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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.)

Purpose: Sodium and water handling by the kidney and the sympathetic nervous system have been implicated in the development of obesity-related hypertension and kidney disease. They have seldom been studied together during stress conditions. The objective of this study was to compare the systemic, renal and hormonal responses to lower body negative pressure (LBNP) in adult healthy participants (H), obese normotensive (OBN) and obese hypertensive patients (OBH)., Materials and Methods: This was a prospective case-control study. Participants from the three groups were exposed to one hour of LBNP. Systemic and renal haemodynamics, sodium and water excretion and hormones were measured before and after LBNP. Intergroup LBNP responses were tested using a Student t-test or a Wilcoxon rank-sum test. An extension of the Wilcoxon rank-sum test was used to test for a trend across the three groups., Results: The study included 54 participants (H: 25, OBN: 16, OBH: 13). LBNP induced a stepwise increase in systolic blood pressure (+2.7 ± 4.7 mmHg (H) vs. +4.7 ± 8.8 mmHg (OBN) vs. +8.0 ± 8.6 mmHg (OBH, p  = .028)) and heart rate (-1.3 ± 4.9 bpm (H) vs. 2.2 ± 6.1 bpm (OBN) vs. 1.9 ± 4.1 bpm (OBH, p  = .041). Urinary output (-2.8 ± 2.1 ml/min vs. -1.4 ± 1.7 ml/min, p  = .028) and free water clearance (-1.9 ± 1.7 mOsm/kg vs. -0.7 ± 1.3 mOsm/kg, p  = .016) responses were more marked in OBN compared to H., Conclusions: These results show that the systemic and the renal response to LBNP differ according to weight and to BP categories. Systolic BP and heart show a progressive increased response form healthy volunteers to OBN and then to obese hypertensive participants while urinary output and free water clearance responses are increased in OBN only, suggesting that the occurrence of hypertension in obese individuals modifies the early kidney responses to stress., Clinicaltrial.gov Identifier: NCT01734096.

Metanephrines (MNs; normetanephrine (NMN), metanephrine (MN) and methoxytyramine (MT)) detected in urine or plasma represent the best biomarker for neuroblastoma (NB) diagnosis, however the metabolism of both catecholamine (CAT) and MNs remains enigmatic in NB. Using patient-derived xenograft (PDX) models derived from primary NB cells, we observed that the plasma levels of MNs in NB-PDX-bearing mice were comparable as in patients. Interestingly, murine plasma displayed an elevated fraction of glucuronidated forms of MNs relative to human plasma where sulfonated forms prevail. In tumors, the concentration ranges of MNs and CAT and the expression levels of the main genes involved in catecholamine metabolism were similar between NB-PDX and human NB tissues. Likewise, plasma and intratumoral profiles of individual MNs, with increased levels of MT and NMN relative to MN, were also conserved in mouse models as in patients. We further demonstrated the downregulation of the Phenylethanolamine N-Methyltransferase gene in NB biopsies and in NB-PDX explaining this biochemical phenotype, and giving a rational to the low levels of epinephrine and MN measured in NB affected patients. Thus, our subcutaneous murine NB-PDX models not only reproduce the phenotype of primary NB tumors, but also the metabolism of catecholamine as observed in patients. This may potentially open new avenues in preclinical studies for the follow up of novel therapeutic options for NB through the quantification of plasma MNs., Competing Interests: CONFLICTS OF INTEREST Authors have no conflicts of interest to declare., (Copyright: © 2021 Abid et al.)

Targeted radionuclide therapy of somatostatin receptor (SST)-expressing tumors is only partially addressed by the established somatostatin analogs having an affinity for the SST subtype 2 (SST2). Aiming to target a broader spectrum of tumors, we evaluated the bis-iodo-substituted somatostatin analog ST8950 ((4-amino-3-iodo)-d-Phe-c[Cys-(3-iodo)-Tyr-d-Trp-Lys-Val-Cys]-Thr-NH 2 ), having subnanomolar affinity for SST2 and SST5, labeled with [ 177 Lu]Lu 3+ via the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). Human Embryonic Kidney (HEK) cells stably transfected with the human SST2 (HEK-SST2) and SST5 (HEK-SST5) were used for in vitro and in vivo evaluation on a dual SST2- and SST5-expressing xenografted mouse model. nat Lu-DOTA-ST8950 showed nanomolar affinity for both subtypes (IC 50 (95% confidence interval): 0.37 (0.22-0.65) nM for SST2 and 3.4 (2.3-5.2) for SST5). The biodistribution of [ 177 Lu]Lu-DOTA-ST8950 was influenced by the injected mass, with 100 pmol demonstrating lower background activity than 10 pmol. [ 177 Lu]Lu-DOTA-ST8950 reached its maximal uptake on SST2- and SST5-tumors at 1 h p.i. (14.17 ± 1.78 and 1.78 ± 0.35%IA/g, respectively), remaining unchanged 4 h p.i., with a mean residence time of 8.6 and 0.79 h, respectively. Overall, [ 177 Lu]Lu-DOTA-ST8950 targets SST2-, SST5-expressing tumors in vivo to a lower extent, and has an effective dose similar to clinically used radiolabeled somatostatin analogs. Its main drawbacks are the low uptake in SST5-tumors and the persistent kidney uptake.

