Quantification Of Gamma-aminobutyric Acid Research Articles

An Enzyme Assay Kit for GABA Quantification in Plant Tissues.

Gamma-aminobutyric acid (GABA) is an amino acid that has a role as a signaling molecule. In plants, its involvement in stress responses is widely investigated. A newly developed method of quantification of GABA is described in this chapter. The assay kit consisting of three bacterial enzymes enables easy but accurate measurement of GABA (~200mg/mL) based on the serial enzymatic reaction leading to dye formation. The method was successfully applied to measure the GABA content in several plant tissues.

Gamma aminobutyric acid production by commercially available probiotic strains.

Certain bacteria can produce gamma aminobutyric acid (GABA) from glutamate in the human intestinal tract, leading to the possibility of altering GABA levels through diet. To this end, we assessed the ability of seven commercially available probiotic supplements to produce GABA. Probiotic strains were compared for GABA production in pure culture. The bacteria were inoculated at a concentration of 107 CFU ml-1 in 10 ml MRS supplemented with monosodium glutamate (1%w/v), both with and without oligofructose-enriched inulin (OFI) (1%w/v). Two strains with the highest production of GABA were further assessed for 48 h in pH-controlled anaerobic batch cultures inoculated with faecal bacteria. Liquid chromatography-mass spectrometry (LC-MS) was used for quantification of GABA and microbiota composition was determined through 16S rRNA gene sequencing. Levilactobacillus brevis LB01 (CGMCC 16921) and Lactiplantibacillus plantarum 299v (DSM 9843) were the most efficient producers of GABA. High GABA levels (28.32 mmol l-1±0.29) were produced by the probiotic strain L. brevis LB01 at pH 5.4-5.6. This was significantly higher than the levels of GABA produced by L. plantarum (4.8 mmol l-1±6.8) and a negative control (2.9 mM±3.1). The addition of OFI did not further stimulate GABA production under the conditions tested. The ability of these strains to produce GABA in-vitro was further evaluated in a faecal microbiota environment. Once again, L.brevis LB01 produced the highest levels of GABA (40.24 mmol l-1±20.98). L. brevis LB01 was found to be the most efficient probiotic strain, of those tested, for GABA production.

Quantifying GABA in Addiction: A Review of Proton Magnetic Resonance Spectroscopy Studies.

Gamma-aminobutyric acid (GABA) signaling plays a crucial role in drug reward and the development of addiction. Historically, GABA neurochemistry in humans has been difficult to study due to methodological limitations. In recent years, proton magnetic resonance spectroscopy (1H-MRS, MRS) has emerged as a non-invasive imaging technique that can detect and quantify human brain metabolites in vivo. Novel sequencing and spectral editing methods have since been developed to allow for quantification of GABA. This review outlines the clinical research utilization of 1H-MRS in understanding GABA neurochemistry in addiction and summarizes current literature that reports GABA measurements by MRS in addiction. Research on alcohol, nicotine, cocaine, and cannabis addiction all suggest medications that modulate GABA signaling may be effective in reducing withdrawal, craving, and other addictive behaviors. Thus, we discuss how improvements in current MRS techniques and design can optimize GABA quantification in future studies and explore how monitoring changes to brain GABA could help identify risk factors, improve treatment efficacy, further characterize the nature of addiction, and provide crucial insights for future pharmacological development.

Development and validation of a UPLC-MS/MS method for the simultaneous determination of gamma-aminobutyric acid and glutamic acid in human plasma

