Browsing by Author "Van Rooyen, Desmaré"

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    An investigation into the biosynthesis and metabolism of adrenal C11-oxy steroids
    (2020-12) Van Rooyen, Desmaré; Swart, Amanda C.; Verhoog, Nicolette J. D.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: This study describes: ▪ an in vitro investigation into the biosynthesis and metabolism of 11β-hydroxyprogesterone (11OHP4) and 21-deoxycortisol (21dF) by steroidogenic enzymes which include the 5α- reductases, 3α-reductases and cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1), as well as the interconversion of the C11-oxy C21 steroids by the 11β-hydroxysteroid dehydrogenase isozymes. The CYP17A1-catalysed conversion of the C11-oxy C21 steroids showed that while 11OHP4 and 11KP4 were readily hydroxylated at C17, negligible lyase activity was detected towards 21dF, 21-deoxycortisone (21dE) and the 5α-reduced C11-oxy C21 metabolites, while the 3α,5α- reduced C11-oxy C21 metabolites were converted to the 3α,5α-reduced C11-oxy C19 steroids. Homology modelling of 11OHP4, 5α-pregnan-11β-ol-3,20-dione (11OH-DHP4), and their keto derivatives, showed that these steroids have similar orientations in the active site of CYP17A1 compared to P4 while the orientation of 5α-pregnan-3α,11β-diol-20-one and 5α-pregnan-3α-ol- 11-20-dione was similar to 17α-hydroxypregnenolone. ▪ the transactivation by the androgen- (AR) and progesterone receptor (PR) isoform A and B in response to the C11-oxy C21 steroids, and transactivation by the PR isoforms in response to C11-oxy C19 steroids. At 100 nM, 11OHP4, 11OH-DHP4 and 21dF exhibited agonist activity towards the AR, PR-A and PR-B, with comparable agonist activity at 10 nM with the exception of 21dF. The 11-keto derivatives exhibited negligible agonist activity towards the AR and PR. The drawbacks of commonly used luciferase reporter promoter assay protocols were highlighted. ▪ steroid profile analysis of serum and salivary samples of male athletes provided evidence of C11-oxy backdoor pathway activity in vivo. Analysis of unconjugated circulating steroids generated comprehensive steroid profiles. No significant differences in steroid levels were detected between the two age groups and the ethnic groups. Fewer steroids were detected in salivary samples with the C11-oxy steroids being the predominant steroids. Inter-individual variation in steroid levels highlighted the clinical benefits of comprehensive steroid profiling for the diagnosis and monitoring of endocrine conditions. ▪ the development and validation of ultra-performance convergence chromatography tandem mass spectrometry (UPC²-MS/MS) methods which were used for the detection and quantification of C19, C21, C11-oxy C19 and/or C11-oxy C21 steroids in cell culture medium, in serum and in salivary samples.