Evaluation of salivary vasopressin as an acute stress biomarker in healthy dogs with stress due to noise and environmental challenges.

BACKGROUND: Stress is associated with various detrimental changes in physiological health that affect an animal's quality of life. The hypothalamus-pituitary-adrenal (HPA) axis and the sympathetic-adreno-medullar (SAM) axis are two main physiological pathways that constitute the stress response of an organism. Arginine vasopressin (AVP) is a mediator of the HPA axis and is known to be related to social behaviours and stress. The serum concentration of AVP is higher in more aggressive dogs and humans with post-traumatic stress disorder. Salivary biomarker analysis is a non-invasive method to assess stress. The purpose of this study was to evaluate the possibility of using salivary AVP as an acute stress biomarker in dogs. Salivary AVP concentration was measured before and after exposure to all relevant environmental stimuli (i.e. car trip to the lab, physical examination by the veterinarian, and sampling procedure,) and then after 30 min of vacuum noise exposure. Behavioural assessments, physiologic parameter assessments, and serum cortisol analysis were conducted in combination. Statistical analysis was conducted separately in the total study population, the less stressed group, and the more stressed group, respectively. RESULTS: Based on stress behaviour analysis scores, 28 dogs were classified into less or more stressed groups. All four physiologic parameters (blood pressure, body temperature, heart rate, and respiratory rate) were significantly increased after noise and environmental challenges, in the more stressed group. Serum cortisol did not show any significant change. Salivary AVP significantly decreased after noise and environmental stimulation in the more stressed group but not in the less stressed group. Salivary AVP and blood pressure changes were negatively correlated in the more stressed group. CONCLUSION: Salivary AVP may be a potential acute stress biomarker in dogs.

Neonatal CSF vasopressin concentration predicts later medical record diagnoses of autism spectrum disorder.

Autism spectrum disorder (ASD) is a brain disorder characterized by social impairments. ASD is currently diagnosed on the basis of behavioral criteria because no robust biomarkers have been identified. However, we recently found that cerebrospinal fluid (CSF) concentration of the "social" neuropeptide arginine vasopressin (AVP) is significantly lower in pediatric ASD cases vs. controls. As an initial step in establishing the direction of causation for this association, we capitalized upon a rare biomaterials collection of newborn CSF samples to conduct a quasi-prospective test of whether this association held before the developmental period when ASD first manifests. CSF samples had been collected in the course of medical care of 0- to 3-mo-old febrile infants (n = 913) and subsequently archived at -70 °C. We identified a subset of CSF samples from individuals later diagnosed with ASD, matched them 1:2 with appropriate controls (n = 33 total), and quantified their AVP and oxytocin (OXT) concentrations. Neonatal CSF AVP concentrations were significantly lower among ASD cases than controls and individually predicted case status, with highest precision when cases with comorbid attention-deficit/hyperactivity disorder were removed from the analysis. The associations were specific to AVP, as ASD cases and controls did not differ in neonatal CSF concentrations of the structurally related neuropeptide, OXT. These preliminary findings suggest that a neurochemical marker of ASD may be present very early in life, and if replicated in a larger, prospective study, this approach could transform how ASD is detected, both in behaviorally symptomatic children, and in infants at risk for developing it.

Impurity identification and quantification for arginine vasopressin by liquid chromatography/high-resolution mass spectrometry.

RATIONALE: For pharmaceutical quality control, impurities may have unexpected pharmacological or toxicological effects on quality, safety, and efficacy of drugs. Arginine vasopressin (AVP) is an important cyclic peptide drug that is mainly used for the treatment of diabetes insipidus and esophageal varices bleeding. With the advancement made in analytical techniques, liquid chromatography/high-resolution mass spectrometry (LC/HRMS) has emerged as a critical technique for the identification and quantification of structurally related peptide impurities in AVP. METHODS: An LC/HRMS/MS-based method using a quadrupole ion trap-Orbitrap mass spectrometer operated in the positive ion electrospray ionization mode was developed for the determination and quantification of structurally related peptide impurities in AVP. RESULTS: Under optimized experimental conditions, three deamidation products, ([Glu4 ]AVP, [Asp5 ]AVP, and AVP acid), two amino acid deletion impurities (des-Pro7 -AVP and des-Gly9 -AVP), one amino acid insertion impurity (endo-Gly10a -AVP), one end chain reaction product (N-acetyl-AVP), and one AVP isomer were detected. Subsequent quantification using an external standard method estimated the total mass fraction of all structurally related peptide impurities in the AVP study material to be 30.3 mg/g with an expanded uncertainty of 3.0 mg/g (k = 2). CONCLUSIONS: This study complements the AVP impurity profile and improves the separation and discovery of other potential impurities in vasopressin analogues.

