Acid In Brain Tissue Research Articles

Cinnabarinic Acid, an Endogenous Metabolite of the Kynurenine Pathway, Activates Type 4 Metabotropic Glutamate Receptors

Cinnabarinic acid is an endogenous metabolite of the kynurenine pathway that meets the structural requirements to interact with glutamate receptors. We found that cinnabarinic acid acts as a partial agonist of type 4 metabotropic glutamate (mGlu4) receptors, with no activity at other mGlu receptor subtypes. We also tested the activity of cinnabarinic acid on native mGlu4 receptors by examining 1) the inhibition of cAMP formation in cultured cerebellar granule cells; 2) protection against excitotoxic neuronal death in mixed cultures of cortical cells; and 3) protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice after local infusion into the external globus pallidus. In all these models, cinnabarinic acid behaved similarly to conventional mGlu4 receptor agonists, and, at least in cultured neurons, the action of low concentrations of cinnabarinic acid was largely attenuated by genetic deletion of mGlu4 receptors. However, high concentrations of cinnabarinic acid were still active in the absence of mGlu4 receptors, suggesting that the compound may have off-target effects. Mutagenesis and molecular modeling experiments showed that cinnabarinic acid acts as an orthosteric agonist interacting with residues of the glutamate binding pocket of mGlu4. Accordingly, cinnabarinic acid did not activate truncated mGlu4 receptors lacking the N-terminal Venus-flytrap domain, as opposed to the mGlu4 receptor enhancer, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC). Finally, we could detect endogenous cinnabarinic acid in brain tissue and peripheral organs by high-performance liquid chromatography-tandem mass spectrometry analysis. Levels increased substantially during inflammation induced by lipopolysaccharide. We conclude that cinnabarinic acid is a novel endogenous orthosteric agonist of mGlu4 receptors endowed with neuroprotective activity.

Determination of l-serine- O-phosphate in rat and mouse brain tissue using high-performance liquid chromatography and fluorimetric detection

l-Serine- O-phosphate (L-SOP), the precursor of l-serine, is an agonist at group III metabotropic glutamate receptors. Despite the interest in L-SOP, very few articles have reported its brain levels. Here we report a convenient and reproducible method for simultaneous analysis of L-SOP and several other important amino acids in brain tissue using high-performance liquid chromatography (HPLC) with fluorimetric detection after derivatization with o-phthaldialdehyde and N-isobutyl- l-cysteine. Analyses were carried out in rat whole brain and cerebellum and in mouse whole brain, forebrain, amygdala, and prefrontal cortex. The method should be useful for future comprehensive neurochemical and pharmacological studies on neuropsychiatric disorders.

Simultaneous determination of amino acids in discrete brain areas in Suncus murinus by high performance liquid chromatography with electrochemical detection

An improved and simple reversed-phase high performance liquid chromatography method with electrochemical detection for the simultaneous determination of amino acids in brain tissue of Suncus murinus was developed. Homogenates from 5 different brain areas were derivatized with o-phthalaldehyde in the presence of sodium sulphite. Subsequent separation was achieved using linear gradient elution over 30 min. The derivatives were stable for up to 20 h at 4 °C. The method was accurate, reproducible, and showed good linearity. The recoveries were >88% for aspartate, glutamine, glutamate, glycine and γ-aminobutyric acid, with the limit of quantification varying from 5 to 30 pmol. The method was successfully applied for the measurement of amino acids under fed and fasted conditions.

The free fatty acid metabolome in cerebral ischemia following human mesenchymal stem cell transplantation in rats

