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Context Most patients with adrenal incidentaloma have nonfunctional lesions that do not require treatment, while others have functional or malignant tumors that require intervention. The plasma steroid metabolome may be useful to assess therapeutic need. Objective This work aimed to establish the utility of plasma steroid profiling combined with metanephrines and adrenal tumor size for the differential diagnosis of patients with adrenal incidentaloma. Methods This retrospective cross-sectional study, which took place at 7 European tertiary-care centers, comprised 577 patients with adrenal incidentaloma, including 19, 77, 65, 104 and 312 respective patients with adrenocortical carcinoma (ACC), pheochromocytoma (PHEO), primary aldosteronism (PA), autonomous cortisol secretion (ACS), and nonfunctional adrenal incidentaloma (NFAI). Mesaures of diagnostic performance were assessed (with [95% CIs]) for discriminating different subgroups of patients with adrenal incidentaloma. Results Patients with ACC were characterized by elevated plasma concentrations of 11-deoxycortisol, 11-deoxycorticosterone, 17-hydroxyprogesterone, androstenedione, and dehydroepiandrosterone-sulfate, whereas patients with PA had elevations of aldosterone, 18-oxocortisol, and 18-hydroxycortisol. A selection of those 8 steroids, combined with 3 others (cortisol, corticosterone, and dehydroepiandrosterone) and plasma metanephrines, proved optimal for identifying patients with ACC, PA, and PHEO at respective sensitivities of 83.3% (66.1%-100%), 90.8% (83.7%-97.8%), and 94.8% (89.8%-99.8%); and specificities of 98.0% (96.9%-99.2%), 92.0% (89.6%-94.3%), and 98.6% (97.6%-99.6%). With the addition of tumor size, discrimination improved further, particularly for ACC (100% [100%-100%] sensitivity, 99.5% [98.9%-100%] specificity). In contrast, discrimination of ACS and NFAI remained suboptimal (70%-71% sensitivity, 89%-90% specificity). Conclusion Among patients with adrenal incidentaloma, the combination of plasma steroid metabolomics with routinely available plasma free metanephrines and data from imaging studies may facilitate the identification of almost all clinically relevant adrenal tumors.

Publisher Summary Monoamine oxidase A (MAO-A) and B (MAO-B) catalyze the oxidative deamination of biogenic monoamines. The genes encoding MAO-A and MAO-B are located close to the gene for Norrie disease (ND). Exclusive deletion of the ND gene results in X-linked congenital blindness, and in 30–50% patients, to progressive hearing loss and mild mental retardation. Loss of MAO-A and MAO-B activity was reflected neurochemically by severely reduced plasma and urinary concentrations of catecholamine deaminated metabolites, including dihydroxyphenylglycol (DHPG), the deaminated metabolite of norepinephrine and epinephrine. MAO-A knock-out mice demonstrated aggressive behavior, probably related to impaired metabolic degradation of 5-HT in an experiment. These mice showed significantly increased 5-HT levels in several brain areas and architectural alterations in the somatosensory cortex. Apart from different affinities for monoamine substrate, the importance of MAO-A for catecholamine metabolism likely reflects selective expression of MAO-A in catecholaminergic neurons, the cellular compartment where most catecholamine deamination takes place. Two patients have been identified with deletions of MAO-B and ND genes, but with the MAO-A gene intact.

The impact of dietary sodium reduction on mouse models of type 2 diabetes is not well understood. Therefore, we analyzed the effect of a low-salt diet on obesity and parameters of type 2 diabetes in db/db mice. Five-week-old male db/db and lean db/m mice were fed a normal salt (0.19% Na+, NS) or a low-salt diet (

Pheochromocytomas/paragangliomas are characterized by a unique molecular landscape that allows their assignment to clusters based on underlying genetic alterations. With around 30% to 35% of Caucasian patients (a lower percentage in the Chinese population) showing germline mutations in susceptibility genes, pheochromocytomas/paragangliomas have the highest rate of heritability among all tumors. A further 35% to 40% of Caucasian patients (a higher percentage in the Chinese population) are affected by somatic driver mutations. Thus, around 70% of all patients with pheochromocytoma/paraganglioma can be assigned to 1 of 3 main molecular clusters with different phenotypes and clinical behavior. Krebs cycle/VHL/EPAS1-related cluster 1 tumors tend to a noradrenergic biochemical phenotype and require very close follow-up due to the risk of metastasis and recurrence. In contrast, kinase signaling–related cluster 2 tumors are characterized by an adrenergic phenotype and episodic symptoms, with generally a less aggressive course. The clinical correlates of patients with Wnt signaling–related cluster 3 tumors are currently poorly described, but aggressive behavior seems likely. In this review, we explore and explain why cluster-specific (personalized) management of pheochromocytoma/paraganglioma is essential to ascertain clinical behavior and prognosis, guide individual diagnostic procedures (biochemical interpretation, choice of the most sensitive imaging modalities), and provide personalized management and follow-up. Although cluster-specific therapy of inoperable/metastatic disease has not yet entered routine clinical practice, we suggest that informed personalized genetic-driven treatment should be implemented as a logical next step. This review amalgamates published guidelines and expert views within each cluster for a coherent individualized patient management plan.

Contains fulltext : 235325.pdf (Publisher’s version ) (Open Access) BACKGROUND: Phaeochromocytoma and paraganglioma (PPGL) in pregnancy, if not diagnosed antepartum, pose a high risk for mother and child. OBJECTIVE: To examine the clinical clues of antepartum and postpartum/postmortem diagnosis of PPGL. SEARCH STRATEGY: Case reports on PPGL in pregnancy published between 1 January 1988 and 30 June 2019 in English, German, Dutch or French. SELECTION CRITERIA: Case reports containing a predefined minimum of clinical data on PPGL and pregnancy. DATA COLLECTION AND ANALYSIS: Two authors independently performed data extraction and assessed data quality. We calculated odds ratios (OR) (with 95% confidence intervals) and used uni- and multivariable logistic regression analysis. MAIN RESULTS: Maternal and fetal/neonatal mortalities were 9.0% (18/200) and 14.2% (29/204), respectively. Maternal mortality was 42-fold higher with PPGL diagnosed postpartum/postmortem (17/58; 29.3%) than antepartum (1/142; 0.7%) (adjusted OR 45.9, 95% CI 5.67-370, P = 0.0003). Offspring mortality was 2.6-fold higher with PPGL diagnosed postpartum/postmortem than antepartum (OR 3.1, 95% CI 1.38-6.91, P = 0.0044). Hypertension at admission (OR 2.29, 95% CI 1.12-4.68, P = 0.022), sweating (OR 3.14, 95% CI 1.29-7.63, P = 0.014) and a history of PPGL, a known PPGL-associated gene mutation or adrenal mass (OR 8.87, 95% CI 1.89-41.64, P = 0.0056) were independent factors of antepartum diagnosis. Acute onset of symptoms (OR 8.49, 95% CI 3.52-20.5, P

Primary aldosteronism is a frequent form of endocrine hypertension caused by aldosterone overproduction from the adrenal cortex. Regulation of aldosterone biosynthesis has been studied in rodents despite differences in adrenal physiology with humans. We, therefore, investigated pig adrenal steroidogenesis, morphology, and transcriptome profiles of the zona glomerulosa (zG) and zona fasciculata in response to activation of the renin-angiotensin-aldosterone system by dietary sodium restriction. Six-week-old pigs were fed a low- or high-sodium diet for 14 days (3 pigs per group, 0.4 g sodium/kg feed versus 6.8 g sodium/kg). Plasma aldosterone concentrations displayed a 43-fold increase ( P =0.011) after 14 days of sodium restriction (day 14 versus day 0). Low dietary sodium caused a 2-fold increase in thickness of the zG ( P CYP11B ( P

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 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.