Background: Somatostatin receptor (SST) targeting, specifically of the subtype 2 (SST2), with radiolabeled somatostatin analogs, is established for imaging and treatment of neuroendocrine tumors. Owing to the concomitant and heterogeneous expression of several subtypes on the same tumor, analogs targeting more subtypes than SST2 potentially target a broader spectrum of tumors and/or increase the uptake of a given tumor. The analog ST8950 ((4-amino-3-iodo)-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH 2 ), bearing 2 iodo-amino acids, exhibits sub-nanomolar affinity to SST2 and SST5. We report herein the development and preclinical evaluation of DOTA-ST8950 labeled with 68 Ga, for imaging SST2- and SST5-expressing tumors. Comparative in vitro and in vivo studies were performed with the de-iodinated DOTA-ST8951 ((4-amino)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH 2 ) and with the reference compounds DOTA-TATE (SST2 selective) and DOTA-NOC (for SST2 and SST5)., Results: Compared with nat Ga-DOTA-NOC, nat Ga-DOTA-ST8950 exhibited higher affinity to SST2 and SST5 (IC 50 (95%CI), nM = 0.32 (0.20-0.50) and 1.9 (1.1-3.1) vs 0.70 (0.50-0.96) and 3.4 (1.8-6.2), respectively), while nat Ga-DOTA-ST8951 lost affinity for both subtypes. nat Ga-DOTA-ST8950 had the same potency for inducing SST2-mediated cAMP accumulation as nat Ga-DOTA-TATE and slightly better than nat Ga-DOTA-NOC (EC 50 , nM = 0.46 (0.23-0.92) vs 0.47 (0.15-1.5) vs 0.59 (0.18-1.9), respectively). [ 67 Ga]Ga-DOTA-ST8950 had a similar internalization rate as [ 67 Ga]Ga-DOTA-NOC in SST2-expressing cells (12.4 ± 1.6% vs 16.6 ± 2.2%, at 4 h, p = 0.0586). In vivo, [ 68 Ga]Ga-DOTA-ST8950 showed high and specific accumulation in SST2- and SST5-expressing tumors, comparable with [ 68 Ga]Ga-DOTA-NOC (26 ± 8 vs 30 ± 8 %IA/g, p = 0.4630 for SST2 and 15 ± 6 vs 12 ± 5 %IA/g, p = 0.3282, for SST5, 1 h p.i.) and accumulation in the SST-positive tissues, the kidneys and the liver. PET/CT images of [ 68 Ga]Ga-DOTA-ST8950, performed in a dual HEK-SST2 and HEK-SST5 tumor xenografted model, clearly visualized both tumors and illustrated high tumor-to-background contrast., Conclusions: [ 68 Ga]Ga-DOTA-ST8950 reveals its potential for PET imaging SST2- and SST5-expressing tumors. It compares favorably with the clinically used [ 68 Ga]Ga-DOTA-NOC in terms of tumor uptake; however, its uptake in the liver remains a challenge for clinical translation. In addition, this study reveals the essential role of the iodo-substitutions in positions 1 and 3 of [ 68 Ga]Ga-DOTA-ST8950 for maintaining affinity to SST2 and SST5, as the de-iodinated [ 68 Ga]Ga-DOTA-ST8951 lost affinity for both receptor subtypes.

Neuropeptide Y (NPY) is a 36-amino acid peptide circulating at a subpicomolar concentration participating in multiple physiological and pathological processes. NPY is prone to peptidolysis, generating metabolites with modified affinity for the five known receptors of NPY that mediate distinct effects. It is, therefore, crucial to distinguish each metabolite to understand the multiple functions of NPY. Since immunoassays are not able to distinguish NPY from its metabolites, we have validated a microliquid chromatography tandem mass spectrometry (micro-LC-MS/MS) assay for the quantification of endogenous NPY, NPY2-36, NPY3-36, NPY1-35, and NPY3-35 in human plasma. Sample preparation relies on immunoextraction in 96-well plates, followed by solid-phase extraction prior to micro-LC-MS/MS. The LLOQ ranged from 0.03 to 0.16 pM, intra- and inter-assay precision were <27% and trueness <22%. We determined reference intervals in 155 healthy volunteers and 40 hypertensive patients. We found that NPY3-36 is the main circulating peptide in resting conditions and that NPY and catecholamines are simultaneously increased during orthostasis. We also showed that the concentrations of NPY and its metabolites are similar in healthy volunteers and hypertensive patients. NPY is the prototype peptide that circulates in concentrations expected to be beyond instrumental capacities. We have been successful in developing a high-throughput specific and sensitive assay by including a deep knowledge of the physicochemical properties of these peptides to an efficient multistep sample preparation, and a micro-LC chromatography. We believe that our methodological approach opens the possibility to selectively quantify other endogenous peptides cleaved by peptidases whose concentrations are below 1 pM.

