The diagnostic accuracy of increased late night salivary cortisol for Cushing's syndrome: a real-life prospective study.

INTRODUCTION AND AIM: A prompt diagnosis of Cushing's Syndrome (CS) in high-risk populations is mandatory: 1-mg dexamethasone suppression test (1-mg DST), late night salivary cortisol (LNSC), and urinary-free cortisol (UFC) are recommended, despite thresholds calculated in retrospective studies. Our aim was to study the diagnostic accuracy of LNSC measured with chemiluminescence assay in a prospective study, confirming discrepancies with mass spectrometry (MS). MATERIALS AND METHODS: We enrolled 117 controls and 164 suspected CS (CS = 47, non-CS = 117). In case of increased LNSC, high clinical suspicion of CS or adrenal incidentaloma, patients were hospitalized to exclude/confirm CS. RESULTS: LNSC levels were higher in patients with suspected CS, CS, and non-CS than controls. Considering 16 nmol/L as threshold for CS, overall LNSC revealed SE 97% and SP 84% in the whole group of subjects considered, achieving positive/negative likelihood ratio of 5.56/0.045, respectively. 35 out of 81 subjects with increased LNSC were non-CS (15 diabetic and 20 obese): considering only those patients with increased likelihood to have a CS (the non-CS patients) SP decreased to 70%, and further reduced to 60% if we discharged subjects with adrenal incidentaloma. MS analyses reduced partially the number of false-positive LNSC. CONCLUSIONS: LNSC measured in automated chemiluminescence is reliable in clinical practice: it present a high diagnostic accuracy to exclude hypercortisolism in patients with normal cortisol levels. MS could be used to reduce the number of false-positive results; nevertheless, some non-CS subjects with functional hypercortisolism could have a mild impairment of cortisol rhythm.

A Single-Center 10-Year Experience with Pasireotide in Cushing's Disease: Patients' Characteristics and Outcome.

Pasireotide is the first pituitary-directed drug approved for treating patients with Cushing's disease (CD). Our 10-year experience with pasireotide in CD is reported here. Twenty patients with de novo, persistent, or recurrent CD after pituitary surgery were treated with pasireotide from December 2003 to December 2014. Twelve patients were treated with pasireotide in randomized trials and 8 patients with pasireotide sc (Signifor(®); Novartis AG, Basel, Switzerland) in clinical practice. The mean treatment duration was 20.5 months (median 9 months; range, 3-72 months). Urinary free cortisol (UFC) levels mean percentage change (± SD) at last follow-up was-40.4% (± 35.1; range, 2-92%; median reduction 33.3%) with a normalization rate of 50% (10/20). Ten patients achieved sustained normalized late night salivary cortisol (LNSC) levels during treatment. LNSC normalization was associated with UFC normalization in 7/10 patients. Serum cortisol and plasma ACTH significantly decreased from baseline to last follow-up. Body weight decrease and blood pressure improvement during pasireotide treatment were independent from UFC response. Glucose profile worsening was observed in all patients except one. The frequency of diabetes mellitus increased from 40% (8/20) at baseline to 85% (17/20) at last follow-up requiring initiation of medical treatment only in 44% of patients. Pasireotide treatment was associated with sustained biochemical and clinical benefit in about 60% of CD patients. Glucose profile alteration is a frequent complication of pasireotide treatment; however, it seems to be easy to manage with diet and lifestyle intervention in almost half of the patients.

Bone complications in patients with Cushing's syndrome: looking for clinical, biochemical, and genetic determinants.

SUMMARY: This is the first study examining the impact of both clinical, biochemical, and genetic determinants in the occurrence of bone complications in patients with overt Cushing's syndrome (CS). It demonstrates that the degree and duration of hypercortisolism seem to play a major role in bone loss and fractures development in these patients. INTRODUCTION: Bone loss and fractures are a common complication of CS. We investigate the role of gender, disease etiology, duration, and degree of hypercortisolism as well as the impact of glucocorticoid receptor (GR) polymorphisms on the development of bone complications in CS. METHODS: Fifty-two patients with active CS (38 Cushing's disease and 14 with cortisol-secreting adrenal adenoma) were genotyped for GR polymorphisms (BclI, N363S, ER22/23EK, and A3669G). In all patients, clinical, hormonal, and biochemical markers of bone turnover, densitometric parameters [lumbar spine and left femur bone mineral density (BMD), T-score, Z-score] as well as the prevalence of bone demineralization and both vertebral and peripheral fractures were assessed. RESULTS: No differences were found in bone complications according to gender, disease etiology, and genetic variants distribution. Fractured patients compared to non-fractured ones showed increased levels of urinary free cortisol (UFC) and a more compromised densitometric profile. UFC levels correlated with the occurrence of vertebral fractures (r = 0.43, p = 0.009) while midnight serum cortisol correlated with L1-L4 BMD values (r = -0.35, p = 0.04). Disease duration correlated with the presence of peripheral fractures (r = 0.36, p = 0.04). CONCLUSIONS: While GR gene variants as well as gender and disease etiology seem not to play a role, the degree and duration of hypercortisolism seem to be the major determinants of bone loss and fractures in this group of patients. More investigations are needed to understand the real impact of these determinants on the development of bone complications in patients with hypercortisolism.

