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Aldo-keto reductase 1C3 (AKR1C3) is a key enzyme in the activation of both classic and 11-oxygenated androgens. In adipose tissue, AKR1C3 is co-expressed with 11ß-hydroxysteroid dehydrogenase type 1 (HSD11B1), which catalyzes not only the local activation of glucocorticoids but also the inactivation of 11-oxygenated androgens, and thus has the potential to counteract AKR1C3. Using a combination of in vitro assays and in silico modeling we show that HSD11B1 attenuates the biosynthesis of the potent 11-oxygenated androgen, 11-ketotestosterone (11KT), by AKR1C3. Employing ex vivo incubations of human female adipose tissue samples we show that inhibition of HSD11B1 results in the increased peripheral biosynthesis of 11KT. Moreover, circulating 11KT increased 2-3 fold in individuals with type 2 diabetes after receiving the selective oral HSD11B1 inhibitor AZD4017 for 35 days, thus confirming that HSD11B1 inhibition results in systemic increases in 11KT concentrations. Our findings show that HSD11B1 protects against excess 11KT production by adipose tissue, a finding of particular significance when considering the evidence for adverse metabolic effects of androgens in women. Therefore, when targeting glucocorticoid activation by HSD11B1 inhibitor treatment in women, the consequently increased generation of 11KT may offset beneficial effects of decreased glucocorticoid activation.
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Andrógenos , Diabetes Mellitus Tipo 2 , Humanos , Femenino , Andrógenos/metabolismo , Glucocorticoides , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1 , Tejido Adiposo/metabolismoRESUMEN
Chronic psychosocial stress is a recognized, yet understudied risk factor for heart disease, with potential sex-specific effects. We investigated whether chronic stress triggers sex-dependent cardiac dysfunction in isolated Wistar rat hearts subjected to ischemia-reperfusion injury. The experimental cohort underwent 1 hour of daily restraint stress for four weeks versus matched controls, followed by euthanasia (sodium pentobarbitone) and heart excision for ex vivo perfusion. Blood analysis revealed sex-specific alterations in stress hormones and inflammatory markers. Compared to controls, chronic restraint stress (CRS) males displayed decreased plasma brain-derived neurotrophic factor (BDNF) levels (p<0.05), while CRS females exhibited elevated plasma adrenocorticotropic hormone (ACTH) (p<0.01) and reduced corticosterone (p<0.001) alongside lower serum estradiol (p<0.001) and estradiol/ progesterone ratio (p<0.01). Of note, CRS females showed increased serum cardiac troponin T (p<0.05) and tumor necrosis factor-alpha (TNF-a) (p<0.01) with suppressed interleukin (IL)-1a, IL-1ß, IL-6, and IL-10 levels (p<0.05) when compared to controls. Ex vivo Langendorff perfusions revealed that CRS female hearts displayed impaired post-ischemic functional recovery for baseline stroke volume (p<0.01), work performance (p<0.05), aortic output (p<0.05), coronary flow (p<0.01), and overall cardiac output (p<0.01) when compared to matched controls and CRS males (p<0.05). Our findings reveal intriguing sex-specific responses at both the systemic and functional levels in stressed hearts. Here, the dysregulation of stress hormones, pro-inflammatory state, and potential underlying cardiomyopathy in females following the stress protocol renders them more prone to damage following myocardial ischemia. This study emphasizes the importance of incorporating sex as a biological variable in cardiac research focusing on stress-related cardiomyopathy.
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Sex steroids are pervasive in mammals and evolutionarily conserved, but differences in the nuances of endocrine profiles characterize distinct species. Two sex steroids, testosterone and progesterone, feature prominently in the life history of mammalian taxa, but neither one has been analyzed from wild giraffes. Our study was designed to address this gap in knowledge by examining how these sex steroids are related to biological features and giraffe life history. We conducted the research at Rooipoort Nature Reserve a 44,000 ha private nature reserve in South Africa on a population of South African giraffes (Giraffa camelopardalis giraffa). Eleven adult giraffe cows and seven adult bulls were immobilized and various biological samples and morphological measurements were obtained. We analyzed both testosterone and progesterone using Ultra High Performance Liquid Chromotography - Tandem Mass Spectrometry (UHPLC-MS/MS). We found that non-pregnant females had lower progesterone concentrations than pregnant females and that those in the last trimester of pregnancy showed a slight drop in progesterone, as well as an increase in testosterone. Among males, chronological age was correlated with testicle size, testosterone concentrations, and ossicone volume. We propose that the progesterone decline functions partly to accelerate resumption of ovulation because giraffes become pregnant while lactating, and that the testosterone elevation provides an endocrine mileu for female defense of neonatal calves, given that lion predation is a major threat to calf survival. We suggest that male reproductive strategies are mediated by the age-related impacts of testosterone on growth in both body mass and ossicone volume as a consequence of sexual selection. We conclude by noting that the robust and solid ossicones of male giraffes function in mate competition as wedges that are used to topple opponents by raising their legs and placing them in an off-balanced position that can seriously injure them when falling to the ground.
