ABSTRACT
The determination of amino acids and monoamine with actions like neurotransmitters or modulators has become increasingly important for studying the relationship between the dysfunction of neurotransmitters and the pathogenesis of diabetic encephalopathy. Here, a high-performance liquid chromatography with fluorescence detection method was developed to simultaneously determine nine monoamines and amino acids including three excitatory neurotransmitters (aspartate, glutamate, and serotonin), four inhibitory neurotransmitters (glycine, γ-aminobutyric acid, taurine, dopamine), a precursor of 5-HT (tryptophan) and methionine using homoserine as the internal standard. The separation was performed on a BDS column with methanol-buffer solution of 35 mmol/L sodium acetate and 5 mmol/L citric acid (pH 6.0) using a simple gradient elution. Several parameters including specificity, precision, and recovery were validated after optimization of the analytical conditions. The developed method was successfully applied to determine the cortex and the hippocampus samples from Sprague-Dawley rats. Our results showed that various neurotransmitters involved in diabetes mellitus may tend to be differentially modulated and present a different alteration tendency at different time course, which might be associated with the duration of diabetes mellitus.
Subject(s)
Brain Diseases/metabolism , Chromatography, High Pressure Liquid/methods , Diabetes Complications/metabolism , Excitatory Amino Acids/analysis , Hippocampus/metabolism , Neurotransmitter Agents/analysis , Animals , Limit of Detection , Linear Models , Male , Rats , Rats, Sprague-Dawley , Reproducibility of ResultsABSTRACT
INTRODUCTION: To comprehend the normal function and pathological characteristics of certain neurological disorders it is important to evaluate the neuroactive amino acids levels in animal models. METHODS: This work describes a simple liquid chromatography-fluorescence detection (LC-FLD) method for the simultaneous determination of aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), taurine (Tau) and γ-aminobutyric acid (GABA), using methyl-L-arginine as internal standard, in samples of rat brain tissue. The five target analytes (Asp, Glu, Gln, Tau and GABA) were determined in a single chromatographic run of less 11â¯min after a derivatization step with o-phthalaldehyde. The derivatives were separated on a reversed-phase C18 column and detected by fluorescence at excitation and emission wavelengths of 340 and 448â¯nm, respectively. RESULTS: The method was validated in accordance with international guidelines on bioanalytical methods validation and it presented limits of quantification in the range of 25-50â¯ng mL-1 and calibration curves with determination coefficients (r2) equal to or higher than 0.9920. In addition, the precision (coefficient variation, %) and accuracy (bias, %) of the method meet the established criteria, and the stability of the analytes at the sample handling and storage conditions was demonstrated. DISCUSSION: Unlike other similar bioanalytical assays, the current method was validated using diluted biological matrix, which is advantageous in order to ensure the derivatization process integrity. Moreover, this LC-FLD method was successfully applied for the determination of the compounds of interest in different rat brain tissue regions (frontal cortex, amygdala, hippocampus, cerebellum and striatum). Thus, this bioanalytical assay represents a useful tool to support multiple nonclinical studies in the field of neurosciences, requiring the quantitative profiling and pattern analysis of neuroactive amino acids.
Subject(s)
Brain Chemistry , Chromatography, High Pressure Liquid/methods , Excitatory Amino Acids/analysis , Neurotransmitter Agents/analysis , Animals , Brain/anatomy & histology , Calibration , Feasibility Studies , Fluorescence , Male , Models, Animal , Rats , Rats, Wistar , Reference StandardsABSTRACT
Chiral CE method has been developed for quantitative determination of d-amino acid modulators of NMDA glutamate receptor; d-serine and d-aspartate along with l-glutamate and l-aspartate in biological samples. These ligands are suggested to be involved in regulation of NMDA receptor related brain functions, such as neurogenesis, neuronal plasticity, and memory formation. For sensitive determination of the amino acids LIF detection was chosen, and a fluorogenic reagent, 7-fluoro-4-nitro-2,1,3-benzoxadiazole was used for derivatization. An amino-modified ß-CD, 6-monodeoxy-6-mono(3-hydroxy)propylamino-ß-CD (HPA-ß-CD) was applied as chiral selector. Determinations were accomplished in a polyacrylamide coated capillary and reverse polarity was used for the analysis of the negatively charged analytes. The method was optimized and validated; 6 mM HPA-ß-CD in 50 mM HEPES buffer, pH 7 was appropriate to achieve baseline separation of the analytes. The limit of quantification with acceptable accuracy is 0.05 µM for both d-amino acids. The method was used for the determination of d-aspartate and d-serine content in various brain regions of adult mice.
