Analysis Of Amino Acid Neurotransmitters Research Articles

A new approach for the analysis of amino acid neurotransmitters in mouse brain tissues using DESI imaging

Mass spectrometry imaging using desorption electrospray ionization (DESI imaging) is a method with the potential to investigate the distribution of neurotransmitters (biogenic amines and amino acids) in brain tissues. Because of the low ionization efficiency of neurotransmitters in direct tissue analysis by DESI, neurotransmitters can be analyzed using derivatization protocols that insert a known compound into their structures through a chemical reaction due to signal suppression by lipids. In this sense, this work aims to combine a chemical derivatization protocol with DESI imaging in order to improve the analysis sensitivity of the amino acids glycine (Gly), alanine (Ala), dopamine (Dop), and gamma-aminobutyric acid (GABA) in mouse brain tissues, as well as to apply this protocol to analyze mice with Parkinson's disease. Derivatization took place using the reagent 2,4,6-triphenylpyrylium tetrafluoroborate (TPP+) and was first investigated in a solvent to ensure the detection of the expected products. Subsequently, the brain tissue surface was subjected to the derivatization solution through spraying. Then, the tissue was analyzed by DESI, and the derivatized Gly, Ala, Dop, and GABA were detected. DESI imaging analysis demonstrated distinct spatial distributions for these derivatized amino acid neurotransmitters. The application of the protocol in brain sections from mice with Parkinson's disease demonstrated differences in the intensities and anatomical distribution for the derivatized amino acids compared with control tissues (unaffected by Parkinson's). The protocol combining derivatization with DESI imaging described in this work emerges as a potential tool for analyzing Parkinson's disease biomarkers in mouse brain tissues.

Combining the Katritzky Reaction and Paper Spray Ionization Mass Spectrometry for Enhanced Detection of Amino Acid Neurotransmitters in Mouse Brain Sections.

This work describes the development of a system that combines a derivatization protocol based on the Katritzky reaction with paper spray ionization mass spectrometry (PSI-MS) for the analysis of amino acid neurotransmitters in mouse brain tissues. The system is relatively simple, consisting of spraying the derivatization solution onto a mouse brain section mounted on a glass slide, applying a small volume of solvent to moisten the sample, pressing a triangular paper onto the sample surface to transfer the sample constituents to the paper surface, and using the paper as a substrate for PSI-MS analysis. The Katritzky reaction facilitated the ionization of the amino acids by reacting a pyrylium salt with the amino group of the analytes, forming very stable pyridinium cations, which greatly increased the sensitivity of the PSI-MS analysis. Most of the intensities of the amino acids modified by the Katritzky reaction were more than 10 times greater than the nonderivatized ones. The system was applied for the analysis of brain sections obtained from mice with Parkinson's disease, and the amino acids gamma-aminobutyric acid (GABA) and glycine (Gly), two compounds very well-known in studies of Parkinson's disease, were readily detected. The results suggest that the Katritzky reaction combined with PSI-MS might offer a significant advance in the knowledge on protocols that improve the sensitivity of detection of crucial biological compounds.

Analysis of amino acid neurotransmitters from rat and mouse spinal cords by liquid chromatography with fluorescence detection

A RP-LC-FL detection method has been developed to identify and quantitate four amino acid neurotransmitters including glutamic acid, glycine, taurine and γ-aminobutyric acid in rat and mouse spinal cord tissue. 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) was employed for the derivatization of these neurotransmitters prior to RP-LC-FL analysis. Different parameters which influenced separation and derivatization were optimized. Under optimum conditions, linearity was achieved within the concentration ranges of 0.50–50.00μM for all analytes with correlation coefficients from 0.9912 to 0.9997. The LODs ranged from 0.03μM to 0.06μM. The proposed method has been successfully applied to the determination of amino acid neurotransmitters in biological samples such as rat and mouse spinal cord with satisfactory recoveries.

Chiral analysis of amino acid neurotransmitters and neuromodulators in mouse brain by CE-LIF.

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 β-CD, 6-monodeoxy-6-mono(3-hydroxy)propylamino-β-CD (HPA-β-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-β-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.

Development of a PDMS‐based microchip electrophoresis device for continuous online in vivo monitoring of microdialysis samples

A PDMS-based microfluidic system for online coupling of microdialysis sampling to microchip electrophoresis with fluorescence detection for in vivo analysis of amino acid neurotransmitters using naphthalene-2,3-dicarboxaldehyde and sodium cyanide as the derivatization reagents is described. Fabricating chips from PDMS rather than glass was found to be simpler and more reproducible, especially for chips with complex designs. The microchip incorporated a 20-cm serpentine channel in which sample plugs were introduced using a "simple" injection scheme; this made fluid handling and injection on-chip easier for the online system compared with gated or valve-based injection. The microchip was evaluated offline for the analysis of amino acid standards and rat brain microdialysis samples. Next, precolumn derivatization was incorporated into the chip and in vivo online microdialysis-microchip electrophoresis studies were performed. The system was employed for the continuous monitoring of amino acid neurotransmitters in the extracellular fluid of the brain of an anesthetized rat. Fluorescein was dosed intravenously and monitored simultaneously online as a marker of in vivo blood-brain barrier permeability. The microdialysis-microchip electrophoresis system described here will be employed in the future for simultaneous monitoring of changes in blood-brain barrier permeability and levels of amino acid neurotransmitters in the rat stroke model.

