Simultaneous determination of dehydroepiandrosterone, its 7-hydroxylated metabolites, and their sulfates in rat brain...

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.