University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Website: @unf

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Classification: Biological tissue -> brain -> rat

Citations 24

"Liquid Chromatography - Electrochemical Detector For The Determination Of Glucose In Rat Brain Combined With In Vivo Microdialysis"
Anal. Chim. Acta 2000 Volume 413, Issue 1-2 Pages 131-136
Guoyue Shi, Jiaxing Lu, Fang Xu, HongGuo Zhou, Litong Jin and Jiye Jin

Abstract: In this paper, microdialysis sampling was combined with liquid chromatography with electrochemical detection (LC-ECD) and glucose in rat brain was determined. A cobalt tetraaminophthalocyanine (CoTAPC)/glucose oxidase (GOD)/Nafion film-coated glassy carbon electrode was used to selectively detect the hydrogen peroxide generated enzymatically in the enzyme reactors, without any interference from electroactive species such as L-ascorbate, mate, proteins. The signal current was linearly related to the glucose concentration in the range of 1.0 x 10^-6-5.0 x 10^-3 mol/l at +0.5 V (versus Ag/AgCl) with a detection limit of 5.0 x 10^-7 mol/l. To measure glucose in rat brain, microdialysis sampling was employed. The dialysate from the microdialysis system was injected into system. The result was 0.74±0.10 mmol/l (n=4).
Glucose Electrode Potentiometry Interferences

"Direct Coupling Of Intracerebral Dialysis With Flow Injection Analysis Based On Enzymatic/fluorescence Detection Of Lactic Acid"
Anal. Chim. Acta 1988 Volume 205, Issue 1-2 Pages 53-59
Werner G. Kuhr and Jakob Korf

Abstract: Dialysis was carried out in the striatum of a live rat with use of Hertz medium (equilibrated with 95% of O - 5% of CO2) at 10 µL min-1. The dialysate was mixed with a reagent stream (50 µL min-1) containing 5 µg mL-1 of lactate dehydrogenase and 0.5 mM NAD+ in Na2CO3 buffer of pH 9.5, and the NADH produced was determined fluorimetrically at 450 nm (excitation at 340 nm). The calibration graph was rectilinear in the range 16 to 160 µM-lactic acid. Response time was ~40 s, and real-time measurement of transient changes in anaerobic metabolism was thus possible, as illustrated after electrically induced seizures.
Lactic acid Clinical analysis Fluorescence Dialysis

"Enzyme-modified Amperometric Sensors For Choline And Acetylcholine With Tetrathiafulvalene Tetracyanoquinodimethane As The Electron-transfer Mediator"
Anal. Chim. Acta 1997 Volume 341, Issue 1 Pages 43-51
Quan Xin and R. Mark Wightman*

Abstract: Amperometric biosensors for choline and acetylcholine were prepared by immobilizing choline oxidase and choline oxidase/acetylcholinesterase, respectively, over tetrathiafulvalene tetracyanoquinodimethane crystals packed into a cavity at the tip of a carbon-fiber electrode (16.5 µm radius). The immobilization matrix consisted of dialdehyde starch/glutaraldehyde, and the sensors were covered with an outer Nafion membrane. The amperometric performance of the sensors was studied with use of an FIA system, an applied potential of +100 mV vs. SCE (Pt-wire auxiliary electrode) and a carrier flow rate of 1 ml/min. The choline and acetylcholine biosensors exhibited linear responses up to 100 µM and 50 µM, respectively, and the response times were ~8.2 s. The choline biosensor was implanted into a rat-brain slice (400 µm thickness) immersed in Krebs buffer and used to monitor the diffusion of choline into the tissue.
Choline Acetylcholine Amperometry Electrode Electrode Sensor Electrode Apparatus

"Characterization Of Amperometry For In Vivo Measurement Of Dopamine Dynamics In The Rat Brain"
Talanta 1994 Volume 41, Issue 6 Pages 865-874
Kirk T. Kawagoe and R. Mark Wightman*

Abstract: A comparison of constant-potential amperometry and fast-scan cyclic voltammetry for in vivo monitoring is reported. The carbon fiber micro-electrodes (diameter 10 µm) were coated with 2.5% Nafion solution and dried before use in conjunction with a Na-saturated SCE in a FIA system for measuring the response of dopamine neurons to electrical stimulation at 10^-60 MHz. Amperometric response was more rapid than voltammetric, but interpretation of the in vivo data based on in vitro calibration was more difficult. Amperometry also had the disadvantage that the diffusion layer was less well defined. The effect of ascorbate was greater in amperometry that in fast-scan voltammetry.
Dopamine Amperometry Voltammetry Electrode In vivo monitoring