Subject(s)
Brain Chemistry , Electrophoresis, Capillary/methods , Excitatory Amino Acids/analysis , Excitatory Amino Acids/chemistry , Animals , Linear Models , Male , Mice , Mice, Inbred C57BL , Reproducibility of Results , Sensitivity and Specificity , StereoisomerismABSTRACT
An optimized HPLC/MS/MS method was established to quantify glutamate (Glu) and aspartic acid (Asp) in rat hippocampus with glutamate-d5 (Glu-d5) as internal standard. The mass spectrometry was operated under the multiple reaction monitoring mode using electrospray ionization in the positive ion mode for Glu and negative ion mode for Asp. The retention times of Glu, Asp and Glu-d5 were 1.53, 2.07 and 1.52 min, respectively. The linearity of calibration curves was good, with r(2) > 0.99 and a lower limit of quantitation of 10 ng/mL. The intra-day precisions (relative standard deviation, RSD) of Glu and Asp were in the range of 3.61-8.17 and 4.22-10.09%, respectively; the inter-day precisions (RSD) of Glu and Asp were in the range of 3.57-5.19 and 2.49-5.04%, respectively. The accuracies of Glu and Asp were in the range of -2.10-6.20 and -0.90-10.00%, respectively. The recovery rates of 10, 100 and 1000 ng/mL were found to be 0.89 ± 0.24, 1.01 ± 0.10 and 0.90 ± 0.12 for Glu and 0.99 ± 0.26, 0.93 ± 0.07 and 1.13 ± 0.13 for Asp, respectively. This optimized method was successfully applied to quantify the concentration of Glu and Asp in rat hippocampus in brain ischemia/reperfusion research.
Subject(s)
Brain Ischemia/metabolism , Chromatography, High Pressure Liquid/methods , Excitatory Amino Acids/analysis , Hippocampus/chemistry , Tandem Mass Spectrometry/methods , Animals , Biomedical Research , Drug Stability , Excitatory Amino Acids/chemistry , Excitatory Amino Acids/metabolism , Linear Models , Male , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
INTRODUCTION: Planaria present a unique model organism for studying primitive central nervous systems. The major mammalian excitatory neurotransmitters, glutamate and aspartate, have previously been measured in planaria via high pressure liquid chromatography (HPLC). A faster extraction and analysis procedure using capillary electrophoresis (CE) was developed which confirms the presence of these amino acids in single planaria homogenates. METHOD: Following homogenization and centrifugation of individual planaria in hydrochloric acid/acetonitrile, glutamate and aspartate were derivatized with naphthalene-2, 3-dicarboxaldehyde (NDA). The labeled amino acids were measured using capillary electrophoresis with laser-induced fluorescence (CE-LIF). RESULTS: CE-LIF electropherograms were generated in less than 1 min. The mean ± S.D. amounts of glutamate and aspartate were 1200 ± 500 and 1900 ± 700 pmol/mg-planarian (n=22), respectively. Spiked average recoveries of glutamate and aspartate were 96% and 91%, respectively. DISCUSSION: The high-throughput method provides the ability to quantitate changes in excitatory neurotransmitters under developmental or stimulatory conditions. The capability to monitor multiple neurotransmitter levels offers the opportunity to correlate behavioral responses with biochemical changes in planaria.