Background Mesenchymal stem cells (MSCs) have the potential to promote brain repair and improve recovery following stroke. We investigated changes in free fatty acids (FFAs) following intravenous human MSC (hMSC) transplantation into rats that had undergone transient middle cerebral artery occlusion (MCAo). Methods Rats were subjected to 2-hours MCAo, followed by intravenous transplantation of hMSC or phosphate-buffered saline (PBS) at one day after MCAo. All rats were sacrificed 5 days after MCAo. Metabolic profiling of free fatty acids (FFAs) level was assessed in plasma and brain from control rats ( n = 8), PBS-treated MCAo rats ( n = 6), and hMSC-treated MCAo rats (MCAo + hMSC, n = 6). Results The levels of some FFAs in plasma and brain samples of the MCAo and MCAo + hMSC groups were significantly different from those of the control group. The percentage composition of myristic acid in plasma and those of myristic acid, linoleic acid, and eicosenoic acid in brain tissues of the MCAo + hMSC group were significantly reduced compared to those in the untransplanted MCAo group. Conclusion Our metabolic approach has provided insights into understanding the complexity of biochemical and physiological events that occur in ischemic brain injury and the transplantation effects of MSCs in stroke.

Phospholipase A2 reduction ameliorates cognitive deficits in a mouse model of Alzheimer's disease

Neuronal expression of familial Alzheimer's disease (AD)-mutant human amyloid precursor protein (hAPP) and hAPP-derived amyloid-β (Aβ) peptides causes synaptic dysfunction, inflammation, and abnormal cerebrovascular tone in transgenic mice. Fatty acids may be involved in these processes, but their contribution to AD pathogenesis is uncertain. A lipidomics approach to broadly profile fatty acids in brain tissues of hAPP mice revealed an increase in arachidonic acid and its metabolites, suggesting increased activity of the group IV isoform of phospholipase A2 (GIVA-PLA2). Levels of activated GIVA-PLA2 in the hippocampus were increased in AD patients and hAPP mice. Aβ caused a dose-dependent increase in GIVA-PLA2 phosphorylation in neuronal cultures. Inhibition of GIVA-PLA2 diminished Aβ-induced neurotoxicity. Genetic ablation or reduction of GIVA-PLA2 protected hAPP mice against Aβ-dependent deficits in learning and memory, behavioral alterations, and premature mortality. Inhibition of GIVA-PLA2 may be of benefit in the treatment and prevention of AD.

Prevention of cognitive deficits and brain oxidative stress with superoxide dismutase/catalase mimetics in aged mice

Continuous decline in cognitive performance accompanies the natural aging process in humans, and multiple studies in both humans and animal models have indicated that this decrease in cognitive function is associated with an age-related increase in oxidative stress. Treating aging mammals with exogenous free radical scavengers has generally been shown to attenuate age-related cognitive decline and oxidative stress. The present study assessed the effectiveness of the superoxide dismutase/catalase mimetics EUK-189 and EUK-207 on age-related decline in cognitive function and increase in oxidative stress. C57/BL6 mice received continuous treatment via osmotic minipumps with either EUK-189 or EUK-207 for 6 months starting at 17 months of age. At the end of treatment, markers for oxidative stress were evaluated by analyzing levels of free radicals, lipid peroxidation and oxidized nucleic acids in brain tissue. In addition, cognitive performance was assessed after 3 and 6 months of treatment with fear conditioning. Both EUK-189 and EUK-207 treatments resulted in significantly decreased lipid peroxidation, nucleic acid oxidation, and reactive oxygen species (ROS) levels. In addition, the treatments also significantly improved age-related decline in performance in the fear-conditioning task. Our results thus confirm a critical role for oxidative stress in age-related decline in learning and memory and strongly suggest a potential usefulness for salen–manganese complexes in reversing age-related declines in cognitive function and oxidative load.

Spinal Morphine Administration Reduces the Fatty Acid Contents in Spinal Cord and Brain by Increasing Oxidative Stress

It is well known that oxidative stress damages biomolecules such as DNA and lipids. No study is available on the morphine-induced oxidative damage and fatty acids changes in brain and spinal tissues. The aim of this work was to determine the effects of morphine on the concentrations and compositions of fatty acid in spinal cord segments and brain tissues in rabbits as well as lipid peroxidation (LP) and glutathione (GSH) levels in cortex brain. Twelve New Zealand albino rabbits were used and they were randomly assigned to two groups of 6 rabbits each. First group used as control although morphine administrated to rats in second group. Cortex brain and (cervical, thoracic, lumbar) samples were taken. The fatty acids between n:18.0 and 21.0 were present in spinal cord sections and n:10 fatty acids in control animals were present in the brain tissues. Compared to n:20.0-24.0 fatty acids in spinal cord sections and 8.0 fatty acids in the brain tissues of drug administered animals. The concentration and composition of the fatty acid methyl esters in spinal cord and brain tissues was decreased by morphine treatments. LP levels in the cortex brain were increased although GSH levels were decreased by the morphine administration. In conclusion, unsaturated fatty acids contents in brain and spinal cord sections and GSH were reduced by administrating spinal morphine although oxidative stress as LP increased. The inhibition oxidative damage may be a useful strategy for the development of a new protection for morphine administration as well as opiate abuse.

