Association of adrenal steroids with metabolomic profiles in patients with primary and endocrine hypertension.

Introduction: Endocrine hypertension (EHT) due to pheochromocytoma/paraganglioma (PPGL), Cushing's syndrome (CS), or primary aldosteronism (PA) is linked to a variety of metabolic alterations and comorbidities. Accordingly, patients with EHT and primary hypertension (PHT) are characterized by distinct metabolic profiles. However, it remains unclear whether the metabolomic differences relate solely to the disease-defining hormonal parameters. Therefore, our objective was to study the association of disease defining hormonal excess and concomitant adrenal steroids with metabolomic alterations in patients with EHT. Methods: Retrospective European multicenter study of 263 patients (mean age 49 years, 50% females; 58 PHT, 69 PPGL, 37 CS, 99 PA) in whom targeted metabolomic and adrenal steroid profiling was available. The association of 13 adrenal steroids with differences in 79 metabolites between PPGL, CS, PA and PHT was examined after correction for age, sex, BMI, and presence of diabetes mellitus. Results: After adjustment for BMI and diabetes mellitus significant association between adrenal steroids and metabolites - 18 in PPGL, 15 in CS, and 23 in PA - were revealed. In PPGL, the majority of metabolite associations were linked to catecholamine excess, whereas in PA, only one metabolite was associated with aldosterone. In contrast, cortisone (16 metabolites), cortisol (6 metabolites), and DHEA (8 metabolites) had the highest number of associated metabolites in PA. In CS, 18-hydroxycortisol significantly influenced 5 metabolites, cortisol affected 4, and cortisone, 11-deoxycortisol, and DHEA each were linked to 3 metabolites. Discussions: Our study indicates cortisol, cortisone, and catecholamine excess are significantly associated with metabolomic variances in EHT versus PHT patients. Notably, catecholamine excess is key to PPGL's metabolomic changes, whereas in PA, other non-defining adrenal steroids mainly account for metabolomic differences. In CS, cortisol, alongside other non-defining adrenal hormones, contributes to these differences, suggesting that metabolic disorders and cardiovascular morbidity in these conditions could also be affected by various adrenal steroids.

Unilateral Primary Aldosteronism: Long-Term Disease Recurrence After Adrenalectomy.

BACKGROUND: Primary aldosteronism (PA) is frequently caused by a unilateral aldosterone-producing adenoma with a PA-driver mutation. Unilateral adrenalectomy has a high probability of short-term biochemical remission, but long-term postsurgical outcomes are relatively undefined. Our objective was to investigate the incidence of long-term recurrence of PA in individuals with postsurgical short-term biochemical remission. METHODS: Adrenalectomized patients for unilateral PA were included from a single referral center. Histopathology and outcomes were assessed according to international histopathology of unilateral primary aldosteronism and PASO (Primary Aldosteronism Surgical Outcome) consensuses. Genotyping was performed using CYP11B2 (aldosterone synthase)-guided sequencing. RESULTS: Classical adrenal histopathology, exemplified by a solitary aldosterone-producing adenoma, was observed in 78% of 90 adrenals, compared with 22% with nonclassical histopathology. The classical group displayed higher aldosterone-to-renin ratios (P=0.013) and lower contralateral ratios (P=0.008). Outcome assessments at both short (12 months [7; 12]) and long (89 months [48; 124]) terms were available for 57 patients. At short-term assessment, 53 (93%) displayed complete biochemical success (43 classical and 10 nonclassical), but long-term assessment demonstrated biochemical PA recurrence in 12 (23%) with an overrepresentation of the nonclassical histopathology (6 [60%] of 10 nonclassical histopathology versus 6 [14%] of 43 classical histopathology; P=0.005). PA-driver mutations were identified in 97% of 64 aldosterone-producing adenomas; there was no association of the aldosterone-producing adenoma genotype with PA recurrence. CONCLUSIONS: A substantial proportion of individuals display postsurgical biochemical recurrence of PA, which is related to the histopathology of the resected adrenal gland. These findings emphasize the role of histopathology and the requirement for continued outcome assessment in the management of surgically treated patients for PA.

