Masters Degrees (Biochemistry)

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Browsing Masters Degrees (Biochemistry) by Author "Barnard, Lise"

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    The biosynthesis of adrenal C 11-oxy C21 steroids, implicated in 21-hydroxylase deficiency-21-desoxycortisol and 21 desoxycortisone and their downstream metabolism
    (Stellenbosch : Stellenbosch University, 2017-03) Barnard, Lise; Swart, Amanda C.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Adrenal C19 steroids are often implicated in numerous androgen dependent disease conditions. Androgen excess is the hallmark of 21-hydroxylase deficiency (21OHD) fuelled by an increase in the production of adrenal androgens and androgen precursors. In addition, increased levels of progesterone (P4) and 17α-hydroxyprogesterone (17OHP4), the substrates of the defective cytochrome P450 steroid 21-hydroxylase, have also been reported. Adrenal steroids are secreted into circulation, for further downstream metabolism. Conversion of adrenal steroids to active androgens in peripheral target tissue is dependent on the tissue specific expression of key enzymes, which significantly influence steroid profiles at cellular level, ultimately influencing homeostasis in androgen responsive tissue. In 21OHD, the conversion of P4 and 17OHP4 in an alternative metabolic pathway to the potent androgen, dihydrotestosterone, is reported, which has been described as the backdoor pathway. The accumulation of P4 and 17OHP4 furthermore present substrates for 11β-hydroxylation by the cytochrome P450 11β-hydroxylase (CYP11B) isozymes. In this study the catalytic activity of the CYP11B isozymes towards P4 and 17OHP4 are presented. CYP11B1 and CYP11B2, also termed cytochrome P450 aldosterone synthase, transiently expressed in HEK293 cells, effectively utilised P4 and 17OHP4 as substrates, yielding 11β-hydroxyprogesterone (11OHP4) and 21-desoxycortisol (DOF). Catalytic conversions of DOF and 21-desoxycortisone (DOE), the C11-keto derivative of DOF, together with P4 and 17OHP4, were assayed in HEK293 cells transiently transfected with steroid 5α-reductase type 1 or type 2 (SRD5A1 or SRD5A2). Conversion of DOF, DOE, P4 and 17OHP4 by the SRD5A isozymes, yielded 5α-pregnan-11β, 17α-diol-3, 20-dione (11OHPdione) and 5α-pregnan-17α-ol-3, 11, 20-trione (11KPdione), dihydroprogesterone and 5α-pregnan-17α-ol-3, 20-dione (Pdione), respectively. We identified these novel steroids, 11OHPdione and 11KPdione, which were converted to the novel products 5α-pregnan-3α, 11β, 17α-triol-20-one and 5α-pregnane-3α, 17α-diol-11, 20-dione by 3α-hydroxysteroid dehydrogenase type 3 (AKR1C2) catalysed conversions in transiently transfected HEK293 cells by accurate mass determination. Metabolism of DOF in LNCaP cells, yielded DOE, 11β-hydroxyandrosterone and 11-ketoandrosterone, indicative of the conversion by endogenous 11β-hydroxysteroid dehydrogenase type 2, SRD5A, AKR1C2 and cytochrome P450 17α-hydroxylase/17, 20 lyase (CYP17A1) enzymes. These findings shows that DOF, produced in 21OHD, can be metabolised via the C11-oxy Pdione en Pdiol (5α-pregnan-3α, 17α-diol-20-one) intermediates to suitable substrates for the lyase activity of CYP17A1 thus leading to the production of C11-oxy C19 steroids. Taken together, the biosynthesis of C11-oxy C21 steroids, together with their metabolism by the enzymes in the backdoor pathway, yielded novel steroid metabolites contributing to the pool of potent androgens in 21OHD, with said steroids also presenting possible biomarkers in disease identification.