Analysis of amino acid neurotransmitters in hypothalamus of rats during cerebral ischemia–reperfusion by microdialysis and capillary electrophoresis

In this work, focal cerebral ischemia and reperfusion were induced by the model of middle cerebral artery occlusion. The dialysate of extracellular fluid in the hypothalamus of rats were obtained by using brain microdialysis technique. An efficient and sensitive MEKC method for the simultaneous determination of multiple amino acid neurotransmitters in microdialysate was developed by capillary electrophoresis with laser-induced fluorescence detection and 5-(4, 6-dichloro-s-triazin-2-ylamino) fluorescein derivatization. Different parameters that influenced derivatization reaction and CE separation were studied and optimized. This method was used to investigate the dynamic change of fourteen amino acid neurotransmitters in microdialysates during cerebral ischemia/reperfusion period. Our results reveal that MCAO and reperfusion elicited significant increases in the extracellular levels of Arg, Lys, Trp, Phe, Gln, GABA, Asn, Pro, Ser, Ala, Tau, Gly, Glu and Asp. The excitatory/inhibitory neurotransmitter balance was disturbed during ischemia/reperfusion. The dynamic changes and functional status of releasable neurotransmitters during ischemia/reperfusion were discussed.

In-capillary derivatization and capillary electrophoresis separation of amino acid neurotransmitters from brain microdialysis samples

A new in-capillary derivatization method with naphtalene-2,3-dicarboxyaldehyde (NDA)/CN − has been developed for capillary electrophoresis with laser-induced fluorescence detection of brain microdialysate amino acids. Samples are sandwiched between two plugs of reagent mixture at the capillary inlet and subsequently separated. Highest derivatization yields are obtained by using a reagent to sample plug length ratio equal to 4, performing a first electrophoretic mixing followed by a zero potential amplification step before applying the separation voltage and using a NaCN to NDA concentration ratio equal to 1. This new single-step methodology allows the analysis of amino acid neurotransmitters in rat brain microdialysis samples.

Analysis of amino acid neurotransmitters by capillary electrophoresis and laser-induced fluorescence using a new fluorescein-derived label

A capillary electrophoresis laser-induced fluorescence detection method (CE-LIF) was developed for the separation of eight neurotransmitters tagged on their amino function with 6-oxy-(N-succinimidyl acetate)-9-(2′-methoxycarbonyl) fluorescein (SAMF), a new fluorescent reagent synthesized in our lab. Derivatization was performed in boric acid buffer (pH = 7.75) at 37 °C over 15 min. The pH-independent fluorescence of SAMF (pH 4–9) permits background buffers over a wide range of pH. It was demonstrated that an acidic running buffer offers a better resolution compared to basic medium in terms of resolution and peak shapes. Employing Cu2+ as the additive, the molecules were baseline-separated using a running buffer consisting of 40 mM sodium acetate and 2 mM Cu2+ (pH 6.0). The detection limits ranged from 1 to 2 × 10−10 M. The method has been validated for the characterization of lymphocyte samples. The results obtained illustrate the advantages of combining SAMF derivatization with CE-LIF for determining neurotransmitters.

Analysis of trace amino acid neurotransmitters in hypothalamus of rats after exhausting exercise using microdialysis

A simple but effective coupling of microdialysis and capillary electrophoresis with laser induced fluorescence detection technique was applied to analysis of amino acid neurotransmitters in the hypothalamus of rats after acute exhausting exercise. The separation of amino acids was achieved using an uncoated fused-silica capillary (57 cm×75 μm I.D.) with a buffer of 10 m M disodium tetraborate at pH 10 and an applied voltage of 12.5 kV. The detection limit was 10 −10 M for each amino acid. It is sufficiently sensitive and rapid for the determination of amino acids in a 5-μl Microdialysate. In comparison to pre-exercise, a significant increase in the levels of six hypothalamic amino acids (arginine, glycine, lysine, glutamic acid, alanine, γ-amino- n-butyric acid) was found after exercise. These results demonstrate that the increase of metabolic amino acids in the hypothalamus of rats can be induced by exhausting exercise and suggests that amino acid neurotransmitters may play functional roles in the central effects of exercise.