Rapid method for the assay of 4-aminobutyric acid (GABA), glutamic acid and aspartic acid in brain tissue and subcellular fractions

The thin-layer electrophoretic separation at pH 4.8 of brain extracts and a procedure for fluorescent staining of the plates with fluorescamine are described for the rapid routine determination of 4-aminobutyric acid (GABA), glutamic acid and aspartic acid in brain extracts and in particulate fractions of brain tissue. Automated sample application, electrophoretic separation using two chambers, and quantitation by in situ fluorescence scanning allows the assay of 280 samples within three working days. The method is reproducible (S.D. less than 8% of the mean) within the range of 0.2--2 nmole per spot. The staining procedure can be applied to a variety of related analytical problems. The method has proved useful for the determination of the specific radioactivities of GABA, glutamic acid and aspartic acid in metabolic studies.     

Use of reversed-phase high-performance liquid chromatography for simultaneous determination of glutamine synthetase and glutamic acid decarboxylase in crude extracts

Glutamine and gamma-aminobutyric acid (GABA), formed from glutamic acid in crude tissue extracts by glutamine synthetase and glutamic acid decarboxylase respectively, were separated by derivatization with dansyl chloride followed by reversed-phase high-performance liquid chromatography on the Altex Ultrasphere ODS-5 column. The mobile phase was a gradient of 100 mM potassium dihydrogen phosphate (pH 2.1) with 0-40% acetonitrile. The amounts of glutamine and GABA formed from glutamic acid were determined under different reaction conditions.     

Use of high performance liquid chromatography in defining the abnormalities in the free amino acid patterns in the cerebrospinal fluid of patients with aseptic meningitis.

Free amino acids were quantitatively determined in cerebrospinal fluid (CSF) and plasma samples from patients with aseptic meningitis by a newly developed high performance liquid chromatographic (HPLC) method. The method of analysis was based on precolumn derivatization of orthophthaladehyde in the presence of 2-mercaptoethanol and detection was made at Eex = 340 nm and Eem = 450 nm. The method was sensitive and the limit for detection was less than 1 pmol for most of the amino acids. It took 45 min to separate 26 amino acids with highly reproducible results, giving a coefficient of variance for retention times and integrated areas less than 0.4% and 2%, respectively, after five replicate runs. The results accumulated in 10 patients were compared statistically with 11 age-matched healthy controls. Among the amino acids almost all the neurotransmitter candidates, such as aspartic acid, glutamic acid, glutamine, glycine, tyrosine, phenylalanine and gamma-aminobutyric acid (GABA), were significantly increased in the patients' CSF, whereas arginine and threonine were low. No change was observed in plasma amino acids in patients as compared to healthy controls. The higher levels of most of the neurotransmitters, especially GABA, aspartic acid and glutamic acid, could be used diagnostically in assessing the progression and remission in aseptic meningitis.     

高效液相色谱法测定鼠脑谷氨酸脱羧酶活性

应用高效液相色谱-电化学检测技术建立了鼠脑谷氨酸脱羧酶(GAD)活性的测定方法.方法快速,灵敏.总分析时间为9min.γ-氨基丁酸的最低柱上检测限为50fmol.天内及天间相对标准差分别小于4.3%和9.8%.测得CAD最大催化速率为0.723nmol/min/mg蛋白.讨论了最佳衍生化pH及色谱条件.     

Rapid determination of 4‐aminobutyric acid and L‐glutamic acid in biological decarboxylation process by capillary electrophoresis‐mass spectrometry

4‐Aminobutylic acid (GABA) is a monomer of plastic polyamide 4. Bio‐based polyamide 4 can be produced by using GABA obtained from biomass. The production of L ‐glutamic acid (Glu) from biomass has been established. GABA is produced by decarboxylation of Glu in biological process. High‐performance liquid chromatography (HPLC) with derivatization is generally used to determine the concentration of GABA and Glu in reacted solution samples for the efficient production of GABA. In this study, we have investigated the rapid determination of GABA and Glu by capillary electrophoresis‐mass spectrometry (CE‐MS) without derivatization. The determination was achieved with the use of a shortened capillary, a new internal standard for GABA, and optimization of sheath liquid composition. Determined concentrations of GABA and Glu by CE‐MS were compared with those by pre‐column derivatization HPLC with phenylisothiocyanate. The determined values by CE‐MS were close to those by HPLC with pre‐column derivatization. These results suggest that the determination of GABA and Glu in reacted solution is rapid and simplified by the use of CE‐MS.     

