C‰數值。因此，本實驗調查臺灣素食者尿液中之δ13C‰數值是否與葷食者有差異，並進一步檢測是否會造成陽性結果。實驗收集50名素食者及50名葷食者的尿液，利用GC-IRMS檢測尿液中testosterone的前驅物及代謝物δ13C‰數值。結果發現，素食者尿液中testosterone的前驅物及代謝物δ13C‰數值皆顯著低於葷食者。調查發現，造成素食者之δ13C‰數值較低是有較高的頻率食用豆類的食品，另外也比較常吃δ13C‰數值較低的蔬菜。雖然有些素食者代謝物之δ13C‰數值小於-28‰，藉由GC-MS檢測testosterone、epitestosterone、etiocholanolone、androsterone及DHEA，並計算Etiocholanolone/Testosterone及Androsterone/Testosterone比值，發現並未有任一數值超過陽性閾值或建議之陽性閾值。因此，本實驗所有的素食者皆為陰性結果。

以文找文

Dehydroepiandrosterone (DHEA) is a doping substance on World Anti-Doping Agency (WADA) prohibited list. However, DHEA is one of the few anabolic steroid precursors that are not banned by Anabolic Steroid Control Act. Therefore, DHEA is a legally available dietary/nutritional supplement over the counter. The long term use and misuse cases of DHEA by athletes are common. Current practice by WADA requires the use of two assays based on different analytical principle for the official “truly positive” endogenous anabolic steroids test. First, gas chromatography–mass spectrometry (GC–MS) is frequently used for urine screening in order to differentiate between negative and presumptively positive samples. Presumptively positive results (testosterone or epitestosterone ≥ 200ng/mL; T/E ratio ≥4; androsterone or etiocholanolone ≥ 10,000ng/mL; DHEA ≥ 100ng/mL) must be confirmed by gas chromatography- isotope ratio mass spectrometric (GC-IRMS) method. For the official “truly positive” endogenous anabolic steroids test, the δ13C‰ values of the testosterone’s precursors and metabolites in urine samples should be below -28‰.
No article has been reported that the δ13C‰ values of testosterone and its metabolites (etiocholanolone, androsterone) and epitestosterone in urine after long term DHEA ingestion. This study investigated the levels and δ13C‰ values of DHEA and its metabolites in urine by GC-MS and GC-IRMS after one-week DHEA ingestion. Volunteers took a single dose of 50 mg DHEA in DHEA group and multiple doses of 50 mg DHEA each morning for 7 day in week-DHEA group, respectively. In GC-MS results, the concentrations of testosterone and epitestosterone were not different in both DHEA and week-DHEA groups. For the GC-MS results, the urine specimens’ etiocholanolone, androsterone, and DHEA concentrations exceeded the thresholds of presumptively positive results in both groups. We suggest that the positive threshold value of etiocholanolone/testosterone and androsterone/testosterone ratio be 550 and 650, respectively. For the GC-IRMS results, the δ13C‰ values of testosterone’s precursor and metabolites were significantly lower than -28‰ from 2 to 72 hour after DHEA ingestion. Positive results were observed in both DHEA and week-DHEA groups.
Testis is the major organ producing the androgen hormone. Chicken testis is the popular food for the eastern people. In this study, we detected the urinary concentrations of testosterone, epitestosterone, etiocholanolone, androsterone and DHEA by GC-MS and the δ13C‰ value of testosterone’s precursor and metabolites by GC-IRMS after 500 g chicken testis ingestion. To understand whether chicken testis ingestion would result in positive. The results showed that the total excretion amounts of etiocholanolone and androsterone significantly increased after chicken testis ingestion, but none was over positive threshold. The etiocholanolone/testosterone and androsterone/testosterone ratio was below 550 and 650, respectively. Therefore, chicken testis ingestion should not cause the positive result.
Gas chromatography isotope ratio mass spectrometry is an important instrument in doping analysis. As we know that C3 plants have lower δ13C ‰ values. The diet pattern might change the δ13C‰ values of testosterone and its metabolites when one ingests C3 plants majorly. The δ13C‰ values probably decrease in the vegetarian urine. This study investigated whether the δ13C‰ values for vegetarians and non- vegetarians were different. The urines were collected from 50 vegetarians and 50 non- vegetarians. The δ13C‰ values of testosterone’s precursor and metabolites were measured in vegetarians’ and non- vegetarians’ urines by GC-IRMS. All δ13C‰ values of testosterone’s precursor and metabolites were significantly lower for vegetarian group than non- vegetarian group. The high frequency of bean eating does have lower δ13C‰ value in vegetarian group. However, the concentrations of testosterone, epitestosterone, etiocholanolone, androsterone and DHEA were not beyond the positive threshold although some vegetarians do have the δ13C‰ values of testosterone metabolites below -28‰. This study also showed negative result by calculating etiocholanolone/testosterone and androsterone/testosterone ratio. Thus, the diet pattern for the vegetarians will not cause the doping positive result.

以文找文

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