Cardiac glycosides in man inhibit renin secretion, probably through a direct effect at the renal level (i.e. inhibition of juxtaglomerular cell Na/K ATPase). Since there is evidence that the human adrenal possesses an intrinsic renin-angiotensin system, we investigated the effect of digoxin on the in vitro generation of renin and angiotensin II/III, as well as of aldosterone, by the human adrenal gland. Minced normal adrenal tissues were studied in a superfusion system, measuring in the 15-min superfusate fractions active renin by immunoradiometric assay and angiotensin II/III and aldosterone by radioimmunoassay, respectively. In a first set of four experiments using different concentrations of digoxin in sequence for 45 min periods, digoxin 10−5, but not 10−8 and 10−6 mol/l, significantly reduced renin and angiotensin II/III output from adrenals, while no change in aldosterone was observed. In a second set of three experiments, the addition of digoxin 10−5 mol/l for 120 min caused a sustained reduction of renin and angiotensin II/III, but not of aldosterone. In the final experiment, the decrease of renin and angiotensin II/III during superfusion with digoxin 10−5 mol/l was significantly greater than that observed during superfusion with digoxin in the presence of antidigoxin antibodies. Our data indicate that digoxin at high doses reduces renin and angiotensin II/III but not aldosterone secretion by the human adrenal gland. This suggests two different effects of digoxin, probably both mediated by inhibition of the Na/K ATPase activity, on the adrenal renin-angiotensin- and aldosterone-secreting cells.
Matteo Pistorello, Margherita Cimolato, Francesco Pedini, Donatella Piovan, Marco Boscaro and Francesco Fallo
Nicoletta Sonino, Maria E Girelli, Marco Boscaro, Francesco Fallo, Benedetto Busnardo and Giovanni A Fava
Contradictory findings have been reported about a possible causal relationship of life stress to Graves' disease. We evaluated this issue by investigating the occurrence of stressful life events in the year before the first signs of disease onset, using methods that have been found to be valid and reliable in psychosomatic research. Seventy consecutive patients with Graves' disease and a control group of 70 healthy subjects, matched for sociodemographic variables, were studied. Paykel's Interview for Recent Life Events (a semistructured research interview covering 64 life events) was administered to patients, not during the acute phase of illness but while on remission, by antithyroid drug treatment. Patients with Graves' disease reported significantly more life events compared to controls (p<0.001). They also had more independent events (p<0.001) and events that had an objective negative impact (p <0.001) according to an independent rater, unaware whether the events had occurred in patients or controls. All categories of events were found to be significantly more frequent in patients suffering from Graves' disease than in controls. By rigorous methods (inclusion of patients with Graves' disease only, careful dating of the onset of symptoms, accurate event definition, delay of the interview upon disease remission, use of a blind rater for judging independence and objective negative impact), our results support the concept of an excess of life events in Graves' disease. Stressful life events may affect the regulatory mechanisms of immune function in a number of ways. Within the extreme complexity of the phenomena implicated in the pathogenesis of autoimmune thyroid hyperfunction, our findings emphasize the role of emotional stress.
Francesco Fallo, Matteo Pistorello, Francesco Pedini, Domenico D'Agostino, Franco Mantero and Marco Boscaro
The adrenal gland of various mammalian species has been shown to contain all the components of a functional renin-angiotensin system. We investigated the existence of this local system in human adrenal tissues surgically obtained. Eight normal adrenals (cortex and medulla) and 6 aldosterone-producing adenomas (aldosteronomas) were examined. Minced tissues were superfused over 270 min, and 15-min fractions were collected. In the perfusates, active renin was measured by immunoradiometric assay with human anti-renin monoclonal antibodies; immunoreactive angiotensin II/III and aldosterone were measured by radioimmunoassay. Adrenal tissues, either normal or pathological, were found concomitantly to release renin, angiotensin II/III and aldosterone. The pattern of this spontaneous release exhibited a pulsatile character. The total amount of renin and angiotensin II/III secreted during superfusion clearly exceeded the tissue content (determined by extraction). Addition of the angiotensin-converting enzyme inhibitor quinaprilat (4×10−5 mol/l) in the superfusion caused a concomitant decrease of angiotensin II/III and aldosterone secretion by 3 normal tissues, and no change in 2 aldosteronomas. These data provide evidence that the human adrenal gland in vitro generates and releases both renin and angiotensin II/III, and support the hypothesis that locally formed angiotensin II/III may play a role as a paracrine regulator of physiological aldosterone secretion.
