Nociceptin is present in synovial fluid of patients undergoing total knee arthroplasty.

BACKGROUND: Osteoarthritis is a mechanical abnormality characterized by chronic joint pain associated with degeneration of the articular cartilage, synovitis, and local inflammation, leading to loss of function and pain. A connection exists between the peripheral nervous system and inflammatory joint degeneration. The process by which inflammation is influenced by the nervous system is known as neuroinflammation. One of the neuropeptides involved in peripheral neuroinflammation is nociceptin, a peptide related to the opioid class of substances. Nociceptin has both pro- and anti-inflammatory effects. Some studies show that nociceptin can be measured in synovial fluid, while other studies have not been able to detect it. The presence of nociceptin in synovial fluid could imply a molecular role for the neuropeptide in the joint, both physiologically as well as pathophysiologically. The goal of this pilot study was to determine whether nociceptin was present in the synovial fluid of osteoarthritic knees. METHODS: Patients undergoing primary total knee arthroplasty were enrolled after Institutional Review Board approval was obtained. Synovial fluid was aspirated from patients' operative knee joints and blood samples were obtained. A commercially available enzyme Immunoassay kit was used to test for nociceptin. A linear mixed-effects model was developed to account for the repeated measurements and baseline covariates. Least squares (adjusted) means were derived from the model to compare the sample types and to compare subgroups. RESULTS: Twenty patients were included in this study. Nociceptin was detected in the synovial fluid and plasma of all patients. The mean concentration (± standard deviation) of nociceptin in synovial fluid was 28.7 ± 18.2 pg/ml. The mean concentration of nociceptin in plasma was 45.2 ± 24.3 pg/ml pre-procedure, and 40.1 ± 20.6 pg/ml post-tourniquet deflation. The nociceptin concentration in synovial fluid was significantly lower than the nociceptin concentration in plasma, both pre-procedure and post-tourniquet deflation (p = 0.002 and p = 0.016 respectively). The nociceptin concentration in both plasma and synovial fluid was significantly lower in females versus males (p = 0.012). CONCLUSION: We demonstrated that nociceptin is present in synovial fluid and plasma of patients undergoing total knee arthroplasty. This implies a potential role for nociceptin in modulating inflammation in osteoarthritis. TRIAL REGISTRATION: ClinicalTrials.gov , NCT02528916 . Retrospectively registered on August 19, 2015.

Dynorphin and Enkephalin Opioid Peptides and Transcripts in Spinal Cord and Dorsal Root Ganglion During Peripheral Inflammatory Hyperalgesia and Allodynia.

Understanding molecular alterations associated with peripheral inflammation is a critical factor in selectively controlling acute and persistent pain. The present report employs in situ hybridization of the 2 opioid precursor mRNAs coupled with quantitative measurements of 2 peptides derived from the prodynorphin and proenkephalin precursor proteins: dynorphin A 1-8 and [Met5]-enkephalin-Arg6-Gly7-Leu8. In dorsal spinal cord ipsilateral to the inflammation, dynorphin A 1-8 was elevated after inflammation, and persisted as long as the inflammation was sustained. Qualitative identification by high performance liquid chromatography and gel permeation chromatography revealed the major immunoreactive species in control and inflamed extracts to be dynorphin A 1-8. In situ hybridization in spinal cord after administration of the inflammatory agent, carrageenan, showed increased expression of prodynorphin (Pdyn) mRNA somatotopically in medial superficial dorsal horn neurons. The fold increase in preproenkephalin mRNA (Penk) was comparatively lower, although the basal expression is substantially higher than Pdyn. While Pdyn is not expressed in the dorsal root ganglion (DRG) in basal conditions, it can be induced by nerve injury, but not by inflammation alone. A bioinformatic meta-analysis of multiple nerve injury datasets confirmed Pdyn upregulation in DRG across different nerve injury models. These data support the idea that activation of endogenous opioids, notably dynorphin, is a dynamic indicator of persistent pain states in spinal cord and of nerve injury in DRG. PERSPECTIVE: This is a systematic, quantitative assessment of dynorphin and enkephalin peptides and mRNA in dorsal spinal cord and DRG neurons in response to peripheral inflammation and axotomy. These studies form the foundational framework for understanding how endogenous spinal opioid peptides are involved in nociceptive circuit modulation.

