Screening of hypercortisolism among patients with hypertension: an Italian nationwide survey.

PURPOSE: Screening of Cushing Syndrome (CS) and Mild Autonomous Cortisol Secretion (MACS) in hypertensive patients is crucial for proper treatment. The aim of the study was to investigate screening and management of hypercortisolism among patients with hypertension in Italy. METHODS: A 10 item-questionnaire was delivered to referral centres of European and Italian Society of Hypertension (ESH and SIIA) in a nationwide survey. Data were analyzed according to type of centre (excellence vs non-excellence), geographical area, and medical specialty. RESULTS: Within 14 Italian regions, 82 centres (30% excellence, 78.790 patients during the last year, average 600 patients/year) participated to the survey. Internal medicine (44%) and cardiology (31%) were the most prevalent medical specialty. CS and MACS were diagnosed in 313 and 490 patients during the previous 5 years. The highest number of diagnoses was reported by internal medicine and excellence centres. Screening for hypercortisolism was reported by 77% in the presence of specific features of CS, 61% in resistant hypertension, and 38% in patients with adrenal mass. Among screening tests, the 24 h urinary free cortisol was the most used (66%), followed by morning cortisol and ACTH (54%), 1 mg-dexamethasone suppression test (49%), adrenal CT or MRI scans (12%), and late night salivary cortisol (11%). Awareness of referral centres with expertise in management of CS was reported by 67% of the participants, which reduced to 44% among non-excellence centres. CONCLUSIONS: Current screening of hypercortisolism among hypertensive patients is unsatisfactory. Strategies tailored to different medical specialties and type of centres should be conceived.

Familial hyperaldosteronism type III.

Primary aldosteronism is the most common form of endocrine hypertension. This disorder comprises both sporadic and familial forms. Four familial forms of primary aldosteronism (FH-I to FH-IV) have been described. FH-III is caused by germline mutations in KCNJ5, encoding the potassium channel Kir3.4 (also called GIRK4). These mutations alter the selectivity filter of the channel and lead to abnormal ion currents with loss of potassium selectivity, sodium influx and consequent increased intracellular calcium that causes excessive aldosterone biosynthesis. To date, eleven families have been reported, carrying six different mutations. Although the clinical features are variable, FH-III patients often display severe hyperaldosteronism with an early onset, associated with hypokalemia and diabetes insipidus-like symptoms. In most cases FH-III patients are resistant to pharmacological therapy and require bilateral adrenalectomy to control symptoms. In the present manuscript, we review the genetics and pathological basis of FH-III, the diagnostic work-up, clinical features and therapeutic management. Finally, we will describe a new case of FH-III of an Italian patient carrying a Gly151Arg mutation.

ARMC5 mutation analysis in patients with primary aldosteronism and bilateral adrenal lesions.

Idiopathic hyperaldosteronism (IHA) due to bilateral adrenal hyperplasia is the most common subtype of primary aldosteronism (PA). The pathogenesis of IHA is still unknown, but the bilateral disease suggests a potential predisposing genetic alteration. Heterozygous germline mutations of armadillo repeat containing 5 (ARMC5) have been shown to be associated with hypercortisolism due to sporadic primary bilateral macronodular adrenal hyperplasia and are also observed in African-American PA patients. We investigated the presence of germline ARMC5 mutations in a group of PA patients who had bilateral computed tomography-detectable adrenal alterations. We sequenced the entire coding region of ARMC5 and all intron/exon boundaries in 39 patients (37 Caucasians and 2 black Africans) with confirmed PA (8 unilateral, 27 bilateral and 4 undetermined subtype) and bilateral adrenal lesions. We identified 11 common variants, 5 rare variants with a minor allele frequency <1% and 2 new variants not previously reported in public databases. We did not detect by in silico analysis any ARMC5 sequence variations that were predicted to alter protein function. In conclusion, ARMC5 mutations are not present in a fairly large series of Caucasian patients with PA associated to bilateral adrenal disease. Further studies are required to definitively clarify the role of ARMC5 in the pathogenesis of adrenal nodules and aldosterone excess in patients with PA.

