Mass spectrometry-based steroid profiling in primary bilateral macronodular adrenocortical hyperplasia.

Biochemical characterization of primary bilateral macronodular adrenocortical hyperplasia (PBMAH) by distinct plasma steroid profiles and its putative correlation to disease has not been previously studied. LC-MS/MS-based steroid profiling of 16 plasma steroids was applied to 36 subjects (22 females, 14 males) with PBMAH, 19 subjects (16 females, 3 males) with other forms of adrenal Cushing's syndrome (ACS), and an age and sex-matched control group. Germline ARMC5 sequencing was performed in all PBMAH cases. Compared to controls, PBMAH showed increased plasma 11-deoxycortisol, corticosterone, 11-deoxycorticosterone, 18-hydroxycortisol, and aldosterone, but lower progesterone, DHEA, and DHEA-S with distinct differences in subjects with and without pathogenic variants in ARMC5. Steroids that showed isolated differences included cortisol and 18-oxocortisol with higher (P < 0.05) concentrations in ACS than in controls and aldosterone with higher concentrations in PBMAH when compared to controls. Larger differences in PBMAH than with ACS were most clear for corticosterone, but there were also trends in this direction for 18-hydroxycortisol and aldosterone. Logistic regression analysis indicated four steroids - DHEA, 11-deoxycortisol, 18-oxocortisol, and corticosterone - with the most power for distinguishing the groups. Discriminant analyses with step-wise variable selection indicated correct classification of 95.2% of all subjects of the four groups using a panel of nine steroids; correct classification of subjects with and without germline variants in ARMC5 was achieved in 91.7% of subjects with PBMAH. Subjects with PBMAH show distinctive plasma steroid profiles that may offer a supplementary single-test alternative for screening purposes.

Development of a Function-Integrative Sleeve for Medical Applications.

Function-integrative textiles bear the potential for a variety of applications in the medical field. Recent clinical investigations suggest that the application of a function-integrative fabric could have a positive impact on currently applied diagnostic procedures of a specific type of tumour. In this context, the fabric should enable local warming of a patient's upper extremity as well as blood flow measurement. Existing solutions comprise a warming a warming system but lack a measuring apparatus for blood flow determination. With regard to the quality of results of current diagnostic procedures, the local warming of the patients' upper extremity and the simultaneous determination of the blood flow plateau are crucial. In the present paper, the development process of a function-integrative sleeve is introduced. Besides the development of an adaptable sleeve-design, the manufacturing process of an integrated warming system was also addressed. Furthermore, the identification of crucial physiological effects, using a Laser Doppler Perfusion Monitor, is introduced. During testing of the function-integrative sleeve, modulation of the desired physiological effects was observed. The results support the initial assumptions and dictate further investigations on increasing user-friendliness and cost-efficiency during adjusting and determining the physiological effects in the course of tumour diagnosis.

A steady state system for in vitro evaluation of steroidogenic pathway dynamics: Application for CYP11B1, CYP11B2 and CYP17 inhibitors.

Disorders featuring dysregulated adrenal steroidogenesis, such as primary aldosteronism, can benefit from targeted therapies. The aldosterone and cortisol producing enzymes, aldosterone synthase (CYP11B2) and 11-beta-hydroxylase (CYP11B1), share 93% homology requiring selective drugs for pharmacological treatment. Herein, we introduce an effective in vitro assay for evaluation of steroidogenic enzyme kinetics based on intracellular flux calculations. H295RA cells were cultured in chambers under constant medium flow. Four hourly samples were collected (control samples), followed by collections over an additional four hours after treatment with fadrozole (10 nM), metyrapone (10 µM), SI_191 (5 nM), a novel CYP11B2 inhibitor or SI_254 (100 nM), a newly synthesized 17-alpha-hydroxylase/17,20-lyase inhibitor. Mass spectrometric measurements of multiple steroids combined with linear system computational modeling facilitated calculation of intracellular fluxes and changes in rate constants at different steroidogenic pathway steps, enabling selectivity of drugs for those steps to be evaluated. While treatment with fadrozole, metyrapone and SI_191 all reduced fluxes of aldosterone, corticosterone and cortisol production, treatment with SI_254 led to increased flux through the mineralocorticoid pathway and reduced production of steroids downstream of 17-alpha-hydroxylase/17,20-lyase. Drug-induced decreases in rate constants revealed higher selectivity of SI_191 compared to other drugs for CYP11B2 over CYP11B1, this reflecting additional inhibitory actions of SI_191 on catalytic steps of CYP11B2 downstream from the initial 11-beta-hydroxlase step. By culturing cells under perfusion the described system provides a realistic model for simple and rapid calculations of intracellular fluxes and changes in rate constants, thereby offering a robust procedure for investigating drug or other effects at specific steps of steroidogenesis.