Gilberta Giacchetti, Vanessa Ronconi, Silvia Rilli, Mario Guerrieri, Federica Turchi and Marco Boscaro
Primary aldosteronism (PA) due to aldosterone-producing adenoma (APA) is the most common curable form of secondary hypertension.
In order to evaluate blood pressure outcome after adrenalectomy for APA and to identify new favorable prognostic factors, data from 42 consecutive APA patients who underwent adrenalectomy were collected from 2005 to 2007.
Renin-angiotensin-aldosterone system (upright and postsaline infusion test), serum and urinary electrolytes, office and ambulatory blood pressure monitoring were evaluated at baseline and after a follow-up of 2.7±2.2 years. Drug history and adenoma size at morphological evaluation were also collected.
Multiple regression analysis showed that, before surgery, patients with a small adenoma (diameter <20 mm) displayed higher postsaline aldosterone values (P=0.0001), and lower serum potassium levels (P=0.020), than patients with adenoma >20 mm. Before surgery, mineralocorticoid receptor (MR) antagonists were used in patients with small APA in greater percentage than patients with bigger adenomas (64 vs 30% respectively, P=0.037). At follow-up, blood pressure normalized in 63% of the subjects. Recovered patients had a shorter duration of hypertension (P=0.038), and a smaller adenoma size (P=0.035). Receiver operating characteristic curves showed that a duration of hypertension ≤6 years and an APA size <20 mm were the best predictors of blood pressure normalization. Patients with APA <20 mm showed the complete restoration of blood pressure circadian rhythm.
The presence of APA <20 mm, duration of hypertension equal or less than 6 years, and preoperative MR antagonists use are favorable prognostic factors for hypertension recovery after adrenalectomy.
Giorgio Arnaldi, Giacomo Tirabassi, Roberta Papa, Giorgio Furlani, Laura Trementino, Marina Cardinaletti, Emanuela Faloia and Marco Boscaro
Corticotropin releasing hormone (CRH) test does not reliably distinguish Cushing's disease (CD) from normality or pseudo-Cushing state (PC). We assessed whether this could be achieved with a novel approach while preserving the ability of the test to distinguish CD from ectopic ACTH syndrome (EAS).
Subjects and methods
We studied 51 subjects with CD, 7 with EAS, 26 with PC, and 31 controls (CT). Human CRH (hCRH) test was performed at 0830 h by measuring plasma ACTH and serum cortisol at −15, 0, 15, 30, 45, 60, 90, and 120 min.
The area under the curve–ACTH exhibited a significant negative correlation with baseline serum cortisol in CT and PC, but not in CD or EAS patients. ACTH response to hCRH was blunted in PC compared with CT, whereas peak serum cortisol was higher in PC than in CT subjects. These findings suggested that ACTH-dependent Cushing's syndrome can be diagnosed by the presence of two hCRH test parameters and excluded if either or both are absent. Application of i) basal serum cortisol >12 μg/dl and peak plasma ACTH >54 pg/ml, or ii) peak serum cortisol >21 μg/dl and peak plasma ACTH >45 pg/ml, had 91.3% (95% confidence intervals (CI) 81–97.1) and 94.8% (CI 85.6–98.9) sensitivity and 98.2% (CI 90.6–99.9) and 91.2% (CI 80.7–97) specificity respectively, in diagnosing ACTH-dependent Cushing's syndrome. The >14% serum cortisol increase from mean baseline values to the mean of 15 and 30 min values in patients who were positive for the test completely discriminated between CD and EAS.
Simultaneous plasma ACTH and serum cortisol analysis enables the hCRH test to distinguish CD from PC and from normality, while preserving its ability to discriminate CD from EAS.