Gamma-aminobutyric acid (GABA) and its precursor glutamic acid are important neurotransmitters. Both are also present in peripheral tissues and the circulation, where abnormal plasma concentrations have been linked to specific mental disorders. In addition to endogenous synthesis, GABA and glutamic acid can be obtained from dietary sources. An increasing number of studies suggest beneficial cardio-metabolic effects of GABA intake, and therefore GABA is being marketed as a food supplement. The need for further research into their health effects merits accurate and sensitive methods to analyze GABA and glutamic acid in plasma. To this end, an ultra-pressure liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantification of GABA and glutamic acid in human plasma. Samples were prepared by a protein precipitation step and subsequent solid phase extraction using acetonitrile. Chromatographic separation was achieved on an Acquity UPLC HSS reversed phase C18 column using gradient elution. Analytes were detected using electrospray ionization and selective reaction monitoring. Standard curve concentrations for GABA ranged from 3.4 to 2500 ng/mL and for glutamic acid from 30.9 ng/mL to 22,500 ng/mL. Within- and between-day accuracy and precision were <10% in quality control samples at low, medium and high concentrations for both GABA and glutamic acid. GABA and glutamic acid were found to be stable in plasma after freeze–thaw cycles and up to 12 months of storage. The validated method was applied to human plasma from 17 volunteers. The observed concentrations ranged between 11.5 and 20.0 ng/ml and 2269 and 7625 ng/ml for respectively GABA and glutamic acid. The reported method is well suited for the measurement of plasma GABA and glutamic acid in pre-clinical or clinical studies.

Spatial variability and reproducibility of GABA‐edited MEGA‐LASER 3D‐MRSI in the brain at 3 T

The reproducibility of gamma‐aminobutyric acid (GABA) quantification results, obtained with MRSI, was determined on a 3 T MR scanner in healthy adults. In this study, a spiral‐encoded, GABA‐edited, MEGA‐LASER MRSI sequence with real‐time motion–scanner‐instability corrections was applied for robust 3D mapping of neurotransmitters in the brain. In particular, the GABA+ (i.e. GABA plus macromolecule contamination) and Glx (i.e. glutamate plus glutamine contamination) signal was measured. This sequence enables 3D‐MRSI with about 3 cm3 nominal resolution in about 20 min. Since reliable quantification of GABA is challenging, the spatial distribution of the inter‐subject and intra‐subject variability of GABA+ and Glx levels was studied via test–retest assessment in 14 healthy volunteers (seven men–seven women).For both inter‐subject and intra‐subject repeated measurement sessions a low coefficient of variation (CV) and a high intraclass correlation coefficient (ICC) were found for GABA+ and Glx ratios across all evaluated voxels (intra−/inter‐subject: GABA+ ratios, CV ~ 8%–ICC > 0.75; Glx ratios, CV ~ 6%–ICC > 0.70). The same was found in selected brain regions for Glx ratios versus GABA+ ratios (CV varied from about 5% versus about 8% in occipital and parietal regions, to about 8% versus about 10% in the frontal area, thalamus, and basal ganglia).These results provide evidence that 3D mapping of GABA+ and Glx using the described methodology provides high reproducibility for application in clinical and neuroscientific studies.

In vitro/in vivo evaluation of gamma-aminobutyric acid-loadedN,N-dimethylacrylamide-based pegylated polymeric nanoparticles for brain delivery to treat epilepsy

Objectives of this study were the delivery of gamma aminobutyric acid (GABA) into the brain by means of developing brain targeted, nanosized, non-toxic and biocompatible polymeric nanoparticles, and investigating their effectiveness in epilepsy. For this purpose, GABA conjugated N,N-dimethylacrylamide-based pegylated nanoparticles were designed and characterised for particle size, zeta potential, pH, morphology, DSC, XRD, FTIR, GABA quantification and in vitro release. Formulations showed smaller particle size, cationic zeta potential characteristic, possible GABA polymeric matrix interaction and prolonged release pattern. Brain responses were examined using epileptic rats. Both formulations prepared were found to increase latency of seizure, decrease ending time of convulsion, duration of severe convulsion and mortality rate significantly compared with GABA solution. When GABA concentration was measured in Stratum corsatum, there was no statistical difference between GABA solution and formulations. All findings suggested enhancement in all phases of seizures indicating efficient delivery of GABA into the brain via formulations.

