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The article describes the case of suspected pheochromocytoma and pituitary pars intermedia dysfunction in a 27-year-old Shetland pony. Topics discussed include brief information on the cause of excessive secretion of catecholamines, clinical symptoms that led to the diagnosis of suspected pheochromocytoma, and the usefulness of measurement of catecholamines and their metabolites in plasma and urine in detecting suspected pheochromocytoma.

Background: Patients who experience severe brain injuries are at risk of secondary brain damage, because of delayed vasospasm and edema. Traditionally, many of these patients are kept on prolonged bed rest in order to maintain adequate cerebral blood flow, especially in the case of subarachnoid hemorrhage. On the other hand, prolonged bed rest carries important morbidity. There may be a clinical benefit in early mobilization and our hypothesis is that early gradual mobilization is safe in these patients. The aim of this study was to observe and quantify the changes in sympathetic activity, mainly related to stress, and blood pressure in gradual postural changes by the verticalization robot (Erigo®) and after training by a lower body ergometer (MOTOmed-letto®), after prolonged bed rest of minimum 7 days.Methods: Thirty patients with severe neurological injuries were randomized into 3 groups with different protocols of mobilization: Standard, MOTOmed-letto® or Erigo® protocol. We measured plasma catecholamines, metanephrines and blood pressure before, during and after mobilization.Results: Blood pressure does not show any significant difference between the 3 groups. The analysis of the catecholamines suggests a significant increase in catecholamine production during Standard mobilization with physiotherapists and with MOTOmed-letto® and no changes with Erigo®.Conclusions: This preliminary prospective randomized study shows that the mobilization of patients with severe brain injuries by means of Erigo® does not increase the production of catecholamines. It means that Erigo® is a well-tolerated method of mobilization and can be considered a safe system of early mobilization of these patients. Further studies are required to validate our conclusions.Trial Registration: The study was registered in the ISRCTN registry with the trial registration number ISRCTN56402432 . Date of registration: 08.03.2016. Retrospectively registered. [ABSTRACT FROM AUTHOR]

Background Diagnosis of pheochromocytoma ( PC) is based on a combination of clinical suspicion, finding an adrenal mass, increased plasma, and urine concentrations of catecholamine metabolites and is finally confirmed with histopathology. In human medicine, it is controversial whether biochemically testing plasma is superior to testing urine. Objectives To measure urinary and plasma catecholamines and metanephrines in healthy dogs, dogs with PC, hypercortisolism (HC), and nonadrenal diseases (NAD) and to determine the test with the best diagnostic performance for dogs with PC. Animals Seven PC dogs, 10 dogs with HC, 14 dogs with NAD, 10 healthy dogs. Methods Prospective diagnostic clinical study. Urine and heparin plasma samples were collected and stored at −80°C before analysis using high-pressure liquid chromatography ( HPLC) coupled to electrochemical detection or tandem mass spectrometry were performed. Urinary variables were expressed as ratios to urinary creatinine concentration. Results Dogs with PC had significantly higher urinary normetanephrine and metanephrine : creatinine ratios and significantly higher plasma-total and free normetanephrine and plasma-free metanephrine concentrations compared to the 3 other groups. There were no overlapping results of urinary normetanephrine concentrations between PC and all other groups, and only one PC dog with a plasma normetanephrine concentration in the range of the dogs with HC and NAD disease. Performances of total and free plasma variables were similar. Overlap of epinephrine and norepinephrine results between the groups was large with both urine and plasma. Conclusion and clinical importance Measurement of normetanephrine is the preferred biochemical test for PC and urine was superior to plasma. [ABSTRACT FROM AUTHOR]

To investigate the signals governing routing of biologically active peptides to the regulated secretory pathway, we have expressed mutated and non-mutated proneuropeptide Y (ProNPY) in pituitary-derived AtT20 cells. The mutations were carried out on dibasic cleavage site and or ProNPY C-terminal sequence. Targeting to the regulated secretory pathway was studied using protein kinase A (8-BrcAMP), protein kinase C (phorbol myristate acetate) specific activators and protein synthesis inhibitor cycloheximide, and by pulse chase. The analysis of expressed peptides in cells and culture media indicated that: neuropeptide Y (NPY) and ProNPY were differently secreted, whilst NPY was exclusively secreted via regulatory pathway; ProNPY was secreted via regulated and constitutive-like secretory pathways. ProNPY secretion behaviour was not Proteolytic cleavage efficiency-dependent. The dibasic cleavage was essential for ProNPY and NPY cAMP-dependent regulated secretion and may have function as a retention signal. [ABSTRACT FROM AUTHOR]

