Kor Venema

Amino Acids in Rat Striatal Dialysates: Methodological Aspects and Changes After Electroconvulsive Shock

Abstract: Extracellular levels of amino acids were estimated in dialysates of the rat striatum that were collected 1,2, and/or more than 5 days after surgery, before (resting release) and during exposure to high K concentrations (50 mM) or electroconvulsive shocks. The resting release of several amino acids (Glu, Asn, Thr, Tau, Tyr, Gly, and Ala) was higher 9 days as compared to 1 day after surgery. In the 1‐day preparation the resting release correlated highly with that observed with push‐pull cannulas. The correlation with the tissue content of the amino acids was high only when they were divided into two groups (putative transmitters and metabolic intermediates). High K exposure produced increased output of Ala, ethanolamine (Eam). Asp, Glu. Tau, and Gly and a decrease in the egress of Gln 1 or 2 days after surgery. The effects on Asp and Glu had disappeared, and that on Gln reversed after 4–9 days. Electrically induced convulsions produced increased output of Ala, Gln, and Eam 1 or 2 days and 2 weeks after implantation of the probe. Changes were seen not only during but also (and some cases even more prominent) after the seizure. This study shows the usefulness of dialysis to monitor extracellular transmitter amino acids in the striatum of conscious rats (also bilateral dialysis was possible) for only a limited time after implantation of the probe. The dialysis method is suitable for longer time, when metabolic changes in amino acids are to be followed. In addition to transmitter release, glycolysis can be monitored by the measurement of Ala in the dialysate.

Increases in Striatal and Hippocampal Impedance and Extracellular Levels of Amino Acids by Cardiac Arrest in Freely Moving Rats

Abstract: The time course of changes in the tissue impedance and the levels of extracellular transmitter and non‐transmitter amino acids was studied in the striatum and hippocampus of the unanesthetized rat after cardiac arrest. Electrodes were implanted for the continuous measurement of tissue impedance so that a measure of the volume of extracellular space was provided. Alternatively, bilateral dialysis probes were used for monitoring levels of extracellular amino acids in subsequent 30‐s samples using an automated precolumn derivatization technique for reversed‐phase HPLC analysis and fluorimetric detection. The impedance started to rise ∼1.2 min following cardiac arrest, increased rapidly during the first 5 min, and increased almost linearly thereafter. After 15 min, a decrease of ∼50% in the extracellular space was calculated. The impedance rose more steeply in the striatum than in the hippocampus. The extracellular levels of taurine, which increased >300% within 5 min after cardiac arrest, most closely resembled the time course of the change in impedance. Glutamate and aspartate levels did not increase until 5 min after circulatory arrest, and at 15 min they had risen to a level of 465 and 265% for the striatum and 298 and 140% for the hippocampus of the resting release, respectively. The release of γ‐aminobutyric acid (GABA) was multiphasic and did not resemble that of any of the other—putative—transmitter amino acids. Fifteen minutes after cardiac arrest, the levels of GABA were 617 and 774% of the resting release in the striatum and hippocampus, respectively. Glycine and ala‐nine efflux substantially increased (232 and 151% in striatum and 141 and 154% in hippocampus, respectively) 15 min postmortem, whereas the glutamine level was slightly increased and levels of asparagine, histidine, threonine, ethanolamine, serine, arginine, and tyrosine were inconsistently higher in the two brain regions. At this time, the extracellular levels of glutamate, GABA, and aspartate were only slightly lower, as expected from the tissue levels and from levels of the other amino acids, an observation indicating that all the amino acids may diffuse through postmortem brain tissue to a nearly similar extent. This study provides evidence that extracellular levels of taurine reflect changes in distribution of electrolytes (and in membrane potentials), that the postmortem release of transmitter amino acids is multiphasic with a delay of at least 1 min, that postmortem shrinkage in extracellular volume cannot account for the increase in the content of transmitter amino acids in the dialysate, and that the massive overflow of glutamate, aspartate, GABA, and taurine seen during ischemia is the result of both release and the failure of uptake. Possible implications of the present findings for excitotoxic damage of the brain are discussed.