GC-MS and LC-MS/MS pilot studies on the guanidine (NG)-dimethylation in native, asymmetrically and symmetrically NG-dimethylated arginine-vasopressin peptides and proteins in human red blood cells.

Protein-arginine methyltransferases catalyze the methylation of the guanidine (NG) group of proteinic L-arginine (Arg) to produce monomethyl and dimethylarginine proteins. Their proteolysis releases the free amino acids monomethylarginine (MMA), symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA), respectively. MMA, SDMA and ADMA are inhibitors of the nitric oxide synthase (NOS) activity. High circulating and low urinary concentrations of ADMA and SDMA are considered risk factors in the cardiovascular and renal systems, mainly due to their inhibitory action on NOS activity. Identity, biological activity and concentration of NG-methylated proteins are largely unknown. The present study addressed these issues by using GC-MS and LC-MS/MS approaches. GC-MS was used to quantify free ADMA released by classical HCl-catalyzed hydrolysis of three synthetic Arg-vasopressin (V) peptides and of unknown endogenous NG-dimethylated proteins. The cyclic (c) disulfide forms of Arg-vasopressin analogs, i.e., Arg-vasopressin (cV-Arg-Gly-NH2), asymmetrically NG-dimethylated vasopressin (cV-ADMA-Gly-NH2) and symmetrically NG-dimethylated vasopressin (cV-SDMA-Gly-NH2) were used as model peptides in quantitative GC-MS analyses of ADMA, SDMA and other expected amino acids from the hydrolyzed Arg-vasopressin analogs. cV-ADMA-Gly-NH2 and cV-SDMA-Gly-NH2 were discriminated from cV-Arg-Gly-NH2 by LC-MS and LC-MS/MS, yet they were indistinguishable from each other. The same applies to the respective open (o) reduced and di-S-acetamide forms of oV-ADMA-Gly-NH2, oV-SDMA-Gly-NH2 and oV-Arg-Gly-NH2. Our LC-MS and LC-MS/MS studies suggest that the Arg-vasopressin analogs form [(M-H)]+ and [(M-H)+H]+ in the positive ESI mode and undergo in part conversion of their terminal Gly-NH2 (NH2, 16 Da) group to Gly-OH (OH, 17 Da). The product ion mass spectra of the di-S-acetamide forms are complex and contain several intense mass fragments differing by 1 Da. cV-ADMA-Gly-NH2 and cV-SDMA-Gly-NH2 induced platelet aggregation in platelet-rich human plasma with moderately different initial velocity and maximal aggregation rates compared to cV-Arg-Gly-NH2. Previous studies showed that human red blood cells are rich in large (>50 kDa) ADMA-containing proteins of unknown identity. Our LC-MS/MS proteomic study identified several membrane and cytosolic erythrocytic NG-dimethylated proteins, including spectrin-α (280 kDa), spectrin-ß (247 kDa) and protein 4.1 (80 kDa). Being responsible for the stability of the erythrocyte membrane, the newly identified main targets for NG-dimethylation in human erythrocytes should be given a closer look in erythrocytic diseases like hereditary spherocytosis.

A target-driven DNA-based molecular machine for rapid and homogeneous detection of arginine-vasopressin.

Rapid detection of physiological changes of neuropeptides is of great importance as they are involved in a wide range of physiological processes and behaviors. Abnormalities in their expression level are correlated with various neurological diseases. However, current methods such as radioimmunoassay, enzyme-linked immunosorbent assays and liquid chromatography tandem mass spectrometry relied on cumbersome operation steps and could not rapidly provide the information of their concentration fluctuations. Thus motivated, we developed a target-driven DNA-based molecular machine that could be triggered only in the presence of a specific target neuropeptide. Using arginine-vasopressin (AVP) as a model neuropeptide, we integrated the DNA-based molecular machine with fluorescence signal transduction and amplification technology. The assay was rapid and homogeneous, which offered a linear range of 75-700 pM and a limit-of-detection as low as 75 pM. It holds great potential for further applications in real-time monitoring of the variations of the AVP level in biological samples.

Diagnosis and management of central diabetes insipidus in adults.