Use of an Optimum Wavelength to Detect Glutamic Acid and its Metabolites in Human Serum by Reversed-Phase HPLC

Abstract

Dansylated glutamic acid, glutamine and γ-amino butyric acid (GABA) show maximum absorption at 221 nm. Using this wavelength, the detection limits for dansylated amino acids studied by reversed-phase HPLC are similar to those reported by fluorescence. This technique was used to look foe the presence of glutamic acid and its metabolites in human serum. Glutamic acid and glutamine were present in significant amounts and their levels were 2.5 and 6.1 nmoles/ml respetively, while GABA was present in trace amounts, less than 0.3 nmoles/ml.     

Design and concise synthesis of fully protected analogues of l-gamma-carboxyglutamic acid

The design and synthesis of four nonnaturally occurring amino acid analogues of l-gamma-carboxyglutamic acid (Gla), appropriately protected for Fmoc-based solid-phase peptide synthesis (SPPS), is described. These amino acids are Bu-Mal 2, BCAH 3, Pen-Mal 4, and Cm-Gla 5. These Gla analogues have been designed to replace the glutamic acid of position 1 in the cyclic decapeptide G1TE, which is a potent inhibitor of tyrosine kinase, to further enhance binding to the Grb2-SH2 domain of signal transduction receptors. In the new amino acids, the propionic acid side chain of Glu has been replaced by a malonyl or a carboxymethylmalonyl moiety located at different distances from the alpha-carbon to optimize interactions in the phosphotyrosine-binding cavity of the Grb2-SH2 domain. Additionally, a direct and efficient synthetic route for the preparation of Fmoc-protected l-gamma-carboxyglutamic acid, which is amenable to large-scale production, has been developed to provide this important and unique amino acid(1) in 55% overall yield.     

Effect of additives on the amination of 2-oxoglutaric acid by gamma-radiation

The effect of added substances was studied on the yield of glutamic acid produced by gamma-ray irradiation of 2-oxoglutaric acid and ammonia in aqueous solution. The contents of amino acids in the irradiated solutions were determined with amino acids analyzer. Sodium nitrate, allyl alcohol or sodium formate was used as an added substance. The yield of glutamic acid significantly decreased by the addition of nitrate, and it was little affected by the addition of allyl alcohol. In the presence of formate the yield increased from G = 0.4 (2-oxoglutaric acid 0.05M and ammonium hydroxide 2M) to G = 1.1. As a result, it was found that hydrated electron contributes on the formation of glutamic acid, but hydroxyl radical does not. The yield showed a maximum at ca. 0.1 M ammonium hydroxide concentration. These facts indicate that NH2 radical does not contribute to the formation of glutamic acid. As a reaction mechanism, it can be explained that 2-oxoglutaric acid which had been reduced by hydrated electron reacts with ammonia.     

A peptide containing aspartic acid, glutamic acid and serine in calf brain synaptic vesicles

Free amino acids and other amino compounds in calf brain synaptic vesicles were identified and determined by thin-layer chromatography and ion-exchange chromatography. The vesicles contained ten identified amino acids with glutamic acid, aspartic acid, taurine and gamma-aminobutyric acid in the highest concentrations, and also cysteic acid (or cysteinesulfinic acid), glutamine, alanine, serine, glycine and lysine. The vesicles also contained certain unknown acid-labile, ninhydrin-positive compounds, one of which was a peptide yielding, after acid hydrolysis, about 40% aspartic acid, 30% serine, 15% glutamic acid, 10% glycine and possibly some alanine and lysine. The concentration of the peptide in the vesicles was as high as that of all the other amino compounds together.     

Role of liquid membrane phenomenon in the biological actions of thioridazine

Role of surface activity in the mechanism of action of thioridazine (THR) has been studied. THR has been shown to generate liquid membrane it self and also in association with the relevant membrane lipids, sphingomyelin and cholesterol in series with a supporting membrane. Transport of relevant biogenic amines e.g. dopamine, nor-adrenaline, adrenaline, serotonin, gamma amino butyric acid (GABA) and glutamic acid and ions viz. sodium, potassium, and calcium has been studied in the presence of liquid membranes generated by THR and THE in association with sphingomyelin-cholesterol. The data on modifications in the permeability of relevant biogenic amines and ions indicate that the liquid membranes generated by THR may contribute to the mechanism of action of THR.     

Structural and spectroscopical study of glutamic acid in the nonzwitterionic form

The molecular structure of glutamic acid in the non-zwitterionic form has been optimized by using the semiempirical MNDO and AMI methods and an ab initio calculation at the 4–31G level. The results were compared with previous reported data for other amino acids. The ab initio optimized structure was used as the starting point for a further force field calculation, and vibrational frequencies and theoretical assignments were thus obtained. In order to compare these results with experiments, the FT-IR spectrum of glutamic acid in an argon matrix was recorded and the measured frequencies were successfully compared with theoretical values, which were previously scaled by using a set of scaling factors.     