Subject(s)
Aspartic Acid/analysis , Electrophoresis, Capillary/methods , Excitatory Amino Acids/analysis , Glutamic Acid/analysis , Neurotransmitter Agents/analysis , Planarians/chemistry , Animals , Calibration , Central Nervous System/metabolism , Models, AnimalABSTRACT
Memory performance, brain excitatory amino acid and acetylcholinesterase activity of chronically aluminum (Al) exposed mice in response to soy isoflavones (SI) treatment was investigated in the study. Forty eight mice were allotted randomly into a control group, an Al exposed group (100 mg/kg Al) and an Al exposed group treated with SI (100 mg/kg Al + 60 mg/kg SI) for 60 days. Chronic Al exposure significantly impaired long memory performance in mice as assessed using a passive avoidance task test (χ(2) analysis, p < 0.05). Interestingly, SI treatment markedly improved the memory performance score in the Al exposed mice. This improvement was associated with a total reversal of Al-induced increases in acetylcholinesterase activity in the cerebral cortex and hippocampus of mice. The Al exposure also led to significant decreases in brain levels of aspartic and glutamic acids, two excitatory amino acid neurotransmitters; whereas SI treatment partially reversed the decreased aspartic and glutamic acid contents in the hippocampus. The results suggest that SI can improve long memory performance in the Al exposed mice, possibly by modulating the metabolism of brain acetylcholine and amino acid neurotransmitters.
Subject(s)
Acetylcholinesterase/analysis , Aluminum/adverse effects , Brain/metabolism , Excitatory Amino Acids/analysis , Isoflavones/pharmacology , Memory/drug effects , Animals , Avoidance Learning/drug effects , Male , Mice , Glycine max/chemistryABSTRACT
OBJECTIVES: Some individually-housed male mice behave aggressively during encounters with strange males, while others are timid or sociable in the same situation. The objective of the present study was to examine concentrations of glutamate, aspartate, and GABA in the brain of aggressive, timid, and sociable mice. METHODS: Random-bred albino mice were housed individually for three weeks and then classified in three groups (aggressive, timid, and sociable mice) according to their behavior during social interaction with non-aggressive group-housed male mice in a neutral cage. One week after categorization, by means of the social conflict test, levels of glutamate, aspartate, and GABA were measured by in vivo microdialysis of the medial prefrontal cortex (mPFC) of the isolated and group-housed mice. RESULTS: Sociable mice had almost triple the levels of GABA in their mPFC than aggressive or timid mice. No significant differences in aspartate and glutamate levels were found in these three types of individually-housed mice. Forebrain chemistry of group-housed mice did not differ from that of individually-housed mice with the exception of levels of glutamate and GABA which were significantly lower in group-housed mice than in sociable individually-housed mice. CONCLUSION: The present results suggest that GABA might play a role in sociable behavior. Results also corroborate other findings indicating that the GABAergic system represents an important molecular and neuronal substrate for the selective attenuation of anxiety and aggression.
Subject(s)
Behavior, Animal/physiology , Brain/metabolism , Excitatory Amino Acids/metabolism , Social Behavior , gamma-Aminobutyric Acid/metabolism , Aggression/physiology , Animals , Aspartic Acid/analysis , Aspartic Acid/metabolism , Excitatory Amino Acids/analysis , Glutamic Acid/analysis , Glutamic Acid/metabolism , Male , Mice , Mice, Inbred ICR , Microchemistry/methods , Microdialysis/methods , Models, Biological , Shyness , Social Desirability , gamma-Aminobutyric Acid/analysisABSTRACT
Hypoxic-ischemic encephalopathy is a severe complication of perinatal asphyxia and causes lifelong deficits in infants and children. Multiple mechanisms acting in serial or parallel fashion are likely to be involved in this procedure. The neuronal injury is strongly related to iron-catalysed oxygen radical production and subsequent peroxidative damage to lipids and protein. Excessive release of excitatory amino acids (EAA) glutamate and aspartate, with consequent overstimulation of glutamate receptors, is also thought to be an important mechanism in this brain injury. Deferoxamine (DFO), a chelator of non-protein-bound iron, has been shown to inhibit lipid peroxidation and hydroxyl radical production via the Fenton reaction and to decrease hypoxic-ischemic and reperfusion associated brain injury. However, the exact mechanism of neuroprotection of DFO and its possible effect on the neurotransmitters' release is currently being investigated. In the present study, a well-established model of perinatal asphyxia was used to investigate the effect of DFO on hypoxic-ischemic-induced damage to different hippocampal brain structures. DFO was administrated subcutaneously immediately after the asphyctic insult. Histological examination of the hippocampus was conducted and the tissue levels of glutamate and aspartate in the same area were determined. A remarkable reduction of hypoxia-ischemia-evoked neurons in the CA1 hippocampal region and a decrease in the asphyxia-induced hippocampal tissue levels of glutamate and aspartate was noted after DFO treatment. These findings suggest a complex action of DFO, which could be neuroprotective when administrated in the immature brain immediately after hypoxia-ischemia.