Chlamydia trachomatis (CT) infection is the most frequent notifiable sexually transmitted infection (STI) in Switzerland. The infection is most frequently observed in 15 to 24 year-old-women and in 25 to 34 year-old-men. 50-75 % of the Chlamydia trachomatis carriage are asymptomatic, making the infection difficult to diagnose and increasing the untreated specimen, leading to complications like infertility, ectopic pregnancy or pelvic inflammatory disease. Despite having a sexual prevention at school, the youths seem to have a lack of knowledge about CT, her transmission and her complications. We performed a survey, which showed that 60.5 % of the participants ignored that this bacteria is mostly asymptomatic. We also found that 11 % of the participants believed that there is no possible relapse of the infection. The prevention must be strengthened, mostly because there is no program in Switzerland, letting every physician to his own beliefs. The medical consultation is an ideal opportunity for this prevention and the youths shared their wish to discuss more about it with health professionals., Competing Interests: Les auteurs n’ont déclaré aucun conflit d’intérêts en relation avec cet article.

Context: Dipeptidyl peptidase 4 (DPP4) inhibitors may increase the risk of heart failure. Decreased degradation of vasoactive peptides like substance P [also degraded by angiotensin-converting enzyme (ACE)] and Y1 agonists peptide YY (PYY 1-36) and neuropeptide Y (NPY 1-36) could contribute., Objective: This study tested the hypothesis that there is an interactive effect of DPP4 inhibition and ACE inhibition (vs antihypertensive control subjects) on vasoactive peptides after a mixed meal., Participants and Design: Fifty-three patients with type 2 diabetes and hypertension were randomized to double-blind treatment with ramipril, valsartan, or amlodipine for 15 weeks in parallel groups. During the 5th, 10th, and 15th weeks, participants also received placebo + placebo, sitagliptin 100 mg/d + placebo, and sitagliptin + aprepitant 80 mg/d in random order. On the last day of each crossover treatment, participants underwent a mixed-meal study., Results: Sitagliptin increased postprandial glucagon-like peptide-1 and decreased glucose in all antihypertensive groups. Sitagliptin increased NPY 1-36 and decreased Y2 agonists NPY 3-36 and PYY 3-36 in all groups. During ramipril or valsartan, but not amlodipine, sitagliptin increased postprandial norepinephrine; substance P receptor blockade with aprepitant prevented this effect. Despite increased norepinephrine, sitagliptin decreased postprandial blood pressure during ACE inhibition., Conclusion: DPP4 inhibition increases postprandial concentrations of the Y1 agonist NPY 1-36. During treatment with an ACE inhibitor or angiotensin receptor blocker, DPP4 inhibition increased postprandial norepinephrine through a substance P receptor-dependent mechanism. Increased NPY 1-36 and norepinephrine could increase risk of heart failure but did not result in higher postprandial blood pressure., Competing Interests: Disclosure Summary: J.W. was supported by DK007061, GM007569, and TR001879. S.H. was supported by GM108554. S.J. was supported by GM007569. N.B. was supported by American Heart Association 17SFRN33520059.All data generated or analyzed during this study are included in this published article or in the data repositories listed in the references.

Teriparatide (PTH 1-34, Forsteo ® ) is a bioactive N-terminal fragment of the native endogenous parathyroid hormone (PTH 1-84) recommended for the treatment of osteoporosis in patients with high risk of fracture. Since PTH 1-34 may undergo proteolysis we have validated an ultra-high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for unambiguously measuring intact PTH 1-34 with the same sensitivity as ELISA, at subpicomolar level (LLOQ at 0.4 pM). The full chromatographic run was achieved in 16.5 min. The method validation showed satisfactory intra- and inter-assay precision (CV < 13%) and excellent trueness (<5%), and almost no matrix effect (recoveries 78-92%). We found that after subcutaneous injection in two volunteers, PTH 1-34 half-life was shorter with UHPLC-MS/MS and that ELISA was overestimating PTH 1-34 late concentrations in both volunteers. Qualitative mass spectrometry was performed and led to the discovery of PTH 1-33, a fragment of PTH 1-34 with unknown function. This study emphasized the importance of switching from immunoassays to mass spectrometry when measuring bioactive peptides prompt to proteolysis into fragments that may exhibit altered bioactivity and duration of action., (Copyright © 2019 Elsevier B.V. All rights reserved.)