Barbara Mariniello, Vanessa Ronconi, Silvia Rilli, Paolo Bernante, Marco Boscaro, Franco Mantero and Gilberta Giacchetti
Objective: To evaluate the expression of 11β-hydrxysteroid dehydrogenase type 1 (11β-HSD1) in omental adipose tissue of patients with Cushing’s syndrome and simple obesity, compared with normal weight controls.
Design and methods: We have performed a case-control study and studied omental adipose tissue from a total of 24 subjects (eight obese subjects, ten patients with Cushing’s syndrome due to adrenal adenoma, and six normal weight controls). Body mass index, blood pressure, plasma glucose, plasma insulin, plasma cortisol, urinary free cortisol and post dexamethasone plasma cortisol were measured with standard methods. 11β-HSD1 mRNA and protein expression were evaluated in real-time PCR and western blot analysis respectively.
Results: 11β-HSD1 mRNA was 13-fold higher in obese subjects compared with controls (P=0.001). No differences were found between Cushing’s patients and controls. Western blot analysis supported the mRNA expression results.
Conclusions: Our data show the involvement of 11β-HSD1 enzyme invisceral obesity, which is more evident in severely obese patients than in Cushing’s syndrome patients. The lack of increase of 11β-HSD1 expression in Cushing’s syndrome could suggest downregulation of the enzyme as a result of long-term overstimulation.
Laura Trementino, Gloria Appolloni, Carolina Concettoni, Marina Cardinaletti, Marco Boscaro and Giorgio Arnaldi
Glucocorticoid receptor (GR) polymorphisms alter glucocorticoid (GC) sensitivity and have been associated with altered metabolic profiles. We evaluate the prevalence of the four GR (NR3C1) polymorphisms BclI, N363S, ER22/23EK, and A3669G in patients with Cushing's syndrome (CS) compared with healthy controls (HC) and we investigate their role in the development of metabolic abnormalities in patients with CS according to their hormonal profile.
Patients and methods
Sixty-one patients with CS and 71 sex- and age-matched HC were genotyped.
BclI variant was markedly higher in patients with CS compared with HC (62 vs 41%, P<0.05) while no significant differences were found among other polymorphisms. A very low frequency of N363S and the ER22/23EK was observed.
In CS patients, despite the significantly increased levels of morning serum cortisol in BclI carriers compared with wild type no clinical or metabolic differences were found.
In contrast, A3669G GR carriers showed a significantly reduced prevalence of type 2 diabetes mellitus compared with wild type (19 vs 68%, P=0.001) despite the higher levels of both serum morning (21.7±6 vs 27.3±8.6 μg/dl, P=0.009) and midnight cortisol (18.8±5.8 vs 24.0±8.0 μg/dl, P=0.01). The negative association between diabetes and A3669G GR polymorphism remained significant when data were adjusted for potential confounding factors.
The A3669G polymorphism of the GR gene plays a protective role in patients with CS, attenuating the effects of GC excess on glucose metabolism as shown by their reduced risk of diabetes.
Filippo Ceccato, Mattia Barbot, Nora Albiger, Marialuisa Zilio, Pietro De Toni, Giovanni Luisetto, Martina Zaninotto, Nella Augusta Greggio, Marco Boscaro, Carla Scaroni and Valentina Camozzi
Patients with 21-hydroxylase deficiency (21OHD) assume a lifelong glucocorticoid (GC) therapy. Excessive GC treatment increases the risk of osteoporosis and bone fractures, even though the role of substitutive therapy is not fully established: we analyzed the effect of GC dose on bone metabolism and bone mineral density (BMD) over time in patients with 21OHD.
We studied bone metabolism markers and BMD in 38 adult patients with 21OHD (19–47 years, 24 females and 14 males) and 38 matched healthy control. In 15 patients, BMD data were available at both baseline and after a long-term follow-up.
