BACKGROUND:Weight loss after gastric bypass is associated with blood pressure (BP) reduction. However, the precise role of the sympathetic nervous system (SNS) and renin-angiotensin-aldosterone system (RAAS) in this reduction remains unclear. We investigated the effect of RYGB-induced weight loss on the hemodynamic, hormonal and renal responses to an orthostatic stress induced by lower body negative pressure (LBNP). METHODS:We conducted a monocentric study comparing response to LBNP (-30 mbar) in two groups of obese participants the first group underwent RYGB surgery (RYGB group) and the second group received lifestyle counselling (control group). We studied urinary sodium excretion, hemodynamic and hormonal responses before, during and 1 h after orthostatic stress induced by LBNP at three timelines: one month before planned RYGB (intervention group), and 3 and 12 months after the intervention. RESULTS:Thirty-seven adult participants were enrolled: 25 patients (72% women, age: 42.1 ± 10.5 years old, BMI 43.0 ± 5.1 kg/m2) in the RYGB group and 12 in control group (58% women, age: 44.8 ± 13.6 years old, BMI 43.3 ± 5.3 kg/m2). At 12 months, mean weight decreased from 126.3 ± 23.2 kg to 116 ± 20.7 kg in the control group and from 120.9 ± 19.4 kg to 78.6 ± 14.0 kg in the RYGB group (p value < 0.01 between groups). During LBNP, the reduction in urinary sodium excretion (-1.98 mmol/h; CI95%: -3.72 to -0.30, p value = 0.02) and the increase in plasma aldosterone concentration (PAC; +9.94 pg/ml, CI 95%: 0.317-19.569, p value = 0.043) were more pronounced in the RYGB group. CONCLUSIONS:Our study suggests that weight loss induced by RYGB increases aldosterone responsiveness to orthostatic stress and enhances the sodium tubular response during orthostatic stress. CLINICALTRIALS.GOV ID:NCT02218112.

We report a patient with autonomic dysfunction following acute SARS-CoV-2 infection, presenting progressively worsening severe orthostatic hypotension to the point where she could no longer sit or stand. The patient experienced a delay in diagnosis after an initial misdiagnosis of a functional neurological disorder. Persistent orthostatic symptoms prompted us to re-examine the diagnosis and explore other diagnostic tools, which ultimately allowed us to identify and treat severe immune-mediated orthostatic hypotension (OH). We identified autoantibodies (AAB) targeting the autonomic nervous system. Intravascular immunoglobulin therapy, along with early, specific multi-disciplinary rehabilitation, completely resolved the symptoms. Hard-to-assess patients are often penalized by suboptimal care due to the lack of a comprehensive patient history and physical examination, resulting in unnecessary and costly ancillary examinations that lead to delays in diagnosis or misdiagnoses. Furthermore, a lack of awareness of rare complications with new diseases may also hamper proper patient care. In the present case, this includes the wide range of SARS-CoV-2 infection manifestations, including immune-mediated autonomic complications.

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.

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.

Background/ObjectiveDue 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 ReportA 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 two 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 (MRI) identified a 29 mm right adrenal mass. The patient underwent right adrenalectomy resulting in resolution of the hypertensive crises.DiscussionThe 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.ConclusionPPGL 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.

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.

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. 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 123I-mIBG uptake. 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 123I-mIBG by 2.3- and 1.9-fold at 4 and 24 h post-injection, respectively, compared to the untreated group. Upregulation of the NET by CUDC-907 lead to a better internalization of mIBG in vitro and in vivo.

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 Graphical abstract

Das Phäochromozytom ist in der Schwangerschaft selten und kann unerkannt zu fatalen Verläufen führen. Anhand von Fallvignetten werden die diagnostischen und therapeutischen Herausforderungen beschrieben mit Diskussion der Epidemiologie.

Le phéochromocytome est très rare pendant la grossesse et peut avoir une évolution fatale s’il n’est pas détecté. Les défis diagnostiques et thérapeutiques sont décrits à l’aide de quatre vignettes de cas, et l’épidémiologie est discutée.

IntroductionNeuroblastoma (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.MethodCatecholamines 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.ResultsPlasma 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.ConclusionThe 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.

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.

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.

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.

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 (V̇O2), 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.