Subject(s)
Deferoxamine/pharmacology , Excitatory Amino Acids/analysis , Hippocampus/drug effects , Hypoxia-Ischemia, Brain/drug therapy , Neuroprotective Agents/pharmacology , Animals , Animals, Newborn , Aspartic Acid/analysis , Female , Glutamic Acid/analysis , Hippocampus/chemistry , Hippocampus/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/analysis , Hypoxia-Ischemia, Brain/metabolism , Hypoxia-Ischemia, Brain/pathology , Male , Oxidative Stress , Rats , Rats, WistarABSTRACT
OBJECTIVE: To observe changes of contents of excitatory amino acids in focal cerebral ischemic tissue after electroacupuncture (EA) treatment at different time courses in cerebral ischemia and reperfusion (CI/R) rats so as to provide experimental evidence for selecting the best duration of EA stimulation in the treatment of apoplexy. METHODS: Forty SD rats were randomized into control, model, EA-10 min, EA-30 min and EA-60 min groups. CI/R model was established by middle cerebral artery occlusion (MCAO) and reperfusion. EA (2 Hz, 3.5 mA and continuous waves) was applied to "Baihui" (GV 20) and "Shuigou" (GV 26) for 10 min, 30 min and 60 min respectively. Cerebral glutamate (Glu) and aspartic acid (Asp) concentrations were detected by using an amino acid automatic analyzer after homogenate and centrifugalization of the right brain tissue. RESULTS: Compared with control group, Asp and Glu contents of model group and EA-10 min group increased significantly (P < 0.01); while in comparison with model group, Asp level in EA-60 min group, and Glu level in EA-10 min, EA-30 min and EA-60 min groups decreased considerably (P < 0.05). No significant differences were found between control and EA-30 min groups, and between control and EA-60 min groups in Asp and Glu levels (P > 0.05). CONCLUSION: EA of GV20 and GV26 for 60 min can markedly reduce Glu and Asp levels in the topical cerebral infarct area in CI/R rats, which may contribute to its effect in relieving cerebral ischemic injury. Suitable duration of EA stimulation is definitely necessary for inducing a better therapeutic effect.
Subject(s)
Brain Chemistry , Brain Ischemia/therapy , Electroacupuncture , Excitatory Amino Acids/analysis , Reperfusion Injury/therapy , Animals , Brain Ischemia/metabolism , Male , Rats , Rats, Sprague-Dawley , Reperfusion Injury/metabolismABSTRACT
A sensitive fluorescence detection system with an Hg-lamp as the excitation source and a photon counter as the detector for microchip CE (MCE) has been developed. O-Phthaldialdehyde (OPA, lambda(ex) = 340 nm) was employed to label the catecholamine neurotransmitters such as dopamine (DA), norepinephrine (NE), and amino acid neurotransmitters including alanine (Ala), taurine (Tau), glycine (Gly), glutamic acid (Glu), and aspartic acid (Asp). The separation of seven derivatized neurotransmitters was successfully performed in MCE and the detection limits (S/N = 3) for DA, NE, Ala, Tau, Gly, Glu, and Asp were 0.85, 0.49, 0.23, 0.15, 0.13, 0.18, and 0.29 fmol, respectively. The system was then successfully applied for separation and determination of neurotransmitters in rat pheochromocytoma (PC 12) cells, and the average amounts of analyte per cell from a cell population were 2.5 fmol for DA, 3.3 fmol for Ala, 8.2 fmol for Tau, 4.0 fmol for Gly, and 1.9 fmol for Glu, respectively. By single-cell injection mode, electrophoresis separation and quantitative measurement of Glu in individual PC 12 cells was obtained. The average value of Glu per cell from single PC 12 cells analysis was found to be 3.5 +/- 3.1 fmol.
