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

Abstract

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.