Pipecolic acid concentrations in brain tissue of nutritionally pyridoxine‐deficient rats

Elevated concentrations of pipecolic acid have been reported in plasma and CSF of patients with pyridoxine-dependent epilepsy, but its molecular background is unclear. To investigate any further association of pyridoxine and pipecolic acid metabolism, we have performed an animal trial and have measured the concentration of pipecolic acid in brain tissue of rats with nutritional pyridoxine deficiency and in control littermates. Concentrations of pyridoxal phosphate were significantly reduced in brain tissue of pyridoxine-deficient rats (p < 0.001), while concentrations of pipecolic acid were not significantly different from the normally nourished control group (p = 0.3). These data indicate that a direct association of pyridoxine and pipecolic acid metabolism is unlikely. We therefore assume that the characteristic elevation of pipecolic acid in pyridoxine-dependent epilepsy could rather be a secondary phenomenon with the primary defect of pyridoxine-dependent epilepsy being located outside the pipecolic acid pathway.

Development of a protocol for the automated analysis of amino acids in brain tissue samples and microdialysates

An automated precolumn derivatisation method has been developed for the measurement of fourteen amino acids in brain tissue and microdialysate samples. The method involves labelling amino acids with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide (CN −). The resulting highly stable N-substituted 1-cyanobenz[ f]isoindole (CBI) derivatives were separated using a binary gradient elution profile and detected fluorometrically. The order of elution of the derivatised amino acids was confirmed by using liquid chromatography with fluorescence and mass spectrometric detection in tandem. Linear calibration plots were obtained for all amino acids in the range studied (0.2–12.5 μ M). The limit of detection for CBI derivatives of amino acids was in the range 5–20 fmol ( S/N=2) using a 5 μl injection volume. The method has been used for the measurement of amino acids in microdialysates from rat brain and tissue homogenates from different regions of mouse brain.

Distribution volume of radiolabeled large neutral amino acids in brain tissue.

Variations in the cerebellum to plasma ratio at late times in 6-[18F]fluoro-L-DOPA studies are shown to be consistent with competitive binding of large neutral amino acids for a common transporter in the blood-brain barrier and the stability of brain tissue large neutral amino acid level in the presence of plasma level changes. The distribution volume of an inert large neutral amino acid can be estimated from plasma and tissue large neutral amino acid levels and apparent half-saturation concentrations (Km) of the transporter in the blood-brain barrier. Stability of brain large neutral amino acid levels is supported by literature findings and can be explained by high saturation of the large neutral amino acid transporter at physiologic conditions.

Ascorbic acid during cerebral ischemia in newborn piglets.

We measured ascorbic acid (reduced and oxidized) in brain, CSF and blood, before, during and after cerebral ischemia in newborn piglets. Bilateral carotid ligation induced a 54% decrease in cerebral blood flow (p < 0.01) and a 43% decrease in the cerebral metabolic rate of oxygen (p < 0.01). After ischemia and reperfusion, we obtained a 60% decrease (p < 0.01) in total brain ascorbic acid content. CSF ascorbic acid increased during reperfusion: +60% at 30 min (p < 0.001) and +160% at 120 min (p < 0.05). Blood ascorbic acid content did not change. These changes and the absence of massive oxidation of ascorbic acid in brain tissue suggest release of ascorbic acid by the brain during ischemia.