AP102 is a di-iodinated octapeptide somatostatin agonist (SSA) designed to treat acromegaly and neuroendocrine tumors. A sensitive and selective method was validated for the quantification of AP102 in plasma following the European Medicines Agency (EMA) and Food and Drug Administration (FDA) guidelines. Sample preparation was performed using solid-phase extraction microplates. Chromatographic separation was achieved on an ultra-high pressure liquid chromatography (UHPLC) C18 column in 6.0 minutes. The compounds were quantified using multiple reaction monitoring on a tandem quadrupole mass spectrometer with 13 C, 15 N-labeled AP102 as internal standard. Calibration ranged from 50 to 10000 pg/mL. The lower limit of quantification (LLOQ) was measured at 20 pg/mL, and robust analytical performances were obtained with trueness at 99.2%-100.0%, intra-assay imprecision at 2.5%-4.4%, and inter-assay imprecision at 8.9%-9.7%. The accuracy profiles (total error) built on the 3 concentrations levels showed accuracy within the 70%-130% range. AP102 is remarkably stable since no proteolytic fragments were detected on plasma samples analyzed by Orbitrap-MS. Pharmacokinetic studies were conducted in rats, after single dose (1, 3, and 10 μg/kg, sc) and continuous subcutaneous administration (osmotic minipumps for 28 days, 3.0 or 10.0 μg/kg/h). AP102 showed a rapid absorption by the subcutaneous route (T max : 15-30 minutes) and a fast elimination (t 1/2 : 33-86 minutes). The PK profile of AP102 exhibited a mean clearance of 1.67 L/h and a mean distribution volume at steady state of 7.16 L/kg, about 10-fold higher than those observed with other SSA or non- and mono-iodinated AP102. LogD 7.4 determination confirmed the lipophilic properties of AP102 that might influence its distribution in tissues., (Copyright © 2018 John Wiley & Sons, Ltd.)

DPP (dipeptidyl peptidase)-4 inhibitors are antidiabetic drugs that may increase heart failure in high-risk patients. NPY (neuropeptide Y) is coreleased with norepinephrine, causes vasoconstriction via the Y1 receptor, and is degraded by DPP4 to NPY (3-36) in vitro. NPY (3-36) decreases release of norepinephrine via the Y2 receptor. We tested the hypothesis that DPP4 inhibition would potentiate the vasoconstrictor effect of NPY. Eighteen nonsmokers (12 healthy controls and 6 with type 2 diabetes mellitus) participated in 1 of 2 randomized, double-blind, placebo-controlled crossover studies. First, subjects were randomized to order of treatment with sitagliptin 100 mg/d versus placebo for 7 days separated by 4-week washout. On the last day of treatment, NPY was infused by brachial artery and forearm blood flow was measured using plethysmography. Blood samples were collected after each dose. NPY infusions were repeated after 90-minute washout and intra-arterial enalaprilat. Second, 5 healthy subjects were randomized to crossover treatment with sitagliptin 100 mg/d plus valsartan 160 mg/d versus placebo plus valsartan. NPY infusions were performed on the seventh day of treatment. NPY caused dose-dependent vasoconstriction. During enalaprilat, sitagliptin significantly potentiated NPY-induced vasoconstriction in controls and diabetics ( P≤0.02 for forearm blood flow in either group). Baseline norepinephrine release was increased during sitagliptin and enalaprilat, but not further by NPY. Sitagliptin increased the ratio of NPY to NPY (3-36). During valsartan, sitagliptin also significantly potentiated NPY-induced vasoconstriction ( P=0.009 for forearm blood flow). Potentiation of endogenous NPY could contribute to cardiovascular effects of DPP4 inhibitors in patients taking an angiotensin-converting enzyme inhibitor or angiotensin receptor blocker.

Background: Urinary vanillylmandelic acid (VMA) and homovanillic acid (HVA) are biomarkers for the diagnosis and follow-up of neuroblastoma, whereas urinary 5-hydroxyindoleacetic acid (5-HIAA) is used to assess a carcinoid tumor. These analytes are conventionally analyzed in a single run by chromatography (LC) coupled with electrochemical detection (LC-ECD) using commercial kits. A rapid dilute-and-shoot LC tandem mass spectrometry (LC-MS/MS) assay was validated in order to replace the LC-ECD method and therefore improve analytical specificity and throughput., Methods: Sample preparation was carried out by dilution of the urine sample with a solution containing the deuterated internal standards. The separation was achieved on an ultra-high pressure LC system with MS detection using a triple quadrupole mass spectrometer. The method was validated according to the current EMA and FDA guidelines., Results: The full chromatographic run was achieved in 8 min. The method validation showed excellent linearity (r2>0.999 for all three analytes), precision (CV <15%), negligible matrix effect (recoveries >90%), low carryover (<1%) and LLOQ of 0.25, 0.4 and 0.4 μM for VMA, HVA and 5-HIAA, respectively. Deming fits and Bland-Altman analyses showed no significant differences between the values obtained between the two assays., Conclusions: The LC-MS/MS method proposed in this study is fast and robust, and the simple sample preparation saves time and avoids the additional costs of dedicated kits used for the LC-ECD assays by switching to LC-MS/MS. Additionally, the near-perfect correlation observed herein between both assays allows the previously established reference ranges to be maintained.