Primary Aldosteronism: Spatial Multiomics Mapping of Genotype-Dependent Heterogeneity and Tumor Expansion of Aldosterone-Producing Adenomas.

BACKGROUND: Primary aldosteronism is frequently caused by an adrenocortical aldosterone-producing adenoma (APA) carrying a somatic mutation that drives aldosterone overproduction. APAs with a mutation in KCNJ5 (APA-KCNJ5MUT) are characterized by heterogeneous CYP11B2 (aldosterone synthase) expression, a particular cellular composition and larger tumor diameter than those with wild-type KCNJ5 (APA-KCNJ5WT). We exploited these differences to decipher the roles of transcriptome and metabolome reprogramming in tumor pathogenesis. METHODS: Consecutive adrenal cryosections (7 APAs and 7 paired adjacent adrenal cortex) were analyzed by spatial transcriptomics (10x Genomics platform) and metabolomics (in situ matrix-assisted laser desorption/ionization mass spectrometry imaging) co-integrated with CYP11B2 immunohistochemistry. RESULTS: We identified intratumoral transcriptional heterogeneity that delineated functionally distinct biological pathways. Common transcriptomic signatures were established across all APA specimens which encompassed 2 distinct transcriptional profiles in CYP11B2-immunopositive regions (CYP11B2-type 1 or 2). The CYP11B2-type 1 signature was characterized by zona glomerulosa gene markers and was detected in both APA-KCNJ5MUT and APA-KCNJ5WT. The CYP11B2-type 2 signature displayed markers of the zona fasciculata or reticularis and predominated in APA-KCNJ5MUT. Metabolites that promote oxidative stress and cell death accumulated in APA-KCNJ5WT. In contrast, antioxidant metabolites were abundant in APA-KCNJ5MUT. Finally, APA-like cell subpopulations-negative for CYP11B2 gene expression-were identified in adrenocortical tissue adjacent to APAs suggesting the existence of tumor precursor states. CONCLUSIONS: Our findings provide insight into intra- and intertumoral transcriptional heterogeneity and support a role for prooxidant versus antioxidant systems in APA pathogenesis highlighting genotype-dependent capacities for tumor expansion.

Prevalence of Functioning Adrenal Incidentalomas: A Systematic Review and Meta-analysis.

CONTEXT: Adrenal hyperfunction is associated with an increased risk of cardiometabolic complications in subjects with adrenal incidentaloma (AI). Reliable prevalence estimates of functioning AIs are important to direct resources allocations. OBJECTIVE: To assess the prevalence of autonomous/possible autonomous cortisol secretion (ACS), primary aldosteronism (PA), pheochromocytoma (PHEO), and Cushing syndrome (CS) in patients with AI. METHODS: We performed a comprehensive search of multiple databases (PubMed, Ovid MEDLINE, Web of Science) for potentially relevant studies without language restriction, up to February 2022. Of the 1661 publications evaluated at title and abstract levels, 161 were examined as full text and 36 were included. Study level clinical data were extracted by 3 independent reviewers. RESULTS: The overall prevalence of functioning AIs was 27.5% (95% CI 23.0, 32.5). ACS/possible ACS, with a prevalence of 11.7% (95% CI 8.6, 15.7), was the most frequent hormonal alteration, while PA occurred in 4.4% of the patients (95% CI 3.1, 6.2). Subgroup analysis showed that PA was more prevalent in patients from Asia than in patients from Europe/America; in contrast, ACS/possible ACS had a lower prevalence in Asian countries. At meta-regression analysis, the prevalence of ACS/possible ACS was influenced by the proportion of female patients, while the prevalence of PA was positively associated with the proportion of patients with hypertension and the publication year. Finally, PHEO and CS prevalence were 3.8% (95% CI 2.8, 5.0) and 3.1% (95% CI 2.3, 4.3) respectively. CONCLUSION: This meta-analysis provides extensive data on the prevalence of functioning AIs and the factors affecting heterogeneity in prevalence estimates.