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Jirafas , Embarazo , Animales , Bovinos , Masculino , Femenino , Sudáfrica , Lactancia , Progesterona , Selección Sexual , Espectrometría de Masas en Tándem , Rumiantes , TestosteronaRESUMEN
In the last decades in vitro studies highlighted the potential for crosstalk between Hypoxia-Inducible Factor-(HIF) and glucocorticoid-(GC) signalling pathways. However, how this interplay precisely occurs in vivo is still debated. Here, we use zebrafish larvae (Danio rerio) to elucidate how and to what degree hypoxic signalling affects the endogenous glucocorticoid pathway and vice versa, in vivo. Firstly, our results demonstrate that in the presence of upregulated HIF signalling, both glucocorticoid receptor (Gr) responsiveness and endogenous cortisol levels are repressed in 5 days post fertilisation larvae. In addition, despite HIF activity being low at normoxia, our data show that it already impedes both glucocorticoid activity and levels. Secondly, we further analysed the in vivo contribution of glucocorticoids to HIF activity. Interestingly, our results show that both glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) play a key role in enhancing it. Finally, we found indications that glucocorticoids promote HIF signalling via multiple routes. Cumulatively, our findings allowed us to suggest a model for how this crosstalk occurs in vivo.
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Glucocorticoides/farmacología , Factor 1 Inducible por Hipoxia/fisiología , Receptor Cross-Talk/fisiología , Pez Cebra , Animales , Animales Modificados Genéticamente , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Glucocorticoides/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Larva/genética , Larva/metabolismo , Receptor Cross-Talk/efectos de los fármacos , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteínas Supresoras de Tumor/genética , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
Androgens are critical drivers of prostate cancer. In this chapter we first discuss the canonical pathways of androgen metabolism and their alterations in prostate cancer progression, including the classical, backdoor and 5α-dione pathways, the role of pre-receptor DHT metabolism, and recent findings on oncogenic splicing of steroidogenic enzymes. Next, we discuss the activity and metabolism of non-canonical 11-oxygenated androgens that can activate wild-type AR and are less susceptible to glucuronidation and inactivation than the canonical androgens, thereby serving as an under-recognized reservoir of active ligands. We then discuss an emerging literature on the potential non-canonical role of androgen metabolizing enzymes in driving prostate cancer. We conclude by discussing the potential implications of these findings for prostate cancer progression, particularly in context of new agents such as abiraterone and enzalutamide, which target the AR-axis for prostate cancer therapy, including mechanisms of response and resistance and implications of these findings for future therapy.
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Andrógenos/metabolismo , Neoplasias de la Próstata/metabolismo , Progresión de la Enfermedad , Resistencia a Antineoplásicos , Humanos , Masculino , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Receptores Androgénicos/metabolismoRESUMEN
The biosynthesis of steroid hormones, essential to the survival of all mammals, is dependent on the activity of 3ß-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3ßHSD). 3ßHSD activity is, in turn, influenced by cytochrome-b(5) (Cyt-b(5)). However, the mechanism through which this occurs is unknown. In this study, we investigated this mechanism by evaluating the influence of Cyt-b(5) on the dehydrogenase and isomerase activities of 3ßHSD. Capra hircus 3ßHSD was overexpressed in SF-9 cells, using a baculovirus expression system, and purified. Substrate and cofactor kinetics were determined spectrophotometrically in the presence and absence of purified Ovis aries liver Cyt-b(5). Nonspecific enzyme activity was evaluated by zero-enzyme, -substrate, and -cofactor blanks. Fusion proteins, 3ßHSD-eCFP, and Cyt-b(5)-eYFP were subsequently coexpressed in COS-1 cells and analyzed for FRET. A CFP-YFP fusion protein served as positive control, while coexpression of 3ßHSD-eCFP and cytochrome P450 17α-hydroxylase/17,20 lyase-eYFP (CYP17A1-eYFP) served as negative control. Results showed Cyt-b(5) to decrease the K(m,)(NAD(+)) value of 3ßHSD ≈3.5-fold while increasing the V(max,app) of the dehydrogenase reaction ≈17%. FRET analysis showed COS-1 cells coexpressing 3ßHSD-eCFP and Cyt-b(5)-eYFP to exhibit a FRET signal ≈9-fold greater than that of the negative control. These results indicate that Cyt-b(5) augments 3ßHSD activity via an allosteric mechanism by increasing the affinity of the enzyme toward NAD(+).