Central diabetes insipidus (CDI) is characterized by hypotonic polyuria due to impairment of AVP secretion from the posterior pituitary. In clinical practice, it needs to be distinguished from renal resistance to the antidiuretic effects of AVP (nephrogenic DI), and abnormalities of thirst appreciation (primary polydipsia). As nephrogenic diabetes insipidus is rare in adults, unless they are treated with lithium salts, the practical challenge is how to differentiate between CDI and clinical disorders of excess thirst. The differential diagnosis is usually straight forward, but the recommended gold standard test, the water deprivation test, is not without interpretative pitfalls. The addition of the measurement of plasma AVP concentrations improves diagnostic accuracy, but the radioimmunoassay for AVP is technically difficult, and is only available in a few specialized centres. More recently, the measurement of plasma copeptin concentrations has been claimed to provide a reliable alternative to measurement of plasma AVP, without the sampling handling challenges. In addition, the measurement of thirst ratings can help the differentiation between CDI and primary polydipsia. Once the diagnosis of CDI is biochemically certain, investigations to determine the cause of AVP deficiency are needed. In this review, we will outline the diagnostic approach to polyuria, revisit the caveats of the water deprivation test and review recent data on value of adding AVP/copeptin measurement. We will also discuss treatment strategies for CDI, with analysis of potential complications of treatment.

Measuring peripheral oxytocin and vasopressin in nonhuman primates.

Studying the neural and hormonal changes that modulate behavior is critical to understanding social relationships. Of particular interest is measuring oxytocin (OT) and arginine vasopressin (AVP) peripherally, and preferably, non-invasively, in nonhuman primates. Due to these peptides' neural origin and their stimulation of brain areas that influence social behavior, there has been debate whether peripheral measures in blood, urine, and saliva reflect central levels in the brain. This review elucidates the challenges of OT measurement and the solutions that provide valuable data on OT's role in social behavior. This review discusses the recent studies in rhesus macaques which indicate that exogenous OT delivered by nasal spray results in increased OT in cerebrospinal fluid, and it notes the new methodologies that can measure both endogenous and exogenous OT simultaneously, which thereby determine the source of measured OT in biological fluids. Next, this review highlights the utility of measuring urinary OT by summarizing the results of clearance rate studies in humans and marmosets, which characterize the timing that circulating OT enters urine and illustrate that endogenous releasers of OT also increase urinary OT. With the ability to reliably measure OT and AVP in urine and in blood, we can now study free-ranging captive, and non-captive primates to answer questions about the biology of social bonding that were not possible before. One procedural concern that this review also highlights is whether extraction of the peptides prior to assay is needed, as the values are higher in samples that have not been extracted. Studies indicate that extractions eliminate the interfering compounds that cause higher values. Across studies, to ensure the reliability of measuring OT for nonhuman primates, this review makes suggestions based on empirical evidence for how to correctly preserve samples and emphasizes the need to validate each assay for individual species.

Mirror-image aptamer kissing complex for arginine-vasopressin sensing.

The recently reported aptamer kissing complex (AKC) strategy has allowed for the development of a new kind of sandwich-like sensing tools. Currently AKC assays have been only applied to low molecular weight molecules and their functionality in complex matrices remains challenging. The objective of the present study broken down into two sub-aims; exploring the propensity to broaden the scope of detectable analytes and designing a more robust system for potential applications to realistic samples. An all L-configuration aptaswitch module derived from a hairpin spiegelmer specific to a larger target, i.e. the arginine-vasopressin (AVP) hormone, was elaborated. The target-induced AKC formation in presence of a specific mirror-image RNA hairpin (L-aptakiss) probe were analyzed by using fluorescence anisotropy. The mirror-image kissing complex was successfully formed when the L-AVP target bound to the engineered L-aptaswitch element. It was also established that the use of methanol as cosolvent significantly improved the assay sensitivity through the stabilization of the ternary complex. Finally, the capability of the mirror-image method to operate in 10-fold diluted, untreated human serum was illustrated. The current work revealed that the AKC concept can be expanded to a wider range of targets and converted to a L-configuration sensing platform especially suitable for bioanalysis purposes.

Anti-nausea effects and pharmacokinetics of ondansetron, maropitant and metoclopramide in a low-dose cisplatin model of nausea and vomiting in the dog: a blinded crossover study.

