Methods for the Determination of Stable Isotopes of Carbon and Nitrogen Directly in Valine, Proline, Glutamine, and Glutamic Acid

Amino acids are very important compounds for the body and are involved in important functions that keep us healthy. Amino acids are essential components such as valine, proline, glutamine and glutamic acid. They can be synthesized either naturally or artificially. To examine the metabolism and regulate the synthesis process, compounds labeled with nitrogen or carbon isotopes need to be used. These isotopic compounds allow for more extensive research and enable studies that would otherwise be impossible. However, their use is dependent on the availability of simple, efficient methods for isotopic analysis. Currently, the determination of the atomic fraction of carbon and nitrogen isotopes is only possible through their conversion into molecular nitrogen or carbon monoxide or carbon dioxide. This leads to the loss of information about isotopic enrichment in specific centers of the molecule. This article explores a new direct approach to determining the atomic fraction of carbon and nitrogen isotopes in the isotope-modified or identical centers of these compounds. This method eliminates the transfer process and dilution due to nitrogen and carbon impurities. It is now possible to simultaneously determine the atomic fraction of nitrogen and carbon isotopes in the research substance. This method can be applied to amino acids, making it an effective tool for proposing new research methods. Several articles [1] [2] [3] have proposed similar methods for organic compounds and amino acids.

Adsorption of glutamic acid from aqueous solution with calcined layered double Mg-Fe-CO_3 hydroxide

Layered double Mg-Fe-CO3 hydroxide （Mg-Fe-LDH） with a mole ratio of Mg to Fe of 3 was synthesized by coprecipitation method and calcined product Mg-Fe-CLDH was obtained by heating Mg-Fe-LDH at 500 ℃ for 6 h. The as prepared Mg-Fe-LDH and calcined Mg-Fe-CLDH were used for removal of glutamic acid （Glu） from aqueous solution, respectively. Batch studies were carried out to address various experimental parameters such as contact time, pH, initial glutamic acid （Glu） concentration, co-existing anions and temperature. Glu was removed effectively （99.9%） under the optimized experimental conditions with Mg-Fe-CLDH. The adsorption kinetics follows the Ho’s pseudo second-order model. Isotherms for adsorption with Mg-Fe-CLDH at different solution temperatures were well described using the Langmuir model with a good correlation coefficient. The intraparticle diffusion model fitted the data well, which suggests that the intraparticle diffusion is not only the rate-limiting step.

Tanshinone IIa antagonizes spinal ischemia/reperfusion injury by interfering with upstream glutamic acid factors

Based on the hypothesis that upstream factor inhibition results in better treatment effects than downstream factor inhibition,the present study interfered with glutamic acid（Glu）-released upstream factors,such as Glu transporter function and Na＋-K＋-adenosine triphosphatases（ATPase）activity relativly.Rats with spinal cord ischemia/reperfusion injury received intraperitoneal injections of tanshinone Ila and Glu uptake and Na＋-K＋-ATPase activity were increased.Results showed that tanshinone Ila influenced Glu-released upstream factors following spinal ischemia/reperfusion injury and protected against spinal ischemia/reperfusion injury.

Granulocyte colony-stimulating factor increases extracellular glutamic acid uptake and suppresses free radicals in an experimental model of amyotrophic lateral sclerosis

Excitatory amino acid toxicity and free radical damage play important roles in amyotrophic lateral sclerosis. Granulocyte colony-stimulating factor （G-CSF） protects nerve cells exposed to high-concentrations of glutamic acid, suggesting positive effects in the treatment of amyotrophic lateral sclerosis. The present study induced in vitro motor neuron injury using glutamic acid excitotoxicity, and the biochemical effects of G-CSF on glutamic acid concentration were determined. In addition, the effects of G-CSF on superoxide dismutase, glutathione peroxidase activity in motor neurons, and malondialdehyde and nitric oxide contents were analyzed. Immunohistochemistry was performed to measure neuronal survival. Results revealed that G-CSF significantly suppressed free radical activity, inhibited excitotoxicity, and reduced apoptosis and loss of motor neurons in the anterior horn of the spinal cord.