Effect of allopurinol on uric acid levels and brain cell membrane Na +,K +-ATPase activity during hypoxia in newborn piglets

Oxygen-free radicals generated by xanthine oxidase during hypoxia-ischemia may result in cellular injury through harmful effects on membrane phospholipids. The present study investigated the effect of administration of allopurinol, an inhibitor of xanthine oxidase, on free-radical generation and brain cell membrane injury during hypoxia by inhibiting the breakdown of hypoxanthine to uric acid. Brain cell membrane Na +,K +-ATPase activity and lipid peroxidation products (conjugated dienes and fluorescent compounds) were determined as indices of brain membrane function and structure. Cerebral oxygenation was continuously monitored during hypoxia by 31P-NMR spectroscopy. Plasma and brain tissue levels of uric acid were measured to evaluate xanthine oxidase activity and purine degradation. Na +,K +-ATPase activity decreased significantly in both hypoxic groups; however, the allopurinol-treated hypoxic group showed a smaller decrease than the untreated hypoxic group (47.3 ± 4.9vs. 42.0 ± 2.7 μmol P i/mg protein/h, P < 0.05), respectively. Conjugated dienes increased significantly in the untreated hypoxic compared to control animals (0.070 ± 0.045vs. 0.004 ± 0.006 μmol/g brain, P < 0.05), with the allopurinol-treated animals having intermediate values (0.053 ± 0.039 μmol/g brain). Fluorescent compounds were lower in the allopurinol-treated hypoxic group compared to the untreated hypoxic group (0.79 ± 0.19vs. 1.06 ± 0.60 μg/quinine sulfate/g brain, P < 0.05). Measurements of serum and brain tissue uric acid were significantly lower during hypoxia in the allopurinol-treated compared to the untreatedgroup (30.3 ± 15.6vs. 45.7 ± 10.6 μM( P < 0.05)and1.69 ± 0.97vs. 4.27 ± 2.37nmol/g( P < 0.05), respectively. Allopurinol is an effective inhibitor of xanthine oxidase and reduces the formation of uric acid in brain tissue under hypoxic conditions. In addition, the present data suggest that allopurinol reduces lipid peroxidation during cerebral hypoxia in newborn piglets. Allopurinol, while unable to offer complete protection from the effects of hypoxia, can attenuate hypoxic brain cell membrane injury.

Quantitative analysis of neuroactive amino acids in brain tissue by liquid chromatography using fluorescent pre-column labelling with o-phthalaldehyde and N-acetyl-L-cysteine.

A high-performance liquid chromatographic method for the determination of aspartic acid, glutamic acid, glutamine, glycine, taurine, and gamma-aminobutyric acid in brain tissue is described. Amino acids were derivatized with o-phthalaldehyde/N-acetyl-L-cysteine, a fluorescent labelling mixture, in the presence of 0.1 M borate buffer pH 9.5. The derivatization reaction was sensitive to the pH and concentration of borate buffer. A drift in the fluorescent response less than 4% was obtained with the reported conditions after 4 h of reaction. The resolution of the amino acid derivatives was accomplished in a reversed-phase column with a methanol gradient in 50 mM acetate buffer pH 5.5. These conditions also allowed the separation of the major tissue free physiological amino acids. L-Norvaline was used as an internal standard for both peak identification and quantification. Within-day and between-day precision were less than 6.2%, and the accuracy ranged from 99.1 to 104%. The applicability of the method was demonstrated in a study in rats in which the levels of the assayed amino acids in discrete areas of brain were examined.

Analysis of Femtomole Concentrations of α-Ketoisocaproic Acid in Brain Tissue by Precolumn Fluorescence Derivatization with 4,5-Dimethoxy-1,2-diaminobenzene

An extremely sensitive fluorimetric HPLC method used to determine in vivo cerebral α-ketoisocaproic acid (KIC) concentration with less than 2 g of tissue (e.g., a single rat brain, ∼1.8 g) is reported. Removal of unwanted lipids and amino acids and isolation of α-ketoacids in an optimal derivatization buffer are achieved by C18 solid-phase extraction of the acid-soluble fraction of brain tissue. Quantitation of KIC to the femtomole level is accomplished by reversed-phase HPLC using 4, 5-dimethoxy-1,2-diaminobenzene precolumn fluorescence derivatization and on-line fluorescence detection. These techniques are applicable to femtomole quantitation of other α-ketoacids in various tissue and blood matrices. Combination of this fluorimetric method with simple nonchromatographic procedures to measure in vivo cerebral [1-14C]α-ketoisocaproic acid radioactivities in tissue provides estimates of specific activities.

