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Molecular characterisation of steroids in the mammalian brain

Ebner, Martin Johannes; (2001) Molecular characterisation of steroids in the mammalian brain. Doctoral thesis (Ph.D.), University College London (United Kingdom). Green open access

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Steroid hormones from endocrine glands are known to regulate mammalian brain development and function. They act not only by regulation of gene transcription, but also via modulation of neurotransmitter receptors. More recently, steroids have been found to be synthesised within the brain, in addition to entry from the circulation. However, there is little information on the full complement of steroid molecules present in the mammalian brain. In order to characterise brain steroid content, an improved procedure for the extraction and fractionation of steroids from nervous tissue was developed and used to generate samples from adult male rat brain suitable for analysis by gas capillary chromatography-electron impact mass spectrometry (GC-EIMS). After extraction of free steroids and sulphate conjugates with acetic acid/ethanol, samples were purified by solvent partitioning followed by hydrophilic-lipophilic balance extraction on polymer based sorbents. Free steroids and sulphate ester fractions could then be separated using mixed mode anion exchange chromatography, which can also be used to separate steroid glucuronides. For GC-EIMS, steroids were derivatised with methoxyamine (MO) and trimethylsilyl-imidazole (TMSI), heptafluorobutyric acid anhydride (HFBA) or N-methyl-N-(tert-butyl-dimethylsilyl)trifluoroacetamide. Diagnostic ion screening procedures were developed for a wide range of free and sulphate conjugated steroids likely to be encountered in mammalian tissue. In initial screening, two diagnostic ions were monitored for the MO/ TMSI derivatives. Further possible generic steroids were screened with 32 ion monitoring. Confirmation employed three ion monitoring of MO/TMSI and/or two ion monitoring of HFBA derivatives. The results unequivocally confirm the free steroids dehydroepiandrosterone, pregnenolone, progesterone, 20α-dihydropregnenolone, 20β-dihydropregnenolone, 3α,5α-tetrahydroprogesterone, 3α,5α-tetrahydrodeoxy- corticosterone, corticosterone, testosterone and 5α-pregnan-3α,17-diol-20-one as present in male rat brain. Further evidence was obtained for the unconjugated steroids 5α-dihydroprogesterone, 20α-dihydroprogesterone, 5α,20α-tetrahydroprogesterone, 5α- pregnane-3α,20α-diol, 3β,5α-tetrahydroprogesterone, 3β,5α-tetrahydrodeoxy-corticosterone and 5α-pregnan-3α,l lβ-diol-20-one. In the sulphate fraction, dehydroepiandrosterone was identified but not the previously described pregnenolone sulphate. The results provide further implications for steroids in brain function.

Type: Thesis (Doctoral)
Qualification: Ph.D.
Title: Molecular characterisation of steroids in the mammalian brain
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: (UMI)AAI10013862; Biological sciences; Steroid hormones
URI: https://discovery.ucl.ac.uk/id/eprint/10104164
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