Analysis of Glutamic Acid in Cerebrospinal Fluid by Capillary Electrophoresis with High Frequency Conductivity Detection

A rapid method to determine glutamic acid (Glu) in cerebrospinal fluid (CSF) by capillaryelectrophoresis with high frequency conductivity detection (contactless conductivity detection) wasdescribed. The CSF sample was pretreated with silver cation resin to remove high concentration ofCl- ions in CSF. The separation was achieved in the buffer solution of 10 mmol/L Tris and 8mmol/L boric acid at the separation voltage of 20.0 kV. Glu showed linear response in the range of5.0×10-6 to 6.0×10-3 mol/L, the limit of detection was 1.0×10-6 mol/L. The method was used foranalysis Glu in CSF satisfactorily with a recovery of 97.8-98.8%.

In Vitro Biomineralization of Glutaraldehyde Crosslinked Chitosan/Glutamic Acid Films

In vitro biomineralization of glutaraldehyde crosslinked chitosan/glutamic acid films were studied. IR and ESCA （electron spectroscopy for chemical analysis） determinations confirm that chitosan and glutamic acid are successfully crosslinked by glutaraldehyde to form chitosan-glutamic acid surfaces. Composite films were soaked in saturated Ca（OH）2 solution for 8 d and then immersed in simulated body fluid （SBF） for more than 20 d. Morphological characterizations and structure of cal-cium phosphate coatings deposited on the films were studied by SEM, XRD, and EDAX （energy dispersive X-ray analysis）. Initially, the treatment in SBF results in the formation of single-layer cal-cium phosphate particles over the film surface. As immersion time increases, further nucleation and growth produce the simulated calcium-carbonate hydroxyapatite coating. ICP results show Ca/P ratio of calcium phosphate coating is a function of SBF immersion time. The inducing of glutamic acid improves the biomineralization property of chitosan films.

Tb(lll) and Ca(ll) Ion Equilibria in the Presence of Glutamic Acid and Glutamine

Tb(111) and Ca(11) ion equilibria in the presence of glutamic acid and glutamine were studied by potentiometric titration at 37'C and an ionic strength of 0. 15mol/L(NaCl) .The stability constants for Tb(111) and Ca(11)complexes in the systems were obtained. The species and their distribution in the systems were discussed.

Determination of Enantiomeric Excess of Glutamic Acids by Lab-made Capillary Array Electrophoresis

Simulated enantiomeric excess of glutamic acid was determined by a lab-made sixteen-channel capillary array electrophoresis with confocal fluorescent rotary scanner. The experimental results indicated that the capillary array electrophoresis method can accurately determine the enanfiomefic excess of glutamic acid and can be used for high-throughput screening system for combinatorial asymmetric catalysis.

Double Labeling Immulloelectron Microscopic Study on the Synaptic Connections between Glutamic Acid Neurons and GABA Neurons in the Hippocampus of Rats

Summary: In order to explore the roles of different neurotransmitters in epileptic pathogenesis, the synaptic connections between glutamic acid (Gin) neurons and GABA neurons in normal rat hippocampus were studied by pre-embedding double labeling immunoelectron microscopy. The GABA immunoreaction was first demonstrated by chromogen DAB, then the Gin immunoreaction was demonstrated by molybdic acid-TAB method. After being stabilized by DAB-cobalt chloride. the sections were processed for electron microscopic embedding. Under electron microscope, there were many Gin immunoreaction-positive neurons in the pyramidal layer of hippocampal CA1 area and some GABA immunoreaction-positive neurons with pyramidal or polygonal perikarya in the pyramidal, polymorphic and radiant layer of CA1 area. There were also symmetric dendro-axonic synapses formed by GABA-positive dendrites and Glu-positive a-cons in the polymorphic layer and symmetric axo-dendritic synapses formed by GABA-positive axons and Glu-positive dendrites in the radiant layer. In addition, there were symmetric autoregulatory axo-dendritic synapses be- tween Gin-positive axons and dendrites and autoregulatory axo-axonic synapses (both symmetric and asymmetric) between GABA-positive axons. Above mentioned results, for the first time, showed that there were complex synaptic regulatory relationships between excitatory Glu neurons and inhibitory GABA neurons in the hippocampal CA1 area, thereby, providing ultrastructural evidence for different neurotransmitters participating in epileptic pathogenesis.