COVID-19 was declared a global pandemic by the WHO and has affected millions of patients around the world. COVID-19 disproportionately affects persons with endocrine conditions, thus putting them at an increased risk for severe disease. We discuss the mechanisms that place persons with endocrine conditions at an additional risk for severe COVID-19 and review the evidence. We also suggest precautions and management of endocrine conditions in the setting of global curfews being imposed and offer practical tips for uninterrupted endocrine care.

Purpose Paraganglioma of the urinary bladder (UBPGL) is a rare neuroendocrine tumor diagnosed in many patients only after surgery. We, therefore, assessed clinical clues relevant to presurgical diagnosis and clinical consequences in patients with a missed presurgical diagnosis of UBPGL. Materials and methods Case reports describing a UBPGL (published from 1–1–2001 and 31–12–2020) were identified in Pubmed. Two authors independently performed data extraction and assessed data quality according to the PRISMA guideline. Patients were divided into two groups: UBPGL diagnosis before and after surgery. Results We included 177 articles reporting 194 cases. In 90 (46.4%) patients, the UBPGL was diagnosed before and in 104 (53.6%) after surgery. In presurgically diagnosed UBPGL, hypertension and catecholamine-associated symptoms were 2- to 3-fold (p p = 0.003) more prevalent in those with postsurgical diagnosis. Hypertension was an independent factor for presurgical biochemical testing (OR 4.45, 95% CI 1.66–11.94) while hematuria (OR 0.23, 95% CI 0.09–0.60) was an independent factor for not performing presurgical biochemical testing. Most patients diagnosed after surgery were not pretreated with alpha-adrenoceptor blockade (95.2%), underwent more frequently transurethral resection instead of cystectomy (70.2% vs. 23.1%) and had more frequent peroperative complications and residual tumor mass. Conclusions In nearly half of all patients with a UBPGL, the diagnosis was not established before surgery. Hypertension and hematuria contributed independently to a presurgical diagnosis. Postsurgical diagnosis, which was associated with suboptimal presurgical and surgical management, resulted in more peroperative complications and incomplete tumor resections.

The protein kinase A (PKA) signaling system mediates the effects of numerous hormones, neurotransmitters, and other molecules to regulate metabolism, cardiac function, and more. PKA defects may lead to diverse phenotypes that largely depend on the unique expression profile of the affected subunit. Deletion of the Prkarcb gene, which codes for PKA catalytic subunit β (Cβ), protects against diet-induced obesity (DIO), yet the mechanism for this phenotype remains unclear. We hypothesized that metabolic rate would be increased in Cβ knockout (KO) mice, which could explain DIO resistance. Male, but not female, CβKO mice had increased energy expenditure, and female but not male CβKO mice had increased subcutaneous temperature and increased locomotor activity compared with wild-type (WT) littermates. Urinary norepinephrine (NE) and normetanephrine were elevated in female CβKO mice. CβKO mice had increased heart rate (HR); blocking central NE release normalized HR to that of untreated WT mice. Basal and stimulated PKA enzymatic activities were unchanged in adipose tissue and heart and varied in different brain regions, suggesting that Prkacb deletion may mediate signaling changes in specific brain nuclei and may be less important in the peripheral regulation of PKA expression and activity. This is a demonstration of a distinct effect of the PKA Cβ catalytic subunit on catecholamines and sympathetic nerve signaling. The data provide an unexpected explanation for the metabolic phenotype of CβKO mice. Finally, the sexual dimorphism is consistent with mouse models of other PKA subunits and adds to the importance of these findings regarding the PKA system in human metabolism.

Endogenous steroid hormones, especially glucocorticoids and mineralocorticoids, derive from the adrenal cortex, and drastic or sustained changes in their circulatory levels affect multiple organ systems. Although hypoxia signaling in steroidogenesis has been suggested, knowledge on the true impact of the HIFs (Hypoxia-Inducible Factors) in the adrenocortical cells of vertebrates is scant. By creating a unique set of transgenic mouse lines, we reveal a prominent role for HIF1α in the synthesis of virtually all steroids in vivo. Specifically, mice deficient in HIF1α in adrenocortical cells displayed enhanced levels of enzymes responsible for steroidogenesis and a cognate increase in circulatory steroid levels. These changes resulted in cytokine alterations and changes in the profile of circulatory mature hematopoietic cells. Conversely, HIF1α overexpression resulted in the opposite phenotype of insufficient steroid production due to impaired transcription of necessary enzymes. Based on these results, we propose HIF1α to be a vital regulator of steroidogenesis as its modulation in adrenocortical cells dramatically impacts hormone synthesis with systemic consequences. In addition, these mice can have potential clinical significances as they may serve as essential tools to understand the pathophysiology of hormone modulations in a number of diseases associated with metabolic syndrome, auto-immunity or even cancer. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-020-03750-1.

Item does not contain fulltext PURPOSE: Current evidence regarding renal involvement in pheochromocytoma and paraganglioma (PPGL) is scant. More accurate diagnostic methods, such as renal Doppler ultrasound for intrarenal hemodynamic studies, may provide more detailed information on renal function. It might be postulated that renal function in PPGL patients might be altered by high blood pressure and excess secretion of catecholamines. The aim of this prospective study was to assess intrarenal blood flow parameters in PPGL patients included in the prospective monoamine-producing tumour (PMT) study and to evaluate the effects of normalisation of catecholamine production after surgical treatment on long-term renal function. MATERIALS AND METHODS: Seventy consecutive patients (aged 46.5 ± 14.0 years) with PPGL were included. Forty-eight patients from the PMT study cohort, matched for age, gender, blood pressure level and presence of hypertension, served as a control group. Renal artery doppler ultrasound spectral analysis included mean resistance index (RRI) and pulsatility index (PI). Forty-seven patients completed 12 months follow-up. RESULTS: There were no differences in renal parameters such as RRI, PI and kidney function between PPGL and non-PPGL patients as assessed by renal ultrasound, serum creatinine, eGFR and albumin excretion rate. No correlations between kidney function parameters, intrarenal doppler flow parameters and plasma catecholamines were observed in PPGL patients. At 12 months after surgery, no differences in creatinine level, eGFR, albumin excretion rate, RI and PI were found as compared to baseline results. CONCLUSIONS: In contrast to patients with other forms of secondary hypertension, our study did not show differences in intrarenal blood flow parameters and renal function between PPGL and non-PPGL subjects. Intrarenal hemodynamics and renal function did not change after normalisation of catecholamine levels by surgical treatment.