Background/aims: In patients with resistant hypertension, renal denervation (RDN) studies have mainly focused their outcomes on blood pressure (BP). The aim of this study was to evaluate the long-term effect of RDN on neurohormonal profiles, renal hemodynamics and sodium excretion in a resting state and during stress induced by lower body negative pressure (LBNP)., Materials and Methods: This was a single center prospective observational study. Norepinephrine, plasma renin activity (PRA), glomerular filtration rate (GFR), renal plasma flow (RPF) and sodium excretion were measured in unstimulated conditions (rest) and after one hour of LBNP at three different time points: before (M0), one (M1) and twelve months (M12) after RDN., Results: Thirteen patients with resistant hypertension were included. In the resting state, no differences were observed in norepinephrine, PRA, sodium excretion and mean BP levels after RDN. GFR (78 ± 32 ml/min at M0 vs 66 ± 26 ml/min at M12 ( p = 0.012) and filtration fraction (22.6 ±5.4% at M0 vs 15.1 ±5.3% at M12 ( p = 0.002)) both decreased after RDN. During LBNP, the magnitude of the mean BP increase was reduced from +6.8 ± 6.6 mm Hg at M0 to +2.3 ± 1.3 mm Hg at M12 ( p = 0.005). The LBNP-induced increase in norepinephrine and decrease in GFR and sodium excretion observed before RDN were blunted after the procedure., Conclusion: A decrease in GFR and filtration fraction was observed one year after RDN. In addition, our results suggest that RDN blunts not only the norepinephrine but also the mean BP, the GFR and the sodium excretion responses to an orthostatic stress one year after the intervention., Registry Number: NCT01734096.

Background: Somatostatin analogs (SSAs) are first-line medical therapy for the treatment of acromegaly and neuroendocrine tumors that express somatostatin receptors (SSTR). Somatostatin suppresses secretion of a large number of hormones through the stimulation of the five SSTR. However, unbalanced inhibition of secretion as observed with the highly potent SSAs pasireotide causes hyperglycaemia mainly by inhibiting insulin secretion. In contrast, AP102 a new SSAs has neutral effect on blood glucose while suppressing GH secretion. Our objective was to establish the cellular effects of AP102 on SSTR2 and SSTR5 that may explain the differences observed between AP102 and other SSAs., Methods: We compared the binding and agonist activity of AP102 with somatostatin-14, octreotide and pasireotide in HEK293 cells transfected with human SSTR2 and SSTR5 receptors. SSAs signal transduction effects (cAMP concentrations) were measured in forskolin-treated cells in the presence of SSAs. Proliferation and apoptotic effects were determined and binding assays were performed using 125 I- somatostatin-14., Results: AP102 has comparable affinity and agonist effect to octreotide at SSTR2 (IC50's of 112 pM and 244 pM, respectively; EC50's of 230 pM and 210 pM, respectively) in contrast to pasireotide that exhibits a 12-27 fold higher IC50 (3110 pM) and about 5-fold higher EC50 (1097 pM). At SSTR5, AP102 has much higher affinity and stimulating effect than octreotide (IC50's of 773 pM and 16,737 pM, respectively; EC50's of 8526 pM and 26,800 pM), and an intermediate affinity and agonist effect between octreotide and pasireotide. AP102, octreotide and pasireotide have variable anti-proliferative effects on HEK cells transfected with SSTR2 and SSTR5., Conclusion: AP102 is a new SSA that better reduces signaling at SSTR2 than SSTR5 and prevents cell proliferation at both receptors. The euglycaemic effect of AP102 observed in preclinical studies may be related to this intermediate agonistic potency between pasireotide and octreotide at SSTR2 and SSTR5., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Purpose: Somatostatin analogs are widely used to treat conditions associated with hormonal hypersecretion such as acromegaly and metastatic neuroendocrine tumors. First generation somatostatin analogs, such as octreotide and lanreotide, have high affinity for somatostatin receptor subtype 2 (SSTR2), but have incomplete efficacy in many patients. Pasireotide targets multiple SSTRs, having the highest affinity for SSTR5, but causes hyperglycemia and diabetes mellitus in preclinical and clinical studies. AP102 is a new somatostatin analogs with high affinity at both SSTR2 and SSTR5. We aimed to characterize the effects of AP102 vs. pasireotide on random and dynamic glucose levels, glucoregulatory hormone concentrations and growth axis measures in healthy Sprague-Dawley rats., Methods: Three doses of each compound were evaluated under acute conditions (1, 10, and 30 µg/kg s.c.), and two doses during a chronic (4-week) infusion (3 and 10 µg/kg/h s.c.)., Results: Neither acute nor chronic AP102 administration altered blood glucose concentrations or dynamic responses following an intraperitoneal glucose tolerance test. In contrast, acute and chronic pasireotide dosing increased random and post-intraperitoneal glucose tolerance test blood glucose measures, compared to vehicle-treated controls. Both AP102 and pasireotide acutely suppressed growth hormone levels, although insulin-like growth factor-1 and somatic growth was suppressed to a greater extent with pasireotide., Conclusions: AP102 is a new dual SSTR2/SSTR5-specific somatostatin analog that acutely reduces growth hormone but does not cause hyperglycemia during acute or chronic administration in a healthy rat model. Further studies in diabetic animals and in humans are necessary to determine the potential utility of AP102 in the clinical setting.

