Browsing by Author "Barnard, Lise"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    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.
  • Thumbnail Image
    Item
    Human steroid biosynthesis, metabolism and excretion are differentially reflected by serum and urine steroid metabolomes : a comprehensive review
    (Elsevier, 2019-07-27) Schiffer, Lina; Barnard, Lise; Baranowski, Elizabeth S.; Gilligan, Lorna C.; Taylor, Angela E.; Arlt, Wiebke; Shackleton, Cedric H. L.; Storbeck, Karl-Heinz
    Advances in technology have allowed for the sensitive, specific, and simultaneous quantitative profiling of steroid precursors, bioactive steroids and inactive metabolites, facilitating comprehensive characterization of the serum and urine steroid metabolomes. The quantification of steroid panels is therefore gaining favor over quantification of single marker metabolites in the clinical and research laboratories. However, although the biochemical pathways for the biosynthesis and metabolism of steroid hormones are now well defined, a gulf still exists between this knowledge and its application to the measured steroid profiles. In this review, we present an overview of steroid hormone biosynthesis and metabolism by the liver and peripheral tissues, specifically highlighting the pathways linking and differentiating the serum and urine steroid metabolomes. A brief overview of the methodology used in steroid profiling is also provided.
  • Thumbnail Image
    Item
    An investigation into the biosynthesis, metabolism and activity of 11-oxygenated estrogens
    (Stellenbosch : Stellenbosch University, 2020-04) Barnard, Lise; Storbeck, Karl-Heinz; Arlt, Wiebke; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Within recent years, a growing number of studies have implicated 11-oxygenated androgens as important role players in women’s health. Significantly, 11-ketotestosterone (11KT) circulates at levels equal to or greater than that of testosterone (T) in healthy women, with elevated levels observed in patients with polycystic ovary syndrome (PCOS) or congenital adrenal hyperplasia (CAH). Given that androgens are obligatory precursors to produce estrogens, a conversion catalysed by cytochrome P450 aromatase (aromatase), it is important to consider whether 11-oxygenated androgens can be aromatized into a corresponding group of 11-oxygenated estrogens. This study shows for the first time that 11-oxygenated androgens are indeed substrates for aromatase leading to the production of 11-oxygenated estrogens, a novel group of bioactive estrogens not previously considered in human physiology. Using several aromatase expressing systems, we also show that 11-oxygenated estrogens can be produced from the aromatase catalysed 11β-hydroxylation of classic estrogens, a reaction never before reported for this enzyme. We go on to show that the 11-oxygenated estrogen, 11β-hydroxyestradiol (11OHE2), binds to and activates both subtypes of the human estrogen receptor (ERα and ERβ), stimulates ER-dependent proliferation and upregulates ER-regulated gene expression in estrogen sensitive breast cancer cell lines. In addition, this study investigated the metabolism of 11KT by steroid 5α-reductase isozymes (SRD5A1 and SRD5A2), and steroid 5β-reductases (AKR1D1). We show for the first time that AKR1D1 catalyses the inactivation of 11KT committing its metabolism to a pathway that feeds into the production of the urinary steroid 11-ketoetiocholanolone (11KEt), which has to date only been considered a metabolite of the abundant glucocorticoid, cortisone. Collectively, this study provides valuable insights into the metabolism of 11-oxygenated androgens. The identification of a novel class of 11-oxygenated estrogens further challenges long-established dogmas in the field of endocrinology and shows that the contribution of the 11-oxygenated androgens, and by extension, 11-oxygenated estrogens, can no longer be disregarded.
  • Thumbnail Image
    Item
    Steroid metabolome analysis in disorders of adrenal steroid biosynthesis and metabolism
    (Endocrine Society, 2019-07-11) Storbeck, Karl-Heinz; Schiffer, Lina; Baranowski, Elizabeth Sarah; Chortis, Vasileios; Prete, Alessandro; Barnard, Lise; Gilligan, Lorna; Taylor, Angela, E.; Idkowiak, Jan; Arlt, Wiebke; Shackleton, Cedric H. L.
    Steroid biosynthesis and metabolism are reflected by the serum steroid metabolome and, in even more detail, by the 24-hour urine steroid metabolome, which can provide unique insights into alterations of steroid flow and output indicative of underlying conditions. Mass spectrometry–based steroid metabolome profiling has allowed for the identification of unique multisteroid signatures associated with disorders of steroid biosynthesis and metabolism that can be used for personalized approaches to diagnosis, differential diagnosis, and prognostic prediction. Additionally, steroid metabolome analysis has been used successfully as a discovery tool, for the identification of novel steroidogenic disorders and pathways as well as revealing insights into the pathophysiology of adrenal disease. Increased availability and technological advances in mass spectrometry–based methodologies have refocused attention on steroid metabolome profiling and facilitated the development of high-throughput steroid profiling methods soon to reach clinical practice. Furthermore, steroid metabolomics, the combination of mass spectrometry–based steroid analysis with machine learning–based approaches, has facilitated the development of powerful customized diagnostic approaches. In this review, we provide a comprehensive up-to-date overview of the utility of steroid metabolome analysis for the diagnosis and management of inborn disorders of steroidogenesis and autonomous adrenal steroid excess in the context of adrenal tumors.