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17-Hidroxiesteroide Deshidrogenasas/metabolismo , Citocromos b5/metabolismo , Esteroide Isomerasas/metabolismo , Regulación Alostérica , Animales , Secuencia de Bases , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Transferencia Resonante de Energía de Fluorescencia , Reacción en Cadena de la Polimerasa , Unión Proteica , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Espectrometría de Masas en TándemRESUMEN
Epitestosterone is a stereoisomer of the active androgen testosterone and its circulating concentrations are similar to those of testosterone in women and children. However, its biological function and pathways of metabolism remain unknown. The structural similarity to testosterone suggests a potential function in the modulation of androgen receptor signalling. It is well established that the conversion of testosterone to 5α-dihydrotestosterone enhances local androgen receptor signalling. In this study, we show that epitestosterone is metabolized to 5α-dihydroepitestosterone by both human steroid 5α-reductase isoforms, SRD5A1 and SRD5A2. Using two different variations of a reporter assay for transactivation of the human androgen receptor, we show that epitestosterone is a partial AR agonist and that the 5α-reduction of epitestosterone increases its androgenic activity. In line with this, we show that 5α-reduction of epitestosterone reduces its ability to antagonize 5α-dihydrotestosterone-induced androgen receptor transactivation. In conclusion, we provide evidence that steroid 5α-reductases regulate the modulatory effect of epitestosterone on androgen receptor signalling.
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3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa , Epitestosterona , Proteínas de la Membrana , Receptores Androgénicos , Activación Transcripcional , Humanos , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , Receptores Androgénicos/metabolismo , Receptores Androgénicos/genética , Activación Transcripcional/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Epitestosterona/metabolismo , Dihidrotestosterona/metabolismo , Andrógenos/metabolismo , Oxidación-ReducciónRESUMEN
Testosterone biosynthesis from its precursor androstenedione is thought to be exclusively catalysed by the 17ß-hydroxysteroid dehydrogenases-HSD17B3 in testes, and AKR1C3 in the ovary, adrenal and peripheral tissues. Here we show for the first time that the glucocorticoid activating enzyme 11ß-hydroxysteroid dehydrogenase type 1 (HSD11B1) can also catalyse the 17ß-reduction of androstenedione to testosterone, using a combination of in vitro enzyme kinetic assays, mathematical modelling, and molecular docking analysis. Furthermore, we show that co-expression of HSD11B1 and AKR1C3 increases testosterone production several-fold compared to the rate observed with AKR1C3 only, and that HSD11B1 is likely to contribute significantly to testosterone production in peripheral tissues.
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Androstenodiona , Testosterona , Femenino , Humanos , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Glucocorticoides , Simulación del Acoplamiento Molecular , Hidroxiprostaglandina Deshidrogenasas , 3-Hidroxiesteroide Deshidrogenasas , 17-Hidroxiesteroide Deshidrogenasas/genéticaRESUMEN
INTRODUCTION: This study aimed to, first, determine the clusters of sex hormones, liver enzymes, and cardiometabolic factors associated with postprandial glucose (PPG) and, second to evaluate the variation these clusters account for jointly and independently with polygenic risk scores (PRSs) in South Africans of African ancestry men and women. RESEARCH DESIGN AND METHODS: PPG was calculated as the integrated area under the curve for glucose during the oral glucose tolerance test (OGTT) using the trapezoidal rule in 794 participants from the Middle-aged Soweto Cohort. Principal component analysis was used to cluster sex hormones, liver enzymes, and cardiometabolic factors, stratified by sex. Multivariable linear regression was used to assess the proportion of variance in PPG accounted for by principal components (PCs) and type 2 diabetes (T2D) PRS while adjusting for selected covariates in men and women. RESULTS: The T2D PRS did not contribute to the PPG variability in both men and women. In men, the PCs' cluster of sex hormones, liver enzymes, and cardiometabolic explained 10.6% of the variance in PPG, with PC1 (peripheral fat), PC2 (liver enzymes and steroid hormones), and PC3 (lipids and peripheral fat) contributing significantly to PPG. In women, PC factors of sex hormones, cardiometabolic factors, and liver enzymes explained a similar amount of the variance in PPG (10.8%), with PC1 (central fat) and PC2 (lipids and liver enzymes) contributing significantly to PPG. CONCLUSIONS: We demonstrated that inter-individual differences in PPG responses to an OGTT may be differentially explained by body fat distribution, serum lipids, liver enzymes, and steroid hormones in men and women.