"Strategies For Low Detection Limit Measurements With Cyclic Voltammetry"
Anal. Chem. 1991 Volume 63, Issue 24 Pages 2965-2970
Donna J. Wiedemann, Kirk T. Kawagoe, Robert T. Kennedy, Edward L. Ciolkowski, and R. Mark Wightman

Abstract: Cyclic voltammetry of Nafion-coated, carbon-fiber electrodes is used to detect trace concentrations of dopamine, both in a flow injection apparatus and in the brain of an anaesthetized rat. To improve signal-to-noise ratios, the sources of noise during cyclic voltammetry have been determined and strategies have been developed to decrease the noise. With the potentiostat employed, the measured noise is comparable to that expected for Johnson noise from the feedback resistor of the current transducer. Additional noise arises from the waveform generator employed and, in some cases, line noise. Line noise is discriminated against by starting each cyclic voltammogram either in phase or 180 degrees out of phase with the line frequency. When used in vivo, additional noise also arises from the physiological activity of the animal. Detection limits are found to closely correspond to those predicted on the basis of simulation of the voltammetric shape and the measured noise. Detection limits are improved by the use of appropriate analog and digital filtering, ensemble averaging, and appropriate timing of repetitive cyclic voltammograms. The combined use of these techniques enables the in vivo detection of approximately 100 nM of dopamine with a signal-to-noise ratio of 25. Carbon-fiber micro-electrodes coated with Nafion were used in conjunction with a calomel reference electrode for the cyclic voltammetric determination of dopamine, both by flow injection analysis with 150 mM NaCl - 20 mM HEPES buffer of pH 7.4 (for calibration) and in vivo in rat brain. For in vivo measurements, voltammograms were recorded at 100-ms intervals at a scan rate of 300 V s-1, line noise being discriminated against. By optimum procedures of filtering, averaging and timing, ~100 nM-dopamine could be detected in vivo with a signal-to-noise ratio of 25.
Dopamine Electrode Electrode Voltammetry Signal to noise ratio Digital filter

"Enzyme Packed Bed System For The Online Measurement Of Glucose, Glutamate And Lactate In Brain Microdialysate"
Anal. Chem. 1992 Volume 64, Issue 17 Pages 1790-1794
Martyn G. Boutelle, Lesley K. Fellows, and Colin Cook

Abstract: An automated immobilized enzyme-based flow injection assay system is described which uses oxidase and peroxidase to convert the analyte into an oxidized ferrocene species which is assayed by HPLC with reductive electrochemical detection. Glucose oxidase, glutamate oxidase, lactate oxidase and horse-radish peroxidase were immobilized on to tresyl-activated silica beads (10 µm, 500 and 1000 Å). A HPLC guard column (2 cm x 2 mm) was packed with the beads and connected to a HPLC pump and an online microdialysis system, used to control sampling. The mobile phase was 0.05 M Na2HPO4 - 0.5 mM ferrocenemonocarboxylic acid - 1 mM EDTA adjusted to pH 7 with HCl. The assays have detection limits of 1.8 to 20 pmol and were configured to have linear calibration responses over the range of basal and stimulated levels of glucose, glutamate and lactate in microdialysate samples from rat striatum. The assays were carried out at 2.5-min intervals. Rapid measurement of glucose, glutamate, and lactate is important in understanding the dynamics of the energy balance of the brain. Glutamate is also the main excitatory neurotransmitter. A general immobilized enzyme-based flow injection assay system is described which uses oxidase and peroxidase enzymes to convert the analyte into an oxidized ferrocene species which is detected electrochemically by reduction. The enzymes glucose oxidase, glutamate oxidase, lactate oxidase, and horseradish peroxidase are immobilized with near 100% efficiency onto 10-microns tresyl-activated silica beads (1000- and 500-A pore size). The beads are slurry-packed into 2- x 20-mm columns to give beds for glucose, glutamate, or lactate which are stable for greater than 40 days. The flow injection assays described have detection limits from 1.8 to less than 20 pmol and have been configured to have linear calibration responses over the range of basal and stimulated levels of the three compounds found in 5 µL microdialysate samples from the rat striatum. The assays are used for automated online measurement of glucose, glutamate, and lactate in striatal microdialysate at 2.5-min intervals.
Glucose Glutamate Lactate Electrochemical analysis Immobilized enzyme Dialysis