TSPAN12 (Tetraspanin 12) Is a Novel Negative Regulator of Aldosterone Production in Adrenal Physiology and Aldosterone-Producing Adenomas.

BACKGROUND: Aldosterone-producing adenomas (APAs) are a major cause of primary aldosteronism, a condition of low-renin hypertension, in which aldosterone overproduction is usually driven by a somatic activating mutation in an ion pump or channel. TSPAN12 is differentially expressed in different subgroups of APAs suggesting a role in APA pathophysiology. Our objective was to determine the function of TSPAN12 (tetraspanin 12) in adrenal physiology and pathophysiology. METHODS: APA specimens, pig adrenals under dietary sodium modulation, and a human adrenocortical cell line HAC15 were used for functional characterization of TSPAN12 in vivo and in vitro. RESULTS: Gene ontology analysis of 21 APA transcriptomes dichotomized according to high versus low TSPAN12 transcript levels highlighted a function for TSPAN12 related to the renin-angiotensin system. TSPAN12 expression levels in a cohort of 30 APAs were inversely correlated with baseline plasma aldosterone concentrations (R=-0.47; P=0.009). In a pig model of renin-angiotensin system activation by dietary salt restriction, TSPAN12 mRNA levels and TSPAN12 immunostaining were markedly increased in the zona glomerulosa layer of the adrenal cortex. In vitro stimulation of human adrenocortical human adrenocortical cells with 10 nM angiotensin II for 6 hours caused a 1.6-fold±0.13 increase in TSPAN12 expression, which was ablated by 10 µM nifedipine (P=0.0097) or 30 µM W-7 (P=0.0022). Gene silencing of TSPAN12 in human adrenocortical cells demonstrated its inverse effect on aldosterone secretion under basal and angiotensin II stimulated conditions. CONCLUSIONS: Our findings show that TSPAN12 is a negative regulator of aldosterone production and could contribute to aldosterone overproduction in primary aldosteronism.

Predicting Hypertension Subtypes with Machine Learning Using Targeted Metabolites and Their Ratios.

Hypertension is a major global health problem with high prevalence and complex associated health risks. Primary hypertension (PHT) is most common and the reasons behind primary hypertension are largely unknown. Endocrine hypertension (EHT) is another complex form of hypertension with an estimated prevalence varying from 3 to 20% depending on the population studied. It occurs due to underlying conditions associated with hormonal excess mainly related to adrenal tumours and sub-categorised: primary aldosteronism (PA), Cushing's syndrome (CS), pheochromocytoma or functional paraganglioma (PPGL). Endocrine hypertension is often misdiagnosed as primary hypertension, causing delays in treatment for the underlying condition, reduced quality of life, and costly antihypertensive treatment that is often ineffective. This study systematically used targeted metabolomics and high-throughput machine learning methods to predict the key biomarkers in classifying and distinguishing the various subtypes of endocrine and primary hypertension. The trained models successfully classified CS from PHT and EHT from PHT with 92% specificity on the test set. The most prominent targeted metabolites and metabolite ratios for hypertension identification for different disease comparisons were C18:1, C18:2, and Orn/Arg. Sex was identified as an important feature in CS vs. PHT classification.

Primary aldosteronism: Pathophysiological mechanisms of cell death and proliferation.

Primary aldosteronism is the most common surgically curable form of hypertension. The sporadic forms of the disorder are usually caused by aldosterone overproduction from a unilateral adrenocortical aldosterone-producing adenoma or from bilateral adrenocortical hyperplasia. The main knowledge-advances in disease pathophysiology focus on pathogenic germline and somatic variants that drive the excess aldosterone production. Less clear are the molecular and cellular mechanisms that lead to an increased mass of the adrenal cortex. However, the combined application of transcriptomics, metabolomics, and epigenetics has achieved substantial insight into these processes and uncovered the evolving complexity of disrupted cell growth mechanisms in primary aldosteronism. In this review, we summarize and discuss recent progress in our understanding of mechanisms of cell death, and proliferation in the pathophysiology of primary aldosteronism.