Subject(s)
Brain Chemistry , Electrophoresis, Microchip/methods , Excitatory Amino Acids/analysis , Neurotransmitter Agents/analysis , Spectrometry, Fluorescence/methods , Animals , Cell Extracts , Dietary Proteins/analysis , Dopamine/analysis , Electrophoresis, Microchip/instrumentation , Fluorescence , Microscopy, Fluorescence , Norepinephrine/analysis , PC12 Cells , Rats , Sensitivity and SpecificityABSTRACT
OBJECTIVE: Our present study examined the effect of intra-articular cyclooxygenase-2 (COX-2) inhibitor parecoxib on osteoarthritis (OA) progression and the concomitant changes in excitatory amino acids' (EAAs) levels of the anterior cruciate ligament-transected (ACLT) knee joint dialysates. METHODS: OA was induced in Wistar rats by anterior cruciate ligament transection of the knee of one hindlimb, the other was left unoperated and untreated. Rats were placed into four groups: Group ACLT/P received intra-articular parecoxib injection (100 microg) in the ACLT knee once a week for 5 consecutive weeks starting at 8 weeks after surgery. Group ACLT/S received the same procedure as group ACLT/P with saline injection instead. Naïve (Naïve/P) rats received only intra-articular parecoxib injection in one knee once a week for 5 consecutive weeks without surgery. The sham-operated rats underwent arthrotomy only without treatment. Twenty weeks after surgery, knee joint dialysates were collected and EAAs' concentration was assayed by high-performance liquid chromatography, and gross morphology and histopathology (Mankin and synovitis grading) were examined on the medial femoral condyles and synovia. RESULTS: Parecoxib alone had no effect on cartilage and synovium of normal knees in Naïve/P rats. In ACLT/P rats, parecoxib treatment showed a significant inhibition of cartilage degeneration of the medial femoral condyle at both the macroscopic level (1.15+/-0.17 vs 2.55+/-0.12, P<0.05) and the Mankin scores (3.03+/-0.28 vs 8.82+/-0.43, P<0.05). Intra-articular parecoxib injection also suppressed the synovial inflammation of ACLT joint compared to the ACLT/S group (3.92+/-0.41 vs 9.25+/-0.32, P<0.05). Moreover, glutamate and aspartate levels were also significantly reduced in the ACLT/P group compared to the ACLT/S group by parecoxib treatment (91.2+/-9.4% vs 189.5+/-17.0%, P<0.05 and 98.2+/-11.6% vs 175.3+/-12.4%, P<0.05, respectively). CONCLUSION: This study shows that intra-articular injection of COX-2 inhibitor parecoxib inhibits the ACLT-induced OA progression; it was accompanied by a reduction of glutamate and aspartate concentration in the ACLT joint dialysates. From our present results, we suggested that intra-articular parecoxib injection, in addition to the anti-inflammatory effect, inhibiting the EAAs' release, may also play a role in inhibiting the traumatic knee injury induced OA progression.
