An isotope dilution based-targeted and non-targeted carbonyl neurosteroid/steroid profiling

Sheila Sharp, Scott Mitchell, Monique Vallée, Elena Kuzmanova, Michelle Cooper, Delia Belelli, Jeremy Lambert, Jeffrey Huang (Lead / Corresponding author)

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Abstract

Neurosteroids are brain-derived steroids, capable of rapidly modulating neuronal excitability in a non-genomic manner. Dysregulation of their synthesis, or metabolism has been implicated in many pathological conditions. Here, we describe an isotope dilution based targeted and non-targeted (ID-TNT) profiling of carbonyl neurosteroids/steroids. The method combines stable isotope dilution, hydroxylamine derivatization, high-resolution MS scanning and data dependent MS/MS analysis, allowing absolute quantification of pregnenolone, progesterone, 5α-dihydroprogesterone, 3α,5α-tetrahydroprogesterone and 3β,5α-tetrahydroprogesterone, and relative quantification of other carbonyl containing steroids. The utility and validity of this approach was tested in an acute stress mouse model and via pharmacological manipulation of the steroid metabolic pathway with finasteride. We report that brain levels of 3α,5α-tetrahydroprogesterone, a potent enhancer of GABAA receptor (GABAAR-mediated inhibitory function, from control mice is in the 5-20 pmol/g range, a value greater than previously reported. The approach allows the use of data from targeted analysis to guide the normalization strategy for non-targeted data. Furthermore, novel findings, including a striking increase of brain pregnenolone following finasteride administration were discovered in this study. Collectively, our results indicate that this approach has distinct advantages for examining targeted and non-targeted neurosteroid/steroid pathways in animal models, and could facilitate a better understanding of the physiological and pathological roles of neurosteroids as modulators of brain excitability.
Original languageEnglish
Pages (from-to)5247-5255
Number of pages9
JournalAnalytical Chemistry
Volume90
Issue number8
Early online date21 Mar 2018
DOIs
Publication statusPublished - 17 Apr 2018

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Isotopes
Dilution
Neurotransmitter Agents
Steroids
Brain
Finasteride
Pregnenolone
Hydroxylamine
GABA-A Receptors
Metabolism
Modulators
Progesterone
Animals
Scanning

Cite this

Sharp, Sheila ; Mitchell, Scott ; Vallée, Monique ; Kuzmanova, Elena ; Cooper, Michelle ; Belelli, Delia ; Lambert, Jeremy ; Huang, Jeffrey. / An isotope dilution based-targeted and non-targeted carbonyl neurosteroid/steroid profiling. In: Analytical Chemistry. 2018 ; Vol. 90, No. 8. pp. 5247-5255.
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abstract = "Neurosteroids are brain-derived steroids, capable of rapidly modulating neuronal excitability in a non-genomic manner. Dysregulation of their synthesis, or metabolism has been implicated in many pathological conditions. Here, we describe an isotope dilution based targeted and non-targeted (ID-TNT) profiling of carbonyl neurosteroids/steroids. The method combines stable isotope dilution, hydroxylamine derivatization, high-resolution MS scanning and data dependent MS/MS analysis, allowing absolute quantification of pregnenolone, progesterone, 5α-dihydroprogesterone, 3α,5α-tetrahydroprogesterone and 3β,5α-tetrahydroprogesterone, and relative quantification of other carbonyl containing steroids. The utility and validity of this approach was tested in an acute stress mouse model and via pharmacological manipulation of the steroid metabolic pathway with finasteride. We report that brain levels of 3α,5α-tetrahydroprogesterone, a potent enhancer of GABAA receptor (GABAAR-mediated inhibitory function, from control mice is in the 5-20 pmol/g range, a value greater than previously reported. The approach allows the use of data from targeted analysis to guide the normalization strategy for non-targeted data. Furthermore, novel findings, including a striking increase of brain pregnenolone following finasteride administration were discovered in this study. Collectively, our results indicate that this approach has distinct advantages for examining targeted and non-targeted neurosteroid/steroid pathways in animal models, and could facilitate a better understanding of the physiological and pathological roles of neurosteroids as modulators of brain excitability.",
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An isotope dilution based-targeted and non-targeted carbonyl neurosteroid/steroid profiling. / Sharp, Sheila; Mitchell, Scott; Vallée, Monique; Kuzmanova, Elena; Cooper, Michelle; Belelli, Delia; Lambert, Jeremy; Huang, Jeffrey (Lead / Corresponding author).

In: Analytical Chemistry, Vol. 90, No. 8, 17.04.2018, p. 5247-5255.

Research output: Contribution to journalArticle

TY - JOUR

T1 - An isotope dilution based-targeted and non-targeted carbonyl neurosteroid/steroid profiling

AU - Sharp, Sheila

AU - Mitchell, Scott

AU - Vallée, Monique

AU - Kuzmanova, Elena

AU - Cooper, Michelle

AU - Belelli, Delia

AU - Lambert, Jeremy

AU - Huang, Jeffrey

N1 - We would like to thank Ms Karolina Wrobel at Biomarker and Drug Analysis Core Facility, as well as the Analytical Chemistry Platform (Neurocentre Magendie, Bordeaux for technical assistance.

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Y1 - 2018/4/17

N2 - Neurosteroids are brain-derived steroids, capable of rapidly modulating neuronal excitability in a non-genomic manner. Dysregulation of their synthesis, or metabolism has been implicated in many pathological conditions. Here, we describe an isotope dilution based targeted and non-targeted (ID-TNT) profiling of carbonyl neurosteroids/steroids. The method combines stable isotope dilution, hydroxylamine derivatization, high-resolution MS scanning and data dependent MS/MS analysis, allowing absolute quantification of pregnenolone, progesterone, 5α-dihydroprogesterone, 3α,5α-tetrahydroprogesterone and 3β,5α-tetrahydroprogesterone, and relative quantification of other carbonyl containing steroids. The utility and validity of this approach was tested in an acute stress mouse model and via pharmacological manipulation of the steroid metabolic pathway with finasteride. We report that brain levels of 3α,5α-tetrahydroprogesterone, a potent enhancer of GABAA receptor (GABAAR-mediated inhibitory function, from control mice is in the 5-20 pmol/g range, a value greater than previously reported. The approach allows the use of data from targeted analysis to guide the normalization strategy for non-targeted data. Furthermore, novel findings, including a striking increase of brain pregnenolone following finasteride administration were discovered in this study. Collectively, our results indicate that this approach has distinct advantages for examining targeted and non-targeted neurosteroid/steroid pathways in animal models, and could facilitate a better understanding of the physiological and pathological roles of neurosteroids as modulators of brain excitability.

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JO - Analytical Chemistry

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