A GC-ECD method for estimation of free and bound amino acids, gamma-aminobutyric acid, salicylic acid, and acetyl salicylic acid from Solanum lycopersicum (L.)

A gas chromatograph with electron capture detection method for estimation of selected metabolites--amino acids (free and bound), gamma-aminobutyric acid (GABA), salicylic acid (SA), and acetyl salicylic acid (ASA) from tomato--is reported. The method is based on nitrophenylation of the metabolites by 1-fluoro-2, 4-dinitrobenzene under aqueous alkaline conditions to form dinitophenyl derivatives. The derivatives were stable under the operating conditions of GC. Analysis of bound amino acids comprised perchloric acid precipitation of protein, alkylation (carboxymethylation) with iodoacetic acid, vapor-phase hydrolysis, and derivatization with 1-fluoro-2,4-dinitrobenzene in that order. The metabolites were resolved in 35 min, using a temperature-programmed run. The method is rapid, sensitive, and precise. It easily measured the typical amino acids (aspartate, asparagine, glutamate, glutamine, alanine, leucine, lysine, and phenylalanine) used for identification and quantification of a protein, resolved amino acids of the same mass (leucine and isoleucine), satisfactorily measured sulfur amino acid (methionine, cystine, and cysteine), and quantified GABA, SA, and ASA, as well. The developed method was validated for specificity, linearity, and precision. It has been applied and recommended for estimation of 25 metabolites from Solanum lycopersicum (L.).     

Use of high-performance liquid chromatography for assay of glutamic acid decarboxylase. Its limitation in use for post-mortem brain.

Rat brain, obtained 10 min after death, contained high levels of endogenous gamma-aminobutyric acid (GABA) and glutamic acid. Incubation of this brain homogenate at 37 degrees C indicated decrease of GABA with time due to degradation by GABA-transaminase. Reported high-performance liquid chromatographic (HPLC) methods for glutamic acid decarboxylase (GAD) assay depend on the difference between the GABA content of the reaction mixture after and before the incubation period. None of the methods considered the degradation of GABA during incubation. Furthermore, during determination of the Michaelis constant (KM) for the reaction none of them considered the endogenous substrate. Here we have focused on these factors which seriously affect the maximum velocity (Vmax) and KM values during GAD assay by the HPLC technique. By a simple and rapid HPLC technique we have measured GAD activity in post-mortem rat brain after removing endogenous glutamic acid by charcoal treatment and using gabaquline to prevent GABA degradation during incubation period. By this method a Vmax value of 46 +/- 4 nmol/h/mg protein and a KM value of 7.5 +/- 0.6 mM were observed for GAD activity of crude brain homogenate. For a comparative study, we have carried out radiometric assay of GAD activity from the same sample and observed a Vmax of 48 +/- 6 nmol/h/mg protein and KM of 6.9 +/- 0.4 mM.     

Preferred conformers of proteinogenic glutamic acid

The molecular shape of proteinogenic glutamic acid has been determined for the first time. Vaporization of the solid amino acid by laser ablation in combination with Fourier transform microwave spectroscopy made possible the detection of five different structures in a supersonic jet. These structures have been identified through their rotational and (14)N quadrupole coupling constants. All conformers show hydrogen bonds linking the amino and alpha carboxylic group through N-H···O═C (type I) or N···H-O (type II) interactions. In three of them there are additional hydrogen bonds established between the amino group and the carboxylic group in the gamma position. Entropic effects related to the side chain have been found to be significant in determining the most populated conformations.     

Amino acid analysis by ion-exchange chromatography using a lithium elution gradient. Influence of methanol concentration and sample pH.

The separation of amino acids has been achieved on a short column of Chromo-Beads C2 resin, with a lithium gradient-elution system. The analysis took 8 h. The separation of asparagine and glutamine from glutamic acid was highly dependent on the sample pH and on the methanol concentration in the first buffer of the gradient. The method has been applied to analysis of human plasma and granulocytes for amino acids.     

