Isotope Dilution-Based Targeted and Nontargeted Carbonyl Neurosteroid/Steroid Profiling
Anal. Chem.. 2018-03-21; 90(8): 5247-5255
DOI: 10.1021/acs.analchem.8b00055
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Sharp S(1), Mitchell SJ(1), Vallée M(2), Kuzmanova E(1), Cooper M(1), Belelli D(1), Lambert JJ(1), Huang JT(1).
Author information:
(1)School of Medicine , University of Dundee , Dundee , DD1 9SY , United
Kingdom.
(2)Université Bordeaux , Neurocentre Magendie, Physiopathologie de la Plasticité
Neuronale , INSERM U1215, F-33000 , Bordeaux , France.
Neurosteroids are brain-derived steroids, capable of rapidly modulating neuronal
excitability in a nongenomic manner. Dysregulation of their synthesis or
metabolism has been implicated in many pathological conditions. Here, we
describe an isotope dilution based targeted and nontargeted (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 5-40 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 nontargeted
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 nontargeted 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.
DOI: 10.1021/acs.analchem.8b00055
PMID: 29561593