Development of a novel photoaffinity probe for labeling nocistatin receptor.

Nocistatin (NST) is a neuropeptide produced from the same precursor protein of opioid peptide nociceptin/orphanin FQ, and it is involved in a broad range of central functions including pain transmission in the nervous system. However, the composition and structure of the receptor(s) for NST remain unclear. Here, we developed NST photoaffinity probe to identify NST receptor. The NST photoaffinity probe contains an azide moiety for the tagging of the binding protein as well as biotin for protein detection. Intrathecal administration of a NST photoaffinity probe, biotin-(AC5)2-[Y6,azF14]bNST, inhibited the nociceptin/orphanin FQ-evoked tactile pain allodynia in a manner similar to that of NST. The biotin-(AC5)2-[Y6,azF14]bNST-binding proteins were primarily localized in the gray matter of the spinal cord. After photo-crosslinking of the protein complex with biotin-(AC5)2-[Y6,azF14]bNST, two dominant binding protein bands were observed at 58 and 64 kDa. Thus, biotin-(AC5)2-[Y6,azF14]bNST has pharmacological activity and is useful for characterizing the NST receptor.

All-Electronic Quantification of Neuropeptide-Receptor Interaction Using a Bias-Free Functionalized Graphene Microelectrode.

Opioid neuropeptides play a significant role in pain perception, appetite regulation, sleep, memory, and learning. Advances in understanding of opioid peptide physiology are held back by the lack of methodologies for real-time quantification of affinities and kinetics of the opioid neuropeptide-receptor interaction at levels typical of endogenous secretion (<50 pM) in biosolutions with physiological ionic strength. To address this challenge, we developed all-electronic opioid-neuropeptide biosensors based on graphene microelectrodes functionalized with a computationally redesigned water-soluble µ-opioid receptor. We used the functionalized microelectrode in a bias-free charge measurement configuration to measure the binding kinetics and equilibrium binding properties of the engineered receptor with [d-Ala2, N-MePhe4, Gly-ol]-enkephalin and ß-endorphin at picomolar levels in real time.

Opioid peptides and gastrointestinal symptoms in autism spectrum disorders

Autism spectrum disorders (ASDs) are characterized by deficits in the individual’s ability to socialize, communicate, and use the imagination, in addition to stereotyped behaviors. These disorders have a heterogenous phenotype, both in relation to symptoms and regarding severity. Organic problems related to the gastrointestinal tract are often associated with ASD, including dysbiosis, inflammatory bowel disease, exocrine pancreatic insufficiency, celiac disease, indigestion, malabsorption, food intolerance, and food allergies, leading to vitamin deficiencies and malnutrition. In an attempt to explain the pathophysiology involved in autism, a theory founded on opioid excess has been the focus of various investigations, since it partially explains the symptomatology of the disorder. Another hypothesis has been put forward whereby the probable triggers of ASDs would be related to the presence of bacteria in the bowel, oxidative stress, and intestinal permeability. The present update reviews these hypotheses.

Opioid peptides and gastrointestinal symptoms in autism spectrum disorders.

Autism spectrum disorders (ASDs) are characterized by deficits in the individual's ability to socialize, communicate, and use the imagination, in addition to stereotyped behaviors. These disorders have a heterogenous phenotype, both in relation to symptoms and regarding severity. Organic problems related to the gastrointestinal tract are often associated with ASD, including dysbiosis, inflammatory bowel disease, exocrine pancreatic insufficiency, celiac disease, indigestion, malabsorption, food intolerance, and food allergies, leading to vitamin deficiencies and malnutrition. In an attempt to explain the pathophysiology involved in autism, a theory founded on opioid excess has been the focus of various investigations, since it partially explains the symptomatology of the disorder. Another hypothesis has been put forward whereby the probable triggers of ASDs would be related to the presence of bacteria in the bowel, oxidative stress, and intestinal permeability. The present update reviews these hypotheses.

Food Proteins as Source of Opioid Peptides-A Review.