Endocan is a soluble molecule secreted from vascular endothelial cells of various organs. Its exact function in humans remains to be elucidated, though it has been postulated that increased tissue expression or serum levels of this molecule may be an indicator of endothelial activation and neovascularization. In the realm of forensic pathology, studies pertaining to endothelial activation following exposure to cold exclusively focused on thrombomodulin, a transmembrane protein specific to endothelial cells. In the study herein described, endocan concentrations were determined in postmortem serum, urine and vitreous humor samples collected during autopsy in a series of cases that underwent medicolegal investigations. A total of 76 autopsy cases were selected and three study groups (hypothermia group, sepsis group and non-hypothermia/non-sepsis group) prospectively formed during the study period. The obtained results seem to indicate that exposure to cold and subsequent death is not distinguished by significant endothelial dysfunction causing enhanced endocan secretion., (Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

Prolyl peptidases of the MEROPS S28 family are of particular interest because they are key enzymes in the digestion of proline-rich peptides. A BLAST analysis of the Aspergillus oryzae genome revealed sequences coding for four proteases of the S28 family. Three of these proteases, AoS28A, AoS28B, and AoS28C, were previously characterized as acidic prolyl endopeptidases. The fourth protease, AoS28D, showed high sequence divergence with other S28 proteases and belongs to a phylogenetically distinct cluster together with orthologous proteases from other Aspergillus species. The objective of the present paper was to characterize AoS28D protease in terms of substrate specificity and activity. AoS28D produced by gene overexpression in A. oryzae and in Pichia pastoris was a 70-kDa glycoprotein with a 10-kDa sugar moiety. In contrast with other S28 proteases, AoS28D did not hydrolyze internal Pro-Xaa bonds of several tested peptides. Similarly, to human lysosomal Pro-Xaa carboxypeptidase, AoS28D demonstrated selectivity for cleaving C-terminal Pro-Xaa bonds which are resistant to carboxypeptidases of the S10 family concomitantly secreted by A. oryzae. Therefore, AoS28D could act in synergy with these enzymes during sequential degradation of a peptide from its C-terminus.

The renal tissular renin-angiotensin and bradykinin-kallikrein systems control kidney function together with the renal sympathetic innervation but their interaction is still unclear. To further elucidate this relationship, we investigated these systems in rats 6 days after left kidney denervation (DNX, n = 8) compared to sham-operated controls (CTR, n = 8). Plasma renin concentration was unchanged in DNX vs. CTR (p = NS). Kidney bradykinin (BK) and angiotensin (Ang) I and II concentrations decreased bilaterally in DNX vs. CTR rats (~20 to 40%, p < 0.05) together with Ang IV and V concentrations that were extremely low (p = NS). Renin, Ang III and dopamine concentrations decreased by ~25 to 50% and norepinephrine concentrations by 99% in DNX kidneys (p < 0.05) but were unaltered in opposite kidneys. Ang II/I and KA were comparable in DNX, contralateral and CTR kidneys. Ang III/II increased in right vs. DNX or CTR kidneys (40-50%, p < 0.05). Ang II was mainly located in tubular epithelium by immunocytological staining and its cellular distribution was unaffected by DNX. Moreover, the angiotensinergic and catecholaminergic innervation of right kidneys was unchanged vs. CTR. We found an important dependency of tissular Ang and BK levels on the renal innervation that may contribute to the resetting of kidney function after DNX. The DNX-induced peptide changes were not readily explained by kidney KA, renin or plasma Ang I generation. However, tissular peptide metabolism and compartmentalization may have played a central role. The mechanisms behind the concentration changes remain unclear and deserve further clarification.

The counterregulatory response to hypoglycemia, which restores normal blood glucose levels to ensure sufficient provision of glucose to the brain, is critical for survival. To discover underlying brain regulatory systems, we performed a genetic screen in recombinant inbred mice for quantitative trait loci (QTL) controlling glucagon secretion in response to neuroglucopenia. We identified a QTL on the distal part of chromosome 7 and combined this genetic information with transcriptomic analysis of hypothalami. This revealed Fgf15 as the strongest candidate to control the glucagon response. Fgf15 was expressed by neurons of the dorsomedial hypothalamus and the perifornical area. Intracerebroventricular injection of FGF19, the human ortholog of Fgf15, reduced activation by neuroglucopenia of dorsal vagal complex neurons, of the parasympathetic nerve, and lowered glucagon secretion. In contrast, silencing Fgf15 in the dorsomedial hypothalamus increased neuroglucopenia-induced glucagon secretion. These data identify hypothalamic Fgf15 as a regulator of glucagon secretion., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

