Studies on neurosteroids XXVI. Fluoxetine-evoked changes in rat brain and serum levels of neuroactive androgen, 5 alpha-androstane-3 alpha,17 beta-diol.

It is well known that fluoxetine (Fluox), a selective serotonin reuptake inhibitor, increases the brain content of allopregnanolone (AP), a potent neuroactive steroid that positively modulates the action of gamma-aminobutyric acid (GABA) at the GABA type A receptors, but the influence of Fluox on the brain and serum levels of a neuroactive androgen, 5alpha-androstane-3alpha,17beta-diol (3alpha,5alpha-Adiol), is poorly understood. In this study, we examined the Fluox-evoked changes in the 3alpha,5alpha-Adiol levels and compared the level changes of 3alpha,5alpha-Adiol with those of AP. The brain and serum 3alpha,5alpha-Adiol and AP levels were determined using previously developed LC-MS/MS. The ratio of the brain 3alpha,5alpha-Adiol to the serum 3alpha,5alpha-Adiol concentrations (B/S value) was significantly elevated in the rats intraperitoneally administered Fluox (10 mg/kg). Although the magnitude of the Fluox-evoked level change in 3alpha,5alpha-Adiol was much lower than that in AP, this study demonstrated that the 3alpha,5alpha-Adiol content is also influenced by Fluox. The most probable cause for the increase in the B/S value by the Fluox treatment is the activation of the 3alpha-hydroxysteroid dehydrogenase enzyme followed by the promotion of the de novo biosynthesis of 3alpha,5alpha-Adiol in the brain.

Studies on neurosteroids XXV. Influence of a 5alpha-reductase inhibitor, finasteride, on rat brain neurosteroid levels and metabolism.

In this study, we examined the influence of finasteride (FIN), a 5alpha-reductase inhibitor, on the brain levels and metabolism of neurosteroids [allopregnanolone (AP), 3alpha-dihydroprogesterone (3alpha-DHP), progesterone (PROG), 20alpha-dihydroprogesterone and 11-deoxycorticosterone (DOC)] in rats exposed to immobilization stress. For this purpose, the sensitive, reproducible and accurate liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) methods that enable the quantification of trace amounts of brain neurosteroids were first developed. The animal study using these methods demonstrated that FIN dose-dependently inhibits the stress-induced elevation of the brain AP, a potent positive modulator of the gamma-aminobutyric acid (GABA) type A receptors, and a 10 mg/kg dose of FIN can almost completely deplete AP in the brains. The study also found that the 20alpha-reduction of PROG is enhanced when its 5alpha-reduction pathway is inhibited in the brains. No change was found in the brain levels of 3alpha-DHP, another GABAergic neurosteroid, and DOC by the administration of FIN.

Studies on neurosteroids XXIV. Determination of neuroactive androgens, androsterone and 5alpha-androstane-3alpha,17beta-diol, in rat brain and serum using liquid chromatography-tandem mass spectrometry.

The development and validation of liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) methods that enable the quantification of neuroactive androgens, androsterone (5alpha-androstan-3alpha-ol-17-one, 3alpha,5alpha-A) and 5alpha-androstane-3alpha,17beta-diol (3alpha,5alpha-Adiol), in the rat brain and serum are presented. The androgens were extracted with methanol-acetic acid, purified using solid-phase extraction cartridges, derivatized with an ESI-active reagent, isonicotinoyl azide (INA), and then subjected to LC-ESI-MS/MS. The quantifications were based on selected reaction monitoring mode using the characteristic transitions of the INA derivatives. The methods allowed the reproducible and accurate quantification of the brain and serum neuroactive androgens using a 100 mg or 100 microL sample; the intra- and inter-assay relative standard deviations were below 3.6%, and the percentage accuracy values were 97.1-103.7% for both androgens. The animal study using the methods suggests that most of 3alpha,5alpha-Adiol found in the brain is derived from the periphery, while 3alpha,5alpha-A is not only transported from the periphery into the brain, but also synthesized in the brain by the oxidation of 3alpha,5alpha-Adiol. The androgens in the rats intraperitoneally administered finasteride, a 5alpha-reductatse inhibitor, were also measured; this treatment significantly reduced the brain 3alpha,5alpha-A and 3alpha,5alpha-Adiol levels and increased only the brain level of androstenedione, the precursor of 3alpha,5alpha-A.