Subject(s)
Cartilage, Articular/drug effects , Cyclooxygenase 2 Inhibitors/therapeutic use , Excitatory Amino Acids/analysis , Isoxazoles/therapeutic use , Osteoarthritis/drug therapy , Animals , Anterior Cruciate Ligament/surgery , Cartilage, Articular/pathology , Excitatory Amino Acids/metabolism , Injections, Intra-Articular , Osteoarthritis/physiopathology , Rats , Rats, WistarABSTRACT
The capillary electrophoresis (CE) system with optical fiber light-emitting diode (optical fiber LED) induced fluorescence detector was developed for the analysis of the excitatory amino acids (EAAs) tagged with naphthalene-2,3-dicarboxaldehyde (NDA). The separation of EAAs was carried out in an uncoated fused-silica capillary (50 cm x 75 microm i.d.) with a buffer of 10 mM borate at pH 9.3 and an applied voltage of 20 kV. High sensitivity was obtained by the use of optical fiber LED induced fluorescence detector with a violet LED as the excitation light source. The limits of detection (S/N = 3) for glutamic acid (Glu) and aspartic acid (Asp) were 2.1 x 10(-8) and 2.3 x 10(-8) M, respectively. The detection approach was successfully applied to the analysis of Glu and Asp in biological fluids including human serum, rabbit serum and human cerebrospinal fluid (CSF) with satisfactory results.
Subject(s)
Electrophoresis, Capillary/methods , Excitatory Amino Acids/analysis , Spectrometry, Fluorescence/methods , Animals , Excitatory Amino Acids/blood , Excitatory Amino Acids/cerebrospinal fluid , Humans , Optics and Photonics , Rabbits , Sensitivity and Specificity , Spectrophotometry, UltravioletABSTRACT
INTRODUCTION: The major excitatory neurotransmitters in the mammalian central nervous system are glutamate and aspartate. We developed a rapid and efficient method for the extraction and measurement of these amino acids in Planaria--a valuable model for mammalian processes because of their simple, centralized nervous system and similar neurotransmitter systems. METHOD: The method utilized buffer extraction (perchloric acid containing 0.025% of L-cystine and Na2EDTA), simple derivatization, high-pressure liquid chromatography (HPLC), and fluorescence detection. RESULTS: The mean+/-S.E.M. amounts of glutamate and aspartate were 322.6+/-43.6 and 188.6+/-27.6 pmol/mg-planarian, respectively. DISCUSSION: The method provides the ability to investigate changes in glutamate and aspartate in response to drug administration or withdrawal.
Subject(s)
Aspartic Acid/analysis , Excitatory Amino Acids/analysis , Glutamic Acid/analysis , Planarians/chemistry , Animals , Aspartic Acid/isolation & purification , Calibration , Chromatography, High Pressure Liquid , Excitatory Amino Acids/isolation & purification , Glutamic Acid/isolation & purification , Reference Standards , Spectrometry, Fluorescence , Time FactorsABSTRACT
Changes in excitatory amino acid (EAA) levels were examined in the knee joint dialysates of rats with early osteoarthritis (OA). Early OA was induced by anterior cruciate ligament (ACL) transection in one knee and the contralateral knee was used as the sham-operated control, the side for ACL transection being assigned randomly. Twenty weeks after operation, knee joint dialysates were collected by microdialysis and assayed for EAAs by high performance liquid chromatography. The rats were then sacrificed for histopathological examination. Hematoxylin/eosin and Safranin-O staining showed cartilage fibrillation, clustering of chondrocytes, and a reduction in matrix proteoglycans at week 20 in the ACL-transected knee, but not in the sham-operated knee. Levels of glutamate and aspartate in dialysates from the ACL-transected knee were significantly increased by 92 +/- 20.3% or 57 +/- 17.5%, respectively, compared to those in the contralateral sham-operated knee. This increase may contribute to the pathogenesis of early OA.