Chemically enhanced liquid chromatography/tandem mass spectrometry determination of glutamic acid in the diffusion medium of retinal cells

A rapid, reproducible and highly sensitive method, based on liquid chromatography mass spectrometry, was developed for the determination of the excitatory amino acid glutamic acid released in the diffusion medium of control, ischemic and mutant cells from retinas. Signal intensity of glutamic acid was enhanced by dansyl chloride derivatization giving rise to a detection limit in the order of pmol/mL. Further, in HPLC-ESI-MS detection an MS-friendly dansyl group to glutamic acid enhanced both ionization efficiency in the ESI source and collision-activated dissociation in the collision cell. The sample processing procedure included liquid-liquid extraction, derivatization with dansyl chloride and a final cation-exchange extraction to generate clean extracts for LC/MS/MS analysis. This approach has been validated as sensitive, linear (20-300 ng/mL), accurate and precise for the differential quantification of glutamic acid in the diffusion medium of retina cells. This is the first report of using chemical derivatization to enhance MS/MS detection of the glutamic acid released in the diffusion medium of wild-type and mutant retina cells, under ischemic conditions.     

Determination of Glutamic Acid Decarboxylase Activity in Subregions of Rat Brain by High Pressure Liquid Chromatography

Abstract

A quantitative high pressure liquid chromato-graphic (HPLC) assay has been developed for the determination of glutamic acid decarboxylase (GAD) activity in subregions of rat brain. GAD activity was determined indirectly by measurement of gamma-aminobutyric acid (GABA). Fluorimetric detection was made possible by derivatization with ortho-phthalaldehyde and the limit of detection was 11 ng GABA.     

Thermoanalytical study of acid-treated clay containing amino acid immobilized on its surface

The aim of this study is to investigate the incorporation of amino acid molecules in an acid-activated montmorillonite by means of solid characterization after the incorporation of these biomolecules. The acid activation procedure was carried out for the purpose of increasing the acid sites in the clay as well as the impurity elimination in the mineral. Cysteine, aspartic, and glutamic acids were adsorbed on montmorillonite K10 which was previously treated with a hydrochloric acid solution. The clay was put in contact with amino acid solutions at two different concentrations. Each amino acid was adsorbed at identical conditions, with the pH fixed to ensure the charge of molecules and surface clay. The solid was characterized by means of X-ray diffraction, infrared spectroscopy, thermogravimetric analysis, and nitrogen adsorption at 77 K. After the amino acid adsorption, the powders showed changes in their characteristics as well as in their thermal behavior, which depended on both the concentration and the nature of the adsorbed amino acid. The thermal decomposition and elimination of cysteine occurred at a higher temperature than the aspartic and glutamic acid; the complete removal of glutamic acid molecules was not observed at 850 °C. The differences observed in the solid characteristic after the adsorption of each amino acid were discussed. Both the thermoanalytical study and characterization of materials after the interaction with amino acid molecules can be useful to understand the adsorption mechanism of biomolecules on solid surfaces.     

In vivo simultaneous monitoring of gamma-aminobutyric acid, glutamate, and L-aspartate using brain microdialysis and capillary electrophoresis with laser-induced fluorescence detection: Analytical developments and in vitro/in vivo validations

gamma-Aminobutyric acid (GABA), glutamate (Glu), and L-aspartate (L-Asp) are three major amino acid neurotransmitters in the central nervous system. In this work, a method for the separation of these three neurotransmitters in brain microdialysis samples using a commercially available capillary electrophoresis (CE) system has been developed. Molecules were tagged on their primary amine function with the fluorogene agent naphthalene-2,3-dicarboxaldehyde (NDA), and, after separation by micellar electrokinetic chromatography, were detected by laser-induced fluorescence using a 442 nm helium-cadmium laser. The separation conditions for the analysis of derivatized neurotransmitters in standard solutions and microdialysates have been optimized, and this method has been validated on both pharmacological and analytical basis. The separation of GABA, Glu, and L-Asp takes less than 10 min by using a 75 mmol/L borate buffer, pH 9.2, containing 70 mmol/L SDS and 10 mmol/L hydroxypropyl-beta-cyclodextrin and + 25 kV voltage. The detection limits were 3, 15 nmol/L and, 5 nmol/L for GABA, Glu, and L-Asp, respectively. Moreover, submicroliter samples can be analyzed. This method allows a simple, rapid and accurate measurement of the three amino acid neurotransmitters for the in vivo brain monitoring using microdialysis sampling.     

Hydrogen bonding between phosphate and amino acid side chains

Hydrogen bonds between polar groups of amino acid side chains (histidine, lysine, glutamic acid) and phosphate ions have been studied by infrared spectroscopy. Proton transfer from amino acid groups to phosphate occur mainly in case that tribasic and dibasic phosphate ions take part in hydrogen bonds. Conformational changes and continuum are strongly related to the degree of proton transfer and hydration. It is pointed out that the aforementioned properties should be of great significance for nucleation and growth of prostatic and renal stones.