Basics of androgen synthesis and action.

Androgens are essential sex steroid hormones for both sexes. Testosterone (T) is the predominant androgen in males, while in adult females, T concentrations are about 15-fold lower and androgen precursors are converted to estrogens. T is produced primarily in testicular Leydig cells in men, while in women precursors are biosynthesised in the adrenal cortex and ovaries and converted into T in the periphery. The biosynthesis of T occurs via a series of enzymatic reactions in steroidogenic organs. Notably, the more potent androgen, dihydrotestosterone, may be synthesized from T in the classic pathway, however, alternate metabolic pathways also exist. The classic action of androgens on target organs is mediated through the androgen receptor, which regulates nuclear receptor gene transcription. However, the androgen-androgen receptor complex may also interact directly with membrane proteins or signaling molecules to exert more rapid effects. This review summarizes the current knowledge of androgen biosynthesis, mechanisms of action and endocrine effects in human biology, and relates these effects to respective human congenital and acquired disorders

Development and function of the fetal adrenal.

The adrenal cortex undergoes multiple structural and functional rearrangements to satisfy the systemic needs for steroids during fetal life, postnatal development, and adulthood. A fully functional adrenal cortex relies on the proper subdivision in regions or 'zones' with distinct but interconnected functions, which evolve from the early embryonic stages to adulthood, and rely on a fine-tuned gene network. In particular, the steroidogenic activity of the fetal adrenal is instrumental in maintaining normal fetal development and growth. Here, we review and discuss the most recent advances in our understanding of embryonic and fetal adrenal development, including the known causes for adrenal dys-/agenesis, and the steroidogenic pathways that link the fetal adrenal with the hormone system of the mother through the fetal-placental unit. Finally, we discuss what we think are the major open questions in the field, including, among others, the impact of osteocalcin, thyroid hormone, and other hormone systems on adrenal development and function, and the reliability of rodents as models of adrenal pathophysiology

Parallel targeted and non-targeted quantitative analysis of steroids in human serum and peritoneal fluid by liquid chromatography high-resolution mass spectrometry

We developed and validated a liquid chromatography high-resolution mass spectrometry method for the absolute quantification of 51 steroids for clinical analysis of human serum and, for the first time, peritoneal fluid. Data acquisition was performed in both targeted and untargeted mode simultaneously, thus allowing the accurate and precise quantification of the main components of the classical steroid pathways (17 steroids) as well as the analysis of 34 additional non-classical steroids. For targeted analysis, validation was performed according to FDA guidelines, resulting, among other parameters, in accuracy < 13% RSD and precision < 10% relative error, for both inter- and intra-day validation runs. By establishing steroid-specific response factors, the calibration curves of the targeted analytes can be extended to untargeted analytes. This approach opens novel possibilities for the post hoc analysis of clinical samples as the data can be examined for virtually any steroid even after data acquisition, enabling facile absolute quantification once a standard becomes available. We demonstrate the applicability of the approach to evaluate the differences in steroid content between peripheral serum and peritoneal fluid across the menstrual cycle phases, as well as the effect of the synthetic gestagen dienogest on the steroid metabolome. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00216-022-03881-3

The serum steroid signature of PCOS hints at the involvement of novel pathways for excess androgen biosynthesis.