Amino Acids in the Substantia Nigra of Rats with Striatal Lesions Produced by Kainic Acid

In an attempt to estimate the pool size of glutamate and other amino acids in γ‐aminobutyric acid (GABA)‐containing neurons, we determined the content of 12 amino acids in the bilateral substantia nigra of rats, in which unilateral striatal lesions had been made with kainic acid two weeks earlier. The assay of the amino acids (including glutamate, aspartate, glutamine, asparagine, glycine, and GABA) and ethanolamine was based on HPLC and fluorimetric detection after precolumn derivatization with o‐phthaldialdehyde. The levels of all measured amino acids (except those of tyrosine, threonine, and ethanolamine) were decreased in the affected striatum, but only the levels of aspartate, taurine, and GABA were lowered in the ipsilateral substantia nigra. These results indicate that the pool size of the various amino acids in the striatonigral GABAergic pathway is small compared to their nigral content, and that in addition to GABA a significant fraction of aspartate and taurine may be confined to nerve terminals in the substantia nigra.

AN ENZYME-REACTOR FOR ELECTROCHEMICAL MONITORING OF CHOLINE AND ACETYLCHOLINE - APPLICATIONS IN HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY, BRAIN-TISSUE, MICRODIALYSIS AND CEREBROSPINAL-FLUID

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 on-line 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.

DESMETHYLIMIPRAMINE ENHANCES THE RELEASE OF ENDOGENOUS GABA AND OTHER NEUROTRANSMITTER AMINO-ACIDS FROM THE RAT THALAMUS

The influence of desmethylimipramine (DMI) on the release of endogenous γ‐aminobutyric acid (GABA) and some other amino acids from the rat thalamus was studied with a push‐pull perfusion technique. Following HPLC the amino acids were fluorimetrically estimated. Added to the perfusion medium at a concentration of 10 μmol L‐1, DMI caused a 5‐ to 10‐fold increase in the release of GABA. Similar effects were found with imipramine, trimeprimine, haloperidol, and propranolol. The elevation of GABA release induced by DMI was Ca dependent. The release of aspartate and glutamate was also increased by DMI, but in contrast to K ions, DMI did not reduce the thalamic output of glutamine.

AUTOMATED PRECOLUMN DERIVATIZATION DEVICE TO DETERMINE NEUROTRANSMITTER AND OTHER AMINO-ACIDS BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A device to derivatize amino acids with o-phthaldialdehyde, which is directly connected to high-performance liquid chromatographic equipment is described. Its principle is that a sample (10-500 microliters) is mixed with a reagent (containing o-phthaldialdehyde, 2-mercaptoethanol and sodium hydrogen carbonate buffer), using a peristaltic pump. This mixture is pumped into a loop of a pneumatically controlled injection valve at atmospheric pressure. When the derivatization is complete the valve switches, so that the sample is applied to a column and the amino acid derivatives are separated with a gradient of methanol-phosphate buffers. The reproducibility is such that brain perfusates or tissue extracts can be analyzed for the amino acid transmitter content and no internal standard is necessary. The major advantages of the present device are that it produces thorough mixing of reagent and sample, so that a high and constant degree of derivatization occurs (thus producing high sensitivity; less than 0.1 pmol can be detected) and its low cost.

