The in vitro inhibition of adrenal steroidogenic enzymes and modulation of adrenal hormone production by Sceletium tortuosum

dc.contributor.advisorSwart, Amandaen_ZA
dc.contributor.advisorSmith, Carineen_ZA
dc.contributor.authorJohannes, Ingeen_ZA
dc.contributor.otherStellenbosch University. Faculty of Sciences. Dept. of Biochemistry.en_ZA
dc.descriptionThesis (MSc)--Stellenbosch University, 2018.en_ZA
dc.description.abstractENGLISH ABSTRACT: This study describes: • The development and validation of an ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS) analytical method for the separation, detection and quantification of 17 adrenal steroid hormones, including two C11-oxy C21 steroids; and the application thereof in the kinetic characterisation of cytochrome P450 11β-hydroxylase (CYP11B1) towards androstenedione (A4) and testosterone (T): Kinetic characterisation showed that CYP11B1 exhibits an apparent Km towards A4 and T of 0.21 ± 0.07 μM and 0.34 ± 0.13 μM, respectively and Vmax values of 315.77 ± 22.46 and 158.89 ± 15.22 pmol/min/mg protein, respectively. • The investigation into the influence of a mesembrine-enriched extract of Sceletium tortuosum at 0.01 and 1 mg/mL (0.345 μM and 34.5 μM) on the catalytic activity of key adrenal steroidogenic enzymes: cytochrome P450 17β- hydroxylase/17,20-lyase (CYP17A1), 3β-hydroxysteroid dehydrogenase (3βHSD2), cytochrome P450 21- hydroxylase (CYP21A2), cytochrome P450 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2), expressed in HEK-293 cells: • The influence of S. tortuosum in comparison to abiraterone (Abi), 10 μM, on adrenal steroid hormone production in the H295R adrenal model endogenously expressing the full complement of steroidogenic enzymes, which catalyse the production of mineralocorticoids, glucocorticoids and adrenal androgens: UPC2-MS/MS analyses of the basal steroid metabolites showed that while Abi resulted in a greater inhibition of the steroid shunt in the androgen pathway than in the glucocorticoid pathway due to the potent inhibition of CYP17A1, S. tortuosum, resulted in a greater decrease in the steroid shunt in the glucocorticoid pathway in comparison to the androgen pathway, due to the inhibition of 3βHSD2, CYP21A2 and CYP11B1.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Hierdie studie beskryf: • Die ontwikkeling en validering van ‘n UPC2-MS/MS-analitiese metode vir die analise van 17 byniersteroïede, insluitend twee C11-oksi-C21-steroïede; die metode is vervolgens gebruik in die kinetiese karakterisering van sitochroom P450 11β- hidroksilase (CYP11B1) met androstenedioon (A4) en testosteroon (T) as substraat: Kineties karakterisering van CYP11B1 lewer ‘n oënskynlike Km van 0.21 ± 0.07 μM en 0.34 ± 0.13 μM vir A4 en T met Vmax waardes van 315.77 ± 22.46 en 158.89 ± 15.22 pmol/min/mg proteïen vir A4 en T, onderskeidelik. • Die ondersoek na die invloed van ‘n mesembrien-verykte ekstrak van S. tortuosum, teen 0.01 en 1 mg/mL (0.345 μM and 34.5 μM), op die aktiwiteit van bynierensieme wat steroïedbiosintese kataliseer: sitochroom P450 17β- hidroksilase/17,20-liase (CYP17A1), 3β-hidroksisteroïed dehidrogenase (3βHSD2), sitochroom P450 21- hidroksilase (CYP21A2), sitochroom P450 11β-hidroksilase (CYP11B1) en aldosteroonsintase (CYP11B2), uitgedruk in HEK-293 selle; S. tortuosum inhibeer die omsetting van natuurlike steroïedsubstrate gekataliseer deur 3βHSD2, CYP11B1 en CYP21A2. • Die invloed van S. tortuosum vergeleke met abirateroon (Abi), 10 μM, op steroïedhormoon produksie in H295R selle – ‘n menslike bynierkarsinoomsellyn wat die volle kompliment steroïed-kataliseerende ensieme bevat wat die produksie van mineralokortikoïede, glukokortikoïede en bynierandrogene kataliseer: UPC2-MS/MS analise van basale steroïedmetaboliete toon dat Abi (10uM) ‘n groter inhibisie op androgeen produksie het as gevolg van die inhibisie van CYP17A1 vergelykend met die produksie van glukokortikoïede, terwyl S. tortuosum ‘n afname in die produksie van glukokortikoïede tot gevolg het met ‘n kleiner invloed op die produksie van androgene, as gevolg van die inhibisie van 3βHSD2, CYP21A2 en CYP11B1.af_ZA
dc.format.extent94 pages : illustrationsen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.subjectAdrenal steroidogenic enzymesen_ZA
dc.subjectSceletium tortuosumen_ZA
dc.subjectMass spectrometryen_ZA
dc.titleThe in vitro inhibition of adrenal steroidogenic enzymes and modulation of adrenal hormone production by Sceletium tortuosumen_ZA
dc.rights.holderStellenbosch Universityen_ZA

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