CONTEXT Polycystic ovary syndrome (PCOS) is defined by androgen excess and ovarian dysfunction in the absence of a specific physiological diagnosis. The best clinical marker of androgen excess is hirsutism, while the best biochemical parameter is still a matter of debate. Current consensus guidelines recommend, among other hormones, serum free testosterone as an important serum parameter to measure androgen excess. Recently, however, novel active androgens and androgen metabolic pathways have been discovered. OBJECTIVE To assess the contribution of novel androgens and related steroid biosynthetic pathways to the serum steroid pool in PCOS women in comparison to healthy controls. DESIGN This is a case control study, wherein PCOS was diagnosed according to the AE-PCOS 2009 criteria. Serum steroid profiling was performed by liquid chromatography high-resolution mass spectrometry. SETTING Yeditepe University and associated clinics in Istanbul, Turkey, together with Bern University Hospital Inselspital, Bern, Switzerland. PARTICIPANTS 42 PCOS women and 42 matched, healthy control women. MAIN OUTCOME MEASURES Assessment of 34 steroids compartmentalized in four androgen related pathways: the classic androgen pathway, the backdoor pathway, the C11-oxy backdoor pathway, and the C11-oxy (11β-hydroxyandrostenedione) pathway. RESULTS Metabolites of all four pathways were identified in healthy and PCOS women. Highest concentrations were found for progesterone in controls and androstenedione in PCOS. Lowest levels were found for 11-ketotestosterone in controls compared to PCOS, and for 20α-hydroxyprogesterone in PCOS compared to controls. PCOS also had higher serum testosterone levels compared to the controls. PCOS women had overall higher levels of steroid metabolites of all four androgen pathways compared to healthy controls. CONCLUSIONS Novel alternative pathways contribute to the androgen production in healthy and PCOS women. Hyperandrogenism in PCOS is characterized by an overall increase of serum androgens in the classic, backdoor and C11-oxy pathways. While monogenetic disorders of steroid biosynthesis can be recognized by a specific pattern in the steroid profile, no diagnostic pattern or classifier was found in the serum for PCOS

Falsely elevated plasma testosterone concentrations in neonates : importance of LC-MS/ MS measurements

CITATION: Hamer, H.M. et al. 2018. Falsely elevated plasma testosterone concentrations in neonates : importance of LC-MS/ MS measurements. Clinical Chemistry and Laboratory Medicine (CCLM), 56(6):e141–e143, doi:10.1515/cclm-2017-1028.The original publication is available at https://www.degruyter.com/view/j/cclmIn 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.Publishers versio

The metabolism of 11β-hydroxyandrostenedione by steroidogenic enzymes yields metabolites contributing to the androgen pool in prostate cancer

Thesis (PhD)--Stellenbosch University, 2018.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.AFRIKAANSE OPSOMMING: Hierdie studie beskryf die volgende: • Die ontwikkeling en validering van drie UPC2-MS/MS analitiese metodes wat vervolgens toegepas is in die skeiding en kwantifisering van steroïedmetaboliete. • Die ondersoek na 11OHA4 and 11OHT se bydrae tot die aktiewe androgeen poel in die prostaat; Androgeen metabolisme is in normale epiteel prostaat PNT2, BPH-1 en prostaat kanker LNCaP, C4-2B en VCaP selmodelle ondersoek. Die steroïdprofiele bevestig die teenwoordigheid van 11βHSD2 aktiwiteit in al die modelle in die omsetting van 11OHA4 na 11KA4, sowel as die aktiwiteit van 17βHSD wat vervolgens die omsetting van 11KA4 na 11KT gekataliseer het. • Die in vitro inaktivering, heraktivering en konjugering van 11OHA4 en 11OHT metaboliete; In prostaatkanker selmodelle was 11KT en 11KDHT konjugering oneffektief in vergelyking met T en DHT, en die inaktivering van die C11-oksie C19 steroïede was nie optimaal in vergelyking met die C19 steroïede in BPH-1 selmodelle nie. • Die in vivo steroïedprofiele in prostaatkankerweefsel, BPH weefsel en kastrasieweerstandige prostaatkankerweefsel en in plasma van gesonde normale individuë en prostaatkanker pasïente, asook die teenwoordigheid van ongekonjugeerde en gekonjugeerde steroïedmetaboliete in sirkulasie; Analiese toon dat ongekonjugeerde 11KT en 11KDHT hoër was in die plasma van pasïente met prostaatkanker; 11OHA4, 11KT en 11KDHT vlakke was abnormaal hoog in die prostaatkankerweefsel, terwyl onaktiewe C11-oksie 3α-gereduseerde metaboliete geïdentifiseer was in BPH weefsel en in kastrasie-weerstandige prostaatkankerweefsel