Subtype Diagnosis of Primary Aldosteronism: Approach to Different Clinical Scenarios.

Identification and management of patients with primary aldosteronism are of utmost importance because it is a frequent cause of endocrine hypertension, and affected patients display an increase of cardio- and cerebro-vascular events, compared to essential hypertensives. Distinction of primary aldosteronism subtypes is of particular relevance to allocate the patients to the appropriate treatment, represented by mineralocorticoid receptor antagonists for bilateral forms and unilateral adrenalectomy for patients with unilateral aldosterone secretion. Subtype differentiation of confirmed hyperaldosteronism comprises adrenal CT scanning and adrenal venous sampling. In this review, we will discuss different clinical scenarios where execution, interpretation of adrenal vein sampling and subsequent patient management might be challenging, providing the clinician with useful information to help the interpretation of controversial procedures.

Renin and aldosterone measurements in the management of arterial hypertension.

Renin-angiotensin-aldosterone system (RAAS) is recognized as the main regulatory system of hemodynamics in man, and its derangements have a key role in the development and maintenance of arterial hypertension. Classification of the hypertensive states according to different patterns of renin and aldosterone levels ("RAAS profiling") allows the diagnosis of specific forms of secondary hypertension and may identify distinct hemodynamic subsets in essential hypertension. In this review, we summarize the application of RAAS profiling for the diagnostic assessment of hypertensive patients and discuss how the pathophysiological framework provided by RAAS profiling may guide therapeutic decision-making, especially in the context of uncontrolled hypertension not responding to multi-therapy.

Aldosterone does not modify gene expression in human endothelial cells.

The toxic effects of aldosterone on the vasculature, and in particular on the endothelial layer, have been proposed as having an important role in the cardiovascular pathology observed in mineralocorticoid-excess states. In order to characterize the genomic molecular mechanisms driving the aldosterone-induced endothelial dysfunction, we performed an expression microarray on transcripts obtained from both human umbilical vein endothelial cells and human coronary artery endothelial cells stimulated with 10 - 7 M aldosterone for 18 h. The results were then subjected to qRT-PCR confirmation, also including a group of genes known to be involved in the control of the endothelial function or previously described as regulated by aldosterone. The state of activation of the mineralocorticoid receptor was investigated by means of a luciferase-reporter assay using a plasmid encoding a mineralocorticoid and glucocorticoid-sensitive promoter. Aldosterone did not determine any significant change in gene expression in either cell type both in the microarray and in the qRT-PCR analysis. The luciferase-reporter assay showed no activation of the mineralocorticoid receptor following aldosterone stimulation. The status of nonfunctionality of the mineralocorticoid receptor expressed in cultured human umbilical and coronary artery endothelial cells does not allow aldosterone to modify gene expression and provides evidence against either a beneficial or harmful genomic effect of aldosterone on healthy endothelial cells.

Primary aldosteronism: who should be screened?

Primary aldosteronism (PA) has a prevalence in the general hypertensive population from 5 to 10%, and is widely recognized as the most frequent form of secondary hypertension. The 2 main PA subtypes are aldosterone producing adenoma (APA) and bilateral adrenal hyperplasia (BAH) that account for 95% of all PA cases. The diagnosis of PA is a 3-step process that comprises screening, confirmatory testing, and subtype differentiation. The different categories of patients at an increased risk of PA who should thus undergo a screening test were described in the first Endocrine Society (ES) Practice Guidelines for diagnosis and treatment of PA published in 2008. These categories include patients with Joint National Committee Stage 2, Stage 3, or drug-resistant hypertension; hypertension, and spontaneous or diuretic-induced hypokalemia; hypertension with adrenal incidentaloma; hypertension and a family history of early-onset hypertension or cerebrovascular accident at a young age and all hypertensive first degree relatives of patients with PA. Recently, a growing number of studies have linked PA with the metabolic syndrome, diabetes, and obstructive sleep apnea that may be partly responsible for the higher rate of cardio and cerobrovascular accidents in PA patients. The aim of this review is to discuss, which patients should be screened for PA, focusing not only on the well-established categories of the ES Guidelines, but also on additional other group of patients with a potentially high prevalence of PA that has emerged from recent research.