Preanalytical Pitfalls in Untargeted Plasma Nuclear Magnetic Resonance Metabolomics of Endocrine Hypertension.

Despite considerable morbidity and mortality, numerous cases of endocrine hypertension (EHT) forms, including primary aldosteronism (PA), pheochromocytoma and functional paraganglioma (PPGL), and Cushing's syndrome (CS), remain undetected. We aimed to establish signatures for the different forms of EHT, investigate potentially confounding effects and establish unbiased disease biomarkers. Plasma samples were obtained from 13 biobanks across seven countries and analyzed using untargeted NMR metabolomics. We compared unstratified samples of 106 PHT patients to 231 EHT patients, including 104 PA, 94 PPGL and 33 CS patients. Spectra were subjected to a multivariate statistical comparison of PHT to EHT forms and the associated signatures were obtained. Three approaches were applied to investigate and correct confounding effects. Though we found signatures that could separate PHT from EHT forms, there were also key similarities with the signatures of sample center of origin and sample age. The study design restricted the applicability of the corrections employed. With the samples that were available, no biomarkers for PHT vs. EHT could be identified. The complexity of the confounding effects, evidenced by their robustness to correction approaches, highlighted the need for a consensus on how to deal with variabilities probably attributed to preanalytical factors in retrospective, multicenter metabolomics studies.

Aldosterone as a Mediator of Cardiovascular Damage.

Besides the physiological regulation of water, sodium, and potassium homeostasis, aldosterone modulates several physiological and pathological processes in the cardiovascular system. At the vascular level, aldosterone excess stimulates endothelial dysfunction and infiltration of inflammatory cells, enhances the development of the atherosclerotic plaque, and favors plaque instability, arterial stiffness, and calcification. At the cardiac level, aldosterone increases cardiac inflammation, fibrosis, and myocardial hypertrophy. As a clinical consequence, high aldosterone levels are associated with enhanced risk of cardiovascular events and mortality, especially when aldosterone secretion is inappropriate for renin levels and sodium intake, as in primary aldosteronism. Several clinical trials showed that mineralocorticoid receptor antagonists reduce cardiovascular mortality in patients with heart failure and reduced ejection fraction, but inconclusive results were reported for other cardiovascular conditions, such as heart failure with preserved ejection fraction, myocardial infarction, and atrial fibrillation. In patients with primary aldosteronism, adrenalectomy or treatment with mineralocorticoid receptor antagonists significantly mitigate adverse aldosterone effects, reducing the risk of cardiovascular events, mortality, and incident atrial fibrillation. In this review, we will summarize the major preclinical and clinical studies investigating the cardiovascular damage mediated by aldosterone and the protective effect of mineralocorticoid receptor antagonists for the reduction of cardiovascular risk in patients with cardiovascular diseases and primary aldosteronism.

Clinical Score and Machine Learning-Based Model to Predict Diagnosis of Primary Aldosteronism in Arterial Hypertension.

[Figure: see text].

Primary Aldosteronism: Metabolic Reprogramming and the Pathogenesis of Aldosterone-Producing Adenomas.

