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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: 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.)

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.)

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.

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.)

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.

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.

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.

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.

The detection and early identification of natural products (NPs) for dereplication purposes require efficient, high-resolution methods for the profiling of crude natural extracts. This task is difficult because of the high number of NPs in these complex biological matrices and because of their very high chemical diversity. Metabolite profiling using ultra-high pressure liquid chromatography coupled to high-resolution mass spectrometry (UHPLC–HR-MS) is very efficient for the separation of complex mixtures and provides molecular formula information as a first step in dereplication. This structural information alone or even combined with chemotaxonomic information is often not sufficient for unambiguous metabolite identification. In this study, a representative set of 260 NPs containing C, H, and O atoms only was analysed in generic UHPLC–HR-MS profiling conditions. Two easy to use quantitative structure retention relationship (QSRR) models were built based on the measured retention time and on eight simple physicochemical parameters calculated from the structures. First, an original approach using several partial least square (PLS) regressions according to the phytochemical classes provided satisfactory results with an easy calculation. Secondly, a unique artificial neural network (ANN) model provided similar results on the whole set of NPs but required dedicated software. The retention prediction methods described in this study were found to improve the level of confidence of the identification of given analytes among putative isomeric structures. Its applicability was verified for the dereplication of NPs in model plant extracts., (Copyright © 2014 Elsevier Ltd. All rights reserved.)