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Enfermedades Cardiovasculares , Diabetes Mellitus Tipo 2 , Masculino , Persona de Mediana Edad , Humanos , Femenino , Glucosa , Diabetes Mellitus Tipo 2/epidemiología , Diabetes Mellitus Tipo 2/genética , Glucemia , Sudáfrica/epidemiología , Hormonas Esteroides Gonadales , Esteroides , Hígado , LípidosRESUMEN
OBJECTIVE: To explore depot-specific functional aspects of adipose tissue, examining the putative role for menopause and HIV status on insulin sensitivity (SI) and beta-cell function in Black South African women. METHODS: Women (n = 92) from the Middle-Aged Soweto Cohort, including premenopausal HIV-negative (n = 21); premenopausal women living with HIV (WLWH; n = 11); postmenopausal HIV-negative (n = 42); postmenopausal WLWH (n = 18) underwent the following tests: body composition (dual energy x-ray absorptiometry); fasting bloods for sex hormones, inflammation and adipokines; frequently sampled intravenous glucose tolerance test for SI and beta-cell function (disposition index, DI); abdominal (aSAT) and gluteal subcutaneous adipose tissue (gSAT) biopsies for cell size and mRNA expression of adipokines, inflammation, and estrogen receptors [ER]. RESULTS: Depot-specific associations between gene expression and insulin parameters did not differ by HIV or menopause status. Pooled analysis showed significant models for SI (P = 0.002) and DI (P = 0.003). Higher SI was associated with lower leptin and CD11c expression in aSAT and higher adiponectin in gSAT. Higher DI was associated with higher aSAT and gSAT expression of adiponectin, LPL, ERα, and PPARγ, and lower leptin in aSAT. WLWH had higher expression of adiponectin and lower expression of leptin in both aSAT (P = 0.002 and P = 0.005) and gSAT (P = 0.004 and P = 0.002), respectively, and a larger proportion of smaller cells in aSAT (P < 0.001). CONCLUSION: Insulin sensitivity and beta cell function were distinctively associated with aSAT and gSAT. While menopause did not influence these relationships, HIV had a significant effect on adipose tissue, characterised by variations in cell size distribution and transcript levels within the depots.
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Objectives: To investigate longitudinal changes in SHBG and free testosterone (free T) levels among Black middle-aged African men, with and without coexistent HIV, and explore associations with incident dysglycaemia and measures of glucose metabolism. Design: This longitudinal study enrolled 407 Black South African middle-aged men, comprising primarily 322 men living without HIV (MLWOH) and 85 men living with HIV (MLWH), with normal fasting glucose at enrollment. Follow-up assessments were conducted after 3.1 ± 1.5 years. Methods: At baseline and follow-up, SHBG, albumin, and total testosterone were measured and free T was calculated. An oral glucose tolerance test at follow-up determined dysglycaemia (impaired fasting glucose, impaired glucose tolerance, type 2 diabetes) and glucose metabolism parameters including insulin sensitivity (Matsuda index), insulin resistance (homeostasis model assessment of insulin resistance), and beta(ß)-cell function (disposition index). The primary analysis focussed on MLWOH, with a subanalysis on MLWH to explore whether associations in MLWOH differed from MLWH. Results: The prevalence of dysglycaemia at follow-up was 17% (n = 55) in MLWOH. Higher baseline SHBG was associated with a lower risk of incident dysglycaemia (odds ratio 0.966; 95% confidence interval 0.945-0.987) and positively associated with insulin sensitivity (ß = 0.124, P < .001) and ß-cell function (ß = 0.194, P = .001) at follow-up. Free T did not predict dysglycaemia. In MLWH, dysglycaemia prevalence at follow-up was 12% (n = 10). Neither baseline SHBG nor free T were associated with incident dysglycaemia and glucose metabolism parameters in MLWH. Conclusion: SHBG levels predict the development of dysglycaemia in middle-aged African men but do not exhibit the same predictive value in MLWH.
