Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography–mass spectrometry

Lière, Philippe; Akwa, Yvette; Weill-Engerer, S.; Eychenne, Bernard; Pianos, Antoine; Röbel, P; Sjövall, Jan; Schumacher, Michaël; Baulieu, Étienne-Émile

doi:10.1016/s0378-4347(99)00563-0

Cited by 150 publications

(112 citation statements)

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“…According to the brain levels of PREGS previously reported (more than 5 ng/g tissue), [4][5][6] the ELISA is able to determine them with less than 20 mg of tissue. However, anticipating that the concentration was lower, 7) 200 mg of tissue was used except for the study on the influence of sample volume.…”

Section: Applicability Of the Elisa To The Determination Of Pregs In mentioning

confidence: 98%

“…The levels of PREG sulfate (PREGS) have conventionally been evaluated by measuring its genin formed after solvolysis by GC-MS 4,5) or radioimmunoassay (RIA). 6) However, these methods have some shortcomings, such as requirement of complicated pretreatment and solvolysis steps and a decline in the recovery rate of the steroid.…”

mentioning

confidence: 99%

“…To overcome these problems, we employed liquid chromatography (LC)-electrospray ionization (ESI)-MS-MS to determine PREGS in rat brains without deconjugation, 7) but contrary to our expectations, its levels were less than one tenth of those previously reported (more than 5 ng/g tissue). [4][5][6] One potential explanation for this discrepancy is that the LC-MS-MS method is more specific for brain samples than the GC-MS or RIA assay combined with solvolysis; the GC-MS may be detecting PREG derived from PREGS as well as that from other conjugated forms, such as sulfolipid conjugate, by solvolysis, and the RIA employs an antibody that reacts with other steroids having structures closely related to that of PREG. Although our LC-MS-MS method was specific and did not need a solvolysis procedure, it did not have sufficient sensitivity for the analysis of PREGS in the brain (limit of quantitation: ca.…”

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confidence: 99%

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Studies on Neurosteroids XVI. Levels of Pregnenolone Sulfate in Rat Brains Determined by Enzyme-linked Immunosorbent Assay Not Requiring Solvolysis

Higashi

Sugitani

Yagi

et al. 2003

Biological & Pharmaceutical Bulletin

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In the 1980s, Baulieu and co-workers demonstrated that some steroids, such as pregnenolone (PREG), dehydroepiandrosterone and their sulfates, are present in higher concentrations in the brain than in blood and are synthesized de novo in the central nervous system. 1) Such steroids are now universally referred to as neurosteroids. They act as modulators of several neurotransmitter receptors (g-aminobutyric acid A , N-methyl-D-aspartate and s 1 receptors) either as stimulators or inhibitors 1,2) and are involved in learning and memory performance.3) For these reasons, there is a considerable interest in determining the location and levels of different neurosteroids in the brain.The levels of PREG sulfate (PREGS) have conventionally been evaluated by measuring its genin formed after solvolysis by 5) or radioimmunoassay (RIA). 6) However, these methods have some shortcomings, such as requirement of complicated pretreatment and solvolysis steps and a decline in the recovery rate of the steroid. To overcome these problems, we employed liquid chromatography (LC)-electrospray ionization (ESI)-MS-MS to determine PREGS in rat brains without deconjugation, 7) but contrary to our expectations, its levels were less than one tenth of those previously reported (more than 5 ng/g tissue).4-6) One potential explanation for this discrepancy is that the LC-MS-MS method is more specific for brain samples than the GC-MS or RIA assay combined with solvolysis; the GC-MS may be detecting PREG derived from PREGS as well as that from other conjugated forms, such as sulfolipid conjugate, by solvolysis, and the RIA employs an antibody that reacts with other steroids having structures closely related to that of PREG. Although our LC-MS-MS method was specific and did not need a solvolysis procedure, it did not have sufficient sensitivity for the analysis of PREGS in the brain (limit of quantitation: ca. 160 pg/injection), and the obtained data using ca. 400 mg of tissue were not so reliable.7) Based on the results, we developed an enzyme-linked immunosorbent assay (ELISA) for PREGS using an antibody raised against 11a-hemiglutaryloxy-PREGS-bovine serum albumin conjugate. 8) This ELISA is sensitive (limit of quantitation: 1 pg/well) and does not require solvolysis procedure. The present paper describes the application of the ELISA to the determination of PREGS in rat brains and some data concerning the source of the brain PREGS. MATERIALS AND METHODS Materials and ReagentsStandard PREGS, PREG and all other reagents were those used in the previous study. 8)Oasis HLB cartridges (60 mg: Waters Assoc., Milford, MA, U.S.A.) were successively washed with AcOEt (1 ml), EtOH (2 ml) and H 2 O (2 ml) prior to use.Rats and Treatment Wistar strain rats (7 weeks old, male, 190-200 g) obtained from Japan S.L.C. (Hamamatsu, Japan) were used and euthanized by decapitation. Brain samples were collected from the rats assigned either to a control group (without injection, nϭ10), a group of animals receiving an intrapertioneal (i.p.) vehicle injection (5% EtOH/ s...