Salivary aldosterone as a diagnostic aid in primary aldosteronism.

Recent evidence demonstrates an increased incidence of primary aldosteronism (PA) in approximately 10% of the hypertensive population, making noninvasive and simple screening methods necessary. The aim of the present study was to apply a time-resolved fluorescence immunoassay for the measurement of aldosterone in saliva and the establishment of a cut-off to identify patients with a high likelihood for PA requiring subsequent screening with the aldosterone to renin ratio. Saliva was collected (AM and PM) to ascertain an optimum time with best discriminating power between healthy and disease states. Plasma aldosterone, after overnight recumbency and 4 h later, was collected for posture testing. The participants included 53 PA patients (aged 14-78), 54 with essential hypertension (EH, aged 19-82), and 38 healthy volunteers (aged 19-56). Saliva aldosterone (SA) (median, 25-75(th)%) in PA was found at 90 pg/ml (61-139) compared to 53 pg/ml (40-85) in EH, with discrimination between PA versus EHs best in the morning (cutoff: 81 pg/ml, 77% sensitivity, 82% specificity). Saliva aldosterone decreases throughout the day in patients with adenomas [APA AM: 123 pg/ml (92-213) vs. PM: 79 pg/ml (41-116)], but not in those with bilateral hyperplasia [BAH AM: 85 pg/ml (59-115)] vs. pm 69 pg/ml (57-114). Morning SA alone allows discrimination between PA and controls, though with significant overlap against EHs, leading to a high number of false positives. More promising is the use of diurnal variation in SA in distinguishing between APA and BAH. The decline in SA seen in patients with APA presents a more constant finding compared to posture testing, which fails to correctly classify a large number of patients.

Confirmatory tests in the diagnosis of primary aldosteronism.

Primary aldosteronism is the most common form of secondary hypertension and patients with hyperaldosteronism are more prone to premature cardiovascular complications compared to essential hypertensives. The diagnostic flow-chart for the diagnosis of PA is performed in three steps: a) screening; b) confirmation; and c) subtype differentiation. Instead of proceeding directly to subtype classification, the recently published Endocrine Society Guidelines recommend that patients with a positive ARR should undergo a confirmatory test, in order to definitively confirm or exclude the diagnosis of PA. The Guidelines recognize four testing procedures: oral sodium loading, saline infusion, fludrocortisone suppression, and captopril challenge. Herein we discuss the diagnostic protocols for these confirmatory tests and highlight both the advantages and contraindications and we discuss studies in which these confirmatory tests have been compared.

Impairment of cardiovascular autonomic pattern in obese adolescents with Type 2 diabetes mellitus.

