Studies on neurosteroids XXII. Liquid chromatography–tandem mass spectrometric method for profiling rat brain 3‐oxo‐4‐ene‐neuroactive steroids

Biological & Pharmaceutical Bulletin

et al. 2008

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Neurosteroids affect neurotransmission through action at the membrane ion-gated receptors and at other neurotransmitter receptors. 3a-Hydroxy-5a-pregnan-20-one (allopregnanolone, AP), a representative neurosteroid, binds to the gaminobutyric acid type A (GABA A ) receptors with a high affinity and positively modulates the action of GABA at these receptors, and hence elicits marked anticonvulsant, antidepressant and anxiolytic effects.1) Under stressful conditions, the brain rapidly synthesizes progesterone (PROG) and subsequently converts it to AP to raise the threshold of brain excitability.2)The two-step metabolism of PROG produces AP through the actions of the enzymes, 5a-reductase (rate-limiting step) and 3a-hydroxysteroid dehydrogenase (3a-HSD). Two distinct 5a-reductase isozymes, types 1 (predominant form in the brain) and 2 (predominant form in the periphery), are found across mammalian species.3) Finasteride [17a-(N-tbutyl)carbamoyl-4-aza-5a-androst-1-en-3-one, FIN] is the 5a-reductase inhibitor that has received clinical approval for the treatment of human benign prostatic hyperplasia and androgenetic alopecia. Both isozymes of the 5a-reductase in the rodent demonstrate comparable inhibition following FIN exposure; FIN can inhibit the 5a-reductase activity in the central nervous system as well as in the periphery.4) Based on this, FIN has also been used to manipulate the brain AP level for characterization of its physiological functions and the mechanism by which it affects the brain functions, and to analyze the exact mechanisms of action of psychotropic agents, whose antidepressant and anxiolytic effects are inferred to occur through an increase in the brain AP synthesis.5,6) Thus, FIN is an indispensable tool in the development of new psychotropic agents targeting neurosteroidogenesis as well as in basic neurosteroid research. On the contrary, the treatment with FIN may induce alternative metabolic pathways, such as 3a-reduction, 20a-reduction and 21-hydroxylation, in the PROG metabolism (Fig. 1), and the generated metabolites may consequently affect the central nervous system and modulate the emotional state. However, there is no report that examines the changes in the brain levels of the PROG and its metabolites following treatment with FIN. Even for AP, a detailed FIN-evoked change in the brain concentration has not been elucidated.Based on this background information, the objectives of this study are to develop liquid chromatography-tandem mass spectrometric (LC-MS/MS) methods that enable the determination of trace amounts of the brain PROG and its down stream conversion products [AP, 3a-dihydroprogesterone (3a-DHP), 20a-dihydroprogesterone (20a-DHP) and 11-deoxycorticosterone (DOC)] (Fig. 1) and to determine the influence of FIN on the brain steroid levels and metabolism in rats under the stressful condition. MATERIALS AND METHODS Materials andReagents AP, 3a-DHP, 3b-DHP and 20a-DHP were obtained from Steraloids (Newport, RI, U.S.A.). PROG and DOC were purchased from Tokyo Kasei Kogyo (Tokyo, Ja...

“…It has been known that the brain PROG, 20a-DHP and DOC levels are also increased by acute stress 8,11) and similar results were reproduced in this study (Figs. 3c-e).…”

Section: Resultssupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

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Studies on Neurosteroids XXV. Influence of a 5.ALPHA.-Reductase Inhibitor, Finasteride, on Rat Brain Neurosteroid Levels and Metabolism

Mukai

Biological & Pharmaceutical Bulletin

et al. 2008

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“…[6][7][8] Industries (Osaka, Japan). The derivatization reagents, isonicotinoyl azide (INA) 6) and 2-hydrazino-1-methylpyridine (HMP), 9) were synthesized in our laboratories.…”

Section: Materials and Reagentsmentioning

confidence: 99%

Studies on Neurosteroids XXVI. Fluoxetine-Evoked Changes in Rat Brain and Serum Levels of Neuroactive Androgen, 5.ALPHA.-Androstane-3.ALPHA.,17.BETA.-diol

Biological & Pharmaceutical Bulletin

Shimada

et al. 2009

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Neuroactive steroids affect neurotransmission by action at the membrane ion-gated receptors and at other neurotransmitter receptors. 3a-Hydroxy-5a-pregnan-20-one (allopregnanolone, AP, Fig. 1), a representative neuroactive steroid, binds to the g-aminobutyric acid type A (GABA A ) receptors with a high affinity and positively modulates the action of GABA at these receptors, and hence elicits marked antidepressant, anxiolytic and anticonvulsant effects. 1) AP is synthesized from progesterone (PROG) via 5a-dihydroprogesterone (5a-DHP) by sequential reduction mediated by 5a-reductase and 3a-hydroxysteroid dehydrogenase (3a-HSD).Recent evidence also suggests that AP may be relevant to antidepressant drug action and depression pathophysiology.2,3) Fluoxetine (Fluox) is a typical selective serotonin (5-HT) reuptake inhibitor (SSRI) and is clinically used for the treatment of depression. However, Pinna et al. demonstrated that Fluox increases the brain AP content and subsequent activation of GABA A receptors at doses that are inactive toward the 5-HT reuptake, but does effectively treat depression. 3)Based on this result, Pinna et al. further stated that the term "SSRI" may be misleading in describing the pharmacological profile of Fluox and suggested the term "selective brain steroidogenic stimulant (SBSS)" as a better descriptor.3) Furthermore, rodent studies examining the Fluox-induced elevation in the brain AP level demonstrated that the effect is present to the same extent in both adrenalectomized and intact animals, suggesting that the Fluox-induced elevation in the brain AP level is due to de novo AP biosynthesis in the brain.2) 5a-Androstane-3a,17b-diol (3a,5a-Adiol, Fig. 1) is the best-characterized neuroactive androgen and derived from testosterone (T) by the action of 5a-reductase and 3a-HSD. 4) 3a,5a-Adiol is structurally similar to AP and is also a potent positive modulator of GABA A receptors with anxiolytic and anticonvulsant effects. 4,5) The anticonvulsant profile of 3a,5a-Adiol is highly consistent with AP, which have been proven by several animal experiments. 4) However, it is not currently known if Fluox influences the brain and serum levels of 3a,5a-Adiol. Examination of this point can be useful in evaluating the exact mechanisms of action of Fluox. Based on this background information, the objectives of this study are to determine the Fluox-evoked changes in the brain and serum 3a,5a-Adiol levels and to compare the level changes of 3a,5a-Adiol to those of AP. MATERIALS AND METHODS Materials and ReagentsAll the steroids including the deuterium-labeled internal standards (ISs) used in this study are those used in the previous studies.6-8) Each steroid was dissolved in and diluted with ethanol to prepare the standard solutions. Fluox was obtained from Wako Pure Chemical 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 g g-aminobutyric acid (G...

“…Determination of brain and serum AD and T. The brain and serum AD and T were quantified using a previously reported method (Higashi et al, 2008a).…”

Section: Animalsmentioning

confidence: 99%

Studies on neurosteroids XXIV. Determination of neuroactive androgens, androsterone and 5α‐androstane‐3α,17β‐diol, in rat brain and serum using liquid chromatography–tandem mass spectrometry

Yokoi

Biomedical Chromatography

et al. 2008

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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.