Kazihnitková Hana, Tejkalová Hana, Benesová Olga, Bicíková Marie, Hill Martin, Hampl Richard
Institute of Endocrinology, Národní 8, 11694 Praha 1, Czech Republic.
Steroids. 2004 Sep;69(10):667-74. doi: 10.1016/j.steroids.2004.06.002.
A method is described for simultaneous assessment of dehydroepiandrosterone (DHEA), its sulfate (DHEAS), and their 7-hydroxylated metabolites in cortex and subcortex of the rat brain. The procedure for determination of unconjugated steroids and DHEAS involved diethyl ether extraction of the homogenized tissue, solvent partition of the dry extract, and final quantification by specific radioimmunoassays. In addition, determination of 7-hydroxy-dehydroepiandrosterone sulfates required solvolysis, followed by high-performance liquid chromatography for separation of 7-hydroxylated metabolites from their precursor. The losses during this process were monitored by measurement of spiked radioactivity of [(3)H]testosterone or [(3)H]dehydroepiandrosterone sulfate. The content of dehydroepiandrosterone sulfate in both brain tissues was of the order of ten(s) nmol/g tissue irrespective its type (cortex or subcortex), while concentrations of other steroids were about 10 times lower in both tissues. In contrast to the ratio of sulfated/unconjugated DHEA, the levels of unconjugated 7-hydroxylated metabolites and their sulfates were close to each other. The reproducibility of the method with respect to coefficients of variation varied from 12 to 25%. An age-related decrease of sulfated dehydroepiandrosterone in the cortex of animals was also observed.
本文描述了一种同时评估大鼠脑皮质和皮质下脱氢表雄酮（DHEA）、其硫酸盐（DHEAS）及其7-羟基化代谢物的方法。测定未结合类固醇和DHEAS的步骤包括用乙醚提取匀浆组织、对干燥提取物进行溶剂分配，最后通过特异性放射免疫测定法进行定量。此外，测定7-羟基脱氢表雄酮硫酸盐需要进行溶剂解，然后通过高效液相色谱法将7-羟基化代谢物与其前体分离。在此过程中的损失通过测量[³H]睾酮或[³H]脱氢表雄酮硫酸盐的加标放射性来监测。两种脑组织中脱氢表雄酮硫酸盐的含量约为每克组织十（几）纳摩尔，与组织类型（皮质或皮质下）无关，而两种组织中其他类固醇的浓度约低10倍。与硫酸化/未结合DHEA的比例相反，未结合的7-羟基化代谢物及其硫酸盐的水平彼此接近。该方法的变异系数重现性在12%至25%之间。还观察到动物皮质中硫酸化脱氢表雄酮随年龄的下降。