Abstract The adrenal gland and its hormones regulate numerous fundamental biological processes; however, the impact of hypoxia signaling on adrenal function remains poorly understood. Here, we reveal that deficiency of HIF (hypoxia inducible factors) prolyl hydroxylase domain protein-2 (PHD2) in the adrenal medulla of mice results in HIF2α-mediated reduction in phenylethanolamine N-methyltransferase (PNMT) expression, and consequent reduction in epinephrine synthesis. Simultaneous loss of PHD2 in renal erythropoietin (EPO)-producing cells (REPCs) stimulated HIF2α-driven EPO overproduction, excessive RBC formation (erythrocytosis), and systemic hypoglycemia, which is necessary and sufficient to enhance exocytosis of epinephrine from the adrenal medulla. Based on these results, we propose that the PHD2-HIF2α axis in the adrenal medulla regulates the synthesis of epinephrine, whereas in REPCs, it indirectly induces the release of this hormone. Our findings are also highly relevant to the testing of small molecule PHD inhibitors in phase III clinical trials for patients with renal anemia. Key messages HIF2α and not HIF1α modulates PNMT during epinephrine synthesis in chromaffin cells. The PHD2-HIF2α-EPO axis induces erythrocytosis and hypoglycemia. Reduced systemic glucose facilitates exocytosis of epinephrine from adrenal gland.

Reduced expression of the plasma membrane citrate transporter INDY (acronym I’m Not Dead, Yet) extends life span in lower organisms. Deletion of the mammalian Indy (mIndy) gene in rodents improves metabolism via mechanisms akin to caloric restriction, known to lower blood pressure (BP) by sympathoadrenal inhibition. We hypothesized that mIndy deletion attenuates sympathoadrenal support of BP. Continuous arterial BP and heart rate (HR) were reduced in mINDY-KO mice. Concomitantly, urinary catecholamine content was lower, and the decreases in BP and HR by mIndy deletion were attenuated after autonomic ganglionic blockade. Catecholamine biosynthesis pathways were reduced in mINDY-KO adrenals using unbiased microarray analysis. Citrate, the main mINDY substrate, increased catecholamine content in pheochromocytoma cells, while pharmacological inhibition of citrate uptake blunted the effect. Our data suggest that deletion of mIndy reduces sympathoadrenal support of BP and HR by attenuating catecholamine biosynthesis. Deletion of mIndy recapitulates beneficial cardiovascular and metabolic responses to caloric restriction, making it an attractive therapeutic target., Deletion of mIndy reduces blood pressure and heart rate by attenuating catecholamine biosynthesis and recapitulates beneficial cardiovascular and metabolic responses to caloric restriction.

Purpose Pheochromocytomas and Paragangliomas (PPGL) result in chronic catecholamine excess and serious health complications. A recent study obtained a metabolic signature in plasma from PPGL patients; however, its targeted nature may have generated an incomplete picture and a broader approach could provide additional insights. We aimed to characterize the plasma metabolome of PPGL patients before and after surgery, using an untargeted approach, and to broaden the scope of the investigated metabolic impact of these tumors. Design A cohort of 36 PPGL patients was investigated. Blood plasma samples were collected before and after surgical tumor removal, in association with clinical and tumor characteristics. Methods Plasma samples were analyzed using untargeted nuclear magnetic resonance (NMR) spectroscopy metabolomics. The data were evaluated using a combination of uni- and multi-variate statistical methods. Results Before surgery, patients with a nonadrenergic tumor could be distinguished from those with an adrenergic tumor based on their metabolic profiles. Tyrosine levels were significantly higher in patients with high compared to those with low BMI. Comparing subgroups of pre-operative samples with their post-operative counterparts, we found a metabolic signature that included ketone bodies, glucose, organic acids, methanol, dimethyl sulfone and amino acids. Three signals with unclear identities were found to be affected. Conclusions Our study suggests that the pathways of glucose and ketone body homeostasis are affected in PPGL patients. BMI-related metabolite levels were also found to be altered, potentially linking muscle atrophy to PPGL. At baseline, patient metabolomes could be discriminated based on their catecholamine phenotype.

Purpose Ampreloxetine is a novel, selective, long-acting norepinephrine reuptake (NET) inhibitor being investigated as a once-daily oral treatment for symptomatic neurogenic orthostatic hypotension (nOH) in patients with autonomic synucleinopathies. The purpose of this study was to characterize the pharmacokinetic and pharmacodynamic profiles of ampreloxetine in this target population. Methods Patients with nOH were enrolled in a multicenter, phase II clinical trial of ampreloxetine (NCT02705755). They received escalating doses over 5 days in the clinical research unit, followed by 20 weeks of open-label treatment and then a 4-week withdrawal. As neurochemical biomarkers of NET inhibition, we assayed plasma concentrations of norepinephrine (NE) and its main intraneuronal metabolite 3,4-dihydroxyphenylglycol (DHPG) pre- and post-ampreloxetine. Results Thirty-four patients with nOH were enrolled. Plasma ampreloxetine concentrations increased with repeated escalating doses, with peak concentrations observed 6–9 h post-drug administration. The median ampreloxetine dose in the 20-week treatment phase was 10 mg once daily. Plasma ampreloxetine concentrations reached steady state by 2 weeks, with stable plasma levels over 24 h. No influence of age or renal function on ampreloxetine plasma concentrations was observed. On treatment, compared to baseline, plasma NE significantly increased by 71% (p

Objectives Plasma free metanephrines are commonly used for diagnosis of pheochromocytoma and paraganglioma (PPGLs), but can also provide other information. This multicenter study prospectively examined whether tumor size, location, and mutations could be predicted by these metabolites. Methods Predictions of tumor location, size, and mutation type, based on measurements of plasma normetanephrine, metanephrine, and methoxytyramine were made without knowledge of disease in 267 patients subsequently determined to have PPGLs. Results Predictions of adrenal vs. extra-adrenal locations according to increased plasma concentrations of metanephrine and methoxytyramine were correct in 93 and 97% of the respective 136 and 33 patients in who these predictions were possible. Predicted mean tumor diameters correlated positively (pNF1 or RET mutations were correctly predicted with those mutations according to increased plasma metanephrine, whereas no or minimal increase in metanephrine correctly predicted all 71 patients with either VHL or SDHx mutations; furthermore, among the latter group increases in methoxytyramine correctly predicted SDHx mutations in 93% of the 29 cases for this specific prediction. Conclusions Extents and patterns of increased plasma O-methylated catecholamine metabolites among patients with PPGLs allow predictions of tumor size, adrenal vs. extra-adrenal locations and general types of mutations. Predictions of tumor location are, however, only possible for patients with clearly increased plasma methoxytyramine or metanephrine. Where possible or clinically relevant the predictions are potentially useful for subsequent clinical decision-making.

Aim The aim of this study was to systematically appraise the quality of a sample of COVID-19-related systematic reviews (SRs) and discuss internal validity threats affecting the COVID-19 body of evidence. Design We conducted a scoping review of the literature. SRs with or without meta-analysis (MA) that evaluated clinical data, outcomes or treatments for patients with COVID-19 were included. Main outcome measures We extracted quality characteristics guided by A Measurement Tool to Assess Systematic Reviews-2 to calculate a qualitative score. Complementary evaluation of the most prominent published limitations affecting the COVID-19 body of evidence was performed. Results A total of 63 SRs were included. The majority were judged as a critically low methodological quality. Most of the studies were not guided by a pre-established protocol (39, 62%). More than half (39, 62%) failed to address risk of bias when interpreting their results. A comprehensive literature search strategy was reported in most SRs (54, 86%). Appropriate use of statistical methods was evident in nearly all SRs with MAs (39, 95%). Only 16 (33%) studies recognised heterogeneity in the definition of severe COVID-19 as a limitation of the study, and 15 (24%) recognised repeated patient populations as a limitation. Conclusion The methodological and reporting quality of current COVID-19 SR is far from optimal. In addition, most of the current SRs fail to address relevant threats to their internal validity, including repeated patients and heterogeneity in the definition of severe COVID-19. Adherence to proper study design and peer-review practices must remain to mitigate current limitations.

