High-Throughput and Sensitive Quantitation of Plasma Catecholamines by Ultraperformance Liquid Chromatography–Tandem Mass Spectrometry Using a Solid Phase Microwell Extraction Plate

Plasma catecholamines provide a reliable biomarker of sympathetic activity. The low circulating concentrations of catecholamines and analytical interferences require tedious sample preparation and long chromatographic runs to ensure their accurate quantification by HPLC with electrochemical detection. Published or commercially available methods relying on solid phase extraction technology lack sensitivity or require derivatization of catecholamine by hazardous reagents prior to tandem mass spectrometry (MS) analysis. Here, we manufactured a novel 96-well microplate device specifically designed to extract plasma catecholamines prior to their quantification by a new and highly sensitive ultraperformance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method. Processing time, which included sample purification on activated aluminum oxide and elution, is less than 1 h per 96-well microplate. The UPLC–MS/MS analysis run time is 2.0 min per sample. This UPLC–MS/MS method does not require a derivatization step, reduces the turnaround time by 10-fold compared to conventional methods used for routine application, and allows catecholamine quantification in reduced plasma sample volumes (50–250 μL, e.g., from children and mice)

Data_Sheet_1_Brain fog in neuropathic postural tachycardia syndrome may be associated with autonomic hyperarousal and improves after water drinking.PDF

BackgroundBrain 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.MethodsThirteen 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.ResultsWorking 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.ConclusionBrain 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.</p

<i>Aspergillus</i> Protein Degradation Pathways with Different Secreted Protease Sets at Neutral and Acidic pH

Aspergillus fumigatus grows well at neutral and acidic pH in a medium containing protein as the sole nitrogen source by secreting two different sets of proteases. Neutral pH favors the secretion of neutral and alkaline endoproteases, leucine aminopeptidases (Laps) which are nonspecific monoaminopeptidases, and an X-prolyl dipeptidase (DppIV). Acidic pH environment promotes the secretion of an aspartic endoprotease of pepsin family (Pep1) and tripeptidyl-peptidases of the sedolisin family (SedB and SedD). A novel prolyl peptidase, AfuS28, was found to be secreted in both alkaline and acidic conditions. In previous studies, Laps were shown to degrade peptides from their N-terminus until an X-Pro sequence acts as a stop signal. X-Pro sequences can be then removed by DppIV, which allows Laps access to the following residues. We have shown that at acidic pH Seds degrade large peptides from their N-terminus into tripeptides until Pro in P1 or P′1 position acts as a stop for these exopeptidases. However, X-X-Pro and X-X-X-Pro sequences can be removed by AfuS28 thus allowing Seds further sequential proteolysis. In conclusion, both alkaline and acidic sets of proteases contain exoprotease activity capable of cleaving after proline residues that cannot be removed during sequential digestion by nonspecific exopeptidases

Dipeptidyl Peptidase 4 Inhibition Increases Postprandial Norepinephrine via Substance P (NK1R) during RAAS Inhibition Supplemental Figure 1

Consort Diagram<br

Image_1_Accurate Location of Catheter Tip With the Free-to-Total Metanephrine Ratio During Adrenal Vein Sampling.tif

BackgroundThe 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.MethodsAdults 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.ResultsOut 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.ConclusionThis 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.</p

Additional file 2 of CUDC-907, a dual PI3K/histone deacetylase inhibitor, increases meta-iodobenzylguanidine uptake (123/131I-mIBG) in vitro and in vivo: a promising candidate for advancing theranostics in neuroendocrine tumors

Additional file 2. Additional data

Additional file 1 of CUDC-907, a dual PI3K/histone deacetylase inhibitor, increases meta-iodobenzylguanidine uptake (123/131I-mIBG) in vitro and in vivo: a promising candidate for advancing theranostics in neuroendocrine tumors

Additional file 1: Table S1. Patients and tumors characteristics: Sp: sporadic, Mutation investigated: RET: Rearranged after transfection (Multiple Endocrine Neoplasia type 2 syndrome), SDH: succinate dehydrogenase subunits A, B, C and D, SDHAF2: succinate dehydrogenase complex assembly factor 2, VHL: von Hippel-Lindau, MAX: MYC-associated factor X. Table S2. List of the different inhibitors used and respective references. Table S3. Primers used for RT-qPCR. Table S4. Chromatographic gradient used for the separation of mIBG by LC–MS/MS. Table S5. Biodistribution data and SPECT/CT images of 123I-mIBG in IGR-NB8 xenografts at 4 h and 24 h p.i. Figure S1. Effect of the treatment with different inhibitors on cell viability in IGR-NB8, SK-N-Be2C, LAN-1 and PC-12 cells. Figure S2. Experimental design of the in vivo studies

Quantification of Neuropeptide Y and Four of Its Metabolites in Human Plasma by Micro-UHPLC-MS/MS

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

DataSheet_1_Low number of neurosecretory vesicles in neuroblastoma impairs massive catecholamine release and prevents hypertension.pdf

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.</p

Image_3_Accurate Location of Catheter Tip With the Free-to-Total Metanephrine Ratio During Adrenal Vein Sampling.tif

BackgroundThe 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.MethodsAdults 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.ResultsOut 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.ConclusionThis 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.</p