"Determination Of Acetylcholine And Choline In Rat Brain Tissue By FIA With Immobilized Enzymes And Chemiluminescence Detection"
Microchem. J. 1996 Volume 53, Issue 3 Pages 290-295
Wenzhe Fan and Zhujun Zhang

Abstract: Brain tissue (prep. details given) was homogenized with a 10-fold volume of 0.6 M HClO4, the homogenates were kept on ice for 30 min and centrifuged at 2000 g for 20 min. The pellets were discarded and the supernatants were neutralized with 2 M K2CO3 of pH 4.2. Portions of neutralized supernatant were injected into a carrier stream of 0.05 M Tris hydrochloride buffer (0.6 ml/min) in a flow injection manifold (schematic shown) and the stream was passed through two reactors containing acetylcholinesterase and choline oxidase immobilized on glass beads (30 x 2 mm i.d.). The resultant H2O2 (H2O2)-containing analyte stream was merged with streams of 1 mM luminol in 0.1 M NaOH and 25 µM-CoCl2 (both at 2.5 ml/min) and the chemiluminescence intensity of the H2O2 was measured. The calibration graphs were linear from 1 pmol to 1 nmol and 1 pmol to 10 nmol, respectively, for acetylcholine (I) and choline (II), and the corresponding detection limits were 600 and 500 fmol. Interference levels for eight compounds (listed) commonly found in neuronal tissue in the analysis are listed. Results for I and II in total brain were in good agreement with those obtained by HPLC.
Choline Acetylcholine Chemiluminescence Immobilized enzyme Interferences Method comparison Glass beads

"An Enzyme-reactor For Electrochemical Monitoring Of Choline And Acetylcholine: Applications In High Performance Liquid Chromatography, Brain Tissue, Microdialysis And Cerebrospinal Fluid"
Anal. Biochem. 1992 Volume 204, Issue 2 Pages 305-310
Frans Flentge, Kor Venema, Tineke Koch and Jakob Korf

Abstract: A sandwich-type enzyme reactor in which the enzymes are physically immobilized in a minimal dead space between two cellulose membranes, resulting in improved sensitivity, was developed for the electro-chemical detection of choline (Ch) and acetylcholine (ACh). The reactor contains the enzymes choline oxidase with or without acetylcholine esterase, for the detection of ACh and Ch, respectively. For the HPLC analysis of Ch and ACh the detection system was coupled post-column. Levels of Ch and ACh of rat striatum tissue and human cerebrospinal fluid were found to be similar to those determined with published methods. Because of low back pressure--a further advantage of the reactor--the detection system could also be directly coupled to the outlet of a microdialysis device, allowing the online real-time measurement of extracellular brain Ch. The versatility of the enzyme reactor for the monitoring of analytes in HPLC eluates, flow injection analysis, with or without prepurification, is emphasized. The usefulness of the reactor-detector system in biomedical applications is illustrated by the measurement of increases of rat striatal extracellular Ch following cardiac arrest.
Choline Acetylcholine HPLC Electrochemical analysis Post-column derivatization Immobilized enzyme Dialysis

"Online Amperometric Assay Of Glucose, L-glutamate, And Acetylcholine Using Microdialysis Probes And Immobilized Enzyme Reactors"
Electroanalysis 1995 Volume 7, Issue 12 Pages 1114-1117
Toshio Yao, Seita Suzuki, Hirohito Nishino, Taketoshi Nakahara

Abstract: A highly selective online, real-time monitoring system is proposed for amperometric assay of glucose, L-glutamate, and acetylcholine. The system includes a microdialysis probe, immobilized enzyme reactor, and poly(1,2-diaminobenzene)-coated platinum electrode. The analyte in the dialysate from the microdialysis probe is enzymatically converted to produce hydrogen peroxide. The hydrogen peroxide is detected selectively at a poly(1,2-diaminobenzene)-coated platinum electrode, without any interference from oxidizable species and proteins. The present method can be successfully applied to in vitro assay of glucose and in vivo monitoring of glucose in rat brains. However, the sensitivity is not sufficient for in vivo monitoring of trace amounts of L-glutamate and acetylcholine in rat brains. (11 references)
Acetylcholine Glucose l-Glutamate Amperometry Electrode Electrode Immobilized enzyme Sensitivity Interferences Dialysis