Aldosterone-producing adenomas (APAs) are characterized by aldosterone hypersecretion and deregulated adrenocortical cell growth. Increased energy consumption required to maintain cellular tumorigenic properties triggers metabolic alterations that shape the tumor microenvironment to acquire necessary nutrients, yet our knowledge of this adaptation in APAs is limited. Here, we investigated adrenocortical cell-intrinsic metabolism and the tumor immune microenvironment of APAs and their potential roles in mediating aldosterone production and growth of adrenocortical cells. Using multiple advanced bioinformatics methods, we analyzed gene expression datasets to generate distinct metabolic and immune cell profiles of APAs versus paired adjacent cortex. APAs displayed activation of lipid metabolism, especially fatty acid ß-oxidation regulated by PPARα, and glycolysis. We identified an immunosuppressive microenvironment in APAs, with reduced infiltration of CD45+ immune cells compared with adjacent cortex, validated by CD45 immunohistochemistry (3.45-fold, p < 0.001). APAs also displayed an association of lipid metabolism with ferroptosis and upregulation of antioxidant systems. In conclusion, APAs exhibit metabolic reprogramming towards fatty acid ß-oxidation and glycolysis. Increased lipid metabolism via PPARα may serve as a key mechanism to modulate lipid peroxidation, a hallmark of regulated cell death by ferroptosis. These findings highlight survival advantages for APA tumor cells with metabolic reprogramming properties.

Characterization of Circulating Extracellular Vesicle Surface Antigens in Patients With Primary Aldosteronism.

[Figure: see text].

BEX1 Is Differentially Expressed in Aldosterone-Producing Adenomas and Protects Human Adrenocortical Cells From Ferroptosis.

[Figure: see text].

Targeted Metabolomics as a Tool in Discriminating Endocrine From Primary Hypertension.

CONTEXT: Identification of patients with endocrine forms of hypertension (EHT) (primary hyperaldosteronism [PA], pheochromocytoma/paraganglioma [PPGL], and Cushing syndrome [CS]) provides the basis to implement individualized therapeutic strategies. Targeted metabolomics (TM) have revealed promising results in profiling cardiovascular diseases and endocrine conditions associated with hypertension. OBJECTIVE: Use TM to identify distinct metabolic patterns between primary hypertension (PHT) and EHT and test its discriminating ability. METHODS: Retrospective analyses of PHT and EHT patients from a European multicenter study (ENSAT-HT). TM was performed on stored blood samples using liquid chromatography mass spectrometry. To identify discriminating metabolites a "classical approach" (CA) (performing a series of univariate and multivariate analyses) and a "machine learning approach" (MLA) (using random forest) were used.The study included 282 adult patients (52% female; mean age 49 years) with proven PHT (n = 59) and EHT (n = 223 with 40 CS, 107 PA, and 76 PPGL), respectively. RESULTS: From 155 metabolites eligible for statistical analyses, 31 were identified discriminating between PHT and EHT using the CA and 27 using the MLA, of which 16 metabolites (C9, C16, C16:1, C18:1, C18:2, arginine, aspartate, glutamate, ornithine, spermidine, lysoPCaC16:0, lysoPCaC20:4, lysoPCaC24:0, PCaeC42:0, SM C18:1, SM C20:2) were found by both approaches. The receiver operating characteristic curve built on the top 15 metabolites from the CA provided an area under the curve (AUC) of 0.86, which was similar to the performance of the 15 metabolites from MLA (AUC 0.83). CONCLUSION: TM identifies distinct metabolic pattern between PHT and EHT providing promising discriminating performance.

Quality of life in primary aldosteronism: A prospective observational study.

BACKGROUND: Previous studies suggested that patients affected by primary aldosteronism (PA) have impaired quality of life (QOL) compared to the general population, but a direct comparison with patients affected by essential hypertension (EH) has never been performed. The aim of the study was to compare the QOL of patients affected by PA to the QOL of patients affected by EH. MATERIAL AND METHODS: We designed a prospective observational study comparing the QOL of patients with PA and carefully matched patients with EH before and after treatment. We recruited 70 patients with PA and 70 patients with EH, matched for age, sex, blood pressure levels and intensity of antihypertensive treatment. We assessed QOL at baseline and after specific treatment for PA or after optimization of medical therapy for patients with EH. RESULTS: Patients with PA displayed impaired QOL compared with the general healthy population, but similar to patients with EH. Both laparoscopic adrenalectomy and treatment with mineralocorticoid receptor antagonist allowed an improvement of QOL in patients with PA, that was more pronounced after surgical treatment. Optimization of blood pressure control by implementation of antihypertensive treatment (without MR antagonists) allowed a minimal improvement in only one of eight domains in patients with EH. CONCLUSIONS: Patients with PA have impaired QOL, which is likely caused by uncontrolled hypertension and the effects of intensive antihypertensive treatment. Surgical and medical treatment of PA allows a significant improvement of QOL, by amelioration of blood pressure control and, after surgical treatment, by reduction of antihypertensive treatment.