BACKGROUND: Nausea is a subjective sensation which is difficult to measure in non-verbal species. The aims of this study were to determine the efficacy of three classes of antiemetic drugs in a novel low dose cisplatin model of nausea and vomiting and measure change in potential nausea biomarkers arginine vasopressin (AVP) and cortisol. A four period cross-over blinded study was conducted in eight healthy beagle dogs of both genders. Dogs were administered 18 mg/m2 cisplatin intravenously, followed 45 min later by a 15 min infusion of either placebo (saline) or antiemetic treatment with ondansetron (0.5 mg/kg; 5-HT3 antagonist), maropitant (1 mg/kg; NK1 antagonist) or metoclopramide (0.5 mg/kg; D2 antagonist). The number of vomits and nausea associated behaviours, scored on a visual analogue scale, were recorded every 15 min for 8 h following cisplatin administration. Plasma samples were collected to measure AVP, cortisol and antiemetic drug concentrations. RESULTS: The placebo treated group vomited an average number of 7 times (range 2-13). None of the dogs in either the ondansetron or maropitant treated groups vomited during the observation period. The onset of nausea-like behaviour in the placebo-treated group occurred at t3.5h and peaked at t4.75h with nausea behaviour score of 58.5 ± 4.6 mm. Ondansetron and maropitant reduced overall the area under the curve of nausea behaviour score by 90% and 25%, respectively. Metoclopramide had no effect on either vomiting or nausea. Cisplatin-induced nausea and vomiting caused concomitant increases in AVP and cortisol. In the placebo-treated group, AVP and cortisol increased from t2.5h, peaked at t5h (11.3 ± 2.9 pmol L-1 and 334.0 ± 46.7 nmol/L, respectively) and returned to baseline by t8h. AVP and cortisol increases were completely prevented by ondansetron and only partially by maropitant, while metoclopramide had no effect. The terminal half-lives (harmonic mean ± pseudo SD) for ondansetron, maropitant and metoclopramide were 1.21 ± 0.51, 5.62 ± 0.77 and 0.87 ± 0.17 h respectively. CONCLUSIONS: 5-HT3 receptor antagonist ondansetron demonstrates the greatest anti-emetic and anti-nausea efficacy of the three drugs. AVP and cortisol appear to be selective biomarkers of nausea rather than emesis, providing a means of objectively measuring of nausea in the dog.

Evaluación de una estrategia diagnóstica combinada con copeptina y troponina T ultrasensibles en el infarto de miocardio sin elevación del segmento ST en los servicios de urgencias / Combined high-sensitivity copeptin and troponin T evaluation for the diagnosis of non-ST elevation acute coronary syndrome in the emergency department

Objetivo. Estudio fue evaluar la capacidad diagnóstica de la copeptina de elevada sensibilidad (copep-es), de forma aislada o conjuntamente con troponina cardiaca T de elevada sensibilidad (Tnc T-es), en el diagnóstico de infarto agudo de miocardio sin elevación del segmento ST (IAMSEST) en los pacientes atendidos por dolor torácico con sospecha de infarto de miocardio en los servicios de urgencias (SU), y seguidamente la capacidad pronóstica a los 12 meses. Método. Estudio observacional retrospectivo de una serie de pacientes atendidos por dolor torácico sugestivo de isquemia miocárdica en 5 SU españoles. Se midieron centralizadamente copep-es y Tnc T-es en la primera muestra sanguínea extraída a la llegada al SU. El rendimiento diagnóstico se evaluó mediante la sensibilidad, la especificidad, los valores predictivos, las razones de verosimilitud, y el área bajo la curva (ABC) de la característica operativa del receptor (COR). Se realizó un análisis separado en el subgrupo de pacientes con presentación precoz (< 3 h desde el inicio de los síntomas). Se registraron las complicaciones, mortalidad o reinfarto, ocurridas a los 12 meses desde el evento índice. Resultados. Se incluyeron 297 pacientes. Se diagnosticaron 63 (21,2%) IAMSEST. La mediana de edad fue 69 (RIC 70- 76) y 199 (67%) fueron varones. Las ABC COR fueron 0,89 (IC 95% 0,85-0,94) para Tnc T-es, 0,58 (IC 95% 0,51- 0,66) para copep-es y 0,90 (IC 95% 0,86-0,94) para la determinación conjunta. El ABC COR de la medida conjunta no mejoró a la de Tnc T-es aislada (p = 0,89). El análisis de los pacientes con presentación precoz mostró el mismo patrón de resultados. Un 60% de las complicaciones ocurrió en los pacientes con ambos biomarcadores elevados. Los incrementos aislados de copep-es no aportaron información pronóstica adicional a la proporcionada por Tnc T-es (p = 0,56). Conclusión. La medida de copep-es no mejora el valor diagnóstico o pronóstico de la Tnc T-es en los pacientes con sospecha de IAMSEST atendidos en los SU (AU)