Simultaneous measurement of monoamines, metabolites and amino acids in brain tissue and microdialysis perfusates

A high-performance liquid chromatographic method with coulometric array electrochemical detection is described for the simultaneous analysis of monoamines, their metabolites and o-phthalaldehyde (OPA)-derivatized amino acids. This method has been used to examine metabolite levels in both striatal tissue homogenates and striatal microdialysis perfusates. An aliquot of sample was initially analyzed for monoamines and metabolites by isocratic elution and electrochemical detection on a serial electrode array of eight coulometric flow-through graphite electrodes (0 to 490 mV; 70-mV increment). The remaining sample was derivatized pre-column with OPA—β-mercaptoethanol and after column switching was analyzed for amino acids on a second isocratic system with electrochemical detection on four electrodes. Metabolites were then identified based on their retention time as well as electrochemical behavior across the arrays. The analysis, derivatization procedure, column switching, data reduction and peak identification were fully automated. The limit of detection for striatal tissue homogenates was approximately 1.38 ng/g wet weight for the monoamines and 8.25 ng/g wet weight for amino acids. The limit of detection for striatal perfusates was approximately 2.5 pg per 20-μl sample for the monoamines and 15 pg per 20-μl sample for the amino acids with analysis completed within 25 min making it ideal for microdialysis samples.

Determination of 3-hydroxykynurenine in human brain and plasma by high-performance liquid chromatography with electrochemical detection: Increased concentrations in hepatic encephalopathy

A simple and specific method was developed for the determination of 3-hydroxykynurenine in brain tissue and blood plasma using high-performance liquid chromatography with electrochemical detection. This involved an extraction procedure using strong cation-exchange columns and also permitted the determination of 3-hydroxyanthranilic acid in brain tissue. The method was applied to the investigation of post mortem brain tissue from patients with hepatic encephalopathy. Cortical 3-hydroxykynurenine concentrations were substantially increased in such patients above control values, providing evidence for a dysfunction of tryptophan metabolism in this disease.

Gamma-aminobutyrate-like immunoreactivity in the thalamus of the cat

Serial sections of the cat's thalamus were incubated with a purified antiserum raised against gamma-aminobutyric acid conjugated to bovine serum albumin by distilled glutaraldehyde. This serum has been extensively characterized and appears to react selectively with fixed gamma-aminobutyric acid in brain tissue treated with glutaraldehyde. Adjoining sections were stained with thionin and served as invaluable guides for a correct evaluation of the immunolabelling pattern. In the neuropil the intensity of the immunostaining varies considerably between thalamic nuclei and even between nuclear subdivisions. The neuropil staining appears particularly dense in the nuclei parataenialis, periventricularis, centralis medialis, reuniens, rhomboideus, habenularis lateralis, centrum medianum, parafascicularis, subparafascicularis, submedius, dorsal and ventral parts of the lateral geniculate body, the dorsal part of the medial geniculate body, the posterior complex, suprageniculate nucleus, pulvinar and parts of the lateral posterior nucleus. The pulvinar/lateralis posterior complex shows a particularly well-differentiated staining pattern which closely matches Updyke's [Updyke (1983) J. comp. Neurol. 219 143–181] parcellation of this region. In several thalamic nuclei or subareas—and notably in those relay nuclei which are known to project upon non-primary sensory cortical areas—the immunostained neuropil is characterized by many puncta encircling an unstained profile. With few exceptions all thalamic nuclei displayed immunoreactive nerve cell bodies. Several examples were found of a mismatch between the number of such cells and the staining intensity of the neuropil. Thus the nuclei periventricularis, parafascicularis, subparafascicularis, parataenialis, limitans and centrum medianum although being very rich in neuropil staining have practically no immunostained perikarya. Rough estimates were made of the size and the proportion of gamma-aminobutyric acid labelled neurons in all major—and some minor—thalamic nuclei and their subdivisions. In some thalamic nuclei, notably the nuclei reticularis, anterior dorsalis, lateralis dorsalis, centralis lateralis, ventralis posterior and the dorsal lateral geniculate body, the population of immunoreactive neurons is distinctly heterogeneous with regard to soma size. The findings are discussed with regard to previous immunocytochemical studies of the distribution of gamma-aminobutyric acid and its synthesizing enzyme in the thalamus. Particular emphasis is put on the great species differences which appear to exist in this respect.