The Aldosterone Synthase Inhibitor FAD286 is Suitable for Lowering Aldosterone Levels in ZDF Rats but not in db/db Mice.

Inhibition of aldosterone synthase is an alternative treatment option to mineralocorticoid receptor antagonism to prevent harmful aldosterone actions. FAD286 is one of the best characterized aldosterone synthase inhibitors to date. FAD286 improves glucose tolerance and increases glucose-stimulated insulin secretion in obese and diabetic ZDF rats. However, there is limited knowledge about the dose-dependent effects of FAD286 on plasma aldosterone, corticosterone, and 11-deoxycorticosterone in ZDF rats and in db/db mice, a second important rodent model of obesity and type 2 diabetes. In addition, effects of FAD286 on plasma steroids in mice and rats are controversial. Therefore, obese Zucker diabetic fatty (ZDF) rats and db/db mice were treated with FAD286 for up to 15 weeks and plasma steroids were evaluated using highly sensitive liquid chromatography-tandem mass spectrometry. In ZDF rats, FAD286 (10 mg/kg/d) treatment resulted in nearly complete disappearance of plasma aldosterone while corticosterone levels remained unaffected and those of 11-deoxycorticosterone were increased ~4-fold compared to vehicle control. A lower dose of FAD286 (3 mg/kg/d) showed no effect on plasma aldosterone or corticosterone, but 11-deoxycorticosterone was again increased ~4-fold compared to control. In contrast to ZDF rats, a high dose of FAD286 (40 mg/kg/d) did not affect plasma aldosterone levels in db/db mice although 11-deoxycorticosterone increased ~2.5-fold. A low dose of FAD286 (10 mg/kg/d) increased plasma aldosterone without affecting corticosterone or 11-deoxycorticosterone. In conclusion, the aldosterone synthase inhibitor, FAD286, lowers plasma aldosterone in obese ZDF rats, but not in obese db/db mice.

Aldosterone Synthase Inhibition Improves Glucose Tolerance in Zucker Diabetic Fatty (ZDF) Rats.

Plasma aldosterone is elevated in type 2 diabetes and obesity in experimental and clinical studies and can act to inhibit both glucose-stimulated insulin secretion by the ß-cell and insulin signaling. Currently mineralocorticoid receptor antagonism is the best characterized treatment to ameliorate aldosterone-mediated effects. A second alternative is inhibition of aldosterone synthase, an approach with protective effects on end-organ damage in heart or kidney in animal models. The effect of aldosterone synthase inhibition on metabolic parameters in type 2 diabetes is not known. Therefore, male Zucker diabetic fatty (ZDF) rats were treated for 11 weeks with the aldosterone synthase inhibitor FAD286, beginning at 7 weeks of age. Results were compared with the mineralocorticoid receptor antagonist eplerenone. Plasma aldosterone was abolished by FAD286 and elevated more than 9-fold by eplerenone. The area under the curve calculated from an oral glucose tolerance test (OGTT) was lower and overall insulin response during OGTT was increased by FAD286. In contrast, eplerenone elevated blood glucose levels and blunted insulin secretion during the OGTT. Fasting glucose was lowered and fasting insulin was increased by FAD286 in the prediabetic state. Glycated hemoglobin was lowered by FAD286, whereas eplerenone showed no effect. We conclude that aldosterone synthase inhibition, in contrast to mineralocorticoid receptor antagonism, has the potential for beneficial effects on metabolic parameters in type 2 diabetes.