Preparation and Characterization of Orally Fast-Disintegrating Mini-Tablets Containing Diphenhydramine Hydrochloride and Aspartic or Glutamic Acid as an Umami Amino Acid

The aim of this study was to prepare diphenhydramine hydrochloride (DPH)-loaded orally fast-disintegrating mini-tablets (OFDMTs) containing either L-aspartic acid (Asp) or L-glutamic acid (Glu) as bitterness-suppressant, to characterize the prepared tablets and to evaluate their bitterness under conditions mimicking those of the oral cavity. The preparation of five formulation batches of the OFDMTs involved mixing DPH, with or without two different concentrations of Asp or Glu, and a premix containing a disintegrating agent. When all ingredients were well mixed, the mixture was directly compacted to form small (4 mm diameter) DPH-loaded OFDMTs. There were only small differences between the tablets with respect to mass, diameter, width and hardness. The disintegration times of the five formulation batches of DPH-loaded OFDMTs were measured using the OD-mate, a disintegration test apparatus in which conditions resemble those of the oral cavity. The disintegration times were all within 10 s of exposure to a medium representing the inside of the oral cavity. Rapid release profiles were observed for DPH, Asp and Glu in these dissolution tests. The taste sensor outputs of samples taken at different times (5 - 30 s) from the dissolution test solutions of the four DPH-loaded OFDMTs containing Asp or Glu were significantly inhibited compared with those of control DPH-loaded OFDMT. These results suggest that the inclusion of Asp or Glu in DPH-loaded OFDMTs is sufficient to mask bitterness in the oral cavity for the first 30 s after the tablet is placed in the mouth. It is anticipated that swallowing will have taken place within 30 s.

SYNTHESIS AND CATALYTIC PROPERTY OF POLYSTYRENE SUPPORTED GLUTAMIC ACID SCHIFF BASE COMPLEX OF Mn(Ⅱ) IN AEROBIC OXIDATION OF CYCLOHEXENE

The polystyrene supported glutamic acid Schiff base complex of Mn （ Ⅱ ） （PS-Sal-Glue-Mn） was prepared with chloromethylated styrene polymer beads, 2,4-dihydroxybenzaldehyde, L-glutamic acid and manganese （ Ⅱ ） acetate tetrahyrate. The polymeric ligand and the complex were characterized by FT-IR, small area X-ray photoelectron spectroscopy （XPS） and 1CP-AES. In the presence of the manganese complex, cyclohexene （1） was effectively oxidized by molecular oxygen without reductant. The major products of the reaction were 2-cyclohexen-l-ol （2）, 2-cyclohexen-l-one （3） and 2-cyclohexen-1- hydroperoxide （4）, which was different with typical oxidation of cyclohexene. The influence of reaction temperature and additive for oxidation had been studied. The selectivity of 2-cyclohexen-1-hydroperoxide varied with reaction time and different additives. The mechanism of cyclohexene oxidation had also been discussed.