Traditional opioids, mainly alkaloids, have been used in the clinical management of pain for a number of years but are often associated with numerous side-effects including sedation, dizziness, physical dependence, tolerance, addiction, nausea, vomiting, constipation and respiratory depression which prevent their effective use. Opioid peptides derived from food provide significant advantages as safe and natural alternative due to the possibility of their production using animal and plant proteins as well as comparatively less side-effects. This review aims to discuss the current literature on food-derived opioid peptides focusing on their production, methods of detection, isolation and purification. The need for screening more dietary proteins as a source of novel opioid peptides is emphasized in order to fully understand their potential in pain management either as a drug or as part of diet complementing therapeutic prescription.

Behavioral effects of food-derived opioid-like peptides in rodents: Implications for schizophrenia?

Dohan proposed that an overload of dietary peptides, such as those derived from wheat gluten and milk casein, could be a factor relevant to the development or maintenance of schizophrenia (SZ) symptoms in at least a subset of vulnerable individuals. Rodent behavioral models may offer insight into the plausibility of Dohan's exorphin hypothesis by providing a means to directly study the effects of such peptides. Accordingly, a review of the literature on the behavioral effects of food-derived opioid-like peptides in rodents was undertaken. Studies using a variety of behavioral tests to examine the effects of several classes of food-derived opioid-like peptides were identified and reviewed. Peptides derived from casein (ß-casomorphins; BCMs, n=19), spinach (rubiscolins; RCs, n=4), and soy (soymorphins; SMs, n=1) were behaviorally active in various paradigms assessing nociception, spontaneous behavior, and memory. Surprisingly, only a single study evaluating a gluten-derived peptide (gliadorphin-7; GD-7, n=1) was identified and included in this review. In conclusion, food-derived peptides can affect rodent behavior, but more studies of GDs using diverse behavioral batteries are warranted. Assuming they occur in sufficient quantities during protein digestion and can access central opioid receptors (which entails crossing both the gastrointestinal and blood-brain barriers intact), these peptides may affect human behavior. Although BCMs and GDs may not be directly pathogenic in SZ, documented associations of casein and gluten sensitivity with SZ justify increased patient screening and dietary intervention where necessary.

Imaging endogenous opioid peptide release with [11C]carfentanil and [3H]diprenorphine: influence of agonist-induced internalization.

Understanding the cellular processes underpinning the changes in binding observed during positron emission tomography neurotransmitter release studies may aid translation of these methodologies to other neurotransmitter systems. We compared the sensitivities of opioid receptor radioligands, carfentanil, and diprenorphine, to amphetamine-induced endogenous opioid peptide (EOP) release and methadone administration in the rat. We also investigated whether agonist-induced internalization was involved in reductions in observed binding using subcellular fractionation and confocal microscopy. After radioligand administration, significant reductions in [(11)C]carfentanil, but not [(3)H]diprenorphine, uptake were observed after methadone and amphetamine pretreatment. Subcellular fractionation and in vitro radioligand binding studies showed that amphetamine pretreatment only decreased total [(11)C]carfentanil binding. In vitro saturation binding studies conducted in buffers representative of the internalization pathway suggested that µ-receptors are significantly less able to bind the radioligands in endosomal compared with extracellular compartments. Finally, a significant increase in µ-receptor-early endosome co-localization in the hypothalamus was observed after amphetamine and methadone treatment using double-labeling confocal microscopy, with no changes in δ- or κ-receptor co-localization. These data indicate carfentanil may be superior to diprenorphine when imaging EOP release in vivo, and that alterations in the ability to bind internalized receptors may be a predictor of ligand sensitivity to endogenous neurotransmitter release.

[Analytical validation of a chromatographic method dedicated to search and identify natural and semi-synthetic opiates]. / Validation analytique d'une méthode chromatographique destinée à rechercher et à identifier les opiacés naturels ou (semi) synthétiques.

The identification of a product absorbed by an opiate consumer is sometimes problematic since there is no specific biomarker for all molecules. We developed an ultra-high pressure liquid chromatography coupled to tandem mass spectrometry technique which allows the identification and the quantification of 25 opiates in plasma. The sample preparation consists in a solid-phase extraction on Oasis MCX cartridges (Waters). The method has been validated according to FDA criteria completely for 21 substances and with some reservations for the remaining 4 analytes. This method has been applied to 80 patients treated at the University Hospital of Liege for whom the screening of opiates was positive. The identification of the product consumed was effective in 86% of cases.