Subject(s)
Anterior Cruciate Ligament/surgery , Excitatory Amino Acids/analysis , Knee Joint/chemistry , Osteoarthritis/metabolism , Animals , Knee Joint/pathology , Male , Microdialysis , Osteoarthritis/pathology , Rats , Rats, WistarABSTRACT
A sensitive and rapid reversed-phase high-performance liquid chromatographic method using pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and fluorescence detection is reported. By directly derivatizing microdialysate samples with AQC, an automatic and rapid simultaneous measurement of aspartate, serine, glutamate, glycine and histidine was developed. Excellent linearity (r2 > or = 0.998) was achieved for the standard mixture used for the validation experiments. Within-day and between-day precision was less than 6.2%, and the accuracy ranged from 95 to 105.2% in standards. This method is suitable for single run analysis of a high number of small volume microdialysate samples from rat hippocampus. Amino acids from microdialysate samples were quantified with RSD for reproducibility below 2%, and at approximately 0.1% for retention time.
Subject(s)
Aminoquinolines/chemistry , Carbamates/chemistry , Chromatography, High Pressure Liquid/methods , Excitatory Amino Acids/analysis , Animals , Excitatory Amino Acids/chemistry , Male , Microdialysis/methods , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Sensitivity and Specificity , Spectrometry, FluorescenceABSTRACT
It was found that native amino acids enhanced the chemiluminescence (CL) reaction between luminol and BrO(-) in an alkaline aqueous solution. This has led to the development of a facile and highly sensitive CL detection scheme for the determination of amino acids in biological samples after capillary electrophoretic (CE) separation. The CE-CL conditions were optimized. An electrophoretic buffer of 2.5 x 10(-2) M sodium borate (pH 9.4) containing 1 x 10(-4) M luminol was used. The oxidizer solution of 8 x 10(-4) M NaBrO in 0.1 M sodium carbonate buffer solution (pH 12.5) was introduced post-column. Under the optimal conditions, the detection limits were 1.0 x 10(-7) M for glutamic acid (Glu) and 1.3 x 10(-7) M (S/N = 3) for aspartic acid (Asp). The relative standard deviations (RSDs) of peak area and migration time were in the ranges of 3.8-4.3% and 1.4-1.6%, respectively. The present method was applied to the determination of excitatory amino acids (i.e., Asp and Glu) in rat brain tissue and monkey plasma. The levels of these major excitatory amino acids in monkey plasma were quantified for the first time and found to be 1.17 +/- 0.17 x 10(-5) M (mean +/- SD, n = 6) for Glu and 1.64 +/- 0.19 x 10(-6) M for Asp, which were comparable with the levels in human plasma.
Subject(s)
Electrophoresis, Capillary , Excitatory Amino Acids/analysis , Luminescent Measurements/methods , Animals , Brain Chemistry , Excitatory Amino Acids/blood , Excitatory Amino Acids/isolation & purification , Haplorhini , Luminol/chemistry , RatsABSTRACT
The aim of this study was to assess the synovial fluid (SF) neurotransmitter excitatory amino acid (EAA) levels, including glutamate (Glu) and aspartate (Asp), in the context of SF levels of other amino acids, TNF-alpha and chemokines from patients with active arthropathies. The SF was collected from patients with active rheumatoid arthritis (RA), gout, or osteoarthritis (OA). The SF samples were analysed for levels of neurotransmitters glutamate and aspartate, tumour necrosis factor-alpha (TNF-alpha), Regulated upon Activation Normally T-cell Expressed and Secreted (RANTES), macrophage inhibitory factor-1 alpha (MIP-1alpha) and interleukin 8 (IL-8). SF WBC counts were also determined. Correlations between SF EAA, TNF-alpha and chemokines were determined by the Pearson product-moment correlation. Primary cultures derived from SF from active RA and gout patients were incubated with added l-glutamate, to assess if exposure to Glu could increase TNF-alpha levels. There were significant elevations in SF EAA, SF TNF-alpha and SF RANTES in RA patients compared to gout or OA patients. Significant correlations between SF EAA and SF RANTES, MIP-1alpha and IL-8 levels were seen, and SF EAA and SF TNF-alpha or SF WBC levels approached significance. Addition of exogenous neurotransmitter glutamate significantly increased TNF-alpha levels in primary cell cultures derived from RA and gout patients. The SF neurotransmitter EAA levels significantly correlated to selected SF chemokine levels, in clinically active RA, gout and OA patients, independent of disease. Added Glu resulted in significantly increased TNF-alpha levels in primary synovial cell cultures. These data expand the relationship of SF neurotransmitter EAA levels to SF cytokines and chemokines in patients with clinically active arthritis, and suggest that neurotransmitters Glu and Asp contribute to peripheral inflammatory processes.