Aim: To compare endocrine and metabolic counterregulation to postprandial hypoglycemia in patients with postbariatric hypoglycemia (PBH) after gastric bypass (GB) vs. surgical and non-surgical controls. Methods: Thirty-two adults (age 42.9±12.8 years, 13% male, BMI 28.2±4.3kg/m2) of 4 matched groups (PBH patients, GB, sleeve gastrectomy [SG] and non-surgical controls [CON]) ingested 15g glucose with subsequent induction of hypoglycemia (target 2.5mM) at t150-170 min using a constant insulin infusion with controller-guided iv dextrose adjustment. Primary outcome was mean glucagon during hypoglycemia (t150-170) . Further outcomes were responses of catecholamines, cortisol, growth hormone, pancreatic polypeptide (PP) , glucagon-like peptide 1, endogenous glucose production (EGP) and exogenous insulin. Values are shown as mean±SD. Results: Plasma glucose and insulin at t150-170 were 2.6±0.2mM and 891±382pM across all groups. Glucagon was significantly lower in all surgical groups (PBH: 12.0±8.2pM, GB: 13.4±5.7pM and SG: 11.0±6.8pM vs. CON: 23.0±6.2pM, p=0.005) . PP was significantly lower in PBH vs. CON (28.1±49.6pM vs. 146.0±93.5pM, p=0.01) . EGP did not differ significantly between groups, nor did any other hormones. Conclusion: Our results suggest diminished glucagon response to postprandial hypoglycemia after GB and SG compared to CON, with additionally lower PP values in PBH. Disclosure A.Tripyla: None. J.Zehetner: None. D.Giachino: None. S.Del favero: Research Support; Dexcom, Inc. A.Thomas: None. M.Thevis: None. C.Dalla man: Research Support; Becton, Dickinson and Company, Sanofi-Aventis Deutschland GmbH. L.Bally: None. D.Herzig: None. G.Reverter branchat: Employee; Fresenius Medical Care. J.Pavan: None. M.Schiavon: None. P.J.Eugster: None. E.Grouzmann: None. C.T.Nakas: None. S.Valérie: None. Funding Swiss National Science Foundation (PCEGP3_186978)

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.

Clinical EndocrinologyVolume 98, Issue 6 p. 832-833 LETTER The road to reliable peptide assays is paved with good guidelines Jonathan Maurer, Jonathan Maurer orcid.org/0000-0003-1011-3431 Service of Clinical Pharmacology, Lausanne University Hospital and University of Lausanne, Lausanne, SwitzerlandSearch for more papers by this authorEric Grouzmann, Eric Grouzmann orcid.org/0000-0002-3224-2536 Service of Clinical Pharmacology, Lausanne University Hospital and University of Lausanne, Lausanne, SwitzerlandSearch for more papers by this authorPhilippe J. Eugster, Corresponding Author Philippe J. Eugster [email protected] orcid.org/0000-0002-0197-6318 Service of Clinical Pharmacology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland Correspondence Philippe J. Eugster, Service of Clinical Pharmacology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland. Email: [email protected]Search for more papers by this author Jonathan Maurer, Jonathan Maurer orcid.org/0000-0003-1011-3431 Service of Clinical Pharmacology, Lausanne University Hospital and University of Lausanne, Lausanne, SwitzerlandSearch for more papers by this authorEric Grouzmann, Eric Grouzmann orcid.org/0000-0002-3224-2536 Service of Clinical Pharmacology, Lausanne University Hospital and University of Lausanne, Lausanne, SwitzerlandSearch for more papers by this authorPhilippe J. Eugster, Corresponding Author Philippe J. Eugster [email protected] orcid.org/0000-0002-0197-6318 Service of Clinical Pharmacology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland Correspondence Philippe J. Eugster, Service of Clinical Pharmacology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland. Email: [email protected]Search for more papers by this author First published: 15 December 2022 https://doi.org/10.1111/cen.14863Citations: 1Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. REFERENCES 1Kowalka AM, Alexiadou K, Cuenco J, et al. The post-prandial secretion of peptide YY1-36 and 3-36 in obesity is differentially increased after gastric bypass versus sleeve gastrectomy. Clin Endocrinol. 2022. Published online November 7, 2022. doi:10.1111/cen.14846 2Reverter-Branchat G, Eugster PJ, Kuenzli C, et al. Multiplexed assay to quantify the PP-fold family of peptides in human plasma using microflow liquid chromatography–tandem mass spectrometry. Clin Chem. 2022; 68(4): 584-594. doi:10.1093/clinchem/hvab229 3Toräng S, Veedfald S, Rosenkilde MM, Hartmann B, Holst JJ. The anorexic hormone peptide YY3-36 is rapidly metabolized to inactive peptide YY3-34 in vivo. Physiol Rep. 2015; 3(7):e12455. doi:10.14814/phy2.12455 4Wong PF, Gall MG, Bachovchin WW, McCaughan GW, Keane FM, Gorrell MD. Neuropeptide Y is a physiological substrate of fibroblast activation protein: enzyme kinetics in blood plasma and expression of Y2R and Y5R in human liver cirrhosis and hepatocellular carcinoma. Peptides. 2016; 75: 80-95. doi:10.1016/j.peptides.2015.11.004 5Johnson JR. Methods for handling concentration values below the limit of quantification in PK studies. PhUSE US Connect. 2018. Citing Literature Volume98, Issue6June 2023Pages 832-833 ReferencesRelatedInformation

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.