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Section: Applicability Of the Elisa To The Determination Of Pregs In mentioning

confidence: 98%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Studies on Neurosteroids XVI. Levels of Pregnenolone Sulfate in Rat Brains Determined by Enzyme-linked Immunosorbent Assay Not Requiring Solvolysis

Higashi

Sugitani

Yagi

et al. 2003

Biological & Pharmaceutical Bulletin

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“…Intratesticular contents of T and DHT were measured by gas chromatography-mass spectrometry as previously described (Liere et al, 2000;Meffre et al, 2007).…”

Section: Measurements Of Hormones and Igf-i Levels And Gonadotropinrementioning

confidence: 99%

Conditional Inactivation of Androgen Receptor Gene in the Nervous System: Effects on Male Behavioral and Neuroendocrine Responses

Raskin¹,

Gendt²,

Duittoz³

et al. 2009

J. Neurosci.

191

221

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Testosterone (T) profoundly influences central sexual differentiation and functions. In the brain, T signals either directly through androgen receptor (AR) or indirectly through estrogen receptor (ER) following aromatization into E2 (17-␤-estradiol).As T, through AR, also controls peripheral male sexual differentiation, the relative contribution of central AR in T-mediated regulation of behavioral and neuroendocrine responses still remains unclear. To address this question, we generated, by using Cre-loxP technology, mice selectively lacking AR expression in the nervous system. The mutant male urogenital tract was normally developed, and mice were able to produce offspring. Nonetheless, sexual motivation and performance as well as aggressive behaviors were affected. Only a low percentage of males displayed a complete sexual behavior and offensive attacks. The latency to show masculine behaviors was increased and copulation length prolonged. Erectile activity during mating was also altered. These alterations occurred despite increased levels of T and its metabolites, and an unaffected number of ER␣-immunoreactive cells. Olfactory preference and neuronal activation, mapped by Fos immunoreactivity, following exposure to estrus female-soiled bedding were also normal. At comparable T levels, greater differences in masculine behaviors were observed between gonadectomized control and mutant males. AR invalidation in the nervous system also disrupted the somatotropic axis since mutant males exhibited growth retardation and decreased serum levels of insulin-like growth factor I. Our findings show that central AR is required in T-induced regulation of male-typical behaviors and gonadotrope and somatotropic axes. This genetic model offers a unique opportunity in the understanding of AR's role in cerebral functions of T.

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“…While widely used, a number of reports have questioned the accuracy of RIA assessments, noting that while RIAs provided highly sensitive assays, cross-reactivity of antibodies, interference with antibody binding by contaminating substances and limitations imposed by extraction procedures are confounds that are likely to underlie the appreciable variation in neurosteroid concentrations reported even within a given sex and species (for discussion, see Cheney et al, 1995;Liu et al, 2003;Liere et al, 2000;2004). Two alternative approaches, enzyme-linked immunosorbent (ELISA) assays performed following separation of crossreactive steroids, and liquid or gas chromatography coupled with mass spectrometry or mass fragmentographic analyses, have provided revised and appreciably lower estimates of brain neurosteroid concentrations.…”

Section: Neurosteroid and Anabolic Androgenic Steroid Levels In The Cnsmentioning

confidence: 99%

Steroid modulation of GABAA receptor-mediated transmission in the hypothalamus: Effects on reproductive function

Henderson

2007

Neuropharmacology

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The hypothalamus, the seat of neuroendocrine control, is exquisitely sensitive to gonadal steroids. For decades it has been known that androgens, estrogens and progestins, acting through nuclear hormone receptors, elicit both organizational and activational effects in the hypothalamus and basal forebrain that are essential for reproductive function. While changes in gene expression mediated by these classical hormone pathways are paramount in governing both sexual differentiation and the neural control of reproduction, it is also clear that steroids impart critical control of neuroendocrine functions through nongenomic mechanisms. Specifically, endogenous neurosteroid derivatives of deoxycorticosterone, progesterone and testosterone, as well and synthetic anabolic androgenic steroids that are self-administered as drugs of abuse, elicit acute effects via allosteric modulation of γ-aminobutyric acid type A receptors. GABAergic transmission within the hypothalamus and basal forebrain is a key regulator of pubertal onset, the expression of sexual behaviors, pregnancy and parturition. Summarized here are the known actions of steroid modulators on GABAergic transmission within the hypothalamus/basal forebrain, with a focus on the medial preoptic area and the supraoptic/paraventricular nuclei that are known to be central players in the control of reproduction.

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Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography–mass spectrometry

Cited by 150 publications

References 33 publications

Studies on Neurosteroids XVI. Levels of Pregnenolone Sulfate in Rat Brains Determined by Enzyme-linked Immunosorbent Assay Not Requiring Solvolysis

Studies on Neurosteroids XVI. Levels of Pregnenolone Sulfate in Rat Brains Determined by Enzyme-linked Immunosorbent Assay Not Requiring Solvolysis

Conditional Inactivation of Androgen Receptor Gene in the Nervous System: Effects on Male Behavioral and Neuroendocrine Responses

Steroid modulation of GABAA receptor-mediated transmission in the hypothalamus: Effects on reproductive function

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