"High Performance Liquid Chromatographic Determination Of Substance P-like Arginine-containing Peptide In Rat Brain By Online Post-column Fluorescence Derivatization With Benzoin"
J. Chromatogr. B 1989 Volume 490, Issue 1 Pages 301-310
Masahiro Ohno, Masaaki Kai and Yosuke Ohkura

Abstract: Brain homogenate, containing [D-Phe11]neurotensin as internal standard, was deproteinized by centrifugation with acetone. The supernatant solution was mixed with 1 M NaHCO3 (0.22 ml) and 0.1 M Na2EDTA (0.5 ml), the ppt. was removed by centrifugation, the acetone was evaporated off and the residue was dissolved in water (2 ml). The solution was cleaned up on a Bond Elut C18 cartridge (details given), the eluate was evaporated and the residue was dissolved in water. A portion (100 µL) of solution was analyzed by HPLC on a column (20 cm x 4 mm) of TSKgel ODS-120T (5 µm) with gradient elution (1 mL min-1) with acetonitrile - 0.3 M sodium phosphate buffer (pH 2.3) - water. The eluate was mixed with a stream (1 mL min-1) of 2 mM benzoin - 1.6 M KOH - 0.7 M 2-mercaptoethanol in a PTFE coil (15 m x 0.3 mm) at 76°C and then with a stream (0.4 mL min-1) of 0.5 M Tris - 2.1 M HCl before fluorimetric detection at 435 nm (excitation at 325 nm). The calibration graph was rectilinear up to 320 pmol of substance P. The detection limit for substance P-like arginine-containing peptide was 580 fmol mg-1 of protein.
Arginine Peptides HPLC Fluorescence Post-column derivatization Buffer Heated reaction

"Analysis Of 6R- And 6S-tetrahydrobiopterin And Other Pterins By Reversed-phase Ion-pair Liquid Chromatography With Fluorimetric Detection By Post-column Sodium Nitrate Oxidation"
J. Chromatogr. B 1993 Volume 617, Issue 2 Pages 249-255
Yoshihiro Tani* and Tomochika Ohno

Abstract: A mixture of D-neopterin, biopterin, pterin, 7,8-dihydropterin, 6R-L-erythro-5,6,7,8-tetrahydrobiopterin and 6S-L-erythro-5,6,7,8-tetrahydrobiopterin was analyzed by HPLC at 40°C on a column (25 cm x 4.6 mm) of Cosmosil 5C18 equipped with a guard column (5 cm x 4.6 mm) of the same packing. Elution (1 ml/min) was effected with 0.1 M phosphate buffer of pH 3 containing 5% methanol, 3 mM sodium octylsulfate, 0.1 mM Na2EDTA and 0.1 mM ascorbic acid. Post-column derivatization was achieved by mixing the eluate with a stream of 5 mM NaNO2 (1 ml/min) before passing through a reaction coil (no dimensions given) at 80°C; fluorescent detection was performed at 440 nm (excitation at 350 nm). The calibration graphs were linear from 0.02-5 ng of each compound injected, with detection limits of 10^-20 pg. Recoveries from tissues ranged from 84.7-99.6% and the within-assay RSD were 0.7-3.7%. The method was applied to the analysis of homogenates of rat liver, kidney and 6 regions of the brain.
d-Neopterin Biopterin Pterin 7,8-Dihydropterin 6R-L-erythro-5,6,7,8-tetrahydrobiopterin 6S-l-erythro-5,6,7,8-tetrahydrobiopterin Fluorescence HPLC Post-column derivatization Heated reaction

"Determination Of Seven Opioid Peptides In Rat Brain By High Performance Liquid Chromatography With Online Post-column Fluorescence Derivatization"
Anal. Sci. 1990 Volume 6, Issue 5 Pages 671-676

Abstract: Sample solution (prep. described; 60 µL) was incubated with 10 µL of carboxypeptidase A or trypsin in 50 mM phosphate buffer (pH 7.8) and phosphate buffer (40 µL) for 30 min at 37°C. A 100 µL portion was analyzed by HPLC on a column (15 cm x 6 mm) of Asahipak ODP-50 (5 µm) with gradient elution (1 mL min-1) with acetonitrile - 50 mM sodium borate buffer (pH 10.0) - water (1:4:15 and 3:1:1). The column eluate was mixed with 0.3 M borate buffer (pH 8.5) and 8 mM hydroxylamine oxalate - 0.2 mM Co(II) acetate, the mixture was passed through a reaction coil (10 m x 0.5 mm) heated to 100°C, and the fluorescence was monitored at 430 nm (excitation at 330 nm). The method was used for the simultaneous determination of seven endogenous opioid peptides at the pmol level in brain tissue. Detection limits were 0.5 to 1.5 pmol injected.
Peptides HPLC Fluorescence Post-column derivatization Buffer pH Column Mixing Detection limit

