RESUMEN
Cytochrome P450 19 (CYP19, aromatase) catalyzes the conversion of androgens to estrogens in a sequence of three reactions that each depend on NADPH and O2. Aromatase is a phylogenetically-ancient enzyme and its breadth of expression in other species has highlighted distinct physiological functions. In songbirds, estrogen production is required for programming the neural circuits controlling song and in the determination of sex in fish and reptiles. This work describes the expression, purification, and biophysical characterization of Aptenodytes forsteri (Emperor penguin, af) aromatase. Using human cytochrome P450 reductase as a redox partner, afCYP19 displayed similar substrate turnover and LC/MS/MS confirmed that afCYP19 catalyzes the transformations through the intermediates 19-hydroxy- and 19-oxo-androstenedione. Androstenedione and anastrozole had the highest affinity for the enzyme and were followed closely by 19-hydroxyandrostenedione and testosterone. The affinity of 19-oxo-androstenedione for afCYP19 was ten-fold lower. The time-dependent changes in the Soret bands observed in stopped-flow mixing experiments of the steroidal ligands and the inhibitor anastrozole with afCYP19 were best described by a two-step binding mechanism. In summary, these studies describe the first biophysical characterization of an avian aromatase that displays strikingly similar enzyme kinetics and ligand binding properties to the human enzyme and could serve as a convenient model system for studies of the enigmatic transformation of androgens to estrogens.
Asunto(s)
Aromatasa/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Anastrozol/metabolismo , Androstenodiona/análogos & derivados , Androstenodiona/metabolismo , Espectrometría Raman , Testosterona/metabolismoRESUMEN
BACKGROUND: The pathophysiology of adrenal insufficiency, common in surgical intensive care units, has not been fully elucidated. METHODS: Patients at risk (age > 55 years, in the surgical intensive care unit >1 week, baseline cortisol < 20 µg/dL) were enrolled. After measuring cortisol and adrenocorticotropic hormone (ACTH), corticotropin-releasing hormone (CRH) was administered. ACTH and cortisol were measured over 120 minutes. Short and long cosyntropin stimulation tests determined adrenal function. Area under the curve (AUC) and mixed linear models were used to compare cortisol and ACTH responses. Patients were grouped according to survival and response to stimulation testing. Chi-square and t tests were performed, and P values < .05 were considered statistically significant. RESULTS: Six of 25 patients responded poorly to cosyntropin, and 5 died compared with 3 after a normal response (P < .01). ACTH (AUC) and ACTH peak were increased in nonsurvivors after CRH administration. Cortisol peak and AUC were not different. CONCLUSIONS: ACTH responsiveness was increased in nonsurvivors and may predict mortality.
Asunto(s)
Insuficiencia Suprarrenal/sangre , Hormona Adrenocorticotrópica/sangre , Cosintropina/administración & dosificación , Hidrocortisona/sangre , Sistema Hipotálamo-Hipofisario/metabolismo , Sistema Hipófiso-Suprarrenal/metabolismo , Complicaciones Posoperatorias/sangre , Insuficiencia Suprarrenal/etiología , Insuficiencia Suprarrenal/mortalidad , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Hormona Liberadora de Corticotropina/administración & dosificación , Enfermedad Crítica , Femenino , Humanos , Unidades de Cuidados Intensivos , Modelos Lineales , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/mortalidad , PronósticoRESUMEN
BACKGROUND: Recent findings suggest that BPH has an inflammatory component. Clinical trials have documented that therapy with LHRH antagonist Cetrorelix causes a marked and prolonged improvement in LUTS in men with symptomatic BPH. We investigated the mechanism of action and effect of Cetrorelix in a rat model of BPH. METHODS: Adult male Wistar rats were used. BPH was induced in rats by subcutaneous injections of TE 2 mg/day for 4 weeks. Control animals received injections of corn oil. After induction of BPH, rats received depot Cetrorelix pamoate at the doses of 0.625, 1.25, and 12.5 mg/kg on days 1 and 22 and TE-control rats received vehicle injections. Whole prostates were weighed and processed for RNA and protein. Real-time RT-PCR assays for numerous inflammatory cytokines and growth factors were performed. Quantitative analyses of prostatic LHRH receptor, LHRH, androgen receptor (AR) and 5α-reductase 2 were done by real-time RT-PCR and immunoblotting; serum DHT, LH, PSA, and IGF-1 by immunoassays. RESULTS: mRNA levels for inflammatory cytokines IFN-γ, IL-3, IL-4, IL-5, IL-6, IL-8, IL-13, IL-15, and IL-17 and for growth factors EGF, FGF-2, FGF-7, FGF-8, FGF-14, TGF-ß1, and VEGF-A were significantly reduced by Cetrorelix 0.625 mg/kg (P < 0.05). Prostate weights were also significantly lowered by any dose of Cetrorelix. CONCLUSIONS: This study suggests that Cetrorelix reduces various inflammatory cytokines and growth factors in rat prostate and, at doses which do not induce castration levels of testosterone, can lower prostate weights. Our findings shed light on the mechanism of action of LHRH antagonists in BPH.
Asunto(s)
Citocinas/genética , Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Antagonistas de Hormonas/farmacología , Próstata/efectos de los fármacos , Hiperplasia Prostática/tratamiento farmacológico , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Hormona Liberadora de Gonadotropina/farmacología , Masculino , Próstata/metabolismo , Próstata/patología , Hiperplasia Prostática/inducido químicamente , Hiperplasia Prostática/patología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores LHRH/efectos de los fármacos , Receptores LHRH/genética , Receptores LHRH/metabolismo , Testosterona/toxicidadRESUMEN
BACKGROUND: Assessment of hypothalamic-pituitary-adrenal (HPA) axis function in stress-related health problems in humans is frequently carried out as a dynamic test by measuring the profile of increment in adrenocortical hormone (ACTH) and/or cortisol level in plasma in response to corticotropin-releasing hormone (CRH) administration. However, obtaining multiple blood samples for this type of test is not only an invasive procedure but also problematic to use in individuals with constricted or damaged veins which collapse during the blood draw such as the injecting drug users (IDUs) and HIV-1-infected individuals. Salivary cortisol measurement presents a non-invasive alternate approach to evaluate HPA axis activity in different situations. In order to validate the efficacy of salivary cortisol measurement for a dynamic test in IDUs and HIV-1-infected individuals, the present study was carried out to evaluate the cortisol profile in matched samples of plasma and saliva in healthy young men in response to ovine CRH (oCRH) administration. METHODS: Cortisol levels were measured in matched samples of plasma and saliva of healthy young men at baseline and over a 90-min period after administration of a single low dose of oCRH (1 microg/kg). RESULTS: Salivary cortisol levels were found to follow the profile similar to that of plasma, increasing significantly at each time point after oCRH administration from their respective baseline values (all Sign tests, p < 0.05). The peak level of cortisol occurred at 30 min in both fluids. Although salivary cortisol concentration was a fraction of the total plasma cortisol levels at all time points, there was a significant correlation in the values between the two fluids at baseline (r = 0.87, p < 0.02) as well as at 90 min (r = 0.70, p < 0.03). CONCLUSION: The findings support the earlier studies and substantiate the efficacy of using salivary free cortisol measurement for assessment of dynamic function of pituitary-adrenal axis in healthy young men and its application in individuals such as IDUs and HIV-infected individuals who may have difficulty in donating multiple blood samples.