Context Radiological characterization of adrenal size in primary bilateral macronodular adrenocortical hyperplasia (PBMAH) has not been previously investigated. Objective We hypothesized that volumetric modeling of adrenal gland size may correlate with biochemical disease severity in patients with PBMAH. Secondary analysis of patients with concurrent primary aldosteronism (PA) was performed. Design A retrospective cross-sectional analysis of 44 patients with PBMAH was conducted from 2000 to 2019. Setting Tertiary care clinical research center. Patients Patients were diagnosed with PBMAH based upon clinical, genetic, radiographic and biochemical characteristics. Intervention Clinical, biochemical, and genetic data were obtained. Computed tomography scans were used to create volumetric models by manually contouring both adrenal glands in each slice using Vitrea Core Fx v6.3 software (Vital Images, Minnetonka, Minnesota). Main Outcome and Measures 17-hydroxycorticosteroids (17-OHS), ARMC5 genetics, and aldosterone-to-renin ratio (ARR) were retrospectively obtained. Pearson test was used for correlation analysis of biochemical data with adrenal volume. Results A cohort of 44 patients with PBMAH was evaluated, with a mean age (±SD) of 53 ± 11.53. Eight patients met the diagnostic criteria for PA, of whom 6 (75%) were Black. In the Black cohort, total adrenal volumes positively correlated with midnight cortisol (R = 0.76, P = 0.028), urinary free cortisol (R = 0.70, P = 0.035), and 17-OHS (R = 0.87, P = 0.0045), with a more pronounced correlation with left adrenal volume alone. 17-OHS concentration positively correlated with total, left, and right adrenal volume in patients harboring pathogenic variants in ARMC5 (R = 0.72, P = 0.018; R = 0.65, P = 0.042; and R = 0.73, P = 0.016, respectively). Conclusions Volumetric modeling of adrenal gland size may associate with biochemical severity in patients with PBMAH, with particular utility in Black patients.

The adrenal gland and its hormones regulate numerous fundamental biological processes; however, the impact of hypoxia signalling on its function remains scarcely understood. Here, we reveal that deficiency of HIF (Hypoxia Inducible Factors) prolyl hydroxylase domain protein-2 (PHD2) in the adrenal medulla of mice results in HIF2α-mediated reduction in phenylethanolamine N-methyltransferase (PNMT) expression, and consequent reduction in epinephrine synthesis. Concomitant loss of PHD2 in renal erythropoietin (EPO) producing cells stimulated HIF2α-driven EPO overproduction, excessive RBC formation (erythrocytosis) and systemic hypoglycaemia. Using mouse lines displaying only EPO-induced erythrocytosis or anaemia, we show that hypo- or hyperglycaemia is necessary and sufficient to respectively enhance or reduce exocytosis of epinephrine from the adrenal gland. Based on these results, we propose that the PHD2-HIF2α axis in the adrenal medulla and beyond regulates both synthesis and release of catecholamines, especially epinephrine. Our findings are also of great significance in view of the small molecule PHD inhibitors being tested in phase III global clinical development trials for use in renal anaemia patients.

Multiple stressors, including 24-h-shifts characterise the working environment of physicians, influencing well-being, health and performance. We aimed to evaluate the effect of the stressor 24-h-shift on the adrenal medullary and sympathoneural system in physicians with the hypothesis that shift work might have different impacts on both systems. Twenty-two physicians collected two 12-h-urine samples (“daytime” and “nighttime”) during a 24-h shift (“on-duty”) and on a free weekend (“off-duty”), respectively. Urinary excretion rates per m2 body surface area were assessed for the catecholamines epinephrine, norepinephrine and their respective free O-methylated metabolites metanephrine and normetanephrine by LC-MS/MS-analysis. The stressor provoked differential responses of epinephrine and norepinephrine. Epinephrine excretion rates showed significant increases from off to on duty. The largest proportional change (off-duty to on-duty) for epinephrine was observed for nighttime (205%), the increase for daytime was 84%. An increase in norepinephrine from off to on duty was only visible for nighttime collections. For the catecholamine metabolites, normetanephrine paralleled norepinephrine and exhibited an increase in excretion from off to on duty during nighttime collections of 53% whereas there was no change during daytime collections (3%). In conclusion: Whilst the 24-h-shift-work stressor in physicians activates the sympatho-adrenomedullary system, represented by epinephrine, the sympathoneural response through norepinephrine reflects mainly an ambulatory position during working hours.

Background: Cross-sectional imaging regularly results in incidental discovery of adrenal tumours, requiring exclusion of adrenocortical carcinoma (ACC). However, differentiation is hampered by poor specificity of imaging characteristics. We aimed to validate a urine steroid metabolomics approach, using steroid profiling as the diagnostic basis for ACC. Methods: We did a prospective multicentre study in adult participants (age ≥18 years) with newly diagnosed adrenal masses. We assessed the accuracy of diagnostic imaging strategies based on maximum tumour diameter (≥4 cm vs

Phaeochromocytoma and paraganglioma (PPGL) are chromaffin cell tumours that require timely diagnosis because of their potentially serious cardiovascular and sometimes life- threatening sequelae. Tremendous progress in biochemical testing, imaging, genetics and pathophysiological understanding of the tumours has far-reaching implications for physicians dealing with hypertension and more importantly affected patients. Because hypertension is a classical clinical clue for PPGL, physicians involved in hypertension care are those who are often the first to consider this diagnosis. However, there have been profound changes in how PPGLs are discovered; this is often now based on incidental findings of adrenal or other masses during imaging and increasingly during surveillance based on rapidly emerging new hereditary causes of PPGL. We therefore address the relevant genetic causes of PPGLs and outline how genetic testing can be incorporated within clinical care. In addition to conventional imaging (CT, MRI), new functional imaging approaches are evaluated. The novel knowledge of genotype-phenotype relationships, linking distinct genetic causes of disease to clinical behaviour and biochemical phenotype, provides the rationale for patient-tailored strategies for diagnosis, follow-up and surveillance. Most appropriate preoperative evaluation and preparation of patients are reviewed, as is minimally invasive surgery. Finally we discuss risk factors for developing metastatic disease and how they may facilitate personalised follow-up. Experts from the European Society of Hypertension have prepared this position document which summarises the current knowledge in epidemiology, genetics, diagnosis, treatment and surveillance of PPGL. CONDENSED ABSTRACT: Phaeochromocytomas and paragangliomas (PPGL) are rare causes of secondary hypertension. The unpredictable serious effects of tumoural surges of catecholamines into the circulation result in significant cardiovascular mortality and morbidity when the diagnosis is delayed or missed. After publication of the Endocrine Society Clinical Practice Guideline on PPGL in 2014, relevant new data have been published reflecting further scientific advances impacting on patient care. This position statement, written by the working group on Endocrine Hypertension of the European Society of Hypertension, provides an update in epidemiology, genetics, diagnosis and therapeutic and surveillance strategies for patients with PPGLs.