UNLABELLED: The aim of this study was to assess the behaviour of insulin sensitivity and insulin resistance (IR) indexes in a group of obese adolescents with Type 2 diabetes mellitus (T2DM) in comparison to obese adolescents without diabetes and normal controls, moreover to compare these parameters with the cardiac autonomic pattern. Seven T2DM obese (12.7 ± 0.5 yr), 18 obese without T2DM, and 10 nonobese control adolescents age matched were studied. In all subjects we performed oral glucose tolerance test (OGTT) with insulin and glucose determination, 24-h electrocardiogram Holter, blood pressure monitoring, ecohocardiogram. RESULTS: serum lipids were significantly higher in obese and T2DM. Insulin sensitivity was significantly reduced in T2DM and obese vs controls; T2DM showed a more pronounced oral glucose insulin sensitivity (OGIS) reduction vs obese. Both obese and T2DM presented an higher IR. T2DM showed an impaired ß-cell function, with insulin areas under the curve and disposition index significantly reduced in comparison to controls and obese who showed similar values. A progressive reduction of vagal indexes and an increase of sympathetic indexes were found in obese adolescents and were more pronounced in T2DM. These parameters were correlated with OGIS and ß-cell function parameters in both obese and T2DM adolescents. T2DM showed a significant relative wall thickness increase suggesting a trend toward concentric remodeling. In conclusion, T2DM adolescents are characterized by a more marked IR reduced ß-cell function in comparison to non-diabetic obese. These modifications may lead to an early impairment of the autonomic pattern.

Saliva as a medium for aldosterone measurement in repeated sampling studies.

BACKGROUND: Saliva is a readily available biological fluid, making it convenient in diagnosis of diseases and in multi-sampling protocols. Several salivary steroids give a useful index of free plasma levels. Increased incidence of primary aldosteronism (PA) in approximately 10% of the hypertensive population has increased interest in the mineralocorticoid aldosterone. METHODS: A biotinylated-aldosterone tracer and a commercially available antibody are used in a time-resolved fluorescence immunoassay (TR-FIA) to measure salivary aldosterone (SA). Saliva was collected in various multi-sampling protocols: Investigation of diurnal rhythm in healthy and PA patients, ACTH stimulation test and posture test in healthy subjects. RESULTS: Method validation showed a sensitivity of 19 ng/L and intra-/inter-assay precision between 7.2-10.1% and 8.7-15.7%, respectively. SA correlated significantly (y = 0.2995x +/- 0.01, r(2)=0.60) to plasma aldosterone measured by a commercial radioimmunoassay. SA (median; 95%CI) was at 111 (95-127)ng/L in PA (n=84) and 50 (44-56)ng/L in healthy subjects (n=60). After change in posture, aldosterone increased in both, saliva (57 (47-63)ng/L to 95 (84-117)ng/L) and plasma (26 (26-41)ng/L to 135 (110-181)ng/L). Peak levels were reached after 1h, and were higher in females than in males. CONCLUSIONS: SA correlates well to plasma aldosterone and mirrors responses during conditions of stress. SA is significantly higher in PA, and the diurnal rhythm seen in the healthy is blunted in PA. We additionally found gender-dependent differential responses to posture, with higher increases in females. Measurement of aldosterone in saliva presents a useful and convenient method for application in multi-sampling studies.

Blood pressure levels, left ventricular mass and function are correlated with left atrial volume in mild to moderate hypertensive patients.

Arterial hypertension is associated with an increased risk of atrial fibrillation (AF), and leads to a pronounced increase in morbidity and mortality. Left atrial volume (LAV) is an important prognostic marker in the older populations. The aim of our study was to identify the clinical and echocardiographic determinants of LAV in middle-aged (<70 years old) essential hypertensive patients.We evaluated cardiac structure and function in 458 patients, 394 treated and untreated mild to moderate essential hypertensives patients (mean+/-s.d. age 48.4+/-11.1 years) with no associated clinical condition and 64 normotensive control participants (age 45.7+/-12.8 years; P=0.12). A multivariate analysis was performed to calculate the relative weight of each of the variables considered able to predict LAV. The LAV index (LAVi) was significantly increased in the essential hypertensive group vs the control group and was significantly dependent on blood pressure levels (SBP and DBP, P<0.05 for both) and body mass index (BMI) (P<0.0001). Considering the left ventricular (LV) variables, the LV mass index (LVMI) (R(2)=0.19, P<0.001) and LAV were increased in essential hypertensive patients with left ventricular hypertrophy (LVH), and patients with enlarged LAV showed lower systolic and diastolic function and an increased LVMI. The LAVi is dependent on blood pressure levels and anthropometric variables (age and BMI). Further structural (LVMI) and functional (systolic and diastolic) variables are related to the LAVi; LVMI is the most important variable associated with LAV in mild to moderate essential hypertensive adult patients. These findings highlight the importance of left atrium evaluation in adult, relatively young, essential hypertensive patients.