Expression of substance P, calcitonin gene-related peptide, ß-endorphin and methionine-enkephalin in human dental pulp tissue after orthodontic intrusion: a pilot study.

OBJECTIVE: To determine the levels of two sensory neuropeptides (substance P [SP] and calcitonin gene-related peptide [CGRP]) and two endogenous opioids (methionine-enkephalin [Met-Enk] and ß-endorphin [ß-End]) in dental pulp tissue samples subjected to controlled orthodontic intrusive forces. MATERIALS AND METHODS: Sixteen healthy premolars were selected from eight patients who were undergoing extraction for orthodontic purposes. Eight were randomly used as controls, and the other eight were assigned to an experimental group (controlled orthodontic intrusive forces applied for 24 hours). After this period, teeth were extracted, and pulp samples were obtained. All samples were processed to quantify the expression levels of SP, CGRP, Met-Enk, and ß-End using commercial radioimmunoassay kits. RESULTS: All samples exhibited basal levels of both neuropeptides and endogenous opioids. After 24 hours of the intrusive stimulus, all patients reported a tolerable discomfort localized at the involved premolar. Only SP was significantly increased (P<.05). For the other molecules, no statistically significant differences were observed (P>.05); however, they expressed important increasing trends. CONCLUSIONS: The expression levels of SP and CGRP in dental pulp samples from the experimental group support the positive correlation between the symptomatic clinical scenario and increased expression levels of neuropeptides, clarifying the role of neurogenic inflammation in early injury response.

Detection and quantification of dermorphin and selected analogs in equine urine.

BACKGROUND: Dermorphin, a hepta-peptide with potent analgesic properties, is classified as a doping agent in equine racing. Since its discovery, a number of biologically active structural analogs have been synthesized and made commercially available so there is a need for reliable methods of detection. METHODOLOGY/RESULTS: A sensitive detection method was developed for dermorphin and six analogs in equine urine. Peptide enrichment was achieved using weak cation exchange with subsequent separation and detection by nano-UHPLC-MS/MS. Method validation parameters included: specificity, linearity (5-10000 pg/ml), recovery (58-93%), intra and inter-assay repeatability, LOD (5-50 pg/ml) and matrix effects. CONCLUSION: The presented method will facilitate the control of the abuse of dermorphin and selected analogs in equine sports.

Preparation and evaluation of an immunoaffinity sorbent with Fab' antibody fragments for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis-mass spectrometry.

An immunoaffinity (IA) sorbent with antibody fragments was prepared for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis-mass spectrometry (IA-SPE-CE-MS). The antibody fragmentation was evaluated by MALDI-TOF-MS. Fab' fragments obtained from a polyclonal IgG antibody against Endomorphins 1 and 2 (End1 and End2) were covalently attached to succinimidyl silica particles to prepare the IA sorbent. An IA-SPE-CE-MS methodology was established analyzing standard solutions of End1 and End2 and acceptable repeatability, linearity ranges and LODs (0.5 and 5 ng mL(-1), respectively) were obtained. The LOD of End1 was slightly better than that previously obtained using an IA sorbent with intact antibodies (1 ng mL(-1)). In human plasma samples, End1 and End2 could be detected at 1 and 50 ng mL(-1), respectively, which meant an improvement of 100 and 2-fold with regard to the LODs using an IA sorbent with intact antibodies (100 ng mL(-1)).

Detection, quantification, and identification of dermorphin in equine plasma and urine by LC-MS/MS for doping control.

