鯉魚頭腎皮質類固酮之研究

Abstract

本實驗分成三部份：首先發展解析固酮類的高壓液柱層析法系統。利用碳18逆相管柱，A溶劑（水55/甲醇32/氰甲烷6.5/異丙醇7.5）等濃度滌洗15分鐘,再用B溶劑(水40/甲醇40/正丁醇20)線性濃度梯度增加80%(35分鐘內),可以完全解析至少15種固酮類。同時,為了實驗目的(腎上腺皮質刺激素生物測定法)發展了另一套快速解析可體松及可體醇的溶劑系統(水55/甲醇32/氰甲烷6.5/異丙醇;等濃度滌洗),此項系統10分鐘可以解析一個樣品。 第二部份為腎上腺皮質刺激生物測定法的建立。利用鯉魚離體頭腎為標的組織，合成的腎上腺皮質刺激素1-24為刺激劑,再利用高壓液柱層析法測定可體醇產生的量。其最適溫度為20℃，腎上腺皮質刺激素的劑量關係範圍在25到320ng，此方法可用來測定鯉魚腦下腺酸丙酮萃取物的腎上腺皮質刺激素的活性。 第三部份為探討鯉魚頭腎皮質素生合成之研究。從頭腎固酮類的調查,推測可能的途徑,再利用外加受腎及抑制物實驗的資料,提出兩條同時存在的可能途徑,即17α-OH-Pregrencione→17α-OH-Progesterone→11-Deoxycorisoi→Cortisol及Progesterone→11β-OH-Progesterone→21-Deoxycortisol→Cortisol。並討論其可能的調節形式。

Efforts are made in this study to investigate the following three subjects: development of high pressure liquid chromatography (HPLC) for steroid analysis, establishment of bioassay of adrenocorticotropin (ACTH) by using carp head kidney to produce cortisol in vitro, and investigation of the biosynthetic pathway of corticosteroid in carp head kidney. The details of development of HPLC systems are described in Part I. A C18 reverse phase column (4.6 x 250 mm) and a gradient solvent elution system are used. The solvent system consists of two solvent mixtures: solvent A (Water 55/Methanol 32/Acetonitrile 6.5/ Isopropanol 7.5, v/v) and solvent B (Water 40/ Methanol 40/ n-Butanol 20, v/v). Steroids are first eluted isocratically with solvent A for 15 min then followed by gradient solvent elution for 35 min in which the concentration of solvent B is built up to 80% against the solvent A within 35 min. By this HPLC system, 15 steroids are completely separated within 15 min. In this study another HPLC system is also developed for rapid analysis of cortisone and cortisol. This rapid system is suitable for the assay of corticosteroid production by head kidney under ACTH stimulation in vitro. Analysis of sample is completed within 10 min and continuous operation is allowed. This rapid HPLC system consists of a shorter C18 column (4.6x150 mm) and an isocratically elution (Water 55/ Methanol 32/ Acetonitrile 6.5/Isopropanol 6.5, v/v). Estimation of the amount of steroid present in a biological sample is by fitting the peak area of a steroid into the regression equations derived from the amount of authentic steroids injected (25 to 400 ng) and the peak area on the HPLC chromatogram. Identification of steroid present in a biological sample is made by co-chromatography with authentic steroid. In Part II of this study, details of bioassay of ACTH by using carp head kidney to produce cortisol in vitro are described. The incubation mixture consists of 0.25 g minced head kidney and 0.5 ml incubation medium. A giving quality of ACTH is also added. Cortisol is the predominate corticosteroid produced by head kidney under ACTH stimulation in vitro. Head kidney continues to produce cortisol up to 6 hr in vitro. During whole incubation peroid, constant distribution pattern of cortisol in incubation medium and in tissue is observed: 60% in medium and 40% in tissue. The optimal temperature is 20℃. When temperature is rised up to 35℃ synthesis of cortisol is greatly impaired but that of cortisone is enhanced. In contrast with cortisol, most of cortisone is retained in tissue rather than released into incubation medium. The range of dose response curve of ACTH on stimulating head kidney to produce cortisol in vitro is from 25 to 320ng. The acid acetone extract of carp pituitary gland can also stimulate head kidney to produce cortisol in vitro. However, its dose response curve is different from that of mammalian ACTH. In the Part III of this study, we investigate the steroid composition of carp head kidney and the biosynthetic pathway of cortisol. Cortisone and cortisol are the predominate steroid in head kidney. In addition, many steroids believed to be the inter. mediates of cortisol biosynthsis are also observed. Among them, l7α-hydroxyprogesterone is the major steroid, followed by progesterone, 11-deoxycortisol, 21-deoxycortisol then 11β-hydroxy-progesterone. After in vivo ACTH treatment, the content of cortisone and cortisol is greatly increased. In addition, there is a tendency of the increase of the content of l1-deoxycortisol and the decrease of the content of 21-deoxycortisol. For investigation of biosynthetic pathways, following experiments are designed. (1) Incubation of progesterone, l7α-hydroxyprogesterone, 11β-hydroxyprogesterone, 11-deoxycortisol and 21-deoxycortisol with head kidney and then the conversion products are investigated. (2) Investigation of the accumulation of inter mediates when the biosynthesis of cortisol under the stimulation of ACTH is inhibited by metyrapone. The substrate efficiency for cortisol production is 11-deoxycortisol＞17α-hydroxyprogesteron＞21-deoxycortisol＞11β–hydroxyprogesterone＞progesterone. Under the action of metyrapone, 1l-deoxycortisol is accumulated. Therefore, it is proposed that under the action of ACTH, the following biosynthetic pathway is favored: l7α-hydroxyprogesterc →11—deoxycortisol →cortisol. In this study, evidence is also presented to indicate that l7α-hydroxyprogesterone is derive from l7α-hydroxypregnenolone through the action of 38-hydroxy-steroid dehydrogenase/△4, △5-isomerase rather than from progesterone through the action of 17α-hydroxylase. Furthermore, another pathway of cortisol biosynthesis is also proposed: progesterone → 11β-hydroxyprogesterone→ 21-deoxycortisol→cortisol. The possible regulatory role of this pathway in the biosynthesis of cortisol is discussed.