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  • Excessive extracellular glutamate can be removed from brain


    Excessive extracellular glutamate can be removed from Pyrimethamine receptor interstitial fluids to the blood plasma for the maintenance of proper extracellular glutamate homeostasis in the mammalian central nervous system [[5], [6], [7]]. The glutamate concentration in the blood plasma increases in case of ischemic stroke and other neurological disorders [8]. The traditional methods of glutamate determination include high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and spectrophotometry. These techniques are sensitive and powerful, but they require very expensive and complex equipment that limits their application in the laboratory work and monitoring kinetics of neurotransmitter release/uptake in clinics [9]. The electrochemical biosensors for the glutamate determination are faster, more user-friendly and cheaper than the traditional methods [10]. Furthermore, the biosensors can be miniaturized for the detection of glutamate in living tissues that cannot be achieved by other methods [11]. Thus, the development and application of the glutamate-sensitive biosensors is a new perspective for simplifying analysis procedure and decreasing its price that can result in their wider involvement in the neurochemical research. Currently, a number of glutamate-sensitive biosensors were developed. They are based on glutamate oxidase (GluOx) [12,13] or glutamate dehydrogenase (GLDH) [14,15]. Both enzymes oxidize glutamate to ketoglutarate, although the first enzyme also generates hydrogen peroxide, whereas the second one reduces a cofactor (NAD+). Both biosensor types appeared to be efficient in the determination of glutamate concentration in biological and food samples. However, GLDH requires the addition of the factor to the working buffer or its co-immobilization with the enzyme. This fact makes the GLDH-based biosensor more complex in comparison with the GluOx-based one. Furthermore, the stability of the GluOx-based biosensor is much better [[16], [17], [18], [19]]. In our previous study, we developed a biosensor-based method for monitoring the rate of glutamate uptake that takes into consideration the extracellular level of endogenous glutamate in the preparations of nerve terminals [20].
    Materials and methods
    Discussion In comparison to the classical analytical methods of glutamate determination (i.e. spectrophotometry, different types of chromatography and radiolabeled technique), the proposed glutamate biosensor-based approach for the analysis of glutamate release has several advantages. The biosensor does not require radiolabeled L-[14C]glutamate preloading or other additional procedures, and thus the synaptosomal samples can be analyzed without pretreatment. The biosensor directly measures changes in the absolute values of endogenous glutamate concentrations in nerve terminals in response to different stimulation. Beside the experimental laboratory work, the glutamate biosensor-based approach can be applied in clinics for neuromonitoring the glutamate-related parameters and extracellular glutamate concentration in the brain samples, liquids and also in the blood plasma. The cost of overall measuring setup (including the biosensor, a potentiostat and auxiliary equipment) is quite small (about 4000 €) that is far lower than a cost of any classical method. The measuring setup is portative, and can be transported to another institution for on-site determination of the glutamate concentration in order to avoid storage and transportation of the synaptosomal samples. In medical practice during neuromonitoring, the transportability and portability of the biosensor is also an advantage. The biosensor analysis is very quick (several minutes for one sample). The biosensor is very specific to glutamate because the enzyme, GluOx, specifically catalyzes only glutamate oxidation, and the working electrode is covered with a semipermeable membrane that prevents oxidation of interfering compounds. This high selectivity was confirmed by comparison of the results with the traditional scintillation method (based on L-[14C]glutamate), spectrofluorimetry (glutamate dehydrogenase-based) and amino acid analyzer in the present and our previous works [20].