Effects of Glutamic Acid on C-type Inactivation of Kv1.4ΔN Channel

Objectives Acidosis has an inhibitory effect on the inactivation of Kv1.4 ΔN channel through the position H508. So in order to show the effects of glutamic acid on the mutant Kv 1.4 channel that lacks N-type inactivation （Kv1.4 Δ2-146）, we studied in the expression system of the Xenopus oocytes. Methods The two-electrode voltage-clamp technique （TEV） was used to record the currents. Results Acidosis increased fKv1.4 Δ2-146 C-type inactivation. After application of glutamic acid （1 mmol/L） to Kv1.4 Δ2-146 increased C-type inactivation further, changed inactivation time constants from （2.02 ± 0.39 s ） to （1.71 ± 0.23 s） （P〈 0.05） at ＋50mv, and shifted the steady-state inactivation curves of Kv1.4 ΔN to positive potential, which was from （-44.30 ± 0.59 mV） to （-39.88 ± 0.29 mV）（P〈0.05）. and slowed the rate of recovery from inactivation, which was from （1.64 ± 0.19 s） to （1.91 ± 0.23 s）（P〈 0.05）. Conclusions Together, these results suggest that 1 mmol/L glutamic acid accelerates the C-type inactivation of Kv1.4 ΔN in pH 6.8.

STUDY ON THE SEPARATION OF GLUTAMIC ACID BY ION-EXCHANGE

The feasibility of recavering glutamic acid by ion exchange method with macroporous resins was investigated. Their adsorption properties in static state and the effective factors,such as pH, concentration of feed and the ratio of ammonium ion toglutamic acid,were systematically explored. The best conditions of separating glutamic acid from mother liquid were obtained.

RAPID DETERMINATION OF L-GLUTAMIC ACID WITH AN ENZYME REACTOR OF L-GLUTAMIC DECARBOXYLASE IMMOBILIZED ON ION EXCHANGE RESIN

The preparation and characterization of an immobilized L-glutamic decarboxylase (GDC) were studied. This work is to develop a sensitive method for the determination of L-glutamate using a new biosensor, which consists of an enzyme column reactor of GDC immobilized on a novel ion exchange resin (carboxymethyl-copolymer of allyl dextran and N.N?methylene-bisacrylamide CM-CADB) and ion analyzer coupled with a CO2 electrode. The conditions for the enzyme immobilization were optimized by the parameters: buffer composition and concentration, adsorption equilibration time, amount of enzyme, temperature, ionic strength and pH. The properties of the immobilized enzyme on CM-CADB were studied by investigating the initial rate of the enzyme reaction, the effect of various parameters on the immobilized GDC activity and its stability. An immobilized GDC enzyme column reactor matched with a flow injection system-ion analyzer coupled with CO2 electrode-data collection system made up the original form of the apparatus of biosensor for determining of L-glutamate acid. The limit of detection is 1.0×10-5 M. The linearity response is in the range of 5×10 -2-5×10 -5 M . The equation of linear regression of the calibration curve is y= 43.3x + 181.6 (y is the milli-volt of electrical potential response, x is the logarithm of the concentration of the substrate of L-glutamate acid). The correlation coefficient equals 0.99. The coefficient of variation equals 2.7%.

Construction and functional activity of a recombinant vector expressing rat glutamic acid decarboxylase 65

Objective Glutamic acid decarboxylase 2（GAD65） is a gamma-aminobutyric acid（GABA） synthetase.This study aimed to construct a recombinant lentivirus-rGAD65（rLV-rGAD65） vector containing the cDNA of rat GAD65（rGAD65） and assess its functional activity in vitro and in vivo.Methods cDNA of rGAD65 was amplified by RT-PCR and subcloned into the LV vector,forming the rLV-GFP-rGAD65 plasmid.The recombinant lentivirus particles（rLVrGAD65） were packaged by the LV Helper-Free System and the titer was measured.Primary rat lung fibroblasts were transfected with rLV-rGAD65.The expression of rGAD65 in fibroblasts was detected by immunocytochemistry and western blot and the level of GABA in the medium was assessed by high-performance liquid chromatograph（HPLC）.In vivo,rLV-rGAD65 was injected into the subthalamic nucleus（STN） of Sprague-Dawley rats using stereotaxic methods,and rGAD65 protein levels in the STN were assessed by immunohistochemistry and Western blot,while the GABA concentration in the substantia nigra pars reticulata（SNr） was assayed by HPLC.Results The sequence of rGAD65 cDNA was in accord with that in GenBank.The amino-acid sequence of rGAD65 had no mutations and the titer of rLVrGAD65 reached 6.8 × 108/mL.The efficiency of infection of fibroblasts was 80%,and the concentration of GABA in the medium was（48.14 ± 9.35） nmol/L.In vivo,rGAD65 expression was detected in the STN,and the concentration of GABA in the SNr increased from（5.95 ± 1.09） to（12.44 ± 3.79） nmol/g tissue.Conclusion The recombinant LVGFP-rGAD65 vector was successfully constructed.rLV-rGAD65-infected primary fibroblasts in vitro and the expressed rGAD65 catalyzed the formation of GABA from glutamic acid.In vivo,the concentration of GABA in the SNr was increased after rLV-rGAD65 injection into the STN.