Objectives. To assess the diagnostic yield of a high-sensitivity copeptin (hs-copep) assay alone or in combination with a high-sensitivity cardiac troponin T (hs-cTnt) assay for the diagnosis of non-ST segment elevation acute coronary syndrome (NSTEMI) in patients with chest pain in the emergency department (ED). The secondary aim was to assess the 1-year prognostic utility of these biomarkers in this clinical context. Material and methods. Retrospective observational study of a series of patients attended for chest pain suggesting myocardial ischemia in 5 Spanish ED. The first blood drawn in the ED was used for hs-copep and hs-cTnt assays, which were processed in a single laboratory serving all centers. Diagnostic utility was assessed by sensitivity, specificity, positive and negative predictive values and likelihood ratios, and the area under the receiver operating characteristic curve (ROC). We also performed a separate analysis with data for the subgroup of patients with early detection of symptoms (3 h of onset of symptoms). We recorded complications, mortality or reinfarction occurring within a year of the index event. Results. We included 297 patients; 63 (21.2%) with NSTEMI. The median age was 69 years (interquartile range, 70-76 years), and 199 (67%) were men. The ROC was 0.89 (95% CI, 0.85-0.94) for the hs-cTnt assay, 0.58 (95% CI, 0.51-0.66) for the hscopep assay, and 0.90 (95% CI, 0.86-0.94) for the 2 assays combined. The ROC for the 2 assays combined was not significantly better than the ROC for the hs-cTnt by itself (P=.89). We saw the same pattern of results when we analyzed the subgroup of patients who presented early. Sixty percent of the complications occurred in patients with elevated findings on both assays. Elevated hs-copep findings did not provide prognostic information that was not already provided by hs-cTnt findings (P=.56). Conclusion. The hs-copep assay does not increase the diagnostic or prognostic yield already provided by the hs-cTnt assay in patients suspected of myocardial infarction in the ED (AU)

Transient receptor potential vanilloid 5 (TRPV5), a highly Ca2+ -selective TRP channel in the rat brain: relevance to neuroendocrine regulation.

Recent studies suggest an important role for transient receptor potential vanilloid (TRPV) ion channels in neural and neuroendocrine regulation. The TRPV subfamily consists of six members: TRPV1-6. While the neuroanatomical and functional correlates of TRPV1-4 have been studied extensively, relevant information about TRPV5 and TRPV6, which are highly selective for Ca2+ , is limited. We detected TRPV5 mRNA expression in the olfactory bulb, cortex, hypothalamus, hippocampus, midbrain, brainstem and cerebellum of the rat. TRPV5-immunoreactive neurones were conspicuously seen in the hypothalamic paraventricular (PVN), supraoptic (SON), accessory neurosecretory (ANS), supraoptic nucleus, retrochiasmatic part (SOR), arcuate (ARC) and medial tuberal nuclei, hippocampus, midbrain, brainstem and cerebellum. Glial cells also showed TRPV5-immunoreactivity. To test the neuroendocrine relevance of TRPV5, we focused on vasopressin, oxytocin and cocaine- and amphetamine-regulated transcript (CART) as representative candidate markers with which TRPV5 may co-exist. In the hypothalamic neurones, co-expression of TRPV5 was observed with vasopressin (PVN: 50.73±3.82%; SON: 75.91±2.34%; ANS: 49.12±4.28%; SOR: 100%) and oxytocin (PVN: 6.88±1.21; SON: 63.34±5.69%; ANS: 20.4±4.14; SOR: 86.5±1.74%). While ARC neurones express oestrogen receptors, 17ß-oestradiol regulates TRPV5, as well as CART neurones and astrocytes, in the ARC. Furthermore, ARC CART neurones are known to project to the preoptic area, and innervate and regulate GnRH neurones. Using double-immunofluorescence, glial fibrillary acidic protein-labelled astrocytes and the majority of CART neurones in the ARC showed TRPV5-immunoreactivity. Following iontophoresis of retrograde neuronal tracer, cholera toxin ß (CtB) into the anteroventral periventricular nucleus and median preoptic nucleus, retrograde accumulation of CtB was observed in most TRPV5-equipped ARC CART neurones. Next, we determined the response of TRPV5-elements in the ARC during the oestrous cycle. Compared to pro-oestrus, a significant increase (P<.001) in the percentage of TRPV5-expressing CART neurones was observed during oestrus, metoestrus, and dioestrus. TRPV5-immunoreactivity in the astrocytes, however, showed a significant increase during metoestrus and dioestrus. We suggest that the TRPV5 ion channel may serve as an important regulator of neural and neuroendocrine pathways in the brain.

Oestradiol effects on neuroendocrine responses induced by water deprivation in rats.