Over long periods, feeding and metabolism are tightly regulated at the central level. The total amount of nutrients ingested is thought to result from a delicate balance between orexigenic and anorexigenic factors expressed and secreted by specialized hypothalamic neuronal populations. We have developed a system of perifused hypothalamic neurons to characterize the relationships existing between the orexigenic peptide galanin and two other physiological modulators of feeding: neuropeptide Y (NPY) and corticotropin-releasing hormone (CRH). We demonstrated that galanin stimulates CRH and NPY secretion from hypothalamic neurons in a dose-dependent manner. Exposure to leptin for 24 h before galanin stimulation decreased NPY secretion by 30%, leaving the responsiveness of CRH neurons intact. These results suggest that CRH and NPY neurons participate to the intrahypothalamic signaling pathway of galanin, an observation that can explain the lower potency of galanin to stimulate food intake in vivo compared with NPY. The differential effects exerted by leptin on CRH and NPY suggest that there exists a subset of NPY neurons that are exquisitely sensitive to marked variations in leptin levels, and that the CRH neurons are less responsive to increases in leptin concentrations. [ABSTRACT FROM AUTHOR]

Neuropeptide Y (NPY) and related peptides have been proposed as promising biomarkers for the diagnosis of prostate cancer by previous immunoassays and immunohistochemical studies. In this study, we evaluated the additional value of NPY and related peptides compared with prostate-specific antigen (PSA). We performed a comprehensive analysis of NPY, its precursors, and metabolite concentrations in both plasma and tissue samples from 181 patients using a highly specific liquid chromatography tandem mass spectrometry method. Compared with PSA, NPY and related peptides (NPYs) were less accurate at diagnosing significant prostate cancer. Combinations of NPYs in a stepwise approach did not improve a model that would be beneficial for patients. NPY may be beneficial for patients presenting with a PSA concentration in the gray area between 4 and 9 ng/ml, but the strength of this conclusion is limited. Thus, the use of NPYs as standalone or in combination with other variables, such as PSA, prostate volume, or age, to improve the diagnosis is not supported by our study., Patient Summary: This study evaluated neuropeptide Y (NPY) of the family of endogenous peptides as a new biomarker to diagnose prostate cancer. We found that NPY in a patient's blood was not more helpful at diagnosing prostate cancer than the standard prostate-specific antigen blood test. Further research is needed to explore the potential of NPY and related peptides in specific subgroups of patients., (© 2024 The Author(s).)

Angiotensin peptides (ANGs) play a central role in the renin-angiotensin-aldosterone system, rendering them interesting biomarkers associated with hypertension. Precise quantification of circulating ANGs holds the potential to assess the activity of angiotensin-converting enzyme (ACE), a key protease targeted by widely prescribed drugs, namely ACE inhibitors. This ability could pave the way for personalised medicine, offering insights into the prescription of inhibitors targeting either the proteases or the receptors within the system. Despite recent developments in liquid chromatography-mass spectrometry (LC-MS) methods for measuring circulating ANG concentrations, comprehensive stability studies of ANGs in human plasma are absent in the literature, raising concerns about the reliability of measured concentrations and their link to clinical conditions. To address this critical gap, we conducted an exhaustive evaluation of the pre-analytical stability of ANG1-10, ANG1-9, ANG1-8, ANG1-7, and ANG1-5. By employing surfactants to mitigate non-specific adsorption and a dedicated mix of protease inhibitors to limit protease activity, we established an MS-based assay for these five peptides. We used this method to quantify circulating concentrations of ANGs in the plasma of 11 healthy donors and 3 patients under kidney dialysis. Our findings revealed that ANG1-10 and ANG1-8 circulate at concentrations ranging from 1 to 10 pM in healthy subjects and exhibit a high degree of correlation. Notably, ANG1-9, ANG1-7, and ANG1-5 were undetectable in any of the 14 patients, despite a sub-picomolar limit of detection. This strikingly contrasts with the reference concentrations reported in the literature, which typically fall within the picomolar range. In light of these discrepancies, we strongly advocate for rigorous pre-analytical considerations and comprehensive stability studies to ensure reliable results. We emphasise the pivotal role of heightened pre-analytical awareness within the clinical chemistry community, and we hope for continued growth in this critical area., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Due to the high prevalence of hypertension in patients with autosomal dominant polycystic kidney disease (ADPKD) and advanced chronic kidney disease, diagnosing secondary hypertension poses challenges. We present a rare case of pheochromocytoma in an ADPKD patient to highlight the diagnostic difficulties in identifying secondary hypertension due to pheochromocytoma/paraganglioma (PPGL) in end-stage renal disease (ESRD) patients., Case Report: A 48-year-old female with ADPKD and ESRD experienced recurrent hypertensive crises (up to 220/135 mmHg) accompanied