An investigation into the influence of rooibos (Aspalathus linearis) on androgen metabolism in normal and prostate cancer cells

Thesis (MSc)--Stellenbosch University, 2015.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.AFRIKAANSE OPSOMMING: In hierdie studie was die invloed van rooibos ten opsigte van die katalitiese aktiwiteite van die ensieme 17β-hidroksi-steroïed-dehidrogenase tipe 2, tipe 3 en tipe 5 (17βHSD2, 17βHSD3, AKR1C3), asook 5α-reduktase tipe 1 en tipe 2 (SRD5A1, SRD5A2) ondersoek. Hierdie ensieme is betrokke in die produksie van androgene in die prostaat. Rooibos het die katalitiese aktiwiteit van 17βHSD3 en AKR1C3 in CHO-K1 en HEK293 selle beïnvloed en het vermindere omskakeling van androstenedioon (A4) en 11keto-androstenedioon (11KA4) na testosteroon (T) en 11-ketotestosteroon (11KT), afsonderlik, veroorsaak. Die katalitiese aktiwiteit van 17βHSD2 teenoor T, 11-hidroksie-testosteroon (11OHT) en 11KT was ook beïnvloed in die teenwoordigheid van rooibos in HEK293 selle. Die katalitiese aktiwiteit van SRD5A1 teenoor A4 en T is nie beïnvloed deur rooibos nie, alhoewel dit voorkom asof rooibos die omsettingstempo van T na dihidrotestosteroon (DHT) deur SRD5A2, getransfekteer in HEK293 selle, verminder het. Verdere ondersoeke is in normale prostaat epiteel selle, in die teenwoordigheid van rooibos uitgevoer. Rooibos het geen invloed op die katalitiese aktiwiteit van SRD5A teenoor A4 gehad nie, alhoewel vermindere omskakeling van T na DHT aangetoon kon word. Rooibos het ook die omskakeling van A4 na T in beide PNT2 en BPH-1 selle tot „n mate geïnhibeer. Die omskakeling van 11KA4 na 11KT was ook verminder in BPH-1, PC-3 en LNCaP selle. Die omskakeling van 11KA4 na 11KT was beduidend laer in PNT2 selle en kon die invloed van rooibos nie aangetoon word nie. Bykomende data toon dat rooibos ook die omskakeling van 5α-androstenedioon (5α-dione) na androsteroon (AST), gekataliseer deur 3α-hidroksi-dehidrogenase tipe 3 (AKR1C2), verminder, gesamentlik met die vermindere omskakeling van T na A4, deur 17βHSD2, in PNT2 selle. Hierdie studie het ook ondersoek ingestel, na die metabolisme van androgene in LNCaP selle. Vermindere A4 metabolisme is in die teenwoordigheid van rooibos asook in die teenwoordigheid van indometasien, „n bekende AKR1C3 inhibitor, gevind. Rooibos verminder dus die aktiwiteit van reduktiewe 17βHSD in LNCaP selle. Verandering in die metabolisme van T, DHT en AST in LNCaP selle, in die teenwoordigheid van rooibos, is ook gevind. Verdere ondersoek in LNCaP selle het gewys dat rooibos „n vermindering in die produksie van gekonjugeerde T, DHT en AST veroorsaak. Die studie het die invloed van rooibos op prostaat-spesifieke antigeen (PSA) ook ondersoek. Daar is vasgestel dat rooibos die vlakke van PSA verminder in die afwesigheid en teenwoordigheid van DHT in LNCaP selle. Hierdie resultaat dui op moontlike interaksie van rooibos met die androgeen (AR) of estrogeen-reseptor-β (ERβ), teenwoordig in LNCaP selle. Rooibos het die katalitiese aktiwiteit van ensieme, wat bydra tot androgeen produksie, geïnhibeer, asook die konjugasie van androgene. Op „n sellulêre vlak, het rooibos die vlakke van PSA-sekresie verminder, wat moontlike interaksie met die AR en ERβ aandui. Hierdie bevindings dui daarop dat rooibos wel n rol het om te speel in die modulasie van aktiewe androgene in die prostaat