Water deprivation (WD) induces changes in plasma volume and osmolality, which in turn activate several responses, including thirst, the activation of the renin-angiotensin system (RAS) and vasopressin (AVP) and oxytocin (OT) secretion. These systems seem to be influenced by oestradiol, as evidenced by the expression of its receptor in brain areas that control fluid balance. Thus, we investigated the effects of oestradiol treatment on behavioural and neuroendocrine changes of ovariectomized rats in response to WD. We observed that in response to WD, oestradiol treatment attenuated water intake, plasma osmolality and haematocrit but did not change urinary volume or osmolality. Moreover, oestradiol potentiated WD-induced AVP secretion, but did not alter the plasma OT or angiotensin II (Ang II) concentrations. Immunohistochemical data showed that oestradiol potentiated vasopressinergic neuronal activation in the lateral magnocellular PVN (PaLM) and supraoptic (SON) nuclei but did not induce further changes in Fos expression in the median preoptic nucleus (MnPO) or subfornical organ (SFO) or in oxytocinergic neuronal activation in the SON and PVN of WD rats. Regarding mRNA expression, oestradiol increased OT mRNA expression in the SON and PVN under basal conditions and after WD, but did not induce additional changes in the mRNA expression for AVP in the SON or PVN. It also did not affect the mRNA expression of RAS components in the PVN. In conclusion, our results show that oestradiol acts mainly on the vasopressinergic system in response to WD, potentiating vasopressinergic neuronal activation and AVP secretion without altering AVP mRNA expression.

Development of a High-Sensitivity Quantitation Method for Arginine Vasopressin by High-Performance Liquid Chromatography Tandem Mass Spectrometry, and Comparison with Quantitative Values by Radioimmunoassay.

Human plasma arginine vasopressin (AVP) levels serve as a clinically relevant marker of diabetes and related syndromes. We developed a highly sensitive method for measuring human plasma AVP using high-performance liquid chromatography tandem mass spectrometry. AVP was extracted from human plasma using a weak-cation solid-phase extraction plate, and separated on a wide-bore octadecyl reverse-phase column. AVP was quantified in ion-transition experiments utilizing a product ion (m/z 328.3) derived from its parent ion (m/z 542.8). The sensitivity was enhanced using 0.02% dichloromethane as a mobile-phase additive. The lower limit of quantitation was 0.200 pmol/L. The extraction recovery ranged from 70.2 ± 7.2 to 73.3 ± 6.2% (mean ± SD), and the matrix effect ranged from 1.1 - 1.9%. Quality-testing samples revealed interday/intraday accuracy and precision ranging over 0.9 - 3% and -0.3 - 2%, respectively, which included the endogenous baseline. Our results correlated well with radioimmunoassay results using 22 human volunteer plasma samples.

Profiling of adrenocorticotropic hormone and arginine vasopressin in human pituitary gland and tumor thin tissue sections using droplet-based liquid-microjunction surface-sampling-HPLC-ESI-MS-MS.

Described here are the results from the profiling of the proteins arginine vasopressin (AVP) and adrenocorticotropic hormone (ACTH) from normal human pituitary gland and pituitary adenoma tissue sections, using a fully automated droplet-based liquid-microjunction surface-sampling-HPLC-ESI-MS-MS system for spatially resolved sampling, HPLC separation, and mass spectrometric detection. Excellent correlation was found between the protein distribution data obtained with this method and data obtained with matrix-assisted laser desorption/ionization (MALDI) chemical imaging analyses of serial sections of the same tissue. The protein distributions correlated with the visible anatomic pattern of the pituitary gland. AVP was most abundant in the posterior pituitary gland region (neurohypophysis), and ATCH was dominant in the anterior pituitary gland region (adenohypophysis). The relative amounts of AVP and ACTH sampled from a series of ACTH-secreting and non-secreting pituitary adenomas correlated with histopathological evaluation. ACTH was readily detected at significantly higher levels in regions of ACTH-secreting adenomas and in normal anterior adenohypophysis compared with non-secreting adenoma and neurohypophysis. AVP was mostly detected in normal neurohypophysis, as expected. This work reveals that a fully automated droplet-based liquid-microjunction surface-sampling system coupled to HPLC-ESI-MS-MS can be readily used for spatially resolved sampling, separation, detection, and semi-quantitation of physiologically-relevant peptide and protein hormones, including AVP and ACTH, directly from human tissue. In addition, the relative simplicity, rapidity, and specificity of this method support the potential of this basic technology, with further advancement, for assisting surgical decision-making. Graphical Abstract Mass spectrometry based profiling of hormones in human pituitary gland and tumor thin tissue sections.

The effect of urocortin I on the hypothalamic ACTH secretagogues and its impact on the hypothalamic-pituitary-adrenal axis.