by palpitations and tremors that recurred over the past 2 years. Introduction of a betablocker to the antihypertensive therapy aggravated her symptoms. The initial documentation of elevated urinary metanephrines was interpreted as false positive finding due to renal failure. Subsequent measurements of free plasma metanephrines revealed significant elevations raising suspicion of PPGL. Magnetic resonance imaging identified a 29 mm right adrenal mass. The patient underwent right adrenalectomy resulting in resolution of the hypertensive crises., Discussion: The diagnosis of PPGLs can present significant challenges and is further complicated in ESRD due to nonspecific clinical symptoms and diagnostic pitfalls. Less than 20 PPGL cases have been reported in patients with ESRD. The intolerance of beta-blocker therapy, as well as the use of a scoring system for the likelihood of PPGL should have raised suspicion., Conclusion: PPGL should be considered in all patients with uncontrolled hypertension and beta-blockers intolerance, even in the presence of other etiologic mechanisms such as ESRD. Measuring free plasma metanephrines provides the most reliable biochemical screening in the context of impaired renal function., Competing Interests: The authors have no multiplicity of interest to disclose., (© 2024 AACE. Published by Elsevier Inc.)

Background: Neuroblastoma (NB) and pheochromocytoma/paraganglioma (PHEO/PGL) are neuroendocrine tumors. Imaging of these neoplasms is performed by scintigraphy after injection of radiolabeled meta-iodobenzylguanidine (mIBG), a norepinephrine analog taken up by tumoral cells through monoamine transporters. The pharmacological induction of these transporters is a promising approach to improve the imaging and therapy (theranostics) of these tumors., Methods: Transporters involved in mIBG internalization were identified by using transfected Human Embryonic Kidney (HEK) cells. Histone deacetylase inhibitors (HDACi) and inhibitors of the PI3K/AKT/mTOR pathway were tested in cell lines to study their effect on mIBG internalization. Studies in xenografted mice were performed to assess the effect of the most promising HDACi on 123 I-mIBG uptake., Results: Transfected HEK cells demonstrated that the norepinephrine and dopamine transporter (NET and DAT) avidly internalizes mIBG. Sodium-4-phenylbutyrate (an HDACi), CUDC-907 (a dual HDACi and PI3K inhibitor), BGT226 (a PI3K inhibitor) and VS-5584 and rapamycin (two inhibitors of mTOR) increased mIBG internalization in a neuroblastoma cell line (IGR-NB8) by 2.9-, 2.1-, 2.5-, 1.5- and 1.3-fold, respectively, compared with untreated cells. CUDC-907 also increased mIBG internalization in two other NB cell lines and in one PHEO cell line. We demonstrated that mIBG internalization occurs primarily through the NET. In xenografted mice with IGR-NB8 cells, oral treatment with 5 mg/kg of CUDC-907 increased the tumor uptake of 123 I-mIBG by 2.3- and 1.9-fold at 4 and 24 h post-injection, respectively, compared to the untreated group., Conclusions: Upregulation of the NET by CUDC-907 lead to a better internalization of mIBG in vitro and in vivo., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The recent increase in peptidomimetic-based medications and the growing interest in peptide hormones has brought new attention to the quantification of peptides for diagnostic purposes. Indeed, the circulating concentrations of peptide hormones in the blood provide a snapshot of the state of the body and could eventually lead to detecting a particular health condition. Although extremely useful, the quantification of such molecules, preferably by liquid chromatography coupled to mass spectrometry, might be quite tricky. First, peptides are subjected to hydrolysis, oxidation, and other post-translational modifications, and, most importantly, they are substrates of specific and nonspecific proteases in biological matrixes. All these events might continue after sampling, changing the peptide hormone concentrations. Second, because they include positively and negatively charged groups and hydrophilic and hydrophobic residues, they interact with their environment; these interactions might lead to a local change in the measured concentrations. A phenomenon such as nonspecific adsorption to lab glassware or materials has often a tremendous effect on the concentration and needs to be controlled with particular care. Finally, the circulating levels of peptides might be low (pico- or femtomolar range), increasing the impact of the aforementioned effects and inducing the need for highly sensitive instruments and well-optimized methods. Thus, despite the extreme diversity of these peptides and their matrixes, there is a common challenge for all the assays: the need to keep concentrations unchanged from sampling to analysis. While significant efforts are often placed on optimizing the analysis, few studies consider in depth the impact of pre-analytical steps on the results. By working through practical examples, this solution-oriented tutorial review addresses typical pre-analytical challenges encountered during the development of a peptide assay from the standpoint of a clinical laboratory. We provide tips and tricks to avoid pitfalls as well as strategies to guide all new developments. Our ultimate goal is to increase pre-analytical awareness to ensure that newly developed peptide assays