Adrenal endothelin-1 levels are not associated with aldosterone secretion in primary aldosteronism.

BACKGROUND: Endothelin-1 (ET-1) may function as an aldosterone secretagogue and, in turn, aldosterone can upregulate ET-1 expression. Hence, the existence of a feedforward loop involving ETs and aldosterone has been speculated in primary aldosteronism (PA). In the present study, we sought to examine ET-1 secretion from the adrenal glands in patients with PA. DESIGN: We determined ET-1 levels in blood samples obtained during adrenal venous sampling of patients affected by PA (n=17). Furthermore, we examined the mRNA expression of the ET system in tissue samples from aldosterone-producing adenomas (APAs, n=9) and control normal adrenals (n=3). METHODS: Blood ET-1 levels were determined by RIA. Tissue mRNA expression of the ET system was assayed with Affymetrix microarrays. RESULTS: ET-1 levels did not differ between inferior vena cava and adrenal vein blood in both bilateral adrenal hyperplasia and APA patients. Moreover, cortisol-normalized ET-1 levels did not show lateralized adrenal ET-1 secretion in APAs. Through gene expression profiling with microarray performed in a distinct set of APA individuals (n=9), we confirmed the adrenal expression of a complete ET system, but we did not detect a significant upregulation of ET components within the APA tissue compared with normal adrenals. CONCLUSIONS: The present data argue against the hypothesis of increased ET-1 secretion from APAs and do not support a general role for adrenal ET-1 in the vascular pathophysiology of PA.

Characteristics of the patients referred to a Hypertension Unit between 1989 and 2003.

The level of blood pressure, the type of antihypertensive treatment and the prevalence of resistant hypertension at the first examination were evaluated in 6254 patients referred to a hospital Hypertension Unit from 1989 to 2003. From 1989-1993 to 1999-2003, we observed a reduced prevalence of grade 2 and grade 3 hypertension, and an increase in the prevalence of grade 1 hypertension, the proportion of treated subjects, the average number of antihypertensive drugs per patient and the prevalence of resistant hypertension.

Conceptual basis and methodology of the SOPHIA study.

To clarify the role of gene polymorphisms on the effect of losartan and losartan plus hydrochlorothiazide on blood pressure (primary end point) and on cardiac, vascular and metabolic phenotypes (secondary end point) after 4, 8, 12, 16 and 48 weeks treatment, an Italian collaborative study - The Study of the Pharmacogenomics in Italian hypertensive patients treated with the Angiotensin receptor blocker losartan (SOPHIA) - on never-treated essential hypertensives (n = 800) was planned. After an 8 week run-in, losartan 50 mg once daily will be given and doubled to 100 mg at week +4 if blood pressure is more than 140/90 mmHg. Hydroclorothiazide 25 mg once daily at week +8 and amlodipine 5 mg at week +16 will be added if blood pressure is more than 140/90 mmHg. Cardiac mass (echocardiography), carotid intima-media thickness, 24 h ambulatory blood pressure, homeostatic model assessment (HOMA) index, microalbuminuria, plasma renin activity and aldosterone, endogenous lithium clearance, brain natriuretic peptide and losartan metabolites will be evaluated. Genes of the renin-angiotensin-aldosterone system, salt sensitivity, the beta-adrenergic system and losartan metabolism will be studied (Illumina custom arrays). A whole-genome scan will also be performed in half of the study cohort (1M array, Illumina 500 GX beadstation).

Baroreflex sensitivity is impaired in essential hypertensives with central obesity.