Urocortin I (UCN I) is a structural analogue of corticotropin-releasing factor (CRF), which, together with arginine-vasopressin (AVP), are the principle adrenocorticotropic hormone (ACTH) secretagogues in mammals. The aim of the present study was to investigate the effects of UCN I on the hypothalamic CRF and AVP concentration and its impact on the hypothalamic-pituitary-adrenal (HPA) axis. First, male Wistar rats were injected intracerebroventricularly (ICV) with 0.5, 1, 2 and 5 µg of UCN I. After 30 min hypothalamic CRF and AVP concentrations were determined by immunoassays. In parallel, the trunk blood was collected and plasma ACTH and corticosterone concentration was determined by ELISA and chemofluorescent assay, respectively. Second, rats were pretreated ICV with selective antagonists of receptors being implicated in the regulation of the HPA axis (0.1 µg antalarmin for CRFR1, 1 µg astressin 2B for CRFR2 or 0.1 µg deamino-Pen1,Tyr2,Arg8-vasopressin for AVPR3) and treated ICV with the most effective dose of UCN I (5 µg). After 30 min plasma corticosterone concentration was determined by chemofluorescent assay. UCN I induced dose-dependent augmentation of the hypothalamic CRF and AVP concentration, associated with dose-dependent elevation of the plasma ACTH and corticosterone concentration. The most significant effect of UCN I on the plasma corticosterone concentration was inhibited by antalarmin, but was not influenced by astressin 2B or deamino-Pen1,Tyr2,Arg8-vasopressin. The present study demonstrates that UCN I modulates the concentration of the hypothalamic ACTH secretagogues in parallel with the concentration of the plasma ACTH and corticosterone. Our results suggest that UCN I may activate the HPA axis by stimulation of the hypothalamic CRF production, and this process is mediated by CRFR1, and not by CRFR2. UCN I may stimulate the AVP production, as well, but, based on the results with AVPR3 antagonist, this effect is not involved in the regulation of the HPA axis.

[Biomarkers of cardiorenal syndrome]. / Biomarqueurs du syndrome cardiorénal.

Complex interactions existing between cardiac and renal diseases led to define 5 types of so-called cardiorenal syndromes. This classification is based on the organ primarily involved and the acute or chronic failure. The mutual impact of renal and cardiac functions makes it difficult to evaluate and manage patients with cardiorenal syndromes and worsen morbidity and mortality. This review seeks to discuss the place of biomarkers in diagnosis, management and follow-up of patients with cardiorenal syndromes. Biomarkers can be classified as functional (creatinine, cystatin C…) or lesional (neutrophil gelatinase-associated lipocalin, urinary cystatin C…) renal markers and functional (natriuretic peptides…) or lesional (troponin, fatty acid binding protein) cardiac markers. A last kind of biomarkers reflects the dialogue between heart and kidney (renin-angiotensin-aldosteron-system, indicators of activation of arginine vasopressin system) or the systemic impact (inflammation, oxidative stress…). In order to evaluate accurately the complex interactions that are the basis of cardiorenal syndromes, a multi-marker approach seems nowadays necessary.

Synaptic innervation to rat hippocampus by vasopressin-immuno-positive fibres from the hypothalamic supraoptic and paraventricular nuclei.

The neuropeptide arginine vasopressin (AVP) exerts a modulatory role on hippocampal excitability through vasopressin V(1A) and V(1B) receptors. However, the origin and mode of termination of the AVP innervation of the hippocampus remain unknown. We have used light and electron microscopy to trace the origin, distribution and synaptic relationships of AVP-immuno-positive fibres and nerve terminals in the rat hippocampus. Immuno-positive fibres were present in all areas (CA1-3, dentate gyrus) of the whole septo-temporal extent of the hippocampus; they had the highest density in the CA2 region, strongly increasing in density towards the ventral hippocampus. Two types of fibres were identified, both establishing synaptic junctions. Type A had large varicosities packed with immuno-positive large-granulated peptidergic vesicles and few small clear vesicles forming type I synaptic junctions with pyramidal neuron dendrites, dendritic spines and with axonal spines. Type B had smaller varicosities containing mostly small clear vesicles and only a few large-granulated vesicles and established type II synaptic junctions mainly with interneuron dendrites. The AVP-positive axons in stratum oriens appeared to follow and contact metabotropic glutamate receptor 1α (mGluR1α)-immuno-positive interneuron dendrites. Fluoro-Gold injection into the hippocampus revealed retrogradely labelled AVP-positive somata in hypothalamic supraoptic and paraventricular nuclei. Hypothalamo-hippocampal AVP-positive axons entered the hippocampus mostly through a ventral route, also innervating the amygdala and to a lesser extent through the dorsal fimbria fornix, in continuation of the septal AVP innervation. Thus, it appears the AVP-containing neurons of the magnocellular hypothalamic nuclei serve as important sources for hippocampal AVP innervation, although the AVP-expressing neurons located in amygdala and bed nucleus of the stria terminalis reported previously may also contribute.

Sexually dimorphic effects of melatonin on brain arginine vasotocin immunoreactivity in green treefrogs (Hyla cinerea).