produce robust and accurate results., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Aims/hypothesis: Post-bariatric hypoglycaemia is an increasingly recognised complication of bariatric surgery, manifesting particularly after Roux-en-Y gastric bypass. While hyperinsulinaemia is an established pathophysiological feature, the role of counter-regulation remains unclear. We aimed to assess counter-regulatory hormones and glucose fluxes during insulin-induced postprandial hypoglycaemia in patients with post-bariatric hypoglycaemia after Roux-en-Y gastric bypass vs surgical and non-surgical control individuals., Methods: In this case-control study, 32 adults belonging to four groups with comparable age, sex and BMI (patients with post-bariatric hypoglycaemia, Roux-en-Y gastric bypass, sleeve gastrectomy and non-surgical control individuals) underwent a postprandial hypoglycaemic clamp in our clinical research unit to reach the glycaemic target of 2.5 mmol/l 150-170 min after ingesting 15 g of glucose. Glucose fluxes were assessed during the postprandial and hypoglycaemic period using a dual-tracer approach. The primary outcome was the incremental AUC of glucagon during hypoglycaemia. Catecholamines, cortisol, growth hormone, pancreatic polypeptide and endogenous glucose production were also analysed during hypoglycaemia., Results: The rate of glucose appearance after oral administration, as well as the rates of total glucose appearance and glucose disappearance, were higher in both Roux-en-Y gastric bypass groups vs the non-surgical control group in the early postprandial period (all p<0.05). During hypoglycaemia, glucagon exposure was significantly lower in all surgical groups vs the non-surgical control group (all p<0.01). Pancreatic polypeptide levels were significantly lower in patients with post-bariatric hypoglycaemia vs the non-surgical control group (median [IQR]: 24.7 [10.9, 38.7] pmol/l vs 238.7 [186.3, 288.9] pmol/l) (p=0.005). Other hormonal responses to hypoglycaemia and endogenous glucose production did not significantly differ between the groups., Conclusions/interpretation: The glucagon response to insulin-induced postprandial hypoglycaemia is lower in post-bariatric surgery individuals compared with non-surgical control individuals, irrespective of the surgical modality. No significant differences were found between patients with post-bariatric hypoglycaemia and surgical control individuals, suggesting that impaired counter-regulation is not a root cause of post-bariatric hypoglycaemia., Trial Registration: ClinicalTrials.gov NCT04334161., (© 2023. The Author(s).)

Introduction: Neuroblastoma (NB) is a pediatric cancer of the developing sympathetic nervous system. It produces and releases metanephrines, which are used as biomarkers for diagnosis in plasma and urine. However, plasma catecholamine concentrations remain generally normal in children with NB. Thus, unlike pheochromocytoma and paraganglioma (PHEO/PGL), two other non-epithelial neuroendocrine tumors, hypertension is not part of the usual clinical picture of patients with NB. This suggests that the mode of production and secretion of catecholamines and metanephrines in NB is different from that in PHEO/PGL, but little is known about these discrepancies. Here we aim to provide a detailed comparison of the biosynthesis, metabolism and storage of catecholamines and metanephrines between patients with NB and PHEO., Method: Catecholamines and metanephrines were quantified in NB and PHEO/PGL patients from plasma and tumor tissues by ultra-high pressure liquid chromatography tandem mass spectrometry. Electron microscopy was used to quantify neurosecretory vesicles within cells derived from PHEO tumor biopsies, NB-PDX and NB cell lines. Chromaffin markers were detected by qPCR, IHC and/or immunoblotting., Results: Plasma levels of metanephrines were comparable between NB and PHEO patients, while catecholamines were 3.5-fold lower in NB vs PHEO affected individuals. However, we observed that intratumoral concentrations of metanephrines and catecholamines measured in NB were several orders of magnitude lower than in PHEO. Cellular and molecular analyses revealed that NB cell lines, primary cells dissociated from human tumor biopsies as well as cells from patient-derived xenograft tumors (NB-PDX) stored a very low amount of intracellular catecholamines, and contained only rare neurosecretory vesicles relative to PHEO cells. In addition, primary NB expressed reduced levels of numerous chromaffin markers, as compared to PHEO/PGL, except catechol O-methyltransferase and monoamine oxidase A. Furthermore, functional assays through induction of chromaffin differentiation of the IMR32 NB cell line with Bt2cAMP led to an increase of neurosecretory vesicles able to secrete catecholamines after KCl or nicotine stimulation., Conclusion: The low amount of neurosecretory vesicles in NB cytoplasm prevents catecholamine storage and lead to their rapid transformation by catechol O-methyltransferase into metanephrines that diffuse in blood. Hence, in contrast to PHEO/PGL, catecholamines are not secreted massively in the blood, which explains why systemic hypertension is not observed in most patients with NB., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mühlethaler-Mottet, Uccella, Marchiori, La Rosa, Daraspe, Balmas Bourloud, Beck Popovic, Eugster, Grouzmann and Abid.)