The Dominant Glutamic Acid Metabolic Flux to Produce γ-Amino Butyric Acid over Proline in Nicotiana tabacum Leaves under Water Stress Relates to its Significant Role in Antioxidant Activity

γ-Amino butyric acid （GABA） and proline play a crucial role in protecting plants during various environmental stresses. Their synthesis is from the common precursor glutamic acid, which is catalyzed by glutamate decarboxylase and △1-pyrroline-5-carboxylate synthetase respectively. However, the dominant pathway under water stress has not yet been established. To explore this, excised tobacco leaves were used to simulate a water-stress condition. The results showed GABA content was much higher than that of proline in leaves under water-deficit and non-water-deficit conditions. Specifically, the amount of GABA significantly increased compared to proline under continuous water loss for 16 h, indicating that GABA biosynthesis is the dominant pathway from glutamic acid metabolism under these conditions. Quantitative reverse transcription polymerase chain reaction and protein Western gel-blot analysis further confirmed this. To explore the function of GABA accumulation, a system producing superoxide anion （O 2 - ）, peroxide hydrogen （H 2 O 2 ）, and singlet oxygen （ 1 O 2 ） was employed to investigate the scavenging role on free-radical production. The results demonstrated that the scavenging ability of GABA for O 2 - , H 2 O 2 , and 1 O 2 was significantly higher than that of proline. This indicated that GABA acts as an effective osmolyte to reduce the production of reactive oxygen species under water stress.

Glutamic acid decarboxylase 65 autoantibody levels discriminate two subtypes of latent autoimmune diabetes in adults

Objective To compare the clinical characteristics between type 2 diabetes mellitus (T2DM) and latent autoimmune diabetes in adults ( LADA) with different titers of glutamic acid decarboxylase autoantibody (GADA) and to define the two distinct subtypes of LADA.Methods Sera of 750 patients with an initial diagnosis of T2DM from central south of China were screened for GADA using a radioligand assay. The distribution and frequency of GADA levels were described. Two hundred and ninety-five patients were divided into the T2DM group (n =233) and the LADA group ( n = 62) to compare the age of onset, body mass index, HbA1c, C-peptide, hypertension, dyslipidemia and chronic diabetic complications. Furthermore, LADA patients with different GADA titers were subdivided to analyze the same indexes as the above.Results The prevalence of LADA (defined as GADA≥0. 05, namely GADA positive) was 9. 7% in the 750 initially diagnosed type 2 diabetic patients. Compared with T2DM, LADA patients were younger at their ages of onset, had lower C-peptide and body mass index, and also had less cases with hypertension and with dyslipidemia. However, only patients with high titer of GADA had poorer beta cell functions and less diabetic complications compared to T2DM and low GADA titer of LADA patients. Patients with low GADA titer were similar to T2DM patients, except that they were prone to develop ketosis more frequently.Conclusions Two clinically distinct subtypes of LADA can be identified by GADA levels in patients initially-diagnosed as type 2 diabetes. Patients with high titer of GADA (GADA≥0. 5) subsequently develope more insulin dependency, which are classified as LADA-type 1; while those with lower GADA titer (0.05≤GADA < 0. 5) and having clinical and metabolic phenotypes of type 2 diabetes are classified as LADA-type 2.

