Latif S A, Camara P, Rosen M P, Morris D J
Department of Pathology, Miriam Hospital, Brown University, Providence, RI 02912.
Steroids. 1987 Jun;49(6):589-600. doi: 10.1016/0039-128x(87)90098-5.
Specific methods are described for the enzymatic synthesis of each of the six possible 3H-labeled Ring-A reduced metabolites of aldosterone (5 alpha- and 5 beta-DHAldo; 3 alpha,5 alpha-THAldo; 3 beta,5 alpha-THAldo; 3 alpha,5 beta-THAldo; and 3 beta,5 beta-THAldo; see footnote 1 for full names). Use of heated jacketed columns (C8-reverse phase) and two HPLC solvent systems, with isocratic aqueous methanol or acetonitrile, respectively, have been developed which resolve all six Ring-A reduced metabolites of aldosterone. The relative retention times and elution order of each reduced metabolite are different with each solvent system and hence help confirm the identities of Ring-A reduced metabolites made in vivo from physiological quantities of [3H]aldosterone. The use of an on-line beta-radioactivity detector (Berthold LB-504) enhanced the sensitivity of detection and markedly improved the resolution of these metabolites, compared with that obtained by off-line scintillation counting. Thus, the use of increased temperature with these two solvent systems, together with an on-line radioactivity detector, provide a useful and efficient analytical tool for the separation and identification of each reduced metabolite of aldosterone.
文中描述了具体方法，用于酶促合成醛固酮六种可能的3H标记的A环还原代谢产物（5α-和5β-双氢醛固酮；3α,5α-四氢醛固酮；3β,5α-四氢醛固酮；3α,5β-四氢醛固酮；以及3β,5β-四氢醛固酮；全名见脚注1）。已开发出使用加热夹套柱（C8反相柱）和两种HPLC溶剂系统，分别采用等度甲醇水溶液或乙腈，可分离醛固酮的所有六种A环还原代谢产物。每种还原代谢产物在每种溶剂系统中的相对保留时间和洗脱顺序都不同，因此有助于确认从生理量的[3H]醛固酮体内生成的A环还原代谢产物的身份。与离线闪烁计数相比，使用在线β放射性检测器（Berthold LB - 504）提高了检测灵敏度，并显著改善了这些代谢产物的分离度。因此，将这两种溶剂系统与在线放射性检测器一起使用，提高温度，为醛固酮各还原代谢产物的分离和鉴定提供了一种有用且高效的分析工具。