Neuropeptide Ys (NPYs) contribute to sympathetic-adreno stimulation: NPY1-36 potentiates the effects of catecholamines (CATs), whereas NPY3-36 inhibits CAT release. We sought to investigate whether inhibiting dipeptidyl-peptidase-4 (DPP4), cleaving NPY1-36 into NPY3-36, leads to increased NPY1-36 potentiating effects and reduced NPY3-36 inhibitory effects on CATs, thereby improving endurance performance. Seven male participants (age 27 ± 3 years, BMI 23.1 ± 2.4 kg/m 2 ) performed time-to-exhaustion cycling exercise at 95% of peak power output with either placebo, or saxagliptin, a DPP4 inhibitor. Oxygen consumption (V̇O 2 ), heart rate variability, NPY1-36, NPY3-36, catecholamines, and lactate were measured at several time points before, during, and after exercise. With saxagliptin, DPP4 activity (12.7 ± 1.6 vs. 0.2 ± 0.3 U/L, p = 0.001; d = 10.7) was decreased at rest, while NPY3-36 (1.94 ± 0.88 vs. 0.73 ± 0.22 pm; p < 0.001; d = 2.04) decreased and NPY1-36 increased during exercise (2.64 ± 2.22 vs. 4.59 ± 2.98 pm; p < 0.01; d = 0.19). CATs were unchanged. Time-to-exhaustion was 32% higher with saxagliptin. The difference in time-to-exhaustion between placebo and saxagliptin was correlated with NPY1-36 differences (R = 0.78, p < 0.05). Peak V̇O 2 and other cardio-respiratory values were not different, whereas peak NPY concentrations were higher with saxagliptin. DPP4 blockade improved performance, increased NPY1-36, and decreased NPY3-36 concentrations which may have potentiating effects on the influences of CATs. However, DPP4 is involved in many different actions, thus NPYs are one group of factors that may underly its performance-enhancing effects; further studies are required to determine the exact mechanisms., (© 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Serotonin is transformed into melatonin under the control of the light/dark cycle, representing a cornerstone of circadian rhythmicity. Serotonin also undergoes extensive metabolism to produce 5-hydroxyindoleacetic acid (5-HIAA), a biomarker for the diagnosis and monitoring of serotonin secreting neuroendocrine tumors (NETs). While serotonin, melatonin and their metabolites are part of an integrated comprehensive system, human observations about their respective plasma concentrations are still limited. We report here for the first time a multiplex UHPLC-MS/MS assay for the quantification of serotonin, 5-HIAA, 5-hydroxytryptophol (5-HTPL), N-acetyl-serotonin (NAS), Mel, 6-OH-Mel, 5-methoxytryptamine (5-MT), 5-methoxytryptophol (5-MTPL), and 5-methoxyindoleacetic acid (5-MIAA) in human plasma. Analytes were extracted by protein precipitation and solid phase extraction. Plasma concentrations for these analytes were determined in 102 healthy volunteers. The LLOQ of the assay ranges from 2.2 nM for serotonin to 1.0 pM for 6-OH-Mel. This sensitivity enables the quantification of circulating serotonin, 5-HIAA, NAS, Mel, and 5-MIAA, even at their lowest diurnal concentrations. This assay will enable specific, precise and accurate measurement of serotonin, Mel and their metabolites to draw a detailed picture of this complex pineal metabolism, allowing a dynamic understanding of these pathways and providing promising biomarkers and a metabolic signature for serotonin-secreting NETs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Neuropeptide Y (NPY1-36) is a vasoconstrictor peptide co-secreted with catecholamines by sympathetic nerves, the adrenal medulla, and neoplasms such as pheochromocytomas