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The biological significance of 11ß-hydroxyandrostenedione (11OHA4) has eluded researchers for the past six decades. It is now known that 11OHA4 is biosynthesized in the androgen arm of the adrenal steroidogenesis pathway and subsequently metabolized by steroidogenic enzymes in vitro, serving as precursor to recognized and novel androgenic steroids. These in vitro findings extend beyond the adrenal, suggesting that 11OHA4 could be metabolized in steroid-responsive peripheral tissues, as is the case for androgen precursor metabolites of adrenal origin. The significance thereof becomes apparent when considering that the metabolism of 11OHA4 in LNCaP androgen dependent prostate cancer cells yields androgenic steroid metabolites. It is thus possible that 11OHA4 may be metabolized to yield ligands for steroid receptors in not only the prostate but also in other steroid-responsive tissues. Future investigations of 11OHA4 may therefore characterize it as a vital steroid with far-reaching physiological consequences. An overview of the research on 11OHA4 since its identification in 1953 will be presented, with specific focus on the most recent works that have advanced our understanding of its biological role, thereby underscoring its relevance in health and disease.
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Androstenodiona/análogos & derivados , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Androstenodiona/biosíntesis , Androstenodiona/metabolismo , Dihidrotestosterona/metabolismo , Humanos , Hidroxiesteroide Deshidrogenasas/metabolismo , Masculino , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/metabolismo , Esteroide 11-beta-Hidroxilasa/metabolismoRESUMEN
For many decades, the prevailing paradigm in endocrinology was that testosterone and 5α-dihydrotestosterone are the only potent androgens in the context of human physiology. The more recent identification of adrenal derived 11-oxygenated androgens and particularly 11-ketotestosterone have challenged these established norms, prompting a revaluation of the androgen pool, particularly in women. Since being recognized as bone fide androgens in humans, numerous studies have focused their attention on understanding the role of 11-oxygenated androgens in human health and disease and have implicated them as role players in conditions such as castration resistant prostate cancer, congenital adrenal hyperplasia, polycystic ovary syndrome, Cushing's syndrome, and premature adrenarche. This review therefore provides an overview of our current knowledge on the biosynthesis and activity of 11-oxygenated androgens with a focus on their role in disease states. We also highlight important analytical considerations for measuring this unique class of steroid hormone.
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Hiperplasia Suprarrenal Congénita , Síndrome del Ovario Poliquístico , Neoplasias de la Próstata , Masculino , Humanos , Femenino , Andrógenos , Testosterona , EsteroidesRESUMEN
Progestin-only injectable contraceptives, mainly depo-medroxyprogesterone acetate intramuscular (DMPA-IM), are the most widely used contraceptive methods in sub-Saharan Africa. Insufficient robust data on their relative side-effects and serum concentrations limit understanding of reported outcomes in contraception trials. The WHICH clinical trial randomized HIV-negative women to DMPA-IM (n = 262) or norethisterone enanthate (NET-EN) (n = 259) at two South African sites between 2018-2019. We measured serum concentrations of study and non-study progestins at initiation (D0) and peak serum levels, one week after the 24-week injection [25 weeks (25W)], (n = 435) and investigated associations between study progestin levels, and BMI and weight of participants. Peak median serum concentrations were 6.59 (IQR 4.80; 8.70) nM for medroxyprogesterone (MPA) (n = 161) and 13.6 (IQR 9.01; 19.0) nM for norethisterone (NET) (n = 155). MPA was the most commonly quantifiable non-study progestin at D0 in both arms (54%) and at 25W in the NET-EN arm (27%), followed by NET at D0 in both arms (29%) and at 25W in the DMPA-IM arm (19%). Levonorgestrel was quantifiable in both arms [D0 (6.9%); 25W (3.4%)], while other progestins were quantifiable in ≤ 14 participants. Significant negative time-varying associations were detected between MPA and NET concentrations and weight and BMI in both contraceptive arms and a significant increase was detected for peak serum progestin concentrations for normal weight versus obese women. Contraceptive-related reported outcomes are likely confounded by MPA, more so than NET, with reported DMPA-IM effects likely underestimated, at sites where DMPA-IM is widely used, due to misreporting of contraceptive use before and during trials, and 'tail' effects of DMPA-IM use more than six months before trial enrolment. Peak serum levels of MPA and NET are negatively associated with BMI and weight, suggesting another source of variability between trial outcomes and a potential increase in side-effects for normal weight versus overweight and obese women. Trail registration: The clinical trial was registered with the Pan African Clinical Trials Registry (PACTR 202009758229976).