"Assay For Enkephalin-generating Enzyme In Rat Brain Tissues By High Performance Liquid Chromatography With Post-column Fluorescence Derivatization"
Anal. Sci. 1991 Volume 7, Issue 4 Pages 561-565

Abstract: A simple and sensitive method is described, for assaying methionine-enkephalin (I) and leucine-enkephalin (II) generating enzyme using a modified HPLC method involving post-column derivatization using hydroxylamine, Co(II) and borate as fluorogenic reagents. The detection limit for the I and II generating activities of the enzyme are 2.5 and 3.0 pmol-1 per mg of protein, respectively. The distributions of the enkephalin generating enzymes in regional tissues of rat brain as well as characterization of the enzyme are discussed.
Leucine Methionine HPLC Fluorescence Post-column derivatization

"Liquid Chromatographic Determination Of Indolamine, Catecholamines And Their Precursors And Metabolites In Rat Brain Tissues With Post-column Fluorescence Derivatization"
Anal. Sci. 1994 Volume 10, Issue 1 Pages 5-9

Abstract: A method is presented for the determination of indolamine (5-hydroxytryptamine), its precursor 5-hydroxytryptophan and metabolites 5-hydroxyindoleacetic acid and 5-hydroxytryptophol simultaneously with norepinephrine, epinephrine and dopamine, their precursor L-3, 4-dihydroxyphenylalanine and nine of their metabolites. LC was performed on columns (25 cm x 4.6 mm i.d.) of TKSgel ODS-80TM (5 µm), with 30 mM citrate buffer of pH 2.5/0.4 mM sodium octanesulfonate/5.5% acetonitrile as the mobile phase (0.8 ml/min). Post-column derivatization was performed at pH 6.5 with aqueous 10 mM NaIO4/3 mM potassium hexacyanoferrate(III) and aqueous 70% ethanolic 30 mM meso-1,2-diphenylethylenediamine/0.14 M sodium methylate (0.3 ml/min). The fluorescence was measured at 480 nm (excitation at 350 nm). The calibration graphs were linear from 0.3-3000 pmol/ml depending on the compound, the detection limits were 13-570 fmol on column and the RSD were 3.1%. The method was used to determine these compounds in tissue from discrete regions of rat brains, treated with saline, the monoamine oxidase inhibitor pargyline and the acid transport inhibitor probenecid, with isopreterenol as internal standard.
Indolamine Catecholamines LC Fluorescence Post-column derivatization

"Rapid Plasmenylethanolamine-selective Fusion Of Membrane Bilayers Catalyzed By An Isoform Of Glyceraldehyde-3-phosphate Dehydrogenase: Discrimination Between Glycolytic And Fusogenic Roles Of Individual Isoforms"
Biochemistry 1995 Volume 34, Issue 38 Pages 12193-12203
Paul E. Glaser and Richard W. Gross

Abstract: Recently we demonstrated that the unique stereoelectronic relationships inherent in the structure of plasmenylethanolamine facilitate membrane fusion, and we postulated the existence of a membrane fusion protein which could exploit the propensity of plasmenylethanolamine molecular species to adapt an inverted hexagonal phase [Glaser and Gross (1994) Biochemistry 33, 5805-5812]. We now report a cryptic membrane fusion activity in rabbit brain cytosol, which requires separation from an endogenous inhibitor to express its activity, and demonstrate that vesicle fusion catalyzed by this protein is highly selective for membrane vesicles containing plasmenylethanolamine. The cytosolic protein catalyzing membrane fusion activity was purified to apparent homogeneity by sequential column chromatographies, revealing a single 38-kDa protein band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Automated Edman degradation demonstrated that the purified protein is an isoform of glyceraldehyde- 3-phosphate dehydrogenase (GAPDH), which was confirmed by Western blot analysis utilizing polyclonal antibodies and by solution-state inactivation of membrane fusion activity by a monoclonal antibody directed against GAPDH. Both GTP-affinity and Mono Q chromatographies resolved GAPDH isoforms that catalyzed dehydrogenase activity from the GAPDH isoform that catalyzed membrane fusion activity. The purified fusion protein was calcium-independent, resistant to treatment with N- ethylmaleimide, and possessed an obligatory requirement for plasmenylethanolamine and cholesterol. High-resolution stopped-flow kinetic analysis of plasmenylethanolamine-facilitated membrane fusion demonstrated that one tetramer of the GAPDH isoform catalyzed one fusion event between two vesicles containing plasmenylethanolamine every millisecond (on average). Collectively, these results constitute the first description of a protein which can catalyze the fusion of vesicles at a rate which satisfies the mathematical constraints imposed by the observed rates of fusion of synaptic vesicles with the presynaptic membrane in vivo.
Plasmenylethanolamine Stopped-flow

