Isotope Dilution-Based Targeted and Nontargeted Carbonyl Neurosteroid/Steroid Profiling

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.