Introduction Polycythemia vera (PV) is a rare myeloproliferative disease associated with an increased prevalence of hypertension and increased risk of cardiovascular complications. However, the precise mechanisms leading to the elevation of blood pressure (BP) and secondary target organ damage remain poorly understood. Objectives The study aimed to evaluate BP profile, assess the activity of the sympathetic nervous system and the renin‑angiotensin system, and provide a comprehensive assessment of subclinical target organ damage in patients with PV. Patients and methods Twenty consecutive patients with newly diagnosed PV and 20 control subjects were included. The following were assessed: BP, levels of catecholamines, urinary and plasma O‑methylated catecholamine metabolites, concentrations of aldosterone and renin. We also assessed microneurography sympathetic nervous system activity (MSNA) and baroreflex control of heart rate as well as subclinical target organ damage. Results At similar levels of BP, BP variability was decreased in the PV group (mean [SD] 24‑hour systolic BP, 9 [3] vs 12 [3] mm Hg; P = 0.003). Patients with PV had lower norepinephrine excretion (mean [SD], 16.54 [6.32] vs 25.46 [12.88] μg/d; P = 0.03) as well as decreased MSNA as assessed by microneurography compared with controls (mean [SD] MSNA, 30.7 [8.7] bursts/min vs 38.7 [5.4] bursts/min; P = 0.007 and MSNA 51.8 [11] bursts/100 beats vs 61.1 [11.3] bursts/100 heart beats; P = 0.04). Baroreflex control of HR was unaltered in the PV group. Increased hemoglobin levels and red blood cell count correlated with decreased retinal capillary flow in patients with PV. Conclusions Patients with PV, characterized by high hemoglobin concentrations and hematocrit levels had lower sympathetic nervous activity and decreased BP variability as compared with controls. There was no relationship between hemoglobin plasma concentration, hematocrit level, and target organ damage.

The adrenal glands regulate multiple physiological processes including the stress response, the immune system and metabolism. The adrenal is composed of an outer cortex that produces steroids, and an inner medulla that produces catecholamines. Tissue-specific stem/progenitor populations have been identified in the adrenal cortex, while the presence of a functional stem/progenitor population in the adrenal medulla is unclear. The adrenal medulla derives from the neural crest and contains chromaffin cells, neurons and sustentacular (support) cells. Establishing cell hierarchy and elucidating mechanisms of regulation of the different cell types is important to understand normal homeostasis and disease pathogenesis, such as of pheochromocytomas. Using genetic approaches in mouse, we have established that a subpopulation of sustentacular cells express the stem/progenitor marker SOX2. Through genetic lineage-tracing using the Sox2-CreERT2 strain, we demonstrate that these are an expanding population, capable of giving rise to chromaffin cells and neurons throughout life, consistent with a stem/progenitor role in vivo. We further demonstrate the self-renewal and differentiation potential of SOX2+ cells through in vitro isolation and expansion. Through analysis of FFPE sections of human adrenals, we confirm the presence of SOX2+ cells in the normal adult organ, as well as in pheochromocytomas. Taken together, our data support the identification of a previously undescribed stem/progenitor cell in the mammalian adrenal medulla, and confirm its functional relevance.

OBJECTIVE Structural abnormalities in resistance arteries are a hallmark of patients with hypertension. In hypertensive patients with pheochromocytoma or paraganglioma (PPGL), it is still a matter of debate whether structural vascular changes are because of elevated blood pressure (BP) or to toxic effects of elevated circulating catecholamines. Hence, the aim of our study was to assess whether catecholamine excess and/or elevated BP affect the structure of small retinal arteries in patients with catecholamine-producing tumors. METHODS The study included 27 patients with PPGL and 27 hypertensive patients. All patients underwent biochemical tests for catecholamine excess, echocardiography and analyses of scanning-laser-Doppler-flowmetry (SLDF) both at baseline and 12 months following surgical resection of PPGL. RESULTS Baseline retinal arterial diameter, arterial wall thickness and wall cross sectional area (WCSA) were higher in patients with PPGL as compared with subjects without PPGL (arterial diameter: 110 ± 16.5 vs. 99.5 ± 10.8 μm, wall thickness: 16.3 ± 6.0 vs. 13.5 ± 4.0 μm, WCSA: 4953.9 ± 2472.8 vs. 3784.1 ± 1446.3 μm, P

Context Pheochromocytomas and paragangliomas (PPGLs) are characterized by distinct genotype-phenotype relationships according to studies largely restricted to Caucasian populations. Objective To assess for possible differences in genetic landscapes and genotype-phenotype relationships of PPGLs in Chinese versus European populations. Design Cross-sectional study. Setting 2 tertiary-care centers in China and 9 in Europe. Participants Patients with pathologically confirmed diagnosis of PPGL, including 719 Chinese and 919 Europeans. Main Outcome Measures Next-generation sequencing performed in tumor specimens with mutations confirmed by Sanger sequencing and tested in peripheral blood if available. Frequencies of mutations were examined according to tumor location and catecholamine biochemical phenotypes. Results Among all patients, higher frequencies of HRAS, FGFR1, and EPAS1 mutations were observed in Chinese than Europeans, whereas the reverse was observed for NF1, VHL, RET, and SDHx. Among patients with apparently sporadic PPGLs, the most frequently mutated genes in Chinese were HRAS (16.5% [13.6-19.3] vs 9.8% [7.6-12.1]) and FGFR1 (9.8% [7.6-12.1] vs 2.2% [1.1-3.3]), whereas among Europeans the most frequently mutated genes were NF1 (15.9% [13.2-18.6] vs 6.6% [4.7-8.5]) and SDHx (10.7% [8.4–13.0] vs 4.2% [2.6–5.7]). Among Europeans, almost all paragangliomas lacked appreciable production of epinephrine and identified gene mutations were largely restricted to those leading to stabilization of hypoxia inducible factors. In contrast, among Chinese there was a larger proportion of epinephrine-producing paragangliomas, mostly due to HRAS and FGFR1 mutations. Conclusions This study establishes Sino-European differences in the genetic landscape and presentation of PPGLs, including ethnic differences in genotype-phenotype relationships indicating a paradigm shift in our understanding of the biology of these tumors.

Background Pheochromocytomas (PCCs) and paragangliomas (PGLs) are neuroendocrine tumors that are mostly benign. Metastatic disease does occur in about 10% of cases of PCC and up to 25% of PGL, and for these patients no effective therapies are available. Patients with mutations in the succinate dehydrogenase subunit B (SDHB) gene tend to have metastatic disease. We hypothesized that a down-regulation in the active succinate dehydrogenase B subunit should result in notable changes in cellular metabolic profile and could present a vulnerability point for successful pharmacological targeting. Methods Metabolomic analysis was performed on human hPheo1 cells and shRNA SDHB knockdown hPheo1 (hPheo1 SDHB KD) cells. Additional analysis of 115 human fresh frozen samples was conducted. In vitro studies using N1,N11-diethylnorspermine (DENSPM) and N1,N12- diethylspermine (DESPM) treatments were carried out. DENSPM efficacy was assessed in human cell line derived mouse xenografts. Results Components of the polyamine pathway were elevated in hPheo1 SDHB KD cells compared to wild-type cells. A similar observation was noted in SDHx PCC/PGLs tissues compared to their non-mutated counterparts. Specifically, spermidine, and spermine were significantly elevated in SDHx-mutated PCC/PGLs, with a similar trend in hPheo1 SDHB KD cells. Polyamine pathway inhibitors DENSPM and DESPM effectively inhibited growth of hPheo1 cells in vitro as well in mouse xenografts. Conclusions This study demonstrates overactive polyamine pathway in PCC/PGL with SDHB mutations. Treatment with polyamine pathway inhibitors significantly inhibited hPheo1 cell growth and led to growth suppression in xenograft mice treated with DENSPM. These studies strongly implicate the polyamine pathway in PCC/PGL pathophysiology and provide new foundation for exploring the role for polyamine analogue inhibitors in treating metastatic PCC/PGL. Precis Cell line metabolomics on hPheo1 cells and PCC/PGL tumor tissue indicate that the polyamine pathway is activated. Polyamine inhibitors in vitro and in vivo demonstrate that polyamine inhibitors are promising for malignant PCC/PGL treatment. However, further research is warranted.