"Peroxyoxalate Chemiluminescence Detection Of Condensates Of Malondialdehyde With Thiobarbituric Acids Using A Flow System"
Biomed. Chromatogr. 1992 Volume 6, Issue 2 Pages 55-58
Kenichiro Nakashima, Masahiko Nagata, Masakatsu Takahashi, Shuzo Akiyama

Abstract: The peroxyoxalate chemiluminescence(CL) detection method for the evaluation of the CL intensity of malondialdehyde(MDA) condensates with seven 2-thiobarbituric acid derivatives is described. The method consists of a flow injection technique together with a CL detection system using bis(2,4,6-trichlorophenyl) oxalate(TCPO) and hydrogen peroxide as chemiluminogenic reagents. Linear correlations between CL intensity and concentration are obtained for pmol levels of condensates. Among the condensates, 1,3-diethyl-2-thiobarbituric acid(DETBA)-MDA shows the largest CL intensity. High performance liquid chromatography (HPLC)/CL detection of DETBA-MDA and 1,3-diphenyl-2-thiobarbituric acid(DPTBA)-MDA using a mixture of TCPO and hydrogen peroxide in acetonitrile as a post-column reagent solution is also described. The detection limits for DETBA-MDA and DPTBA-MDA are 20 and 200 fmol, respectively, per 20 µL injection at a signal-to-noise ratio of 2. This HPLC/CL detection system was applied to the determination of MDA in rat brains by using DETBA as a fluorescent derivatizing reagent.
Malondialdehyde Chemiluminescence HPLC Post-column derivatization

"Serotonin And 5-hydroxyindole-3-acetic Acid In Human Plasma And Rat Brain Determined By Liquid Chromatography With Post-column Derivatization And Fluorescence Detection"
Clin. Chem. 1993 Volume 39, Issue 11 Pages 2355-2356
Junichi Ishida, Ryuji Iizuka and Masatoshi Yamaguchi

Abstract: Plasma or rat brain homogenate (5-hydroxyindole-3-acetamide as internal standard) was deproteinized with HClO4 and analyzed by HPLC [cf. Analyst (London), 1993, 118, 165] with post-column derivatization with benzylamine in the presence of K3Fe(CN)6 in weakly alkaline medium and fluorescence detection of the oxazole derivatives at 480 nm (excitation at 345 nm). Response was linear up to 500 pmol injected. Detection limits were 1.6 nM-5-hydroxytryptamine (serotonin) and 3.3 nM-5-hydroxyindole-3-acetic acid. Recovery was 67.7-95.0% and within-day RSD (n = 10) were 2.2-3.9%.
Serotonin 5-Hydroxyindole-3-acetic acid HPLC Fluorescence Clinical analysis Post-column derivatization Letter

"Design And Development Of An Amperometric Biosensor For Acetylcholine Determination In Brain Microdialyzates"
Electrochim. Acta 1998 Volume 43, Issue 23 Pages 3541-3554
N. Larsson, T. Ruzgas, L. Gorton, M. Kokaia, P. Kissinger and E. Csöregi*

Abstract: An amperometric three-enzyme based biosensor for determination of acetylcholine has been developed with possible use for monitoring of brain microdialyzates by co-immobilizing acetylcholinesterase (AchE), choline oxidase (ChOx) and horseradish peroxidase (HRP) in an Os-based redox polymer on solid graphite electrodes. The redox hydrogel was formed by crosslinking the appropriate enzymes and the Os-polymer (PVI13-dmeOs) working as a non-diffusing mediator between the electrode and HRP. The sensor was used in a flow injection system at an applied potential of -50 mV vs. Ag/AgCl. A detection limit of 0.3 µM (twice the S/N ratio) for acetylcholine was obtained, thus representing a sensitive detection system. By adapting the electrode into a microsystem, the release of acetylcholine in real samples (rat brain dialyzates) could be shown. Electrode design, optimization steps and characteristics for the optimized electrode configuration are presented.
Acetylcholine Amperometry Sensor Electrode Electrode Optimization Apparatus Detector