Arginine vasotocin (AVT) and its mammalian homologue, arginine vasopressin (AVP), regulate a variety of social and reproductive behaviors, often with complex species-, sex- and context-dependent effects. Despite extensive evidence documenting seasonal variation in brain AVT/AVP, relatively few studies have investigated the environmental and/or hormonal factors mediating these seasonal changes. In the present study, we investigated whether the pineal hormone melatonin alters brain AVT immunoreactivity in green treefrogs (Hyla cinerea). Reproductively active male and female frogs were collected during the summer breeding season and a melatonin-filled or blank silastic capsule was surgically implanted subcutaneously. The duration of hormone treatment was 4 weeks, at which time frogs were eutha-nized and the brains and blood collected and processed for AVT immunohistochemistry and steroid hormone assay. We quantified AVT-immunoreactive (AVT-ir) cell bodies in the nucleus accumbens (NAcc), caudal striatum and amygda- la (AMG), anterior preoptic area, suprachiasmatic nucleus (SCN) and infundibular region of the ventral hypothalamus. Sex differences in AVT-ir cell number were observed in all brain regions except in the anterior preoptic area and ventral hypothalamus, with males having more AVT-ir cells than females in the NAcc, amygdala and SCN. Brain AVT was sensitive to melatonin signaling during the breeding season, and the effects of melatonin varied significantly with both region and sex. Treatment with melatonin decreased AVT immunoreactivity in both the NAcc and SCN in male H. cinerea. In contrast, brain AVT was relatively insensitive to melatonin signaling in females, indicating that the regulation of the AVT/AVP neuropeptide system by melatonin may be sexually dimorphic. Finally, melatonin did not significantly influence testosterone or estradiol concentrations of male or female frogs, respectively, suggesting that the effects of melatonin on AVT immunoreactivity are independent of changes in gonadal sex steroid hormones. Collectively, our results indicate that the AVT/AVP neuronal system may be an important target for melatonin in facilitating seasonal changes in reproductive physiology and social behavior.

Simultaneous oxytocin and arg-vasopressin measurements in microdialysates using capillary liquid chromatography-mass spectrometry.

Oxytocin (OXT) and arg-vasopressin (AVP) are nonapeptides with many important functions both peripherally and centrally. Intracerebral microdialysis has helped characterize their importance in regulating complex social and emotional processes. Radioiummunoassay is the most commonly used analytical method used for OXT and AVP measurements in microdialysates. These measurements have several well-known issues including single peptide per assay limit, possible cross-reactivity between structurally related peptides, and laborious sample preparation with radioactive materials. Here we demonstrate the use of capillary LC-MS(3) for measuring OXT and AVP simultaneously in dialysates at a 10 min sampling frequency. Microdialysate samples required no preparation and instrumentation was commercially available. Microdialysis probes made with polyacrylonitrile membranes were suitable for high level recovery of the peptides in vitro and in vivo. Responses were linear from 1 to 100 pM. Matrix effect was assessed by standard addition experiments and by comparing signal intensities of OXT and AVP standards made in aCSF or dialysate. It was determined that the online washing step used on this setup was adequate for removing contaminants which interfere with electrospray ionization efficiency. In vivo, both peptides were stimulated by high K(+) (75 mM) aCSF perfusion in the paraventricular nucleus (PVN). Also, a systemic injection of high Na(+) (2M) caused a rapid and transient increase in PVN OXT while AVP increased only after 1.5h. Our findings suggest that capillary LC-MS(3) is a straightforward method for monitoring OXT and AVP simultaneously from complex samples such as dialysates.

Enantioselective and label-free detection of oligopeptide via fluorescent indicator displacement.

In this work, a simple and label-free fluorescent method via fluorescent indicator displacement (FID) was proposed for enantioselectively determining d-enantiomer of arginine vasopressin (DV) using DV-specific DNA aptamer (V-apt) and one guanidiniophthalocyanine dye (Zn-DIGP). Zn-DIGP that preferentially binds to single-stranded DNA with fluorescence enhancement rather than duplexes occupies the long internal loop of V-apt and generates intensive fluorescence. Then DV is introduced into the solution containing Zn-DIGP and V-apt, and displaces the Zn-DIGP from the binding site of internal loop, leading to fluorescence decrease. But l-enantiomer cannot induce any fluorescence change due to the selectivity of V-apt. This established FID technique can detect DV with a detection limit of 100 nM and exhibits a broad linear range, and is able to discriminate enantiomers of arginine vasopressin unambiguously. Moreover chiral separation by chromatography, complicated experimental procedures and covalent modification of tags (such as organic dyes, redox-active metal complexes) are avoided in our strategy. This simple and label-free method is promising for fabricating diverse aptasensors to determine other biomolecules and drugs.