Browsing by Author "Du Toit, Therina"

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    Falsely elevated plasma testosterone concentrations in neonates : importance of LC-MS/ MS measurements
    (De Gruyter, 2018-01) Hamer, Henrike M.; Finken, Martijn J. J.; Van Herwaarden, Antonius E.; Du Toit, Therina; Swart, Amanda C.; Heijboer, Annemieke C.
    In newborns with atypical genitalia, suspicious for a disorder of sex development (DSD), measurement of testosterone is an essential part in the diagnostic workup. Previously, direct testosterone immunoassays have proven to be inaccurate because they tend to overestimate testosterone concentrations in the lower ranges, such as those in females and infants, but specifically also in neonates. Based on the concern for cross-reactivity in neonatal samples, the recently revised UK guideline on the initial evaluation of DSD from the UK Society for Endocrinology recommends that steroids in plasma or serum are measured by either LC-MS/MS or immunoassays after organic solvent extraction. The use of LC-MS/MS was considered superior by a recent consensus meeting of DSD experts across Europe, although validation and quality control remain challenging.
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    An investigation into the influence of rooibos (Aspalathus linearis) on androgen metabolism in normal and prostate cancer cells
    (Stellenbosch : Stellenbosch University, 2015-04) Du Toit, Therina; Swart, Amanda C.; Storbeck, Karl-Heinz; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: In this study, the influence of rooibos on the catalytic activity of enzymes 17β -hydroxysteroid dehydrogenase type 3 (17βHSD3), 17β-hydroxysteroid dehydrogenase type 5 (AKR1C3), 17β-hydroxysteroid dehydrogenase type 2 (17βHSD2), 5α-reductase type 1 (SRD5A1) and 5α-reductase type 2 (SRD5A2), which catalyse prostate androgen metabolism, was investigated. The activities of both 17βHSD3 and AKR1C3 heterologously expressed in CHO-K1 and HEK293 cells were inhibited significantly by rooibos, with rooibos reducing the conversion of androstenedione (A4) and 11keto-androstenedione (11KA4) to testosterone (T) and 11ketotestosterone (11KT), respectively. The catalytic activity of 17βHSD2 towards T, 11hydroxytestosterone (11OHT) and 11KT was also significantly inhibited by rooibos in transiently transfected HEK293 cells. In transiently transfected HEK293 cells rooibos did not inhibit SRD5A1 while the rate of T conversion to dihydrotestosterone (DHT) by SRD5A2 was decreased. Analysis of steroid metabolism in PNT2 cells also suggests that rooibos does not modulate the catalytic activity of endogenously expressed SRD5A towards A4, however, the conversion of T to DHT was reduced. In addition, reductive 17βHSD activity towards A4 was inhibited in the presence of rooibos in both PNT2 and BPH-1 cells. In contrast, the conversion of 11KA4 to 11KT was inhibited in BPH-1, PC-3 and LNCaP cells, with negligible conversion of 11KA4 in PNT2 cells. Interestingly, data suggests inhibition of 3α-hydroxysteroid dehydrogenase type 3 (AKR1C2) activity in the production of androsterone (AST) from 5α–androstenedione (5α-dione), as well as the dehydrogenase reaction of T to A4 in PNT2 cells by rooibos. Androgen metabolism pathways were subsequently investigated in LNCaP cells to determine androgen metabolism by endogenous enzymes. Rooibos resulted in the reduced conversion of A4 in LNCaP cells to the same extent as indomethacin, a known AKR1C3 inhibitor. Rooibos also modulated T, DHT and AST metabolism in LNCaP cells. Furthermore, uridine diphosphate glucuronosyltransferase (UGT) activity in LNCaP cells was inhibited by rooibos, decreasing T-, DHT– and AST-glucuronide formation. These data prompted subsequent investigations into the influence of rooibos at cellular level, and prostatespecific antigen (PSA) levels were assayed in the presence of rooibos. PSA was significantly inhibited by rooibos in the absence and presence of DHT, suggesting possible interaction of rooibos with the mutated androgen receptor (AR) or estrogen receptor-β (ERβ) expressed in LNCaP cells. Taken together, rooibos inhibited the catalytic activity of key enzymes that catalyse the activation of androgens in the prostate, as well as inhibiting enzymes involved in the conjugation of androgens. At cellular level, PSA levels were also decreased by rooibos, possibly through AR or ERβ interactions – clearly indicating a modulatory role for rooibos in active androgen production.
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    The metabolism of 11β-hydroxyandrostenedione by steroidogenic enzymes yields metabolites contributing to the androgen pool in prostate cancer
    (Stellenbosch : Stellenbosch University, 2018-03) Du Toit, Therina; Swart, Amanda C.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: This study describes: • The development and validation of three ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS) analytical methods which were applied in the detection and quantification of C19 and C21 steroids, including C11-oxy C19 and C11-oxy C21 steroids; • The investigation into the contribution of adrenal 11β-hydroxyandrostenedione (11OHA4) and 11β-hydroxytestosterone (11OHT) to the pool of active androgens in the prostate, by following androgen metabolism in normal epithelial prostate PNT2, benign prostatic hyperplasia (BPH-1) and prostate cancer LNCaP, C4-2B and VCaP cell models; Steroid profiles revealed 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) activity in all the cell models, confirmed in the conversion of 11OHA4 to 11keto-androstenedione (11KA4), with reductive 17β-hydroxysteroid dehydrogenase (17βHSD) enzymes metabolising 11KA4, ultimately yielding 11keto-testosterone (11KT). • The in vitro investigation into the inactivation, reactivation, glucuronidation and sulfation of 11OHA4, 11OHT and their downstream metabolites; In prostate cancer (PCa) cell models, the conjugation of 11KT and 11ketodihydrotestosterone (11KDHT) were hampered compared to testosterone (T) and dihydrotestosterone (DHT), while the inactivation and reactivation of the C11-oxy C19 steroids were less efficient than the C19 steroids in BPH-1 cells. • The in vivo steroid profiles in PCa, BPH and castration-resistant prostate cancer (CRPC) tissue and plasma of healthy and PCa patients; Analyses of the C19 and C11-oxy C19 steroids, together with glucuronide and sulfate conjugates, showed increased unconjugated levels of 11KT and 11KDHT in plasma of PCa patients compared to a healthy subject, and 11OHA4, 11KT and 11KDHT levels were prominent in PCa tissue, while downstream inactive C11-oxy 3α-reduced metabolites were identified in BPH and CRPC tissue.