and paragangliomas (PPGLs). It is produced by the intracellular cleavage of proNPY and metabolized into multiple fragments with distinct biological activities. NPY immunoassays for PPGL have a diagnostic sensitivity ranging from 33 to 100%, depending on the antibody used. We have validated a multiplex micro-UHPLC-MS/MS assay for the specific and sensitive quantification of proNPY, NPY1-39, NPY1-37, NPY1-36, NPY2-36, NPY3-36, NPY1-35, NPY3-35, and the C-flanking peptide of NPY (CPON) (collectively termed NPYs), and determined the NPYs reference intervals and concentrations in 32 PPGL patients before, during, and after surgery. Depending on the peptide measured, NPYs were above the upper reference limit (URL) in 20% to 67% of patients, whereas plasma free metanephrine and normetanephrine, the gold standard for PPGL, were above the URL in 40% and 87% of patients, respectively. Age, sex, tachycardia, and tumor localization were not correlated with NPYs. Plasma free metanephrines performed better than NPYs in the detection of PPGL, but NPYs may be a substitute for an early diagnosis of PPGL for patients that suffer from severe kidney impairment or receiving treatments that interfere with catecholamine reuptake., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Brain fog is a common and highly disturbing symptom for patients with neuropathic postural tachycardia syndrome (POTS). Cognitive deficits have been measured exclusively in the upright body position and mainly comprised impairments of higher cognitive functions. The cause of brain fog is still unclear today. This study aimed to investigate whether increased autonomic activation might be an underlying mechanism for the occurrence of brain fog in neuropathic POTS. We therefore investigated cognitive function in patients with neuropathic POTS and a healthy control group depending on body position and in relation to catecholamine release as a sensitive indicator of acute stress. The second aim was to test the effect of water intake on cardiovascular regulation, orthostatic symptoms, cognitive function and catecholamine release., Methods: Thirteen patients with neuropathic POTS and 15 healthy control subjects were included. All participants completed a total of four rounds of cognitive testing: two before and two after the intake of 500 ml still water, each first in the supine position and then during head-up tilt. At the end of each cognitive test, a blood sample was collected for determination of plasma catecholamines. After each head-up tilt phase participants were asked to rate their current symptoms on a visual analogue scale., Results: Working memory performance in the upright body position was impaired in patients, which was associated with self-reported symptom severity. Patients had elevated plasma norepinephrine independent of body position and water intake that increased excessively in the upright body position. The excessive increase of plasma norepinephrine was related to heart rate and symptom severity. Water intake in patients decreased norepinephrine concentrations and heart rate, and improved symptoms as well as cognitive performance., Conclusion: Brain fog and symptom severity in neuropathic POTS are paralleled by an excessive norepinephrine secretion. Bolus water drinking down-regulates norepinephrine secretion and improves general symptom severity including brain fog., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rodriguez, Hochstrasser, Eugster, Grouzmann, Müri and Z’Graggen.)