Prevalence of Hypokalemia and Primary Aldosteronism in 5100 Patients Referred to a Tertiary Hypertension Unit.

Primary aldosteronism (PA) was considered a rare disorder almost always associated with hypokalemia. The widespread screening of patients with hypertension unveiled an increased prevalence of PA with normokalemic hypertension the prevailing phenotype. Many studies have reported the prevalence of hypokalemia in patients with PA; conversely, the prevalence of PA in patients with hypokalemia is unknown. In this retrospective observational study, we define the prevalence of hypokalemia in referred patients with hypertension and the prevalence of PA in patients with hypokalemia and hypertension. Hypokalemia was present in 15.8% of 5100 patients with hypertension, whereas 76.9% were normokalemic, and 7.3% hyperkalemic. The prevalence of PA in patients with hypokalemia was 28.1% and increased with decreasing potassium concentrations up to 88.5% of patients with spontaneous hypokalemia and potassium concentrations <2.5 mmol/L. A multivariate regression analysis demonstrated the association of hypokalemia with the occurrence of cardiovascular events independent of PA diagnosis. An association of PA with the occurrence of cardiovascular events and target organ damage independent of hypokalemia was also demonstrated. In conclusion, our results confirm that PA is a frequent cause of secondary hypertension in patients with hypokalemia, and the presence of hypertension and spontaneous hypokalemia are strong indications for PA diagnosis. Finally, we show that PA and hypokalemia are associated with an increased risk of cardiovascular events.

The spectrum of low-renin hypertension.

Low-renin hypertension (LRH) is a frequent condition in patients with arterial hypertension, accounting for 30% of patients. Monogenic forms can cause LRH in a minority of cases. However, in the large majority of patients, LRH is caused by the combined effects of congenital and acquired factors, comprising dietary habits. Several genetic variants have been proposed as co-factors in the pathogenesis of LRH with normal-low serum aldosterone. Emerging evidences support the hypothesis that a large proportion of LRH with normal-high serum aldosterone is associated with subclinical primary aldosteronism (PA). The recent identification of aldosterone-producing cell clusters (APCCs) as the possible cause of subclinical PA, further supported the concept of a continuous spectrum of autonomous aldosterone secretion, from subclinical forms towards overt PA. In this review we describe the main aspects of LRH, focusing on molecular basis, clinical risk profile and patients' management.

Renin-Angiotensin-Aldosterone System Triple-A Analysis for the Screening of Primary Aldosteronism.

Primary aldosteronism is recognized as the most frequent cause of secondary hypertension, and its screening is expected to become a routine evaluation in most patients with hypertension. The interference of antihypertensive therapies with the aldosterone-to-renin ratio during screening process is a major confounder. Renin-angiotensin-aldosterone system Triple-A analysis is a novel liquid chromatography/tandem mass spectrometry diagnostic assay that allows simultaneous quantification of aldosterone, equilibrium Ang I (angiotensin I), and Equilibrium Ang II in a single sample of serum. We performed a comparative evaluation of the diagnostic performance of the aldosterone-to-Ang II ratio and 5 renin-based diagnostic ratios, differing in methods to determine aldosterone levels and renin activity in a cohort of 110 patients with hypertension (33 patients with confirmed primary aldosteronism and 77 with essential hypertension). All ratios showed comparable areas under the curves ranging between 0.924 and 0.970 without significant differences between each other. The evaluation of the Ang II-to-Ang I ratio revealed persistent drug intake in some patients as cause for suppressed renin-based diagnostic ratios, while aldosterone-to-Ang II ratio remained unaffected. The Youden index optimal cutoff value for the aldosterone-to-Ang II ratio was 6.6 ([pmol/L]/[pmol/L]) with a sensitivity of 90% and a specificity of 93%, proving noninferiority compared with the aldosterone-to-renin ratio while pointing to the potential for an interference-free application in patients under ACE (angiotensin-converting enzyme) inhibitor therapy. This study shows for the first time the accuracy and reliability of renin-angiotensin-aldosterone system triple-A analysis for the screening of primary aldosteronism that can be applied in clinical routine.