Sequential injection for determination of gamma-aminobutyric acid based on its effect on second order light scattering of silver nanoparticles

An automated sequential injection (SI) with second order light scattering (SOS) detection for determination of gamma-aminobutyric acid (GABA) was developed. Quantitation is based on electrostatic interaction between GABA and citrate-capped silver nanoparticles (AgNPs). In acetate buffer at pH 3.8, the positively charged GABA induces the nanoparticles to aggregate. This results in a change of light scattering monitored using a spectrofluorometer. In this work, working standard solutions of GABA were prepared in-line by the SI system pumping appropriate volumes of a stock solution of GABA and acetate buffer into a holding coil. Solution of AgNPs was subsequently drawn into the coil. The reaction zone was then transferred to the spectrofluorometer, set with excitation and detection wavelengths at 300 and 600nm, respectively. Under optimised condition, the SOS intensity was proportional to the concentration of GABA. As a result, a linear curve was obtained in the range of 100–400mgL−1 GABA, with a lower limit of detection of 39.6mgL‐1. Good precision of analysis was achieved, with 0.6 and 3.3% relative standard deviation (RSD) for external calibration (n=5) and standard addition (n=3), respectively. The developed method was successfully applied for quantification of GABA in dietary supplements (2 samples) and samples of instant green tea (2 samples).

Modulation of Corpus Striatal Neurochemistry by Astrocytes and Vasoactive Intestinal Peptide (VIP) in Parkinsonian Rats.

The neurotoxin 6-hydroxydopamine (6-OHDA) is widely used in animal models of Parkinson's disease. In various neurodegenerative diseases, astrocytes play direct, active, and critical roles in mediating neuronal survival and functions. Vasoactive intestinal peptide (VIP) has neurotrophic actions and modulates a number of astrocytic activities. In this study, the effects of VIP on the striatal neurochemistry were investigated in parkinsonian rats. Adult Sprague-Dawley rats were divided into sham-operated, unilaterally 6-OHDA-lesioned, and lesioned + VIP-administered (25ng/kgi.p.) groups. VIP was first injected 1h after the intrastriatal 6-OHDA microinjection and then every 2days throughout 15days. Extracellular striatal concentration of glutathione (GSH), gamma-aminobutyric acid (GABA), glutamate (GLU), and lactate were measured in microdialysates by high-performance liquid chromatography (HPLC). Quantification of GABA and activity dependent neuroprotective protein (ADNP)-expressing cells were determined by glutamic acid decarboxylase (GAD)/ADNP + glial fibrillary acidic protein (GFAP) double immunohistochemistry. Our results demonstrated that a 6-OHDA lesion significantly increased the density of astrocytes in the striatum and VIP treatment slightly reduced the gliosis. Extracellular concentration of GABA, GLU, and lactate levels did not change, but GSH level significantly increased in the striatum of parkinsonian rats. VIP treatment reduced GSH level comparable to sham-operated groups, but enhanced GABA and GLU levels. Our double labeling results showed that VIP primarily acts on neurons to increase ADNP and GAD expression for protection. These results suggest that, in the 6-OHDA-induced neurodegeneration model, astrocytes were possibly activated for forefront defensiveness by modulating striatal neurochemistry.

Effects of Frequency Drift on the Quantification of Gamma-Aminobutyric Acid Using MEGA-PRESS.

The MEGA-PRESS method is the most common method used to measure γ-aminobutyric acid (GABA) in the brain at 3T. It has been shown that the underestimation of the GABA signal due to B0 drift up to 1.22 Hz/min can be reduced by post-frequency alignment. In this study, we show that the underestimation of GABA can still occur even with post frequency alignment when the B0 drift is up to 3.93 Hz/min. The underestimation can be reduced by applying a frequency shift threshold. A total of 23 subjects were scanned twice to assess the short-term reproducibility, and 14 of them were scanned again after 2–8 weeks to evaluate the long-term reproducibility. A linear regression analysis of the quantified GABA versus the frequency shift showed a negative correlation (P < 0.01). Underestimation of the GABA signal was found. When a frequency shift threshold of 0.125 ppm (15.5 Hz or 1.79 Hz/min) was applied, the linear regression showed no statistically significant difference (P > 0.05). Therefore, a frequency shift threshold at 0.125 ppm (15.5 Hz) can be used to reduce underestimation during GABA quantification. For data with a B0 drift up to 3.93 Hz/min, the coefficients of variance of short-term and long-term reproducibility for the GABA quantification were less than 10% when the frequency threshold was applied.