BMD was lower in patients than in controls at lumbar spine (0.961±0.1g/cm2 vs 1.02±0.113g/cm2, P=0.014) and femur neck (0.736±0.128g/cm2 vs 0.828±0.103g/cm2, P=0.02); otherwise, after height correction, only femoral neck BMD was lower in patients (0.458±0.081g/cm2 vs 0.498±0.063g/cm2, P=0.028). In those 21OHD subjects with at least 10 years follow-up, we observed an increase in lumbar BMD (P=0.0429) and a decrease in femur neck BMD values (P=0.004). Cumulative GC dose was not related to bone metabolism or BMD. No patient experienced clinical fragility fractures.
BMD values are decreased in patients with 21OHD, which are in part explained by decreased height, but not by the dose of glucocorticoids. Nevertheless, bone status should be carefully monitored in patients with 21OHD.
Filippo Ceccato, Giorgia Antonelli, Mattia Barbot, Marialuisa Zilio, Linda Mazzai, Rosalba Gatti, Martina Zaninotto, Franco Mantero, Marco Boscaro, Mario Plebani and Carla Scaroni
The Endocrine Society Clinical Guidelines recommend measuring 24-h urinary free cortisol (UFF) levels using a highly accurate method as one of the first-line screening tests for the diagnosis of Cushing's Syndrome (CS). We evaluated the performance of UFF, urinary free cortisone (UFE), and the UFF:UFE ratio, measured using a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method.
Subjects and methods
The LC–MS/MS was used to analyze UFF and UFE levels in 43 surgically confirmed CS patients: 26 with Cushing's disease (CD, 16 de novo and ten recurrences), 11 with adrenal CS and six with ectopic CS; 22 CD patients in remission; 14 eu-cortisolemic CD patients receiving medical therapy; 60 non-CS patients; and 70 healthy controls. Sensitivity and specificity were determined in the combined groups of non-CS patients, healthy controls, and CD in remission.
UFF>170 nmol/24 h showed 98.7% specificity and 100% sensitivity for de novo CS, while sensitivity was 80% for recurrent CD patients, who were characterized by lower UFF levels. The UFF:UFE and UFF+UFE showed lower sensitivity and specificity than UFF. Ectopic CS patients had the highest UFF and UFF:UFE levels, which were normal in the CD remission patients and in those receiving medical therapy.
Our data suggest high diagnostic performance of UFF excretion measured using LC–MS/MS, in detecting de novo CS. UFF:UFE and UFF+UFE assessments are not useful in the first step of CS diagnosis, although high levels were found to be indicative of ectopic CS.
Stephan Petersenn, Albert Beckers, Diego Ferone, Aart van der Lely, Jens Bollerslev, Marco Boscaro, Thierry Brue, Paolo Bruzzi, Felipe F Casanueva, Philippe Chanson, Annamaria Colao, Martin Reincke, Günter Stalla and Stelios Tsagarakis
A number of factors can influence the reported outcomes of transsphenoidal surgery (TSS) for Cushing's disease – including different remission and recurrence criteria, for which there is no consensus. Therefore, a comparative analysis of the best treatment options and patient management strategies is difficult. In this review, we investigated the clinical outcomes of initial TSS in patients with Cushing's disease based on definitions of and assessments for remission and recurrence.
We systematically searched PubMed and identified 44 studies with clear definitions of remission and recurrence. When data were available, additional analyses by time of remission, tumor size, duration of follow-up, surgical experience, year of study publication and adverse events related to surgery were performed.
Data from a total of 6400 patients who received microscopic TSS were extracted and analyzed. A variety of definitions of remission and recurrence of Cushing's disease after initial microscopic TSS was used, giving broad ranges of remission (42.0–96.6%; median, 77.9%) and recurrence (0–47.4%; median, 11.5%). Better remission and recurrence outcomes were achieved for microadenomas vs macroadenomas; however, no correlations were found with other parameters, other than improved safety with longer surgical experience.
The variety of methodologies used in clinical evaluation of TSS for Cushing's disease strongly support the call for standardization and optimization of studies to inform clinical practice and maximize patient outcomes. Clinically significant rates of failure of initial TSS highlight the need for effective second-line treatments.