Background: During clinical trials, researchers rarely question nominal doses specified on labels of investigational products, overlooking the potential for inaccuracies that may result when calculating pharmacokinetic and pharmacodynamic parameters. This study evaluated the disparity between nominal doses and the doses actually administered in two Phase I trials of a biosimilar drug., Methods: In Trial A, 12 healthy volunteers received various doses of an interferon β-1a biosimilar via either subcutaneous or intravenous injection, prepared by partially emptying 0.53 ml syringes supplied by the manufacturer. In Trial B, 12 volunteers received three different formulations of the drug via intravenous injection (biosimilar with and without albumin and a comparator), followed by multiple subcutaneous injections. In both trials, the dose administered was calculated as D = C × V - losses, where C is the drug concentration assessed using ELISA, V is the volume administered calculated using syringe weighing and losses are deduced from in-vitro experiments. Interferon binding to added albumin and infusion lines was evaluated using a (125)I-interferon tracer with gel-filtration chromatography., Results: In Trial A, measured concentrations were close to the nominal strength indicated by the manufacturer (median bias: -6 %), whereas in Trial B they differed significantly for all three formulations (median biases: +67 %, +73 % and +31 % for the biosimilar with albumin, the biosimilar without albumin and the comparator, respectively). In Trial A, the doses actually administered showed large variability and biases, especially at the lowest doses. Indeed, actually injected volumes differed by as much as 74 % from theoretical volumes - a phenomenon mainly attributed to unnoticed fluid re-aspiration through the syringe needle. This was corrected in Trial B. Interferon was not significantly adsorbed on the infusion lines used for intravenous administration. Its binding to albumin was slow, reaching 50 % after a 16 h incubation., Conclusions: These examples illustrate the importance of assessing the actual doses administered in clinical trials, to ensure accuracy in the determination of clearance, distribution volume, bioavailability and dose-response relationships., Trial Registration: Clinicaltrials.gov NCT02515695 (Trial A) and NCT02517788 (Trial B). Registered on 24 July and 5 August 2015, respectively.

The aim of this study was to measure catecholamines and their O-methylated metabolites in urine and vitreous humor collected in cardiac deaths and noncardiac control cases that underwent medicolegal investigations. Our first goal was to assess whether cardiac events of different types are characterized by different catecholamine/metanephrine urine and/or vitreous profiles. Our second goal was to determine whether noncardiac causes of death with different survival intervals are characterized by different catecholamine/metanephrine urine and/or vitreous profiles. Two study groups were prospectively and retrospectively formed, a cardiac death group (including three subgroups, according to the cause of death) and a noncardiac death group (including two subgroups, according to the length of the agonal period). Postmortem angiography, autopsy, histology, toxicology, and biochemistry were performed in all cases. First results seem to indicate that absolute values measured in urine and vitreous for each of the analyzed markers display no significant differences relating to each of the tested cardiac death subgroups. In the control group, absolute concentrations measured in urine and vitreous for each of the analyzed parameters failed to show significant differences relating to the length of agonal period. Our preliminary findings do not seem to confirm the conclusions of former studies and fail to corroborate the usefulness of urine catecholamine and metanephrine analysis to characterize stress response intensity or length of the dying process in the postmortem setting.

Purpose: The aim of this study was to assess the diagnostic value of catecholamines and their O-methylated metabolites in vitreous humor samples in identifying antemortem cold exposure and fatal hypothermia in the forensic casework., Methods: A total of 80 autopsy cases (40 hypothermia fatalities and 40 cases in which hypothermia as the main or contributory cause of death was excluded) were selected for this study. Catecholamines and their O-methylated metabolites were measured in urine and vitreous humor samples collected at autopsy., Results: Urine catecholamine and their O-methylated metabolite concentrations were significantly higher in hypothermia-related deaths. On the other hand, measurements in vitreous humor samples did not reveal statistically significant differences between hypothermia-related deaths and controls., Conclusions: Globally considered, our findings seem to suggest that, contrary to urine catecholamines and their O-methylated metabolites, vitreous levels of these compounds appear to be of limited value in characterizing human antemortem stress reactions due to cold exposure and can hardly be used in the forensic setting to support the diagnosis of hypothermia.

Renal denervation (DNX) is a treatment for resistant arterial hypertension. Efferent sympathetic nerves regrow, but reinnervation by renal afferent nerves has only recently been shown in the renal pelvis of rats after unilateral DNX. We examined intrarenal perivascular afferent and sympathetic efferent nerves after unilateral surgical DNX. Tyrosine hydroxylase (TH), CGRP, and smooth muscle actin were identified in kidney sections from 12 Sprague-Dawley rats, to distinguish afferents, efferents, and vasculature. DNX kidneys and nondenervated kidneys were examined 1, 4, and 12 wk after DNX. Tissue levels of CGRP and norepinephrine (NE) were measured with ELISA and mass spectrometry, respectively. DNX decreased TH and CGRP labeling by 90% and 95%, respectively (P < 0.05) within 1 wk. After 12 wk TH and CGRP labeling returned to baseline with a shift toward afferent innervation (P < 0.05). Nondenervated kidneys showed a doubling of both labels within 12 wk (P < 0.05). CGRP content decreased by 72% [3.2 ± 0.3 vs. 0.9 ± 0.2 ng/gkidney; P < 0.05] and NA by 78% [1.1 ± 0.1 vs. 0.2 ± 0.1 pmol/mgkidney; P < 0.05] 1 wk after DNX. After 12 wk, CGRP, but not NE, content in DNX kidneys was fully recovered, with no changes in the nondenervated kidneys. The use of phenol in the DNX procedure did not influence this result. We found morphological reinnervation and transmitter recovery of afferents within 12 wk after DNX. Despite morphological evidence of sympathetic regrowth, NE content did not fully recover. These results suggest a long-term net surplus of afferent influence on the DNX kidney may be contributing to the blood pressure lowering effect of DNX., (Copyright © 2016 the American Physiological Society.)