Cerebral GABA-ergic and glutamatergic function in hepatic encephalopathy

Measurement of amino acids in brain tissue obtained at autopsy from cirrhotic patients dying in hepatic coma revealed a threefold increase in glutamine and a concomitant decrease in brain glutamate. The GABA levels were found to be unaltered. Studies using an animal model of portal-systemic encephalopathy gave similar results. Glutamic acid decarboxylase (GAD) activities were within normal limits, both in the brains of cirrhotic patients and portocaval-shunted rats. A previous study reported normal [3H]GABA binding to synaptic membrane preparations from cerebral cortex in these animals. Taken together, these findings suggest that cerebral GABA function is not impaired in hepatic encephalopathy associated with chronic liver disease and portal-systemic shunting. On the other hand, there is evidence to suggest that the releasable pool of glutamate may be depleted in brain in hepatic encephalopathy. Data consistent with this hypothesis include: Reduction in the evoked release of endogenous glutamate by superfusion of hippocampal slices with pathophysiological levels of ammonia; ammonia-induced reduction of glutamatergic neurotransmission; and an increase in the number of [3H]glutamate binding sites in synaptic membrane preparations from hyperammonemia rats and from rats with portocaval shunts. Such neurochemical changes may be of pathophysiological significance in hepatic encephalopathy.

Effect of pulse exposure to methoxychlor on brain serotonin levels in american flagfish (Jordanella Floridae, goode and bean) as modified by time after exposure, concentration and gender

AbstractAdult American flagfish (Jordanella floridae) were exposed for 2 h to 1.6, 1.9, 3.1 or 5.1 mg L−1 of the chlorinated hydrocarbon insecticide methoxychlor. Control and ethanol‐carrier control treatments were also maintained. Fish were sampled at 2, 6, 24, 48 and 336 h after exposure, and the concentrations of tryptophan, serotonin and 5‐hydroxyindole acetic acid in brain tissue were determined. Flagfish exposed to the three highest concentrations of methoxychlor had significantly lower concentrations of serotonin, relative to controls, 2, 6 and 24 h after exposure. Serotonin concentrations had returned to control levels by 48 h. The period of serotonin depression corresponded with observed convulsive activity in the exposed fish. Female flagfish had significantly higher brain levels of serotonin than did males. Although tryptophan and 5‐hydroxyindole acetic acid levels were unaffected by methoxychlor exposure, the levels of both were significantly depressed by the concentration of ethanol carrier (270 mg L−1) used. Serotonin levels were unaffected by ethanol. The brain weight of flagfish was found to be related to body weight by the equation brain weight (mg) = 2.53 + 5.72 body weight (g) (n = 139; r2 = 0.74; p &lt; 0.001).

Simple automated gas chromatographic analysis of amino acids and its application to brain tissue and urine

The separation of 21 free amino acids as their isobutyl N(O,S)-pentafluorobenzoyl derivatives by capillary gas chromatography with electron-capture detection is described. This method has been successfully applied to routine analysis of most of these amino acids in brain tissue and urine. The amino acids are converted to their isobutyl N(O,S)-penta-fluorobenzoyl derivatives by acid-catalysed esterification followed by aqueous extractive pentafluorobenzoylation. The 21 amino acids can be separated on a single capillary column in 21 min. The high sensitivity permits analysis in minute quantities of sample and the high stability permits the use of an automatic sample injector at ambient temperatures. Thus, over 50 samples can be readily analyzed in a single day.