Background Diagnosis of primary aldosteronism (PA) involves a multistep process reliant on the accuracy of aldosterone measurements at each step. We report on immunoassay interference leading to a wrongful diagnosis and indication for surgical intervention. Case A 38-year old hypertensive male with a 1.4 cm left adrenal mass was diagnosed with PA based on an elevated aldosterone:renin ratio and a positive saline infusion test. Adrenal venous sampling (AVS) indicated left-sided aldosterone hypersecretion, supporting a decision to remove the left adrenal. The patient was also enrolled in a study to evaluate mass spectrometry-based steroid profiling, which indicated plasma aldosterone concentrations measured in five different peripheral samples averaging only 11% those of the immunoassay. Mass spectrometric measurements did not support left-sided adrenal aldosterone hypersecretion. Two independent laboratories confirmed differences in measurements by immunoassay and mass spectrometry. Lowered concentrations measured by the immunoassay that matched those by mass spectrometry were achieved after sample purification to remove macromolecules, confirming immunoassay interference. Conclusions Although our patient may represent an isolated case of immunoassay interference leading to misdiagnosis of PA, unnecessary AVS and potentially wrongful removal of an adrenal, it is also possible that such inaccuracies may impact the diagnostic process and treatment for other patients.

Coronavirus disease 2019 (Covid-19) has affected millions of people and may disproportionately affect those with hypertension and diabetes. Because of inadequate methods in published systematic reviews, the prevalence of diabetes and hypertension and associated risks of poor outcomes in Covid-19 patients are unknown. We searched databases from December 1, 2019, to April 6, 2020, and selected observational peer-reviewed studies in English of patients with Covid-19. Independent reviewers extracted data on study participants, interventions, and outcomes and assessed risk of bias, and the certainty of evidence. We included 65 (15 794 participants) observational studies at moderate to high risk of bias. Overall prevalence of diabetes and hypertension was 12% (95% confidence interval [CI], 10-15; n = 12 870; I2: 89%), and 17% (95% CI, 13-22; n = 12 709; I2: 95%), respectively. In severe Covid-19, the prevalence of diabetes and hypertension were 18% (95% CI, 16-20; n = 1099; I2: 0%) and 32% (95% CI, 16-54; n = 1078; I2: 63%), respectively. Unadjusted relative risk for intensive care unit admission and mortality were 1.96 (95% CI, 1.19-3.22; n = 8890; I2: 80%; P = .008) and 2.78 (95% CI, 1.39-5.58; n = 2058; I2: 75%; P = .0004) for diabetics; and 2.95 (95% CI, 2.18-3.99; n = 1737; I2: 0%; P PROSPERO registration number CRD42020176582.

Objective Sympathoadrenal activity is decreased during overnight rest. This study assessed whether urinary-free normetanephrine, metanephrine and methoxytyramine in overnight/first-morning urine collections might offer an alternative to measurements in 24-h collections or plasma for diagnosis of pheochromocytoma and paraganglioma (PPGL). Design and methods Prospective multicenter cross-sectional diagnostic study involving 706 patients tested for PPGL, in whom tumors were confirmed in 79 and excluded in 627 after follow-up. Another 335 age- and sex-matched volunteers were included for reference purposes. Catecholamines and their free O-methylated metabolites were measured in 24-h collections divided according to waking and sleeping hours and normalized to creatinine. Plasma metabolites from blood sampled after supine rest were measured for comparison. Results Urinary outputs of norepinephrine, normetanephrine, epinephrine and metanephrine in the reference population were respectively 50 (48–52)%, 35 (32–37)%, 76 (74–78)% and 15 (12–17)% lower following overnight than daytime collections. Patients in whom PPGLs were excluded showed 28 (26–30)% and 6 (3–9)% day-to-night falls in normetanephrine and metanephrine, while patients with PPGLs showed no significant day-to-night falls in metabolites. Urinary methoxytyramine was consistently unchanged from day to night. According to receiver-operating characteristic curves, diagnostic accuracy of metabolite measurements in overnight/first-morning urine samples did not differ from measurements in 24-h urine collections, but was lower for both than for plasma. Using optimized reference intervals, diagnostic specificity was higher for overnight than daytime collections at similar sensitivities. Conclusions Measurements of urinary-free catecholamine metabolites in first-morning/overnight urine collections offer an alternative for diagnosis of PPGL to 24-h collections but remain less accurate than plasma measurements.

BACKGROUND Diagnosis of Cushing syndrome requires a multistep process that includes verification of hypercortisolism followed by identification of the cause of adrenocortical hyperfunction. This study assessed whether pituitary, ectopic, and adrenal subtypes of Cushing syndrome were characterized by distinct plasma steroid profiles that might assist diagnosis. METHODS In this retrospective cross-sectional study, mass spectrometric measurements of a panel of 15 plasma steroids were applied to 222 patient samples tested for Cushing syndrome. Disease was excluded in 138 and confirmed in 51 patients with pituitary Cushing syndrome, 12 with ectopic adrenocorticotropin secretion, and 21 with adrenal disease. Another 277 age- and sex-matched hypertensive and normotensive volunteers were included for comparison. RESULTS Compared with patients without disease, the largest increases in plasma steroids among patients with Cushing syndrome were observed for 11-deoxycortisol (289%), 21-deoxycortisol (150%), 11-deoxycorticosterone (133%), corticosterone (124%), and cortisol (122%). Patients with ectopic disease showed the most prominent increases, but there was considerable variation for other steroids according to subtype. Patients with adrenal disease had the lowest concentrations of androgens, whereas those with ectopic and pituitary disease showed the lowest concentrations of aldosterone. Plasma 18-oxocortisol was particularly low in ectopic disease. With the use of 10 selected steroids, subjects with and without different Cushing syndrome subtypes could be discriminated nearly as closely as with the use of salivary and urinary free cortisol, dexamethasone-suppressed cortisol, and plasma adrenocorticotropin (9.5% vs 5.8% misclassification). CONCLUSIONS Patients with different subtypes of Cushing syndrome show distinctive plasma steroid profiles that may offer a supplementary single-test alternative for screening purposes.