"Flow Injection Determination Of Glutamate In Human Serum And Rat Brain Samples With Immobilized Glutamate Oxidase And Glutamate Dehydrogenase Reactors"
Clin. Chem. Lab. Med. 1994 Volume 32, Issue 10 Pages 767-772
C. D. Stalikas / M. I. Karayannis / Stella M. Tzouwara-Karayanni

Abstract: Two methods are proposed for the determination of regional concentrations of glutamate in the rat brain as well as in human serum. Glutamate oxidase was immobilized on non-porous glass beads and glutamate dehydrogenase was immobilized on glass derivatives. These supports were employed for the construction of Single Bead String Reactors and Packed Bed Reactors, respectively, which in turn were linked to Flow Injection Analysis systems with either photometric or fluorometric detection. Analytical working curves are linear in the range 1-200 µmol/L for packed bed reactors and 10^-500 mmol/l for single bead string reactors. The samples were pretreated depending on their origin and the applied measuring system. Optimal dilution factors were established for the two techniques. Optimal dilution ratios were established and the influence of several added substances was investigated. Recovery and method comparison studies including high performance liquid chromatography verified the accuracy of the proposed methods. Results from within-day and between-day measurements gave relative standard deviations of 4.7 and 5.9% for serum samples and 2.5 and 4.0% for brain samples, respectively.
Glutamate Fluorescence HPLC Clinical analysis Immobilized enzyme Glass beads Single bead string reactor Method comparison

"Physiological Stimulation Increases Nonoxidative Glucose Metabolism In The Brain Of The Freely Moving Rat"
J. Neurochem. 1993 Volume 60, Issue 4 Pages 1258-1263
Fellows LK, Boutelle MG, Fillenz M.

Abstract: The effects of mild stress on nonoxidative glucose metabolism were studied in the brain of the freely moving rat. Extracellular lactate levels in the hippocampus and striatum were monitored at 2.5-min intervals with microdialysis coupled with an enzyme-based flow injection analysis system. Ten minutes of restraint stress led to a 235% increase in extracellular lactate levels in the striatum. A 5-min tail pinch caused an increase of 193% in the striatum and 170% in the hippocampus. Local application of tetrodotoxin in the striatum blocked the rise in lactate following tail pinch and inhibited the subsequent clearance of lactate from the extracellular fluid. Local application of the noncompetitive N-methyl-D-aspartate receptor antagonist MK-801 had no effect on the tail pinch-stimulated increase in lactate in the striatum. These results show that mild physiological stimulation can lead to a rapid increase in nonoxidative glucose metabolism in the brain.
Glucose Dialysis

"ATP-sensitive Potassium Channels And Local Energy Demands In The Rat Hippocampus: An In Vivo Study"
J. Neurochem. 1993 Volume 61, Issue 3 Pages 949-954
Fellows LK, Boutelle MG, Fillenz M.

Abstract: Microdialysis coupled with an enzyme-based flow injection analysis was used to monitor brain extracellular lactate and glucose in the freely moving rat. Glucose levels reflect the balance between supply from the blood and local utilisation, and lactate efflux indicates the degree of local nonoxidative glucose metabolism. Local application of tolbutamide, a blocker of the ATP-sensitive potassium channel, decreased extracellular glucose and lactate levels in the hippocampus but not in the striatum. The increase in glucose and lactate levels following mild behavioural stimulation was also reduced by tolbutamide in the hippocampus. Similar effects on both basal and stimulated lactate levels were obtained with local application of 10 mM glucose. These results indicate that ATP-sensitive potassium channels are active under physiological conditions in the hippocampus and that the effects of tolbutamide can be mimicked by physiological glucose levels.
Lactate Glucose Dialysis In vivo monitoring

"Changes Of Amino Acid And Monoamine Levels After Neonatal 6-hydroxydopamine Denervation In Rat Basal Ganglia, Substantia Nigra, And Raphe Nuclei"
J. Neurosci. Res. 1993 Volume 35, Issue 4 Pages 409-418
E. Molina-Holgado, K. M. Dewar, L Grondin, N. M. van Gelder, T. A. Reader