A novel colorimetric method for detection of gamma-aminobutyric acid based on silver nanoparticles

A novel, simple and rapid colorimetric method for determination of gamma-aminobutyric acid (GABA) was developed using a negatively charged citrate-capped silver nanoparticle (AgNP) probe. At an acidic pH of 3.8, GABA had a positive charge due to a protonation of amine groups. Therefore GABA could induce an aggregation of AgNPs due to electrostatic interaction. This caused a decrease in the surface plasmon resonance spectra of the colloidal solution at wavelength of 390nm with a slight red shift. At the same time, the color of the suspension turned from yellow to green upon aggregation. Quantification of GABA was done spectrophotometrically. Under optimum conditions, the method showed a linear calibration in range of 100–500mgL−1 GABA, with a detection limit of 57.7mgL−1. The method was successfully applied to measure GABA quantities in dietary supplements.

Gamma-aminobutyric acid loaded halloysite nanotubes and in vitro-in vivo evaluation for brain delivery

Gamma-aminobutyric acid (GABA) is a key neurotransmitter where it usually inhibits impulse transmission. GABA release blockage or postsynaptic reaction were determined to provoke epileptic convulsions. The aim of the present study was the development of brain-targeted, nanosized, nontoxic, biocompatible, highly specific formulations. Incorporation of GABA into halloysite nanotubes (HNT) was performed using different methods. Particle size, zeta potential and pH measurements, morphological, thermal, XRD, FTIR analyses and GABA quantification by validated HPLC method were used for the characterization of the systems prepared. Release pattern of GABA from the nanotubes was determined using a dialysis membrane.Following successful incorporation of GABA into HNTs for brain delivery, nanotube formulation coded HNT–GABA H1 was selected for in vivo studies. Smaller particle size with narrow size distribution, possible HNT–GABA interaction indicated by thermal, XRD and FTIR analyses and prolonged release were the parameters considered in this selection. Moreover, HNT–GABA H1 remained stable for 3-month storage period and showed higher cell viability values than GABA. Rats were used in in vivo studies and potential of anticonvulsant effect of GABA was determined in the pentylenetetrazole model of seizure. HNT–GABA H1 was found to increase latency of seizure, decrease ending time of the convulsion, duration of severe convulsion and mortality rate significantly compared to pure GABA. After administration of HNT–GABA H1, GABA concentration in Stratum corsatum measured by enzyme immune assay showed that it was not significantly higher than GABA administered alone.These findings suggest that GABA loaded HNTs reduces the duration of all phases of convulsion indicating efficient delivery of GABA to all brain areas to interfere with epileptic mechanism.

HPLC determination of gamma amino butyric acid (GABA) and some biogenic amines (BAs) in controlled, germinated, and fermented brown rice by pre-column derivatization

The gamma amino butyric acid (GABA) and biogenic amines (BAs) have great physiochemical importance due to their unique functions in human biological systems. The current study describes the investigation and separation of GABA and BAs including putrescine (PUT), cadaverine (CAD), dopamine (DOP), tyramine (TYRA), histamine (HIST), spermine (SPM) and spermidine (SPD) in some Pakistani brown rice. The GABA and BAs were analyzed in controlled, germinated and fermented conditions by using high performance liquid chromatographic (HPLC) technique after pre-column derivatization on a reversed phase C-8 column with diode-array detection at 230 nm. 2-Hydroxynaphthaldehyde was used as a pre-column derivatizing agent and the analytes were detected as Schiff base derivatives. A linear regression was found within 2.25–34.5 μg/mL for the standards GABA and DOP, 1.15–28.5 μg/mL for standards PUT, CAD, HIST, and 2.50–48.5 μg/mL for standards TYRA, SPD, SPM with the correlation coefficient (r2) in the range of 0.997–0.998. GABA was detected in controlled, germinated and fermented samples, whereas analyzed biogenic amines were below the detection limit in controlled conditions and their concentration increased with the passage of germination time and also detected after the fermentation process. This method could be used for separation and quantification of GABA and other BAs simultaneously in food samples especially in cereal seeds during germination and fermentation to examine their levels.