Objective Our objective was to improve molecular diagnostics in patients with hereditary pheochromocytoma and paraganglioma (PPGL) by using next-generation sequencing (NGS) multi-gene panel analysis. Derived from this study, we here present three cases that were diagnosed with NF1 germline mutations but did not have a prior clinical diagnosis of neurofibromatosis type 1 (NF1). Design We performed genetic analysis of known tumor predisposition genes, including NF1, using a multi-gene NGS enrichment-based panel applied to a total of 1029 PPGL patients. We did not exclude genes known to cause clinically defined syndromes such as NF1 based on missing phenotypic expression as is commonly practiced. Methods Genetic analysis was performed using NGS (TruSight Cancer Panel/customized panel by Illumina) for analyzing patients’ blood and tumor samples. Validation was carried out by Sanger sequencing. Results Within our cohort, three patients, who were identified to carry pathogenic NF1 germline mutations, attracted attention, since none of the patients had a clinical suspicion of NF1 and one of them was initially suspected to have MEN2A syndrome due to co-occurrence of a medullary thyroid carcinoma. In these cases, one splice site, one stop and one frameshift mutation in NF1 were identified. Conclusions Since phenotypical presentation of NF1 is highly variable, we suggest analysis of the NF1 gene also in PPGL patients who do not meet diagnostic NF1 criteria. Co-occurrence of medullary thyroid carcinoma and PPGL was found to be a clinical decoy in NF1 diagnostics. These observations underline the value of multi-gene panel NGS for PPGL patients.

BACKGROUNDMeasurements of plasma or urinary metanephrines are recommended for diagnosis of pheochromocytoma and paraganglioma (PPGL). What test offers optimal diagnostic accuracy for patients at high and low risk of disease, whether urinary free metanephrines offer advantages over deconjugated metanephrines, and what advantages are offered by including methoxytyramine in panels all remain unclear.METHODSA population of 2056 patients with suspected PPGLs underwent prospective screening for disease using mass spectrometric-based measurements of plasma free, urinary deconjugated, and urinary free metanephrines and methoxytyramine. PPGLs were confirmed in 236 patients and were excluded in others on follow-up evaluation.RESULTSMeasurements of plasma free metabolites offered higher (P < 0.01) diagnostic sensitivity (97.9%) than urinary free (93.4%) and deconjugated (92.9%) metabolites at identical specificities for plasma and urinary free metabolites (94.2%) but at a lower (P < 0.005) specificity for deconjugated metabolites (92.1%). The addition of methoxytyramine offered little value for urinary panels but provided higher (P < 0.005) diagnostic performance for plasma measurements than either urinary panel according to areas under ROC curves (0.991 vs 0.972 and 0.964). Diagnostic performance of urinary and plasma tests was similar for patients at low risk of disease, whereas plasma measurements were superior to both urinary panels for high-risk patients.CONCLUSIONSDiagnosis of PPGLs using plasma or urinary free metabolites provides advantages of fewer false-positive results compared with commonly measured deconjugated metabolites. The plasma panel offers better diagnostic performance than either urinary panel for patients at high risk of disease and, with appropriate preanalytics, provides the test of choice. Measurements of methoxytyramine in urine show limited diagnostic utility compared with plasma.

Diseases of the adrenal gland are as important for the general practitioner as for the endocrine specialist. The high prevalence of some adrenal endocrinopathies, such as adrenal incidentalomas (1–2% of the population) and primary aldosteronism (6% of hypertensives), which affect millions of patients, makes adrenal diseases a relevant health issue. The high morbidity and mortality of some of the rarer adrenal diseases, i. e., Addison’s disease and Cushing’s syndrome (Table 1), make early detection and appropriate treatment such a challenge for the health care system.

Inhibition of aldosterone synthase (CYP11B2) is an alternative treatment option to mineralocorticoid receptor antagonism to prevent harmful aldosterone effects. FAD286 is the best characterized aldosterone synthase inhibitor. However, to date, no study has used sensitive liquid chromatography-tandem mass spectrometry to characterize in detail the effect of FAD286 on the secreted steroid hormone profile of adrenocortical cells. Basal aldosterone production in NCI-H295R cells was detectable and 9-fold elevated after stimulation with angiotensin II. FAD286 inhibited this increase, showing a maximal effect at 10 nmol/l. Higher concentrations of FAD286 did not further reduce aldosterone concentrations, but showed a parallel reduction in corticosterone, cortisol and cortisone levels, reflecting additional inhibition of steroid-11β-hydroxylase (CYP11B1). Pregnenolone, progesterone and 17-OH-progesterone levels remained unaffected. In conclusion, the aldosterone synthase inhibitor FAD286 lowers angiotensin II-induced aldosterone concentrations in adrenocortical cells but the relative lack of selectivity over CYP11B1 is evident at higher FAD286 concentrations.

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.

Although there is strong evidence linking obesity with increased sympathoneural activity, involvement of the adrenal medulla is less clear. We therefore investigated adrenal medullary function under fasting and feeding conditions in normal weight (NW, n=33), overweight (OW, n=28) and obese (OB, n=36) adults (59% women). Ninety-seven healthy adults participated in a cross-sectional study with recruitment stratified according to BMI. Plasma for catecholamines and metanephrines was sampled in the fasting state, at 30-min intervals during a 120-min glucose tolerance test and during an euglycaemic-hyperinsulinaemic clamp (40 mU m−2 min−1 insulin dose). Body composition was determined by leg-to-leg bioelectrical impedance analysis. Obese subjects had the lowest fasting plasma concentrations of epinephrine (NW: 0.17, 95% confidence interval (CI): 0.14–0.20 nmol l−1; OW: 0.16, 95% CI: 0.12–0.19 nmol l−1; OB: 0.11, 95% CI: 0.08–0.13 nmol l−1; P=0.018) and metanephrine (NW: 0.17, 95% CI: 0.15–0.19 nmol l−1; OW: 0.15, 95% CI: 0.13–0.16 nmol l−1; OB: 0.13, 95% CI: 0.12–0.15 nmol l−1; P=0.022), the latter reflecting adrenal medullary store size. Fasting plasma epinephrine (r=−0.437; P

Phaeochromocytoma localisation is generally reliably achieved with modern imaging techniques, particularly in sporadic cases. On occasion, however, there can be diagnostic doubt due to the presence of bilateral adrenal abnormalities, particularly in patients with mutations in genes predisposing them to the development of multiple phaeochromocytomas. In such cases, surgical intervention is ideally limited to large or functional lesions due to the long-term consequences associated with hypoadrenalism. Adrenal venous sampling (AVS) for catecholamines has been used in this situation to guide surgery, although there are few data available to support diagnostic thresholds. Retrospective analyses of AVS results from 2 centres were carried out. A total of 172 patients (88 men, 84 women) underwent AVS under cosyntropin stimulation for the diagnosis of established primary aldosteronism (PA) with measurement of adrenal and peripheral venous cortisol, aldosterone and catecholamines. Six patients (3 men, 3 women) with phaeochromocytoma underwent AVS for diagnostic purposes with subsequent histological confirmation. Reference intervals for the adrenal venous norepinephrine to epinephrine ratio were created from the PA group. Using the 97.5th centile (1.21 on the left, 1.04 on the right), the false negative rate in the phaeochromocytoma group was 0%. In conclusion, this study describes the largest dataset of adrenal venous catecholamine measurements and provides reference intervals in patients without phaeochromocytoma. This strengthens the certainty with which conclusions related to adrenal venous sampling for catecholamines can be drawn, acknowledging the procedure is not part of the routine diagnostic workup and is an adjunct for use only in difficult clinical cases.