DECARBOXYLATION OF EXOGENOUS l‐5‐HYDROXYTRYPTOPHAN AFTER DESTRUCTION OF THE CEREBRAL RAPHE SYSTEM

Abstract— l‐5‐Hydroxytryptophan (l‐5‐HTP) was administered intravenously to rats (12 mg/kg) after inhibition of the peripheral aromatic l‐amino acid decarboxylase with l‐α‐hydrazino‐α‐methyl dopa (MK 486). The accumulation of 5‐hydroxytryptamine (5‐HT) and 5‐hydroxyindoleacetic acid in the cerebral cortex was measured 1, 2 and 4 h after injection of 5‐HTP with automated assay techniques. Besides controls two groups of rats were studied: rats after inhibition of tryptophan‐5‐hydroxylase with p‐chlorophenylalanine (pcpa) and subjects with a chronic lesion in the area of the raphe nucleus. The net accumulation of both measured 5‐hydroxyindoles was diminished in rat cerebral cortex after degeneration of 5‐HT containing nerve endings, compared with control animals and pcpa‐treated rats. These results indicate that the formation of 5‐HT in the cerebral cortex from exogenous l‐5‐HTP, after inhibition of the peripheral aromatic amino acid decarboxylase, occurs predominantly in 5‐HT containing nerve endings possibly by a specific 5‐HTP‐decarboxylating enzyme.

Utilization of in vivo ultrafiltration in biomedical research and clinical applications

Ultrafiltration (UF) is a filtrate selection method with a wide range of biomedical and clinical applications, including detoxification of blood in hemodialysis and peritoneal dialysis. New is, however, the use of UF as a convenient in vivo sampling method that, for example, has been used in diabetics. Ultrafiltration avoids complicated and time-consuming recovery calculations that are necessary when using in vivo microdialysis, as recoveries of low molecular weight molecules are near 100%. The subcutaneously or intravenously placed UF probes have been studied for off-line sample analysis and for continuous on-line monitoring, in a wide variety of species, including dogs, rats, pigs and humans. This review discusses the potential of in vivo UF as a continuous tissue sampling technique in clinical research areas, and in several major biomedical applications including glucose and lactate monitoring and drug kinetic studies.

Bi-enzyme reactor for electrochemical detection of low concentrations of uric acid and glucose

An enzyme-based flow-injection amperometric analysis system (FIA) for monitoring of uric acid and glucose is described. The oxidase and peroxidase enzymes are physically coimmobilised in a sandwich-type reactor and ferrocene serves as a mediator. The assays are based on the measurement of a reduction current resulting from the enzymatic reactions, at a glassy carbon electrode held at 0.00 mV (vs. Ag/AgCl). The high selectivity (ascorbic acid did not interfere) is coupled to high sensitivity (a detection limit of 30 and 60 nmol/l for uric acid and glucose, respectively; signal/noise = 3) and good stability (the enzymes remained active for more than 6 weeks at 30 degrees C). The usefulness of the assay in clinical chemistry is illustrated by the measurement of human serum uric acid and glucose concentration. The results obtained were in fairly good agreement with those obtained using conventional hospital laboratory methods.

On-line continuous monitoring of glucose or lactate by ultraslow microdialysis combined with a flow-through nanoliter biosensor based on poly(m-phenylenediamine) ultra-thin polymer membrane as enzyme electrode

A miniaturised flow-through biosensor with a cell volume of only a few nanoliters was developed in our laboratory. The biosensor can be directly coupled to a microdialysis or ultrafiltration probe. Sampling and continuous on-line monitoring can thus be carried out at submicroliter levels and as a consequence quantitative recoveries of the analyte of interest are achieved. Via this method excessive calibration procedures, as are necessary with conventional microdialysis, are avoided. Here, the construction and the performance of such a biosensor for the continuous on-line monitoring of glucose and lactate will be presented. The biosensor is based on the amperometric detection of hydrogen peroxide after conversion of the analyte of interest by an immobilised oxidoreductase enzyme. Immobilisation of the enzyme is performed through electropolymerisation of m-phenylenediamine. Strategies to improve the performance (e.g. linearity, selectivity and stability) of the miniaturised biosensor are discussed and ex vivo and in vivo experiments carried out thus far demonstrate the potential of this miniaturised flow-through biosensor.