Simultaneous HPLC determination of gamma amino butyric acid (GABA) and lysine in selected Pakistani rice varieties by pre-column derivatization with 2-Hydroxynaphthaldehyde

A selective and sensitive HPLC method for the simultaneous determination of gamma amino butyric acid (GABA) and lysine in Pakistani rice varieties was developed. Both analytes were detected in rice varieties as Schiff base derivatives with 2-hydroxynaphthaldehyde. The derivatives were analyzed on a reverse phase C-18 column with Diode Array Detector (DAD) at 254 nm. The calibration curves were found linear over a concentration range of 3.83–34.58 μg/mL for GABA and 5.16–48.68 μg/mL for lysine with a correlation coefficient of 0.998 for both standards. GABA and lysine contents were found higher in brown rice varieties (4.1–6.58 mg/100 g for GABA and 15.1–27.6 mg/100 g for lysine) than the polished varieties (0.32–0.47 mg/100 g GABA and 13.1–19.8 mg/100 g lysine). This method could be used for separation and quantification of GABA and lysine simultaneously in food samples, especially in cereal seeds.

Determination of -Aminobutyric Acid in Food Matrices by Isotope Dilution Hydrophilic Interaction Chromatography Coupled to Mass Spectrometry

The estimation of the dietary intake of gamma-aminobutyric acid (GABA) is dependent upon the knowledge of its concentration values in food matrices. To this end, an isotope dilution liquid chromatography-mass spectrometry method has been developed employing the hydrophilic interaction chromatography technique for analyte separation. This approach enabled accurate quantification of GABA in apple, potato, soybeans, and orange juice without the need of a pre- or post-column derivatization reaction. A selective and precise analytical measurement has been obtained with a triple quadrupole mass spectrometer operating in multiple reaction monitoring using the method of standard additions and GABA-d(6) as an internal standard. The concentrations of GABA found in the matrices tested are 7 microg/g of apple, 342 microg/g of potatoes, 211 microg/g of soybeans, and 344 microg/mL of orange juice.

In vivo quantification of intracerebral GABA by single-voxel 1H-MRS—How reproducible are the results?

Gamma aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the human brain. It plays a decisive role in a variety of nervous system disorders, such as anxiety disorders, epilepsy, schizophrenia, insomnia, and many others. The reproducibility of GABA quantification results obtained with a single-voxel spectroscopy J-difference editing sequence with Point Resolved Spectroscopy localization (MEGA-PRESS) was determined on a 3.0 Tesla MR scanner in healthy adults. Eleven volunteers were measured in long- and short-term intervals. Intra- and inter-subject reproducibility were evaluated. Internal referencing of GABA+ to total creatine (tCr) and water (H 2O), as well as two different post-processing methods for the evaluation (signal integration and time-domain fitting) were compared. In all subjects lower coefficient of variation and therefore higher reproducibility can be observed for fitting compared to integration. The GABA+/tCr ratio performs better than the GABA+/H 2O ratio or GABA+ without internal referencing for both fitting and integration (GABA+/tCr: 13.3% and 17.0%; GABA+/H 2O: 15.0% and 17.8%; GABA+: 19.2% and 21.7%). Four-day measurements on three subjects showed higher intra- than inter-subject reproducibility (GABA+/tCr ∼10–12%). With a coefficient of variation of about 13% for inter-subject and 10–12% for intra-subject variability of GABA+/tCr, this technique seems to be a precise tool that can detect GABA confidently. The results of this study show the reproducibility limitations of GABA quantification in vivo, which are necessary for further clinical studies.

The quantification of gamma-aminobutyric acid in the cerebrospinal fluid by a radioreceptorassay

The development of a radioreceptor assay designed to measure gammaaminobutyric acid (GABA) in CSF is described. The method is based on the presence of high affinity and selective GABA binding sites obtained from rat brain membrane preparations treated with 0.05% Triton X-100. The optimum protein concentration in the incubation medium, the duration of incubation to reach equilibrium, the temperature and the optimum pH are discussed. The standard curve permits measurements in the range 35 to 2250 nmol/l GABA. The imprecision of the method calculated from three different concentrations: 1125, 562 and 281 nmol/l shows coefficients of variation for ‘within’ and ‘between’ assays, between 5.2% and 9.3% and between 7.4% and 12.4%, respectively. The percentage of recovery is 102 ± 3.3% ( n = 4). This radioreceptorassay has a sensitivity of 14 nmol/l. The method is easy, rapid and not expensive. It enables analysis of small volumes of CSF (200 μl). Several samples (> 20) can be analysed in the same run in about one hour.