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Acetato de Medroxiprogesterona , Progestinas , Femenino , Humanos , Acetato de Medroxiprogesterona/efectos adversos , Anticonceptivos , Índice de Masa Corporal , Noretindrona/farmacología , ObesidadRESUMEN
BACKGROUND: Multi-steroid profiling is a powerful analytical tool that simultaneously quantifies steroids from different biosynthetic pathways. Here we present an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the profiling of 23 steroids using post-column infusion of ammonium fluoride. METHODS: Following liquid-liquid extraction, steroids were chromatographically separated over 5 min using a Phenomenex Luna Omega C18 column and a water (0.1 % formic acid) methanol gradient. Quantification was performed on a Waters Acquity UHPLC and Xevo® TQ-XS mass spectrometer. Ammonium fluoride (6 mmol/L, post-column infusion) and formic acid (0.1 % (vol/vol), mobile phase additive) were compared as additives to aid ionisation. RESULTS: Post-column infusion of ammonium fluoride enhanced ionisation in a steroid structure-dependent fashion compared to formic acid (122-140 % for 3ßOH-Δ5 steroids and 477-1274 % for 3-keto-Δ4 steroids). Therefore, we analytically validated post-column infusion of ammonium fluoride. Lower limits of quantification ranged from 0.3 to 3 nmol/L; All analytes were quantifiable with acceptable accuracy (bias range -14 % to 11.9 % for 21/23, -21 % to 11.9 % for all analytes). Average recovery ranged from 91.6 % to 113.6 % and average matrix effects from -29.9 % to 19.9 %. Imprecision ranged from 2.3 % to 23 % for all analytes and was < 15 % for 18/23 analytes. The serum multi-steroid profile of 10 healthy men and 10 healthy women was measured. CONCLUSIONS: UHPLC-MS/MS with post-column infusion of ammonium fluoride enables comprehensive multi-steroid profiling through enhanced ionisation particularly benefiting the detection of 3-keto-Δ4 steroids.
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Esteroides , Espectrometría de Masas en Tándem , Compuestos de Amonio , Cromatografía Líquida de Alta Presión/métodos , Femenino , Fluoruros , Formiatos , Humanos , Masculino , Esteroides/análisis , Espectrometría de Masas en Tándem/métodosRESUMEN
OBJECTIVE: Androgens are important modulators of immune cell function. The local generation of active androgens from circulating precursors is an important mediator of androgen action in peripheral target cells or tissues. We aimed to characterize the activation of classic and 11-oxygenated androgens in human peripheral blood mononuclear cells (PBMCs). METHODS: PBMCs were isolated from healthy male donors and incubated ex vivo with precursors and active androgens of the classic and 11-oxygenated androgen pathways. Steroids were quantified by liquid chromatography-tandem mass spectrometry. The expression of genes encoding steroid-metabolizing enzymes was assessed by quantitative PCR. RESULTS: PBMCs generated eight-fold higher amounts of the active 11-oxygenated androgen 11-ketotestosterone than the classic androgen testosterone from their respective precursors. We identified the enzyme AKR1C3 as the major reductive 17ß-hydroxysteroid dehydrogenase in PBMCs responsible for both conversions and found that within the PBMC compartment natural killer cells are the major site of AKRC13 expression and activity. Steroid 5α-reductase type 1 catalyzed the 5α-reduction of classic but not 11-oxygenated androgens in PBMCs. Lag time prior to the separation of cellular components from whole blood increased serum 11-ketotestosterone concentrations in a time-dependent fashion, with significant increases detected from two hours after blood collection. CONCLUSIONS: 11-Oxygenated androgens are the preferred substrates for androgen activation by AKR1C3 in PBMCs, primarily conveyed by natural killer cell AKR1C3 activity, yielding 11-ketotestosterone the major active androgen in PBMCs. Androgen metabolism by PBMCs can affect the results of serum 11-ketotestosterone measurements, if samples are not separated in a timely fashion. SIGNIFICANCE STATEMENT: We show that human peripheral blood mononuclear cells (PBMCs) preferentially activate 11-ketotestosterone rather than testosterone when incubated with precursors of both the classic and the adrenal-derived 11-oxygenated androgen biosynthesis pathways. We demonstrate that this activity is catalyzed by the enzyme AKR1C3, which we found to primarily reside in natural killer cells, major contributors to the anti-viral immune defense. This potentially links intracrine 11-oxygenated androgen generation to the previously observed decreased NK cell cytotoxicity and increased infection risk in primary adrenal insufficiency. In addition, we show that PBMCs continue to generate 11-ketotestosterone if the cellular component of whole blood samples is not removed in a timely fashion, which could affect measurements of this active androgen in routine clinical biochemistry.