Combination of hydrophobically modified γ-poly(glutamic acid)and trehalose achieving high cryosurvival of RBCs

Trehalose is expected to be an alternative for toxic glycerol as a biocompatible cryoprotectant of red blood cells(RBCs).In this work,γ-poly(glutamic acid)(PGA)is modified by grafting hydrophobic phenethylamine,3,4-dimethoxyphenylethylamine and dodecylamine,respectively.The graft-modified PGA can significantly enhance cryosurvival of RBCs in combination with trehalose.Analyses of dynamic light scattering,hemolysis assay,atomic force microscope and confocal laser scanning microscope suggest that the modified PGA polymers can self-assemble into nanoparticles in phosphate buffer saline solutions at the pH range of 6.0–7.4,and exhibit membrane-disruptive activity due to hydrogen bond,conjugation and hydrophobic interactions with cell membranes.It is assumed that the modified PGA polymers can improve the cryosurvival of RBCs by promoting membrane permeability of trehalose.Among the three graft-modified polymers,phenethylamine-grafted PGA(PGA-g-PEA)can significantly increase the intracellular trehalose-loading content to 11.3±2.4 m M at pH 7.4,much higher than the value0.17±0.66 m M when trehalose is used without any polymers.In view of the aforementioned merit,the cryosurvival rate of sheep RBCs is increased to about 90%by incubation with 1.0 mg mL-1 PGA-g-PEA and 0.36 M trehalose.In vitro cell culture of L929 fibroblasts demonstrates low cytotoxicity of PGA-g-PEA.Therefore,hydrophobic PEA-modified PGA with enhanced intracellular trehalose-loading ability can be potentially applied in glycerol-free RBC cryopreservation or other related biomacromolecule delivery systems.

Change of glutamic acid decarboxylase antibody and protein tyrosine phosphatase antibody in Chinese patients with acute-onset type 1 diabetes mellitus

Background Glutamic acid decarboxylase antibody （GADA） and protein tyrosine phosphatase antibody （IA-2A） are two major autoantibodies, which exert important roles in the process of type 1 diabetes mellitus （T1D）. Our study aimed to investigate the changes in positivity and titers of GADA and IA-2A during the course of Chinese acute-onset T1D patients and their relationships with clinical features.

Diagnostic role of antibodies to glutamic acid decarboxylase in latent autoimmune diabetes mellitus in adults

Objective To investigate the diagnostic role of antibodies to glutamic acid decarboxylase (GAD 65 Ab) in latent autoimmune diabetes of adults (LADA) and the frequency of GAD Ab in Chinese patients initially diagnosed as non insulin dependent diabetes mellitus (NIDDM) Methods Forty five control subjects and 195 consecutive inpatients initially classified as NIDDM with ≥35 years of age at onset and nonketotic history for >6 months after diagnosis, were recruited In vitro transcripted and translated recombinant human 35 S GAD 65 was used in radioligand assay of GAD Ab Results The overall prevalence of GAD 65 Ab was 14 8% (29/195) in NIDDM patients and 2 2% (1/45) in control subjects, respectively Of the 29 GAD 65 Ab positive patients, 17 (58 6%) were insulin deficient while 12 (41 4%) were non insulin deficient The prevalence of GAD 65 Ab in NIDDM group with age of <40 years at diabetes onset, ketotic history, body mass index (BMI) <21 kg/m 2, were significantly higher than that of corresponding control diabetic subgroups (2 5, 4 1 and 3 2 times, respectively) The sex, duration, symptoms of polyphagia, polydipsia, polyuria and weight loss at onset of the disease were not related to the prevalence of GAD 65 Ab positivity Conclusions In China, patients initially diagnosed as NIDDM may in many cases suffer from LADA Testing by GAD 65 Ab may be of assistance to identifying LADA at the earliest stage of disease