Dermorphin is a unique opioid peptide that is 30-40 times more potent than morphine. It was misused and went undetected in horse racing until 2011 when intelligence obtained from a few North American race tracks suggested its use. To prevent such misuse, a reliable analytical method became necessary for detection and identification of dermorphin in post-race horse samples. This paper describes the first liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for such a purpose. Equine plasma and urine samples were pre-treated with ethylenediamine tetra-acetic acid and urea prior to solid-phase extraction (SPE) on Oasis MCX cartridges. Resulting eluates were dried under vacuum and analyzed by LC-MS/MS for dermorphin. The matrix effect, SPE efficiency, intra-day and inter-day accuracy and precision, and stability of the analyte were assessed. The limit of detection was 10 pg/mL in plasma and 20 pg/mL in urine, and the limit of confirmation was 20 pg/mL in plasma and 50 pg/mL in urine. Dermorphin in plasma is stable at ambient temperature, but its diastereomer is unstable. With isotopically labeled dermorphin as an internal standard, the quantification range was 20-10,000 pg/mL in plasma and 50-20,000 pg/mL in urine. The intra-day and inter-day accuracy was from 91 % to 100 % for the low, intermediate, and high concentrations. The intra-day and inter-day coefficients of variation were less than 12 %. The method differentiates dermorphin from its diastereomer. This method is very specific for identification of dermorphin in equine plasma and urine, as assessed by BLAST search and targeted SEQUEST search, and by MS/MS spectrum library search. The method has been successfully applied to analysis of samples collected following dermorphin administration to research horses and of official post-race samples.

Early in life bladder inflammation alters opioid peptide content in the spinal cord and bladder of adult female rats.

PURPOSE: Previous research suggests that a failure of opioid inhibition may contribute to chronic bladder pain. We determined how acute adult and/or prior early in life exposure to bladder inflammation alters the adult content of endogenous opioid peptides in the bladder, spinal cord and blood. MATERIALS AND METHODS: Inflammation was induced by intravesical administration of zymosan. Female Sprague-Dawley® rats were exposed to anesthesia only or zymosan early in life (postnatal days 14 to 16) and anesthesia only or zymosan as adults (ages 12 to 17 weeks). Thoracolumbar and lumbosacral segments of the spinal cord, and blood and bladders were collected 24 hours after adult treatment. Opioid peptide content was measured using enzyme-linked immunosorbent assay. RESULTS: Early in life bladder inflammation alone produced a chronic increase in dynorphin A (1-17) in the lumbosacral spinal cord. When early in life inflammation was followed by adult re-inflammation, spinal cord dynorphin remained unchanged but bladder dynorphin was decreased. In addition, early in life inflammation combined with adult bladder inflammation decreased endomorphin-2 content in the thoracolumbar spinal cord. Neither early in life nor adult bladder inflammation affected thoracolumbar dynorphin, serum dynorphin, lumbosacral endomorphin-2 or plasma ß-endorphin. CONCLUSIONS: Several opioid peptides were measured using enzyme-linked immunosorbent assay following early in life and adult bladder inflammation. The changes observed are consistent with the view that early in life bladder inflammation alone can chronically alter spinal cord peptide content. When coupled with adult re-inflammation, these changes could set the neurochemical stage to support bladder hypersensitivity.

Preparation and evaluation of an immunoaffinity sorbent for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis-mass spectrometry.

In this study, we explored a procedure for the preparation of an immunoaffinity (IA) sorbent for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis-mass spectrometry (IA-SPE-CE-MS). We followed a site-specific antibody immobilization approach based on the covalent attachment of the oxidized antibodies through their carbohydrate moieties to hydrazide silica particles, using a polyclonal antibody against Endomorphin 1 and 2 (End1 and End2). The main features of the IA sorbent were studied, such as the amount of hydrazide groups and antibodies attached onto oxidized diol silica particles. Once the procedure was optimized, standard solutions of End1 and End2 were used in order to establish the IA-SPE-CE-MS methodology. Acceptable repeatability, reproducibility and linearity range values were obtained for the proposed methodology. The limits of detection (LODs) of 1 ng mL(-1) were approximately 100-fold better than those obtained by CE-MS. Selectivity of the IA sorbent was good but some cross-reactivity against Dynorphin A (1-7) was observed when a mixture of several opioid peptides was analyzed. Human plasma samples spiked with End1 and End2 were also analyzed and both peptides could be detected down to 100 ng mL(-1).

Quantitative study of [Tyr10]nociceptin/orphanin FQ (1-11) at NOP receptors in rat periaqueductal gray and expressed NOP receptors in HEK293 cells.