Recently, much interest has focussed on the potential interaction between sympathetic nervous system and global cardiovascular risk. We investigated how baroreflex sensitivity (BRS), an index of autonomic function, interacts with central obesity (CO) in an essential hypertensive (EH) population. We selected 170 EHs and 43 normotensives (NT), (median age 47.3+/-11.3 and 49.1+/-13 years, respectively). Anthropometric parameters were measured for each and BRS was evaluated by a non-invasive method using Portapres TNO. The BRS evaluation was made using the sequences method. Systolic blood pressure (SBP) and heart rate were significantly higher in EH (P<0.001 and P=0.007, respectively). BRS was significantly greater in NT (P=0.02), and was associated inversely with waist circumference (WC) (P=0.005), but not with SBP or with other metabolic risk factors. Body mass index, total and high-density lipoprotein cholesterol, age and WC were not significantly different between the two groups. These results were confirmed by age pounded analysis. Finally, a separate analysis of the hypertensive group with CO (n=84) demonstrated a significantly lower BRS compared with the other hypertensive patients (n=86) (P<0.001). BRS is associated with WC but not with arterial pressure values and metabolic risk factors. Hypertensive subjects with CO show an impairment of BRS. Owing to its association with abdominal fat distribution and subsequently insulin resistance, BRS could represent a further and reliable index for evaluation of global cardiovascular risk in hypertensive patients.

Genetic mutation screening in an italian cohort of nonsyndromic pheochromocytoma/paraganglioma patients.

To assess the prevalence of genetic mutations in nonsyndromic pheochromocytoma/paraganglioma (PHEO/PGL) patients we have performed a systematic search for mutations in the succinate dehydrogenase (SDH) B, C, and D subunits, von Hippel-Lindau (VHL), and RET genes by direct bidirectional sequencing. Patients were selected from the medical records of hypertension centers. After exclusion of syndromic patients, 45 patients with familial (F+, n=3) and sporadic (F-, n=42) cases of isolated PHEO/PGL were considered. They included 35 patients with PHEO, 7 with PGL, and 3 with head/neck PGL (hnPGL). Three patients with PHEO (2F-, 1F+) presented VHL mutations (P86A, G93C, and R167W), six with PGL (4F-, 2F+) were positive for SDH or VHL mutations (SDHB R230G in two patients, SDHB S8F, R46Q, R90Q, and VHL P81L in one subject each), and one with hnPGL carried the SDHD 348-351delGACT mutation. We have also detected missense (SDHB S163P, SDHD H50R and G12S), synonymous (SDHB A6A, SDHD S68S), and intronic mutations that have been considered nonpathological polymorphic variants. No mutation was found in SDHC or RET genes. Our data indicate that germline mutations of VHL and SDH subunits are not infrequent in familial as well as in sporadic cases of nonsyndromic PHEO/PGL (overall, 12 of 45 probands, 22%). Accordingly, screening for such mutations seems to be justified. However, a more precise characterization of the functional relevance of any observed sequence variant and of other genetic and environmental determinants of neoplastic transformation is essential in order to plan appropriate protocols for family screening and follow-up.

[Role of aldosterone in the metabolic syndrome]. / Ruolo dell'aldosterone nella sindrome metabolica.

The purpose of this review is to summarize the current knowledge regarding metabolic syndrome prevalence and features in primary aldosteronism. We will also discuss the link between aldosterone and the different metabolic changes typical of the metabolic syndrome. Hypertensive patients have a high prevalence of obesity, dyslipidemia and hyperglycaemia. These are risk factors for the metabolic syndrome, and are associated with an increased cardiovascular risk profile. In particular, insulin resistance seems to be the major alteration in patients affected by primary aldosteronism. We will then describe the experimental and clinical evidences of the role of aldosterone in the pathogenesis of insulin resistance. Higher rates of cardiovascular events have been recently reported in primary aldosteronism: they could be partly due to the increased prevalence of the metabolic syndrome in this disorder.