Introduction: The efficacy of Gamma Knife surgery (GKS) in local tumor control of non-secreting paragangliomas (PGLs) has been fully described by previous studies. However, with regard to secreting PGL, only one previous case report exists advocating its efficacy at a biological level., Case Report: The aims of this study were: 1) to evaluate the safety/efficacy of GKS in a dopamine-secreting PGL; 2) to investigate whether the biological concentrations of free methoxytyramine could be used as a marker of treatment efficacy during the follow-up. We describe the case of a 62-year-old man diagnosed with left PGL. He initially underwent complete surgical excision. Thirty months after, he developed recurrent biological and neuroradiological disease; the most sensitive biomarker for monitoring the disease, concentration of plasma free methoxytyramine, started to increase. GKS was performed at a maximal marginal dose of 16 Gy. During the following 30 months, concentration of free methoxytyramine gradually decreased from 0.14 nmol/l (2*URL) before GKS to 0.09 nmol/l, 6 months after GKS and 0.07 nmol/l at the last follow-up after GKS (1.1*URL), confirming the efficacy of the treatment. Additionally, at 30 months there was approximately 36.6% shrinkage from the initial target volume., Conclusion: The GKS treatment was safe and effective, this being confirmed clinically, neuroradiologically and biologically. The case illustrates the importance of laboratory tests taking into account methoxytyramine when analyzing biological samples to assess the biochemical activity of a PGL. In addition, the identification of methoxytyramine as a unique positive biomarker could designate it for the monitoring of tumor relapse after treatments, including Gamma Knife surgery.

Prolyl endopeptidases are key enzymes in the digestion of proline-rich proteins. Fungal extracts rich in prolyl endopeptidases produced by a species such as Aspergillus oryzae used in food fermentation would be of particular interest for the development of an oral enzyme therapy product in patients affected by intolerance to gluten. Two major A. oryzae secreted prolyl endopeptidases of the MEROPS S28 peptidase family, AoS28A and AoS28B, were identified when this fungus was grown at acidic pH in a medium containing soy meal protein or wheat gliadin as the sole source of nitrogen. AoS28B was produced by 12 reference A. oryzae strains used in food fermentation. AoS28A was secreted by six of these 12 strains. This protease is the orthologue of the previously characterized Aspergillus fumigatus (AfuS28) and Aspergillus niger (AN-PEP) prolyl endopeptidases which are encoded by genes with a similar intron-exon structure. Large amounts of secreted AoS28A and AoS28B were obtained by gene overexpression in A. oryzae. AoS28A and AoS28B are endoproteases able to cleave N-terminally blocked proline substrates. Both enzymes very efficiently digested the proline-rich 33-mer of gliadin, the most representative immunotoxic peptide deriving from gliadin, with some differences in terms of specificity and optimal pH. Digestion of the gliadin peptide in short peptides with both enzymes was found to occur from its N terminus.

Background: After no research in humans for >40 years, there is renewed interest in using lysergic acid diethylamide (LSD) in clinical psychiatric research and practice. There are no modern studies on the subjective and autonomic effects of LSD, and its endocrine effects are unknown. In animals, LSD disrupts prepulse inhibition (PPI) of the acoustic startle response, and patients with schizophrenia exhibit similar impairments in PPI. However, no data are available on the effects of LSD on PPI in humans., Methods: In a double-blind, randomized, placebo-controlled, crossover study, LSD (200 μg) and placebo were administered to 16 healthy subjects (8 women, 8 men). Outcome measures included psychometric scales; investigator ratings; PPI of the acoustic startle response; and autonomic, endocrine, and adverse effects., Results: Administration of LSD to healthy subjects produced pronounced alterations in waking consciousness that lasted 12 hours. The predominant effects induced by LSD included visual hallucinations, audiovisual synesthesia, and positively experienced derealization and depersonalization phenomena. Subjective well-being, happiness, closeness to others, openness, and trust were increased by LSD. Compared with placebo, LSD decreased PPI. LSD significantly increased blood pressure, heart rate, body temperature, pupil size, plasma cortisol, prolactin, oxytocin, and epinephrine. Adverse effects produced by LSD completely subsided within 72 hours. No severe acute adverse effects were observed., Conclusions: In addition to marked hallucinogenic effects, LSD exerts methylenedioxymethamphetamine-like empathogenic mood effects that may be useful in psychotherapy. LSD altered sensorimotor gating in a human model of psychosis, supporting the use of LSD in translational psychiatric research. In a controlled clinical setting, LSD can be used safely, but it produces significant sympathomimetic stimulation., (Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)