AIM: The nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor was reported to be functionally heterogeneous. We investigated if [Tyr(10)]N/OFQ(1-11), a peptide ligand reported to selectively bind to the high affinity site of (125)I-[Tyr(14)]N/OFQ in rodent brains, can be a tool for revealing the NOP receptor heterogeneity. We have previously founded an NOP receptor subset insensitive to Ro 64-6198 and (+)-5a Compound, two non-peptide NOP agonists, in rat ventrolateral periaqueductal gray (vlPAG) neurons. Here, we examined if [Tyr(10)]N/OFQ(1-11) differentiated (+)-5a Compound-sensitive and -insensitive vlPAG neurons. Certain mu-opioid (MOP) receptor ligands highly competing with [Tyr(10)]N/OFQ(1-11) in binding studies also showed high affinity at expressed heteromeric NOP-MOP receptors. We also examined if [Tyr(10)]N/OFQ(1-11) distinguished heteromeric NOP-MOP receptors from homomeric NOP receptors. MAIN METHODS: The NOP receptor activity was evaluated by G-protein coupled inwardly rectifying potassium (GIRK) currents in rat vlPAG slices, and by inhibition of cAMP accumulation in HEK293 cells expressing NOP receptors or co-expressing NOP and MOP receptors. KEY FINDINGS: In vlPAG neurons, [Tyr(10)]N/OFQ(1-11), like N/OFQ, induced GIRK currents through NOP receptors. It was less potent (EC(50): 8.98µM) but equi-efficacious as N/OFQ. [Tyr(10)]N/OFQ(1-11) displayed different pharmacological profiles as (+)-5a Compound, and was effective in both (+)-5a Compound-sensitive and -insensitive neurons. In NOP-expressing HEK293 cells and NOP- and MOP-co-expressing cells, [Tyr(10)]N/OFQ(1-11) displayed similar concentration-response curves in decreasing cAMP accumulation. SIGNIFICANCE: [Tyr(10)]N/OFQ(1-11) is an NOP full agonist and less potent than N/OFQ. However, it can neither reveal the functional heterogeneity of NOP receptors in vlPAG neurons nor differentiate heteromeric NOP-MOP and homomeric NOP receptors.

Innovative management of pruritus.

Pruritus remains a significant problem facing dermatologists and can be associated with various dermatoses and systemic derangements. At times, one can treat the underlying cutaneous or systemic process to alleviate itch. However, it is frequently challenging to identify the cause of a patient's itch and, in this situation, even more difficult to manage the symptom effectively. In this article, the authors discuss the approach to a patient with generalized pruritus without clinically obvious dermatoses. They also addresses mechanisms and management modalities of itch in common systemic diseases, including cholestasis, uremia, and neuropathic dysfunction.

Analysis of opioid and amyloid peptides using time-of-flight secondary ion mass spectrometry.

The imaging capability and high detection sensitivity of time-of-flight secondary ion mass spectrometry (ToF-SIMS) makes it a potentially attractive complement to other mass spectrometry methods, such as ESI and MALDI, for the analysis of proteins and peptides. We have explored this possibility by performing a systematic analysis of synthetic opioid and amyloid peptides with ToF-SIMS using Bi(3)(+) and Au(3)(+) primary ions. In the low mass region of the spectra, a number of single amino acid ion peaks were detected, providing information about the amino acid content in each peptide. In the medium and high mass range of the spectra, peaks corresponding to multiple amino acid ions (backbone cleavage ions) as well as molecular ions were detected, allowing for the determination of the amino acid sequence and the molecular mass of the entire peptide, respectively. Detection efficiencies were determined for the molecular ions of some of the peptides, indicating detection limits in the attomole range. The fragmentation patterns observed in the ToF-SIMS analysis of opioid and amyloid peptides showed interesting similarities with collision-induced dissociation (CID) studies using other mass spectrometry methods. The present work provides important progress toward ToF-SIMS proteomics.

D-amino acid detection in peptides by MALDI-TOF-TOF.

Detection of a D-amino acid residue in natural peptides by mass spectrometry remains a challenging task, as this post-translational modification does not induce any change in molecular mass. To our knowledge, the present article is the first report using matrix-assisted laser desorption/ionization (MALDI) for the discrimination and the quantification of peptide isomers. In this work, we used synthetic hepta- and decapeptides of biological relevance and their isomers. All-L sequences and some isomers containing a D-residue in various positions were analyzed.