Neuropeptide Y 1-36 (NPY1-36) is a vasoconstrictor peptide co-secreted with norepinephrine (NE) by nerve endings during sympathetic activation. NPY1-36 potentiates NE action post-synaptically through the stimulation of the Y1 receptor, whereas its metabolite NPY3-36 resulting from DPP4 action activates Y2 presynaptic receptors, inhibiting NE and acetylcholine secretion. The secretions of NPY1-36 and NPY3-36 in response to sympathetic nervous system activation have not been studied due to the lack of analytical techniques available to distinguish them. We determined in healthy volunteers NPY1-36, NPY3-36 and catecholamine kinetics and how these neurotransmitters modulate the physiological stress response during and after moderate- and heavy-intensity exercises. Six healthy males participated in this randomized, double-blind, saxagliptin vs placebo crossover study. The volunteers performed an orthostatic test, a 30-min exercise at moderate intensity and a 15-min exercise at heavy intensity each followed by 50 min of recovery in two separate sessions with saxagliptin or placebo. Oxygen consumption (V̇O 2 ), ventilation and heart rate were continuously recorded. NE, epinephrine, NPY1-36 and NPY3-36 were quantified by tandem mass spectrometry. We found that exercise triggers NPY1-36 and NE secretion in an intensity-dependent manner and that NE returns faster to the baseline concentration than NPY1-36 after exercise. NPY3-36 rises during recovery parallel to the decline of NPY1-36. Saxagliptin reverses the NPY1-36/NPY3-36 ratio but does not affect hemodynamics, nor NPY1-36 and catecholamine concentrations. We found that NPY1-36 half-life is considerably shorter than previously established with immunoassays. NPY1-36 and NE secretions are finely regulated to prevent an excessive physiological Y1 stimulating response to submaximal exercise., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Peptide Tyr-Tyr (PYY1-36), pancreatic polypeptide (PP1-36) and neuropeptide Y (NPY1-36) constitute the PP-fold family of peptides that is involved in metabolic regulation. Very low plasma concentrations and cleavage into active 3-36 fragments challenge bioanalytical assays used for the quantification of these peptides., Methods: We developed a multiplexed isotopic dilution assay to quantify PYY1-36, PP1-36, and NPY1-36 and their dipeptidyl peptidase-4 (DPP4)-derived metabolites PYY3-36, PP3-36 and NPY3-36. All peptides were immunocaptured from plasma using a monoclonal antibody and quantified by micro-ultra-HPLC-MS/MS. Blood samples from healthy volunteers were collected fasting and 30 min after nutrient stimulation. Method comparison was performed with commercial immunoassays., Results: Linearity was shown in the measured intervals (r2 > 0.99). The lower limit of quantification (LLOQ) with a CV at 20% was 1.5 pM for PYY1-36 and PYY3-36, 3.0 pM for PP1-36 and PP3-36, 0.8 pM for NPY1-36 and 0.5 pM for NPY3-36. In all cases, intra- and inter-assay bias and imprecision were <21%. Pre-analytical stability required addition of a protease inhibitor cocktail. Physiological concentrations of PYY3-36, NPY3-36, PP1-36 and PP3-36 were above the LLOQ in 43% to 100% of the samples. PYY1-36 and NPY1-36 were above the LLOQ in 9% and 0% of the samples, respectively. Immunoassays showed higher concentrations of measurands and poor agreement when compared with micro-UHPLC-MS/MS., Conclusions: The assay allowed for specific multiplexed analysis of the PP-fold family of peptides and their DPP4-cleaved fragments in a single sample, thereby offering new perspectives to study the role and therapeutic potential of these essential peptide hormones in health and metabolic disease., (© American Association for Clinical Chemistry 2022.)

Background: The selectivity index (SI) of cortisol is used to document correct catheter placement during adrenal vein sampling (AVS) in patients with primary aldosteronism (PA). We aimed to determine the cutoff values of the SIs based on cortisol, free metanephrine, and the free-to-total metanephrine ratio (FTMR) using an adapted AVS protocol in combination with CT., Methods: Adults with PA and referred for AVS were recruited in two hypertension centers. The cortisol and free metanephrine-derived SIs were calculated as the concentration of the analyte in adrenal veins divided by the concentration of the analyte in the distal vena cava. The FTMR-derived SI was calculated as the concentration of free metanephrine in the adrenal vein divided by that of total metanephrine in the ipsilateral adrenal vein. The AVS was classified as an unequivocal radiological success (uAVS) if the tip of the catheter was seen in the adrenal vein. The SI cutoffs of each index marker were established using receiver operating characteristic curve analysis., Results: Out of 125 enrolled patients, 65 patients had an uAVS. The SI cutoffs were 2.6 for cortisol, 10.0 for free metanephrine, 0.31 for the FTMR on the left side, and 2.5, 9.9, and 0.25 on the right side. Compared to free metanephrine and the FTMR, cortisol misclassified AVS as unsuccessful in 36.6% and 39.0% of the cases, respectively., Conclusion: This study is the first to calculate the SIs of cortisol, free metanephrine, and the FTMR indices for the AVS procedure. It confirms that free metanephrine-based SIs are better than those based on cortisol., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Christou, Pivin, Denys, Abid, Zingg, Matter, Pechère-Bertschi, Maillard, Grouzmann and Wuerzner.)