Abstract: The effects of a neonatal dopaminergic deafferentation with the neurotoxin 6-hydroxydopamine (6-OHDA) on endogenous tissue levels of catecholamines, indoleamines, and amino acids were investigated in discrete rat brain regions. After producing the lesion at postnatal day 3 by intraventricular injections of 6-OHDA, with a desipramine pretreatment to protect noradrenaline neurons, the animals were kept for 3 months. Their brains were dissected to obtain samples of neostriatum, Globus pallidus, Substantia nigra, and Raphe nuclei, which were then analyzed by high performance liquid chromatography, coupled either to electrochemical detection for aromatic monoamines, or to post-column ninhydrin derivatization with spectrophotometry for amino acids. The neonatal 6-OHDA treatment depleted dopamine (DA) levels in neostriatum, Globus pallidus, and Substantia nigra, but in Raphe nuclei DA was increased. The main metabolites of DA were also decreased in neostriatum, Globus pallidus, and Substantia nigra but remained unchanged in Raphe nuclei. Serotonin (5-HT) and its metabolite 5-hydroxy-indole-3-acetic acid increased in neostriatum and Raphe nuclei; in Substantia nigra there was a slight increase in 5-HT only. The 6-OHDA lesion caused heterogeneous alterations in amino acid contents, which varied according to the region. In the neostriatum there were increases of γ-aminobutyric acid (GABA), aspartic acid, and glycine. In the Globus pallidus taurine, GABA, glutamic acid, glutamine, aspartic acid, serine, and alanine were elevated. In the Substantia nigra only increases in taurine, GABA, glutamic acid, and glutamine could be documented. This study shows important changes in amino acid levels and in some of their ratios, occurring in different anatomical subdivisions of the basal ganglia and related brainstem nuclei following a neonatal treatment with 6-OHDA. The results thus demonstrate major biochemical modifications in amino acids in the aftermath of a DA denervation and/or a 5-HT hyperinnervation during an early developmental period.
Amino Acids

"Ultra-micro-analysis Of Bioactive Substances: An Approach Based On The Development Of Highly Selective Fluorogenic Reagents"
J. Pharm. Soc. Jpn. 1988 Volume 108, Issue 1 Pages 22-38

Abstract: A series of mono- and di-functional fluorogenic reagents has been developed for the selective derivatization of various bioactive substances and as enzyme substrates. These have been used for fluorimetric determination and for HPLC with pre- or post-column derivatization of the substances in biological materials. Methods are described for HPLC of neuraminic acids in serum, of catecholamines in plasma and urine, of peptides in, e.g., rat brain, of reducing sugars in serum and urine, of fatty acids in serum, of prostaglandins in seminal fluid and of fluorouracil in serum.
Acids, fatty Catecholamines Fluorouracil Neuraminic acids Peptides Prostaglandins Sugars, reducing HPLC Fluorescence Post-column derivatization

"Micro-flow In Vivo Analysis Of L-Glutamate With An On-line Enzyme Amplifier Based On Substrate Recycling"
Anal. Sci. 2001 Volume 17, Issue 6 Pages 703-708
Toshio Yao, Youko Nanjyo And Hirohito Nishino

Abstract: A micro-flow enzyme system with a microdialysis probe is proposed for the amperometric detection of trace amounts of neurotransmitter L-glutamate released from rat brain cells. The L-glutamate oxidase (EC dehydrogenase (EC coimmobilized reactor was used to enhance the sensitivity of L-glutamate as an on-line amplifier based on substrate recycling. A poly(1,2-diaminobenzene) film-coated platinum electrode was also used to selectively detect only the hydrogen peroxide generated into a upstream enzyme reactor, without interference from oxidizable species, such as L-ascorbate, the adsorption of low molecular-weight proteins in a dialysate, and NADPH added to the carrier solution to initiate substrate recycling. By the present in vivo system, L-glutamate was selectively assayed with about a 600-fold increase in sensitivity compared with the unamplified responses. The detection limit was 0.08 µmol L-1. This method was applied to an in vivo assay of L-glutamate in the extracellular space of rat brain; also, monitoring of the L-glutamate level changed after a continuous stimulation of KCl to demonstrate the reliability of the system.
l-Glutamate Amperometry Dialysis In vivo monitoring Immobilized enzyme Reactor