Digital video assessment (dva) as catalyst for skill transfer in the road and airport visual condition survey environment in rsa

Government clients require that 30% of work be done by Exempt Micro Enterprise (EME) and Qualifying Small Enterprise (QSE) sub-consultants and can be difficult to achieve in high technology environments such as specialist pavement condition assessment. Tender requirements for experience with a proven track record as pre-qualification qualification work against such capacity-building objectives. Multi-Functional-Vehicles (MFV’s) used for instrument surveys typically already have accurate Geographic Positioning Systems (GPSs). The addition of high definition video cameras enables a technology coupling innovation approach to the normal manual visual survey methodology. The Digital Video Assessment (DVA) software system was developed with pre-calibrated camera settings to enable accurate measurements of grabbed photos at pre-set length intervals. The TMH 9 for Visual Condition Index (VCI) and also the American Society Test Method (ASTM) for Pavement Condition Index (PCI) survey templates are used to record the various forms of distress in terms of degree and extent. The survey data is analysed by the DVA software in the safety and comfort of an office environment. Surveys can thus be done with initially inexperienced EME and QSE sub-consultants. The DVA allows “on the job training” as their surveys can be checked by a qualified experienced visual surveyor numerous times with no additional travel and stay cost until they are trained and quality ensured. A recent comparative study using manual visual surveys versus this DVA survey methodology found that this DVA survey is not only more accurate and better quantifiable but can provide more detail with geo-referenced image records than what is required at project-level investigations. This DVA software and survey approach offer tremendous opportunity for capacity building of new entrants to this specialized field of visual survey with accuracy in a safe survey mode.Papers presented virtually at the 39th International Southern African Transport Conference on 05 -07 July 202

No extra-adrenal aldosterone production in various human cell lines.

Extra-adrenal de-novo aldosterone (Aldo) production has been described inconsistently. Systematic data based upon state-of-the-art technology including validated controls are sparse. We hypothesized that aldosterone synthase (CYP11B2) expression and de-novo Aldo production are absent in non-adrenal human cell lines, either immortalized cell lines or commercially available primary cell lines, including peripheral blood mononuclear cells (PBMCs) of individuals without and with primary hyperaldosteronism (PA). CYP11B2-transfected COS-7 and endogenous CYP11B2 expressing adrenal H295R cells served as positive controls. Various well-characterized, purchased, immortalized (BeWo, HEK293, HTR-8/SVneo, JEG-3) and primary (HAEC, HLEC, HRGEC, HRMC, HUAEC, HUVEC, PBMC) cell lines as well as self-isolated PBMCs from PA patients (n=5) were incubated with the steroid hormone substrates progesterone, deoxycorticosterone, corticosterone or 18-OH-corticosterone with and without Ang II for 24h to assess CYP11B2 enzymatic activity. CYP11B2 expression was analyzed by Real-time PCR and liquid chromatography-mass spectrometry (LC-MS) was used to quantify Aldo production. Pronounced CYP11B2 mRNA expression and Aldo production were observed in both positive controls, which followed an incremental time course. Neither substrates alone nor co-incubation with Ang II significantly stimulated CYP11B2 expression or Aldo production in various immortalized and primary cell lines and PBMCs of PA patients. These results strongly support the absence of a relevant de-novo extra-adrenal Aldo production in non-adrenal cells including, blood mononuclear cells irrespective of the absence or presence of autonomous adrenal Aldo production