Institutionen för medicinsk biokemi och biofysik (MBB) / Department of Medical Biochemistry and Biophysics
Abstract
Neurosteroids are steroids synthesised in the central and peripheral
nervous systems. Known neurosteroids include pregnenolone,
dehydroepiandrosterone (DHEA), progesterone and its reduced metabolites.
It has been demonstrated that neurosteroids modulate neurotransmission by
binding to neurotransmitter receptors, and exert physiological functions
that are clearly different from those of endocrine steroids. The effects
of neurosteroids on improving the memory of cognitively impaired aged
rats, on the inhibition of aggressiveness in castrated male mice, and
trophic effects on neuronal regeneration and remyelination have been
documented. The local synthesis, selective interaction with
neurotransmitter receptors and behavioural effects of neurosteroids
strongly suggests that they may have important physiological or
pathophysiological roles. There is an increasing need to develop methods
to analyse these hormones with high sensitivity and high specificity. In
this thesis I focused on the development of methods combining
nano-electrospray (ES) mass spectrometry with capillary column liquid
chromatography (CLC) for the analysis of profiles of neurosteroids in rat
brain. It was also an aim to make the methods applicable to a broad range
of lipophilic biomolecules.
Initially, synthetic steroid sulphates and unconjugated oxosteroids
(ketosteroids) were studied by nano-ES and tandem mass spectrometry.
Steroid sulphates could be detected as deprotonated molecules in full
range scanned spectra at a level of 1 pg/µL. Information about steroid
structure was obtained from collision-induced dissociation (CID) spectra
of 1 ng of steroid sulphate, while characterisation of the sulphate ester
group required only 3 pg of material. Unconjugated oxosteroids were
converted into their oximes which were detected as protonated molecules
with 20 times higher sensitivity than the underivatised steroids. The
detection limits for the oximes of 3-oxo-delta4, 20-oxo and 17-oxo
steroids were 2.5, 5, and 25 pg/µL, respectively in full range scans. CID
spectra of the protonated oximes provided valuable information regarding
the position of oxo and hydroxyl group(s). These studies established a
basis for determination and structure characterisation of neurosteroids
from brain samples
A procedure for CLC-ES mass spectrometry was then developed. A double
splitter method was introduced which made it possible to use a pre-column
for analyte focusing from large sample volumes. It also made it possible
to operate the solvent pumps at flow rates compatible with gradient
elution while the flow rates through the analytical column were
compatible with micro-electrospray. The method was designed to be
generally applicable to the analysis of biomolecules and its utilities
were demonstrated by the analysis of steroid sulphates, in human plasma.
In the course of these studies, certain CLC-ES conditions were found to
cause on-column chemical transformations of 3beta- hydroxy-delta5 steroid
sulphates. Radical species generated from electrolysis of water and
methanol in the solvent are proposed to be responsible for the formation
of oxidised and methoxylated products of these steroids. Other analytes
with double bonds were also transformed under these conditions. Thus,
on-column electrochemistry can be an important source of artefacts in
analyses by CLC-ES mass spectrometry. The reactions could be prevented by
appropriate grounding.
The analysis of neurosteroids in rat brain required the development of an
extraction, purification and subfractionation procedure. Brain steroids
were extracted, and unconjugated neutral steroids and sulphated steroids
were separated. The steroid sulphate fraction was then analysed by CLC-ES
mass spectrometry. Endogenous sulphates of pregnenolone and DHEA were not
detected at levels above the detection limit, 0.3 ng/g wet brain, while
pregnenolone sulphate, added to brain extract at a level of 6.6 ng/g, was
easily detected. The unconjugated oxosteroids were converted to their
oximes, selectively isolated on a cation exchanger, and analysed by
CLC-ES tandem mass spectrometry. The chromatograms showed the presence of
progesterone, pregnenolone, pregnanolone isomers, DHEA and testosterone
in rat brain. These steroids were characterised by tandem mass
spectrometry, Based on the results of CLC-ES tandem mass spectrometry,
the levels Of C21 and C19 steroids were estimated in the range of 0.04 -
20 ng/g wet brain. The levels of progesterone and testosterone showed a
sex difference.
During the development of the above analytical methods, nano-ES mass
spectrometry was applied to the characterisation of a lipophilic
modulatory factor isolated from mouse brain. The factor, which activated
the retinoid X receptor (RXR), was extracted from mouse brain incubates,
purified by HPLC and analysed by nano-ES and tandem mass spectrometry.
Accurate mass measurement and CID spectra of the purified active compound
revealed that it was cis-4,7,10,13,16,19docosahexaenoic acid.
In conclusion, the methods developed and described in this thesis are
suitable for the analysis of sulphated steroids and oxosteroids, as well
as other related compounds. With their high sensitivity the methods
enable highly specific analysis of these important compounds from small
amounts of sample