Subject(s)
Arthritis/metabolism , Chemokines/analysis , Excitatory Amino Acids/analysis , Synovial Fluid/chemistry , Tumor Necrosis Factor-alpha/analysis , Adult , Aged , Arthritis/immunology , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/metabolism , Chemokine CCL3 , Chemokine CCL4 , Chemokine CCL5/analysis , Chromatography, High Pressure Liquid/methods , Female , Gout/immunology , Gout/metabolism , Humans , Interleukin-8/analysis , Macrophage Inflammatory Proteins/analysis , Male , Middle Aged , Osteoarthritis/immunology , Osteoarthritis/metabolismABSTRACT
Spinal dynorphin is hypothesized to contribute to the hyperalgesia that follows tissue and nerve injury or sustained morphine exposure. We considered that these dynorphin actions are mediated by a cascade involving the spinal release of excitatory amino acids and prostaglandins. Unanesthetized rats with lumbar intrathecal injection and loop dialysis probes received intrathecal NMDA, dynorphin A(1-17), or dynorphin A(2-17). These agents elicited an acute release of glutamate, aspartate, and taurine but not serine. The dynorphin peptides and NMDA also elicited a long-lasting spinal release of prostaglandin E2. Prostaglandin release evoked by dynorphin A(2-17) or NMDA was blocked by the NMDA antagonist amino-5-phosphonovalerate as well the cyclooxygenase (COX) inhibitor ibuprofen. To identify the COX isozyme contributing to this release, SC 58236, a COX-2 inhibitor, was given and found to reduce prostaglandin E2 release evoked by either agent. Unexpectedly, the COX-1 inhibitor SC 58560 also reduced dynorphin A(2-17)-induced, but not NMDA-induced, release of prostaglandin E2. These findings reveal a novel mechanism by which elevated levels of spinal dynorphin seen in pathological conditions may produce hyperalgesia through the release of excitatory amino acids and in part by the activation of a constitutive spinal COX-1 and -2 cascade.
Subject(s)
Dinoprostone/metabolism , Dynorphins/pharmacology , Excitatory Amino Acids/metabolism , Isoenzymes/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Spinal Cord/drug effects , Animals , Cyclooxygenase 1 , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Cyclooxygenase Inhibitors/pharmacology , Dinoprostone/analysis , Drug Antagonism , Excitatory Amino Acid Antagonists/pharmacology , Excitatory Amino Acids/analysis , Glutamic Acid/analysis , Glutamic Acid/metabolism , Hyperalgesia/etiology , Injections, Spinal , Isoenzymes/antagonists & inhibitors , Membrane Proteins , Microdialysis , N-Methylaspartate/pharmacology , Peptide Fragments/pharmacology , Rats , Spinal Cord/metabolismABSTRACT
1. Although microdialysis is a widely used approach for in vivo monitoring extracellular neurotransmitter concentrations, it has been previously limited in many cases by its poor temporal resolution. It is clear that when 10-30-min sampling is performed, short-lasting changes in extracellular neurotransmitter concentrations can be overlooked. Such a low sampling rate is necessary when combining microdialysis with the conventional analytical methods like high performance liquid chromatography. 2. Since capillary electrophoresis coupled to laser-induced fluorescence detection (CE-LIFD) allows the detection of attomoles of neurotransmitters, the temporal resolution of microdialysis may be significantly improved: high sampling rates, in the range of 5 s to 1 min, have been already reported by our group and others using CE-LIFD for simultaneously analyzing catecholamines and amino acids in microdialysates. 3. The power of combining microdialyis and CE-LIFD is shown, using examples of physiological and pharmacological studies dealing with the dynamics of in vivo efflux processes and/or interactions between neurotransmitters.