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Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas/metabolismo , Andrógenos/metabolismo , Leucocitos Mononucleares/metabolismo , Cromatografía Liquida , Humanos , Masculino , Espectrometría de Masas en Tándem , Testosterona/análogos & derivados , Testosterona/metabolismoRESUMEN
This study demonstrates the application of a mathematical steroidogenic model, constructed with individual in vitro enzyme characterisations, to simulate in vivo steroidogenesis in a diseased state. This modelling approach was applied to the South African Angora goat, that suffers from hypocortisolism caused by altered adrenal function. These animals are extremely vulnerable to cold stress, leading to substantial monetary loss in the mohair industry. The Angora goat has increased CYP17A1 17,20-lyase enzyme activity in comparison with hardy livestock species. Determining the effect of this altered adrenal function on adrenal steroidogenesis during a cold stress response is difficult. We developed a model describing adrenal steroidogenesis under control conditions, and under altered steroidogenic conditions where the animal suffers from hypocortisolism. The model is parameterised with experimental data from in vitro enzyme characterisations of a hardy control species. The increased 17,20-lyase activity of the Angora goat CYP17A1 enzyme was subsequently incorporated into the model and the response to physiological stress is simulated under both control and altered adrenal steroidogenic conditions.
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Hidrocortisona/metabolismo , Modelos Moleculares , Esteroide 17-alfa-Hidroxilasa/metabolismo , Esteroides/biosíntesis , Animales , Simulación por Computador , Cabras , Funciones de Verosimilitud , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Androgens are the obligatory precursors of estrogens. In humans, classic androgen biosynthesis yields testosterone, thought to represent the predominant circulating active androgen both in men and women. However, recent work has shown that 11-ketotestosterone, derived from the newly described 11-oxygenated androgen biosynthesis pathway, makes a substantial contribution to the active androgen pool in women. Considering that classic androgens are the obligatory substrates for estrogen biosynthesis catalyzed by cytochrome P450 aromatase, we hypothesized that 11-oxygenated androgens are aromatizable. Here we use steroid analysis by tandem mass spectrometry to demonstrate that human aromatase generates 11-oxygenated estrogens from 11-oxygenated androgens in 3 different cell-based aromatase expression systems and in human ex vivo placenta explant cultures. We also show that 11-oxygenated estrogens are generated as a byproduct of the aromatization of classic androgens. We show that 11ß-hydroxy-17ß-estradiol binds and activates estrogen receptors α and ß and that 11ß-hydroxy-17ß-estradiol and the classic androgen pathway-derived active estrogen, 17ß-estradiol, are equipotent in stimulating breast cancer cell line proliferation and expression of estrogen-responsive genes. 11-oxygenated estrogens were, however, not detectable in serum from individuals with high aromatase levels (pregnant women) and elevated 11-oxygenated androgen levels (patients with congenital adrenal hyperplasia or adrenocortical carcinoma). Our data show that while 11-oxygenated androgens are aromatizable in vitro and ex vivo, the resulting 11-oxygenated estrogens are not detectable in circulation, suggesting that 11-oxygenated androgens function primarily as androgens in vivo.