Background and Objectives: The pathophysiological mechanisms linking weight loss to blood pressure (BP) reduction are not completely understood. The objective of this study was to compare the effect of weight loss after Roux-en-Y gastric bypass (RYGB) on BP, renin-angiotensin-aldosterone system (RAAS), and urinary electrolytes excretion to those of dietary advice., Methods: This was a case-control prospective study including obese patients referred for RYGB (cases) and obese receiving diet advice only (controls). Ambulatory BP, plasma renin activity (PRA), plasma aldosterone concentration (PAC), and urinary electrolytes were measured before (M0) and after intervention (M3: 3 months and M12: 12 months)., Results: Twenty-five patients were included in the RYGB group and twelve patients in the control group. After 12 months, weight loss (-42 ± 11.5 vs -12.3 ± 6.3 kg in the control group, p=0.001) and decrease in PAC were more pronounced in the RYGB group (-34 ± 76 vs +14 ± 45 pg/ml in the control group, p=0.002). There was no difference in PRA between both groups (-0.08 ± 1.68 vs 0.01 ± 0.37 ng/ml/h, p=0.31). Sodium excretion was more marked in the RYGB group after 3 months only (-89 ± 14.9 vs -9.9 ± 27.9 mmol/day, p=0.009). The decrease in SBP was similar between both groups (-6.9 ± 9.9 vs -7.1 ± 11.9 mmHg in the control group, p=0.96)., Conclusions: Bariatric-induced weight loss induces a progressive decrease in PAC independently of PRA and sodium excretion. Whether this decrease in PAC affects target organ damage in the long term remains to be determined., Clinical Trial Registration: ClinicalTrials.gov, identifier NCT02218112., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Berney, Vakilzadeh, Maillard, Faouzi, Grouzmann, Bonny, Favre and Wuerzner.)

Background: Plasma-free normetanephrine and metanephrine (metanephrines) are the recommended biomarkers for testing of pheochromocytoma and paraganglioma (PPGL). This study evaluated the status of harmonization of liquid chromatography-tandem mass spectrometry-based measurements of plasma metanephrines and methoxytyramine and clinical interpretation of test results., Methods: 125 plasma samples from patients tested for PPGLs were analyzed in 12 laboratories. Analytical performance was also assessed from results of a proficiency-testing program. Agreement of test results from different laboratories was assessed by Passing-Bablok regression and Bland-Altman analysis. Agreement in clinical test interpretation based on laboratory specific reference intervals was also examined., Results: Comparisons of analytical test results by regression analysis revealed strong correlations for normetanephrine and metanephrine (R ≥ 0.95) with mean slopes of 1.013 (range 0.975-1.078), and 1.019 (range 0.963-1.081), and intercepts of -0.584 (-53.736 to 54.790) and -3.194 (-17.152 to 5.933), respectively. The mean bias between methods was 1.2% (-11.6% to 16.0%) for metanephrine and 0.1% (-18.0% to 9.5%) for normetanephrine. Measurements of 3-methoxytyramine revealed suboptimal agreement between laboratories with biases ranging from -32.2% to 64.0%. Interrater agreement in test interpretation was >94% for metanephrine and >84% for normetanephrine; improvements in interrater agreement were observed with use of harmonized reference intervals, including age-specific cut-offs for normetanephrine., Conclusions: Analytical methods for metanephrines are well harmonized between laboratories. However, the 16% disagreement in test interpretation for normetanephrine suggests use of suboptimal method-dependent reference intervals for clinical decision-making for this metabolite. Improved analytical methods and reference interval harmonization are particularly required for 3-methoxytyramine., (© American Association for Clinical Chemistry 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Urinary 5-hydroxyindoleacetic acid (5-HIAA), vanillylmandelic (VMA), homovanillic acid (HVA), catecholamines and metanephrines are produced in excess by catecholamine-producing tumors. These biogenic amines are unstable at low or high pH and require hydrochloric acid (HCl) to prevent their degradation. However, HCl addition may result in very low pH causing degradation or deconjugation of several metabolites. This study evaluated the buffering properties of sodium citrate to stabilize all biogenic amines. The metabolite concentrations were measured by LC-MS/MS or by a coulometric assay in 22 urine samples collected native and with HCl or sodium citrate. We studied the effect of pH, time (48 h, four weeks) and storage temperature at 22 °C, 4 °C, and -20 °C. We found that catecholamines degradation was prevented by HCl and citrate and that 5-HIAA was degraded in 5 out of 22 samples collected with HCl. All biogenic amines were efficiently stabilized by citrate for four weeks at 22 °C, except epinephrine (48 h at 4 °C, or four weeks at -20 °C). Sodium citrate did not cause quantification or analytical artefacts concerns. In conclusion, sodium citrate is a non-hazardous alternative to HCl for patients to send unfrozen urine samples to the laboratory which may safely store the sample for four weeks., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)