To the Editor: The diagnostic work-up of pheochromocytoma/paraganglioma (PPGL) among patients with chronic kidney disease (CKD) is a clinical challenge. Impaired renal elimination of catecholamines and their metabolites confounds urinary tests, while increased plasma concentrations of free metanephrines in patients with CKD also complicate interpretation of plasma tests.1, 2, 3 We therefore aimed to establish optimal reference intervals for plasma free metanephrines and 3-methoxytyramine in patients with CKD stages 3 and 4 or receiving hemodialysis (HD). This prospective multicenter study included 234 patients (79 CKD3, 48 CKD4, and 107 receiving HD) and an aged-matched reference population of 173 individuals (Table 1; Item S1). Measurements of plasma free normetanephrine, metanephrine, and 3-methoxytyramine were performed using liquid chromatography–tandem mass spectrometry (LC-MS/MS).4

BACKGROUND Neuroblastoma, the most common extracranial solid tumor of childhood, produces catecholamines that are metabolized within tumor cells. Homovanillic acid (HVA) and vanillylmandelic acid (VMA), the end products of catecholamine metabolism, have limited accuracy for testing of the tumors. This study assessed whether metabolites produced in earlier steps of catecholamine metabolism might offer improved diagnostic accuracy over urinary HVA and VMA. PROCEDURE Plasma concentrations of 3-methoxytyramine, normetanephrine, and metanephrine were measured in two pediatric cohorts: (i) 96 children with confirmed neuroblastoma and (ii) 41 children with signs and symptoms of a catecholamine-producing tumor or other neoplasms and in whom neuroblastoma was excluded. Additional measurements of plasma 3-O-methyldopa and relationships of metabolites to MYCN amplification were examined in patient subgroups. RESULTS Overall, 94 of the 96 patients with neuroblastoma had concentrations of 3-methoxytyramine or normetanephrine above age-specific upper limits of reference intervals, providing a diagnostic sensitivity of 97.9% that was higher (P

There are no officially approved therapies for metastatic pheochromocytomas apart from ultratrace 131I-metaiodbenzylguanidine therapy, which is approved only in the United States. We have, therefore, investigated the antitumor potential of molecular-targeted approaches in murine pheochromocytoma cell lines [monocyte chemoattractant protein (MPC)/monocyte chemoattractant protein/3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)], immortalized mouse chromaffin Sdhb−/− cells, three-dimensional pheochromocytoma tumor models (MPC/MTT spheroids), and human pheochromocytoma primary cultures. We identified the specific phosphatidylinositol-3-kinase α inhibitor BYL719 and the mammalian target of rapamycin inhibitor everolimus as the most effective combination in all models. Single treatment with clinically relevant doses of BYL719 and everolimus significantly decreased MPC/MTT and Sdhb−/− cell viability. A targeted combination of both inhibitors synergistically reduced MPC and Sdhb−/− cell viability and showed an additive effect on MTT cells. In MPC/MTT spheroids, treatment with clinically relevant doses of BYL719 alone or in combination with everolimus was highly effective, leading to a significant shrinkage or even a complete collapse of the spheroids. We confirmed the synergism of clinically relevant doses of BYL719 plus everolimus in human pheochromocytoma primary cultures of individual patient tumors with BYL719 attenuating everolimus-induced AKT activation. We have thus established a method to assess molecular-targeted therapies in human pheochromocytoma cultures and identified a highly effective combination therapy. Our data pave the way to customized combination therapy to target individual patient tumors.

Pheochromocytomas and paragangliomas (PCC/PGLs) are rare, mostly catecholamine-producing neuroendocrine tumors of the adrenal gland (PCCs) or the extra-adrenal paraganglia (PGL). They can be separated into three different molecular clusters depending on their underlying gene mutations in any of the at least 20 known susceptibility genes: The pseudohypoxia-associated cluster 1, the kinase signaling-associated cluster 2, and the Wnt signaling-associated cluster 3. In addition to tumor size, location (adrenal vs. extra-adrenal), multiplicity, age of first diagnosis, and presence of metastatic disease (including tumor burden), other decisive factors for best clinical management of PCC/PGL include the underlying germline mutation. The above factors can impact the choice of different biomarkers and imaging modalities for PCC/PGL diagnosis, as well as screening for other neoplasms, staging, follow-up, and therapy options. This review provides a guide for practicing clinicians summarizing current management of PCC/PGL according to tumor size, location, age of first diagnosis, presence of metastases, and especially underlying mutations in the era of precision medicine.

Diagnosis and surveillance of gastroenteropancreatic neuroendocrine tumors (GEP-NETs)2 involve a repertoire of several different biochemical tests with some differences in utility according to classifications and guidelines from the North American Neuroendocrine Tumor Society (1) and the European Neuroendocrine Tumor Society (2). Measured biomarkers include plasma chromogranin A, urinary or plasma 5-hydroxyindole acetic acid, platelet or whole blood serotonin, serum neuron-specific enolase, and, depending on presentation, a variety of peptides such as gastrin, vasoactive intestinal peptide, and several pancreatic-specific peptides (3). Measured alone for screening, these biomarkers have limited clinical sensitivity, but when positive they can be further useful for patient management, including therapeutic monitoring. Some of the more commonly measured biomarkers, such as chromogranin A, suffer from poor clinical specificity or may be rendered pointless to measure by medications (e.g., proton pump inhibitors) or associated clinical conditions (e.g., renal failure). At the routine laboratory level, maintaining multiplicities of tests for such relatively rare tumors can be cost-prohibitive. Thus, measurements available for clinicians to make the diagnosis or monitor patients can be limited. To address the above issues, van Faassen and colleagues present in this issue of Clinical Chemistry a novel LC-MS/MS method for the measurement of four indole biomarkers in platelet-rich plasma (4). The method is made possible by a derivatization step utilizing propionic anhydride. The question is why use derivatization when one of the advantages of liquid over gas chromatographic front ends in mass spectrometry is to escape such extra steps. First, however, …

BACKGROUND Lewy body diseases, a family of aging-related neurodegenerative disorders, entail loss of the catecholamine dopamine in the nigrostriatal system and equally severe deficiency of the closely related catecholamine norepinephrine in the heart. The myocardial noradrenergic lesion is associated with major non-motor symptoms and decreased survival. Numerous mechanisms determine norepinephrine stores, and which of these are altered in Lewy body diseases has not been examined in an integrated way. We used a computational modeling approach to assess comprehensively pathways of cardiac norepinephrine synthesis, storage, release, reuptake, and metabolism in Lewy body diseases. Application of a novel kinetic model identified a pattern of dysfunctional steps contributing to norepinephrine deficiency. We then tested predictions from the model in a new cohort of Parkinson disease patients. METHODS Rate constants were calculated for 17 reactions determining intra-neuronal norepinephrine stores. Model predictions were tested by measuring post-mortem apical ventricular concentrations and concentration ratios of catechols in controls and patients with Parkinson disease. RESULTS The model identified low rate constants for three types of processes in the Lewy body group-catecholamine biosynthesis via tyrosine hydroxylase and L-aromatic-amino-acid decarboxylase, vesicular storage of dopamine and norepinephrine, and neuronal norepinephrine reuptake via the cell membrane norepinephrine transporter. Post-mortem catechols and catechol ratios confirmed this triad of model-predicted functional abnormalities. CONCLUSION Denervation-independent impairments of neurotransmitter biosynthesis, vesicular sequestration, and norepinephrine recycling contribute to the myocardial norepinephrine deficiency attending Lewy body diseases. A proportion of cardiac sympathetic nerves are "sick but not dead," suggesting targeted disease-modification strategies might retard clinical progression. TRIAL REGISTRATION This study was not a clinical trial. FUNDING The research reported here was supported by the Division of Intramural Research, NINDS.