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Estrógenos/análogos & derivados , Estrógenos/sangre , Oxígeno/química , Animales , Aromatasa/metabolismo , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Estradiol/análogos & derivados , Estradiol/química , Estradiol/metabolismo , Estrógenos/química , Femenino , Sangre Fetal/química , Sangre Fetal/metabolismo , Células HEK293 , Humanos , Recién Nacido , Células MCF-7 , Placenta/química , Placenta/metabolismo , Embarazo/sangre , Unión Proteica/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Testosterona/análogos & derivados , Testosterona/sangre , Testosterona/químicaRESUMEN
Steroid hormones, including glucocorticoids and androgens, exert a wide variety of effects in the body across almost all tissues. The steroid A-ring 5ß-reductase (AKR1D1) is expressed in human liver and testes, and three splice variants have been identified (AKR1D1-001, AKR1D1-002, AKR1D1-006). Amongst these, AKR1D1-002 is the best described; it modulates steroid hormone availability and catalyses an important step in bile acid biosynthesis. However, specific activity and expression of AKR1D1-001 and AKR1D1-006 are unknown. Expression of AKR1D1 variants were measured in human liver biopsies and hepatoma cell lines by qPCR. Their three-dimensional (3D) structures were predicted using in silico approaches. AKR1D1 variants were overexpressed in HEK293 cells, and successful overexpression confirmed by qPCR and Western blotting. Cells were treated with either cortisol, dexamethasone, prednisolone, testosterone or androstenedione, and steroid hormone clearance was measured by mass spectrometry. Glucocorticoid and androgen receptor activation were determined by luciferase reporter assays. AKR1D1-002 and AKR1D1-001 are expressed in human liver, and only AKR1D1-006 is expressed in human testes. Following overexpression, AKR1D1-001 and AKR1D1-006 protein levels were lower than AKR1D1-002, but significantly increased following treatment with the proteasomal inhibitor, MG-132. AKR1D1-002 efficiently metabolised glucocorticoids and androgens and decreased receptor activation. AKR1D1-001 and AKR1D1-006 poorly metabolised dexamethasone, but neither protein metabolised cortisol, prednisolone, testosterone or androstenedione. We have demonstrated the differential expression and role of AKR1D1 variants in steroid hormone clearance and receptor activation in vitro. AKR1D1-002 is the predominant functional protein in steroidogenic and metabolic tissues. In addition, AKR1D1-001 and AKR1D1-006 may have a limited, steroid-specific role in the regulation of dexamethasone action.
Asunto(s)
Empalme Alternativo/genética , Oxidorreductasas/genética , Secuencia de Aminoácidos , Andrógenos/metabolismo , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Glucocorticoides/metabolismo , Células HEK293 , Humanos , Hígado/metabolismo , Masculino , Proteínas Mutantes/química , Proteínas Mutantes/genética , Oxidorreductasas/química , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Testículo/metabolismoRESUMEN
BACKGROUND: Given the rarity of 11ß-hydroxylase deficiency (11ßOHD), there is a paucity of data about the differences in clinical and biochemical characteristics of classic (C-11ßOHD) and nonclassic 11ßOHD (NC-11ßOHD). OBJECTIVE: To characterize a multicenter pediatric cohort with 11ßOHD. METHOD: The clinical and biochemical characteristics were retrospectively retrieved. CYP11B1 gene sequencing was performed. Seventeen plasma steroids were quantified by liquid chromatography-mass spectrometry and compared to that of controls. RESULTS: 102 patients (C-11ßOHD, nâ =â 92; NC-11ßOHD, nâ =â 10) from 76 families (46,XX; nâ =â 53) had biallelic CYP11B1 mutations (novel 9 out of 30). Five 46,XX patients (10%) were raised as males. Nineteen patients (19%) had initially been misdiagnosed with 21-hydroxylase deficiency. Female adult height was 152 cm [-1.85 SD score (SDS)] and male 160.4 cm (-2.56 SDS).None of the NC-11ßOHD girls had ambiguous genitalia (C-11ßOHD 100%), and none of the NC-11ßOHD patients were hypertensive (C-11ßOHD 50%). Compared to NC-11ßOHD, C-11ßOHD patients were diagnosed earlier (1.33 vs 6.9 years; Pâ <â 0.0001), had higher bone age-to-chronological age (Pâ =â 0.04) and lower adult height (-2.46 vs -1.32 SDS; Pâ =â 0.05). The concentrations of 11-oxygenated androgens and 21-deoxycortisol were low in all patients. The baseline ACTH and stimulated cortisol were normal in NC-11ßOHD. Baseline cortisol; cortisone; 11-deoxycortisol; 11-deoxycorticosterone and corticosterone concentrations; and 11-deoxycortisol/cortisol, 11-deoxycorticosterone/cortisol, and androstenedione/cortisol ratios were higher in C-11ßOHD than NC-11ßOHD patients (Pâ <â 0.05). The 11-deoxycortisol/cortisol ratio >2.2, <1.5, and <0.1 had 100% specificity to segregate C-11ßOHD, NC-11ßOHD, and control groups. CONCLUSION: NC-11ßOHD can escape from clinical attention due to relatively mild clinical presentation. However, steroid profiles enable the diagnosis, differential diagnosis, and subtyping of 11ßOHD.