Cyclical Cushing's syndrome in a patient with a bronchial neuroendocrine tumor (typical carcinoid) expressing ghrelin and growth hormone secretagogue receptors.

A 56-yr-old woman was referred with a diagnosis of Cushing's disease. Hypertension and severe hypokalemia were present and high urinary free cortisol/cortisone ratio was detected, raising a suspicion of an ectopic ACTH syndrome. Inferior petrosal sinus sampling, thoracic computed tomography, and octreotide scans were negative. Remission and relapse periods lasting 3-4 months were observed during the 3.5 yr of follow-up. Finally a thoracic computed tomography scan showed a basal paracardic nodule in the left lung. After surgery, a well-differentiated neuroendocrine tumor (typical bronchial carcinoid) was diagnosed, staining positively for ACTH. RT-PCR revealed expression of proopiomelanocortin, CRH receptor, and V3 vasopressin receptor. Somatostatin receptor type 1, 2, 3, and 5 mRNA was detected only in tumoral tissue. Interestingly, we observed the simultaneous presence of ghrelin and both GH secretagogue (GHS) receptors (1a and 1b) mRNA in tumoral tissue but not in the normal lung. This finding correlates with the in vivo ACTH hyperresponsiveness to hexarelin (a GHS). This is the first report of a cyclical ectopic ACTH-secreting tumor with an in vivo ACTH response to hexarelin coupled with the tumoral expression of ghrelin and GHS receptors. This finding might imply an autocrine/paracrine modulatory effect of ghrelin in bronchial ACTH-secreting tumors.

Postsynaptic effects of methoctramine at the mouse neuromuscular junction.

Functional studies were performed to evaluate the effects of methoctramine at the neuromuscular junction of the mouse. The presynaptic control of acetylcholine release and the postsynaptic activation of the nicotinic receptor have been analysed by means of the extracellular recording with an EPC7 Patch Clamp amplifier. This electrophysiological method revealed a dose-related inhibitory effect of methoctramine on the studied parameters. The dramatic reduction of the kinetics of the quantal conductance change indicates an action at the postsynaptic level. The effects of methoctramine have been compared with those of the muscarinic agonist oxotremorine. Concentration/response curves for the two drugs were obtained and the apparent EC50 values calculated. The effects of oxotremorine were not antagonized by 1 microM methoctramine. These findings suggest an interaction of some muscarinic agents on the postsynaptic receptor-ion-channel complex at the mouse neuromuscular junction.

Cholinergic effects of cimetidine and ranitidine.

Cimetidine and ranitidine were submitted to eight in vitro assays: their intrinsic activity was evaluated on guinea-pig auricles and ileum, acetylcholine synergism on guinea-pig tracheal muscle, frog rectus abdominis, guinea-pig vas and rat phrenic nerve diaphragm, and antagonism on guinea-pig auricles and rat jejunum estimated at a range of concentrations. Ranitidine alone opposed 1-hyoscyamine antagonism in guinea-pig ileum when acetylcholine was the agonist, but not when the agonist was bethanechol.

Computerized estimation of spontaneous and evoked acetylcholine release at the neuromuscular junction.

A new and automated on-line analysis of the parameters related to the neuromuscular activity is shown. The procedure used to obtain in real time the statistical evaluation of the recorded electrical signals is presented and discussed. The hardware is composed of a PDP-11/73 microcomputer equipped with an analog input/output board. The software is written principally in FORTRAN 77 and also uses MACRO/ASSEMBLY language.

An experimental study on dependence liability of zipeprol.