Characterization and Gene Expression Analysis of Serum-Derived Extracellular Vesicles in Primary Aldosteronism.

Patients affected by primary aldosteronism (PA) display an increased risk of cardiovascular events compared with essential hypertension (EH). Endothelial dysfunction favors initiation and progression of atherosclerosis and circulating extracellular vesicles (EVs), reflecting endothelial cell activity, could represent one of the mediators of endothelial dysfunction in these patients. The aim of this study was to characterize circulating EVs from patients diagnosed with PA and to explore their functional significance. Serum EVs were isolated from 12 patients with PA and 12 with EH, matched by sex, age, and blood pressure, and compared with 8 normotensive controls. At nanoparticle tracking analysis, EVs concentration was 2.2× higher in patients with PA ( P=0.033) compared with EH and a significant correlation between EV number and serum aldosterone and potassium levels was identified. Fluorescence-activated cell sorting analysis demonstrated that patients with PA presented a higher absolute number of endothelial-derived EVs compared with both patients with EH and normotensive controls. Through EV mRNA profiling, 15 up-regulated and 4 down-regulated genes in patients with PA compared with EH were identified; moreover, EDN1 was expressed only in patients with PA. Microarray platform was validated by quantative real-time polymerase chain reaction on 4 genes ( CASP1, EDN1, F2R, and HMOX1) involved in apoptosis, inflammation, and endothelial dysfunction. After unilateral adrenalectomy, EVs number and expression of CASP1 and EDN1 significantly decreased in patients with PA ( P<0.05). Additionally, the incubation with PA-derived EVs reduced angiogenesis and induced apoptosis in vitro. Circulating EVs might not only represent a marker of endothelial dysfunction but also contribute themselves to vascular dysfunction in patients with PA.

Classification of microadenomas in patients with primary aldosteronism by steroid profiling.

In primary aldosteronism (PA) the differentiation of unilateral aldosterone-producing adenomas (APA) from bilateral adrenal hyperplasia (BAH) is usually performed by adrenal venous sampling (AVS) and/or computed tomography (CT). CT alone often lacks the sensitivity to identify micro-APAs. Our objectives were to establish if steroid profiling could be useful for the identification of patients with micro-APAs and for the development of an online tool to differentiate micro-APAs, macro-APAs and BAH. The study included patients with PA (n = 197) from Munich (n = 124) and Torino (n = 73) and comprised 33 patients with micro-APAs, 95 with macro-APAs, and 69 with BAH. Subtype differentiation was by AVS, and micro- and macro-APAs were selected according to pathology reports. Steroid concentrations in peripheral venous plasma were measured by liquid chromatography-tandem mass spectrometry. An online tool using a random forest model was built for the classification of micro-APA, macro-APA and BAH. Micro-APA were classified with low specificity (33%) but macro-APA and BAH were correctly classified with high specificity (93%). Improved classification of micro-APAs was achieved using a diagnostic algorithm integrating steroid profiling, CT scanning and AVS procedures limited to patients with discordant steroid and CT results. This would have increased the correct classification of micro-APAs to 68% and improved the overall classification to 92%. Such an approach could be useful to select patients with CT-undetectable micro-APAs in whom AVS should be considered mandatory.