大鼠脑组织中不同部位单胺类神经递质含量的HPLC测定

单胺类神经递质,多巴胺(DA)、去甲肾上腺素(NA)和肾上腺素(A)是哺乳动物和人类中枢重要的信息传递物质。了解脑组织中各部位单胺类神经递质的含量对于研究其生理功能、有关疾病的诊断和药物及手术疗效的评价均有重要的意义。     

Determination of selected neurotransmitter metabolites using monolithic column chromatography coupled with chemiluminescence detection

The oxidation of selected clinically important neurotransmitter metabolites with acidic potassium permanganate in the presence of polyphosphates evokes chemiluminescence of sufficient intensity to enable the sensitive determination of these species. Limits of detection for 5-hydroxyindole-3-acetic acid (5-HIAA), vanilmandelic acid (VMA; α,4-dihydroxy-3-methoxybenzeneacetic acid), 4-hydroxy-3-methoxyphenylglycol (MHPG), homovanillic acid (HVA, 4-hydroxy-3-methoxyphenylacetic acid) and 3,4-dihydroxyphenylacetic acid (DOPAC) were between 5 × 10⁻⁹ and 4 × 10⁻⁸ M, using flow-injection analysis methodology. In addition, we demonstrate the rapid determination of homovanillic acid and 5-hydroxyindole-3-acetic acid in human urine - without the need for extraction procedures - using monolithic column chromatography with chemiluminescence detection.     

1,4‐Michael Addition – The Analytical Way for Quantitative Sensing of Neurotransmitter at Bare Gold Electrode in Physiological Solution in the Presence of Interfering Biogenic Compounds

Electrooxidation of norepinephrine in the presence a nucleophile was investigated on a bare gold electrode. Electrochemically produced norepinephrinequinone undergoes an attack by morpholine as nucleophile via 1,4‐Michael addition. The reaction products were identified by electrospray ionization mass spectrometry. The procedure used was suitable for quantitative norepinephrine determination in the concentration range from 1×10^?9 M to 8×10^?4 M with a detection limit of 8.7×10^?10 M in a samples containing an excess of ascorbic and uric acids. The proposed method is simple, green which means that it does not require the use of toxic compounds and solvents and thus is promising for detection of norepinephrine in physiological environment.     

Electrogravimetric Analysis by Quartz-Crystal Microbalance on the Consumption of the Neurotransmitter Acetylcholine by Acetylcholinesterase

Electrogravimetric analysis was performed on the consumption of the neurotransmitter Acetylcholine (ACh) by Acetylcholinesterase (AChE) in situ and in real time. Michaelis-Menten assumption was achieved by using an enzyme micro-reactor in which the total enzyme was anchored in a quartz crystal microbalance chip (QCM-chip) with a strategically engineered self-assembled monolayer (SAM) of alkanethiols, which can prevent diffusion-controlled or spatially restricted kinetics. The real-time frequency changes indicated the rate of the products formation from enzymatic reaction. The QCM-chip was tested showing that it could demonstrate AChE inhibition by physostigmine.     

Quantification of Neurotransmitter Amino Acids in Human Serum by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection is developed as a simple and sensitive method for the quantification of arginine (Arg), tyrosine (Tyr) and glutamic acid (Glu) in human serum. The separation conditions and the derivatization conditions with fluoresceinisothiocyanate (FITC) were investigated. Regression equations revealed a linear relationship (correlation coefficients: 0.9927–0.9998) between the peak area and concentration of each analyte. For the amino acids detected, 10^–10M detection limits were reached, and the levels of these amino acids in human serums were easily determined with recoveries of 93.5–106.5%.     