Zipeprol, a piperazine ethanol derivative, is a non-essential but widely used (paediatric) antitussive, which is not legally considered as being capable of creating dependence or abuse liability. A first group of experimental results was obtained assaying the displacement of 1 nM [3H]naloxone by zipeprol vs morphine on the rat brain homogenate fractions. A second group was carried out on the longitudinal muscle of guinea-pig ileum, using the field stimulation technique, either in the absence or in the presence of naloxone 1 x 10(-5)M or in the absence or in the presence of yohimbine 1 x 10(-5)M. Further investigations concerning the pharmacological and the biochemical characterization of the mechanisms involved in abuse liability were carried out by means of the in vitro inhibition of ACh response on the guinea-pig ileum preparation. The results indicate zipeprol as a moderate opioid agonist, which also shows a direct anticholinergic effect, independent of presynaptic alpha 2 interaction.

Muscarinic modulation of neurotransmission: the effects of some agonists and antagonists.

1. Functional studies were performed to evaluate the effects of some muscarinic agents at the neuromuscular junction of the mouse. 2. The presynaptic control of acetylcholine release and the postsynaptic activation of the nicotinic receptor have been analyzed by means of extracellular recording. The amplitude of spontaneous and of evoked acetylcholine release, the frequency of spontaneous acetylcholine release and the time course of the quantal release have been measured by means of an EPC7 patch clamp amplifier. 3. This electrophysiological method revealed multiple dose-related effects of some agonists and antagonists on the above parameters. Concentration-response curves related to the parameters underlying the function of this cholinergic synapse were obtained and the apparent EC50 values calculated. 4. Many of the interactions of the agonists and antagonists could inhibit neuromuscular transmission. The rank order potencies related to the inhibition of the evoked signals were carbachol > oxotremorine > d,l-muscarine for the agonists and methoctramine > 4-DAMP > l-hyoscyamine > AFDX-116 > ipratropium > pirenzepine for the antagonists. 5. These findings suggest a more complicated pattern related to the muscarinic action at the mouse neuromuscular junction with the involvement of some post-synaptic located sites.

Electrophysiological effects of a pumiliotoxin-B-like alkaloid derived from the skin of the Australian frog Pseudophryne coriacea.

Skin extracts of the Australian frog Pseudophryne coriacea (PsC) potentiate and prolong the bioelectric activity of various excitable tissues. When studied by electrophysiological means in a preparation of mouse diaphragm, PsC skin extracts did not affect the spontaneous acetylcholine (ACh) release. However, the indirect stimulation of the preparation in the presence of PsC skin extracts gave rise to a different profile of rhythmic activity showing afterpotentials and variable oscillatory activity. The action potential and the total sodium current recorded in the muscle fibre with the loose patch clamp method were not modified significantly by the alkaloid. Concentrations of PsC skin extract that did not cause repetitive activity, seemed to reduce slightly the quantal content of the evoked release of ACh. The resting potential of muscle fibres was not modified even by the highest PsC skin extract concentrations. These results suggest that the facilitation effects of PsC skin extracts could be due to intracellular mechanisms probably related to the control of the cytosolic calcium concentrations or to an increased excitability of the presynaptic biomembranes.

Characterization of the rabbit aorta endothelium-dependent cholinergic receptor by agonist equipotent molar doses.

The endothelium-dependent acetylcholine, metacholine, carbachol, betanechol, and furtrethonium relaxation values have been measured in vitro for the rabbit aorta, in presence of a high concentration of hexamethonium. The produced EPMR values complete the values that were obtained in a previous experiment under analogous conditions for rat bladder, ileum, iris, stomach, and trachea preparations. The complete set was analyzed by new statistical techniques of data analysis and display, which are both simpler and more precise than the statistical modeling techniques used by us in the past. This has led to new results concerning the clustering of drugs and of receptors, as well as to a characterization of the endothelium receptor.

Some approaches to the pharmacology of multisubstrate enzyme systems.

Analytical and exploratory in vitro, in situ and in vivo, physio-pharmacotoxicology, from enzymology to population epidemiology, now embraces those approaches that correlate complex dynamic multisubstrate kinetics through conventional and more recent non-invasive quantitative methodologies. Basically, substrates may be classed as pertaining to fundamental energy turnovers (first-order cellular metabolic pathways or networks) and to iso- vs allosteric modulator systems (second-order metabolic control network). Pairs of substrates and cofactors set-up the third-order multienzyme-receptor patterns, which in intact, native in vivo structures establish and maintain the compartmentalized, dynamically superimposed overall coordination of local redox and phosphate potentials. Perturbations of the various levels of the metabolic hierarchy induced by drugs, as well their relaxations, can be readily submitted to non-invasive kinetic analysis. Both indirect and direct titrations of substrate levels, their modelling and statistical ad hoc evaluations of their interrelations can lead to the identification of the multiple sites involved in drug effects as structured at the different orders/levels of concomitant functional variations. Fractal geometries contribute towards defining the space- and time-related events.