Rapid labeling of amino acid neurotransmitters with a fluorescent thiol in the presence of o‐phthalaldehyde

LIF detection often requires labeling of analytes with fluorophores; and fast fluorescent derivatization is valuable for high‐throughput analysis with flow‐gated CE. Here, we report a fast fluorescein‐labeling scheme for amino acid neurotransmitters, which were then rapidly separated and detected in flow‐gated CE. This scheme was based on the reaction between primary amines and o‐phthalaldehyde in the presence of a fluorescent thiol, 2‐((5‐fluoresceinyl)aminocarbonyl)ethyl mercaptan (FACE‐SH). The short reaction time (<30 s) was suited for on‐line mixing and derivatization that was directly coupled with flow‐gated CE for rapid electrophoretic separation and sensitive LIF detection. To maintain the effective concentration of reactive FACE‐SH, Tris(2‐carboxyethyl)phosphine was added to the derivatization reagents to prevent thiol loss due to oxidation. This labeling scheme was applied to the detection of neurotransmitters by coupling in vitro microdialysis with online derivatization and flow‐gated CE. It is also anticipated that this fluorophore tagging scheme would be valuable for on‐chip labeling of proteins retained on support in SPE.     

Direct Quantification of Nanoplastics Neurotoxicity by Single-Vesicle Electrochemistry

Nanoplastics are recently recognized as neurotoxic factors for the nervous systems. However, whether and how they affect vesicle chemistry (i.e., vesicular catecholamine content and exocytosis) remains unclear. This study offers the first direct evidence for the nanoplastics-induced neurotoxicity by single-vesicle electrochemistry. We observe the cellular uptake of polystyrene (PS) nanoplastics into model neuronal cells and mouse primary neurons, leading to cell viability loss depending on nanoplastics exposure time and concentration. By using single-vesicle electrochemistry, we find the reductions in the vesicular catecholamine content, the frequency of stimulated exocytotic spikes, the neurotransmitter release amount of single exocytotic event, and the membrane-vesicle fusion pore opening-closing speed. Mechanistic investigations suggest that PS nanoplastics can cause disruption of filamentous actin (F-actin) assemblies at cytomembrane zones and change the kinetic patterns of vesicle exocytosis. Our finding shapes the first quantitative picture of neurotoxicity induced by high-concentration nanoplastics exposure at a single-cell level.     

Specific glycine to alanine mutation eliminates dynamic interaction of polymeric GlyT1a N‐terminus with Coomassie Brilliant Blue G‐250

We previously found that multimeric GlyT1aN16 protein exhibits increased diffusion in a polyacrylamide gel and shows an unusual time‐dependent absorbance rearrangement, as revealed by the Bradford assay. Here, we find that glycine to alanine mutation eliminates the absorbance shift, but not the altered diffusion properties of GlyT1aN16, indicating that these two phenomena are not interconnected. The absorbance shift is apparent with both native and urea‐denatured GlyT1aN16, suggesting that the effect is either not dependent on protein structure, or the required structure is restored very quickly following denaturant removal. In the far‐UV spectra, circular dichroism (CD) curves for both wild‐type and mutated GlyT1aN16 are under the zero line, indicating largely unstructured character. However, a significant shift of the mutant CD curve suggests possible microstructural heterogeneity. Deconvolution of the CD data indicates a potential 3‐fold increase in isolated helical content, which would inhibit an absorbance shift, as we demonstrated previously. These results suggest that, in addition to protein quantification, Coomassie Brilliant Blue G‐250 can be used to reveal certain properties of the secondary structure of proteins.     

Studies on the Electrochemistry of Dopamine at a Pyrocatechol Sulfonephthalein Modified Glassy Carbon electrode

IntroductionThere has been a considerable interest in developing the methods to measure the secretionneurotransmitters. Electrochemical teChniques have proven to be significantly advantageous tothe biosciencesLlj. The application of ultramicroelectrodes to neuroscien.ce, which has been pioneered by Adams[2], to monitor the concentration of neurotransmitters in the central nervesystem has had a special impact. Several neurotransmitters, e. g., dopamine(DA) are electroactlve and therefore can …